Covid Commentaries January 2022-December 2022 by Access-Health

The COVID Commentaries A Chronicle of a Plague Personal Commentaries on COVID-19 from January to December 2022

WILLIAM A. HASELTINE, PhD

Copyright © 2023 by William A. Haseltine, PhD All rights reserved. No part of this book may be used or reproduced by any means, graphic, electronic, or mechanical, including photocopying, recording, taping, or by any information storage retrieval system, without the written permission of the publisher except in the case of brief quotations embodied in critical articles and reviews. All author proceeds from the sale of the COVID Commentaries will be donated to the nonprofit global think tank ACCESS Health International.

Thank you for purchasing the COVID Commentaries: A Chronicle of a Plague. This book is a Living eBook. It will be updated regularly with new resources and information about COVID-19. When you purchased this book, you also purchased access to every subsequent edition of the book, as it is released. To access future editions, please visit: www.williamhaseltine.com/commentaries You’ll be taken to a special section of our website where you can download the most up to date version.

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Introduction

In early 2020, I was working on the final chapters of my autobiography, My Lifelong Fight Against Disease. It was then that I heard the first rumblings of concern about a new pneumonia-like illness coming from Wuhan, China. COVID-19 has gone on to infect at least twenty five million people worldwide and, as of this writing, can be tied to nearly one million deaths. Despite the breadth of damage COVID-19 has already wrought, we are only beginning to glimpse what its long term toll might be. There is no doubt though that the impact of the pandemic will be massive, indelibly imprinting and changing the behaviors of our rising generations. My personal losses have been far fewer than what many have suffered. Yet COVID-19 has still completely upended my life. From the very beginning of the outbreak, my friends and colleagues have come to me for counsel. Some wanted to know the best way to protect their families. Some wanted to discuss the science behind the virus itself. Still others sought to commiserate over how poorly our leaders seemed to grasp the seriousness of the pandemic. In most all cases, I found I had something to offer. Today after a lifetime spent in science, medicine and pursuing better public health, I find myself once again logging eighteen hour days battling a new and still somewhat unknown disease. I am working closely with three generations of students—my former students, their former students and those who studied under them— to understand the virus and what makes it tick. Together, and along with many in the private sector I have worked with over the years, we are doing what we can to create new diagnostic tests, new drugs and new vaccines. I have also been asked by governments all over the world – in Europe, Asia, the Middle East, Africa and here in the United States – for advice on the best policies and containment measures. Equally important is my effort to advance the public’s understanding of SARS-CoV-2 and how to defeat it. On January 22, a day after the first COVID-19 case was diagnosed in the United iv

States, President Donald Trump declared on national television that the U.S. had the virus “totally under control.” In fact, no coordinated plan was in place to contain an outbreak that by then might have been spreading within our borders for weeks. A week after that hollow declaration, I published an article in Scientific American, “Want to Prevent Another Coronavirus Epidemic? We need to treat it as the deadly biological threat that it is.” Since then, my opinions about ongoing news, research and other developments in the pandemic have been sought regularly, many times a week, in live conversations with anchors at major cable news networks MSNBC, CNN, Fox and international networks like Bloomberg Asia and CGTV. My commentaries on COVID-19 have been published online and in print at the Washington Post, Los Angeles Times, Project Syndicate, CNN.com, Forbes.com and Psychology Today. I have interviewed dozens of physicians, scientists and economists as they published new research or proposed new ideas. Suggestions I offered on their approaches often enabled me to discover new details that in turn deepened my understanding of the virus, our attempts to contain it and the long term effects now unfolding. The COVID Commentaries contain a collection of my writings, research and interviews on COVID-19. This is a Living eBook, updated regularly with new information on the disease and our response as it unfolds. It is organized into three sections. The first contains my published commentaries on the outbreak, giving you an in-depth account of the most pressing topics at the time. The second section provides links to my media interviews and mentions in news articles. Finally, at the end of the book, you’ll find a collection of my social media posts, offering a quick, spur of the moment glimpse into my thinking day to day. For a robust assemblage of COVID-related news, medical and scientific research papers, webinars and online resources from other organizations, feel free to head over to my website: https://covid19resources.org/ I first coined the term Living eBook when I published A Family Guide to COVID: Questions & Answers for Parents, Grandparents & Children in June 2020. At the time, I was acutely aware of how frustrated, confused and frightened many people were over what was happening in the world, their world. My goal in writing the book v

was to simplify the science and make as much information as possible available to anyone looking for answers to those questions and others like them. I also wanted to make sure that the information in the book was as accurate as possible. I knew that as the science and our understanding of the virus evolved, so too would my recommendations of how best to protect ourselves and those we love against infection. I soon found that no publishing house would be able to deliver a book as quickly as we needed or update it as regularly as I wanted so I created a new genre of books. The Family Guide and my subsequent book, A COVID Back To School Guide: Questions and Answers for Parents and Students, were the first books published as Living eBooks. They are free on the ACCESS Health website and available in print on Amazon. Each time new discoveries happen, we update the books immediately on our website and within a few days through the major retailers. The Commentaries follow the same format. When this pandemic is finally over, as it no doubt will be one day, my hope is that my Commentaries will provide the historical perspective to help us reckon with mistakes we have made, especially in the United States, and provide insights into how we might better contain the next contagion and reduce the number of lives ultimately, needlessly lost—a modern day version of Samuel Pepys’s Plague Diary.

Table of Contents Introduction ................................................................ iv Personal Commentaries ................................................. 1 January, 2022 .................................................................................... 2 Difficulties Of Single Monoclonal Antibody Treatment Of SARSCoV-2: The Sotrovimab Experience In Australia ........................... 3 Breaching The Barrier That Protects Children From Serious Covid19-Related Disease ........................................................................ 7 New Insights Into Lung Damage And Repair Relevant To Covid-19 .....................................................................................10 New Potential Covid Virus Variants Of Concern .........................13 The Challenges of Treating COVID-19: Lessons from Gilead’s Remdesivir ..................................................................................19 The Case For Third And Fourth Booster Shots.............................22 Omicron: Less Virulent But Still Dangerous .................................24 Time to Expand Vaccination to Infants and Toddlers and Boosters for Children .................................................................................29 We Can’t Stop Reporting Covid Cases ........................................32 Endemic Disease Does Not Necessarily Mean Mild Disease ..........34 Inhaled Remdesivir for Home Use for Covid Treatment ..............37 New Data Suggests That 50% Of Omicron Infections In Healthy Young Men Remain Transmissible After Five Days ......................41 Major Protease Of SARS-CoV-2 Acts To Suppress Innate Immunity .....................................................................................45 Novel Antiviral Approach To Covid-19 Treatment ......................49 The Case for Vaccination Against the Omicron Variant ................53 The Danger Of Covid-19 Monotherapy: Drug Resistance ............54 New Clues To Long Covid: Prolonged Inflammatory Response ...57 Birth Of The Omicron Family: BA.1, BA.2, BA.3. Each As Different As Alpha Is From Delta..................................................60 Functions Of SARS-CoV-2 NSP12 Polymerase: Replication, Transcription, And Suppression Of Natural Immunity ..................66 Functions Of SARS-CoV-2 NSP13 Helicase: Replication, Transcription, And Suppression Of Natural Immunity ..................69 Understanding Omicron: A Structure-Function Tour De Force ...72 Do Cannabinoids Offer A Treatment For Covid-19? Maybe. .......79 Novel Vaccine Booster Strategy Prevents Covid-19 Infection .......82

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February 2022.................................................................................. 88 'Decoy' Protein Offers New Treatment Approach For Covid-19 .. 89 The Omicron Surprise ................................................................. 92 Covid Infection During Pregnancy: What Are The Risks?.......... 101 Good News: Full Vaccination Protects Against Omicron Hospitalization And Death ......................................................... 104 Quantum Leap In Newborn Whole Genome Sequencing .......... 110 Losing the Sense of Smell: How Covid-19 Infection Induces LongLasting Symptoms ...................................................................... 113 The SARS-CoV-2 Genome: The Importance of the Termini .... 118 Do Not Underestimate The Consequences Of SARS-CoV-2 Escape: The Omicron Example .................................................. 126 Protection In The Present From The Deep Past ......................... 135 Whither the Omicron Family: BA.1, BA.1.1, BA.2, BA.2.H78Y, BA.3?......................................................................................... 138 Pericyte Damage: Surprising Cause of Covid-Related Myocarditis ........................................................ 145 Op-Ed: Will the next coronavirus variant escape our best immune defenses? .................................................................................... 150 March 2022 ................................................................................... 153 Whence And Whither Covid-19: The Many Faces Of Omicron 154 Covid-19 Has Orphaned 5.2 Million Children ........................... 161 Covid-19 Pathogenesis: Spike Protein Alone Can Damage Cells. 163 Covid Ping-Pong: Human To Deer, Deer To Human ............... 168 Antibody-Activated Endothelial Cells Increase the Risk of Blood Clots with Covid-19 .................................................................. 174 Covid-19 Damage To The Male Reproductive Tract ................. 178 Covid-19 Infection Increases Heart Disease Risk, Even In Mild Cases .................................................................... 183 Stripping Covid's Camouflage .................................................... 187 A New Process Of SARS-CoV-2 Variation Uncovered: Intragenomic Recombination..................................................... 195 Covid-19: Long Term Brain Injury ............................................ 202 An Ancient Form Of Immunity Protects Many Against Covid-19 Today ........................................................................................ 206 An Omicron-Omicron Recombinant—BA.4 ............................. 213 A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells.................................................................... 217 En Garde For SARS-CoV-2 Chimeras (Recombinants) ............. 223 Antibodies Team Up Against Omicron ...................................... 228 Innate And Adaptive Immunity In The Human Intestine............ 234 How Your Gut Protects You from Myriads Of Microbes. .......... 238

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A Primary Defense Against SARS-CoV-2: Defensins .................. 243 Imaging the Brain: What Brain Scans Reveal About the Consequences of Covid-19 ........................................................ 248 Innate Lymphoid Cells (ILCs): Guardians Against Infection ........ 254 New And Effective Monoclonal Antibody Treatment For Ebola On The Horizon .............................................................................. 259 Faster And Stronger. ................................................................... 265 Hide And Seek: How Group 3 Innate Lymphoid Cells Help Cloak ACE2 Receptors From SARS-CoV-2 ........................................ 269 April 2022...................................................................................... 275 Coronaviruses Can Recombine With Cellular And Heterologous Viruses To Create Unexpected Variants ...................................... 276 The Artful Dodger SARS-CoV-2: Evading Immunity By Inhibition Of Interferon ............................................................................. 281 Quantitative Markers for Covid-19 Brain Injury ......................... 286 Two More Members Of The Omicron Family To Keep An Eye On ............................................................................................. 291 Innate Lymphoid Cells And Covid-19 Severity: Chicken Or The Egg? ................................................................ 295 Head in the Clouds: Living with Covid-19 ................................. 301 S100s: A Blunderbuss Approach To SARS-CoV-2 Defense ........ 305 In the Eye of the Storm: How Covid-19 Impacts the Eye ........... 310 Two Emerging Viral Adversaries—Nipah And Hendra Virus—May Soon Meet Their Match ............................................................. 314 Remdesivir Resistance Reported Upon Treatment Of An Immunocompromised Patient..................................................... 320 New Analysis Shows Fluvoxamine Has The Potential To Reduce Covid-19 Hospitalizations By More Than 90% ........................... 324 ‘Test-to-treat’ could transform the pandemic .............................. 327 SARS-CoV-2 Actively Infects And Kills Lymphoid Cells ........... 330 Six Ways Fluvoxamine May Act To Prevent Severe Covid-19.... 334 New Members Of The Omicron Family Of Viruses: BA.2.12.1, BA.4, And BA.5 ......................................................................... 339 Moral Injury Is Similar in Healthcare Workers and Veterans ....... 346 SARS-CoV-2 Infection Of Monocytes Triggers Inflammation ... 350 STING: It Takes Two To Tango ............................................... 355 Survival Of The Fittest: The Rise Of BA.2.12.1 ......................... 362 SARS-CoV-2 Mimics Inflammatory Proteins In Our Body ........ 366 STING-Mediated Innate Immunity: How One Discovery Unlocked New Possibilities ........................................................ 371 SARS-CoV-2 Infection Causes Cell Fusion In The Lungs, Triggering Inflammation. ........................................................... 377

The Role Of N Protein Mutant Variants As Determinants Of SARS-CoV-2 Replication And Pathogenesis ............................. 382 The Center For Forecasting And Outbreak Analytics Could Represent A New Era In Infectious Disease Control, But Challenges Lie Ahead ................................................................. 388 Additional Support Arrives For The Covid-19 Test-To-Treat Program ..................................................................................... 391 35B5: A Potent, Broadly-Neutralizing Monoclonal Antibody Effective Against All Known Variants ......................................... 394 May 2022 ...................................................................................... 400 Asthma Medication Points The Way To Drugs To Prevent And Treat Covid-19: Nsp1, A Novel Target ..................................... 401 Structural Analysis Of The Omicron Spike Unveils Houdini-Like Immune Escape.......................................................................... 406 New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part II .................................................. 412 Far-Ultraviolet Light Is Another Way To Keep Our Public Spaces Safe ............................................................... 419 Study Shows Unvaccinated People Are At Increased Risk Of Infecting The Vaccinated ........................................................... 422 Omicron Transmission And Immune Evasion Explained By The Omicron Spike’s Unique Structure............................................. 425 Scientists Discover Genetic Cause of Lupus, Findings May Help Research on Long Covid. .......................................................... 431 Targets for Infection: How SARS-CoV-2 Damages the Kidneys 437 New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part III ................................................ 442 A Dynamic Duo: How ACE-2 And CD147 Mediate Covid-19 Infection In The Kidneys ........................................................... 448 FDA Turns Down Fluvoxamine Emergency Use Authorization Request ..................................................................................... 451 The Growing Threat Of Tick-Borne Disease Part I: Powassan Virus ................................................................ 453 Expelling COVID: It Takes Longer Than You May Think ........ 460 June 2022 ...................................................................................... 464 The Vaccinia Virus That Hopped From Rabbits to Hares ........... 465 Tick-Borne Disease Part II: CrimeanCongo Hemorrhagic Fever ........................................................ 469 Rabbitpox: the Story of a Specialized Killer ................................ 475 Study Finds Previous Covid-19 Infection Doesn’t Protect Children From Omicron .......................................................................... 481

Unraveling Hope For Prion Disease And Other Progressive Neurodegenerative Diseases........................................................ 483 Paxlovid Resistance: Challenges And Opportunities.................... 489 The Virus Is Still Winning .......................................................... 492 This is how we should be diagnosing long COVID .................... 496 There May Be A New Polio Epidemic On Its Way- If So, What We Can Do ............................................................................... 499 Attack and Counterattack: How SARS-CoV-2 Blocks Our Natural Immune Defenses ....................................................................... 505 Is It Safe To Fly? The National Academy Of Sciences, Engineering, And Medicine Weighs In ........................................................... 511 Don’t You Wish You Didn’t Need To Breathe Someone Else’s Exhaled Air While Flying?.......................................................... 514 Flying Under The Radar: How SARS-CoV-2 ORF7a Contributes To Immune Evasion And Inflammation ...................................... 517 Medical Science Must Go Hand In Hand With Social Science For Effective Control Of Covid-19 And Other Pandemics ................ 523 There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part II.................................................................... 525 Biggest Risk Factor for Severe Covid-19 Other than Age? Autoantibodies ........................................................................... 529 The Future Of Aviation In A Covid And Post-Covid World ...... 534 What We Can Learn From Other Poxviruses About Monkeypox ..................................................................... 538 Study Uncovers A New Way To Think About Alzheimer’s Disease .................................................................... 541 Carbon Dioxide Levels May Predict Covid Risk In Your Immediate Surroundings............................................................. 544 SARS-CoV-2 Spike: A Potential Biomarker For Long Covid ..... 546 Covid-19 During Pregnancy: Increased Risk Of Preterm Delivery And Infant Neurodevelopmental Issues ....................................... 550 There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part III .................................................................. 554 Origin Of Virus Variation: Real-Time Evolution Of SARS-CoV-2 In An Immunocompromised Patient........................................... 557 How Common Is Long Covid? More Common Than You’d Think ..................................................................... 561 July 2022 ....................................................................................... 564 The Many Faces Of Omicron..................................................... 565 NYC To Offer Instant Access To Paxlovid At Mobile Testing Sites, But Messaging Around The Drug Needs To Improve ................ 570 Research On Flies Provides Hope For Brain Repair ................... 572

BA.2.75: A Dark Horse In The Covid Pandemic........................ 578 Long Covid And Its Unequal Burden ......................................... 586 More Danger Ahead With BA.5: Covid-19 Reinfection Doubles The Risk For Death, Blood Clots, And Lung Damage ............... 590 Pasteur Institute Scientists Discover SARS-CoV-2 BroadlyNeutralizing Antibody................................................................ 594 Robotic Arms Allow Partially Paralyzed Man To Feed Himself .. 599 Should You Get Vaccinated For Covid-19? Yes. Will It Protect You From Long Covid? Probably Not. ...................................... 603 Are Chronic Infections Responsible For SARS-CoV-2 Variants? 606 New SARS-CoV-2 Variant BA.2.75 Evades All Approved Monoclonal Antibody Therapies ................................................ 612 How to advocate for yourself as you struggle with long COVID 616 Can Inflammation Lead To Cognitive Issues? Past Research Says 'Yes', With Implications For Covid-19 ....................................... 618 Learn from past pandemic mistakes and authorize second boosters for all ......................................................................................... 625 New-Wave Materials Help Create Mini Functional Kidneys ...... 627 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies III............................................................................. 631 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies IV ............................................................................ 635 A Cooling Implantable Device For Pain Relief........................... 640 August 2022 .................................................................................. 648 Even Mild Covid-19 May Cause Lasting Brain Fog (Part 1)........ 649 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies V .............................................................................. 654 Even Mild Covid-19 May Cause Lasting Brain Fog (Part 2)........ 659 Covid-19, Gender And Immune Response: What’s The Relationship? (Part Two) ........................................................... 663 Even Mild Covid-19 May Cause Lasting Brain Fog (Part 3)........ 668 Loss Of Smell Linked To Long Term Covid Cognitive Impairment ................................................................ 673 Advanced Technology (CRISPR) Shows That Mucus Is Your Body’s First Line Of Defense Against Viruses.............................. 675 Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 1) ...................................................................................... 680 Fixing Our Public-Health Data Problem .................................... 686 Controlling COVID-19: What Can We Learn From Previous Pandemics? ................................................................................ 689 An End To HIV In Botswana, Why Can’t We Accomplish The Same In The US? ....................................................................... 696

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Will Covid-19 Vaccines Continue To Protect Us From Hospitalization And Death?? ....................................................... 700 Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 2) ....................................................................................... 706 How To Mend A Broken Heart ................................................. 713 Health Messaging in the Disinformation Age .............................. 719 Solving The Question Of Covid Variant Increased Fitness Is Like Deciphering A Rubik’s Cube ..................................................... 723 T Cells Play An Important Role In Immunity, But Without B Cells Cannot Provide Protection From Covid-19................................ 727 Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 3) ....................................................................................... 734 Increased Disease Potential Of Covid Variant BA.5 Currently Circulating In The United States ................................................ 741 September 2022 ............................................................................. 747 Five Days Is Too Short! Study Says To End Covid-19 Isolation Based On Rapid Tests Results Instead ........................................ 748 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VI ............................................................................. 754 Hopeful New Entry In The Race For A Universal Covid Vaccine ........................................................ 759 Long Covid Is Keeping Millions Of People Out Of Work.......... 763 Covid-19 Infection Increases Risk And Excess Burden Of Cardiovascular Disease ................................................................ 766 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII ........................................................................... 771 Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 4) ....................................................................................... 778 Public-Health Funding Today Keeps the Doctor Away .............. 784 Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VIII .......................................................................... 787 Molecular Jujitsu: SARS-CoV-2 Co-Opts Host Defense ............ 791 Nasal Vaccines May Not Be The Game Changer We Think They Are ............................................................................................ 798 Will The BA.4.6 Variant Drive The Next Wave Of The Covid-19 Pandemic? .................................................................................. 801 Reimagining Alzheimer’s (Part 1) ............................................... 809 Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 5) ....................................................................................... 815 SARS-CoV-2 Main Protease Suppresses Innate Immunity By Cleaving Proteins Required For Interferon Induction And Inflammation .............................................................................. 820

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Waiting In The Wings: A Potential New Variant Of SARS-CoV-2 ........................................................... 828 We need resilient health systems to address the dual crisis of infectious and chronic diseases .................................................... 834 Is A Universal Influenza Vaccine —One Shot For All Strains— On The Horizon? ............................................................................ 837 CDC Study Says Isolating In A Separate Room Is Most Effective Against Household Transmission Of SARS-CoV-2 .................... 846 Covid Virus Accelerates With Each New Variant ....................... 848 CDC Study Says Isolating In A Separate Room Is Most Effective Against Household Transmission Of SARS-CoV-2 .................... 852 October 2022 ................................................................................ 854 Two Are Better Than One: Expanding Our Covid-19 Vaccine Antigens .................................................................................... 855 Saline Nasal Irrigation After Covid-19 Diagnosis Reduces Hospitalization ........................................................................... 861 Reimagining Alzheimer’s (Part 2): Breaking The Barrier ............ 863 Broadly Neutralizing Monoclonal Antibodies For Covid-19 Treatment, Prevention, And Vaccine Design .............................. 868 Supercharging mRNA Vaccines With Self-Amplifying RNA Technology ............................................................................... 877 Self-Amplifying mRNA Vaccine Receives EUA Nod From Indian Regulators ................................................................................. 883 Progress In The Search To Reverse Age-Related Vision Loss ..... 889 Children Experiencing Respiratory Trouble Post-Covid-19 Improve With The Use Of An Inhaler And Exercise Regimen... 894 Reimagining Alzheimer’s (Part 3): The APOE Story In Alzheimer’s And Other Diseases .................................................................... 897 CAR T Therapy [Part III]: A New Direction for Multiple Myeloma Treatment .................................................................................. 902 Introducing A “Naked” Self-Amplifying RNA Vaccine Candidate ..................................................................... 908 How Enhanced Ventilation And Air Filtration Can Fight Covid-19 ................................................................... 914 Broadly Neutralizing SARS-CoV-2 Antibodies From Immunized Macaque Monkeys ..................................................................... 918 Reimagining Alzheimer’s (Part 4): Cautious Optimism For A New Alzheimer’s Disease Treatment ................................................... 924 As Protection From Current Covid-19 Monoclonal Antibody Treatments Fades, The Discovery Of A New Class Of Antibodies Brings Hope .............................................................................. 927

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The Dramatic Cost Of The Covid-19 Pandemic: A Historical Drop In Life Expectancy ..................................................................... 933 How Strength Training Can Help Post-Covid Recovery ............ 937 Salt, Fat And Sugar: How Americans Became Addicted To Eating .................................................................... 940 Covid-19 Infection Linked With Poor Cardiovascular Outcomes And Death ................................................................................. 944 November 2022 ............................................................................. 946 CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example ........................................................................... 947 "Naked" Self-Amplifying RNA Vaccine Shows Promise As A Booster, Inducing Strong Cytotoxic T-Cell Responses ............... 952 New Monoclonal Antibody Cocktail Neutralizes Lassa Virus ...... 963 Study Finds That Regular Physical Activity Enhances Vaccine Effectiveness Against Covid-19 ................................................... 968 Female Healthcare Workers Have Faced Heightened Moral Distress ............................................................................ 972 CAR T Therapy For Cardiac Fibrosis: A New Method .............. 976 The Best Reason To Keep Up To Date With Boosters: Covid-19 Protection From Infection, Hospitalization And Death All Wane Over Time ................................................................................. 984 Reimagining Alzheimer’s (Part 5): Setback For Potential Alzheimer’s Treatment ............................................................... 989 New Monoclonal Antibody For Treatment Of Malaria ............... 992 CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment .................................................................................. 996 Do You Have The “Thin” Gene? ............................................ 1002 Hope For A New Treatment On The Horizon For Zika Virus . 1005 The N Protein, A New Target For Anti-Covid Drugs .............. 1010 Covid-19 And Children: What We Know................................ 1015 December 2022 ........................................................................... 1019 Crimean-Congo Hemorrhagic Fever Virus Monoclonal Antibodies: A Work In Progress.................................................................. 1020 Reimagining Alzheimer’s (Part 6): The Many Effects Of The APOE4 Variant ........................................................................ 1025 Symptomatic Covid-19 Infection Is Associated With An Increased Risk Of Overactive Bladder Symptoms .................................... 1030 Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T .................................................................................... 1033 Covid-19 Treatments Like Paxlovid Are Being Underused ....... 1038

Why The Covid Vaccines Work And How To Make Them Better .................................................................. 1040 The Fat Truth: You Feel It In Your Gut .................................. 1047 Six Potential Causes of Brain Fog in Covid-19 Patients ............ 1051 Extended Antigen Availability Improves Vaccine Protection ..... 1055 Cholesterol Abnormalities May Contribute To Alzheimer’s Disease ............................................................ 1062 CAR T Therapy To Treat And Cure Rheumatoid Arthritis ..... 1068 Successes Like The Covid-19 Vaccines Come From Long-Term Investments In Public Health.................................................... 1073 New Insights Into The Postfusion Structure Of SARS-CoV-2 Spike Protein..................................................................................... 1075 CAR T Therapy, A Promising New Therapy For Multiple Sclerosis? .................................................................................. 1084 New Monoclonal Antibody Fully Approved For The Treatment Of Covid-19 ................................................................................. 1089 African Morgue Data Reveals A More Significant Covid-19 Death Toll Than Official Reporting ................................................... 1092 How Recently Approved Tocilizumab Treats Covid-19........... 1094 Synthetic Gene Circuit To Hone CAR T Therapy .................. 1097

Media Inerviews .......................................................1102 Lifting Mask Mandates ............................................................. 1103 Discovery of New HIV Variant Sends Warning for COVID Pandemic ................................................................................. 1106 Mask Mandates ........................................................................ 1109 Impact of Long Covid on the Climate Crisis ............................ 1114 President Biden's "Cancer Moonshot" Initiative ........................ 1119 How the Pandemic Shortened Life Expectancy and New Drugs on the Horizon: COVID, Quickly, Episode 40 ............................. 1124 The Quest For New COVID-19 Solutions .............................. 1125 Simone Fishburn Interviews William A. Haseltine at BioFuture 2022 ................................................ 1132 Ask the Doctor: Will We See A “Triple-demic” of COVID, Flu, and RSV? Plus, the Latest on the Zero-COVID Policy Protests in China ........................................................... 1136

Media Mentions .......................................................1137 Covid Cases Are Still Rising. Here’s When They Could Peak. . 1138 New HIV Variant Discovered: May be More Infectious and Severe ............................................................... 1139

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4th shots and COVID-19 boosters: Why and when best to get it..................................................... 1140 I'm a Doctor and These COVID Symptoms Alarm Me............. 1145 Worried About Long COVID? You May Be at Less Risk if You’ve Had Omicron Variant .............................................................. 1148 Longtime HIV patient is effectively cured after stem cell transplant ........................................................................... 1151 As Anthony Fauci announces retirement, experts weigh in on how history will remember him ....................................................... 1153 How To Stay Protected Against Polio ...................................... 1155 How President Biden Can Meet His Pledge to End HIV by 2030..................................................................... 1157 Without a nasal vaccine, the U.S. edge in fighting Covid is on the line .................................................................. 1160 Promising Pfizer RSV Vaccine for Pregnancy May Protect Newborns ................................................................................ 1163 Coronavirus variants are dodging antibody treatments. New labmade options may help. ............................................................ 1164

Social Media ........................................................... 1165 January 2022 ................................................................................ 1166 February 2022 .............................................................................. 1174 March 2022 ................................................................................. 1178 April 2022.................................................................................... 1190 May 2022 .................................................................................... 1202 June 2022 .................................................................................... 1210 July 2022 ..................................................................................... 1228 August 2022................................................................................. 1246 September 2022 ........................................................................... 1259 October 2022 .............................................................................. 1276 November 2022 ........................................................................... 1295 December 2022 ........................................................................... 1306

Acknowledgements .................................................. 1319

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Personal Commentaries

January, 2022

Difficulties Of Single Monoclonal Antibody Treatment Of SARS-CoV-2: The Sotrovimab Experience In Australia Forbes | January 4, 2022 | Article

Spike protein with sotrovimab monoclonal antibody in purple. CORTI ET AL.

The SARS-CoV-2 Omicron variant has spread around the globe at unprecedented speed. Neither prior infection nor multiple vaccinations impede transmission. One hope was that early treatment with monoclonal antibodies for those most susceptible to serious disease would reduce hospitalization and death. 3

Unfortunately, Omicron has proved to be resistant to most FDAapproved monoclonal antibody treatments. Laboratory experiments suggested that at least one monoclonal antibody, sotrovimab, retained significant activity. Unfortunately, recent data from Australia raises serious issues of sotrovimab as a stand-alone treatment. Rockett et al. demonstrate that resistance to sotrovimab as a monotherapy rises rapidly in treated patients. Moreover, they worry that such viable variants may also be highly resistant to existing vaccines. Rockett et al. determined the sequence of virus isolated from patients treated with sotrovimab. A patient cohort of 100 received sotrovimab at a treatment center in New South Wales, Australia. The treatment was a single 500mg dose targeted at patients within five days of symptom onset thought to be at risk for severe disease progression. Of the initial 100 patients, 23 tested positive for SARSCoV-2 infection at least 10 days post-infusion, and of these, the preand post-infusion respiratory tract samples of eight were collected. Of these eight, seven were hospitalized, six were partially vaccinated or unvaccinated, and four were given additional antibody treatment (Table 1).

TABLE 1: Patient demographics and details. ROCKETT ET AL.

Viable virus samples obtained from four of the patients contained a mutation at amino acid E340 of the spike protein, either E340K, 4

E340A, or E340V (Table 1). The virus in one patient contained an additional mutation: the P337L mutation. These mutations are predicted to yield virus resistance to sotrovimab. Analyses of mutations in this region of the Spike, the receptor-binding domain, indicate that the observed mutations decrease sotrovimab neutralization by almost 300 fold. Sotrovimab, like many other SARS-CoV-2 monoclonal antibodies in development, targets highly conserved regions of the Spike protein. This strategy is an attempt to create pan-variant neutralizing treatments. Residues 337 and 340 are both among the most highly conserved epitopes in the SARS-CoV-2 Spike protein. Each is only found mutated in a few hundred of the millions of sequences in the GISAID sequence database. Rockett et al. note that in addition to monoclonal antibody resistance, similar resistance may be found in vaccine-elicited neutralizing antibodies. The experimental evidence showing sotrovimab treatment induces dangerous mutations suggests we should conduct similar experiments with current generations of vaccines as the subject. This study provides several sobering lessons: Resistance to singular antibody treatment can develop rapidly in high-risk patients. Mutant viruses that persist for ten days or more may transmit further antibody and vaccine-resistant infections. In vitro neutralization activity provides little assurance for treatment efficacy. Effective therapy will require the use of two or more monoclonal antibodies each targeting different epitopes. The rapid appearance of antibody resistance should come as no surprise for those who follow the literature on the use of convalescent sera to treat persistently infected patients. Variants resistant to the neutralizing activity of such polyclonal sera develop in a matter of days to weeks. Some speculate that Omicron arose from such a patient. The data of Rockett et al. also serve as a warning for the use of monotherapy low molecular weight antiviral drugs such as paxlovid and molnupiravir. Laboratory experiments demonstrate that resistance to these drugs does arise. We should not be surprised if such resistance develops rapidly. 5

We conclude by noting that molnupiravir may create even more vaccine and monoclonal antibody drug-resistant variants than sotrovimab. The drug is a polymerase inhibitor that induces a destructive number of mutations to the virus. The possibility of molnupiravir introducing new mutations to the virus, without neutralizing the virus, seems a significant risk. In vivo human trials akin to the one examined here could shed light on this dangerous possibility. This article originally appeared on Forbes, and can be read online here: Difficulties Of Single Monoclonal Antibody Treatment Of SARS-CoV-2: The Sotrovimab Experience In Australia

Breaching The Barrier That Protects Children From Serious Covid-19-Related Disease Forbes | January 4, 2022 | Article

This is part of a series called “How SARS-CoV-2 Delays, Evades, and Suppresses the Immune System.” Read parts 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. One of the many mysteries of Covid-19 that continues to perplex us is the resistance of young children, following SARSCoV-2 infection, to disease. With data released last week suggesting children may be less protected against Omicron than previous SARS-CoV-2 variants, more than ever we need clarity on what drives risk and protection in younger populations. Why the virus largely spares children was anyone’s guess, until now. A new study, peer-reviewed and published unedited in Nature in December, offers evidence that natural immunity is responsible for children’s enhanced defense against disease. Natural immunity, also known as innate immunity, describes the body’s ability to respond to invasions by novel antigens regardless of prior exposure. Individual infected cells first respond by triggering the production of interferon, which in turn unleashes a veritable storm of antimicrobial genes and proteins, in addition to warning neighboring cells to prepare for impending infection. Over time interferon and other interferon-stimulated genes provide key signals to initiate the adaptive immune response. Since the beginning of the pandemic experts have postulated that innate immunity, like the immune system writ large, becomes less robust and dexterous with age. In this study, Yoshida et al. set out to determine which factors contribute to deterioration in the face of Covid-19 specifically, by comparing the pathogenesis of cohorts of adult and pediatric patients. The findings of Yoshida et al. boil down to three main observations. Firstly, the natural (innate) immune response in children seems to be pre-activated in nasal tissues, enabling immune cells to resist the more dire consequences of infection via robust 7

interferon activity. Interferons are a class of signaling proteins that, upon detecting the presence of a foreign intruder, mobilize legions of interferon-stimulated genes that alert surrounding cells to ongoing infection. Previous studies show that in adults, SARS-CoV-2 deploys an array of immunosuppressive mechanisms that target interferon production by direct and indirect means, delaying the onset of the innate immune response. Suppression of interferon production allows the virus to replicate faster and for longer, reaching higher viral loads in a shorter period of time. According to Yoshida et al., no such delays were observed in pediatric patients, whose nasal immune cells exhibited robust interferon signaling both prior to and during SARS-CoV-2 infection, resulting in “much faster induction and clearance of interferon-stimulated cells.” Secondly, Yoshida et al. noted that adult Covid-19 patients, relative to pediatric patients, exhibited dense clusters of interferonstimulated genes in the blood. The appearance of specific subpopulations of interferon-stimulated genes coincided with symptom onset, suggesting that during the acute phase of infection is when the innate immune response flares up to regain control. This tracks with existing models of disease progression that pinpoint the peak of SARS-CoV-2 viral load right before symptom onset, before the immune system kicks in. According to Yoshida et al., the correlation between interferon-stimulated gene expression and symptom onset is altogether absent in pediatric patients. Interferonstimulated genes still surfaced in abundance in some asymptomatic children, but varied temporally in their manifestation compared to adults, indicating a swift rate of induction and viral clearance. The third and final observation is that pediatric Covid-19 patients have an immunological landscape that is simultaneously more naive and diverse than that of adults, meaning their antibodies differentiate and mature more readily in the face of SARS-CoV-2 infection. The older we get, the more memory cells our bodies accumulate—good for defending against familiar foes, not so good against the unknown. Without as many naive lymphocytes available to differentiate into anti-SARS-CoV-2 antibodies, the adult immune response must harness the brute power of cytotoxic T cells to fight infection. The kinetics of viral replication and clearance thus remain largely localized to the airways in children, whereas in adults

immunosuppression can lead to the development of multi-organ systemic disease and increased cytotoxicity in the blood. Data from South Africa, the United Kingdom, and the United States suggests that Omicron may have developed the ability to breach the natural (innate) immune defenses of children and young adults. It is thus imperative to both understand these defenses in greater detail and to develop new methods of strengthening natural immunity and inactivating the virus’ immunosuppressive activity. By my count, there are more than 20 different mechanisms SARS-CoV-2 deploys to suppress the induction of interferon and interferon-stimulated genes, as well as MHC-I. Each one of these mechanisms makes an attractive target for therapies that can protect children and adults from infection and disease. We must harness the knowledge and technology we have at our disposal to change the course of the pandemic for the better. Otherwise 2022 won’t look much different from 2021, with more and more infectious variants destabilizing our best efforts to recover from the damage of the past two years and forge a way forward. This article originally appeared in Forbes, and can be read online here: Breaching The Barrier That Protects Children From Serious Covid-19Related Disease

New Insights Into Lung Damage And Repair Relevant To Covid-19 Forbes | January 6, 2022 | Article

Lungs that have sustained severe damage from diseases such as Covid-19 or Idiopathic Pulmonary Fibrosis (IPF) are characterized by the abnormal presence of basal cells in the tiny air sacs, known as alveoli, of the lungs. These misplaced basal cells interrupt the healing process, often leading to impaired lung function and even death. Researchers at the University of California, San Francisco set out to learn why and how this happens. Released in Nature Cell Biology and co-led by Jaymin Kathiriya, Ph.D. and Chaoqun Wang, Ph.D., the report details a novel stem cell pathway in seriously injured lungs; specifically, the researchers discovered that human alveolar epithelial type 2 cells (hAEC2s) can turn into basal cells in response to signals sent by damaged and scarred mesenchyme tissue. Our lungs are massively complex organs with many different components. The alveoli represent one part of this equation, helping to exchange oxygen and carbon dioxide between inhaled air and the bloodstream. Within the alveoli, AEC2 cells are responsible for maintenance and regeneration should anything bad happen. Basal cells, in turn, are mostly found in the conducting airways, a different section of the lung. Here they replace damaged cells and remodel the epithelium as needed in response to injury. But the two don’t mix. At least, they shouldn’t. So, it was to the great surprise of the researchers that both in vitro and in vivo models clearly showed hAEC2 transdifferentiation into KRT5+ basal cells—a rare process by which one cell type transforms into another. For the in vivo model, Kathiriya et al. created a 3D organoid. Essentially, a tiny replica of the particular lung tissue in question. They did this by co-culturing hAEC2s with adult human lung mesenchyme. Mesenchyme tissue is responsible for the production of most of our body’s connective tissue, and, in the case of the lungs,

is a critical determinant of their shape and size. By day 14, most of the organoid models contained KRT5+ basal cells. The same result was reflected by an in vivo model, in which researchers transplanted human AEC2s into the damaged lungs of mice. "The first time we saw hAEC2s differentiating into basal cells, it was so striking that we thought it was an error," said Peng. "But rigorous validation of this novel trajectory has provided enormous insight on how the lung remodels in response to severe injury, and a potential path to reverse the damage." Further, when co-cultured with IPF-derived mesenchyme tissue, the rate of hACE2 to KRT5+ basal cell differentiation was noticeably accelerated. Kathiriya et al. also observed that adult human lung mesenchyme co-cultured with hACE2s underwent a dramatic shift in cellular identity, much more closely resembling IPF mesenchyme tissue than fresh adult human lung mesenchyme. Comparative analysis of the hAEC2-derived basal cells with those present in IPF lungs revealed a very similar genetic expression. Although hAEC2-derived basal cells share certain foundational biomarkers with basal cells from a normal lung, they also overexpress many of the same markers previously determined to be upregulated in damaged IPF lungs (Figure 2). As a whole, the hAEC2-derived basal cells had a lot more in common with the IPF lung basal cells than they did with those of the normal, healthy lung. Wanting an even more detailed picture, the researchers performed a single-cell analysis of the cultured adult human lung mesenchyme. They found that BMP antagonists and transforming growth factor-β (TGF-β) ligands were significantly upregulated in the cultured mesenchyme. TGF-β plays a crucial role in cell proliferation and differentiation, so its upregulation would help describe the transdifferentiation of hAEC2s. Along with this, Kathiriya et al. also described seeing intermediate cell types that acted as stepping stones in the transdifferentiation process. These intermediate cell types, as with the aforementioned biomarkers, are usually found in IPF lungs. Seeing the same intermediate cells arise in the researcher’s cultured organoid models suggests that they appear in response to alveolar damage, and are a key component of hAEC2 transdifferentiation. 11

This study shines light onto the intricate and aberrant process by which hAEC2 cells turn into basal cells in response to severe alveolar injuries, a transformation that changes the architecture of the lungs and can lead to further damage as well as impaired healing. Understanding the mechanisms at play is the first step towards developing a therapeutic intervention; with enough research, serious lung damage might become treatable and, at some point, maybe even reversible. This article originally appeared on Forbes, and can be read online here: New Insights Into Lung Damage And Repair Relevant To Covid-19

New Potential Covid Virus Variants Of Concern Forbes | January 7, 2022 | Article

The past year of the Covid-19 pandemic has been driven by variants, now designated as Alpha, Beta, Gamma, Delta, and now Omicron. It is natural to wonder what will be next. Scientists and epidemiologists around the world are alert to the possibility that yet another dangerous variant may arise. This is the context to understand the interest in a recent report of a new variant, B.1.640.2, in France. A variant originally designated B.1.640 was first discovered in southern France in late October 2021. It quickly spread to other areas of Europe, including the United Kingdom, and sections of Africa, including Cameroon and the Republic of Congo. In early December, B.1.640 was divided into two distinct sublineages: B.1.640.1 and B.1.640.2. The first of these, B.1.640.1 is nearly identical to the original, apart from a 3’ end deletion which we will discuss later on. B.1 640.2 shares a large subset of mutations with B.1.640.1. However, they differ sufficiently that it is likely they both are independent descendants of an unidentified common precursor which we call B.1.640-pre (Figure 1).

FIGURE 1: Hypothesized lineage of SARS-CoV-2 variants B.1.640.1 and B.1.640.2. ACCESS HEALTH INTERNATIONAL

When compared to the Omicron variant, these two sublineages share a few important mutations but contain many notable differences, many of which are unique.

TABLE 1: Similar Mutations between Omicron and the B.1.640 sublineage variants. ACCESS HEALTH INTERNATIONAL

Table 1 summarizes some of the salient mutations in the B.1 640 viruses that are cause for concern, specifically those they share with Omicron. Spike Protein Mutations The 640.1 and 640.2 variants carry 14 and 15 mutations in the Spike protein, respectively, as compared to the Wuhan isolates (Figure 2).

FIGURE 2: Spike protein amino acid mutations in B.1.640 sublineages. Mutations in black are in both ... [+] ACCESS HEALTH INTERNATIONAL

The glutamic acid to lysine mutation at position 484 (E484K) and asparagine to tyrosine mutation at position 501 (N501Y) are common to many variants of interest and reduce neutralization and increase the binding affinity of the Spike protein for the ACE2 receptor. The phenylalanine to arginine/serine mutation at position 490 (F490R/S) and tyrosine to asparagine mutation at position 449 (Y449N) may also increase the affinity of the Spike for ACE2. Orf1ab Mutations Non-Spike proteins may also play a significant role in viral pathogenesis, replication, and virulence. Figure 3 summarizes the mutations in the replicative proteins (Orf1ab), the structural proteins other than the spike, namely the membrane protein (M), the envelope protein (E), and the nucleocapsid protein (N), and the accessory regulatory proteins (Orf3a-Orf10).

FIGURE 3: Genomic amino acid mutations in B.1.640 sublineages. Mutations in black are in both ... [+] ACCESS HEALTH INTERNATIONAL

There are a large number (15 in total) of mutations in orf1a of the 640 variants. The deletion of three amino acids of NSP6, 106 to 108 is common to other variants of interest and concern. Structural and Regulatory Protein Mutations The remaining proteins have a role in the pathogenesis and virulence of SARS-CoV-2. These are the structural and regulatory proteins. While mutations in these regions for the sublineages are relatively limited, their impact may be great. One mutation of note is in Orf8, which is a rather unusual protein. It plays a role in the inhibition of the innate immune system but is often deleted in SARS-CoV-2 variants, as it is in both sublineages. Residue Q27 is mutated to a stop codon. The Nucleocapsid threonine to isoleucine mutation at position 205 (T205I) is notable. Recent studies of the protein demonstrate that mutations in the nucleocapsid at this position can increase the titer of virus in culture more than 100 fold. This study indicates that T205I at least doubles transmissibility from the Wuhan wildtype N protein. Noncoding Mutations Figure 4 summarizes changes in the nucleic acid sequence that do not alter viral proteins. Recent studies indicate that such subtle changes in the RNA sequence may dramatically affect the abundance of specific virus messenger RNAs and proteins.

FIGURE 4: Noncoding mutations which do not change an amino acid in the B.1.640 sublineage genome. ... [+] ACCESS HEALTH INTERNATIONAL

Untranslated Region Mutations The sequences at the extreme ends of the genome, the 5’ and 3’UTRs, play a central role in viral replication, messenger RNA synthesis, and genome stability. Both 640 sublineages contain the canonical C241U mutation present in one of the earliest variants identified in early 2020. B.1.640.2 contains an additional mutation: C222U. Both lie in stem-loop 5 (Figure 5). The B.1.640.1 sublineage contains an interesting deletion of 20 nucleotides in the 3’UTR, illustrated below.

ACCESS HEALTH INTERNATIONAL

FIGURE 5: 5’ and 3’ end mutations in the B.1.640 sublineages. Mutations in black are in both ... [+] ACCESS HEALTH INTERNATIONAL

It is too early to know if the B.1.640 variants pose a significant threat. Their significance lies mostly in their existence. It is now clear that SARS-CoV-2 variants pose an ever-present pandemic danger. We are amply forewarned to isolate, characterize, and understand the potential threat that each new variant poses. This article originally appeared on Forbes, and can be read online here: New Potential Covid Virus Variants Of Concern

The Challenges of Treating COVID-19: Lessons from Gilead’s Remdesivir Forbes | January 10, 2022 | Article

As COVID-19 infections continue to peak, researchers around the world are working at unprecedented speeds to manufacture disease prevention and treatment options to combat the evergrowing list of COVID-19 variants. Several candidates have been identified for emergency use authorization, including monoclonal antibodies, but the current surge of infections suggests that the emerging Omicron variant is already resistant to current treatments and vaccines. The administration of these antiviral treatments, particularly to immunocompromised patients, has consequently created new drugresistant mutations to the viral genome. Researchers in Australia, for example, found that patients treated with sotrovimab, a monoclonal antibody therapy, displayed rapid changes in the viral genome and the emergence of new variants. Although current treatments against COVID-19 are unable to slow the spread of infection, several candidates have been beneficial in preventing individuals at high risk of severe disease from being hospitalized. In particular, remdesivir, brand name Velurky, gained significant coverage in the early months of the pandemic as the first and only FDA approved antiviral treatment for COVID-19. Authorized for adults and children 12 years and older, intravenous doses of remdesivir have been shown to shorten recovery time for hospitalized patients by 5 days. Administration to children under 12 years old has not gained full approval, but the drug is authorized for emergency use for hospitalized pediatric patients. Laboratory studies demonstrate that remdesivir acts as a nucleoside ribonucleic acid (RNA) polymerase inhibitor against the COVID-19 virus. Simply put, the antiviral drug works by preventing replication of viral RNA in infected cells, which stops the virus from multiplying. In a majority of COVID-19 cases, the body produces a substantial immune response that slows the spread 19

of infection without the need for medication. Among individuals with significant comorbidities or other risk factors that weaken immune response, however, treatments like remdesivir help the body to fight infection, preventing the progression of severe complications or death. For those at risk of developing severe disease, treatment with remdesivir at early mild to moderate stages of infection could significantly halt disease progression. Starting treatment at the first indication of COVID-19 infection, therefore, would reduce hospitalizations and lessen the strain on our healthcare system. In a recent study published in the New England Journal of Medicine, researchers at the Baylor University Medical Center in Dallas, TX and affiliated institutions found that a three-day course of remdesivir lowered the risk of hospitalization by 87.5% in symptomatic, nonhospitalized COVID-19 patients. Participants met the recruitment criteria if the onset of their symptoms was within the previous 7 days and if they had at least one risk factor for developing severe disease, including pre-existing conditions and age over 60 years. The most common pre-existing conditions were obesity, diabetes, and hypertension. Among the 562 total participants, 0.7% of patients receiving remdesivir were hospitalized or died due to COVID-19related complications, compared to 5.3% in the placebo groups. Researchers are optimistic that remdesivir may play an important role in the campaign to end the COVID-19 pandemic. Eligibility restrictions and safety concerns for certain vulnerable groups, however, narrows the strength of remdesivir’s effectiveness within the larger population. Although the FDA previously determined that remdesivir has an acceptable benefit to risk ratio, studies have yet to consider its efficacy and safety for treating vaccinated individuals and against new variants of concern, including Delta and Omicron. Remdesivir may not in fact be the silver bullet that halts the pandemic. Clinical studies warn that this antiviral drug may induce mild to moderate inflammation of the liver evident by increased enzyme levels. Therefore, remdesivir is not recommended for patients with liver disease or chronic kidney disease, which dysregulates the body’s ability to expel toxins and results in damage to the liver. Remdesivir’s effect on the liver also disqualifies patients taking medications with known side effects to the liver. This includes a 20

wide range of common medications, such as certain steroids, birth control pills, drugs to treat high cholesterol, antibiotics and even over the counter drugs like Tylenol. Many of these drugs are routinely taken together to treat a multitude of conditions, especially among older populations. However, possible interactions with remdesivir may prohibit millions of people at high risk of COVID19 complications from receiving treatment. Remdesivir may also be inaccessible for patients that do qualify for treatment. Clinical studies indicate that this antiviral treatment works best as a three-day intravenous course. Therefore, nonhospitalized patients would need to travel to health centers with the equipment needed for an IV and available supply of remdesivir. Also, having to return for two additional days of treatment increases the burden on symptomatic individuals, as well as the health professions risking exposure. To combat these limitations, alternative methods for administering remdesivir should be considered, such as oral pills that could be taken from home. In the midst of this pandemic, we do not have the luxury of time. Many of the outstanding questions related to remdesivir’s efficacy, safety and accessibility can only be answered with increased clinical trials and case studies, requiring a great deal of time and resources. As long as the COVID-19 virus continues to spread globally, weaknesses in the current campaign to prevent infection may facilitate the emergence of mutations resistant to treatment. Therefore, the race is on to manufacture and approve additional treatments with multidimensional approaches for fighting COVID19. In the meantime, the most effective strategy for slowing the pandemic includes increasing accessibility to testing, so that patients at high risk for severe disease are treated as early as possible. This article originally appeared on Forbes, and can be read online here: The Challenges of Treating COVID-19: Lessons from Gilead’s Remdesivir

The Case For Third And Fourth Booster Shots Forbes | January 11, 2022 | Article

Omicron continues to surge at unprecedented rates, we need to act quickly to avoid exhausting our healthcare system. Tools like rigorous testing, tracing, isolating, masking, and ventilation are critical pillars for our long-term fight against Covid-19. But few interventions, other than lockdowns will have the immediate effect needed to flatten catastrophic hospitalization and death rates. A third dose of the mRNA vaccines is one tool that can be deployed swiftly to save lives and hospital beds. We need to strongly encourage every American, including children to get their third dose immediately. Hospitalizations are occurring mostly among the unvaccinated but also among people whose immunity from primary vaccination or previous infection has waned. While it is clear that our vaccines cannot prevent transmission. Booster shots appear to be highly effective at preventing severe illness and hospitalization. A study published in JAMA also suggests that the waning of vaccine-induced protection against SARS-CoV-2 infection seems to be counteracted in the short-term by a third dose. During December 2021, the daily pace of booster vaccinations was approximately 770,000 doses. New data from the Commonwealth Fund has found that tripling the pace of booster vaccination from 770,000 doses per day to 2.3 million could reduce the expected number of hospitalizations by more than 35% and deaths by nearly 30% through April. The U.S. has the capacity to achieve this increase. During early 2021, the U.S. administered more than 2 million doses of COVID19 vaccine daily for almost three months. The federal government has already mobilized additional vaccination capacity in December due to Omicron. Yet presently only 39.6% of Americans aged 18 years and older have currently received a booster dose and while 88 percent of Americans 65 and older have received their first two doses, only 60 percent have received a booster. 22

Unfortunately, in the U.S. the issue with increasing booster vaccinations is not one of supply but instead poor messaging and confusion. In September 2021, I wrote that the FDA would live to regret their decision to approve boosters only for those who are at high risk of severe Covid and those 65 and older. Israel had given us very clear data about waning immunity and during a pandemic, delays can be deadly and you need to act proactively. This was a major opportunity for the U.S. to get ahead of the Covid-19 pandemic and it was wasted to the detriment of all our lives. As a result of this decision many were infected by Omicron and experienced severe disease. Our public health authorities have some have a pattern of delaying decisions with dire consequences. It is a pattern that needs to be broken and can be with the approval of a fourth shot for those over 60 or other high risk individuals. Once again there is clear evidence from Israel that this can prevent infection and possibly disease and we need to act on it. Early messaging around the booster shots suggested that it was not essential and once that perception is set, it remains difficult to change. By not approving the boosters for all and going against President Biden’s recommendation, the FDA called into question the safety of a booster dose and planted the seeds of doubt. Differing recommendations by the FDA, CDC, and the Biden Administration caused further erosion of trust in the public. We need clear, consistent messaging about the critical nature of booster shots for everyone. If our healthcare systems are overwhelmed with Omicron cases, it won’t just affect patients with Covid, but it will affect the standard of medical care for all health issues. A widespread booster campaign can keep our healthcare systems functioning. This article originally appeared on Forbes, and can be read online here: The Case For Third And Fourth Booster Shots

Omicron: Less Virulent But Still Dangerous Forbes | January 11, 2022 | Article

The rapid rise of Omicron variant infections around the world has led the virological community to take a closer look at the extremely infectious strain. Early data clearly indicates that Omicron is far more transmissible than strains that came before. One study estimates that Omicron is 2.7 to 3.7 times more transmissible than the Delta variant among vaccinated individuals. However, it seems Omicron may not be as virulent, or intense in terms of symptoms, as earlier strains like Alpha and Delta, particularly in vaccinated individuals. The Moderna, Pfizer, Johnson & Johnson, and AstraZeneca vaccines appear to protect against severe disease consequences. However, it is likely that in the unvaccinated, Omicron will still cause severe illness, and as such should not be taken lightly. Director-General of the World Health Organization Tedros Adhanom Ghebreyesus notes that “While Omicron does appear to be less severe compared to Delta, especially in those vaccinated, it does not mean it should be categorized as mild.” Omicron’s infectivity stems from a couple of factors. Firstly, it appears to overcome full vaccination and booster shot protection against infection, meaning the virus has more hosts at its disposal. Secondly, Omicron replicates extremely rapidly in the nasopharynx, more so than the strains that came before. Thirdly, Omicron transmits from asymptomatic individuals at a much higher rate than previous strains. For the Delta variant, asymptomatic transmission rates were between 1% and 2.6%. The rate for Omicron is over 30%, according to a study by Garrett et al. A recent study by Diamond et al. aimed to detail the exact pathogenic nature of Omicron in an effort to understand its greater impact on humanity. Their method was simple. They infected hamsters and mice with the Omicron variant and compared the relative damage caused by the strain to data gathered from rodent subjects previously infected.

They found the Omicron variant to be overall less virulent than previous strains. In particular, rodent subjects did not experience unhealthy weight loss, which was observed in the earlier subjects. Additionally, the viral burden in the upper and lower respiratory tract was 10 to 100-fold lower than Beta variant-infected mice after only three days post-infection. Viral load in nasal turbinates was nearly 1000-fold lower in Omicron-infected subjects. Four additional studies corroborate these findings, including three others investigating Omicron in hamsters and one in mice. All three found that Omicron induced more moderate disease than Delta or earlier strains. We note caution in the extrapolation of animal models to human virus behavior, but these findings are significant. How is this possible? It seems this strain has substantial differences in its pathogenetic process that lead to differing symptomatic outcomes. Omicron is localized to the upper respiratory system: nose, throat, windpipe, and upper lung. Chi-wai et al. found that Omicron infects and multiplies 70-times faster in the upper airways than Delta, supporting a transmission advantage. However, lower lung titers dropped as much as 10-fold in the lower respiratory tract as early as 48 hours post-infection. This data suggests that Omicron is custom-suited to infect rapidly, but does not yield as severe respiratory symptoms as seen in previous variants. This difference between Omicron and its ancestors could be attributed to a mechanistic difference in the Spike protein. The SARS-CoV-2 Spike protein is cleaved into two subunits. This is caused by the Spike furin cleavage sites and attenuates cell fusion. While Omicron contains a cleaved Spike as well, recent studies indicate that it may be less efficient than earlier strains. Dr. Ravindra Gupta of Cambridge University suggests that this may be why we observe less significant lung damage in Omicron than in previous variants. He notes that the reduced cleavage leads to Omicorn being less likely to bind a lung protein called TMPRSS2. This protein is common in the lower respiratory tract, but not so often in the higher airway, potentially explaining the stark difference in virus load. This is without a doubt a positive sign. More people are being infected today than ever before during the pandemic. This data suggests that these infections will be less harsh on the respiratory 25

health of the patients, though less deadly does not equate to not deadly. There are still stark differences in the disease outcomes of vaccinated individuals as compared to the unvaccinated. While not fully researched, there is some residual protection granted by adenovirus and mRNA vaccines. Those that are unvaccinated are much more likely to be hospitalized or succumb to Covid-19, including with the slightly less virulent Omicron variant. Additionally, whether Omicron is more or less transmissible in the unvaccinated remains an open question. The vast majority of infections are among the unvaccinated and hospitalizations follow a similar trend.

Infections over time by vaccination status and age STATISTA

In fact, Omicron is beginning to make a larger mark on hospitalizations and deaths. The sheer amount of infections, which 26

the daily average now counts nearly 700,000 in the United States, means that hospitals are filling up and deaths will come. The elderly and immunocompromised are at particular risk, as decreased virulence in Omicron would likely still overwhelm a lowered immune system. In children, cases of SARS-CoV-2 infection have sharply risen as well in the wake of the Omicron variant. CDC Director Rochelle Walensky mentioned in a previous briefing that “this is the highest number of pediatric hospitalizations we’ve seen throughout the pandemic.” Omicron infections in children are complicated by lower vaccination rates in those under 18, due to parent hesitancy and lack of authorization by the FDA until recent months. Recent reports even indicate that SARS-CoV-2 infections are associated with increased diabetes risk among youth, creating even more danger for unvaccinated children during the Omicron wave.

US trends of cases, hospitalizations, tests, and deaths. NEW YORK TIMES

Hospitalizations are spiking in the United States, at their highest rate since January of 2021, and will only continue to rise. Deaths tend to lag roughly a month behind infections and hospitalizations, so an increase should be expected in the coming weeks. We emphasize that while Omicron may be less virulent than previous strains, everyone should do what they can to avoid infection. As much as possible, continue social distancing and mask27

wearing efforts. In particular, schedule your vaccinations. While less effective against Omicron at preventing infection, reinfection, and illness, vaccines still seem to protect against severe disease as compared to unvaccinated individuals. Now is not the time to let our guard down, especially as this round of high-volume infections only creates greater opportunity for new and potentially more virulent variants in the months to come. This article originally appeared on Forbes, and can be read online here: Omicron: Less Virulent But Still Dangerous

Time to Expand Vaccination to Infants and Toddlers and Boosters for Children Forbes | January 11, 2022 | Article

The current surge in COVID-19 infections is hospitalizing children and adolescents at unprecedented rates. In the early stages of the pandemic, parents were assured that children primarily experience mild flu-like symptoms. Innate immune protection against the COVID-19 virus largely spared young children from contracting previous variants. However, as the predominant variant of COVID-19, Omicron has unique adaptations that penetrate natural immunity, leading to increased infection and hospitalization among children as young as infants. Although rare, multisystem inflammatory syndrome in children (MIS-C) can manifest in pediatric patients vulnerable to severe COVID-19 related complications. Coinciding with positive COVID-19 symptoms, MIS-C is characterized by inflammation of organs and tissues, including the skin, brain, heart, blood vessels, lungs, digestive systems or kidneys. Most children recover from MIS-C with medical care, but for some, symptoms worsen quickly. Clinical data from previous surges related to the Delta variant reveal that unvaccinated children are particularly vulnerable to developing severe COVID-19- related complications. The CDC COVID-19 Response Team recently released a report, tracking the clinical outcomes of 915 children and adolescents hospitalized for COVID-19 across six children’s hospitals during July to August 2021. A disproportionate number of these children were Black or Hispanic, consistent with previous reports on how COVID-19 is exacerbating racial health disparities. 713 patients, or 77.9%, reported COVID-19 as their primary reason for hospitalization, while asymptomatic patients hospitalized for unrelated reasons and those with MIS-C respectively made up 19.3% and 2.7% of the study’s sample. Among the pediatric patients hospitalized primarily by COVID-19, as few as 0.9% of children were fully vaccinated, even though 37.7%, aged 12 to 17, would 29

have been eligible as of May 12, 2021. Children five years old and younger, who remain ineligible for vaccination, accounted for the second largest proportion of identified patients, following adolescents aged 12 years and older. Approximately two thirds of these pediatric patients had at least one underlying medical condition, with the most common being obesity. In particular, 61.4% of patients aged 12 to 17 were diagnosed with obesity. These patients were admitted to the ICU and required oxygen support at higher rates, compared to those without obesity. In contrast to other age groups, adolescent patients were resultingly more likely to be admitted to the ICU for a longer duration and require higher levels of oxygen support. Across all age groups, approximately 1% of hospitalized pediatric patients died of COVID-19 related complications. However, even for the children that were able to return home, the experience of being hospitalized puts tremendous strain on the family. Parents are often forced to take time from work to oversee their child’s hospital stay, which creates a great deal of emotional and financial stress. Though a vast majority of children and adults recover from COVID-19 with mild symptoms, the spread of infection continues to disrupt our social systems. Home quarantines and COVID-19realated school closures have become a regular occurrence for school-aged children across the country. However, few parents have the flexibility in their work schedules to respond to the routine shifts between virtual and in-person schooling. In addition to the fear of contracting the virus themselves, the pandemic is forcing parents to decide whether going to school is a necessary risk for their children. Lessons from previous waves of the COVID-19 pandemic suggest that hospitalization rates rise as variants become more contagious, though not necessarily more deadly. With a particularly contagious variant such as Omicron, unvaccinated individuals are at the greatest risk of developing severe disease and being hospitalized. Consequently, every bed given to unvaccinated patients positive for COVID-19 limits the hospital’s capacity to take in patients with other severe diseases or injuries. As we enter yet another year of this pandemic, reducing hospitalizations should be one of our highest priorities. It is particularly important to vaccine children at risk of severe disease. Although the FDA recently made booster shots available to children 30

12 years and older, the increase in child hospitalization demonstrates that vaccination eligibility needs to be extended to children below five years of age. This article originally appeared on Forbes, and can be read online here: Time to Expand Vaccination to Infants and Toddlers and Boosters for Children

We Can’t Stop Reporting Covid Cases Forbes | January 12, 2022 | Article

With record-breaking Covid case counts sweeping the country once more, many experts have called for a shift from reporting cases to simply recording hospitalizations and deaths. The weak rationale behind this shift is related to the assumption that the Omicron variant is more transmissible but only causes “mild cases” for those who are vaccinated and boosted. Implementing a shift to reporting hospitalizations and deaths would be a grave mistake that endangers many lives. Understanding infection rates in our communities is critical knowledge that keeps us safe by allowing us to modify our behavior based on risk. I like to use a simple weather analogy when assessing personal risk. If it’s sunny outdoors, you can head out and enjoy the day. Similarly, if infection rates are low in your community, you can feel comfortable going about your everyday activities with few precautions. When it’s drizzling, you may pop open an umbrella. When it’s pouring, you slip on a raincoat and rain boots as well. This may be akin to wearing a mask and avoiding indoor or high-risk activities. But when there’s a hurricane warning, you head to the safest part of your home and hunker down until the danger has passed. Unfortunately, Omicron has led us into the path of the hurricane, and if we have the privilege to do so, we should be sheltering at home until it passes. The warning that this variant was highly transmissible was delivered in the form of surging case counts which immediately informed the decision to modify our behavior. That’s how we have to think about this pandemic and how we can protect ourselves. Just imagine if we were only reporting hospitalizations and deaths before the outbreak of Omicron. We would have been caught even more unprepared than we were. We would not have known to modify our behavior, cancel events, distribute resources to healthcare systems and dramatically ramp up testing. This precious 32

data allows planning at the local and federal level, allowing us to understand where to send PPE and testing supplies. Reporting cases internationally allows travelers to make informed decisions and informs international assistance allocations. Not counting cases will also draw focus away from testing as one of the most important tools we have for controlling the pandemic. It may even prevent us from early detection of new variants. Referring to Omicron infections as “mild” in vaccinated patients discounts the experiences of those who are immunosuppressed and those who experience Long Covid even after an asymptomatic infection. A Penn State study from October 2021 found that more than half of the 236 million people who have been diagnosed with Covid-19 worldwide since December 2019 will experience postCovid symptoms. Those with Long Covid face a challenging road to diagnosis and even then have very few treatments available. Symptoms range from psychological to heart issues, breathing trouble, chronic fatigue, gastrointestinal distress, and loss of smell and taste. Many have found themselves unable to work or care for their families. We also cannot neglect the safety and wellbeing of children under the age of 5 who are not yet eligible for vaccinations. Pediatric hospitalizations are up 335% for the week ending Jan 1 in New York. Parents cannot keep their children safe if we are not able to provide them with the data to assess risk. Let us not keep repeating the same painful mistakes in this pandemic. It was not that long ago that the previous administration was discouraging testing, telling the public that it “makes us look bad” and “testing creates cases”. The more knowledge we have about this ever-evolving virus, the more power we will have to control it. We have very little to gain by eliminating the daily report of cases and everything to lose. This article originally appeared on Forbes, and can be read online here: We Can’t Stop Reporting Covid Cases

Endemic Disease Does Not Necessarily Mean Mild Disease Forbes | January 18, 2022 | Article

Many are hoping that the Omicron variant signals a transition into the endemic phase of Covid-19 pandemic. But this hope is based on the flawed logic that endemic equals mild, resulting in less severe disease and fewer deaths. This is not always true, diseases like Malaria, are endemic but according to WHO’s latest World malaria report, there were an estimated 241 million malaria cases and 627,000 malaria deaths worldwide in 2020. Many of the greatest plagues of mankind have become endemic such as tuberculosis, cholera, influenza. Influenza has been endemic for decades but WHO estimates that 290,000 to 650,000 people die of flu-related causes every year worldwide. Many virologists also believe that influenza has the potential to become even more deadly than Covid-19 by undergoing an antigenic shift and transmitting from birds or pigs to humans. An endemic is a disease outbreak that is consistently present, irrespective of severity. Covid-19 is still a very serious disease with many unknown outcomes. Stable endemic disease is a world away from the unpredictable surges and evolving variants of the current pandemic. Once again, public health strategies have suffered across the globe at the hands of poor communication and messaging. Early reports of proportionally lower hospitalization rates characterized the Omicron variant as “mild” when hospitalization rates only tell part of the story. Unfortunately, this early perception has proven difficult to shake, with some people even deliberately seeking out infection to “get it over with” or dispensing with mitigation measures. With record cases around the world, it has never been more clear that herd immunity is a myth and not a viable strategy when it comes to this virus. Infection with Omicron does not guarantee protection against new variants that are likely to arise. 34

Omicron infection is not mild for those who are immunocompromised, unvaccinated or have a risk factor for severe Covid-19, which accounts for a significant fraction of the United States population. Unlike the hopeful initial data that emerged from South Africa, hospitalizations in the US have already hit a new pandemic peak. When Omicron finds a vulnerable host it can still exact SARSCoV-2’s worst and with the increased transmissibility of this variant, it is finding them fast. The burden on our health systems from Omicron’s surging cases will also affect the standard of care for all healthcare. Pediatric hospitalizations are the highest they have ever been throughout the pandemic in the US and Europe, with many children too young to be vaccinated and the increased transmissibility of Omicron. We should also be monitoring pediatric hospitalizations for increases in pediatric multisystem inflammatory syndrome (PIMS) as it is not yet known whether Omicron increases the risk of this condition. There is also much we don't know about Long Covid, beyond the initial infection. A Penn State study from October 2021 found that more than half of the 236 million people who have been diagnosed with Covid-19 worldwide since December 2019 will experience post-Covid symptoms. Those with Long Covid face a challenging road to diagnosis and even then have very few treatments available. Many have found themselves permanently disabled unable to work or care for their families. Hepatitis C is just one example of another endemic disease that causes significant health issues. WHO estimated that in 2019, approximately 290 000 people died from hepatitis C, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer). Globally, an estimated 58 million people have chronic hepatitis C virus infection, with about 1.5 million new infections occurring per year. We can hope for the best, but also we need to prepare for the worst. As pandemic fatigue grows we must still remember to implement the latter part of that maxim. We cannot predict how this virus will change and evolve or what future variants will bring. The only path forward is a Multimodal strategy combining medical

defenses such as antiviral drugs and vaccines and public health defenses such as testing, tracing, isolation, ventilation, and masking. This article originally appeared on Forbes, and can be read online here: Endemic Disease Does Not Necessarily Mean Mild Disease

Inhaled Remdesivir for Home Use for Covid Treatment Forbes | January 18, 2022 | Article

For a patient at high risk of severe COVID-19 related complications, newly released data shows that inhaled remdesivir offers the promise of an effective treatment for home use. Remdesivir is currently the only FDA-approved treatment of Covid-19. Recent data shows that if used early after infection, a three-day course of intravenously administered remdesivir significantly reduces the risk of hospitalization. However, because this antiviral drug has only been approved for intravenous use, there are considerable challenges to treating patients outside hospital settings. Now comes a new study that offers the prospect of home treatment via inhaled remdesivir. Under the current guidelines for remdesivir use, an individual first must know they are positive for COVID-19, which requires timely access to rapid testing. The infected individual then needs to consult their primary care doctor or other health professional to determine their eligibility for remdesivir within the early stages of infection. With a referral for treatment, the patient must then travel to a hospital or health center with a readily available supply of remdesivir and equipment needed for an IV infusion. The most effective treatment plan would require the patient to return for two additional days, putting strain on the infected individual as well as the healthcare worker providing treatment. In reality, information regarding where non-hospitalized COVID-19 patients can go to be treated with remdesivir and the capacity for these locations to take walk-in patients remains largely unclear. The FDA made remdesivir widely available with full authorization of use, but limited appointment slots for IV infusion and shortages in clinical staff, particularly specialized infusion nurses, present major challenges to treatment. Considering that IV infusion centers often cater to immunocompromised patients being treated for other conditions, there is also a significant risk to exposing these 37

patients to the COVID-19 virus. With little federal guidance on who pays for remdesivir treatment in an outpatient setting, additional equity issues restrict universal access. Lastly, persistent challenges to accessing rapid testing delays the start of treatment, thus reducing the drug’s capacity to prevent hospitalization. To overcome these challenges, an inhaled version of remdesivir has been proposed as an alternative method for administering treatment. This innovation would allow patients to be treated outside a hospital or clinical setting, effectively reducing the risk for spreading infection. With a nebulizer machine, commonly found in drug stores, infected individuals could administer their own treatment, or if further assistance is needed, other members of the household could help load the drug into the machine. Several medications, particularly those for young children, already capitalize on the non-invasive mechanics of nebulizer treatment. Once a fluid drug is loaded into the machine, a patient simply wears the attached mask or mouthpiece and slowly inhales a vaporized form of the drug. While intravenous treatment circulates the bloodstream before reaching respiratory pathways, inhalation allows for direct exposure to nasal and respiratory tissues. According to a manufacturersponsored study published by Science, African green monkeys recruited for the study demonstrated that a 20-fold lower dose of remdesivir can be used for inhaled treatment compared to IV infusion. Gilead researchers reported comparable outcomes for the primate subjects treated with inhaled remdesivir to those given traditional IV infusions. Repeated doses of inhaled treatment were shown to be safe and effective among the primate subjects. Under the assumption that the lung function between monkeys and humans is similar, Vermillion et al. suggest that 0.35 mg/kg inhaled dose in the monkey subjects is equivalent to a standard 200 mg IV infusion of remdesivir in humans. Direct administration to respiratory pathways demonstrated high antiviral activity within respiratory tissues, while largely sparing nonrespiratory organs, including the liver and kidneys. Because previous studies have reported increased inflammation of the liver as a major side effect of remdesivir, inhalation reduces the risk of liver damage, especially among patients with liver disease or other metabolic disorders. Additional research is needed to directly explore these benefits in human patients. 38

Inhaled administration was also highly effective in reducing viral load in both the lower and upper portions of the respiratory tract, while IV treatment has a greater effect on lower tissues. This method would be particularly potent against emerging variants like Omicron, which rapidly multiplies in the nose and upper respiratory tract. Remdesivir has already been shown to be effective against previous circulating strains, including Alpha, Beta, Gamma, Epsilon and Delta. It should be noted, however, that the results from these primate animal models may not be easily translatable to human subjects. The nature of this experiment allowed treatment to begin within a few hours after infection, which would be difficult to replicate in human clinical studies or real-world settings. Further refinement of the inhaled remdesivir formula is necessary to ensure effective and safe antiviral treatment in humans. Although this alternative method for treatment allows for the treatment of COVID-19 patients at home, concerns regarding availability and access to the drug itself continue to persist. First and foremost, supply of remdesivir needs to be extended beyond hospitals and IV treatment centers. Individuals should have the option to pick up the medication from their local drive-thru pharmacy, or ideally have the medication delivered directly to their home. Since early treatment has been shown to be the most beneficial, individuals at high risk of hospitalization may be recommended to stock emergency doses in their medicine cabinets. This would require additional regulatory procedures to identify the most vulnerable patients and promptly deliver sufficient doses of remdesivir. Another option may be to set up distribution centers in local communities, but this does not guarantee access to those that need the drug the most. To alleviate financial obstacles, additional policies should consider subsidizing costs for both the drug and nebulizer machinery. At this stage of the pandemic, an inhaled version of remdesivir best serves as part of a larger strategy that expands access to rapid testing, defines stricter isolation procedures, and facilitates greater communication to patients who have tested positive. As more antiviral drugs against COVID-19 are produced and distributed, it is critically important for researchers to work alongside policy makers

and clinical providers to ensure universal access to those among us that are the most vulnerable. This article originally appeared on Forbes, and can be read online here: Inhaled Remdesivir for Home Use for Covid Treatment

New Data Suggests That 50% Of Omicron Infections In Healthy Young Men Remain Transmissible After Five Days Forbes | January 19, 2022 | Article

New data is now available regarding the transmission of the SARS-CoV-2 Omicron variant. With regard to transmission, there are two major concerns. The first is how rapidly following infection people become infectious. The second is how long Omicron hosts remain infectious. Both of the concerns have been addressed by Hay et al., at least for healthy young men. The study gathered PCR tests from basketball players in the National Basketball Association, aiming to elucidate the viral dynamics of Omicron, namely when viral load peaks and how long the general infection lasts. The NBA regularly tests players for Covid-19 to ensure the integrity of its 82-game season. Using the data collected from these samples, Hay et al. quantified the duration of viral proliferation, clearance rate, and peak viral concentration for individual infections. They additionally sequenced the samples to differentiate between Omicron, Delta, and other variants. From their data set, Hay et al. noted 97 Omicron infections and 107 Delta infections. They found that 27 of the 97 Omicron cases tested positive one day or less after a previous negative. Of these, 52% were positive at day five, 25% at day six, and 13% at day seven. For the 70 of 97 testing positive two or more days after a previous negative, 39.1% were positive at day five, 33.3% at day six, and 22.2% at day seven.

FIGURE 1: Solid colored lines show mean Ct value over time, counting negative tests as Ct=40. The horizontal dotted line shows the cutoff of PCR Ct=30 (legend from original study).

This data illustrates that Omicron infected individuals become transmissible within the first few days of infection, with the viral load peaking rather early. The mean infection length for Omicron cases was 9.87 days. For comparison, Delta infections averaged over a day longer: 10.9 days. Peak viral load came slightly later in Delta cases as well, particularly in those that tested positive one or fewer days after testing negative, though there were only six such cases.

FIGURE 2: Mean posterior viral trajectories for Omicron (red) and Delta (blue) infections (legend from original study).

Hay et al. also found that the variant portrayed a lower peak viral load than the Delta variant. Omicron’s mean peak Ct value was 23.3 compared to Delta’s 20.5, emphasizing that the lower the Ct value, the higher the peak viral load. In other words, Delta infections elicit more virus particles in a host than Omicron infections, but only moderately. There is some speculation as to if the Ct value for Omicron should be higher than 30 based on its increased transmission advantage, but for the purposes of this study, the value for Omicron and Delta is set at 30.

FIGURE 3: (A) Peak viral RNA concentration, (B) proliferation time, (C) clearance time, and (D) acute infection duration for omicron (red) and delta (blue) infections (legend from original study)

Additionally, Hay et al. note that Omicron and Delta display similar rates of viral clearance, meaning how fast the virus is expelled from the host. As such, Omicron has a slightly shorter clearance phase, 5.35 days as compared to 6.23 days for Delta, as Omicron has a lower peak viral load. These data points raise a sort of transmission paradox. If the virus is less concentrated in Omicron, infections are cleared faster and mean infection lengths are all lower than those of Delta, then why is Omicron so much more transmissible?

By some estimates, Omicron is 2.7 to 3.7 times more transmissible than the Delta variant. The first potential explanation is that protective convalescent antibodies from the previous infection display little to no protection against Omicron, whereas they provided some extended period of protection against Delta. While this may be the case, I believe it is only part of the story. I believe Omicron is much more successful at transmission, in part, due to its immune-suppressive advantages. Once the virus enters the cell, Omicron may be more efficient at suppressing the innate immune response. The proteins which conduct interaction with innate immunity are those outside the Spike protein and mutations in these proteins may be the hidden key to Omicron’s proliferation. We emphasize the importance of taking this strain seriously. The data indicate that peak viral loads and mean infection duration was greater for the Delta virus, but this is only half the picture. Omicron’s transmission advantage and potent immune suppression make it a real threat, especially as society continues to a pre-pandemic lifestyle, representing a breeding ground for the virus that will yield millions more infections and many deaths. Take caution, receive your vaccinations, and do not take Omicron for granted. This article originally appeared on Forbes, and can be read online here: New Data Suggests That 50% Of Omicron Infections In Healthy Young Men Remain Transmissible After Five Days

Major Protease Of SARS-CoV-2 Acts To Suppress Innate Immunity Forbes | January 19, 2022 | Article

In October 2020, researchers discovered that the SARS-CoV-2 nonstructural protein 5 (NSP5), like many other of its viral proteins, suppresses innate immunity. This has key implications for Covid-19 drug development that have yet to be fully explored. Though some aspects of the Covid-19 pandemic are comparable to previous encounters between humanity and a lethal infectious disease, much about the virus that has claimed the lives of so many in the past two years is without known precedent. Among these are the novel mechanisms SARS-CoV-2 has developed for antagonizing the immune system, which scientists have documented and endeavored to understand since the pandemic began. One critical piece of this highly complex puzzle, outlined by Wu et.al in Signal Transduction and Targeted Therapy in October 2020, is the immunosuppressive function of NSP5, otherwise known as the main protease. The function of NSP5 as a protease, not just in SARS-CoV-2 but other viruses, are well-known. As the main protease, NSP5 drives the enzymatic processes necessary for the virus to reproduce itself, chewing up chunkier polyproteins into discrete nonstructural proteins that assist in the virus’ program of immune suppression. Protease inhibitors based on NSP5 were developed by drugmakers long before SARS-CoV-2, some of which have been approved as treatments for Covid-19. But Wu et.al have discovered that in addition to its protease function, NSP5 is also active in evading and suppressing the immune system. The multifunctionality of this protein increases its appeal as a drug target, as treatments that inhibit the protease function may do the same for its immunosuppressive capacities. SARS-CoV-2 encodes many proteins in its viral genome. One of the most remarkable aspects of these proteins is they have multiple functions, both for enhancing viral replication and counteracting the 45

immune system from multiple angles. The majority of these points of interference coalesce along the signaling pathways that lead to induction of interferon, a protein key to activating the natural (innate) immune response. When interferons detect invading pathogens, they mobilize production of hundreds of interferonstimulated genes that spring surrounding cells into defense mode and kickstart the adaptive immune response. But when interferon induction and signaling is blocked, pathogens can hijack cellular machinery for their own purposes and replicate for a time all but unnoticed, as is the case with SARS-CoV-2. The study conducted by Wu et.al was an early contribution to the body of literature on the immunosuppressive mechanisms of SARS-CoV-2 that has grown considerably since. Though initially promising interferon-based drug treatments for Covid-19 precipitated too many adverse effects in clinical trials to be brought to market, their success at reducing viral replication compelled researchers to identify with greater precision how the virus delayed and inhibited the interferon response. Over a series of experiments, Wu et.al found that SARS-CoV2 NSP5 inhibited interferon signaling across pathways mediated by IRF3 (interferon regulatory factor 3) and NF-κB (nuclear factorκB). They also found that NSP5 expression mitigated phosphorylation of TBK1 (TANK binding kinase 1) and IRF3, a necessary step in interferon induction.

Figure 1. "Schematic representation of SARS-CoV-2 Mpro antagonizing antiviral immunity." "MAIN PROTEASE OF SARS-COV-2 SERVES AS A BIFUNCTIONAL MOLECULE IN RESTRICTING TYPE I INTERFERON ANTIVIRAL SIGNALING"

Wu et.al also examined the effects of NSP5 on RIG-I (retinoic acid-inducible gene I), the RNA helicase that initiates one of the primary interferon induction pathways by looking for recognizable patterns in viral RNA. RIG-I is triggered by double-stranded RNA, the obligate replication immediate for SARS-CoV-2. Once RIG-I picks up on viral RNA, its activation is catalyzed by a ubiquitination process involving K63 (lysine-63) and mediated by TRIM25 (Tripartite Motif Containing 25). Wu et.al speculate that NSP5 might reduce K63-linked ubiquitination of RIG-I as another means of blocking interferon.

Figure 2. "Luciferase activity in 293T cells transfected with IFNβ luciferase reporter and vectors ... [+] "MAIN PROTEASE OF SARS-COV-2 SERVES AS A BIFUNCTIONAL MOLECULE IN RESTRICTING TYPE I INTERFERON ANTIVIRAL SIGNALING"

Additionally, the study highlights SARS-CoV-2 NSP5 interactions with the second leg of the interferon induction pathway, which precipitates the release of interferon-stimulated genes. Wu et.al show that NSP5 likely promotes degradation of STAT1 (Signal Transducer and Activator of Transcription 1) and delays interferonstimulated gene production, allowing the virus to replicate to higher titers in the meantime. SARS-CoV-2 NSP5 is worthy of further study for numerous reasons. One, as illustrated by Wu et.al, is its propensity for suppressing interferon. Another is its multifunctionality. This makes it a compelling target for drugmakers developing treatments that debilitate SARS-CoV-2 before it can debilitate us. Wu et. al found the protease function was not needed because they introduced mutations to the active site. Nonetheless, it will be worthwhile investigating whether protease inhibitors also block the immunosuppressive activity of NSP5. This article originally appeared on Forbes, and can be read online here: Major Protease Of SARS-CoV-2 Acts To Suppress Innate Immunity

Novel Antiviral Approach To Covid-19 Treatment Forbes | January 20, 2022 | Article

Figure 1: Model of the three RBD-targeting DARPin molecules of ensovibep bound to the RBD regions of the Spike ... [+] FROM: “ENSOVIBEP, A NOVEL TRISPECIFIC DARPIN CANDIDATE THAT PROTECTS AGAINST SARS-COV-2 VARIANTS” SYLVIA ROTHENBERGER ET AL. 2021.

As we move into the third year of the Covid-19 pandemic, things continue to look fairly bleak. Caution fatigue combined with the idea that omicron is “mild” have left us with record case numbers and record hospitalizations. But on the treatment front, we’re still lagging, with omicron and other variants resistant to many of our currently available antiviral therapies. That said, Swiss pharmaceutical corporation Novartis, working together with biotech group Molecular Partners, has just thrown its hat in the ring, and early trial results for their new drug, ensovibep, seem promising. Coming off the back of a successful phase 1 safety study in March of 2021, Novartis and Molecular Partners initiated a global clinical trial called EMPATHY. This phase 2 and 3 study aims to test the safety and efficacy of ensovibep in symptomatic Covid-19 patients in a non-hospitalized setting. Ensovibep is administered as a singledose intravenous infusion.

The latest results come from phase 2, which focused on preliminary efficacy and on determining the optimal dose. To this end, 407 adult patients were recruited and randomized into four different arms: one for each dose tested, and one placebo control group. All patients displayed at least two symptoms of mild to moderate Covid-19 —fever, cough, body aches, shortness of breath, etc.— and represented a mix of both vaccinated and unvaccinated individuals. Their Covid-19 status was confirmed via positive rapid antigen test the day of dosing as well as a further PCR test at baseline. Dosing took place within seven days of initial symptom onset. The trial met its primary endpoint, showing a marked reduction in viral load across the span of eight days. It also met the secondary endpoint, with a 78% reduction in hospitalizations, visits to the emergency room, or death when compared to the placebo group. This held true across all of the doses tested: 75mg, 225mg and 600mg. Importantly, all three doses were described as being welltolerated by the patients, offering a good safety profile. Novartis plans on moving forward with the 75mg dose. After the results of the first trial phase came through in May of 2021, Patrick Amstutz, Chief Executive Officer of Molecular Partners, said: “By virtue of its tri-specific design, ensovibep was built to resist viral mutations and indeed shows potent inhibition of all variants of concern to date, with the potential to maintain activity also for future variants.” Mechanism of Action Ensovibep is based on designed ankyrin repeat proteins (DARPins): genetically engineered proteins that mimic an antibody’s ability to bind to, and eventually neutralize, antigens. DARPins are derived from natural ankyrin repeat proteins, a class of binding protein very frequently found in nature. They can be designed around their exact target and their intended effect, allowing for a great deal of specificity. This results in a high-strength, tightfitting bond with their target molecule. In the case of ensovibep, there are five different DARPin domains linked together as one single molecule (FIGURE 1A); two of these bind to human serum albumin (HSA) in order to keep the drug in the body for longer, the other three domains map onto the three-part structure of the SARS-CoV-2 Spike protein. To be more precise, the DARPins simultaneously block the receptor binding 50

domain (RBD) of each spike trimer, allowing them to inhibit both tightly and cooperatively ACE2 interaction (FIGURE 1G). By blocking ACE2 interaction, ensovibep prevents SARS-CoV-2 from binding to our cells and, in so doing, protects them from infection.

FIGURE 1. A) Schematic representation of the five-part ensovibep construct (half life extenders, H1 ... [+] SYLVIA ROTHENBERGER ET AL. 2021.

The three-part structure of ensovibep, combined with the high binding affinity of DARPins, makes it difficult for mutations to buck off. The only mutations that managed to do so, a substitution at F486V and another at N234Q, come at the expense of viral fitness more generally. For example, with the F486V substitution, there was a ~8.5 fold reduction in binding affinity between human ACE2 and the SARS-CoV-2 RBD. That kind of reduction would significantly reduce the virus’ ability to infect our cells, decreasing transmissibility and virulence. The importance of F486 is further reflected by a low frequency of naturally occurring substitutions. In addition to ensovibep’s potential to hold out against variants, it has the benefit of being heat stable —meaning it can be stored and transported easily. Also, because of DARPins’ relatively simple molecular architecture, they can be manufactured in high yields, leading to cost-effective and scalable production. 51

The main drawback is that ensovibep has to be delivered intravenously, restricting its use to a clinical setting and likely raising its cost. It also means it can’t be used prophylactically, to prevent infection and to prevent transmission, which, realistically, is what we most need to control this pandemic. We’ll have to wait and see what picture the results from phase 3 paint —which will include a sample size of 2100 patients and double down on testing the clinical efficacy at 75mg— but so far it’s looking like ensovibep will make a valuable addition to our still-sparse quiver of antiviral drugs. This article is originally featured on Forbes, and can be read online here: Novel Antiviral Approach To Covid-19 Treatment

The Case for Vaccination Against the Omicron Variant WSJ | January 23, 2022 | Opinion Letter

Luc Montagnier and Jed Rubenfeld’s principal argument in “Omicron Makes Biden’s Mandates Obsolete” (op-ed, Jan. 10) is flawed and irresponsible. Vaccines do not need to prevent infection to be highly effective. The Salk inactivated virus polio vaccine, for example, doesn’t prevent infection or transmission but has been responsible for eradicating polio. Vaccination doesn’t prevent infection from the Omicron variant but it reduces the chances substantially. More important, it radically reduces the chance of severe disease, death and hospitalization. Recent data shows that fully vaccinated New Yorkers had between a 90.2% and 95.7% lower chance than unvaccinated New Yorkers of being hospitalized with Covid-19. Vaccination reduces the burden on our healthcare systems, which are stretched to their limits with the Omicron surge. If hospitals are overwhelmed with Covid cases, it affects care for all health issues. William Haseltine President, Access Health International Mr. Haseltine is a former Harvard Medical School professor and founder of the university’s cancer and HIV/AIDS research departments. This opinion letter can be found online here: The Case for Vaccination Against the Omicron Variant - WSJ

The Danger Of Covid-19 Monotherapy: Drug Resistance Forbes | January 24, 2022 | Article

This scanning electron microscope image shows SARS-CoV-2 (round gold objects) emerging from the ... [+] BSIP/UNIVERSAL IMAGES GROUP VIA GETTY IMAGES

The current protocol for Omicron infections dictates that patients are treated with either one monoclonal antibody preparation or one antiviral drug. This is not an optimal strategy but instead, one necessitated by current drug approvals. As welcome as these newly approved treatments are, they come with the potential of long-term dangers. Past experience demonstrates that the use of one drug rather than a combination of drugs means that viruses are very likely to develop resistance over time potentially leading to a pandemic fueled by drug resistant viruses. This is particularly true for immunosuppressed patients who are likely to be chronically infected. Drug-resistant mutations arise quickly in such cases and may persist for weeks or even months. Such events are speculated to have been the origin of some of the most troubling variants of concern. We need to heed lessons from prior Influenza and HIV epidemics about drug resistance. Amantadine (sold under the brand name Gocovri) was an antiviral that was used for the prevention and 54

treatment of influenza A in 1976. However, amantadine-resistant influenza viruses were first reported during the 1980 influenza A epidemic and the frequency of resistant viruses continued to rise into the early 2000s. The drug is no longer recommended for the treatment of flu viruses in the United States due to widespread resistance. During the early days of HIV research, the drug azidothymidine, known as AZT was prescribed to HIV patients as monotherapy with initial positive results. However that approach quickly eventually led to resistance in individual patients and the transmission of an evolved virus. It was this experience that led to the development of the gold standard for HIV treatment; combination antiretroviral therapy. The problem with drug resistance in HIV has become so severe it is strongly recommended that before antiretroviral treatment is initiated, the entire nucleotide sequence of the HIV genome be determined. Telltale mutations that would demonstrate resistance can be detected in the whole genome sequence allowing physicians to use a targeted cocktail of two or more drugs. Clinical availability of rapid DNA and genome sequencing should be a high priority for research and development purposes. Not only for Covid-19 but for all other viral and bacterial diseases to combat another growing problem which is antibiotic drug resistance. We now have the opportunity for very rapid total DNA sequencing that can take less than half an hour. By using the latest methods, the entire human genome can be sequenced within five hours. This provides enough time and data for a physician to make a wise choice on what antiviral drugs could be used in a combination to combat resistance. Presently there is no combination therapy approved for the treatment of Covid-19. As the virus continues to evolve and mutate to evade and suppress our acquired and innate immune systems, I recommend that we explore the use of combination therapy to combat drug resistance. Already the Covid-19 virus has evolved to create the Omicron variant, which is resistant to all FDA approved monoclonal antibodies except sotrovimab. However, recent data from Australia raises serious issues of sotrovimab as a stand-alone treatment. In this study, the researchers determined the sequence of virus isolated from patients treated with sotrovimab from a cohort of 100 55

at a treatment center in New South Wales, Australia. The treatment was a single 500mg dose targeted at patients within five days of symptom onset thought to be at risk for severe disease progression. Of the initial 100 patients, 23 tested positive for SARS-CoV-2 infection at least 10 days post-infusion, and of these, the pre-and post-infusion respiratory tract samples of eight were collected. Of these eight, seven were hospitalized, six were partially vaccinated or unvaccinated, and four were given additional antibody treatment. Viable virus samples obtained from four of the patients contained a mutation at amino acid E340 of the spike protein, either E340K, E340A, or E340V. The virus in one patient contained an additional mutation: the P337L mutation. These mutations are predicted to yield virus resistance to sotrovimab. Analyses of mutations in this region of the Spike, the receptor-binding domain, indicate that the observed mutations decrease sotrovimab neutralization by almost 300 fold. The Australian researchers note that prior to the advent of sotrovimab clinical trials, mutations at amino acid 340 that confer resistance were extremely rare. They comment that the incidence of such 340 mutations is rising in virus isolates from around the world. Whether that is the result of sotrovimab induced mutation or to the greatly increased number of infected persons due to Omicron is an open question. We need to follow the strategies used for HIV and create combinations of powerful, long-acting drugs that target a broad range of functions with a high therapeutic index. There are still many unexplored targets for SARS-CoV-2 drug development. I suggest that we expand our range of targets for drug development far beyond the common protease to the many enzymes and regulatory proteins specific to SARS-CoV-2 that do not exist in humans. As soon as is practical, we need to begin testing combination therapy and conducting rapid sequencing of the virus infecting each patient to determine the best course of therapy. We need to closely monitor viable viruses present in treated patients for drug resistance as well. This article originally appeared on Forbes.com, and can be read online here: The Danger Of Covid-19 Monotherapy: Drug Resistance

New Clues To Long Covid: Prolonged Inflammatory Response Forbes | January 25, 2022 | Article

For many Covid-19 patients, the end of the acute stage of infection is only the beginning of another difficult experience: Long Covid. Defined by the persistence of physical and neuropsychiatric symptoms over a period of 12 weeks or longer, the exact causes of long Covid remain largely elusive. A recent analysis by researchers at the University of New South Wales’ Kirby institute and St Vincent’s Hospital Sydney sheds some light on the topic. In long Covid patients they have uncovered evidence of sustained inflammation and activation of the immune response for at least 8 months after initial infection. These findings provide a framework through which to define more accurately and diagnose long Covid. Phetsouphanh et al. were given a chance to look for “biomarkers” underlying long Covid with help from data gathered as part of St Vincent’s Hospital’s ADAPT study. The study collected blood samples from unvaccinated Australians during the height of the country’s first pandemic wave, Immune biomarkers are measurable indicators that act as a kind of map key, letting researchers know what processes and responses characterize a certain disease. This study represents the first laboratory analysis of long Covid’s impact on the immune system. To pin down exactly what’s happening to Covid “long-haulers,” as they’ve come to be known, Phetsouphanh et al. compared the blood samples from the ADAPT study with those derived from healthy donors unexposed to SARS-CoV-2. The ADAPT cohort was made up of individuals with PCR-confirmed Covid-19 infections, tracked over a period of eight months. Blood samples were drawn two months, four months, and eight months after the initial infection. After four months, 31 of a total 147 participants were classified as having long Covid based on the persistence of one of three major symptoms: fatigue, labored breathing, or chest pain. Those exhibiting long Covid symptoms were matched with 31 57

symptom-free participants of the same trial, used as an additional control cohort. The team of researchers also compared the blood samples with those of individuals infected with other, non-SARS-CoV-2 human coronaviruses. “As immunologists we’re almost like detectives at a crime scene. We have thousands of potential biomarkers – or leads – to investigate, but only a handful of them will reveal something useful. We can use some of our knowledge of what’s been measured in acute COVID and other post-viral fatigue syndromes to narrow the investigation down a little bit, but because long COVID is still a new syndrome, we have to take a broad examination of the evidence and look almost everywhere,” says Dr Phetsouphanh. Of the 28 potential markers the researchers analyzed, six were noticeably elevated in both the long Covid cohort and the asymptomatic control cohort four months after initial infection. All six were proinflammatory cytokines, signaling proteins that help boost inflammation as part of the innate immune response. Two proinflammatory cytokines stood out as particularly elevated in the Covid cohorts vis-à-vis the other two cohorts: interferon β (IFNβ), and interferon λ1 (IFN-λ1). The remaining 22 analytes were the same across all four cohorts. Inflammation is a critical part of recovery, helping the body to get rid of the source of damage and helping it repair injured tissue, but too much of it can have unwanted effects. Especially when the inflammation persists beyond any actual outside threat. Professor Gail Matthews, the study’s senior researcher, mentioned: “But what we’re seeing with long COVID is that even when the virus has completely left the body, the immune system remains switched on. If you measure the same thing after a standard cough or cold, which we did in this study through one of our control groups, this signal is not there. It’s unique to sufferers of long COVID.” The long Covid cohort and the asymptomatic matched control cohort may have had the same readings four months in, but at eight months the two began to come apart. The levels of proinflammatory cytokines in the asymptomatic cohort dropped off, whereas those in the long Covid cohort remained more or less steady, with only a statistically insignificant decrease. 58

Four of the markers, analyzed via a data model, proved to be especially accurate in predicting long Covid: IFN-β, PTX3, IFNλ2/3 and IL-6. Of these, IFN-β was the single most important indicator of long Covid, present 94% of the time when modeled in a set of four markers. As exciting as this data is, the researchers are already looking ahead: how does the rate of long Covid incidence and distribution of biomarkers change depending on vaccination status, the variant with which one was infected, and the severity of one’s infection? The plight of long haulers was dismissed early on in the pandemic, often leaving sufferers to deal with life-altering symptoms on their own, without clinical or institutional support. This analysis by Phetsouphanh et al. helps firmly ground their experiences in biology. Long Covid is a medical condition, often debilitating, and has to be treated as such. This article originally appeared on Forbes.com, and can be read online here: New Clues To Long Covid: Prolonged Inflammatory Response

Birth Of The Omicron Family: BA.1, BA.2, BA.3. Each As Different As Alpha Is From Delta. Forbes | January 26, 2022 | Article

Time course for the evolution of significant SARS-CoV-2 variants, note the considerable divergence ... [+] NEXTSTRAIN.ORG

This is the first in a series on the Omicron variant, specifically discussing its derivative lineages: BA.1, BA.2, and BA.3. We have previously speculated on the potential origin of Omicron in an earlier article. The Omicron variant has continued to surprise by its sudden appearance, its evident ability to evade both vaccines and immune responses, and its rapid spread worldwide. There's another surprise buried in the emergence of Omicron. The variant is not a single strain, but rather a family of three: BA.1, BA.2, and BA.3. We should take all three sublineages seriously. BA.1 is the most prolific sublineage, detected in most countries worldwide and currently accounting for 99% of cases in the United States. BA.2 is less prolific, but has overtaken BA.1 in Denmark, Nepal, and the Philippines as the most frequently detected variant, and has a minor presence in India, the United Kingdom, and several other countries. The third, BA.3, is yet to take off globally, only accounting for several hundred cases at the most. 60

Each of these variants is as different from one another as Alpha, Beta, Gamma, and Delta are from one another. What this means for the current state of the pandemic is uncertain. However, one thing is clear: SARS-CoV-2 has an enormous capacity not only to continue to produce new variants, but variants that surprise us both in their number and their biological properties. Here, we introduce the Omicron family of variants. More detailed descriptions of each strain will follow this introduction. We have already described BA.1 in a previous story under the moniker of “Omicron” before the family was apparent. In this first entry, we will introduce the three, how they diverged from the Wuhan strain, and how they compare to each other. As with any variant of SARS-CoV-2, we return to the source of the virus: the wild-type Wuhan strain. This is the blueprint for all variants to come. The significant variant has no Greek letter name, but rapidly displaced the Wuhan almost everywhere in the world except East Africa. Here we call it the Triad, a name that denotes three mutations, the D614G mutation in the Spike protein, the P323L mutation in the NSP12 polymerase, and the C241U noncoding mutation in the 5’ end. The Triad is the founding variant of all variants of concern viruses.

FIGURE 1: Evolutionary distances between major variants of concern, the Omicron family of viruses, ... [+] ACCESS HEALTH INTERNATIONAL

We note that the Omicron variant significantly differs from other major variants of concern. We identify a precursor, which we call the Omicron parent, likely to have risen about March of 2021. The three sublineages share 39 mutations, which we include as the 61

presumptive "Omicron parent.” The Omicron parent diverged into the Omicron family: BA.1, which contains an additional 20 mutations; BA. 2, which includes an additional 27; and BA.3, which consists of an additional 13. Remarkably, all the family members were detected simultaneously in South Africa, although they likely diverged from one several months previously. This is a unique example of such highly divergent strains appearing in a population simultaneously.

FIGURE 2: Lineage of Omicron family viruses. The date included is when that specific variant was ... [+] ACCESS HEALTH INTERNATIONAL

Of the 39 mutations introduced in the Omicron precursor, most are located in the Spike protein. This is the virus region that makes contact with the host cell ACE2 receptor and plays a significant role in transmissibility and evasion of neutralizing antibodies. Non-Sprotein mutations are sprinkled throughout and may play an equally important role in the transmission and pathogenesis of the virus. Figure 3 displays the similarities and differences amidst the three sublineages.

FIGURE 3: Venn diagram showing the similarities and differences between the three Omicron family ... [+] ACCESS HEALTH INTERNATIONAL

The largest single class of mutations is shared amongst all three Omicron family members. Figure 3 emphasizes the differences amongst the three variants as well. We reiterate that BA.1, BA.2, and BA.3 differ from each other as much as the Alpha, Beta, Gamma, and Delta variants differ from one another. Several mutations differ between the three as well. We suspect that these differences will be reflected in the septic characteristics of each variant affecting growth rate, suppression of innate immunity, virulence, and vaccine evasion. Omicron (BA.1)is already known to have some unusual biological properties relative to the other variants. Most strikingly, it is 2.7-3.7-fold more transmissible than Delta. The origin of the increased transmissibility is somewhat of a mystery, as the concentration of virus in nasal sections is not exceptionally high, and is, in fact, lower than that of Delta. Moreover, the affinity of the BA.1 Spike for the ACE2 receptor is only twice as high as the Wuhan strain. We speculate that the increased transmissibility may be in the mutations found in the nonstructural, structural, and accessory proteins found throughout the genome. Another biological property is how Omicron enters the cell. The Wuhan wildtype and all previous variants enter primary via membrane-to-membrane fusion. Omicron seems to prefer an endosomal route similar to SARS-CoV (the SARS virus) but different from all previously characterized variants. Omicron does not induce cell to cell fusion (syncytial formation) in cell culture, consistent with endosomal entry.

FIGURE 4: Membrane to membrane vs. endosomal entry of SARS-CoV-2 in Delta vs. Omicron GEORGINA BROWN/JOE GROVE/INSIDER

The fundamental change in cell entry may be explained by a set of mutations described by Martin et al. They describe three clusters of mutations that appear in SARS-CoV-2 variants very rarely, if at all, based on the 7.4 million SARS-CoV-2 sequences in GISAID. The first cluster is composed of receptor-binding mutations G339D, S371F/L, S373P, and S375F. The second cluster is composed of receptor-binding domain mutations Q493R, G496S, Q498R, and Y505H. The third is composed of fusion domain mutations N764K, N856K, Q954H, N969K, and L981F. The combination of these clusters may well lead to the modified preference of cell entry. We note that three of these mutations, G496S, N856K, and L981F, are found only in BA.1, not BA.2 or BA.3. This may impact the viral fitness of BA.1 relative to its sibling sublineages. How and to what extent they are affected, we do not yet know.

FIGURE 5: Three Spike cluster mutations in Omicron as described by Martin et al. MARTIN ET AL.

This series aims to understand Omicron and its sublineages in more detail, as they are by far the most transmissible version of SARS-CoV-2 to date, but fortunately less pathogenic—at least for well vaccinated healthy young adults. We will examine the mutations in BA.2 and BA.3 in stories to follow soon.

This article originally appeared on Forbes.org, and can be read online here: Birth Of The Omicron Family: BA.1, BA.2, BA.3. Each As Different As Alpha Is From Delta.

Functions Of SARS-CoV-2 NSP12 Polymerase: Replication, Transcription, And Suppression Of Natural Immunity Forbes | January 27, 2022 | Article

This is part twenty of a series, “How SARS-CoV-2 Delays, Evades, and Suppresses the Immune System.” Many of the proteins SARS-CoV-2 encodes, researchers have found, are multifunctional. Not only do they enable replication of the virus, they antagonize the human immune system as well. In February 2021, a paper published in Cellular & Molecular Immunology by Wang et.al confirmed that NSP12, also known as the RNA-dependent RNA polymerase (RdRp), possesses this dual function. More specifically, it suppresses the activity of a coding gene, IRF3 (interferon regulatory factor 3), key to the production of interferon. Interferon is a key component of the innate immune response, the body’s first line of defense against invading pathogens. Though the general population is far more familiar with adaptive immunity, the antibody-led mode of protection that follows, an increasing amount of evidence suggests that innate immunity is where SARSCoV-2 concentrates its attack. The longer the virus can delay interferon induction, which sets off a cascade of signals alerting surrounding cells to the presence of an intruder, the more it can replicate unnoticed. This ultra-precise replication strategy has been millions of years in the making, the product of a war between humans and microbes that has lasted several millennia. Prior to the preeminence of SARS-CoV-2, the enzymatic function of NSP12 was already well-documented. As the RNAdependent RNA polymerase, NSP12 helps synthesize viral RNA. Its structure consists of an RdRp domain and a nidovirus RdRpassociated nucleotidyltransferase (NiRAN) domain (Figure 1). Studies of the role of NSP12 in the replication cycle of other viruses, such as hepatitis C and enteroviruses, have also shown that the 66

protein has the ability to suppress interferon expression. Until the study conducted by Wang et.al, however, the precise mechanics of this function in SARS-CoV-2 specifically were poorly understood.

IFN regulation by SARS-CoV-2 nsp12 is not related to the NiRAN domain. a & b Schematic diagram of ... [+] "SARS-COV-2 NSP12 ATTENUATES TYPE I INTERFERON PRODUCTION BY INHIBITING IRF3 NUCLEAR TRANSLOCATION" Image Origin

First, Wang et.al set out to determine whether NSP12 stops interferon production. They knew this was true of several other viruses and suspected SARS-CoV-2 would be no exception. After transfecting cells with NSP12-expressing plasmids, Wang et.al found that NSP12 could suppress a receptor kinase that triggers the RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated protein 5) signaling pathways, which mobilize interferon beta production. This confirmed that the protein did indeed inhibit initial activation of interferon. Wang et.al also repeated the experiment to test whether SARSCoV-2 NSP12 could suppress signaling pathways downstream of interferon. This possibility arose when an assay showed that NSP12 suppressed STAT1 phosphorylation, one of the processes that precipitates the expression of interferon-stimulated genes. But NSP12-expressing cells failed to suppress the interferon-stimulated gene reporter, confirming that SARS-CoV-2 NSP12 antagonizes interferon activation, rather than downstream activities. Next, Wang et.al identified the exact point of interference of NSP12-mediated immunosuppression. Assays revealed that the presence of SARS-CoV-2 NSP12 reduced levels of signaling proteins downstream of IRF3 (interferon regulatory factor 3), indicating that IRF3 was likely where inhibition took place.

Both phosphorylation and nuclear translocation of IRF3 are critical steps in interferon production. With IRF3 identified as a likely target, the next question posed by Wang et.al was which of these processes NSP12 antagonized. After determining that the presence of NSP12 didn’t have a notable effect on IRF3 phosphorylation, they repeated the experiment with nuclear translocation and discovered the opposite. In cells that overexpressed NSP12, the ability of IRF3 to translocate to the nucleus was significantly diminished. The final aspect of NSP12-mediated immunosuppression that Wang et.al examined was whether the RdRp domain, which contains the protein’s enzymatic RNA synthesis function, was also the mechanism that inhibited IRF3 nuclear translocation. To do this they used remdesivir, an antiviral Covid-19 drug that inhibits the functionality of the NSP12 polymerase. While remdesivir suppressed viral replication successfully, it failed to prevent NSP12 antagonization of IRF3. Wang et.al found the same to be true of the NiRAN domain, which also facilitates RNA synthesis. When they deleted the NiRAN domain, NSP12-mediated immunosuppression was unaffected. This led them to the conclusion that the inhibitory effects of SARS-CoV-2 NSP12 on IRF3 are enacted independently of the polymerase function. A drug that targets the immunosuppressive capabilities of SARSCoV-2 NSP12 specifically, instead of its enzymatic activity exclusively, could make a promising candidate for treating and preventing Covid-19. Drugmakers should act to exploit the knowledge we now have of the SARS-CoV-2 genome and the viral proteins that inhibit innate immunity. After all, what use is this preponderance of evidence if we don’t use it to help the people this disease hurts most? This article originally appeared on Forbes.org, and can be read online here: Functions Of SARS-CoV-2 NSP12 Polymerase: Replication, Transcription, And Suppression Of Natural Immunity

Functions Of SARS-CoV-2 NSP13 Helicase: Replication, Transcription, And Suppression Of Natural Immunity Forbes | January 28, 2022 | Article

This is part twenty-one of a series, “How SARS-CoV-2 Delays, Evades, and Suppresses the Immune System.” The remarkable transmissibility of SARS-CoV-2, and the new Omicron variant in particular, can be attributed to a wide range of factors, both epidemiological and biological. The trick is figuring out where exactly to intervene to stop the endless cycle of replication and transmission, especially when the protections afforded by vaccination prove insufficient. The more we study how this virus engages the human immune system, the more evident it becomes that the interactions between them—and the molecules involved— hold the key to potentially life-saving drug treatments. Over time researchers have compiled an extensive catalog of viral proteins that SARS-CoV-2 deploys to delay and suppress a robust immune response. Though this multilayered program of immune suppression emerged from an eons-long battle between humans and microbes, meaning we’ve seen this before, in SARS-CoV-2 the mechanisms are alarmingly sophisticated, clearing the way for intensive viral replication before the immune system can react. A new study, published in January 2022 in The Journal of Immunology by Sui et.al, sheds light on one of the roles NSP13 (nonstructural protein 13) plays in this initial attack, which is to degrade a kinase enzyme upstream of interferon, TBK1 (TANK-binding kinase 1). Interferon is the signaling protein largely responsible for activation of the natural (innate) immune response, the body’s first line of defense against invading pathogens. In turn, detection of these pathogens by molecules along immune signaling pathways is what produces interferon. Some SARS-CoV-2 proteins target interferon directly, but many also interfere at other stops along the circuit, obstructing t0 the point of inaction. 69

One modality of immune suppression deployed by SARS-CoV2 is selective degradation. Normally in living organisms, the clearing of damaged cellular debris is key to maintaining a healthy internal ecosystem. This occurs through two main processes. First, through the proteasome pathway, whereby polypeptides known as ubiquitins (otherwise known as the “kiss of death”) mark proteins for degradation via proteolysis, the breakdown of proteins into peptides or amino acids. Second, through the autophagy-lysosome pathway, whereby debris is delivered to the lysosome, a specialized membrane, for degradation. According to Sui et.al, it is via autophagy—which in the original ancient Greek means “self-devouring”—that NSP13 degrades TBK1. TBK1, along with MAVS (mitochondrial antiviral-signaling protein) and IRF3 (interferon regulatory factor 3), form a complex that initiates interferon production once RIG-I (retinoic acidinducible gene I) or MDA5 (melanoma differentiation gene 5) detects viral RNA. Building off evidence from a previous study that SARS-CoV-2 NSP13 interacts with TBK1, Sui et.al first identified that overexpression of NSP13 leads to a decrease in TBK1, but not RIG-I, MDA5, MAVS, or IRF3. This suggests that NSP13 inhibits transcription of interferons by degrading TBK1 specifically. To deduce which protein degradation pathway is involved in decreasing TBK1 levels, Sui et.al used autophagy and proteasome inhibitors to treat cells transfected with NSP13 and TBK1 plasmids. The resulting data showed that autophagy inhibitors kept TBK1 quantities intact, leading Sui et.al to conclude that the autophagy pathway was what NSP13 hijacked to degrade TBK1 in SARSCoV-2-infected cells. Next Sui.et al had to discern whether NSP13 induces autophagy directly, or recruits facilitating cofactors. They found that NSP13 doesn’t induce autophagy directly, but instead recruits TBK1 into autophagosomes selectively. More specifically, NSP13 enlists the help of p62, a cargo receptor that typically regulates adaptors along the interferon and inflammatory response pathways, including STING (stimulator of IFN genes). Sui et.al show that p62 associates with the complex formed by NSP13 and TBK1 that enters the autophagosome. Each of these components—p62, TBK1, and SARS-CoV-2 NSP13—represents a potential target we can use to tip the balance 70

in our favor. Drug developers should take note. Against increasingly infectious variants like Omicron, we need all the reinforcements we can muster. This article originally appeared on Forbes.org, and can be read online here: Functions Of SARS-CoV-2 NSP13 Helicase: Replication, Transcription, And Suppression Of Natural Immunity

Understanding Omicron: A Structure-Function Tour De Force Forbes | January 28, 2022 | Article

This is the second in a series on the Omicron variants. Scientific progress in understanding Omicron has been remarkably swift since its discovery in late November of last year. The newest paper from the University of Washington by McCallum et al. is a tour de force in helping us understand the properties of Omicron in molecular detail. It is a marvel of modern science that such depth and understanding of a new form of a dangerous pathogen can be unraveled in such a short time. Just two weeks ago, we wrote our first story about the structural-functional relationship of the genome and proteins of the Omicron variant infecting the United States and most of Europe at that time. In previous weeks, there was little appreciation of the potential importance not only of the predominant form of the Omicron variant, now called BA.1 but of its sibling variants—BA.2 and BA.3—that arose simultaneously and are now a topic of great interest as BA.2 is on the rise. This series discusses what's known about these three variants in more detail. McCallum and colleagues employed two techniques to understand the structural-functional relationship of mutations in the Spike protein of BA.1: cryo-electron microscopy and X-ray crystallography. Both of these techniques can elucidate the structure of proteins at near-atomic resolution. ACE2 Binding One of the outstanding questions of Omicron is why it is so much more transmissible than the original Wuhan strain or any of the following variants: Alpha, Beta, Gamma, and Delta. The initial thought is that the properties of the Spike protein, particularly the affinity of binding to the ACE2, would reveal the answer. McCallum et al.'s work reflects what other studies have found. Omicron binds the ACE2 receptor less tightly than many previous variants and only

about two and a half times as tightly as the Wuhan strain. Molecular analysis of the Spike protein reveals why this is the case. Some mutations, like lysine to asparagine at position 417 (K417N), glutamine to arginine at position 493 (Q493R), and glutamine to arginine at position 498 (Q498R), are predicted to reduce the binding affinity to ACE2 by modifying the salt bridges in the Spike. Others, like asparagine to tyrosine at position 501 (N501Y) and serine to asparagine at position 477 (S477N), have been shown to increase ACE2 affinity by creating new hydrogen bonds. Collectively, these changes add up to no more than a 2.4fold increase in binding affinity relative to the Wuhan isolates.

FIGURE 1: SARS-CoV-2 Omicron RBD mutations promote escape from a panel of clinical mAbs. (A) RBD ... [+] MCCALLUM ET AL.

In conclusion, the Omicron receptor-binding domain affinity does not fully account for the variant’s increased infectivity. For example, the Beta variant binds to the ACE2 receptor more tightly than Omicron it is not noticeably more but it is substantially less transmissible. Immune Evasion The ability of Omicron to evade the immune response, elicited either by natural infection or by vaccination, is now legendary. Although people who have recently been triple vaccinated have some protection against Omicron infection, the protection from natural or vaccine-induced antibodies fades to close to zero between three and five months. It is essential to distinguish between protection from transmission and serious disease. Most, if not all, vaccines provide substantial protection, in some cases up to 90% or greater, from hospitalization, serious illness, and death for a year or more. The study provides a detailed explanation at an atomic resolution of how viruses evade natural immune defenses and monoclonal 73

antibodies in an elegant atom by atom tour. The work also describes why at least one FDA-approved monoclonal antibody, sotrovimab, maintains much of its activity. McCallum and colleagues describe each of the mutations and how they contribute to escape from monoclonal antibodies that are effective against other variants. They suggest that amino acid mutations threonine to isoleucine at position 95 (T95I), deletion at position 211 (del211), leucine to isoleucine at position 212 (L212I), and insertion of glutamic acid-proline-glutamic acid at position 214 (ins214EPE) modulate recognition by some of the antibodies, specifically S2L20 and S2X333. Glycine to aspartic acid at position 142 (G142D) and deletion of positions 143 to 145 (del143-145) account for evasion from neutralization mediated by antibodies that recognize the N-terminal domain specifically. Amino acid substitutions lysine to asparagine at position 417 (K417N), glycine to serine at position 466 (G446S), serine to asparagine at position 477 (S477N), threonine to lysine at position 478 (T478K), glutamic acid to alanine at position 484 (E484A), glutamine to arginine at position 493 (Q493R), glycine to serine at position 496 (G496S), glutamine to arginine at position 498 (Q498R), and tyrosine to histidine at position 505 (Y505H) are all shown to inhibit at least one binding site from a monoclonal antibody (Figure 1B). These mutations use a combination of steric hindrance, electrostatic contact remodeling, abrogating electrostatic interactions, and so on. These and the rest of the N-terminal domain and receptor-binding domain mutations work similarly to reduce the binding of convalescent and vaccine antibodies.

FIGURE 2: (A) S2L20-bound Omicron NTD with mutated, deleted, or inserted residues rendered or ... [+] MCCALLUM ET AL.

In conclusion, the molecular analysis provides a very firm basis for understanding the ability of Omicron to view evade both natural, vaccine-induced natural, and monoclonal antibody neutralization. Endosomal Entry Another prominent feature, which has come as a great surprise to those who study the virus, is that the route of entry for the Omicron family of viruses is remarkably different from that of previous strains. Those viruses enter membrane-to-membrane, which occurs after binding. Two proteolytic cleavage events release the fusion peptide, forming a membrane bridge between the virus and the cell. This allows the virus nucleic acids to enter the cell and begin replicating. Such is not the case for the Omicron family, which enters via the endosomal pathway. The whole virus enters the cell in a bubble instead of membrane entry. Once within the bubble, the virus can break through and spread.

FIGURE 3: Membrane to membrane vs. endosomal entry of SARS-CoV-2 in Delta vs. Omicron GEORGINA BROWN/JOE GROVE/INSIDER

Why does this occur? Mutations near the furin cleavage site in many variants led some to believe they would enhance cleavage efficiency. In the Omicron variants, there are many more mutations in these regions. However, McCallum et al. show that some of these mutations in the Omicron Spike protein interfere with cleavage, reducing efficiency. The mutations in this region reduce fusion and cleavage efficiency by introducing inter-promoter electrostatic contacts, improving intra-promoter hydrophobic packing, and so on. The mutations, threonine to lysine at position 547 (T547K), histidine to tyrosine at position 655 (H655Y), asparagine to lysine at position 764 (N764K), aspartic acid t tyrosine at position 796 (D796Y), histidine to lysine at position 856 (N856K), glutamine to histidine at position 954 (Q954H), asparagine to lysine at position 969 (N969K), and leucine to phenylalanine at position 981 (L981F), may work synergistically to yield reductions in these processes.

FIGURE 4: SARS-CoV-2 Omicron S mutations outside the NTD and RBD. MCCALLUM ET AL.

This work is a dramatic advance in our knowledge of the details of Omicron. McCallum et al. elucidate some of its most outstanding characteristics, differentiating from its predecessor variants of concern. However, many mysteries remain. Why this virus is so much more transmissible does not seem to be a property solely of the Spike protein. We note that several other mutations in the other proteins in the genome and silent mutations may alter regulatory sequences, potentially accounting for increased transmissibility. This leads to an interesting comparison over time between Omicron and Delta. A recent study by Hay et al. noted that Delta infections yield a greater viral load than Omicron, and infection durations are typically longer in Delta. However, Omicron’s incubation period is shorter than Delta by roughly a day, meaning after initial infection, a host with Omicron is infectious to others faster than Delta. Absence of Cell-to-cell Fusion Another mystery is Omicron’s reduced syncytia formation. Once a cell is infected with SARS-CoV-2 or some other dangerous pathogens, it can fuse with neighboring cells in an evet called syncytia formation. Therefore, syncytia formation is typically associated with pathogenicity. However, the mutations in the Spike protein, particularly those in the furin and fusion regions, lead to 77

reduced fusogenicity, meaning fusion is less efficient. This is likely what leads to the endosomatic entry. But how this virus came to develop these attributes, we do not know, as most variants to this point of the pandemic have continued to grow in pathogenicity, not reduce.

FIGURE 5: Syncytia formation. VIRAL ZONE

The Virulence Mystery A third mystery is why this virus is less likely to induce hospitalization and death. Some speculate that the requirement for endosomal entry and the absence of cell-to-cell fusion may be part of replicating preferentially in the nasopharynx instead of the deep lung. The speculation suggests that the requirement for endosomal entry is one reason omicron is less pathogenic. Although there may be some merit to the argument, recall that SARS-CoV, the virus responsible for the SARS epidemic, also enters cells via the endosome, not by fusion. SARS-CoV is highly pathogenic, killing about 4% of young adults and 45% of those over 65. The deep lung pathology of SARS resembles that of Covid-19. I doubt that the difference in the pathology of Omicron virus earlier variants of SARS-CoV-2 is due solely to differences in the spike protein. We have much more to learn about the function of the exterior glycoproteins and the 29 other proteins of SARS-CoV-2. This article originally appeared on Forbes.org, and can be read online here: Understanding Omicron: A Structure-Function Tour De Force

Do Cannabinoids Offer A Treatment For Covid19? Maybe. Forbes | January 28, 2022 | Article

New studies show that a familiar medication may help to slow the spread of Covid-19 and its variants. Cannabinoids are a chemical found in hemp that can affect the entire body including the central nervous system and the immune system. While most people may recognize cannabinoids for their relaxing and mind-altering properties, they may also be useful for the treatment of some diseases— perhaps even Covid-19. A recent study in the Journal of Natural Products along with a preprinted study from Cold Spring Harbor Laboratory, suggest several potential areas in which cannabinoid molecules may play a role in treating SARS-CoV-2 infections. Hemp, including the hemp flower marijuana, contains a wide variety of cannabinoids. The most well-known of these cannabinoids are tetrahydrocannabinol (THC) and cannabidiol (CBD). CBD has recently been approved for the treatment of severe epilepsy in children but is also widely available in commercial natural products. In addition to these two compounds, there are dozens of minor components that may have medical applications as well. One of the compounds that has been studied by two different research groups is CBD. The first paper explores the role of a CBD precursor in the interaction between the SARS-CoV-2 spike protein and ACE2 receptors found in human epithelial cells.

Chemical structure of cannabidiol (CBD).WIKIMEDIA COMMONS

Surprisingly, when examining the structure of a precursor to CBD called CBDA, along with a precursor to another compound called cannabigerol (CBGA), scientists found that both CBDA and CBGA contained physical properties that allowed them to bind to the SARS-CoV-2 spike protein. When CBDA and CBGA were introduced to a system containing healthy cells and the virus, the cannabinoids competed with the epithelial cells to bind with the spike protein, effectively blocking SARS-CoV-2 from infecting the cells. While the study found that both compounds were able to combat SARS-CoV-2 infections, a very high dosage was required for them to be effective. This suggests that while CBDA and CBGA may not be ideal molecules to treat SARS-CoV-2, there may be variants of the compounds that have a much tighter affinity to the spike protein and would require a smaller dosage, which is worth exploring. The second study provides an entirely different mechanism for the potential action of CBD against SARS-CoV-2 through a response called apoptosis. Apoptosis is a natural consequence of infections and occurs when an infected cell self-destructs to prevent the virus from replicating and spreading to other healthy cells. This occurs without causing an inflammatory reaction, making it a fairly innocuous process with limited side effects. It is one of the fundamental aspects of our body’s natural immunity and apparently, may be potentiated by CBD. Fernandes et al. found that when infected cells were exposed to CBD the cells engaged in apoptosis at a significantly higher 80

frequency than their control counterparts. This indicates that in the presence of CBD, our cells’ innate immune systems are much more effective at defending against SARS-CoV-2 which could lead to a much milder or completely absent course of illness. In this case, significant results occurred in the presence of a much lower concentration of CBD. These two studies are promising. However, they do not suggest that patients should seek over-the-counter CBD products or recreational marijuana as a defense against Covid-19. While companies have already begun using studies like these to promote their CBD products, no clinical trials have been conducted to suggest that these compounds would be effective against Covid-19 in humans. Not to mention, separate studies have found that the presence of THC – a common compound found in marijuana products— actually counteracted any benefits of CBD found in the studies. Overall, though these studies do not suggest an immediate application of CBD for the treatment of Covid-19, they do open the door to a very interesting new area of research for anti-SARS-CoV2 drugs. This could include drugs that have a higher affinity for the SARS-CoV-2 spike protein, or research on a variety of cannabinoids that may strengthen the body’s ability to combat the virus. This article originally appeared on Forbes.org, and can be read online here: Do Cannabinoids Offer A Treatment For Covid-19? Maybe.

Novel Vaccine Booster Strategy Prevents Covid-19 Infection Forbes | January 31, 2022 | Article

A new approach to Covid-19 vaccinations may slow the spread of infection. We all know by now that current vaccines are not effective in preventing infection against Omicron. Although these vaccines may protect people from severe disease for a year or more, they are only effective for preventing infection for a few months. A new report suggests that there may be a way to not only protect against severe disease but also reduce the risk of infection. One weakness of intramuscular injections is their inability to block the virus at the primary site of infection— the nasal passages. As a result, researchers at the University of Hong Kong are exploring a novel vaccine strategy aimed at increasing antibody activity throughout the respiratory system and supporting long-term protection against infection. This approach consists of a two-step process— injection plus intranasal booster. The two-part vaccine strategy first employs an intramuscular DNA vaccine. Similar to the mRNA vaccines produced by Pfizer and Moderna, this injection exposes the immune system to part of the SARS-CoV-2 spike protein used for viral entry into host cells. In the presence of this viral antigen, the immune system produces neutralizing antibodies to protect against future infection. The second part of this two-part strategy is administered through a nasal spray. Researchers constructed a viral vector from plasmid DNA commonly used for intranasal flu vaccines and genetically engineered them to also express part of the SARS-CoV-2 spike protein. In animal studies, intranasal vaccines have been variably effective in blocking viral replication before a virus has an opportunity to infect cells. While the first injection provides some systemic protection against the virus, an intranasal Covid-19 booster can strengthen immunity directly at the site of infection. For the first phase of this vaccine approach, Zhou et al. manufactured an intramuscular injection of a DNA vaccine. A 82

sequence expressing SARS-CoV-2 receptor-binding domain (RBD) joined to a programmed cell death protein (PD1) gene sequence was fused to a pVAX plasmid backbone (RBD- PD1DNA). The SARS-CoV-2 RBD, which appears as spike proteins on the virus’s outer surface, is a short fragment from the virus that binds to ACE2 receptors on host cells to gain entry. Preventing these proteins from binding to cells is an important target for developing neutralizing antibodies. Fusion of the PD-1 domain to the RBD antigen encourages T immune cells to bind onto the viral antigen and induce an immune response. To facilitate protein expression of the RBD antigen, a CMV promoter and human tissue plasminogen activator (tPA) were also inserted, as shown in the figure below.

FIGURE 1: Construction of RBD-PD1-DNA intramuscular vaccine. A (G4S)3 linker sequence was used to fuse Sars-CoV-2 RDB and PD1 domain on a pVAX plasmid backbone. A CMV promoter and tPA secretory signal sequence controlled for protein expression.FROM: “NASAL PREVENTION OF SARS-COV-2 INFECTION BY INTRANASAL INFLUENZA-BASED BOOST VACCINATION IN MOUSE MODELS” RUNHONG ZHOU 2022

Packaging the viral antigen within a plasmid DNA allows the manufactured vaccine to reach the cell’s nucleus for synthesis of the viral RNA to begin. The viral RNA is transported into the cytoplasm, where ribosomes transcribe RNA into proteins. The corresponding viral proteins are then displayed on the surface of immune cells to alert the immune system to begin producing antibodies. If the individual is later exposed to the virus, immune cells recognize the viral spike proteins and readily fight infection before it spreads.

FIGURE 2: Mechanism of action for PD-1 antigen-linked DNA vaccine.IMMUNO CURE BIOTECH LIMITED

Direct injection into the muscle optimizes the vaccine to reach important immune cells in muscle tissues, which then deliver viral antigen to the lymph nodes, the main hubs for initiating immune responses. Over time, however, the immune system’s memory of the spike protein weakens and immunity declines. A booster vaccine can strengthen immunity by reintroducing the viral spike protein into the body. The second phase of this vaccine strategy exploits the influenza virus’s ability to enter the body through the nasal pharynx. Derived from viral vectors commonly used for intranasal flu vaccines, researchers created an intranasal booster spray using strains of live attenuated influenza virus vaccine (LAIV). Against the flu virus, intranasal vaccines have been effective in not only producing an immune response in the nasal passage but also providing systemic immune protection. The mucosal immune system triggers adaptive immune responses that block pathogens from invading nasal tissue. Immunoglobulin A (IgA) antibodies are at the forefront of this defense system, working to bind onto and neutralize foreign pathogens. In contrast, systemic immunity induced by vaccine injections largely protects the inner body and primarily recruits Immunoglobulin G (IgG) antibodies found circulating in the blood. 84

Intranasal LAIV are specifically designed to stimulate the production of mucosal IgA antibodies in the nose, blocking the flu virus from attaching to host cells. Animal studies suggest that nasal vaccines can enhance both mucosal and systemic immunity. To protect against Covid-19 infection, LAIV were modified to express the SARS-CoV-2 RBD gene. Using a pHW2000 viral plasmid backbone, an RBD antigen was fused into a NS1 genedeleted segment between two noncoding regions (NCR). As illustrated in the figure below, the RBD domain was linked to a human plasminogen activator (tPA) and a V5 tag to allow the immune system to better detect the viral gene segment. Two strains of intranasal LAIV DelNS1-RBD were derived from surface protein sequences of corresponding H1N1 and H3N2 influenza subtypes: LAIV-CA4-RBD and LAIV-HK68-RBD.

FIGURE 3: Construction of DelNS1-RDB intranasal vaccine booster. Human tissue plasminogen activator (tPA)-linked SARS-CoV-2-RBD fused to an NS1-gene-deleted segment between the noncoding regions (NCR) and autoproteolytic cleavage site (2A) within a pFROM: “NASAL PREVENTION OF SARS-COV-2 INFECTION BY INTRANASAL INFLUENZA-BASED BOOST VACCINATION IN MOUSE MODELS” RUNHONG ZHOU 2022

This method takes advantage of well-established routes for flu vaccination and modifies it to boost protection against Covid-19 infection. When administered through a fine mist, this harmless viral vector stimulates IgA production in nasal mucus and supports systemic immunity throughout the respiratory system. The most successful vaccination strategy first exposed mice subjects to an intramuscular vaccine injection followed by an intranasal booster 21 days later. This prompted enhanced mucosal immunity, particularly in nasal tissues, and systemic immunity in the upper and lower respiratory pathway. When they were later exposed to Covid-19, mice in this experimental group had no detectable amount of the virus in their nasal passages, compared to mice that 85

received two intramuscular injections or two intranasal vaccines. Furthermore, in contrast to other experimental groups, these mice experienced no loss in weight and displayed only mild injury to respiratory tissue. All vaccinated mice had some protection against the virus, as indicated by increased levels of IgG antibodies found in blood and other bodily fluids. Mice that received a vaccine injection followed by a nasal booster, however, had the greatest protection against Covid-19. These mice displayed the highest and most sustained recruitment of viral antibodies 90 days after the initial injection. It is important to note that only the mice who received the prime boost had enhanced levels of IgA antibodies collected by nasal swab samples. IgA levels were not sustained long-term in mice that received two intranasal vaccinations, suggesting that a heterogenous injection plus nasal booster strategy is most effective in promoting mucosal immunity. Additional experiments from this study confirmed that the mice given a vaccine injection then an intranasal booster have a significantly higher frequency of CD8+ T cells in both the lungs and nose, which positively correlated with an increased prevalence of neutralizing antibodies. The long-term protection of this vaccine and booster regiment correlates with the recruitment of memory CD8+ T cells, a type of white blood cell that induces cell death in infected tissues. These cells continuously circulate throughout the body, so that when a foreign pathogen is encountered, they become activated to prevent the spread of infection. CD8+ T cells are critical for preventing the growth of cancer tumors and defending against viral infection. Because they are antigen specific, encountering the viral antigen again generates an enhanced immune response within these longlived immune cells. CD8+ T cells continue to hold a memory of the viral antigen for a long time, allowing them to readily activate when exposed to the virus in the future. Therefore, vaccine regimens that capitalize on this adaptive immune mechanism are particularly effective at preventing infection anywhere from a few months to years. The next step for research is human trials to capitalize on the double barrel effect of this vaccination strategy. Currently, Covid19 booster shots remain an integral part of our infection prevention 86

strategy, alongside masking in crowded spaces, physical distancing and self-quarantine following a positive test. To control the spread of infection more effectively, future vaccines should aim to boost immunity directly through the nose and upper respiratory system. As long as the virus is able to easily spread throughout the population, emerging variants will continue to overwhelm our healthcare system. This article originally appeared on Forbes.org, and can be read online here: Novel Vaccine Booster Strategy Prevents Covid-19 Infection

February 2022

'Decoy' Protein Offers New Treatment Approach For Covid-19 Forbes | February 2, 2022 | Article

The rise of immune-evasive variants like omicron, and breakthrough infections along with it, has foregrounded the need for antiviral therapies. Particularly antiviral therapies that can stand up to multiple different variants without succumbing to resistance. Researchers at the University of Illinois Chicago have just taken a step in that direction, developing and testing a “decoy” protein that tricks SARS-CoV-2 into binding to it instead of to host cells. SARS-CoV-2 infects us by binding to angiotensin-converting enzyme 2 (ACE2). Located on its outer surface, the virus’ Spike proteins latch onto our ACE2 receptors, which act like cellular doorways of sorts, allowing the virus to enter our cells. Given the importance of this interaction, many variants of concern exhibit mutations to their spike protein that improve their ability to bind to ACE2. Mutations that interfere with the binding process would hinder the variant’s ability to replicate, reducing transmissibility and viral fitness more generally. So, most variants are primed to “recognize” and bind to ACE2. By extension, they are also primed to bind with anything structurally similar to ACE2. Zhang et al. took advantage of this by engineering artificial ACE2 proteins that mimic human ACE2. The engineered decoys are designed with three amino acid substitutions—T27Y, L79T and N330Y— which improve how strongly they can bind to SARSCoV-2’s spike protein: a full 35-fold increase in binding strength when compared to unmutated human ACE2. The drug treatment works by allowing these extra “sticky” engineered ACE2 proteins to compete with human ACE2 in a race to bind to SARS-CoV-2’s Spike protein. Because of their heightened binding affinity, the decoys win out (Figure 1).

FIGURE 1. Schematic representation of ACE2 decoy (red) binding to SARS-CoV-2 before it can get to a ... [+] FROM: “ENGINEERED ACE2 DECOY MITIGATES LUNG INJURY AND DEATH INDUCED BY SARS-COV-2 VARIANTS.” ZHANG ET AL. 2022

This was reflected in vivo by tests the researchers performed on mice. Usually mice aren’t a good measure of Covid-19-related injury since their ACE2 receptors don’t bind well with the Spike proteins of SARS-CoV-2. To circumvent this issue, Zhang et al. used mice that express human ACE2 instead, more accurately modeling the disease progression and damage one would see in humans. They infected the mice with SARS-CoV-2 and then split them into three groups: a control group that received nothing, a group that received the ACE2 decoy 12 hours post-infection, and another that received it 24 hours post-infection. The treatment was delivered intravenously daily for a period of seven days. After two weeks, all of the mice in the control group had died, showing a 30% weight loss. Both treatment groups, on the other hand, boasted survival rates of 50 - 60%, with marked reduction in lung damage and no signs of severe acute respiratory syndrome. Fourteen days post-infection, the treated mice were back to normal. In vitro results showed that the engineered ACE2 also remained effective against the alpha, beta, gamma, and delta variants of concern, maintaining a tight bond with their respective Spike proteins. Omicron had not yet come onto the scene during these trials. To make sure these results carried over to live infection, Zhang et al. exposed mice to the gamma variant. Again, all of the mice in the control group died. The mice that received treatment 24 hours post-infection did not fare much better, their death only delayed but 90

not avoided. Treatment at 12 hours post-infection, however, brought about a 50-60% reduction in death and a return to full health 14 days post-infection. "Considering the emergence of omicron, it is very good news that the ACE2 decoy was able to bind and neutralize several variants, and this reinforces the potential of this drug as a treatment, including against new or future variants of the virus," said Jalees Rehman, colead author of the study. Aside from its ability to remain effective against variants, the engineered ACE2 protein has two other advantages: it can be delivered through inhalation and it can be used as a pre-exposure prophylactic. Intranasal administration significantly reduces time and cost when compared to intravenous delivery and pre-exposure use could help immunocompromised individuals preemptively boost their immune systems, albeit temporarily. Hopefully the results from these mouse models can be replicated in human trials; after all, this engineered ACE2 decoy seems to offer us another promising approach to Covid-19 treatment. The more interventions we have available to us, the more varied and tailored we can make our treatment plans, and the less likely we are to be confronted with viral resistance. This article originally appeared on Forbes.org, and can be read online here: 'Decoy' Protein Offers New Treatment Approach For Covid19.

The Omicron Surprise

Forbes | February 2, 2022 | Article

This is the third in a series on the Omicron variants. The rise of Omicron has come as a surprise. Many were hoping Delta would be the last in a chain of variants of increasing transmission and virulence. Not one but two new forms of Omicron, BA.1 and BA.2, have put a swift end to that hope. Both seem to have arrived as if from nowhere to quickly sweep the world. Omicron first appeared in Southern Africa in late November. Omicron appeared not as a single variant but as three simultaneously denoted BA.1, BA.2, and BA.3. All three are derived from a common parent, which itself derived from the long-vanished B.1 variant, which was the dominant global virus beginning in early 2020 until it was displaced by an alphabet of regional variants: the Alpha to Eta set.

FIGURE 1: Lineage of Omicron family viruses. The date included is when that specific variant was ... [+] ACCESS HEALTH INTERNATIONAL

Despite detection simultaneously, all three Omicron variants are as different from one another as Alpha is from Beta, Beta from Gamma, and Gamma from Delta. The origin is shrouded in mystery. Did all three incubate in an isolated population? Is the parent the result reverse Zoonosis? Did all three arise in one or more immunosuppressed individuals after prolonged incubation? Is there another story to be told? Omicron Dominates 92

As I write in early February 2022, Omicron is now the dominant virus worldwide. BA.1 quickly displaced Delta in mid-rampage in many parts of the world, including the United States and Europe. Concurrently, BA.2 is on the rise or the dominant variant in many countries. When BA.1 and BA.2 are present, BA.2 is out-competing even BA.1. As of now, BA.3 accounts for only a few infections.

FIGURE 2: Regional distribution of cases relative to the frequency of Delta, BA.1, and BA.2 strains. STEBBINGS

Sadly, most countries seem entirely helpless to stop the sweep of the virus. Omicron infections rise and fall not due to our efforts to contain the infection but rather from its own internal dynamics. To state it clearly, Omicron infections are out of our control (with the exception of China, as per usual). The degree of transmissibility of both BA.1 and BA.2 is astonishing, outpacing even our most pessimistic projections by epidemiologists and virologists. Omicron leads to near-vertical increases in cases numbers. In the two months since Omicron was first discovered, the virus has infected at least 90 million people worldwide, more than all other variants for 2020, the first full year of the pandemic. Several key novel characteristics seem to account for Omicron's spread. The virus's ability to evade immune protection conferred by prior infection or vaccination is startling. Both BA.1 and BA.2 rip 93

through even well-vaccinated populations as if they were Covid naive. Once the infection is initiated, the virus takes hold at a record pace. Virus concentrations reach a peak within two days of exposure, assuring those who contract the virus spread it quickly to others. Transmission Just how very transmissible are the Omicron variants? The previously dominant variant Delta is about 1.5 times as transmissible as Alpha, Beta, and Gamma., In their day, the prior variants of concern were about 1.5 more transmissible than the original B.1 variant, which was somewhat more transmissible than the original Wuhan isomers. Recent studies from Denmark where BA.2 rapidly displaces BA.1 as the dominant variant suggest that BA.2 is at least 1.5 times more transmissible than BA.2. After some quick calculations, BA.2 is at least seven and a half times more transmissible than the original Wuhan isolate, on par with the most infectious diseases ever described!

FIGURE 3: Secondary Attack Rate for households infected with the Omicron BA.1, BA.2, and Delta VOC, ... [+] LYNGSE ET AL.

We are fortunate that increased lethality does not track with Omicron increased infectivity. BA.1, though pathogenic, is less virulent than Delta. BA.1 infection of susceptible hamsters and mice causes a much milder disease than with earlier variants. How much less virulent Omicron BA.1 is than another variant—as measured by 94

severe disease incidence, hospitalization, and death—is up for debate. What is clear is that full vaccination (three doses) or prior infection plus vaccination (two doses) reduces serious consequences of Omicron infection five to tenfold. A recent preliminary study from South Africa suggests that the unvaccinated Omicron is still about as virulent as the early Alpha variant. Be that as it may, the BA.1 variant, as of early February 2022, kills between 2,000 and 3,000 people a day in the United States alone. The bright note is that complete vaccination (three or four shots of an mRNA or a combination of an adenovirus-based vaccine with two doses of mRNA vaccines) provides substantial protection against hospitalization and death.

FIGURE 4: Severity of Disease in the Unvaccinated ACCESS HEALTH INTERNATIONAL

Immune escape The vast number of mutations in the Spike protein BA.1 and BA.2 (more than thirty each) alter the immune response to the virus. Sera of susceptible mice infected with BA.1 neutralize BA.1 effectively, but the same neutralize all prior variants poorly. Conversely, most sera induced either by natural infection or vaccination are approximately ten times less effective in neutralizing BA.1 than their activity against other variants. Current vaccines' protection wanes to close to zero over five to six months. Virus Entry Efficient cleavage of the Spike protein while budding from the surface of a virus-producing cell is a hallmark of SARS-CoV-2 compared to SARS-CoV-1. Efficient cleavage of the precursor Spike proteins into the active S1 and S2 proteins allows SARS-CoV2 to enter the cell via fusion of the viral cell membranes. In contrast, the endosomal entry route is typical of SARS CoV-1. Efficient cleavage of the SARS-2 spike is attributed to the furin cleavage site at the S1/S2 boundary. Spike protein cleavage at the cell also underlies syncytial formation (cell-to-cell fusion) of infected cells. A puzzle: BA.1 retains the furin site but is poorly cleaved. Why? The answer must lie in the altered structure, most probably mutations near the cleavage site and in the membrane-associated S2 95

protein. Still, despite elegant work on the structure of the Omicron spike, the details remain a mystery. Analysis of Omicron BA.1 and BA.2 Mutations Orf1ab: Replication Complex The replication complex of SARS-CoV-2 is comprised of 16 nonstructural proteins (NSP 1-16). Between BA.1 and BA.2, there are 21 changes in the amino acids in this otherwise highly conserved region of SARS-CoV-2. Of these seven are shared between the two, six are unique to BA.1, and eight are unique to BA.2 One of these shared mutations is a proline to leucine change at position 323 of the RNA-dependent RNA Polymerase (NSP12 P323L). This NSP12 mutation, together with the nucleotide change C241U in the five prime untranslated regions, is common to all variants of the B.1 lineage, including the Alpha, Beta, Gamma, and Delta. The functional significance of the single amino acid changes in the polymerase proteins is yet to be determined. The single nucleotide change in 5 prime translated regions is reported to create a binding site for the cellular TAR binding protein known to regulate RNA metabolism and increase the rate of virus replication.

FIGURE 5: BA.2 Genome mutations. Those in blue are shared with other Omicron sublineages. Those in ... [+] ACCESS HEALTH INTERNATIONAL

Spike Protein The Spike protein of the Omicron variants is heavily mutated. There are 43 amino acid changes between the sequence of the reference spike protein and that of the BA.1 and BA.2 proteins, far more than any other variants of concern. Of these, 22 are shared, 16 are unique to BA.1, and 5 are unique to BA.2.

FIGURE 6: BA.2 Spike mutations. Those in blue are shared with other Omicron sublineages. Those in ... [+] ACCESS HEALTH INTERNATIONAL

Neutralizing antibodies in sera of infected patients recognize the receptor-binding domain (amino acids 331 to 528) and the N terminal domain (amino acids 13-306). Within the N-terminal domain of BA.1 and BA.2, there are 14 amino acid changes. Only one is shared between the two variants. Twelve are unique to BA.1, and three are unique to BA.2. Such divergence suggests that the two variants evolved resistance under different immune pressure, possibly in different hosts. Antibodies that neutralize the virus via binding Nterminal domain epitopes of BA.1 are unlikely to neutralize BA.2 and vice versa. There are 18 amino acid changes in the receptor-binding domain of the two Omicron variants. Thirteen of these are shared. Two are unique to BA.1, and 3 are unique to BA.2. Likely, most antibodies that bind BA.1 will also bind and neutralize BA.2. I suspect most sera will be cross-neutralizing, whereas many monoclonal antibodies may neutralize only one but not both variants. There are 11 amino acid changes in the Spike protein that lies carboxy-terminal to the receptor-binding domain. Eight are shared, and three are unique to BA.1. I suspect that BA.2 is likely to share the unusual cleavage and membrane fusion properties of BA.1. The Structural Proteins M, E, and N All genes that encode the structural proteins S (spike), M (membrane), E (envelope), and N (nucleocapsid) are located three prime to orf1ab. BA.1 and BA.2 share the same single amino acid mutation in the envelope protein. Two of the three amino acid changes in membrane protein BA.1 contains one additional amino acid change in the membrane protein.

FIGURE 7: Viral schematic of SARS-CoV-2 structural proteins. ACCESS HEALTH INTERNATIONAL

These changes entail either a charge or a change from polar to non-polar residues, potentially affecting the structure and function of the mature virus particle. These changes may increase the stability of the virus particle and therefore prolong the survival of the virus in the air or on surfaces. Increased stability of the virus particle may also increase the transmission rate. There are seven amino acids changes in the nucleocapsid proteins. Six of these are shared between BA.1 and BA.2. One is unique to BA.2. Two of the changes are common to most variants of concern, those at amino acids 203 and 204. Identical changes are found in most variants of concern. These changes have been shown to dramatically increase the titers of virus when green on cells in the laboratory and may account in part for the increase in transmission of all variants as compared to the reference isolates. BA.1 and BA.2 share the same deletion of amino acids 31, 32, and 33. This deletion is unique to Omicron. Accessory Genes SARS-CoV-2 specifies an additional set of accessory genes, many of which regulate the host's innate immune response. These include Orf3a,b,c and d; Orf6, Orf7a and b; Orf8; Orf9b; and Orf10. 98

There are no amino acid changes in these proteins in BA.1 relative to the reference isolate and only two in BA.1 proteins—a single amino acid change in Orf3a and another in Orf6 of BA.2. The lack of mutations in the accessory proteins is usual amnesty variants of interest. Noncoding Mutations We also note many noncoding mutations in the Omicron viral genome. These nucleotide changes that do not change amino acids could still impact the virus. There are sequences throughout the virus that play into processes like replication and transcription. Noncoding mutations have the potential to impact these sequences. Below is the summary of noncoding mutations in BA.2.

FIGURE 8: Noncoding Mutations in Omicron sublineages. ACCESS HEALTH INTERNATIONAL

Ultimately, BA.2 poses a similar, if not more significant, threat than its more pervasive sibling BA.1. All indications suggest at least 1.5 times as transmissible as BA.1. If the virulence of this strain remains the same or is more severe, it could yield more infections, hospitalizations, and deaths than any previous point of the pandemic. As such, we must remain cautious and stringent in our control of Covid-19. The lesson from Omicron is that we should expect the unexpected from this wily virus. We are just beginning to take its full measure. What will come next, a variant that has the lethality of SARS (killing 4% of the young and 45% of the elderly) that transmits like Omicron, or a variant that spreads quickly, evades immunity but causes only cold-like symptoms? Regardless of what is to come, we must do better at controlling the pandemic by all measures possible, 99

public health (detection, contact tracing, and isolation for the incubation period), near-universal vaccination, development of prophylactic and therapeutic drugs, and global equity in access to vaccines, diagnostic tests, and vaccines. This article originally appeared on Forbes.org, and can be read online here: The Omicron Surprise

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Covid Infection During Pregnancy: What Are The Risks? Forbes | February 7, 2022 | Article

Amid the Covid-19 pandemic, the risk of viral infection is a cause for both the mother and child. Although pregnant women are not more likely to contract the virus, infected mothers have an increased risk of severe disease and premature delivery. Key questions are: What are the risks of Covid-19 for the mother and baby? Covid-19 Risks to Mothers The risk of severe Covid-19-related complications is higher among pregnant women, compared to the rest of the population. Pregnant women are more likely to be hospitalized and require respiratory ventilation and critical care. Pregnant women who have Covid-19 also have a greater risk than other expecting mothers of stillbirth and preterm birth, defined as delivering earlier than 37 weeks, as well as an increased risk for developing other pregnancy related complications. In a study from Scotland, unvaccinated women seem to be particularly at risk of these complications if exposed to the virus. Of 4,950 confirmed Covid-19 cases in pregnant women, 77.4% were not vaccinated at the time of infection while 11.5% and 11.1% were partially or fully vaccinated, respectively. Among unvaccinated mothers, 19.5% were admitted to the hospital, in contrast to 8.3% of partially vaccinated women and 5.1% of those that were fully vaccinated. The only documented fetal or infant deaths were seen in unvaccinated women with positive Covid-19 symptoms. Limited data, however, does not explain how direct or indirect effects of viral infection may contribute to preterm births or deaths. Most pregnant women exposed to Covid-19 experience mild to no symptoms, but more severe pregnancy complications are associated with symptomatic unvaccinated patients. Yalda Ashfar, an obstetrician at the Ronald Reagan UCLA Medical Center, warns, “This should

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shake us and really be a call to action…Vaccination is the clear action item to improve health for pregnant women and their babies.” This enhanced risk can be understood by first considering how a woman's body changes as it grows and prepares to birth a new life. Throughout the pregnancy changes in the body alter the maternal immune system to condition tolerance of the fetus. As the fetus grows, the mother’s body enters different stages of immunity. The first trimester is characterized as pro-inflammatory. This phase is important for implanting the embryo and forming the placenta. In the process of adapting to the presence of the fetus, the corresponding innate immune response sends different types of immune cells to the maternal-fetal interface, where the placenta contacts the mother’s blood. Changes in estrogen and progesterone hormone levels in the first trimester simultaneously reduce levels of CD4+ and CD8+ T cells, important for targeting and killing foreign pathogens. Moreover the levels of the ACE2 virus receptor in the placenta, high early on, lower in mid-pregnancy, high again in the last trimester facilitate infection. Emerging data on Covid-19 outcomes suggests that gestational age at the time of infection is one of the greatest predictors for whether the child will be delivered at full term. In a cohort of 18,335 pregnant women who tested positive for Covid-19, researchers found that women infected during the first two trimesters were more likely to deliver pre-term and stillbirth. Minority and low-income communities with high rates of infection have greater risk factors associated with worse maternal and birth outcomes. Piekos et al. stated that women in their Covid-19 positive cohort were more likely to be Hispanic and a race other than White or Asian. Anxieties surrounding the risks of pregnancy during this pandemic also pose significant challenges to mental health. Compared to pre-pandemic incidences, rates of depression among pregnant women have jumped up to 37% and up to 57% for anxiety which persists post delivery. Covid-19 Risk to the Fetus and Infant Current findings indicate that the fetus is largely protected against the infection. There is no evidence that infected mothers transmit the virus to the fetus in utero, though in very rare cases traces 102

of the virus have been detected in fetal blood. Most infants delivered to infected mothers test negative for Covid-19. Understanding this protective mechanism requires a deeper look at the maternal-fetal interface, where the placenta meets the uterus. The placenta is a multi-purpose organ for the fetus, acting as the lungs, heart, kidneys and liver. All the nutrients and resources needed to develop life are transferred from the mother across the placenta and through the umbilical cord. An immune microenvironment is created and sustained at the maternal-fetal interface, serving as a barrier against viral infection. No infection does not mean no consequence for the fetus of the baby. The immune system of the fetus does respond to the mother’s infection. Compared to Covid-19-negative mothers, infected mothers have elevated levels of IL-8, IL-10 and IL-15 cytokines. Researchers hypothesized that some maternal cytokines can pass through the placenta and induce fetal inflammation This neonatal inflammatory response is mostly mild and does not directly threaten the life of the child. Although it is clear that maternal infection does have a measurable impact on the infant, additional research is needed to determine the short-term and long-lasting effects of Covid-19 exposure. Conclusion Expecting mothers need to be vigilant about reducing their risk of exposure to the SARS-CoV-2 virus. This includes upgrading to surgical or N95/KN95 masks, avoiding crowded spaces, and maintaining effective sanitation practices. Women planning to be pregnant should be fully vaccinated against Covid-19 with three standard doses of the Moderna or Pfizer vaccines. Limiting the mother’s risk for infection is our best strategy at protecting the health of the child. Although vaccines are highly recommended for expecting mothers, the persistence of breakthrough cases highlights the importance of greater innovation in vaccine development. Pregnant women, and other vulnerable populations, need vaccines that not only reduce the risk of severe disease but also prevent infection from happening in the first place. The health of the future generation depends on it. This article originally appeared on Forbes.org, and can be read online here: Covid Infection During Pregnancy: What Are The Risks?

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Good News: Full Vaccination Protects Against Omicron Hospitalization And Death Forbes | February 8, 2022 | Article

This is the fourth in a series on the Omicron variants. The current wave of Omicron-based Covid-19 infections has highlighted both the strengths and weaknesses of our current Covidcontrol strategy, which relies primarily on vaccines. The Omicron virus seems to infect most of those vaccinated once or twice, almost as if they were not previously vaccinated at all. Three doses provide some protection, but that protection wanes over four to six months to a very low level. The good news is that two or three doses of currently-available vaccines provide excellent protection against severe disease leading to hospitalization or death. Initial data from the United Kingdom confirms some fears regarding the new strain. A “fully vaccinated” individual with two doses of mRNA vaccine is only protected from Omicron infection roughly 25% of the time. That percentage jumps to 50% in triplevaccinated individuals. Protection from hospitalization and death in those with two and three doses is much greater than protection from infection. Two-dose prevention of hospitalization is roughly 57%, whereas three-dose prevention jumps to approximately 90%. This rise is mirrored in protection from death, as two doses prevent death 59% of the time, while the rate for three doses skyrockets to 95%. If anything, this data is an advertisement to immediately get a third dose of vaccine.

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FIGURE 1: Vaccine effectiveness against Omicron comparing infection, hospitalization, and death ... [+] UK HEALTH SECURITY AGENCY

These risks are exponential in household settings. At present, children and specifically toddlers or newborns are far less likely to be fully vaccinated or boosted based on previous and existing CDC guidelines. That means they are at far greater risk of infection, hospitalization, or worse. Now picture those children in their home setting. If both parents are fully vaccinated or boosted, that child is much less likely to contract Covid-19 at home. However, suppose one is partially or unvaccinated. In that case, the do-diligence of the other is negated, and the children are at much greater risk, not to mention the risk of living with two unvaccinated parents that interact with others at work or society at large.

FIGURE 2: Visual representation of the dangers of Covid-19 infection in a household with one or more ... [+] HAYEK ET AL.

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What accounts for the ability of omicron to infect those who have been previously vaccinated? Molecular studies show that both major Omicron sublineages, BA.1 and BA.2, are heavily mutated in the Spike protein, both in the gene's S1 and S2 subunit portions. These mutations dramatically alter the ability of both convalescent sera and monoclonal antibodies to recognize, bind, and neutralize the Omicron variants. Although BA.1 and BA.2 share many common mutations in the Spike protein, there are several differences. Of the 42 distinct mutations found in the BA.1 and BA.2 sublineages, 21 are shared, while 13 are exclusive to BA.1 and eight are exclusive to BA.2. It seems likely that both BA.1 and BA.2 are similar concerning their diminished sensitivity to neutralizing antibodies from vaccines, monoclonal treatments, or convalescent sera, perhaps accounting for the variant's rapid spread. A thoroughly-vaccinated population such as Denmark and Israel seem to be highly susceptible to infection by Omicron as a consequence. Will Omicron infection provide robust protection against other infections? If current vaccines do not provide reasonable protection against Omicron, will Omicron-specific vaccines provide good protection against previous and new variants to come? The answer is not necessarily yes. Although existing data shows that Omicron infection will protect against reinfection, Omicron sera poorly neutralizes other variants. A simple conclusion would be that Omicron infection is unlikely to protect against future variants to come. A recent study by Gagne et al. on monkeys tested whether Omicron-specific vaccines granted greater protection against the strain than a standard mRNA vaccine booster. Their results suggest that the rush to license variant-specific vaccines may not be justified. They tested an Omicron-specific mRNA booster against a currently-available mRNA vaccine to test the jump in neutralizing antibody titers post-inoculation. Not only did the Omicron booster provide worse protection against the D614G Triad variant than the current mRNA vaccines, but protection against Omicron itself was also worse.

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FIGURE 3: Kinetics of mucosal antibody responses following mRNA-1273 immunization and boost (C-D) ... [+] GAGNE ET AL.

If the virus can infect a previously vaccinated population, the entire population is susceptible. This enables the virus to spread much faster as it has many more targets in a given population. In contrast, previous variants were somewhat limited to those that were partially or unvaccinated against SARS-CoV-2. That, alone, does not account for the rapid acceleration of the rate of transmission as compared to previous variants. The virus is intrinsically more transmissible. Factors that affect the speed of replication may be independent of the Spike protein changes. We know that the Spike structure of the BA.1 variant is more avid to the host ACE2 receptor than the wildtype virus, but that does not entirely account for the rapid spread of Omicron not as avid as Delta. This indicates that changes throughout the genome in non-Spike proteins likely contribute to the rapid increase of transmissibility along with the Spike mutations. The next question is pathogenicity. It is clear that vaccines vigorously protect against Omicron death compared to other variants. However, in the United States, for example, a small portion of the population has received their third dose.

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FIGURE 4: Share of the US population boosted, fully vaccinated, partially vaccinated, and ... [+] NYTIMES

Those partially vaccinated or even wholly unvaccinated are at the most significant risk of infection, hospitalization, and death. In the state of Texas, for instance, the Texas Department of State Human Services estimates that compared to fully vaccinated Texans, those unvaccinated are at least twice as likely of becoming infected with Covid-19 and 17 times as likely to die from Covid-19-related health complications. These rates are consistent with estimations throughout the world. An even more dire comparison is between unvaccinated and boosted individuals. The CDC estimates that unvaccinated adults are 13 times more likely to be infected with Covid-19 and 68 times more likely to die from Covid-19 than boosted individuals. In the United States, as of early December, weekly death rates based on vaccination status were vastly different. The unvaccinated had a weekly death rate of 9.74 per 100,000, those fully vaccinated dropped to 0.71 per 100,000, and those boosted dropped even further to 0.10 per 100,000. In the United States, with a population of roughly 330 million, these rates extrapolate to about 32,000 Covid deaths per week in the unvaccinated, compared to 2,300 for fully vaccinated individuals and 330 for boosted individuals.

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FIGURE 5: Rates of death in the United States among the unvaccinated, fully vaccinated, and boosted ... [+] OUR WORLD IN DATA

In short, if someone is reading this that has not received three doses of mRNA vaccine, we implore them to do so. It is the quickest and most reliable way to protect yourself against the most infectious virus in recent memory. Recent data emerging from Israel suggests that a fourth dose of mRNA vaccine is more effective at preventing severe outcomes. It is very likely that doses of the mRNA Covid-19 vaccine become regular aspects of regular vaccine schedules, akin to the flu, measles, and tetanus shots. We can only hope and encourage people to receive their doses for their protection and that of those around them. Although vaccine protection against infection seems to be more transient, protection against hospitalization and death appears more robust for at least the current period of observation—about one year. It remains an open question how long such protection will last. This article is published on Forbes.org, and can be read online here: Good News: Full Vaccination Protects Against Omicron Hospitalization And Death

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Quantum Leap In Newborn Whole Genome Sequencing Forbes | February 7, 2022 | Article

When a critically ill 3-month-old infant entered a Stanford hospital with unexplained seizures, physicians were mystified by the young patient’s illness. The infant displayed many signs of epilepsy, but brain scans found no abnormalities to indicate what could be causing the infant’s epileptic seizures. Doctors quickly ordered an epilepsy gene panel to determine if the patient had common gene mutations associated with epilepsy. However, results could take weeks and there was a chance that the infant might have a rare disorder that would not be detected by a common gene panel. Luckily, in the same hospital, Stanford researchers were busy creating an optimized method to detect and diagnose diseases— super rapid genome sequencing. Genome sequencing is a process used to analyze a patient’s full DNA makeup. For physicians tasked with diagnosing rare genetic diseases, genome sequencing is an essential tool. It helps doctors determine if their patient’s genes are mutated and what genetic diseases they may have from those mutations. In the past, it has taken weeks to months to receive DNA sequencing results. Now, Stanford scientists have found an improved method of genome sequencing that can diagnose a patient within the span of just eight hours. How did they do this? The first step was to optimize the genome sequencing device. To do so, the Stanford team consulted Oxford Nanopore Technologies. Oxford Nanopore Technologies recently built a device containing 48 DNA sequencing units called flow cells. The device also contained an important feature called “long-read sequencing”. Typically, DNA sequencing occurs by chopping a person’s genome into small DNA fragments. The fragments are then replicated and pieced back together using a standard human genome 110

as reference. However, this approach does not always accurately capture the entirety of a patient’s genome. This means that it can be difficult, if not impossible, to locate mutations that occur over a large chunk of DNA. By using long-read sequencing which preserves much longer stretches of the patient’s genome, the chances of locating these long mutations and accurately diagnosing the patient are much higher. Stanford’s theory was that by using long-read sequencing and all 48 flow units to simultaneously process a single patient’s genome, they could drastically reduce sequencing time, without compromising accuracy. The nanopore device was successful—too successful. The device sequenced patient DNA at such high speeds that the lab’s computational system could not process the data. The Stanford team would have to amend their original approach or find some way to increase their computational power. The team soon found that by funneling the data directly to a cloud-based storage system, they could increase computational power enough to process all the data produced by the nanopore device. Algorithms were then used to scan the sequenced DNA and look for mutations in the patient’s genome that could cause disease. Using this super rapid genome sequencing technique, scientists scanned the 3-month-old patient’s full genome within just eight and a half hours. They found that the infant had a mutated CSNK2B gene. CSNK2B is a gene associated with a rare neurodevelopmental disorder called Poirier-Bienvenu which is characterized by earlyonset epilepsy. Within just a couple of days, doctors diagnosed the patient with Poirier-Bienvenu, prescribed the correct antiseizure medication, and provided the patient’s family with disease-specific counseling and a prognosis. In contrast, the epilepsy gene panel results arrived 2 weeks later and were inconclusive. This advance in genome sequencing marks a significant breakthrough in diagnostic tools. With the power to sequence a person’s full DNA within just hours, super rapid genome testing could become a widely available tool used to identify inheritable diseases in infants. By detecting these diseases at an early stage,

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families and doctors can avoid any further diagnostic testing, begin treatments as early as possible, and improve patient prognosis. Rapid genome sequencing may also be the key to discovering and classifying mystery diseases in adult patients that are undiagnosed. Previous genetic tests relied on scanning a patient’s DNA for a pre-determined set of common genes. However, the algorithms used in super rapid genome testing can scan a patient’s full genome for all mutations suggested by scientific literature, even if the mutations were only discovered the day before. Not only this but as genome testing continues to develop it may be a valuable tool against viruses like Covid-19 or the common cold. Much like the human body, viruses contain nucleic acids. These nucleic acids are either DNA or RNA, but both can be deciphered by genome sequencing. By using rapid genome sequencing, we can quickly identify viruses in a sick patient and offer the most effective medications against that specific virus. For pathogens as virulent and harmful as Covid-19, early identification and treatment through rapid genome testing may be the key to preventing wider spread. This study marks significant progress towards personalized medicine. Now that scientists have discovered a method to make DNA sequencing more available, rapid genome sequencing could soon become a common tool found in every hospital and doctor’s office. As we continue to optimize our ability to sequence DNA, we can anticipate a critical shift towards personalized medical treatments that could increase patient care, improve overall health, and pave the way to new genetic discoveries. This article is originally featured on Forbes.org, and can be read online here: Quantum Leap In Newborn Whole Genome Sequencing

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Losing the Sense of Smell: How Covid-19 Infection Induces Long-Lasting Symptoms Forbes | February 14, 2022 | Article

An illustration of the olfactory bulb and epithelium. Top right: A pericyte (light orange) wraps around a blood vessel (red). Bottom right: Olfactory sensory neurons (light red, orange) surrounded by sustentacular cells (tan) and basal cells (yellow IMAGE: BRANN ET. AL., 2020.

HTTPS://HMS.HARVARD.EDU/NEWS/HOW-COVID19-CAUSES-LOSS-SMELL This is part of a series on bystander SARS-CoV-2 induced damage to organs, tissues and cells. One of the most defining symptoms of Covid-19 is loss of smell without the experience of a stuffy nose, known as anosmia. This distinct feature of Covid-19 affects at least half of those infected with the virus. In most cases, the sense of smell restores itself after a few weeks, but twelve percent of people continue to report complete loss of smell months after the initial infection. A recent study from researchers at the NYU Grossman School of Medicine and

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Columbia University offers a possible explanation for how structural damage to olfactory cells may delay sensory restoration. The Indirect Effects of Covid-19 Infection The first insight into how Covid-19 impairs the sense of smell came with the discovery that the virus does not infect olfactory receptor neurons involved in detecting odors. Rather, the virus binds to and depletes sustentacular support cells, which surround olfactory receptors in the epithelium. Unlike receptor neurons, sustentacular cells express ACE-2 and TmPRSS2 membrane proteins that the SARS-CoV-2 uses to attach to and invade cells. Despite the fact that olfactory receptors are not directly infected, they do sustain significant damage from the infection of neighboring cells. The olfactory bulb is one of the few sensory organs that regenerates its cells. When an olfactory receptor is damaged, the olfactory bulb generates new receptors to replace them in a process called neurogenesis. In contrast to other types of neurons, olfactory receptors regenerate throughout an individual’s life, preserving pathways important for differentiating between various smells. Widespread damage to olfactory neurons, however, can alter or completely erase the sense of smell. In a majority of Covid-19 cases, the sense of smell restores itself with the replacement of damaged olfactory neurons and support cells. Researchers are just beginning to understand the indirect effects of viral infection on the olfactory system. The next question asks: if the virus does not directly infect olfactory neurons, what explains the damage to nerve cells that contributes to long-lasting loss of smell? Understanding these effects in the olfactory system may provide further insight into how the SARS-CoV-2 virus impacts the nervous system as a whole. Damage to Olfactory Receptors To examine the effect, Zazhytska et. al studied infected hamsters, as well as autopsy human samples previously diagnosed with Covid19. Hamsters serve as ideal models because they rely more on their sense of sense and are more susceptible to nasal infection than humans. Researchers intranasally exposed the SARS-CoV-2 virus to anesthetized hamsters. They studied in detail the effects of the virus in hamsters, then examined post-mortem nasal tissue to see if what they found in hamsters was similar in humans. 114

Initial observations of the olfactory epithelium detected viral infection in supporting sustentacular cells but not olfactory receptor neurons. Their second analysis took a closer look at the structure of these nerve cells. Much to their surprise they found disruption of a protein network within the neuron’s nucleus, called the olfactosome. The specialized organization of the olfactosome network facilitates interactions between chromosomes important for generating the sense of smell.

Cartoon of the olfactory epithelium after being exposed to SARS-CoV-2 virus. Loss of smell is attributed to depletion of sustentacular cells (blue) and changes in nuclear architecture of the olfactory receptors (light pink). Destruction of the olfact FROM: “NON-CELL AUTONOMOUS DISRUPTION OF NUCLEAR ARCHITECTURE AS A POTENTIAL CAUSE OF COVID-19 INDUCED ANOSMIA” ZAZHYTSKA ET. AL 2022.

When an odor is detected, several membrane proteins on an olfactory receptor are activated called G-coupled proteins. Binding an odor to these receptors induces a sequence of transduction signals that trigger the activity of various proteins and other molecules. These transduction signals tell the olfactory receptors to alert the brain that an odor has been detected. How these cells are able to coordinate these extensive signaling pathways, however, has been a significant area of research. Current theories suggest that the answer lies within the nucleus, where DNA is folded and organized in specific conformations that make up the olfactosome. Olfactory genes are distributed across different chromosomes. To express these genes, chromosomes need to be in close proximity with each other. The olfactosome facilitates these interactions by bringing together distant chromosomes, through a process that is not fully understood. 115

The destruction of the olfactosome in uninfected neurons explains why the sense of smell disappears. Fortunately, for most people, the sense of smell returns after a couple weeks, though there is no guarantee that it will come back full. The extensive reconstruction of the olfactory epithelium can also prevent receptors from wiring correctly in some cases. This means something that once smelled good may become unpleasant, in a condition called parosmia. After the initial restoration of smell, disordered smell can last anywhere from a few weeks to months. If the virus does not directly infect these neurons, why does the loss of smell or disordered smell last for so long? Another finding suggests that the key signals that regulate gene expression in the olfactory nerve cell associated with the ability of the neuron to respond to odorants are altered. The process of signal transduction involves several genes important for detecting smells. RNAsequencing of hamster nasal tissues revealed that Covid-19 infection downregulates, or reduces the activity, of several genes involved in perceiving different odors. Researchers hypothesized that reorganization of the olfactosome disrupts key transcription factors that determine what genes are expressed and when. These changes then alter the orientation and subsequent expression of olfactory genes, which persist even after elimination of the virus forming a sense of “nuclear memory” that delays sensory restoration. An additional question considered the extent to which viral infection contributes to these abnormalities. In particular, are these changes primarily linked to nearby infection or systemic infection via the bloodstream? By collecting blood serum from infected hamsters and neutralizing the SARS-CoV-2 virus, researchers discovered that immune activity alone induces similar biological responses. This suggests that nearby cells do not need to be infected to see an impact on olfactory neurons. Rather, components of the immune response that circulate the bloodstream, such as cytokines, chemokines, and other immune products, can cause widespread damage. Lastly, researchers asked whether these effects are similar in humans by examining cadaver nasal tissue. Their investigations confirmed the downregulation of olfactory receptor genes and components of signal transductions that contribute to loss of smell. The downregulation of olfactory receptors and signaling genes 116

provides a likely explanation for prolonged loss of smell following Covid-19 infection. However, we cannot exclude possible biological factors specific to hamsters that may interfere with this theory. Future Directions The conclusions from this study demonstrate that “the sense of smell relies on extremely ‘fragile’ genomic interactions between chromosomes,” as stated by co-corresponding author Benjamin tenOever, PhD, professor in the Departments of Medicine and Microbiology at NYU Langone Health. Although this provides an explanation for the persistence of lost sense of smell, further investigations are needed to understand why recovery of olfactory neurons distorts smell and more importantly, how long-haul Covid19 symptoms impacts brain tissues. Studies suggest that some cytokine proteins, which coordinate inflammation in response to a pathogen, can cross the blood-brain barrier and enter the cerebrospinal fluid. Therefore, there are significant concerns that viral infection may interfere with cognitive functions. This could explain why some individuals exposed to the virus experience brain fog, headaches, and other neurological effects anywhere from a couple weeks to months after infection. Similar to the pattern of infection seen in the olfactory system, the neurons themselves are not infected but rather the epithelial support cells neighboring brain cells. Since these effects cannot be ethically studied in humans, exploring these cognitive effects in hamsters and other model organisms provides the best insight into how the virus can impact our brains. This article originally appeared on Forbes.org, and can be read online here: Losing the Sense of Smell: How Covid-19 Infection Induces Long-Lasting Symptoms

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The SARS-CoV-2 Genome: The Importance of the Termini Inside Precison Medicine | February 16, 2022 | Article

Precisely with William Haseltine The severe acute respiratory syndrome (SARS)-coronavirus (CoV)-1 caused an epidemic in 2002 with 8437 cases in 30 countries. Its most recent relative, SARS-CoV-2, is responsible for a pandemic which started in 2019 and continues to linger driven by viral variants with 300 million cases and over 4 million deaths. The ability of SARS-CoV-2 to rapidly evolve into different variants has not ceased to surprise us, as exemplified by the Omicron variant with over 30 mutations in its spike protein that seem to have come from left field. This prompted us to launch a systematic effort to better understand how the virus genomes change. Here we examine the ends or termini of the SARS-CoV-2 genome, which are also known as the 5’ and 3’ untranslated regions. Circularization of the viral genome involving complementary sequences in its ends The replication and transcription of the positive-sense single stranded RNA genome of all coronaviruses including SARS-CoV1 and 2 requires continuous and discontinuous negative-strand synthesis with differing stoichiometry of nucleic acid and protein components without splicing (Figure 1). To create the nested set of messenger RNAs, the elongating negative-sense strand jumps from a set of transcription regulatory sequences (TRS-Bs) to a similar leader transcription regulatory sequence (TRS-L) located in the 5’ genomic terminus. This unique mode of messenger RNA synthesis has prompted the speculation for the need for the proximity of the 5’ and 3’ termini during the synthesis of subgenomic negative-sense strands. We recently reported the presence of complementary terminal segments in SARS-CoV-1 and 2 that could mediate such circularization 1.

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Figure 1. The finding of potential circularization sequences which as far as we can determine are unique to SARS-CoV-1 and 2 as well as closely related viruses of bat and other animal origin, raises the question of how other human coronaviruses replicate without such sequences. Others have proposed that a protein bridge composed of cap binding protein, eIF4E, eIF4G, and poly(A)

The finding of potential circularization sequences which as far as we can determine are unique to SARS-CoV-1 and 2 as well as closely related viruses of bat and other animal origin, raises the question of how other human coronaviruses replicate without such sequences. Others have proposed that a protein bridge composed of cap binding protein, eIF4E, eIF4G, and poly(A) binding protein mediates circularization 2,3. We speculate that the presence of the circularization sequences in SARS-CoV-1 and 2 helps to stabilize such protein bridges. It remains to be determined which viral and cellular factors contribute to the replication complexes and how the putative RNA-RNA interaction described here contributes to facilitating or stabilizing RNA-protein and protein-protein interactions in subgenomic RNA synthesis. Structural flexibility at the ends of SARS-CoV-2 and related viruses Evolution of SARS-CoV-2 variants involves point mutations and small insertions and deletions as well as recombination, in which segments of the genome of the virus are moved around or derived from another source. Tracking of variation in SARS-CoV-2 is 119

focused on mutations, insertions, and deletions in viral structural proteins, most notably the spike protein. Our study of the genomic termini of SARS-CoV-2 also revealed unanticipated structural flexibility and its importance as an inherent source of variation that affects not only the essential functions of genomic termini in viral replication, transmission, gene expression, host pathogenicity, immune evasion, and variation but also those of other viral genomic regions. By analyzing thousands of sequences of SARS-CoV-2 isolates from around the world including cruise ships and from closely related coronaviruses from bats in Laos, Thailand, and Great Britain, we recently reported numerous previously undescribed duplications, inversions, and translocations within the genomic termini and into or from coding regions of the viral genome 4. As illustrated in Figure 2, our analysis revealed a striking variation in the length and composition of 5’ terminal sequences driven by the presence at the proximal end of the viral genome of exact inverted duplications of 5’-UTR sequences of various lengths (~20 to over 100 nucleotides) relative to the Wuhan reference strain.

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Figure 2

Changes in length of viral genomic termini via insertions may serve an adaptive purpose and reflect a compensatory mechanism to address a common problem of linear genomes, namely, that they fray at both ends. Rather than acquiring segments from cellular mRNA as do influenza viruses, SARS-CoV-2 and related bat coronaviruses appear more parsimonious by deriving the insertions from their own genomes, and in the case of the 5’-UTR from the negative-sense strand. The presence of the insertions leads to changes in how the RNA folds itself resulting in long-double stranded stems that encompass sequences that would have otherwise folded into individual stem-loop structures involved in interactions with RNA and proteins. The stem-loop (SL)1 loop in either plus-sense or minus-antisense orientation and the loops of SL5, both consistently present in one copy each, appear to be needed while the loops of SL2 to SL4.5 along with their conserved stems can become part of a long double-stranded stem. These observations call for a 121

reexamination of the biochemical fundamentals of coronavirus replication and gene expression as we know them, and for correlations of these structural changes with pathogenicity, immune evasion, and infectiousness of these variants. 5’-UTR intragenic insertions in SARS-CoV-2 variants Further search of potential insertions of 5’-UTR sequences revealed duplication and translocation of 5’ terminus sequences not only within the 5’-UTR but also to coding regions of the viral genome. We detected an insertion of a 27-nucleotide positive-sense strand segment of the 5’-UTR to the end of ORF8 gene in a SARSCoV-2 variant isolated in Minnesota, USA and encoding an ORF8X protein with a modified carboxyl-terminus (5 last amino acids are replaced by 10 amino acids) (Figure 3). It was previously noted that a longer overlapping 57-nucleotide segment of the 5’UTR duplicated and translocated in place of an 882-nucleotide deletion within the coding portion of the viral genome of a SARSCoV-2 variant isolated from 3 patients in Hong Kong with absent ORF7a, ORF7b, and ORF8 (lineage B.1.36.27) and encoding a Cterminally modified ORF6 product, termed ORF6X 6.

Figure 3

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We note that in both translocations, the end of the inserted 5’UTR sequence corresponds to the leader transcription regulatory sequence (TRS-L) that has been associated with spots with a higher frequency of recombination, and that insertion occurs at the same site immediately proximal to the nucleocapsid (N) and ORF9b genes, thereby altering gene expression regulatory sequences at this location 7. Analysis of the Omicron (B.1.1.528) variant (exemplified by OL672836.1 in Figure 4) revealed a 15-nucleotide match (only one differing nucleotide) which spans up to 16 nucleotides in 44 SARSCoV-2 isolates (exemplified by OV045104 in Figure 4) from around the globe between the negative-sense strand of the 5’-UTR of SARS-CoV-2 Omicron and the region in Omicron’s spike (S) protein with insertion of the amino acids EPE at position 214 (ins214EPE) and proximal deletion of an asparagine at position 210 (N210del) relative to the Wuhan reference strain. Translation of the 5’-UTR anti-sense matching sequence generated an almost identical amino acid sequence (valine [V] and leucine [L] are conservative substitutions) to that present in Omicron’s S protein including N210del and the first glutamic acid (E) in ins214EPE. The remaining 5 nucleotides remain of unknown origin. We think it unlikely that this insertion originates from either cellular or other viral RNAs.

Figure 4

We also reported two instances of duplication, and/or inversion and translocation of coding sequences at the end of the nucleocapsid (N) gene and/or the beginning of ORF10 to the distal end of the 3’-UTRs of two bat coronaviruses. These insertions can form stemloop structures that may affect 3’ terminus-mediated regulation of

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gene expression, minus strand synthesis, and viral RNA stability and turnover as well as viral evolution. Deletions within the genomic termini Another source of variation comes from deletion rather than insertion of sequences in the genomic termini. For instance, the 3’ genomic terminus of SARS-CoV-2 shares with other positive-sense single-stranded RNA viruses including beta, gamma, and delta coronaviruses, picornaviruses and astroviruses from various animals an approximately 40-nucleotide-long stem-loop-like motif (s2m). This sequence is recognized by a human microRNA, hsa-miR1307-3p, as is a similar one in influenza A virus H1N1, which has caused epidemics of severe disease. A single point mutation in the target region of influenza A virus H1N1 adversely affects the binding of hsa-miR-1307-3p thereby weakening the host attack on the virus 5. The s2m motif is deleted in the 3’ UTR of the B.1.640.1 variant from Congo and France but not in that of its close relative B.1.640.2 (IHU variant) which is now spreading in southern France after originating in Cameroon, and in the background of a fast-spreading Omicron variant. Conclusion The relevance of genomic termini to viral evolution, replication and pathogenicity highlighted here calls for careful tracking of the ends of the genome of SARS-CoV-2. This is rendered difficult by limitations secondary to the selection of primers for sequencing which in several cases obviate the first and last 100 nucleotides at the ends of the virus. Most publicly available sequences of the Omicron variant lack detailed information on the extreme termini of the genome. The genomic termini, via their regulatory sequences, contribute to the overall transmissibility, pathogenicity and immune evasion of the virus and study of their variation will continue to shed light on all these clinically relevant areas. Whether the structural rearrangements reviewed here provide an advantage or disadvantage to the viral variants remains to be determined and correlations with viral infectivity, pathogenicity and immune evasion are warranted. As we consider the potential of future variants, we must be mindful of the structural flexibility of genomic termini as an inherent source of variation. In view of the flexibility of the SARS-CoV-2 genome and as more therapeutic agents become available, it is going to become more important to 124

determine the sequence of the SARS-CoV-2 variant affecting a patient to inform the choice of the most appropriate combination of antiviral drugs as is currently done for HIV-1. References 1. Patarca R, Haseltine WA. Circularization via complementary sequences in the 5’ and 3’ termini may facilitate replication of SARS coronaviruses. Authorea. January 04, 2022. DOI: 10.22541/au.164132044.46753705/v1 2. Tarun SZJr, et al. 1997. Proc Natl Acad Sci USA 94: 9046 3. Spagnolo JF, Hogue BG. 2000. J Virol 74:5053 4. Patarca R, Haseltine WA. Structural flexibility of the SARSCoV-2 genome relevant 5. to variation, replication, pathogenicity, and immune evasion. 6. Chan AP, et al. mSphere 2020. 5: e00754 7. Tse H, et al. 2021. J Inf Dis 73, 1696 8. Thorne LG, et al. Nature 2021 Dec 23. doi: 10.1038/s4158621-04352-y William R, Haseltine, PhD. is chair and president of the think tank ACCESS Health International, a former Harvard Medical School and School of Public Health professor and founder of the university’s cancer and HIV/AIDS research departments. He is also the founder of more than a dozen biotechnology companies, including Human Genome Sciences. Roberto Patarca, M.D. PhD. is Chief Medical Officer at ACCESS Health International and a former pharmaceutical and medical device company executive and faculty at Harvard Medical School and the University of Miami. His research has focused on diagnostics, pathophysiology, pharmacogenomics, and immunotherapy of infectious and other diseases. This article is featured on the Inside Precision Medicine Website, and can be read online here: The SARS-CoV-2 Genome: The Importance of the Termini

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Do Not Underestimate The Consequences Of SARS-CoV-2 Escape: The Omicron Example Forbes | February 16, 2022 | Article

From the outset of the Covid pandemic, the extent to which the virus SARS-CoV-2 might vary was seriously underestimated. We all recall when leading national and international healthcare authorities assured us that because the virus had a “proofreading" activity, it was unlikely that the fundamental characteristics of the virus would change. The history of the pandemic—the story of the Omicron family of viruses in particular—has shown just how misguided such early optimism was. Some of us were more cautious. We warned that coronavirus infections typically return year after year to reinfect populations infected just the year before. Such is the case for the four human cold-causing coronaviruses that sweep the globe every year. Such is also the case for chronic infection of long-lived bats, cats, and many other species. Coronaviruses seem uniquely well-adapted to reinfecting immunocompetent adults infected just a few months prior. That is why I was dismayed by the early assurances that we would quickly reach "herd immunity” (a term I intensely dislike), and we would need to worry no more. A few of us warned that not only was the concept of "herd immunity" flawed but deadly dangerous to countries that embraced it. Sadly the idea of “herd immunity” lives on, many suggesting that the Omicron is a blessing in disguise and that the virus will now infect so many we can now relegate Covid to the status of a disease like influenza with which we can live. I argue it is unwise to dismiss the potential of the disaster that may come about with a future virus variant that combines the transmissible characteristics of Omicron with the lethality of SARS and MERS. Is such a scenario guaranteed? By no means. Is such a scenario within the realm of the possible? Absolutely. Each of us may assign a probability to such a catastrophe. I place the odds at about fifteen percent over the next few years. I am sure that is one of the 126

most pessimistic estimates. Given what we know today, no one can, in good faith, place the odds at zero. Three Dimensions of Virus Variation that Determine Pandemic Severity Transmission: How contagious is the virus? How likely is it that a person with the infection will transmit it to others? The Omicron family shows displays a dramatic increase in transmission from earlier variants, demonstrating how SARS-CoV-2 may rapidly alter infectivity via variation. Immune Evasion: Will the virus evade neutralizing antibodies raised by prior infection or vaccination? The Omicron variants are far more immune evasive than previous variants, capable of infecting most of those previously infected or vaccinated. Virulence: How virulent is the infection? How many people infected will fall seriously ill or die? Mercifully, the Omicron variants are less virulent than their immediate predecessor, the Delta variant. However, the very fact that the virulence of SARS-CoV-2 does vary should be cause for concern if not alarm. Protection Against Severe Disease and Death There is some positive news. Vaccination is extraordinarily effective in protecting against severe illness and death for most. Moreover, for many protection outlasts the transient protection against infection offered by neutralizing antibodies The biggest question in predicting how serious the pandemic will become is how long protection against the severe disease a death may last. A second question still outstanding is to what good fortune we owe such protection. Scientists and doctors are working around the clock to unravel both questions. One answer—how long such protection lasts—will only come with time. We have hints but no definitive answer to the second question of exactly how, once vaccinated, our bodies protect us from severe disease. Recent studies show that long-term protection from Omicron may not be universal. Up to 20% of those previously infected, vaccinated, or both fail to mount strong CD8+ T-cell responses to the Omicron Spike protein required to protect against hospitalization and death. SARS-CoV-2 variants have the potential to escape both antibody and T-cell immunity. Until we know how widespread such resistance may arise, we wonder whether the following variant may overpower even our most robust defenses. 127

FIGURE 1: Courtesy of Gaurav D. Gaiha, M.D. T-cell responses from Wuhan wildtype to Omicron variant ... [+] GAIHA ET AL.

Determinants of Virus Variation So now on to the fundamental properties of the virus that determine transmission, immune evasion, and virulence. Transmission Multiple virus characteristics influence the transmission of SARS-CoV-2. These include: Peak Virus Particle Concentration The peak concentration and location of virus particles in an infected person are important determinants of transmission. The sheer number of virus particles in nasal secretions is amazing, varying at the peak from 100 million to ten billion or more. SARS-CoV-2 is also shed in urine and feces. Variants differ in the concentration of shed virus by orders of magnitude. The peak concentration of the Delta within nasal fluids is about one thousand times higher than that of earlier SARS-CoV2 variants. However, a high viral load is not in and of itself a guarantee of greater transmission. Yes, Delta is about twice as transmissible as earlier variants, but Omicron is twice as transmissible as Delta without increasing peak viral loads. We still have more to learn. Speed and Duration of Virus Growth Variants differ in how rapidly they reach peak concentration. They also differ in the number of days a person remains infectious. Omicron replicates much faster than does Delta. In cell culture, 128

recent data shows that Delta and Omicron follow similar trajectories with respect to the kinetics of infection, the peak viral load, and the time to viral clearance in naive and well-vaccinated individuals. Stability The stability of the released virus particle in aerosols, droplets, and surfaces will influence transmission. Recent evidence suggests that Omicron virus particles remain infectious longer than earlier variants. The longer the virus lives once released, the likelier it may infect another. There are reports that infectious virus particles may hang in the air for over half an hour in enclosed spaces. Receptor Avidity Once released, the virus must attach itself to the surface of an infected cell. The primary site of binding of SARS-CoV-2 is a surface protein called ACE2. The receptor is present on the surface of some cells that line nasal passages, upper and lower airways, and the lung. SARS-CoV-2 variants differ in how tightly they bind the receptor. Some of the earlier variants bound ACE2 much more weakly than later variants such as Delta. Omicron binds to ACE2 more tightly than the earliest variants but less tightly than Delta. Can SARS-CoV-2 virions become more avid than those naturally observed? Laboratory experiments demonstrate that the association of the virus to the cell can become tighter several hundredfolds, raising troubling possibilities. I should mention that there is evidence that ACE2 binding is not the only means by which SARS-CoV-2 attaches and enters cells. Cell Entry One of the most surprising characteristics of the Omicron viruses is the mode of entry. The original Wuhan variant, and all prior SARS-CoV-2 variants, enter cells via fusion of the virus and cell membranes. This is not so for the Omicron viruses. After attachment, the virus particles are surrounded by the cell membrane and transported to the interior. The virus then bursts through this enclosure to enter the cell, called endosomal entry. Endosomal entry is the most unanticipated feature of Omicron. By itself, the endosomal entry does not account for all the unusual properties of Omicron. SARS-CoV-1, the cause of the SARS epidemic, enters cells via the endosomal route yet is far less

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transmissible and far more lethal than are any of the SAR-CoV-2 variants. The takeaway from these observations is that SARS-CoV-2 can alter fundamental characteristics relevant to transmission. We have yet to understand the full range and disease implications of such changes. Replication Rate The rate at which a virus replicates once it enters the cells is determined by many factors, some of which we understand, most of which remain opaque. My conservative estimate is that the rate of replication is determined by the concerted action of at least twentyfive of the thirty viral proteins and their interactions with literally hundreds of cellular proteins and RNAs. It will require generations of dedicated work by scientists to understand the replication process in sufficient detail to predict the course of viral evolution. Experiments to date allow us to variation in replication through a glass darkly. One of the earliest variants noted is a single nucleotide change in the 5’ translated region, changing from a C to a U at nucleotide 241. The change occurs at the tip of a conserved stemloop structure. This single base change increases the rate of virus replication in laboratory experiments. Moreover, changes in a small set of amino acids of the viral nucleocapsid proteins (N), at positions 199 to 205, increase the virus's replication rate in culture by over a hundredfold. Such significant changes in a fundamental virus trait, such as replication rate altered by such a slight change in the genome sequence, one nucleotide of 30,000, should give us an appreciation for the power of tiny changes in the genome to affect major changes the pandemic. For comparison, Omicron variants contain least of more than seventy nucleotide changes when compared to the original Wuhan isolates. The takeaway from these observations is that SARS-CoV-2 can alter fundamental characteristics relevant to transmission. We have yet to understand the full range and disease implications of such changes. We should remain humble but observant. Immune Evasion SARS-CoV-2’s ability to evade protraction from an infection acquired by prior infection or vaccination has come as an unpleasant shock. That should not have been the case. As mentioned, we know that the cold-causing coronaviruses infect the global population 130

every year regardless of prior exposure. In that respect, they resemble the influenza viruses. Decades of research have taught us that such viruses deploy several tactics to evade our immune defenses. The concentration of protective antibodies raised either by natural infection or vaccination capable of neutralizing wane with time. Continued protection relies on the rapidly mobilizing antibodies that recognize the invading virus. We recognize two primary means by which viruses can thwart the memory response: The first is antigenic variation. Influenza viruses—and as we now know, coronaviruses also—alter the virus's exterior to avoid recognition by the antibodies made to earlier versions of the virus. These viruses are practically invisible to the earlier antibodies. Despite these changes, the virus retains key properties such as binding and entering cells. As Omicron infections demonstrate, such altered virus can sweep through an entire population regardless of prior infection vaccination status. A virus may also outrace the memory response. The production of high levels of protective antibodies by memory cells requires that they recognize the presence of an invader and proliferate in sufficient numbers to produce enough protective antibodies to make a difference. Two to four days may pass before antibodies reach the required level. Both influenza and SARS-CoV-2 replicate so rapidly that those infected may become infectious within one to one in a half days from exposure. In other words, the virus may enter and exit before the memory response kicks in. Omicron is case-in-point on both accounts. The Spike protein of SARS-CoV-2 is the major target for neutralizing antibodies. The Spike proteins variants contain thirty-six amino acid changes. These mutations reduce the ability of vaccine-induced antibodies to recognize the virus by more than thirtyfold, even after double vaccination followed by a booster. Moreover, protection by current vaccines or prior infection falls dramatically over the ensuing months leaving the population, vaccinated or not, susceptible to Omicron infection. We have yet to understand the extent of possible antigenic variation fully. I am not optimistic that we will ever reach the point where SARS-CoV-2 has reached the limit of possible variation. Witness the decades-long success of coronavirus infections n humans 131

and the millennia-long success of the virus in other species, most notably bats. I am much more optimistic regarding the power of our immune system to protect us from severe disease and death once infected. The best evidence is that T-cell, not B-cell, is the immunity that offers us such protection. The good news is that, at least for now, we have observed only limited variation in the ability of the virus to evade recognition by memory T-cells. However, the observation that CD8+ T-cell recognition of the Omicron Spike protein is diminished in 20% of those multiply vaccinated may be a warning of dangers to come. Virulence My greatest fear of a variant that has the transmission characteristics of Omicron and the lethality of SARS or MERS. Remember that SARS killed about four percent of those under fifty and about forty-five percent of those over sixty-five. MERS killed and still kills about one-third of all those it infects. We remain in the uncomfortable position of not knowing how to account for the striking differences in the pathogenic impact of the viruses. Optimists point to the four cold-causing coronaviruses that, except in rare cases, cause cold-like symptoms. Pessimists highlight the lethality of SAR-CoV-1 and MERS. The history of viruses is replete with examples in which the change of one or a few nucleotides of amino acids convert a mild disease into almost uniformly lethal. Two of the best examples come from studies of coronavirus. The closest being studies of a SARS-1. Nieto-Torres et al. adapted a lethal variant of SARS-CoV-1 to grow in mice. To attenuate the virus for vaccine use, they introduced single point mutations into the envelope protein (E) at two different positions. Both are located within the hydrophobic amino-terminal region ion channel pore-forming sequence. Both mutations inactivate the ion channel activity. One of the mutations is a change from asparagine to arginine at position 15, the other valine to phenylalanine at position 25. Both mutants are viable and infect mice. Both resulted in no or at most very mild disease. The first conclusion is that a single amino acid change in the E protein can attenuate pathogenicity without destroying virus viability.

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Repeated passage of both mutants in culture resulted in rapidly growing revertants. Mice infected with the revertant virus die quickly. Surprisingly the revertants retain the original mutations but contain second site amino acid changes within the short hydrophobic region that restore ion channel activity. The revertant mutations occur at different sites within the hydrophobic channel. All restore ion channel activity. The second conclusion from these experiments is that a single amino acid change with the E protein hydrophobic channel can transform a nonpathogenic strain of SARS-1 into highly lethal in mice. The E proteins of SARS-CoV-1 and SARS-CoV-2 are similar. Omicron contains a mutation in a conserved amino acid immediately amino adjacent to the transmembrane region, a threonine to isoleucine substitution at position 9. Might the T9I mutation contribute to the reduced virulent of Omicron? Might a second site revertant transform SARS-CoV-2 into a virus with lethality characteristic of the SARS-1 variant? Frightening to contemplate.

FIGURE 2: The topology of the SARS-CoV E protein is colored to indicate the different parts. The ... [+] INSIDECORONA.NET

The second example is found in feline coronaviruses. One variant causes only mild disease in kittens. However, a change in three amino acids in the membrane-associated S2 protein transforms the virus into uniformly lethal to adult animals. Mutations in this 133

area of the feline coronavirus Spike may also increase macrophage tropism, as well as alter the mode of entry from fusion to endosomal, as seen with the Omicron variant. I note that the Omicron variants carry 7-9 amino acid changes in S2 protein compared to the Wuhan strain. Some of these mutations likely contribute to the preferred endosomal route of viral entry. Might these Omicron variants affect Omicron pathogenesis as well?

FIGURE 3: Schematic representation of the FCoV spike protein structure. The glycoprotein has an ... [+] ROTTIER ET AL.

Conclusion The take-home message is that highly lethal variants of coronavirus can and do exist. Some of these variants differ only slightly from one another, sometimes by as little as a single amino acid change. It is not farfetched to imagine such changes arising in SARS-CoV-2. After genomes differ from those of the Wuhan parent by more than 50 changes, not including small deletions and insertions, while we hope for the best, we must prepare for the worst. Our recent experience with Omicron teaches us that most countries are are unprepared to contain a highly contagious virus. While our vaccines provide significant protection against severe disease and death against current variants, we are unsure how long that protection will last. We cannot be certain to remain protected against whatever nature may devise. Our future will depend on the success of continued research, drug and vaccine development, and our willingness to institute effective mitigation measures. This article originally appeared on Forbes.org, and can be read online here: Do Not Underestimate The Consequences Of SARS-CoV-2 Escape: The Omicron Example

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Protection In The Present From The Deep Past Forbes | February 21, 2022 | Article

By now, we know that our bodies have many ways to defend themselves from infecting microbes. This includes the innate immune system— the body’s first response to organisms it has never seen before, and the learned or adaptive immune system which allows our bodies to recognize organisms we’ve already encountered. Only recently has there been great progress in understanding our innate immune responses. However, we are still filling out the picture. A recent story, by Johnson et al., adds a new chapter to the book of innate immunity by introducing a protein called gasdermin. Here, we will describe gasdermins and the role they play against invading microbes in innate immunity. Our story actually begins several billion years ago when bacteria were required to fend off viruses and other bacterial predators. These bacteria developed defense mechanisms to protect themselves. Surprisingly, Johnson et al.’s recent paper shows that many of those defense mechanisms developed by our bacterial ancestors are employed by the human body today. Scientists have known of gasdermins for some time. However, this new discovery shows that gasdermins found in the human body are remarkably similar to our bacterial ancestors both in structure and mode of action. In many ways, we owe our protection to these ancient systems and proteins. In humans, gasdermins are part of the innate immune system and prevent the spread of bacteria and viruses by inducing a violent form of cell suicide in infected cells called pyroptosis. These proteins are specialists in warding off disease, but how do they work? The gasdermin structure consists of two terminals: an Nterminal that is lipophilic, meaning it can enter any cell membrane, and a C-terminal that cannot enter the cell membrane. So long as the C-terminal remains attached to the N-terminal, a gasdermin is inhibited from any action. This means that the body keeps the 135

gasdermin dormant and in reserve until it receives a signal that there is disease present in a cell.

Caspase, a protease, cleaves the Gasdermin to activate it.LIU Z, WANG C, YANG J, ET AL. IMMUNITY. 2019....

Once the disease has been detected by another protein called caspase, the caspase will travel to the gasdermins and activate the protein by cutting its inhibitory C-terminal from its attacking Nterminal. This allows the gasdermin’s lipophilic N-terminal to enter the infected cell membrane. Once the gasdermin has entered the cell membrane, it begins a process called oligomerization. During oligomerization, the gasdermin N-terminal recruits 27 other lipophilic gasdermin Nterminals and binds to them. This creates a long snake-like structure that self-assembles into a pore within the membrane. Once multiple pores have been created within the membrane, the contents of the infected cell spill out, effectively killing the cell. In Johnson et al.’s paper, scientists at Harvard’s Department of Microbiology were shocked to discover unfamiliar genes in two species of bacteria: Bradyrhizobium tropiciagri and Vitiosangium. What was unusual about these genes was that they had never been detected in bacteria. However, they were suspiciously similar to genes that encode gasdermins in mammals. After analyzing the structure of the proteins produced by these genes through a process called x-ray crystallography, it was clear—the bacteria contained proteins that were akin to the gasdermins found in mammals. 136

The bacterial gasdermins adopted the same shape as the inactivated proteins in mammals. However, they did not contain the long C-terminal/protein responsible for keeping gasdermins dormant in humans. Instead, scientists found that the bacterial proteins were much simpler. Bacterial gasdermins contain a small hook that stabilizes the gasdermin’s dormant state. Once the hook is cut away, the bacterial gasdermin is activated and is free to induce cell suicide in infected cells. The presence of gasdermins in bacteria indicates that these proteins are a very ancient mechanism of immunity and are a remnant of our evolutionary past. This study is a significant step forward for immunology and infectious diseases. As we begin to uncover more antimicrobial agents that have successfully defended organisms against viruses, diseases, and bacteria for eons, we may begin to see new advances in pharmacology that incorporate these natural defense systems in medications or other medical treatments. This article originally appeared on Forbes.org, and can be read online here: Protection In The Present From The Deep Past

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Whither the Omicron Family: BA.1, BA.1.1, BA.2, BA.2.H78Y, BA.3? Forbes | February 23, 2022 | Article

This is the fifth in a series on the Omicron variants. The Omicron family of SARS-CoV-2 continues to trouble and puzzle. First is the mysterious origin. The Omicron variants emerged fully formed in Southern Africa in late November 2021, sporting many more mutations than all other variants combined. It appeared not as one but three variants, all related to a cryptic parent itself highly divergent from the strain that swept the world beginning early 2020. The Omicron variants raise more questions than they answer. Yamasoba et al. begin to lift the veil, and what they find is not reassuring. Questions Question 1: Where was this cryptic parent hiding? We would dearly love to know the answer. We are uneasy in our ignorance as we do not know where from a new danger may arise. Question 2: How did it happen that three all Omicron variants emerged in the same place at the same time? All are as different from one another as the three previous pandemic viruses Alpha, Beta, and Gamma. Will such triple threats be a common feature of future variants? Question 3: All variants before the appearance of Omicron were neutralized reasonably well by antibodies from those infected or vaccinated. Not so Omicron. Initial studies showed Omicron-BA.1 is resistant to antibodies from those infected with earlier variants as well as to vaccines and many monoclonal antibodies. Moreover, antibodies raised against Omicron BA.1 do not protect against other variants. Does that mean we will need to develop a new vaccine and novel protective monoclonal specific for each new variant? Question 4:

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Omicron BA.1 differs from all previous variants in several fundamental respects. The virus enters cells via endocytosis, not by fusion. BA.1 forms either no or very small syncytia (fused cells). The spike protein is poorly cleaved into the active S1/S2 form on cell exit. Infection is not dependent on the protease TMPRSS2 cleavage as are all previous variants. Are these phenomena linked? What might this mean for pathogenicity and drug development? Question 5: Pathogenicity. Multiple reports suggest that Omicron BA.1 is less virulent than Delta. How much less pathogenic it is remains an open question. However, we know that in animals, hamsters, mice, and monkeys, Omicron BA.1 is far less pathogenic than all previous variants. Many predict new variants will arise and will be less dangerous than those we have already encountered. Is this hope justified? Yamasoba et al. offer no insights on the origins, questions 1 and 2, but do provide new insights regarding immune evasion, virology, and virulence. Their approach is to compare five variants: the B.1.1 early variant (Please note. Do not confuse B.1.1 with BA.1), Delta, as well as Omicron BA.1 and BA.2 in respects that matter for transmission, immune evasion, and disease. Recall BA.1 differs from BA.2 by more nucleotide changes and amino acid changes than distinguish all previous variants from one another [Figures 1 & 2].

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FIGURE 1. Venn diagram illustrating the wealth of shared and differing mutations in the Omicron ... [+] CREDIT: ACCESS HEALTH INTERNATIONAL

FIGURE 2. Proposed Omicron family tree, showing the development of mutations over time from the ... [+] CREDIT: ACCESS HEALTH INTERNATIONAL. CREATED IN: HTTPS://BIORENDER.COM/

Observations and Implications Observation 1: The reproduction of Delta in human populations is greater than that of the B.1.1. The reproduction rate of Delta is greater than that of Alpha, Beta, and Gamma. The reproduction rate of BA.1 is greater still. 140

Many speculated that with BA.1 the virus had reached the limit of its reproduction capacity. Not so The reproduction rate of BA.2 in human populations exceeds that of BA.1. Just as BA.1 replaced Delta as the dominant strain globally, BA.2 displaces BA.1 in countries where they both take hold. Yamasoba et al. calculation agrees with others that BA.2 reproduction rate is about 1.4 times that of BA.1 (range by country 1.29-1.52) [Figures 3]. Recent data from Denmark points to a new variant of BA.2 that is even more transmissible than BA.2, BA.2.H78Y. Implication: We can expect BA.2 and BA.2.H78Y to be the next dominant variants globally.

FIGURE 3. Schematic of the relative transmission rates and pathogenicity of: Wild-type Wuhan-1 ... [+] CREDIT: ACCESS HEALTH INTERNATIONAL. CREATED IN: HTTPS://BIORENDER.COM/

Observation 2: BA.2 is at least as resistant as is BA.1 to neutralization by antibodies in sera of those infected or vaccinated. Moreover, antibodies in hamsters infected by BA.1 are threefold weaker in neutralizing BA.2 than they are BA.1. Sera against BA.1 spike protein from mice exposed to the BA.1 S protein is six-fold less effective in neutralizing BA.2 than it is against BA.1. Sera from those vaccinated and infected with BA.1 show only a slight decrease in activity against BA.2. Several studies find that sera from breakthrough infections with BA.1 neutralizes BA.2 well, with only a slight reduction in potency. 141

Implications: We should develop vaccines that protect against Omicron-like infections, as current vaccines are not effective. We may need to develop vaccines against both Omicron BA.1 and BA.2. Whether prior infection of BA.1 protects against BA.2 is an open question. The current data suggests that BA.2 and BA.2.H78Y may be pathogenic for those naïve to SARS-CoV-2. Prior infection or vaccination is likely to be protective, at least from serious disease and death. Observation 3: Both BA.1 and BA.2 are resistant to most monoclonal antibodies. BA.2 is resistant to the antibody pair casirivimab/imdevimab (REGN-COV2), active against BA.1. Yamasoba et al. report that BA.2 is 35 fold more resistant to Sotrovimab than is BA.1, which itself is threefold more resistant than is B.1.1 or Delta. Implications: We may need to develop new monoclonal antibodies to each new variant. We should expand and accelerate the search for monoclonal antibodies that neutralize all variants. We should accelerate the search for other types of pre- and post-exposure prophylactic drugs. Observation 4: Omicron BA.1 and BA.2 differ in their mode of entry, syncytia formation, and dependence on TMPRSS2 protease. BA.1 enters via endocytosis. BA.2 enters by fusion [Table 1 & Figure 4]. BA.1 forms tiny syncytia if any. BA.2 forms syncytia. A common hypothesis is that the endosomal entry and failure to form syncytia is dictated by poor cleavage of the S protein at the cell surface. Contrary to expectations, the BA.2 S protein is poorly cleaved (less so than other syncytial forming viruses) raising serious doubts regarding the linkage between S protein cleavage and fusion activity. Implication: We need to know more about the requirements for viral entry and cell to cell fusion.

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TABLE 1. Comparison of various viral properties across four SARS-CoV-2 variants, and SARS-1. CREDIT: ACCESS HEALTH INTERNATIONAL

FIGURE 4. Endosomal viral entry of SARS-CoV-2 Omicron BA.1 variant versus membrane-to-membrane ... [+] CREDIT: HTTPS://VIRALZONE.EXPASY.ORG/985 (HEADING TEXT ALTERED BY ACCESS HEALTH INTERNATIONAL).

Observation 5: Pathogenesis. BA.1 is noticeably less pathogenic in animals than in all prior variants including B.1.1, Alpha, Beta, Gamma, and Delta. By contrast, Yamasoba et al. find that BA.2 is highly pathogenic in 143

hamsters. Infection of the animals shows that in contrast to BA.2 induces weight loss, deep lung infection, and alveolar damage comparable two that of Delta. This observation also upsets the hypothesis that S protein cleavage and TMPRSS2 dependence is required for a lower lung infection, at least in this model. Implication: There is a strong likelihood that BA.2, now en route to be the dominant global variant, is capable of deep lung infection and may be considerably more virulent than BA.1. Conclusions: Many are asking what comes after Omicron. The answer in many countries is already clear. What comes next is Omicron BA.2 and BA.2.H78Y The good news is that the incidence of serious disease and death is similar to that observed for infections with Omicron BA.1. That is somewhat surprising given the hamster data. Widespread infection and vaccination may account for data. It may be that despite failure to protect from infection, prior infection or vaccination may protect against BA.2 severe disease and death. Although it seems like forever, we are likely in the early days of the Covid pandemic/epidemic/endemic experience. More questions remain than are answered. We need continued vigilance, surveillance, research, drug and vaccine development to see our way to a better future. This article is featured on Forbes.org, and can be read online here: Whither the Omicron Family: BA.1, BA.1.1, BA.2, BA.2.H78Y, BA.3?

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Pericyte Damage: Surprising Cause of CovidRelated Myocarditis Forbes | February 25, 2022 | Article

A recent study reveals that SARS-CoV-2 may damage blood vessels without infecting cells directly. This is part of a series on bystander SARS-CoV-2 induced damage to organs, tissues and cells. Summary A recent study reveals the details of how SARS-CoV-2 affects pericytes, a cell type critical for the maintenance and repair of blood vessels. Surprisingly, the virus does not need to infect these cells directly to alter their function. Spike protein binding to the surface of the pericyte without virus entry suffices. Pericyte dysfunction may, at least partially, account for Covid-19 related myocarditis and other vascular disease. Myocarditis There continue to be outstanding questions surrounding the extent of damage to tissues and organs as a result of Covid-19 infection. Injury to the lungs and upper respiratory system can be explained by damage to the lung epithelium either by direct infection and by hyperactive antiviral immune reactions. Covid-19 145

frequently damages other organs, including the heart, brain, pancreas, and kidneys, often without evidence of direct virus infection. We recently described how indirect damage to the olfactory neurons due to Covid-19 results in the loss of the sense of smell. Here, we describe experiments by Alovio et al. that explore how SARS-CoV-2 may damage the blood vessels in the absence of direct infection. The investigators began their study with the realization that damage was occurring in the heart, despite scant evidence of direct infections. As Dr Elisa Avolio, the first author of this story from the University of Bristol Medical School, describes, “Microvascular complications are frequent and harmful in patients with Covid-19, with up to eleven percent of those hospitalized in intensive care units having myocardial damage or having suffered a heart attack.” The Pericyte Alovio et al. experiments focus on the pericyte. Pericytes wrap around blood vessels and play an important role in microvasculature maintenance and repair. (Figure 1)

Figure 1: Illustration of a cardiac pericyte wrapped around a capillary vessel. WIKIPEDIA COMMONS- KELVINSONG

Curiously, researchers found that even though heart pericytes express the ACE-2 receptors needed for viral entry, they are resistant to infection. The authors recalled earlier experiments that found the S protein of SARS-CoV-1 stimulates human pneumocytes to produce pro-inflammatory factors in the absence of infection. Specifically, binding of the ACE 2 receptor by the SARS-1 S protein activates the RAF-MEK-ERK1/2 pathway that in turn stimulates production of CCL2 and other inflammatory genes. 146

Avolio et al. asked if similar viral pathways could be uncovered in heart pericytes. They found that exposing these cells to the SARSCoV-2 S protein increased the activity of ERK signal transduction proteins, providing evidence that this viral protein does activate the Raf-MEK-ERK signal transduction pathway. Activating this pathway consequently destabilized heart pericytes, causing them to detach from blood vessels. Significantly fewer pericytes remained attached to the heart tissue in these samples, compared to controls not exposed to the viral proteins. As heart pericytes detached and migrated from blood vessels, researchers also detected abnormalities in the endothelial heart cells that they left behind. Normally, cardiac pericytes and endothelial cells work together to stabilize blood vessels and control the flow of blood. In the presence of the viral S protein, however, the migration of heart pericytes not only disrupted their own function but also induced apoptosis, or programmed cell death, in the corresponding endothelial cells. Endothelial heart cells themselves do not express the receptors needed for the virus to invade and kill the cell. Therefore, to understand how these cells still die, researchers speculated whether functional changes in the pericytes could be producing this effect. In fact, that is exactly what they saw. Binding the viral S protein to heart pericytes and activating the Raf-MEKERK signaling pathway prompted the released pro-apoptotic factors that triggered the death of endothelial cells, as well as generated proinflammatory cytokines that damaged other nearby cells. A surprise finding was that blocking ACE2-S protein binding does not prevent endothelial cells’ death. Prior research suggested that the cell surface protein CD147, another protein found on the surface of pericyte, can serve as a second receptor for SARS-CoV2. They found that antibodies to CD147 that prevent block S protein spare the pericytes. Blocking binding to CD147 prevents the migration of heart pericytes away from blood vessels. The antibodies also block pericyte secretion of pro-apoptotic factors, sparing endothelial cells. Moreover, blocking the S protein-CD147 prevented activation of the Raf-MEK-ERK signaling pathway. Avolio et al. conclude that the primary action of infection is S protein binding to CD147 and associated integrins to send critical signals that compromise pericyte function.

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Figure 2: Summary of how the binding of SARS-CoV-2 S protein (A) increases the migration of cardiac pericytes (B) and disrupts interaction with endothelial cells lining the heart and blood vessels (C). In a mechanism dependent on CD147 receptors, perFROM: “THE SARS-COV-2 SPIKE PROTEIN DISRUPTS HUMAN CARDIAC PERICYTES FUNCTION THROUGH CD147 RECEPTOR-MEDIATED SIGNALLING: A POTENTIAL NON-INFECTIVE MECHANISM OF COVID-19 MICROVASCULAR DISEASE” AVOLIO ET AL. 2021

Circulating S protein To determine if the concentration of circulating S protein was sufficient to trigger the response studied in the laboratory, Avolio et al. measured the level of S protein in Covid-19 patients. Consistent with previous studies, they detected high levels of S protein in the blood of infected individuals, which peaked 5 to 10 days after infection. Researchers reported that these blood samples had an average S protein concentration of 33.5 ng/mL, which is notably lower than the 1000 ng/mL concentrations used for the isolated tissue experiments. Conclusion Avolio et al. demonstrate that circulating S protein in Covid-19 patients may impair vascular function in cardiac and other tissue by triggering pericyte disfunction. This is one of several recent reports that reveal a previously unknown mechanism of SARS-CoV-2 pathogenesis— S protein binding to surface receptors triggering dysregulation and/or cell death in the absence of infection. 148

This article is featured on Forbes.org, and can be read online here: Pericyte Damage: Surprising Cause of Covid-Related Myocarditis

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Op-Ed: Will the next coronavirus variant escape our best immune defenses? LA Times | February 27, 2022 | Op-ed Article

It is now well known that SARS-CoV-2, the virus that causes COVID-19, can mutate to evade vaccine protection against infection. The Omicron variants — BA.1, B1.1 and BA.2 — can infect those who were previously infected by other variants, even when vaccinated. A third booster shot offers some protection from an Omicron infection, but it wanes after three or four months, leaving most people susceptible to reinfection. That said, the immunity conveyed by prior infection or vaccination still dramatically reduces the incidence of hospitalization and death. We have also come to realize that our main saviors against COVID-19 turn out not to be antibodies, but rather another part of the immune system: T cells. Studies show that the strength of our long-lived T-cell response to the virus’ proteins — especially by T cells that recognize the spike protein — strongly correlates with the degree of protection. There are two types of T cells, CD4+ and CD8+, which are distinguished by proteins on their surface. Because CD4+ T cells mostly assist in the production of antibodies, the CD8+ T cells are the real heroes of the story. Once they identify an invader they remember from a previous encounter, they act quickly to move in for the kill, demolishing infected cells and cutting short the life cycle of the virus. Until Omicron, the differences in neutralization by vaccineinduced antibodies and by monoclonal antibodies were relatively minor. But the process by which T cells recognize viral proteins is very different from that of antibodies, which recognize structures on the intact viral protein. We know that these critical structures, particularly those of the exterior spike protein, differ from variant to variant. It is precisely such structural diversity that allows the virus to evade most antibodies made in response to natural infection and vaccination. 150

By contrast, T cells do not recognize intact proteins. Rather, Tcell recognition occurs when a viral protein within a cell is chopped into short segments and cradled in the grip of a cellular protein called MHC type 1. MHC type 1 presents the viral fragment to the T cell at the cell surface, where the T cell can recognize the combination of the viral fragment presented by the MHC type 1 protein. T cells recognize and react to a very broad array of viral protein fragments. For SARS-CoV-2, these fragments overlap very little with the regions of the virus that are sensitive to neutralization by antibodies. That is why T-cell responses to viral infection are generally preserved across variants. Until Omicron, vaccines that use one viral protein raised almost the same T-cell response to all variants. But now the situation has changed. Our MHC type 1 proteins are diverse, and each recognizes a unique set of viral protein fragments. Our reaction to viral proteins thus depends on their sequence and that of our own particular MHC type 1 set of proteins. Consider a recent study by Gaurav D. Gaiha and his colleagues, examining T-cell responses to the Wuhan, Delta and Omicron strains in people who have been either infected, vaccinated and boosted, or infected and vaccinated but not boosted. They found that most people who are infected after vaccination have strong and durable CD4+ and CD8+ responses to all three variants. But there was one worrying discovery. Approximately 20% of those vaccinated showed a decline of greater than 50% in T-cell response to Omicron, compared to the Wuhan and Delta variants. These poor T-cell responses were not correlated with sex or age, and follow-up experiments revealed that the difference was due to lower CD8+ reactivity, rather than to the CD4+ T-cell response. The authors speculate that CD8+ T cells’ inability to respond to Omicron may be due to a lack of recognition of the mutated peptides. Indeed, their theoretical calculations are consistent with the hypothesis that changes in the amino acid sequence of the Omicron spike protein underlie the observed blind spots in T-cell recognition. Inherited differences in the ability to recognize specific protein fragments likely account for some people’s failure to mount antiOmicron defenses. The authors offer the conjecture that “it is possible that these individuals will have reduced protection against severe disease.” 151

One sobering conclusion is that Omicron has drifted so far from the original strain that 20% of people in the study may not be fully protected either from infection or from hospitalization and death. However, the study found that a third vaccine dose increases T-cell responses by 20 times or more. “While the Omicron spike protein was able to escape T cells in a subset of individuals,” Gaiha told me, “what we learned is that this deficiency in T-cell recognition can be overcome by booster vaccination. In addition, we found that non-spike proteins could be attractive targets for second-generation vaccines to protect against future SARS-CoV-2 evolution.” Gaiha espouses an optimistic interpretation. But Omicron is a warning that future variants may escape both antibodies and T-cell immunity. We cannot predict whether a variant will arise that evades the vaccines’ ability to protect against infection and serious illness, but we must be prepared for such a threat, lest we remain unguarded against it. This article is featured on LAtimes.org, and can be read online here: Op-Ed: Will the next coronavirus variant escape our best immune defenses?

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Whence And Whither Covid-19: The Many Faces Of Omicron Forbes | March 1, 2022 | Article

Emergence of Omicron sublineages. Blue is BA.1. Pink is BA.2. Green is BA.2 + H78Y. DR. ERIC DING

What, without asking, hither hurried whence? And, without asking, whither hurried hence! Another and another Cup to drown The Memory of this Impertinence! Omar Khayyam / English version by Edward FitzGerald We are all wondering what comes next for the Covid pandemic. Are we on the brink of the next wave of infections from an as yet unidentified variant? If so, will the new variant be more or less dangerous? Will our vaccines, those we have now and others that will arrive soon, protect us from infection, severe disease, and worse?

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The short answer is that we do not know either whence or whither the SARS-CoV-2 pandemic. Yet, two years of experience provides some insight. We know that new variants start small and multiply. They may begin almost anywhere, such as the UK, India, or South Africa, and spread globally, displacing all those that came before. The new wave may be intrinsically more lethal, as with Delta, or more contagious, as with Omicron. In this context, I observe the Omicron family of SARS-CoV-2 variants. I call it a family as all Omicron variants stem from an as yet undiscovered parent (Figure 1). This cryptic parent gave birth to triplets siblings—Omicron BA.1, BA.2, and BA.3—all born at or near the same time. In the short span of three months, BA.1 spawned BA.1. 1, now carving a path through the US. BA.2 displaced BA.1 in Asia, Southern Africa, and European countries. BA.2 has a new offspring n BA.2 + Orf3a H78Y.

FIGURE 1: Proposed Omicron family tree, showing the development of mutations over time from the ... [+] ACCESS HEALTH INTERNATIONAL

Here, we focus on BA.2 + H78Y, the latest member of the Omicron family. We judge the inherent contagiousness of a new variant by its ability to replace prominent dominant viruses. Omicron BA.1 and BA1.1 displaced Delta as the dominant variant globally. BA.2 then displaced BA.1 and BA.1.1, where both branches occur. Now BA.2 + H78Y is replacing its parent BA.2 in Denmark. Despite our hopes that SARS-CoV-2 reached the limit of contagion with BA.1, both BA.2 and BA.2 + H78Y show such is not the case. BA.2 outcompetes BA.1, and BA.2 + H78Y outcompetes BA.2. We have a powerful lens to view and perhaps understand what accounts for the increase in the replication rate of this newest 155

variant—the sequence of the virus genomes. At first glance, the changes are surprisingly modest; one amino acid change in the Spike protein separates BA.1 from BA.1.1. Another single amino acid change in the Orf3a gene distinguishes BA.2 + H78Y from its parent BA.2 (Figure 2) (This analysis omits differences that might arise from mutations that do not alter viral proteins these called cis-acting mutations).

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FIGURE 2: BA.2 + Orf3a H78Y mutation genome and Spike protein. Mutations in blue are shared between ... [+] ACCESS HEALTH INTERNATIONAL

BA.1.1 differs from BA.1 by changing the amino acid 346 from an arginine to a lysine (R346K). The change is relatively conservative, exchanging one positively-charged amino acid for another. The side chain of arginine is slightly longer than that of lysine. The best interpretations are that this subtle change shields the BA.1.1 Spike protein from recognizing some anti-spike neutralizing antibodies present in those who have recovered from an infection or are vaccinated. Viruses with enhanced immune escape capability will spread through such populations rapidly. This explanation fits the popular narrative comfortably that immune escape mediated by changes in the receptor-binding domain of the spike protein accounts for the replication success of new variants.

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The same narrative does not account for the success of BA.2 + H78Y. The Spike protein of BA.2 + H78Y is identical to BA.2. Rather the difference lies in the accessory protein Orf3a, a change at position 78 of histidine for a tyrosine (H78Y). The shape of the two amino acids is similar, but they differ in charge and polarity. Histidine is polar and positively charged, whereas tyrosine is hydrophobic and uncharged (Figure 3).

FIGURE 3: Amino acid structures with histidine and tyrosine highlighted. Amino acids are sorted ... [+] DR. KAREN STEWARD // TECHNOLOGY NETWORKS

There are two general explanations to account for the difference in the two strains: immune escape or enhanced function, neither of which are mutually exclusive. Orf3a is immunogenic, recognized by both antibodies and T cells. A change in amino acid sequence might alter recognition by either antibodies, T cells, or both, providing the observed replication advantage. Orf3a thwarts the innate immune response of infected cells. Innate immunity is the primary means our bodies protect us from novel pathogens. Innate immunity also limits the success of reinfection. Might the Orf3a H78Y mutation enhance suppression of the innate immune response? There is some evidence that subtle 157

changes in accessory proteins account for the increased transmissibility of some SARS-Cov-2 variants. For example, Nevan Kroger and associates suggest that mutations that increase the production of Orf9b and Orf6, two proteins that also suppress the innate immune response, contribute to the replicative success of the Alpha variant. The structure of the Orf3a may offer a clue regarding the effect of the H78Y mutation on function. Two groups determined the three-dimensional structure of the protein. Orf3a is 275 amino acids long. The amino terminus of the monomer folds into three roughly parallel alpha helices that traverse the cell membrane. The carboxyterminal constitutes the cytoplasmic domain (Figure 4a). The Orf3a monomers dimerize to form a pore in the membrane channel formed by six juxtaposed alpha helices (Figure 4b). The pore serves as a channel for transporting calcium ions (Ca+) and possibly others positively charged. We now add Orf3a to the known viral protein ion channels known as viporins (viral ion channel pore-forming proteins).

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FIGURE 4: 3D rendering of the SARS-CoV-2 Orf3a protein. (A) The solventexcluded surface of 3a ... [+] KERN ET AL.

Amino acid 78 of Orf3a occupies a key site in the channel, facing the aqueous interior of the pore at what may be a choke point that determines the ease of passage of positively charged ions (Figure 5). A change from a positive to a neutral charge may facilitate calcium ion flux through the pore. Experiments to determine whether or not this is so are in progress (Brohawn, personal communication).

FIGURE 5: H78 as a potential choke point that determines the ease of passage of positively charged ... [+] KERN ET AL.

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Another outstanding question is the importance of the Orf3a ion channel activity to virus replication and the suppression of the innate immune response. The answer awaits the results of additional experiments. I note that single mutations in the ion channel of another viporin, the E protein of SARS-CoV-1, have a profound effect on the virulence of the virus in animal studies. We note the H78Y in Orf3a has been sequenced 52,275 times in the GISAID database. While a majority of these sequences are recent, alongside the rise of the Omicron family of variants which began in November 2021, there are previous instances of H78Y in Orf3a. The mutation appeared in the United States in the short-lived “bluebird” variant that appeared in August 2020. In fact, throughout Europe and the United States, there are sequences of H78Y as early as March 2020. This may suggest that interacts with other mutations via epistasis, as the mutation has circulated for nearly two years without major implication until the rise of the Omicron family. We note that as of March 1st, Denmark represents the plurality of sequences with 19,723, followed by other European countries, for example England with 6,330 and Germany with 3,775. Some recent GISAID entries are also from the United States, suggesting that BA.2 + H78Y may have already arrived. BA.2 + H78Y is a prime example of the significance and variability of SARS-CoV-2 variants. A single mutation in a nonSpike protein yields a potentially more transmissible virus that can outcompete its parental lineages. As the virus continues to spread and adapt, we anticipate more such examples appearing throughout the globe, extending the pandemic for the immediate future. This article is featured on Forbes.org, and can be read online here: Whence And Whither Covid-19: The Many Faces Of Omicron

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Covid-19 Has Orphaned 5.2 Million Children Forbes | March 1, 2022 | Article

An updated modeling study in The Lancet shows that number of children globally affected by COVID-19-associated orphanhood and caregiver death is estimated to have increased dramatically from approximately 2.7 million in April 2021 to a devastating 5.2 million in October 2021. To put those numbers into context, the researchers state that is the equivalent of one child every six seconds during the six-month period. With the pandemic far from over, we have both a moral and public health imperative to protect and support these children from direct and secondary harms. Children’s lives are permanently changed by the loss of a mother, father, grandparent, or other primary caregivers. The loss of a parent is an adverse childhood experience that is linked to a greater risk of dropping out of school, lower self-esteem, suicide, violence, sexual abuse, and developing anxiety, depression, and substance abuse problems. These impacts could be compounded further by the circumstances and additional stressors of the pandemic. The study authors defined orphanhood as the death of one or both parents, primary caregiver loss as the death of one or both parents, or of one or both co-residing custodial grandparents aged 60–84 years, and secondary caregiver loss as the death of one or more co-residing grandparents or older kin. The study used mathematical modeling and mortality and fertility data from 21 countries with 76 percent of global deaths from Covid-19 to estimate the number of children who lost a caregiver. These countries included Argentina, Brazil, Colombia, England and Wales, France, Germany, India, Iran, Italy, Kenya, Malawi, Mexico, Nigeria, Peru, Philippines, Poland, Russian Federation, South Africa, Spain, United States, and Zimbabwe. In reality, the number of children who have lost parents is probably far greater than the study estimates due to international coronavirus testing and reporting gaps. In the US, the CDC only 161

records deaths from Covid-19 and not the survivors left behind. We need to establish domestic and global institutions to collect this data and allocate resources to provide evidence-based psychosocial and economic support to children who have lost a caregiver. Children who have been orphaned from AIDS globally are supported through the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR). Services include health and nutrition, education, psychosocial care, and support, household economic strengthening, parental communication skills, and legal protection. Organizations such as FXB International have provided grants, medical care, and psychological and social support, to families directly affected and infected with HIV/AIDS. We need to develop similar programs for those orphaned by Covid-19. The study also demonstrated that countries with low vaccination rates had the highest number of children orphaned by Covid-19. This is yet another critical reason to accelerate the equitable distribution of Covid vaccines globally. We must take action and provide support to these children before this becomes a shadow pandemic whose health impacts echo through decades to come. This article is featured on Forbes.org, and can be read online here: Covid-19 Has Orphaned 5.2 Million Children

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Covid-19 Pathogenesis: Spike Protein Alone Can Damage Cells Forbes | March 2, 2022 | Article

Covid-19 patients have reported a vast array of symptoms, including issues related to the heart, kidneys, liver, brain, and a number of other areas. Neurological problems following SARSCoV-2 infection, ranging from the inability to concentrate to overwhelming fatigue, have been especially concerning. Exactly how this happens, however, remains somewhat of a mystery. By and large, these kinds of symptoms were considered a result of direct viral infection of host cells. Yet increasingly we’re realizing that indirect mechanisms may play a larger role than initially assumed— whether through secondary effects from viral proteins or through the release of inflammatory cytokines and chemokines. A recent study by researchers at Cleveland State University, led by Abhijit Basu with senior author Barsanjit Mazumder, might help shed some light into this dark corner of Covid-19 pathogenesis. The key discovery is that the SARS-CoV-2 virus need not enter a cell to disturb its function in profound ways; it may be sufficient for the spike (S) protein to simply bind to the outside of a cell to cause significant, and even lethal, changes. Nearly two decades ago, Mazumder and his then colleagues were researching how our immune system autoregulates inflammation. They knew that a protein called ceruloplasmin (Cp) was involved in this process, but weren’t sure how. Looking into it, Mazumder et al. discovered that ceruloplasmin messenger RNA contains a special “hairpin” loop structure (figure 1). When this structure gets bound by a specific bundle of proteins, ceruloplasmin production is halted, restricting inflammation and preventing our immune response from accidentally harming our own bodies. All of this happens via signals sent from outside the cell, kickstarted by a small protein called interferon-gamma (IFN-γ).

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The researchers called the mRNA structure a gamma interferon inhibitor of translation (GAIT) element, and the bundle of proteins that binds to it a GAIT complex.

FIGURE 1. Schematic of the GAIT hairpin loop structure of human ceruloplasmin mRNA. FROM: “A STRUCTURALLY CONSERVED RNA ELEMENT WITHIN SARS-COV-2 ORF1A RNA AND S MRNA REGULATES TRANSLATION IN RESPONSE TO VIRAL S PROTEIN-INDUCED SIGNALING IN HUMAN LUNG CELLS” BASU ET AL. 2022

Since then, it has been discovered that some viruses also carry GAIT-like elements in their genome. For example, a common pig coronavirus, transmissible gastroenteritis coronavirus (TGEV), has been shown to have a GAIT-like element that allows it to modulate the host’s innate immune response. Curious to see whether something similar could be seen in SARS-CoV-2, Basu et al. analyzed the entirety of the viral genome. They found two sequences, one located within ORF1a and a second in the S gene, that could form structures akin to those of canonical GAIT elements (Figures 2 & 3), albeit with dissimilar genetic sequences.

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FIGURE 2. Schematic representation showing the location of ORF1a RNA and Spike mRNA within the ... [+] BASU ET AL. 2022

FIGURE 3. GAIT-like hairpin loop structures in SARS-CoV-2 ORF1a RNA and S mRNA. BASU ET AL. 2022

The researchers discovered that the GAIT-like elements in SARS-CoV-2 help silence the production of ORF1a and S. This process kicks off when the SARS-CoV-2 spike protein binds to our lung cells’ ACE2 receptors. They decided to call the hairpin loop structures found in SARS-CoV-2 virus-activated inhibitor of translation (VAIT) elements, emphasizing the fact that genomic silencing takes place on the basis of viral signals rather than signals from IFN-γ. Although Basu et al. aren’t exactly sure of the first steps of the signaling pathway, they suggest that it may happen by way of the ERK1/2 “kinase cascade.” Kinase cascades link extracellular signals 165

to a number of different cellular processes, generally through the phosphorylation of proteins— essentially, they provide a means of giving a cell a set of instructions without needing to enter into it. This activates another kinase cascade, DAPK-ZiPK, which causes the release of L13a from our ribosomes— the cellular “machinery” used in the production of proteins. L13a binds with other proteins, as of yet still unknown, to form the VAIT protein complex that ultimately binds to the VAIT RNA elements. Figure 4 outlines a proposed pathway for VAIT element binding.

FIGURE 4. A model of the VAIT-dependent signaling pathway that silences synthesis of SARS-CoV-2 ... [+] BASU ET AL. 2022

It’s important to note that death-associated kinase 1 (DAPK-1) is involved in apoptosis and autophagy. Apoptosis is a process by which stressed cells commit suicide in such a way as to not induce inflammation; they just quietly digest themselves. But regardless, it leads to cell death. In this sense, the virus may be triggering a very potent reaction by activating DAPK-1. Basu et al.’s research shows that, independent of its role as an entry receptor for SARS-CoV-2, extracellular binding of the spike protein to host ACE2 triggers an intracellular cascade that has the

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potential to alter both cell physiology and expression of viral proteins. An independent study by Elisa Avolio et al. found that SARSCoV-2 spike binding to another receptor, C147, initiates a signaling cascade that disrupts pericyte function and induces death of vascular endothelial cells. Together these studies show that signal transduction induced by sarbecovirus S protein binding to surface receptors are likely to contribute to viral pathogenesis independent of the virus’s ability to enter and to replicate in the target cell. This article is featured on Forbes.org, and can be read online here: Covid-19 Pathogenesis: Spike Protein Alone Can Damage Cells

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Covid Ping-Pong: Human To Deer, Deer To Human Forbes | March 4, 2022 | Article

An ongoing question regarding the SARS-CoV-2 virus is the extent of variation. The Omicron family of viruses are among the most mutated natural variants observed thus far, displaying over fifty amino acid changes throughout the genome. While some may hope that this approaches an upper limit, the unfortunate truth is that the potential number of mutations is unknowable due to the potential of animal cross-infection. Here we describe a recent paper by Pickering et al. that describes a series of SARS-CoV-2 sequences originating in white-tailed deer highly divergent from previous variants, with as many as 76 consensus nucleotide mutations from the original Wuhan virus. Instances of SARS-CoV-2 infecting animals and yielding new variants is not uncommon. We have observed emerging variant strains from Gorillas in California zoos with Epsilon and mink in Denmark and the United States. These variants reflect adaptations to the new host species. This reflects documented transmission from hamsters, mink, and deer into humans. Several previous studies have documented SARS-CoV-2 infection of white-tailed deer. Palmer et al. describe the isolation of multiple independent isolates of SARS-CoV-2 in US deer populations. The sequence of these isolates is very close or identical to that of prevailing human isolates in the same geographical area. That renders the discovery of a new highly-diverged variant in Canadian deer a troubling outlier. Now the story from Ontario, Canada. White-tailed deer regularly come in contact with humans through hunting and foraging. This species is among the most common large mammal in North America and regularly contact humans via hunting activity. Pickering et al. collected SARS-CoV-2 samples from white-tailed deer to examine new mutations and monitor for similar associated human cases, i.e., deer-to-human transmission. Of over 250 sampled 168

deer, roughly 6% had antibodies in the sera samples to SARS-CoV2, indicating previous infection. From five nasal swabs taken from white-tailed deer infected with SARS-CoV-2, Pickering et al. developed three consensus genomes that contained 76 conserved nucleotide mutations. In searching for the deer-to-human transmission, they also found a SARS-CoV-2 genome in the GISAID database derived from a human host in Michigan in November 2020 that closely resembles the deer consensus. The surprise comes with the striking difference between the Ontario white-tailed deer samples and previous variants of concern or interest. Figure 1 illustrates the departure of the 76 consensus deer mutations as compared to other animal derived sequences in the GISAID database.

FIGURE 1: Maximum-likelihood (ML) phylogeny of white-tailed deer-derived viral genomes (and ... [+] PICKERING ET AL.

In tracking down the origin of this strain, the authors note it likely stemmed in late 2020 from Beta, Iota, or Epsilon variant. These variants were also circulating mink populations in Wisconsin and Michigan in late 2020. Pickering et al. believe that at some point thereafter, the strain transmitted into the Ontario deer population via an intermediary unknown species. Pickering et al. note that the large number of mutations specifically occurred in the undetected intermediary for a prolonged period, up until their detection in this 169

study. They entertain the notion that the intermediary may have been humans. Based on the sheer number of species that interact with humans regularly, there are likely several reverse zoonosis strains akin to the white-tailed deer strain waiting to emerge. The potential severity of the strains is uncertain. As outlined in Figure 2, after a long residence in the unknown predecessor, the white-tailed deer strain either made its way into deer and the one human concurrently or sequentially. This gives us some insight into the blueprint of reverse zoonosis strains.

FIGURE 2: Overview of potential zoonotic scenarios underpinning the evolution of the Ontario WTD ... [+] PICKERING ET AL.

We note that this series of events resembles, in some ways, the origin of the Omicron family of viruses (Figure 5). From the original Wuhan strain sampled in humans in late 2019, the B.1 virus which we call the Triad emerged in February and March 2020. This variant contained a triad of mutations: D614G in the Spike, P323L in NSP12, and C241U in the 5’ UTR. We previously proposed that an undetected parent of the Omicron viruses emerged from B.1 containing 31 mutations. From there, at least five distinct strains have been sampled in recent months, comprising the Omicron family.

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FIGURE 3: Proposed Omicron family lineage. ACCESS HEALTH INTERNATIONAL

Of the 76 conserved mutations, Pickering et al. observed that many were not found in human or mink samples in the region at the time of collection, aside from the one human sample taken from Michigan months prior. Of the 76 nucleotide changes, 49 are different from the closest related natural variant. There are 51 changes in Orf1ab, 11 of which are in NSP3 and nine in NSP4. There are nine changes in the Spike protein. The structural proteins E, M, and N, contain six observed mutations and the Orf genes contain ten. Below is a diagram of the sequenced mutations in the deer consensus and human genomes. Additionally, the deer strains have the C241U conserved mutation in the 5’ untranslated region of the genome associated with cellular TAR binding.

FIGURE 4: Deer strain amino acid mutations in the Non-Spike proteins, with those that appear in ... [+] ACCESS HEALTH INTERNATIONAL

The mutations noted, however, were not exceptionally uncommon in animal sequences. Particularly in the Spike protein, which interacts with the human or nonhuman ACE2 receptor, the 171

deer variant contains many mutations shared with bats, cats, hamsters, and mink. Of the consensus Spike protein amino acid mutations (H49Y, T95I, 143-145del, F486L, N501T, and D614G), only F486L is rarely found in any sequence, human or other. There were also three amino acid mutations in only one white-tailed deer genome (T22I, S247G, and V705A) and several cis-acting synonymous mutations that do not change amino acids but could affect RNA transcription sequences.

FIGURE 5: Analysis of variants present in the Ontario white-tailed deer clade relative to other ... [+] PICKERING ET AL.

Note that most mutations in the strain are not in the Spike protein. The majority are in the Orf1ab replicative proteins, structural proteins, and accessory genes. Pickering et al. make the unusual suggestion that the majority of these mutations arise without selection, simply via genetic drift. The difference in mutations between the deer samples and Omicron, particularly in the Spike protein, illustrate the differences in selective pressure, as the paucity of mutations in the deer Spike likely indicate less efficient transmissibility as compared to Omicron. The large number of mutations outside of the Spike may represent either neutral drift or other selective pressures, including T-cell recognition and suppression of innate immune responses.

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TABLE 1: Number of amino acid mutations in SARS-CoV-2 proteins compared between the human sample of ... [+] ACCESS HEALTH INTERNATIONAL

The relative lack of mutations in the Spike protein is also reflected by the comparable neutralization of these viruses by vaccine-elicited antibodies to non-Omicron variants. Additionally, it does not seem that infected deer were significantly affected by symptoms, similar to the Omicron variant in reduced symptom severity concentrated in the nasopharynx. This observation should put us on notice that there may be many other lingering variants in humans or in other species, which at any time may reemerge to cause the kind of pandemic associated with Omicron. Instances of reverse zoonosis are more common than we realize, as many households have domesticated pets and many cities have many wild and feral species. As a final note, hunters in this region should be wary of dressing deer carcasses as this represents a transmission risk. This article was originally featured on Forbs.org, and can be read online here: Covid Ping-Pong: Human To Deer, Deer To Human

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Antibody-Activated Endothelial Cells Increase the Risk of Blood Clots with Covid-19 Forbes | March 4, 2022 | Article

The activity of antiphospholipid antibodies may help explain hypercoagulation associated with late stages of Covid-19 and longterm post-acute sequelae SARS-CoV-2 infection (PASC), also known as long-haul Covid-19. A recent study found that at least forty-five individuals out of every thousand infected with Covid-19, regardless of age, gender, race, and prevalence of pre-existing conditions, experience serious cardiovascular consequences, including widespread blood clots. Forty-five out of a thousand may seem small, but given that there are an estimated 140 million Covid19 cases nationally, over six million people could be at risk. Shi et al., writing for Arthritis & Rheumatology, analyzed blood samples from nearly 250 individuals hospitalized for Covid-19 and found that Covid-19-induced blood clots may be triggered partly by “rouge” autoantibodies. Unlike other antibodies, natural autoantibodies can be generated outside of the immune system and bind to a variety of non-related antigens. These antibodies, by definition, attack the body’s own cells. Exposure to the SARS-CoV-2 virus seems to induce a vigorous antibody response, including an enhanced recruitment of autoantibodies that may attack uninfected cells and tissues. Covid-19-induced thrombosis, or blood clotting, is common among people hospitalized for severe infection, with estimates suggesting that nearly 60% of those that die from the virus are affected. Emerging research reveals that SARS-CoV-2 activates endothelial cells that line blood vessels, making them vulnerable to clotting. As Dr.Hui Shi, lead author and rheumatology research fellow at Michigan Medicine, explains “When endothelial cells are activated, they cause healthy blood vessels to become 'sticky', attracting other cells to the vessel walls and becoming more prone to thrombosis. This can affect many of the body's essential organs,"

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How are endothelial cells activated in the first place? Shi et al. 2022 suggests there may be a connection between increased circulation of antiphospholipid autoantibodies and endothelial activation. In a previous investigation, the team intriguingly found that exposing mice to the autoantibodies from the blood of Covid19 patients produced a “striking amount of clotting.” Their interest in autoantibodies as potential biomarkers for Covid-19 was further supported by evidence that antiphospholipid antibodies mediate a rare autoimmune disorder called antiphospholipid syndrome (APS). In this condition, the body produces antibodies that target its own fat molecules— phospholipids— involved in forming blood clots. The present study not only shed light on how Covid-19 induces thrombosis but may also explain the underlying mechanisms of this life-threatening disease. Shi et al. began their investigation by confirming that Covid-19 does in fact activate endothelial cells. Using blood samples from 118 individuals hospitalized with Covid-19, serum collected after blood cells were left to clot revealed increased expression of cell adhesion molecules, E-selectin, VCAM1 and ICAM-1. These surface proteins are upregulated during immune responses to facilitate the binding and activation of white blood cells on endothelial cells. Overexpression of E-selectin, VCAm-1, and ICAM-1 surface proteins, however, can signify that endothelial cells are being overpowered and subsequently weakened by immune activity. In fact, increased expression of these adhesion proteins was shown to correlate with worse Covid-19 outcomes. For example, after obtaining additional serum samples from 126 hospitalized Covid-19 patients, Shu et al. 2020 found that individuals requiring oxygen ventilation experienced greater upregulation of ICAM-1, compared to unventilated controls. How much does Covid-19 contribute to these effects? Could endothelial activation simply be a symptom of any severe disease? To answer these questions, investigators compared plasma samples from 100 individuals admitted to intensive care for sepsis infections to plasma from their Covid-19 cohort. Unlike serum, plasma is extracted after anti-clotting agents are added to blood samples. Although the individuals with sepsis more often required mechanical 175

ventilation, their ICAM-1 levels were significantly lower compared to plasma samples from individuals hospitalized for Covid-19. This suggests that increased expression of surface adhesion molecules, as well as the corresponding activation of endothelial cells, may be specific to the SAR-CoV-2. The team then asked how antiphospholipid antibodies may mediate these effects. Interestingly, they found a strong correlation between endothelial activation and the prevalence of autoantibody subtypes, anticardiolipin and anti-phosphatidylserine/prothrombin (anti-PS/PT), in immunoglobulin tests. In particular, significantly high levels of anticardiolipin and anti-PS/PT autoantibodies were detected in IgG tests from their Covid-19 cohort with enhanced expression of ICAM-1, compared to healthy controls and sepsis patients. Depleting the IgG samples of anticardiolipin and antiPS/PT also prevented the upregulation of E-selectin, VCAM-1 and ICAM-1 surface proteins. Although the researchers conclude that the presence of antiphospholipid antibodies in Covid-19 IgG samples can activate endothelial cells, the underlying mechanism remains unclear. Further investigations are needed to determine if the prevalence of these autoantibodies could be a useful biomarker for identifying an infected individual's risks for severe Covid-19 compilations, such as thrombosis and respiratory failure. How such high amounts of autoantibodies are created in the first place also remains a mystery. An emerging theory suggests that the heightened activity of autoantibodies may be linked to the immune response of circulating B cells during Covid-19. Naïve B cells generated from bone marrow stem cells circulate the body looking for foregin pathogens, without first being “educated” at the follicles of the lymph nodes. When the body is exposed to SARS-Cov-2, naïve B cells seem to take an extrafollicular route, bypassing the lympoids, to quickly transform into antibodies. As seen in the autoimmune disease lupus, antibodies created outside of lymphoid follicles do not benefit from counterselection against those that may react against the body, aka autoantibodies. As one recent study reports, extrafollicular maturation of B cells may be a hallmark of Covid-19 infection. Our understanding of this mechanism, however, is limited.

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Given the very high incidence of Covid-19-induced coagulation, as well as short and long term cardiovascular disease, understanding the contribution of antiphospholipid autoantibodies is increasingly important. Several likely mechanisms seem to contribute to bystander damage to blood vessels exposed to SARSCoV-2, including direct infection and exaggerated activation of the immune system via a “cytokine storm.” Autoantibodies may only be one piece of a much larger puzzle. This article is featured on Forbes.org, and can be found online here: Antibody-Activated Endothelial Cells Increase the Risk of Blood Clots with Covid-19

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Covid-19 Damage To The Male Reproductive Tract Forbes | March 4, 2022 | Article

Overview of the various male sexual health and fertility issues associated with SARSCoV-2 ... [+] FROM: “AN IMMUNOPET PROBE TO SARS-COV-2 REVEALS EARLY INFECTION OF THE MALE GENITAL TRACT IN RHESUS MACAQUES” MADDEN ET AL. 2022

Two pre-prints, one from Canada the other from Hong Kong and China, provide convincing evidence that SARS-Cov-2 infects and damages male reproductive organs in monkeys and hamsters. The work helps to explain observations why some men with Covid178

19 experience testicular pain, decreased fertility, and have SARSCoV-2 present in their semen. Both studies provide early evidence that vaccination may reduce or prevent damage to the male reproductive tract. The Hong Kong researchers, Li et al., specifically looked for evidence of testicular damage in hamsters, a model often used to study SARS-CoV-2 pathogenesis. They infected hamsters either intranasally or by direct injection into the testes. They then examined the testes for evidence of damage. The original experiments were done using an early SARS-CoV-2 isolate, HK13, and were repeated using the more recent beta and omicron isolates. All three variants induced testicular damage, albeit with minor variations in pathology. Intranasal injection results in substantial damage to the testes. The findings include significantly reduced testicular size and weight, acute histopathological damage including inflammation, hemorrhage, and reduced sperm count (Figure 1A-C). Levels of testosterone, the male sex hormone in charge of sexual development and healthy sexual function, and inhibin B, a protein that helps regulate the production of testosterone, also decreased noticeably (Figure 1D). The reduced testes size and weight persist at least 120 days post-infection as does reduced sperm count. Direct injection of the testes confirmed that the virus could replicate in the testicular tissue as judged by the presence of the nucleocapsid (N) protein and by the presence of sub-genomic viral messenger RNAs.

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FIGURE 1. (A & B) Decreased testicular size and weight 120 days post infection (dpi). (C) Decreased ... [+] FROM: “SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) INFECTIONS BY INTRANASAL OR TESTICULAR INOCULATION INDUCES TESTICULAR DAMAGE PREVENTABLE BY VACCINATION IN GOLDEN SYRIAN HAMSTERS” LI ET AL. 2022

Notably, vaccination with two doses of inactivated whole virus administered 14 days apart prevented testicular damage of hamsters challenged by intranasal infection using the HK-13 strain of SARSCoV-2. The authors conclude that, “Awareness of possible hypogonadism and subfertility is important in managing convalescent males.” The Canadian researchers, Madden et al., used an entirely different novel approach. They designed experiments to identify the distribution of SARS-CoV-2 in living animals. Their tool is an antibody to the SARS-CoV-2 spike protein coupled to the shortlived copper 64 isotope. Rapid decay of the copper isotope signals the presence of the antibody by positron emission tomography, i.e. a PET scan. The researchers infected rhesus macaque monkeys intranasally and intratracheally with the Washington WA-1 or delta variants. The radiolabeled anti-spike protein antibody was injected at several times after infection. The location of the spike protein in 180

the infected animals was revealed by PET scan. These tissues were harvested for examination for presence of virus and evidence of damage. The authors describe their expectation that they would observe virus in the lungs and possibly other tissues such as the heart and intestines. The work provides a detailed picture of infection in the lung over time as expected. The surprise was the intense PET signal in the male reproductive tract, most notably but not exclusively in the testes (Figure 2). A similar string signal was observed for both infections by the WA-1 and delta variants. Madden et al. followed this observation up by a detailed examination of the organs and tissue harvested from the infected animals.

FIGURE 2. (A&B) Whole-body PET/CT scans of LP14 8 days 1167 post-infection. Front view (A) and ... [+] MADDEN ET AL. 2022

Madden et al. write, “Detection of robust and dynamic signals in the male genital tract including the prostate, penis, and testicles… is consistent with clinical observations of orchitis (inflammation of the testicles), oligo/azoospermia (low sperm count), and erectile dysfunction… likely a consequence of direct viral infection of the tissues." The researchers observed the course of infection one week and two weeks post-infection. Infection in the lungs decreased sharply 181

between weeks one and two consistent with the observations on many groups of rhesus macaques infected by SARS-CoV-2. Not so with infection of the male genital tract, which increased in intensity, rather than decreased, between weeks one and two. Examination of the infected organs revealed substantial damage to several discrete tissues. These include inflammation of the testes as suggested by the presence of infiltrating immune cells, disappearance and active apoptosis (cell death) of Sertoli cells (a cell type essential for the formation of sperm), absence of spermatids (nascent sperm), and denuded stretches of the twisted and intertwined seminiferous tubule. Also noted were intense signals from the prostate, the base of the penis, and regions immediately above and below the testes. They attribute the signal above the testes to infection of the vessels of the spermatic cord and the pampiniform plexus (a network of blood vessels that serve to radiate heat to help cool the testes). The signal below the testes is from the epididymis that stores the sperm. Additionally Madden et al. comment on infection of the penis. They write, "SARS-CoV-2 of the penis is potentially associated with the vascular of the corpus cavernous which expressed high levels of ACE2 in the rhesus macaque and human penile tissue. Because the corpus cavernosum plays a key role in erectile function, the inflammation caused by SARSCoV-2 infection of the penile vasculature is hypothesized told to erectile dysfunction.” They conclude with a sobering comment, “Because of the distinct mechanism negatively impacting human male sexual health and fertility…. we feel compelled to report this information at this early stage of study and evaluation.” The good news in these reports, if any, is that the effects may be transient—decreased male fertility lasting three to four months—and may be at least partially mitigated by vaccination. Additional clinical and experimental research is warranted on the consequences of the Covid-19 pandemic for male fertility. This article is featured on Forbes.org, and can be found online here: Covid-19 Damage To The Male Reproductive Tract

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Covid-19 Infection Increases Heart Disease Risk, Even In Mild Cases Forbes | March 7, 2022 | Article

As we enter the third year of the pandemic, it is becoming increasingly clear that Covid-19 infection impacts our health beyond the acute stage of the illness. More than 40% of Covid-19 survivors globally have experienced longer-term symptoms. Now, a new study demonstrates that infection with Covid-19 appears to impact the risk of cardiovascular events up to 12 months post-infection, even in those who weren’t hospitalized or had mild cases. Risks increased regardless of age, race, sex, obesity, smoking, or other cardiovascular disease risk factors. The study found the incidence of serious cardiovascular problems was 4.5% higher in the 12 months after people were diagnosed with Covid-19 compared to those who were not infected. This percentage may seem small until you consider that the CDC recently estimated that over 140 million people in the US have been infected with Covid-19, meaning approximately 6.3 million could be facing cardiovascular problems. Our ongoing case counts could create generations of patients with heart problems. The study published in Nature used data from US Department of Veterans Affairs national health care databases to follow over 153,000 veterans with a history of Covid-19 infection for up to a year after their recovery. The researchers also used a contemporary and a historical control group to estimate risks and 1-year burdens of a set of pre-specified cardiovascular incident outcomes. The contemporary control group compromised of 5,637,647 patients with no evidence of SARS-CoV-2 infection who used VHA services in 2019. The historical control group compromised of 5,859,411 pre-pandemic patients who used VHA services in 2017. The study population was largely made up of older white male patients. The Covid-19 cohort, which averaged 61 years old, included 89 percent males and about 71 percent white individuals. However because the study population was large, it also included 183

almost 17, 000 female patients; approximately 37, 000 Black patients; and almost 8,000 Latino, Asian, American Indian, Native Hawaiian, and patients of other races with Covid-19. The researchers found that in the year after recovering from the illness’s acute phase, patients had starkly increased risks of different cardiovascular problems, including abnormal heart rhythms, heart muscle inflammation, blood clots, strokes, myocardial infarction, and heart failure. At the 12-month mark, for every 1000 people compared to the contemporary control group, Covid-19 infection was associated with: • 45.29 incidents of any prespecified cardiovascular outcome • 23.48 incidents of major adverse cardiovascular events including myocardial infarction, stroke, and all-cause mortality • 19.86 incidents of dysrhythmias, including 10.74 incidents of atrial fibrillation • 12.72 incidents of other cardiovascular disorders including 11.61 incidents of heart failure and 3.56 incidents of nonischemic cardiomyopathy • 9.88 incidents of thromboembolic disorders, including 5.47 incidents of pulmonary embolism and 4.18 incidents of deep vein thrombosis • 7.28 incidents of ischemic heart disease including 5.35 incidents of acute coronary disease, 2.91 incidents of myocardial infarction, and 2.5 incidents of angina • 5.48 incidents of cerebrovascular disorders, including 4.03 incidents of stroke • 1.23 incidents of inflammatory disease of the heart or pericardium, including 0.98 incidents of pericarditis and 0.31 incidents of myocarditis

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Risks and burdens were assessed at 12 months in mutually exclusive groups comprising non-hospitalized individuals with COVID-19 (green), individuals hospitalized for COVID-19 (orange) and individuals admitted to intensive care for COVID-19 during theXIE, Y., XU, E., BOWE, B. ET AL. LONG-TERM CARDIOVASCULAR OUTCOMES OF COVID-19. NAT MED (2022)

While hospitalization did increase the likelihood of future cardiovascular complications, people who avoided hospitalization were still at higher risk for many conditions. Other subgroup analyses found increased risks regardless of age, race, sex, obesity, smoking, hypertension, diabetes, chronic kidney disease, hyperlipidemia, and preexisting cardiovascular disease.

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The study was well designed, not only using extensive control cohorts but also accounting for external factors. Because some Covid-19 vaccines may be associated with very rare cases of myocarditis and pericarditis, the researchers conducted analyses to remove the effect of vaccination. The increased risk of myocarditis and pericarditis remained among people who were not vaccinated and was clear regardless of vaccination status. Based on the results of this study, I recommend that everyone who has been infected with Covid-19, mild or otherwise get a cardiovascular workup within 12 months of infection. I urge anyone who has unexplained cardiovascular symptoms after a Covid-19 infection to take them seriously and seek medical care immediately. Physicians should also be adjusting their screening questions to include past infection with Covid-19 and assess for all Long Covid symptoms including cardiovascular. Early identification, diagnosis, and treatment of heart disease are essential to lessen the risk of adverse health impacts. The cardiovascular disease risk associated with Covid-19 infection further highlights how we need a coordinated global response strategy to urgently address the challenges of dealing with the long-term health effects of Covid-19. This article originally appeared on Forbes.org, and can be read online here: Covid-19 Infection Increases Heart Disease Risk, Even In Mild Cases

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Stripping Covid's Camouflage Forbes | March 8, 2022 | Article

Infection requires that SARS-CoV-2 slip into a cell, unnoticed by multiple alarms, and camouflage itself to resemble cellular genes and hijack the cell's machinery. To this end, the virus is equipped with numerous means to thwart cellular alarms and to disguise its messenger RNAs to resemble that of the cell. Tripping the cellular alarms leads to the destruction of the virus early on. Failure to successfully mimic viral RNA and the viral message is destroyed before essential viral components can be made. SARS-CoV-2 genome encodes 30 proteins. Virtually all of them participate in one way or another the intricate dance of deception and subterfuge. Blocking one more of these essential functions with target-specific drugs will strip Covid’s camouflage, preventing virus replication.

FIGURE 1: SARS-CoV-2 genome with targets indicated for proteins that inhibit the host immune ... [+] ACCESS HEALTH INTERNATIONAL

A recent study by Park et al. unveils the unexpected details of one such trick, the formation of viral RNA's five prime (5’) cap. In so doing, the authors provide a road map to guide the development of multiple anti-viral drugs to prevent and treat SARS-CoV-2 infections. 187

All messenger RNAs require a cap, a structure at the 5 prime termini (the beginning) of the messenger RNAs. The structure of the cap is shown in figure 2. The newly formed messenger RNA is modified first by adding phosphorylated guanosine, then adding methyl groups to the newly added guanosine and the terminal 5' terminal residue of the messenger RNA. Absent the cap, the messenger is not recognized by the synthetic protein machinery and is degraded. Moreover, RNAs that lack the methyl group on the initial nucleotide of the RNA trip cellular alarms that initiate a signaling cascade to activate the innate immune response. The cellular messenger RNAs are made and capped in the cell's nucleus before export to the cytoplasm for translation. The nuclear enzymes include an RNA triphosphatase to remove the terminal phosphate of the nascent messenger RNA), a guanylyltransferase which transfers a guanosine monophosphate from GTP to form the cap core, a guanine-N7-methyltransferase which adds a methyl group to the terminal guanosine at the N7 position and a nucleoside 2-O-methyltransferase which adds a methyl group to the 2’ hydroxyl group of the first nucleotide of the messenger RNA.

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FIGURE 2: (A) the SARS-CoV-2 mRNA cap and (B) the process it is made. PARK ET AL.

As mentioned, all these reactions occur in the cell nucleus. These are not available to the messenger RNAs of SARS-CoV-2, as virus replication takes in the cytoplasm. Viral RNA never enters the nucleus. Prior work on other coronaviruses and SARS-CoV-2 found three of the viral proteins encoded by the initial long open reading frame orf1ab. Nsp13 encodes an RNA triphosphatase necessary to remove the 5' most phosphate of the nascent RNA. N7 methylation of the added guanine to yield is accomplished by nsp14. Nsp16 (in a complex stabilized by nsp10) is required to add the final 2’-O-methyl group to the terminal nucleotide. The process of coronavirus messenger RNA synthesis is unique. The positive-strand genomic RNA is initially copied from a nested set of complementary negative strands. This nested set of negativestrand RNAs serves as a template for synthesizing the positive strand messenger RNAs, all of which contain an identical 5’ leader sequence.

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FIGURE 3: SARS-CoV-2 replication-transcription strategy. ACCESS HEALTH INTERNATIONAL

The SARS-CoV-2 replication transcription complex includes several different enzymes. These include nsp12, the RNAdependent RNA polymerase required for RNA synthesis, a helicase to open double-stranded RNA structures for copying, an errorcorrecting exonuclease through which nascent RNAs must pass, nsp9, and what are described as stabilizing proteins, nsp7, nsp8, and nsp10. In addition to a structure common to all viral RNAdependent RNA polymerases, the SARS-CoV-2 polymerase contains an additional amino-terminal domain called NiRAN (Nidovirus RNA dependent RNA Polymerase Associated Nucleotidyltransferase)

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FIGURE 4: SARS-CoV-2 replication complex. HARTENIAN ET AL.

FIGURE 5: NSP12 domain schematic with start and stop amino acids indicated. Mutations discussed in ... [+] ACCESS HEALTH INTERNATIONAL

Park et al. uncovered an unexpected pathway when used purified viral proteins to reconstruct how the SARS-CoV-2 cap is formed. They expected to find that by analogy with cellular processes, the first step of cap formation would be the removal of the 5’ terminal phosphate of the nascent RNA to yield ppA…mRNA. Note that the initial nucleotide of all SARS-CoV-2 messenger RNAs is adenosine (A). That is not what they found. Using a short RNA (5'’pppA…n10) as a surrogate messenger RNA, they discovered that the very first step in cap formation is the covalent attachment of the RNA to the nsp9 protein. Further investigation revealed that attachment occurs to the amino-terminal asparagine of nsp9. The phosphate closest to adenosine (the alpha phosphate) attaches to the 191

backbone amino group, the terminal asparagine of nsp9. Park et al. call the process of covalent enzymatic attachment of RNA to a protein RNAylation.

FIGURE 6: During transcription, the nascent 5′-pppRNA binds to the NiRAN active site in either a cis ... [+] PARK ET AL.

The authors also show that attachment of the nascent RNA to nsp9 is catalyzed by NiRAN, the amino-terminal domain nsp12 protein. Mutations at positions 73 and 218 that inactivate the phosphotransferase activity of NiRAN fail to attach RNA to nsp9. By contrast, a mutation at 760 of nsp12 that inactivates the polymerase activity does not affect RNAylation of nsp9. NiRAN but not polymerase activity is essential for cap formation. How then is the terminal guanine diphosphate added to the cap? Park et al. find that NiRAN binds guanosine diphosphate (GDP). The addition of guanosine diphosphate occurs via NiRAN catalyzed transfer of the asparagine-RNA complex to guanosine yield the cap's GppA…mRNA core structure. The subsequent addition of an N7methyl group to guanosine is followed by O-methylation of the

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terminal adenosine is mediated by nsp14 and the nsp16/10 to yield the final cap structure. Park et al. also report the 3-dimensional structure of a complex that contains the nsp12/nsp9 together with the stabilizing proteins nsp7 and nsp8. They find that the amino terminus of nsp9 inserts into the active site of the NiRAN domain of nsp12, providing a structural explanation of how the covalent addition of the nascent RNA to nsp9 occurs.

FIGURE 7: (A) Front and back views of nsp12/7/8/9 cryo-EM maps, with respect to the NiRAN domain. ... [+] PARK ET AL.

Park et al. provide two models by which the nascent messenger RNA chain encounters nsp9. In one model, all the reactions occur on a single nsp12/nsp9 complex. This model requires the nascent RNA to twist and bend during synthesis. According to this model, on exit from the polymerase, the growing messenger must first 193

transit the exonuclease, then twist and bend to enter NiRAN. For this reason, they suggest that it is possible and even likely that RNAylation occurs on a second nsp12/nsp9 complex. This work explains how the SARS cap is formed outside of the nucleus. The authors mention several well-known targets for antiviral drugs, the polymerase domain of nsp12 (the target of remdesivir), as well as nsp14 and nsp16/nsp10. They also identify two new targets, the NiRAN domain of nsp12 and the amino terminus of nsp9. Single drugs or combinations of drugs that inhibit these enzymes will likely prevent and treat SARS-CoV-2 infections effectively. I favor efforts to discover drugs that inhibit the nsp12/nsp9 reactions over those that interfere with the activity of the two methylases. The viral methylase proteins closely resemble their cellular counterparts. For that reason, it may be difficult to find drugs that inhibit the viral enzymes that are not toxic to cells as well. By contrast, RNAylation of nsp9 is unique to coronaviruses and, therefore, would seem to be an excellent selective target. Park et al. validate this strategy as they find that mutations in NiRAN that inactive RNAyation also prevent replication, as is an asparagine at the amino terminus nsp9. The paper Park et al. is an elegant demonstration of the power of a novel approach to anti-viral drug development—specifically to the development of drugs that target viral proteins that either camouflage the virus on entry and/or block the innate immune response. Figure 7 highlights the plethora of such targets we identified, which are some among many in the SARS-CoV-2 genome. This article originally appeared on Forbes.org, and can be read online here: Stripping Covid's Camouflage

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A New Process Of SARS-CoV-2 Variation Uncovered: Intragenomic Recombination Forbes | March 10, 2022 | Article

Virus variation has been the bane of Covid-19 control. No sooner has one wave of infection passed than a second arrives, evading prior immunity and even vaccines. Monoclonal antibody drugs effective against an early variant lose potency against the next. Variants even change within a single person developing resistance to antiviral drugs. We now know that contending with this shape shifting virus will be the key to Covid-19 control. How does the virus manage to change so much so quickly? At the outset of the pandemic few believed the coronavirus had the capacity to change as rapidly as it did. Most ignored the obvious clue that the same four common cold-causing coronaviruses return year after year to reinfect those who were infected just the year prior. Over the past year we have come to understand that to survive coronaviruses have adapted to reinfect the same host year after year, despite the host robust immune defenses. We recognize variants by their success in spreading though populations and displacing previous dominating strains. The original Wuhan variant was replaced by the B.1 strain that carried three single point mutations, that in turn was replaced by regional variants such as Alpha (dominant in Europe and the US), Beta (dominant in South Africa) and Gamma (dominant in South America). All were replaced by Delta worldwide. In turn, the Omicron variants BA.1 and BA.2 displaced Delta and new sub-variants of Omicron may now be replacing previously dominant Omicron infections. Currently there are two theories on the origin of variants. Both involve selective pressure forcing the virus to adapt to new circumstances Selection to antibody resistance and rapid replication in individual patients There are now several well documented cases of virus evolution within a single individual. Typically, these are people who cannot 195

clear the virus naturally as they are partially immune suppressed. Over time and in response to treatment new variants arise. Some of the mutations found in coronaviruses isolated from these people are identical to those found later in variants of concern. Reverse Zoonosis Zoonosis is infection of humans by an animal virus. Many suspect Covid-19 began when one or more people were directly infected by a bat or intermediary animal species. Reverse zoonosis is human infection from an animal that was infected by the human virus to begin with. At least 29 different species are known to have been infected by humans carrying SARS-CoV-2. Reverse zoonosis is documented to have occurred from SARS-CoV-2 infected mink, hamsters, and deer. Once a virus infects a different species it adapts to that species via mutations in many of the viral genes. Most of those changes persist once the virus re-enters the human population. These changes may alter fundamental properties of the virus including immune evasion, transmission, and disease. We can now enumerate some of the ways the virus changes. 1. Variants may differ from one another by changes in single RNA nucleotide, akin to a change of a single letter in a word (for example, she—>the). These changes can alter the amino acid sequence and therefore the function of specific proteins. Well-known examples include multiple single nucleotide changes in the region of the spike protein gene that binds the ACE2 receptor. Such changes can increase the strength of binding to the receptor thereby increasing infectivity. Changes in the receptor-binding domain can also render the virus invisible to protective antibodies. Single amino acid changes can also alter any viral proteins, affecting virus replication rate, transmission, stability, and immune evasion. 2. Single nucleotide changes need not change protein sequences to make a difference. They may alter what is called cis-acting control sequences. One such example is a single change from a C to a U in the stem-loop 5 region of the 5’ untranslated region, a change that alters the speed of virus replication. 3. Some variant genomes contain small deletions of one or more nucleotides. Deletions often occur in the N-terminal domain of the spike protein where they eliminate as many as 196

six to ten amino acids. Such deletions may eliminate recognition by several neutralizing antibodies at a single go. 4. Coronaviruses can swap parts with other closely related coronaviruses. Bat virus that closely to SARS-CoV-2 in Laos appear to be mosaics comprised by assemblage of as many as fifteen different parent genomes, the result of multiple recombination events. Recombination events occur at hot spots often dictated by the very special way coronaviruses produce their messenger RNAs. Infection of the same person by two different viruses at the same time is required for recombination. A Danish study recently found several people infected by two SARS-CoV-2 strains at the same time. A new SARS-CoV-2 that appears to be a recombinant between Omicron variants BA.1 and BA.3 has been recently reported in South Africa, the UK, Denmark, Ireland, Puerto Rico, and the US. In other viruses such as influenza, recombinants pose a great danger, as some escape almost all prior immunization and are highly pathogenic. Intragenic Recombination A recent preprint by Patarca et al. reports yet another means of SARS-CoV-2 variation. The new phenomenon involves intragenomic rearrangements in which segments of various lengths of the untranslated region at the proximal 5’ end of the virus, in particular the 5’-leader sequence, are duplicated and translocated into the coding portion of the genome (Figure 1).

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Figure 1. Intragenomic rearrangements affect structural, nonstructural, and accessory genes

IMAGE COURTESY OF ROBERTO PATARCA The 5’-leader sequence includes the leader transcription regulatory sequence (TRS-L) and three stem-loop structures designated SL1, SL2, and SL3 (Figure 2). In SARS-CoV-2, there is a lengthy (44 amino acids long) open reading frame within the 5’untranslated region of the virus overlapping SL1, SL 2 and SL3. The open reading frame is conserved in location and mostly in primary sequence amongst SARS-CoV-2 isolates. The open reading frame on SARS-CoV-2 continues past the end of the TRS-L and includes a methionine that marks the beginning of what has been termed the upstream ORF (uORF; depicted in Figure 2 with a grey rectangle), which is conserved in location and mostly primary sequence among SARS-CoV-1 and -2 isolates and related bat coronaviruses.

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Figure 2. The 5-untranslated region of SARS-CoV-2, and the 5’-leader sequence

IMAGE COURTESY OF ROBERTO PATARCA There is one previous report of duplication and translocation of a nucleotide sequence segment from the 5’-UTR region of the SARS-COV-2 genome (underlined in Figure 2) to the distal end of the accessory ORF6 gene (shown in Figure 1) of a SARS-CoV-2 variant with deleted ORFs 7a, 7b and 8 isolated from 3 patients in Hong Kong, and therefore representing infectious isolates. This observation stimulated Patarca et al. to search for other similar occurrences, and they reported that a shorter portion of the 5’-leader sequence (nucleotides 50-75) is duplicated and translocated to the end of the accessory ORF8 gene of a USA variant generating a modified ORF-8 protein. Patarca et al. then undertook a systematic analysis and reported the presence of such rearrangements in various parts of the coding regions of SARS-CoV-2 (as shown in Figure 1) and other betacoronaviruses, such as the Middle Eastern respiratory syndrome (MERS)-coronavirus for which 2 isolates had rearrangements, and the common cold causing human coronaviruses OC43 and HKU1, with one-third and one-fifth of surveyed isolates, respectively, having rearrangements. The analysis included the two human alpha-coronaviruses, 229E and NL63 that also cause the common cold, and other alpha, gamma, 199

and delta coronaviruses, some involved in large outbreaks such as the swine enteric coronaviruses of the alpha and delta genera and avian infectious bronchitis virus of the gamma genus that have been studied over decades with hundreds of isolates characterized without apparent evidence for intragenomic rearrangements, which bodes well for the specificity of the findings. In many cases, these intragenomic rearrangements change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. In limited instances, there is mounting evidence that these insertions alter the fundamental biological properties of mutant viruses. As illustrated in Figure 3, the rearrangement involving the nucleocapsid protein of SARS-CoV-2 generates changes in 5 of 7 amino acids in the rearrangement region including two mutations known as R203K and G204R that are commonly present together. The latter co-mutations have been associated with the emergence of the SARS-CoV-2 B.1.1.7 (alpha) lineage and are present in the P1 (gamma) lineage. The mutations have been shown to confer a replication advantage over the preceding lineages, and to increase nucleocapsid phosphorylation, as well as viral infectivity, replication, virulence, fitness, and pathogenesis in a hamster model, human cells, and an analysis of association between Covid-19 severity and sample frequency of R203K/G204R co-mutations.

Figure 3. SARS-CoV-2 nucleocapsid protein with sequence derived from 5’-end (in blue) IMAGE COURTESY OF ROBERTO PATARCA

Patarca et al. are careful to point out that the described rearrangements might be secondary to artifacts in the sequencing of the virus. However, the isolates in which they occur have been 200

isolated in diverse countries, laboratories and over the time course of the pandemic, from diverse variants of concern and interest. In the case of rearrangements that alter the tail end of the ORF8 protein, isolates with intragenomic rearrangements were collected from March 2020 to December 2021 in 38 USA states, Bahrain, China, Kenya, and Pakistan, which is not exhaustive of what exists, and USA samples were processed in 28 laboratories in California; 20 in Michigan; 18 in Florida; 17 in Minnesota; 15 in Maryland; and 13 in Pennsylvania, among the most representative. Intragenomic rearrangements add to our appreciation of how SARS-CoV-2 variants may arise. In several cases, they affect highly conserved genes with a low propensity for recombination which may underlie the generation of variants homotypic with those of concern or interest and with differing pathogenic profiles. The mechanisms underlying intragenomic rearrangements warrant further study to understand if they represent additional ‘jumping’ of the replication machinery during the discontinuous synthesis of negative-strand messenger RNAs or deficiencies in repair mechanisms. Understanding the variation that they introduce is of relevance in the design of prophylactic and therapeutic interventions for all coronaviruses, including a pan-beta-coronavirus vaccine. This article originally appeared on Forbes.org, and can be read online here: A New Process Of SARS-CoV-2 Variation Uncovered: Intragenomic Recombination

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Covid-19: Long Term Brain Injury Forbes | March 14, 2022 | Article

This story is part of a larger mosaic of stories on Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part one of this series on loss of smell after Covid-19. SARS-CoV-2 infection can damage many organs other than the lungs. The most troubling is damage to the brain. A series of recent studies document long term brain-damage in as many as one quarter of all those infected regardless of the severity of the initial disease. Those numbers are daunting considering that an estimated 140 million Americans have been infected by SARS-CoV-2. Symptoms, such as brain fog, fatigue, depression and a host of other maladies, may be mild or incapacitating. Several studies warn that treatment of those with long term brain injury will strain the healthcare care system for years to come. Understanding the origin and treatment of Covid-19 related brain injury is a high priority for medical science. A recent study by Frontera et al. of the NYU Grossman School of Medicine evaluated the cognitive function of Covid-19 patients six months after they were hospitalized for Covid-19. To their surprise, over 90% of their total cohort reported at least one neurological symptom. Among those that had not experienced neurological complications while hospitalized, 88% reported new cognitive symptoms. These cognitive impairments seem to be separate and apart from damage due to hypoxia, or the lack of oxygen to the brain, often experienced by those hospitalized for severe Covid-19. Reportedly, some individuals that recover from mild or asymptomatic infection may later develop complications that are not immediately apparent. The table below lists commonly reported neurological symptoms by previously hospitalized Covid-19 patients.

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Table: Summary of incidence of new neurological complications following hospitalization with Covid-19 and commonly reported neurological symptoms from featured articles. ACCESS HEALTH INTERNATIONAL

Through in-person screenings, a team of neurologists diagnosed more than half of those who participated in the NYU study with encephalopathy. Encephalopathy broadly refers to damage or disease that alters the brain’s structure or function. Those diagnosed with encephalopathy had a higher than expected incidence of strokes and seizures. Another 21% had symptoms related to oxygen starvation related to Covid-19 damage to the lung and in some cases to the heart. 203

Frontera et al. found significant correlations between the prevalence of neurological complications and an inability to return to daily activity, even six months after the initial infection. Those that developed neurological complications while hospitalized for Covid-19, in particular, were twice as likely to perform worse on cognitive assessments, compared to individuals not diagnosed during hospitalization. For instance, over 50% reported being unable to return to daily activities and 59% of those that were previously employed are now not able to return to work. Frontera et al. observed that, among those that completed their mental health outcome tests, 62% of individuals previously hospitalized for Covd-19 scored worse for anxiety, sleep, fatigue and depression, in contrast to population averages. When they compared their Covid-19 groups with and without neurological diagnosis, no significant differences were found. This suggests that poor mental health outcomes may be linked to the experience of being severely sick with the virus, but mental health issues alone does not explain why some are unable to return to their daily activities. How long do Covid-19-related neurological complications last? A new study by Rass et al. from Austria attempted to answer this question by interviewing previously hospitalized Covid-19 patients three months after infection and then again one year later. While a few participants experienced some cognitive improvement, 73% showed no difference between three-months and a year. In fact, at one year, almost 60% of respondents continued to report neurological symptoms, including fatigue, concentration difficulties, sleep disturbances, headaches, impaired sensation and loss of smell. To their surprise, the prevalence of these symptoms did not correlate with disease severity. Another NYU Langone study, Valdes et al. found the patients who were Black, unemployed and/or had fewer years of education were more likely to perform worse on cognitive assessments six months after being hospitalized for Covid-19, compared to other demographic groups. The authors speculate that the observed differences are a consequence of social and economic disparities. These observations raise troubling long-term issues for the medical system and for society. Must we now add the millions of people disabled by Covid-19 and in need of chronic care, many of whom are young and in the prime of life, to the rapidly growing 204

ranks of the elderly in need of similar social and medical services? How do we account for the loss of revenue from both the employers and employees perspective? The time is now to begin to understand and plan for this new potential social and medical crisis. • We need to understand how SARS-CoV-2 damages the brain and how such damage may be avoided. • We need to know how many people suffer from Covid-19related brain damage and how long such symptoms last. • And, we need to plan for the social and medical care of those most seriously affected, those who cannot care for themselves, as well as those who cannot return to work. Our social support systems must recognize Covid-19 related long-term disability as a reality and assure those who suffer are protected. It is now clear that our encounter with Covid-19 will not fade with the pandemic but will endure for decades. This article is featured on Forbes.com, and can be read online here: Covid-19: Long Term Brain Injury

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An Ancient Form Of Immunity Protects Many Against Covid-19 Today Forbes | March 15, 2022 | Article

Figure 1: Mannose-binding lectin (blue strands) can bind to sites on the SARS-Cov-2 spike protein on ... [+] STRAVALACI ET AL., NATURE IMMUNOLOGY (2022), DOI: HTTPS://DOI.ORG/10.1038/S41590-021-01114-W

One of the most striking features of the SARS-CoV-2 pandemic is that across all variants, most people who are infected have few if any, serious symptoms. The majority experience mild cold-like symptoms, while the most serious symptoms are confined to only about 10% of those infected. These numbers are even better for those who are vaccinated. What accounts for the fact that unvaccinated people seem to be mostly resistant to a virus that has the potential to kill? The answer lies in our innate immune response. This series of articles is an extension of a previous series which is now available as an anthology in the book, Natural Immunity and Covid-19: What it is and How it Could Save Your Life. Here, we

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extend that series based on new research about yet another aspect of the natural (innate) immune system. The fundamental feature of the innate immune system is that it protects our body from microbes, viruses, bacteria, and parasites that we have not previously encountered. Much of the attention and public awareness of human immunity is focused on adaptive or learned immunity. Learned immunity is developed in our bodies after exposure to prior infection or vaccinations. Only in recent years has there been tremendous progress on what may be our most important defense system of all: innate immunity. This story focuses on an unheralded aspect of innate immunity and describes a category of proteins called lectins. Lectins are believed to have evolved prior to antibodies and continue to persist in our immune system today. It has now been discovered that these lectins may provide two aspects of protection by preventing infection and by limiting the ability of SARS-CoV-2 to replicate and spread. Introducing the Lectin Family Lectins occur ubiquitously across nature and are considered pattern-recognition molecules. This is because lectins have a special ability to recognize foreign molecules in the body and can activate a response to those molecules. The lectin system has many components including mannose-binding lectin, collectins, and three different ficolins, all of whom are pattern recognition molecules that can recognize different molecules and activate different responses. Now, at least two proteins from the lectin family have been implicated in our body’s defense against SARS-CoV-2: mannosebinding lectin and its related enzyme, MASP-2. How Mannose-binding Lectin Recognizes SARS-CoV-2 Infection How does mannose-binding lectin recognize foreign molecules in the body? Most invading microorganisms, cells, and viruses contain a surface coated with complex glycan structures or “sugar” molecules. These structures help the microorganism move, aid in aggregation, and can serve as a shield to conceal the microbe from a host’s antibodies. SARS-CoV-2 is no exception to this. One of the salient features of the SARS-CoV-2 virus is its spike (S) protein. Much like other microbial proteins, the spike protein is covered with its own unique glycan residues (Figure 2). 207

Figure 2: A) SARS-CoV-2 and its spike protein. Each spike protein is covered with a unique pattern ... [+] A) CASALINO ET AL., ACS CENTRAL SCI (2020), DOI: 10.1021/ACSCENTSCI.0C01056. B) STRAVALACI ET AL., NATURE IMMUNOLOGY (2022), DOI: HTTPS://DOI.ORG/10.1038/S41590-021-01114-W

Mannose-binding lectin contains multiple receptors that are used to identify the glycan residues of invading microorganisms. It also contains a stem that activates a defense mechanism called the lectin complement system (Figure 3). Mannose-binding lectin and the lectin complement pathway evolved well before antibodies and are often considered to be proto-antibodies. They are known to play a crucial role in defending our body against viruses and bacteria like HIV, salmonella, and streptococci.

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Figure 3: Mannose-binding lectin (MBL, left) contains a bouquet-like structure with a stem used to ... [+] HAYAT, AZAM. (2012). THE MOLECULAR INTERACTIONS BETWEEN TWO ACTIVATION PATHWAYS OF COMPLEMENT ARE ESSENTIAL FOR A PROTECTIVE INNATE IMMUNE RESPONSE TO NEISSERIA MENINGITIDIS INFECTION.

The Lectin Complement Cascade and the Role of MASP-2 When mannose-binding lectin recognizes an invading pathogen, it initiates the lectin complement cascade by activating MASP-2 and its counterpart, MASP-1. When MASP-2 and MASP-1 are activated, they cleave two other proteins: C2 and C4. The C2 and C4 protein fragments then bind together to form C3 and C5. C3 and C5 can directly kill pathogens by binding together to form holes in the pathogen membrane (lysis). The cleavage of C2 and C4 also releases inflammatory cytokines which alert other immune cells to aid in the attack of the pathogen (Figure 4).

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Figure 4: The lectin complement cascade can lead to several forms of attack against invading ... [+] ANDRADE ET AL., SPRINGER NATURE (2017), DOI: HTTPS://DOI.ORG/10.1007/978-981-10-2513-6_18

MASP-2 is a crucial enzyme within the lectin pathway. Only MASP-2 can cleave both C4 and C2. When MASP-2 is inhibited in the body, the lectin pathway can no longer be activated and cannot defend the body against invading pathogens. Previous studies have found that activation of the lectin complement system was associated with the development of respiratory distress and failure during viral pneumonia—a prominent symptom of severe SARS-CoV-2 infections. In addition, autopsies of patients who were infected with SARS-CoV-2 have shown evidence of lectin complement activation through the presence of C5, C3, C4, and MASP-2 proteins. The question is, are mannose-binding lectin and MASP-2 the proteins responsible for defending our bodies against SARS-CoV-2 through the lectin complement system? Mannose-binding Lectin Prevents SARS-CoV-2 Infection In the first paper, Stravalaci et al. began by determining if any lectins could bind to SARS-CoV-2. By testing the interactions between specific proteins found in SARS-CoV-2 and each lectin, researchers found that of the lectins tested, only mannose-binding lectin consistently bound to the SARS-CoV-2 spike protein.

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While this confirmed that the mannose-binding lectin would interact with the spike protein, researchers needed to be sure that mannose-binding lectin would still bind to the spike protein when it was expressed on the SARS-CoV-2 cell membrane. With this in mind, Stravalaci et al. induced the SARS-CoV-2 spike protein onto other cell membranes and found that the mannose-binding lectin continued to bind to the spike protein in its natural conformation (Figure 1). When mannose-binding lectin interacted with the spike protein, it induced the lectin complement pathway. Researchers incubated the spike protein in a serum that contained mannose-binding lectin as well as other proteins involved in the lectin complement pathway. After running the experiment, the data was clear: the lectin complement was fully activated. Mannose-binding lectin could effectively defend against SARS-CoV-2 infections. Stravalaci et al. then tested mannose-binding lectin against four variants: Gamma, Alpha, Beta, and Delta. Using the same procedures, they found that the mannose-binding lectin was as effective against SARS-CoV-2 variants as it was against the original strain. Mannose-binding Lectin and MASP-2 Prevent SARS-CoV-2 Spread Stravalaci et al. found that mannose-binding lectin could bind to SARS-CoV-2 through its spike protein. However, in a second related story published in Frontiers Immunology, researchers found that lectins may actually have two modes of attack against SARSCoV-2. The second study focused on the interactions of mannosebinding lectin and MASP-2 protein with SARS-CoV-2. To test these interactions, Youssif et al. first placed mannose-binding lectin in a serum of SARS-CoV-2 proteins and incubated the cells for an hour. Using this experimental method, they found that in contrast to the first paper, mannose-binding lectin displayed strong interactions with both the SARS-CoV-2 spike protein and its nucleocapsid protein. The researchers then tested whether mannose-binding lectin could still interact with the spike protein when it was expressed on the cell membrane. After inducing the spike protein on other cell membranes and testing their interactions with the mannose-binding 211

lectin, the results confirmed Stravalaci’s results. The mannosebinding lectin continued to bind to the spike protein when it was expressed on the cell membrane. In addition, they found that this experiment led to the deposition of C3 on the cell membrane, indicating that the lectin complement had been activated. Youssif et al. then explored whether MASP-2 could interact with the SARS-CoV-2 spike and nucleocapsid proteins. After incubating MASP-2 with both proteins, surprisingly, researchers found that MASP-2 bound directly to the nucleocapsid protein. When the MASP-2 was then incubated with C4, the resulting C4 fragments demonstrated that MASP-2 also retained its ability to cleave the C4 protein. This indicated that not only could MASP-2 be activated by the SARS-CoV-2 nucleocapsid protein, but MASP2 continued to promote subsequent steps of the complement cascade after binding with SARS-CoV-2. The nucleocapsid protein is the most abundant and immunogenic protein in SARS-CoV-2. It is a critical component of the virus’s ability to replicate and spread. Recent studies have shown that the nucleocapsid protein also plays a large role in suppressing our innate defenses against SARS-CoV-2. By directly binding to the nucleocapsid protein, this study indicates that mannose-binding lectin and MASP-2 could serve as effective counter defenses against one of SARS-CoV-2’s most dangerous attacks. This article is featured on Forbes.org, and can be read online here: An Ancient Form Of Immunity Protects Many Against Covid-19 Today

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An Omicron-Omicron Recombinant—BA.4 Forbes | March 16, 2022 | Article

Successive waves of Covid-19 have been driven by new variants. In Summer 2020, the B.1 variant swept the globe, receding in the Fall. The Winter of 2020 brought about the Alpha variant, which too fell to the wayside. Then the Delta variant fueled another wave, first in India and then around the globe, again receding in the Fall. Most recently, the BA.1 strain of Omicron fueled the largest spike to date, peaking in late January. A new wave, driven by yet another variant, BA.2, that shares a common undetected parent with BA.1 is triggering yet another wave of infections in Europe (Figure 1) and has established a firm beachhead in the United States.

FIGURE 1: 7-day rolling average of new confirmed Covid-19 cases per million people in various ... [+] OUR WORLD IN DATA

All the variants of concern to date appear to arise by mutations from an earlier parent. Mutations include single nucleic acid substitutions as well as small deletions and insertions. That by no means exhausts the repertoire of coronavirus genomic variation. Betacoronaviruses such as the SARS-CoV-2 genome can undergo internal sequences rearrangements and recombination, the exchange of one part of the genome for that of a closely-related virus. Two 213

such recombinants have been described recently, an OmicronOmicron recombination and a Delta-Omicron recombinant. Here we describe the Omicron-Omicron recombinant, given the preliminary designation Omicron BA.4. As of yet, appearances of BA.4 around the world are very few. There have been four confirmed sequences of the strain in South Africa, one in the US, and one in Puerto Rico through March 16th. This is the number of sequences in the GISAID database. The real number of infections by this recombinant could be much higher, especially as Omicron BA.1 and BA.2 continue to rage. The simplest interpretation of the origin of BA.4 is a recombination event between BA.1 and BA.3. It is likely that BA.3 contributes the portion of the genome extending from the 5’ end halfway through the NSP3 gene of the replication complex (about 2,000 amino acids). The remainder is plausibly contributed by BA.1. The anomaly is a single mutation in NSP6 found in BA.1, but not BA.3 or BA.4. This could also be coincidental, as many SARS-CoV2 viruses are mutated in NSP6 from 105-108, though we cannot be certain. We note two mutations are unique to BA.4 These include the mutations in NSP15 (N11S) and in the N-terminal domain of the Spike protein (L212I). We propose these two mutations arose in BA.4 post-recombination.

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FIGURE 2: Venn Diagram showing the similarities and differences between BA.4 and its sibling ... [+] ACCESS HEALTH INTERNATIONAL

FIGURE 3: Recombination schematic of BA.4. The sections in red are derived from BA.3. The red ... [+] ACCESS HEALTH INTERNATIONAL

Recombination events amongst closely-related coronaviruses are expected. For example, scientists from the Pasteur Institute suggest that a bat virus isolated in Laos closely resembling SARS-CoV-2 is the product of recombination amongst at least fifteen other bat 215

coronaviruses. Recombination requires that a single host be infected simultaneously by two different viruses. Co-infection of Omicron BA.1 and BA.2 has been reported by researchers in Denmark. While rare, the researchers identified 47 instances of rapid reinfection of BA.2 shortly after BA.1, indicating that a recombination event could occur in such hosts. The recent reports are likely to be only the beginning of the next chapter of SARS-CoV-2 in which a swath of variant recombinants emerge. At present, it is too early to predict whether the recombinants will have novel biological properties including increased transmissibility, immune evasion, and pathogenesis. This article is featured on Forbes.org, and can be read online here: An Omicron-Omicron Recombinant—BA.4

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A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells Forbes | March 21, 2022 | Article

This story is part of a larger mosaic of stories on Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part one and two of this series. Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain. Comparing brain volume before and after individuals were exposed to SARS-CoV-2, this study documents significant cortical gray matter loss, equivalent to nearly 10 years of aging. Gweanaelle Douaud, the study’s first author and Professor at the University of Oxford, says that infected individuals display structural “differences over time above and beyond any potential baseline differences.” Most strikingly, individuals that experienced no or only mild symptoms with Covid-19 displayed specifically significant changes, but cortical damage seems to occur regardless of disease severity, age, sex, or vaccination status. It may be years before the long-term consequences of these structural differences are fully understood. Douand et al had unique access to an extensive biomedical database, called the UK BioBank. The UK BioBank provided prepandemic brain scans from 785 individuals that were used as a baseline for normal size and structure relative to each participant. About three years later, in May 2021, the same population of participants returned for new brain scans. From the total cohort, 401 individuals were infected with Covid-19 between scans and the remaining served recruited as controls. Between the first and second brain scans, individuals previously infected with Covid-19 experienced a 0.7% reduction in overall cortical gray matter on average, compared to the control group. To put that into perspective, people middle aged and beyond only lose 0.2% to 0.3% of volume per year. Although it is clear that SARS-CoV-2 can damage the brain, how this damage occurs without directly infecting nerve cells 217

remains a mystery. Current theories suggest there may be multiple factors contributing to these deficits. Structural abnormalities in the brain may in fact be secondary to infection occurring in other sites around the body, including the olfactory epithelium. Considering how close the nose is to the brain, this Oxford University Study considered whether losing the sense of smell may be linked to other neurological damage. Loss of smell is a consistent clinical feature of Covid-19, with recent studies suggesting that 86% of individuals exposed to the virus may experience partial or complete loss of smell. A smaller percentage of people also develop additional neurological complications, including brain fog, fatigue, impaired cognitive function, and encephalography, characterized by impairments in brain structure and function. For some, these symptoms may continue to linger anywhere from a few months to more than a year after the initial infection. These effects, however, do not discriminate between mild and severe disease. First, Douand et al. found reduced gray matter in several regions associated with olfaction, consistent with symptoms of prolonged loss of smell. Individuals exposed to Covid-19 experienced the most significant neural damage to the primary olfactory cortex, or piriform cortex. This structure receives input directly from olfactory receptors that pass through the olfactory bulb, making it the first neural target for processing and perceiving odors. Reduced cortical thickness was also observed in a connected region called the orbitofrontal cortex. Illustrated in Figure 1, the orbitofrontal cortex (OFC) receives inputs from the primary olfactory cortex and is commonly referred to as the secondary olfactory cortex.

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Figure 1: Schematic view of the human olfactory system. The primary and secondary olfactory cortices are represented in blue and green, respectively. Amyg, amygdala; Ento, entorhinal cortex; Hipp, hippocampus; OFC, orbitofrontal cortex; PC, piriform FROM: “ A REVIEW ON THE NEURAL BASES OF EPISODIC ODOR MEMORY: FROM LABORATORY-BASED TO AUTOBIOGRAPHICAL APPROACHES” SAIVE ET AL. 2014

Damage to these areas may be linked to loss of smell, but there is no evidence of causation. Instead, researchers speculate that the loss of volume to brain regions associated with olfaction may be partially attributed to widespread damage to the olfactory epithelium that disrupts neural pathways and impairs function. As with many other neural systems, pathways that are not being used over a long period of time cease to exist, a process neuroscientists often call “use it or lose it” that causes brain tissues to shrink. Surprisingly, Douand et al. also found additional abnormalities in regions not normally associated with the sense of smell. In particular, they observed reduced gray matter volume in some regions of the limbic system, involving several structures important for producing behavioral and emotional responses. The largest differences, ranging from 0.2% to 2% reductions, were seen in the left parahippocampal gyrus and the entorhinal cortex. These regions play an important role in the hippocampal memory system, so gray matter loss could signify future memory impairments. Structures are shown below in Figure 2 for reference.

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Figure 2: Illustration of the limbic system. The parahippocampal gyrus and hippocampus, which contains the entorhinal cortex, are highlighted. BRUCEBLAUS, WIKIPEDIA COMMONS

Are the differences seen in these limbic structures also linked to deficits in the olfactory system during Covid-19? Douand et al. argues that the parahippocampal gyrus, the orbitofrontal cortex and other parts of the limbic system are in some way connected to the olfactory cortex. Since sensory inputs, including those for olfaction, are transmitted and integrated all over the brain to guide a range of behavioral responses, damage to the olfactory epithelium may also have disastrous consequences on regions of the brain not exclusively involved in olfaction. More likely, however, these and other changes in brain structure may be a consequence of a robust immune response occurring all around the brain, albeit the mechanisms underlying inflammation-induced brain damage remain unclear. Additional exploratory analyses found gray matter loss in the amygdala, the insula which borders the temporal cortex, and the front-most portion of the cingulate gyrus, known as the anterior cingulate gyrus. Interestingly, all these regions play a role in emotion processing and regulation. More research is needed to determine whether deficits in these brain regions may be linked to mood disorders associated with long-haul Covid-19, including depression and anxiety.

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Figure 3:Reference locations for amygdala, anterior cingulate cortex, and insula. APOLLO MEDICINE 2020

It is important to note that not every individual infected with Covid-19 will experience a reduction in brain volume, while others will experience much greater losses. Those hospitalized with Covid19, for example, had more widespread tissue damage and atrophy, compared to those not hospitalized for infection. Beyond hospitalized vs. non-hospitalized, there was limited data from this study showing how severity of infection may contribute to these effects. Finally, Douand et al. asked whether these structural changes in the cerebral cortex are linked to new neurological symptoms following Covid-19 infection. Interestingly, they did not find any significant correlations. No correlation between structural changes and the prevalence of new neurological symptoms, however, does not mean that these changes will not impact brain function. For a vast majority of people, the regenerative properties of the olfactory bulb restores the sense of smell within a few weeks or months. What about the rest of the brain? Damage to brain cells cannot be reversed. When tissues die, cerebrospinal fluid and other biomolecules fill the excess space to maintain the integrity of the brain. Perhaps, this explains why neurological complications associated with long-haul Covid-19 show little improvement over time. Years of additional research are needed before the consequences of losing so much gray matter are fully understood. 221

Identifying these changes now will help us to better support and treat what will be a growing class of people with cognitive impairments. Although the loss of smell is often one of the first symptoms of Covid-19 preceding any respiratory complications, the hypothesis that the SARS-CoV-2 damages the brain when it infects cells in the olfactory epithelium remains heavily-debated. Researchers do seem confident that the virus does not directly infect brain cells. If the nose is a window to the brain, it may be time to develop new vaccines that aim to close it off from the SARS-CoV-2 virus. This article is featured on Forbes.org, and can be read online here:A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells

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En Garde For SARS-CoV-2 Chimeras (Recombinants) Forbes | March 22, 2022 | Article

Legend: Ancient Greek representation of the Chimera: a recombinant animal.

SCIENCEABC.COM Variants are driving the Covid-19 pandemic. New variants, mutated to improve transmissibility, immune evasion, and pathogenicity, have emerged to fuel wave after wave of new infections. These new strains, to date, have almost exclusively mutated via point mutations, small deletions, or insertions. There is a new class of SARS-CoV-2 that the scientific community has long contended may soon become a problem. These are SARS-CoV-2 recombinants. Recombinants are the result of exchanging one part of a virus for another. Recombination is possible with SARS-CoV-2 if a host cell is infected with two different variants at the same time. For SARS-CoV-2, this may occur when one variant displaces another over time, such as the current displacement of BA.1 by BA.2 or the recent overtaking of Delta by Omicron.

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The New York Times reports 463 million confirmed global Covid cases since the pandemic's start. This number is likely much higher, perhaps in the billions, with asymptomatic and unreported cases. Millions of cases extrapolated into dozens of significant strains throughout the past two years yield the ideal breeding ground for new recombinant strains. Our previous story on recombinant strains discussed the emergence of an Omicron-Omicron recombinant strain identified in South Africa. This strain combined portions of BA.1 and BA.3, sibling variants in the Omicron family, to create a new variant provisionally named BA.4.

FIGURE 1: BA.2 (purple) displacing BA.1 (pink) in Los Angeles over time.

DEADLINE.COM Here we discuss recent reports of recombinants between Delta and Omicron, almost certainly occurring when both viruses were in circulation in late 2021. The wave of late Spring and Summer 2021 was driven by the Delta variant, which did not cause the same rapid acceleration of cases as Omicron, but was moderately more severe in terms of symptoms. The figure below demonstrates some of the confirmed instances of the Delta-Omicron recombinant, dubbed Deltacron. The figure notes six confirmed sequences of Deltacron, all slightly different. We note that many more Deltacron cases may exist at the time of writing and certainly in the coming weeks. For example, the study by Colson et al. that identified the French Deltacron has since identified the same sequence in as many as 43 cases; 33 samples in France, eight in Denmark, one in Germany, and one in The Netherlands. 224

FIGURE 2: Confirmed recombinants of Delta and BA.1. SARS-CoV-2 proteins are detailed with mutations ... [+]

ACCESS HEALTH INTERNATIONAL Coronaviruses are particularly prone to recombination as template switching, that is polymerase jumping from one strand to another, is required for messenger RNA synthesis. Although most of these jumps occur at sequences called transcription regulatory sequences (TRS), other sites have also been documented as template switching sequences, albeit at a lower frequency. The SARS-CoV2 replication transcription machinery may also switch templates at transcription pause sites when they encounter stable secondary structures. Roberto Patarca (personal communication) identified one such stable secondary structure at the recombination junction of recombinant “GM” pictured in Figure 3 projected to be extremely stable and, therefore, a site for polymerization pausing. It is predicted to have -89kcal stability, where a greater negative value is associated with higher stability.

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FIGURE 3: UK Delta x BA.1 recombinant GM. This strain is Delta up to nucleotide position 17,040 ... [+]

ACCESS HEALTH INTERNATIONAL Although these recombinants have been identified, their significance remains to be determined. To date, none of these seem to show a propensity for displacing current variants. The concern comes chiefly from the fact that recombination of existing SARSCoV-2 viruses actually occurs and they are not so rare as to remain undetected. New recombinants that arise may be able to enhance the three critical virological variables: transmissibility, immune evasion, and virulence. Recombinants may also appear not only by coinfection in humans, but infection in the large reservoir of animal populations SARS-CoV-2 is capable of infecting, namely housepets, mice, deer, and others. We also note detection of highly mutated reverse zoonosis variants in New York City sewers containing nearly sixty amino acid changes, posing another risk of recombination. There is a chance that SARS-CoV-2 could coinfect an animal with another coronavirus, resulting in a recombinant that makes its way back to 226

humans. This is of particular concern as some animal viruses have an extensive set of mutations in the Spike protein and larger genome, for example, deer viruses in Ontario were found with 76 nucleotide mutations throughout the virus. These are the early days in our detection and understanding of recombinants in some was similar to the discovery of the first variant from Wuhan, B.1, in which three mutations granted a strong infectivity advantage. It now seems possible that these examples render the possibility concrete that new variants can arise via recombination, which will make a significant difference to transmission, immune evasion, and disease. Watch with space. This article is featured on Forbes.org, and can be read online here: En Garde For SARS-CoV-2 Chimeras (Recombinants)

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Antibodies Team Up Against Omicron Forbes | March 23, 2022 | Article

Omicron was an unpleasant surprise in many respects. First, the sheer number of mutations in the Spike and throughout the genome yield other significant effects, such as resistance to many vaccines and modified cellular entry. Second, Omicron is now known to be not one, but a family of variants—BA.1, BA.2, BA.3, and the recombinant BA.4. Third, the Omicron family is more infectious than any variant preceding it. BA.1 is more infectious than Delta and BA.2 is more infectious than BA.1.

FIGURE 1: Increasing infectivity of SARS-CoV-2 variants ... [+] ACCESS HEALTH INTERNATIONAL

Here we address another unpleasant surprise of Omicron. The family of variants, in addition to being highly resistant to many vaccines, is also resistant to currently available monoclonal antibodies, which are often the first line of defense for treating people infected, but not yet seriously ill, in hospital settings. Antibodies developed to treat infections resulting from the original Wuhan virus or initial B.1 variant have reduced effectiveness against Omicron.

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FIGURE 2: Omicron family resistance to AstraZeneca monoclonal antibodies and cocktail, ADG-2, ... [+] FENWICK ET AL.

Evushield and bebtelovimab are two examples of antibodies with reduced Omicron neutralization. Both antibody treatments target binding sites that are altered in the Omicron receptor-binding domain. Among Evushield’s targeted residues, S477, T478, E484, and Q493 are all altered in BA.1, BA.1.1, and BA.2. Bebtelovimab’s binding map is also altered at positions N440, G446, Q498, and N501

FIGURE 3: Evushield antibody binding map DONG ET AL.

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FIGURE 4: Bebtelovimab (red) binding the ACE2 receptor-binding domain, avoiding major mutated sites ... [+] WESTENDORF ET AL.

Thankfully, there are a few antibodies that may still have significant neutralization of the Omicron family. These may become the next great tools in the fight against severe Covid-19. Here we describe a study by Fenwick et al. that describes these antibody candidates and how they are capable of neutralizing BA.1, BA.2, and all their subvariants. Screening for the presence of anti-Spike antibodies in over 100 samples of sera donors, Fenwick et al. identified two monoclonal antibody candidates that showed promising results against previous variants of concern, including Alpha, Beta, Gamma, and Delta. They compared these two antibodies against currently available monoclonal treatments, such as the AstraZeneca combination cocktail, Regeneron cocktail, and sotrovimab. The two antibodies, P2G3 and P5C3, strongly neutralized not only the Wuhan strain of SARS-CoV-2, but also Alpha, Beta, Gamma, Delta, and, most notably, BA.1, BA.1.1, and BA.2 remarkably effectively. P2G3 was between 5 and 907-fold more potent at neutralizing the Omicron family Spike proteins as compared to other monoclonal therapies. P5C3 was slightly less potent than P2G3, but still more neutralizing than the other tested therapies. Notably, Fenwick et al. identified upon cryo-electron microscopy that P5C3 bound the virus noncompetitively with P2G3, meaning they could be combined in a single treatment. This 230

combination performed roughly the same as P2G3 on its own, though we note that a combination would be more difficult for the virus to mutate to overcome.

FIGURE 5: Map of P2G3 and P5C3 binding to the Omicron Spike, as well as local maps of their binding ... [+] FENWICK ET AL.

The two are non-competitive because they attach to the Spike receptor-binding domain at different angles. The authors believe that P5C3 takes a common approach as previous monoclonal antibodies, latching to the up-configuration of the receptor-binding domain. P2G3 attacks at an unusual angle, targeting the side of the protein rather than the top, meaning the antibody can bind in the up or down configurations. Together, the antibodies effectively lock the Spike protein in place, limiting its transmission.

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FIGURE 6: P2G3 angle of attack against the SARS-CoV-2 Spike receptor-binding domain. FENWICK ET AL.

Throughout the pandemic, variants have mutated to evade our latest defenses against severe illness. Just as Omicron mutated a number of Spike and genome-wide mutations leading to reduced effectiveness of vaccines and monoclonal treatments, future variants may mutate to overcome P2G3 and P5C3. When administered together, we are essentially delaying that process by giving the virus more targets to mutate against. Recent studies of the sotrovimab antibody indicate that resistance to the treatment, when used as a monotherapy, rises rapidly in treated patients. Particularly in those that are immunosuppressed, Rockett et al. note that over time, infected hosts with persisting infections may develop mutant viruses that reduce sotrovimab neutralization by as much as 300-fold. We suggest avoiding the monotherapy approach of antibodies altogether. There are a number of monoclonal antibodies that attach to relatively conserved sites across SARS-CoV-2 variants. Among these are Vir/GSK’s S2K146 antibody, which uses a wide footprint to overcome heavily mutated sites like N501 and E484. Another is the CV3-1 monoclonal antibody that binds the 485-GFN-487 loop in the receptor-binding domain, which is a highly conserved site in Omicron and other variants. A third is CV3-25, which avoids the receptor-binding domain altogether and inhibits the Spike protein via the S2 region. This one 232

is of particular interest as it binds a different region altogether than the receptor-binding domain.

FIGURE 7: CV3-25 (orange) binding the S2 region of the SARS-CoV-2 Spike protein (blue). WENWEI ET AL.

Now that there are many antibodies available, and more coming soon, we should be combining at least three or more of the described antibodies in one treatment, making the task of overcoming neutralization that much more difficult for the virus. One of the included antibodies should definitely be CV3-25, as it differs from most circulating antibody candidates to date. Ultimately, SARSCoV-2 will continue to mutate and overcome our tools for the months and perhaps years to come. During such time, we must continue to innovate and develop strategies to fight back against severe illness, and combination monoclonal therapies are one such avenue. This article is featured on Forbes.org, and can be read online here: Antibodies Team Up Against Omicron

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Innate And Adaptive Immunity In The Human Intestine Forbes | March 24, 2022 | Article

This is part of a series of stories on innate immune protection in the gut, focusing on SARS-CoV-2 infection. For the most part, discussions surrounding Covid-19 have focused on its impact on the respiratory system, with special emphasis placed on the lungs. This is understandable considering the primary symptoms of infection are respiratory. Now that we’re more than two years into the pandemic, we’re quickly realizing that Covid-19 also affects a variety of other major organs across the body, including the heart, the blood vessels, the brain, the pancreas, and even the male reproductive tract. One area that hasn’t received all that much attention, on the other hand, is the gastrointestinal tract. That is, our gut. We know that Covid-19 can cause gastrointestinal issues, diarrhea being a fairly common symptom of infection. We also know that surveillance and frequent testing of wastewater make for valuable epidemiological strategies that help us track the spread of the virus, including new variants. Still, there have been fewer complaints about Covid-19 gastrointestinal symptoms than there have been for other areas. This poses somewhat of a mystery. Our intestines expose more than 350 square feet of mucosal membrane, the equivalent of around half a badminton court. This mucosal membrane is covered in cells rich in angiotensin-converting enzyme-2 (ACE2) receptors— the main receptor by which SARS-CoV-2 enters into our cells. So how come, despite such a large number of functional ACE2 receptors, Covid-19 isn’t more of an intestinal disease? There are three general explanations. First, the mucosal lining of the intestine is covered with a protective coat of mucous, limiting direct access to the cells’ surface. Second, the intestines are a major source of adaptive immunity, housing approximately one third of all of our immune tissues. Finally, as we will discuss in this series, the

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intestines contain special immune defenses against viruses and other invading pathogens. Only recently have researchers come to realize that one of the major defenses against infection, especially in the gut, but also elsewhere, is mediated by a family of innate immune cells known as innate lymphoid cells (ILCs). Here we focus on one particular subset of this family, group 3 innate lymphoid cells (ILC3s), which have some remarkable and unexpected properties. Innate Immunity, Adaptive Immunity, and … Trained Immunity? All living systems are under constant assault from microorganisms— be they viruses, bacteria, fungi, or parasites. To survive, and thrive, every living being must have an effective way to counter these threats. This is where the immune system comes into play. Humans have two main immune strategies: innate and adaptive. Our innate immune system has developed over thousands of years and is present in all vertebrates, from reptiles to fish to mammals. It is something we are born with and it can protect us from the very get-go. The innate immune response is broad and, as it's sometimes also called, nonspecific. But in this breadth lies its strength; it doesn’t need to learn, through first-hand experience, what’s dangerous and what isn’t, it already has a general sense from the outset. At its core, the innate immune system does a really good job of separating self from other. This is because most of our innate immune cells —macrophages, neutrophils, dendritic cells, and so on— are equipped with a kind of sensor, called pattern recognition receptors (PPR), that help detect molecules often associated with pathogens. These molecules are called pathogen-associated molecular patterns (PAMPs). These small molecular “motifs” are recognized as a threat by our innate immune system because they are highly conserved across many pathogenic microbes. Think of PAMPs as representing those features that, at the most basic level, are shared across most pathogens of a given class— bacterial, fungal, or viral. Even if there is no one essential feature that tethers them all together, there may be a series of overlapping similarities, a “family resemblance” of sorts. Because of how little these foundational features change over time, our innate immune

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system has had the time to build up a rolodex which it can refer to when faced with a pathogenic threat. Where the innate immune system is defined by its breadth, the adaptive immune system is defined by its specificity, by its level of specialization. For example, our innate immune system will recognize only that there is some bacterial threat and deploy its usual antibiotic strategies, but our adaptive immune system will recognize exactly what kind of bacteria is posing the threat and will have the exact tools required to quickly dispose of it. Our adaptive immune system, unlike the innate immune system, starts out with only a very limited rolodex, having to build it up over time. The main way of doing so is by being exposed to pathogens first-hand. It’s for this reason that we’re most at risk from serious disease when we are really young, our adaptive immune system not yet having had the time to build up any experience, and then again as we become older, our adaptive immune system having weakened with age. Our adaptive immune response is carried out mainly by lymphocytes, a kind of white blood cell. They are split up into two main branches: those that produce antibodies, called B cells, and those in charge of getting rid of virus-infected cells, called T cells. The biggest difference between the innate and adaptive immune responses is that the innate immune system sees and reacts almost instantly, kicking in within minutes of exposure to pathogens and triggering a very broad defense. The adaptive immune system can also recognize invading pathogens quickly, but T and B cells can take anywhere from 10 to 15 days to expand and mature fully. This process of maturation builds up their specificity and brings them to full effectiveness— adding that particular pathogen to its rolodex of bad actors. Once the adaptive immune system has been exposed to a pathogen and built up its memory, it is ready to spring into action with a tailor-made immune response almost immediately the next time around. Until this process is complete, the innate immune system is doing the bulk of the work to protect us. In most cases, it alone is capable of clearing the infection before the adaptive immune response is ready to help out. With Covid-19, for example, many of those who are infected have few or mild symptoms, and the virus is cleared thanks almost entirely to innate immunity. It is when innate immunity does not clear the initial infection, and the adaptive 236

immune response hasn’t had time to mature, that serious consequences ensue. There is another kind of immunity that straddles the line between the innate and adaptive immune responses: trained immunity. Trained immunity is the modification of innate immune cells in response to exposure to a pathogen. Once modified, the cells are better able to deal with the pathogen the second time around. Basically, it’s innate immune cells building up immunological “memory”, something previously thought to happen only as part of the adaptive immune response. As we will discuss, new evidence suggests that innate lymphoid cells fall into this category; capable of both responding to threats immediately and also remembering what they have seen. This article is featured on Forbes.org, and can be read online here: Innate And Adaptive Immunity In The Human Intestine

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How Your Gut Protects You from Myriads Of Microbes. Forbes | March 25, 2022 | Article

This is part of a series of stories on innate immune protection in the gut, focusing on SARS-CoV-2 infection. Part one can be read here. The Milky Way Galaxy, the one we have the pleasure of calling home, contains somewhere between 100 to 400 billion stars. That’s a difficult number to wrap your head around. Now, what if I told you you’re home to around 50 times as many “inhabitants”—good, bad, and ugly— in your gut alone? These inhabitants comprise part of what we call the human microbiome. Coined in 2001 by the American molecular biologist Joshua Lederberg, the term refers to “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease.” Essentially, all of the microorganisms that live in and around our bodies. There’s a lot. In fact, despite not being outnumbered 10:1, as often claimed, still only around half of the cells in the human body are actually human cells. Although there are various different microbiome sites, including oral, vaginal, and skin microbiomes, the gut is by far the largest and most complex. Our immune system has the difficult task of presiding over this environment, having to sort the friendly microorganisms from those with the potential to cause us harm. The innate immune system, in particular, plays an important role in keeping things in balance. Our gut is an especially tricky environment for our immune system to keep track of. One big reason for this is that, unlike our skin, the gut needs to allow a certain amount of “trade” between outside and inside: we need to absorb vital nutrients from the food that passes through our intestines while also getting rid of metabolic waste. As such, the immune system in our gut is constantly on its toes, making sure only the things we need are allowed to enter our body. 238

Added to this is the fact that the entire alimentary canal, from your mouth onwards, inhabits a strange in-between space— on the one hand it is inside our body, but on the other it is constantly exposed to the outside world. In a sense it’s like a second skin, just that it’s on the inside and that it’s a lot more permeable. Being exposed to the outside while also being more permeable makes these areas very vulnerable to infection. This second, inside skin is called the mucosa. Here we focus on the small intestine, in which the mucosa is made up of three main layers: the mucus layer, the intestinal epithelium layer, and the lamina propria. The inner wall of our small intestine —the section that faces the food passing through— looks like a mountainous valley, composed of multiple folds of mucosal tissue (Figure 1). These folds are covered in little hair-like projections called villi, which themselves are covered by a single-cell layer of epithelial cells (Figure 2). It is around these folds, and the villi that cover them, that we find the mucus layer.

FIGURE 1. Schematic representation of the anatomy of the small intestine. FROM: “ORAL DRUG DELIVERY, ABSORPTION AND BIOAVAILABILITY.” DAHLGREN ET AL. 2021

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FIGURE 2. Structure of the intestinal mucosa, with the villi clearly marked. FROM: “IMMUNOLOGICAL ASPECTS OF INTESTINAL MUCUS AND MUCINS.” JOHANSSON ET AL. 2016

The mucus layer helps keep food and other large substances from contacting the epithelial cells underneath while still allowing smaller molecules to move through it. Given that it's made up of a sticky gel, the mucus also slows the advance of entering microorganisms, buying our immune system some time should any of them be deemed a threat. The mucus layer also contains defensins —peptides that kill misplaced microorganisms— and a host of antibodies and other antimicrobial substances as a first line of defense. Given these antimicrobial features, it is considered the biochemical barrier of the mucosa (Figure 3).

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FIGURE 3. A diagram of the three layers of the intestinal mucosa: the mucus layer (biochemical ... [+] FROM: “KEEPING BUGS IN CHECK: THE MUCUS LAYER AS A CRITICAL COMPONENT IN MAINTAINING INTESTINAL HOMEOSTASIS” FADERL ET AL. 2015

The next layer, the intestinal epithelium, acts as the main physical barrier to entry; it’s a web of different endothelial cells tightly bound by protein complexes. These tight junctions, as they’re known, give the endothelial layer its structural solidity and make it difficult for unwanted microorganisms to move through. Interspersed amidst enterocytes, the most common type of endothelial cell, you’ll find two other kinds of highly-specialized epithelial cells: Paneth cells and goblet cells. Paneth cells are found in the “valleys” of the intestinal mountainscape, between the villi. The technical name for these valleys is intestinal crypts (as seen in Figure 2). From their location within the crypts, Paneth cells secrete the majority of the antimicrobial content found within the mucus layer— they are the factories of antimicrobial production. Goblet cells, in turn, are located along the trunk of the villi. They’re main purpose is to synthesize and secrete the mucus that acts as a buffer to the epithelial cells. Finally, the lamina propria sits underneath the intestinal endothelial cells. Here, it offers added structural support and assists the prior two layers with host defense. This layer houses the majority of the innate and adaptive immune cells, including macrophages, 241

dendritic cells, B and T cells, and also innate lymphoid cells (ILCs). The immune cells are brought into action with help from specialized intestinal epithelial cells called microfold (M) cells. M cells act as gateways of sorts, ferrying antigens and microbes from the lumen of the gut into the lamina propria (as seen in figure 3). This process helps present antigens to the immune cells below. Upon encountering a foreign antigen, the immune cells can then migrate to lymph nodes and amplify the immune response as needed. All of the aforementioned parts come together to perform the delicate dance of keeping the intestinal environment in balance. Too little or too late of a response and we end up with serious disease; too overzealous of a response and we end up with autoimmune disorders like irritable bowel syndrome (IBS) or Crohn's disease. This article is featured on Forbes.org, and can be read online here: How Your Gut Protects You from Myriads Of Microbes.

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A Primary Defense Against SARS-CoV-2: Defensins Forbes | March 26, 2022 | Article

This article is an extension of a previous series which is now available as an anthology in the book, “Natural Immunity and Covid: What it is and How it Could Save Your Life”. Here, we introduce part 1 of a small series about defensins—small molecules which help protect us against infectious disease. One of the most important layers of our body’s defense against SARS-CoV-2 lies in our innate immune system. The innate immune system protects our body from microbes, viruses, bacteria, and parasites that we have not previously encountered. While much of public attention and awareness of human immunity is focused on adaptive or learned immunity, innate or natural immunity accounts for why most people who are infected with SARS-CoV-2 have few, if any serious symptoms. Now, a family of molecules called defensins are the latest to be implicated in our natural defense against SARSCoV-2. Introducing Defensins Defensins are an ancient family of proteins that can be traced far back into our evolutionary history and are active in most multicellular organisms. In humans, they are found throughout the body and are classified into either alpha defensins or beta defensins based on their structure. Alpha defensins are further classified into human neutrophil peptides (HNPs) or human defensins (HDs). There are four forms of human neutrophil peptides (HNP 1-4) and 2 forms of human defensins (HD 5-6). HNPs 1-4 are produced by neutrophils in the circulatory system, while HD 5-6 are produced by Paneth cells. Paneth cells are specialized epithelial cells that line the surface of the small intestine. Many beta defensin genes have also been discovered in the human genome, however, only three have been classified at the functional level. These are human beta defensins 1-3 (HBD 1-3) and they are produced by epithelial cells in multiple organs. 243

Figure 2: A comparison of alpha defensin and beta defensin structures. THOMAS SHAFEE - OWN WORK, CC BY 4.0, WIKIMEDIA COMMONS

Defensins are considered antimicrobial peptides and are known to defend against bacteria, fungi, and viruses. While some defensins directly kill invading microbes, others are adept at recognizing infected cells and killing them before infection can spread to healthy cells. Now, recent research has shown that some defensins may play a role in suppressing SARS-CoV-2 infection. How do Defensins work? Defensins stave off microbial infections through a wide variety of mechanisms. However, the most frequently cited mechanism is membrane disruption. Defensins can kill cells by inserting themselves into the cell membrane to create holes in the membrane. This causes the contents of the cell to leak out, resulting in cell death. One of the most potent features of defensins is that they are Janus-faced—one side of them is positively charged while the other side is negatively charged. This makes them amphipathic molecules and allows them to interact favorably with any membrane’s charges to cause membrane disruption and cell death.

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Figure 3: Amphipathic molecules can reorient themselves to interact favorably with a cell membrane. This allows them to insert themselves into the membrane. HAZLETT L, WU M. DEFENSINS IN INNATE IMMUNITY. CELL AND TISSUE RESEARCH. 2011;343:175-188. DOI: 10.1007/S00441-010-1022-4

The question that remains is: do any defensins have a similar effect on SARS-CoV-2? Recent research suggests yes. Defensins Inhibit SARS-CoV-2 Infection To investigate this question, Chuan et al. began by inducing the SARS-CoV-2 spike protein onto fluorescent cells. To test whether defensins could inhibit infection, Chuan et al. treated the fluorescent spike protein cells with defensins for 1 hour. They then introduced the spike protein cells to cells with ACE2 receptors. Typically, when SARS-CoV-2 infects a cell, its spike protein will bind to ACE2 receptors to enter a cell and transfer its viral RNA. Researchers could measure how many ACE2 cells were infected, by whether fluorescent material had been transferred to them. Surprisingly, they found that four alpha defensins exhibited significant anti-SARS-CoV-2 activity. These were: HNP-1, HNP2, HNP-3, and HD5. HNPs 1-3 suppressed infection by 50% while HD5 suppressed infection by 60%. All four defensins were effective at physiological concentrations of each molecule. HD6 also blocked SARS-CoV-2 infection, but only at a much higher concentration that was out of physiological range, indicating that it may not be as effective against SARS-CoV-2 in the body. 245

When this experiment was repeated with spike proteins from the SARS-CoV-2 variants Alpha and Gamma, researchers found that the defensins were less effective at suppressing infection, suggesting that these variants were more resistant to the defensins. Defensins Inhibit Viral Entry Next, Chuan et al. sought to deduce whether defensins suppress SARS-CoV-2 infection by inhibiting the virus from entering the cell or by inhibiting the virus’s ability to replicate. To do so, researchers first exposed the ACE2 cells to the spike protein cells, allowing the virus to enter the ACE2 cells. They then treated the already-exposed ACE2 cells with defensins. After incubating the cells for three days, researchers found that the defensins exhibited no anti-SARS-CoV-2 effects, suggesting that defensins block SARSCoV-2 infection by preventing the virus from entering the cell in the first place. So how do defensins prevent SARS-CoV-2 from entering the cell? A separate study conducted by Wang et al. at the Army Medical University in Chongqing, China may have the answer. Wang et al., used computational modeling techniques to determine how the alpha defensin HD5 interacts with SARS-CoV2 and ACE2 receptors. Through computational simulations, they found that HD5 has a higher binding affinity for ACE2 receptors than it does for SARS-CoV-2 proteins. This indicates that HD5 may prevent SARS-CoV-2 from infecting cells by blocking the ACE2 receptors and “cloaking” the healthy cells from the virus. This study marks significant progress in our understanding of how innate immunity protects us against SARS-CoV-2. As we continue to delve deeper into how defensins prevent SARS-CoV-2 infections, we may discover new avenues for medications and treatments. Intestinal Defensins One of the anomalies of SARS-CoV-2 infection is that the lining of the intestine is far richer in ACE2 than the lungs, yet the primary symptoms of SARS-CoV-2 infection are respiratory. Some have speculated that it’s the efficiency of the innate immune defense in the gut that allows the intestine to be resistant to the effects of SARS-CoV-2.

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One of the key defensins identified in both Chuan et al. and Wang et al. is HD5. HD5 is highly abundant in the intestine and its production is triggered by the release of cytokines. In a paper published by researchers at the Pasteur Institute, scientists made an interesting discovery. They found that invading microbes in the gut can activate a molecule called interleukin-3. When interleukin-3 is activated, it produces pro-inflammatory cytokines which trigger the production of HD5. The intestine already contains multiple methods of defense against viral infection. Its mucous membrane surface makes it more difficult for the virus to attach to ACE2 receptors. The adaptive immune system also provides a high level of protection by producing the antibody immunoglobulin A (IgA) which is transmitted across the surface of the gut epithelial cells to prevent infection. Now, these studies indicate that the intestine contains a vigorous innate immune system that responds to SARS-CoV-2 by activating interleukin-3 and triggering the production of HD5. Since HD5 has antiviral effects and can kill infected cells, this innate immune reaction may explain why the intestine seems to be especially protected from the effects of SARS-CoV-2. This article is featured on Forbes.org, and can be read online here: A Primary Defense Against SARS-CoV-2: Defensins

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Imaging the Brain: What Brain Scans Reveal About the Consequences of Covid-19 Forbes | March 28, 2022 | Article

This story is part of a larger mosaic of stories on the neurological consequences of Covid-19 and Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part one, two, and three of this series. Approximately two-thirds of those hospitalized for Covid-19 experience at least one neurological complication. How this damage occurs without directly infecting brain cells remains one of many mysteries regarding the pathology of Covid-19. Even more puzzling is the fact that people who experience mild or asymptomatic infection may later develop new cognitive and psychiatric symptoms, regardless of age and pre existing conditions. A recent report from the Journal of Nuclear Medicine may shed light on some of the structural damage to the brain as a result of Covid-19. Covid-19 and Parkinson's Disease One of the more striking consequences of Covid-19 are the increasing reports of Parkinson's Disease following infection. Therefore, several studies have sought to investigate the structural changes in the brain induced by Covid-19, as well as corresponding symptoms of abnormal movement, that seem to resemble neurodegenerative disease. Meyer et al. suggest that Covid-19 may unmask or worsen neurodegeneration, putting some at risk for Parkinson’s disease and other neurodegenerative diseases. In some cases, symptoms begin only a few weeks after being infected with SARS-CoV-2. Studies imaging the brains of those affected in fact found significant deficits in the dopaminergic nigrostriatal pathway.

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Figure 1: Illustration of dopaminergic pathways in the midbrain. The nigrostriatal pathway begins in the substantia nigra and extends through the caudate nucleus and putamen, as well as the basal ganglia (not shown). WIKIMEDIA COMMONSBRUCEBLAUS

As shown in the figure above, this dopamine pathway begins in the substantia nigra and extends through the caudate and putamen, as well as parts of the basal ganglia. Neurons in this pathway release dopamine neurotransmitters to coordinate voluntary movements. With Parkinson’s disease and related neurodegenerative diseases, some of these neurons begin to degenerate or die. Less neurons releasing dopamine makes initiating movement progressively becomes more difficult. It is unclear whether Parkinson's-like symptoms develop from Covid-19 infection or whether the infection accelerates deficits that are already present. This is not the first time that a viral infection has been linked to Parkinson's Disease. The first recorded example dates all the way back to the 1918 influenza outbreak. People born during this epidemic were two to three times more likely to develop Parkinson's Disease later in life, compared to those born before or after. Similar to Covid-19, having the flu does not guarantee a Parkinson's diagnosis, but there does seem to be a connection between inflammation in the brain and neurodegeneration. Inflammatory Damage to the Brain

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How does SARS-CoV-2 gain access to the brain? One theory points to pro-inflammatory cytokines. These immune cells travel throughout the bloodstream and are able cross the blood-brain barrier. Exposure to SARS-CoV-2 can produce a robust immune response that overwhelms the brain with pro-inflammatory cytokines, even if nerve cells are not directly infected. While cytokines are helpful for fighting infection, high concentrations can severely damage healthy cells. It is possible the stress from proinflammatory cytokines not only induces short-term damage to brain tissues, but also may promote the progression of underlying neurodegenerative conditions, including dementia and Parkinson's. Activation of the microvascular system by the SARS-CoV-2 spike protein alone has also been shown to inflame endothelial cells that line blood vessels. This can have significant consequences throughout the cardiovascular system, including the blood-brain barrier. A dense vascular system travels in between cells throughout the brain to provide nutrients and remove toxins, as shown in the figure below. Inflammation in those epithelial cells, therefore, can impact adjacent neurons and supportive glial cells.

Figure 2: Close up image of microvasculature in the brain. PIXABAY- ENNAZIZ

Another theory suggests that neurodegenerative deficits may be linked to an alternative immune condition: autoimmune encephalitis. Albeit rare, severe cases of Covid-19 can lead to autoimmune encephalitis during which the body’s own immune cells attack healthy cells. PET imaging studies indicate that the consequences of autoimmune activity can vary throughout the brain. One case study, for instance, found reduced activity in the outer 250

cortical layer of the brain, but surprisingly also found increased metabolic activity in the basal ganglia and cerebellum that may be related to the production of specific anti-neuronal antibodies. Therefore, brain damage could be a consequence of self-attacking antibodies weakening structures in the nigrostriatal dopaminergic pathway, as well as the regions of the cerebellum involved in movement. However, cases of autoimmune encephalitis seem to be separate from those that develop neurodegenerative conditions following Covid-19 infection. In fact, treatment with immunoregulatory drugs has been shown to improve symptoms for those with autoimmune encephalitis but not those who developed neurodegenerative conditions. This finding is consistent with the idea that SARS-CoV2 accelerates existing degeneration rather than promotes the development of new deficits. Bystander Damage to the Brain Thirdly, brain tissues may experience bystander damage as a result of infection in a region very close to the brain– the nose. Meyer et al. therefore considered whether olfactory deficits associated with Covid-19 may contribute to structural changes in the brain. Several studies found decreased metabolic activity in regions mostly associated with olfaction and increased activity in the orbitofrontal and parietal cortex important for processing sensory information. Since these findings were not consistent across all individuals reporting lost or disordered sense of smell, hypometabolism in some regions and hypermetabolism in others may instead be a consequence of neurons reorganizing once a sensory input is lost, i.e. olfaction. Hypoxia, which develops when an infection overwhelms the lungs and less oxygen is available to cells, may also generate a broad spectrum of damage to the brain. When the brain goes without sufficient oxygen for an extended period of time, cognitive processes are disrupted and brain cells begin to die. Early autopsies of people that died from Covid-19 suggested that hypoxia may underlie several neurological compilations. This could explain the high prevalence of neurological complications among those hospitalized for the virus. Recent brain imaging, however, reveals that a more diverse set of mechanisms may be contributing to symptoms during infection and long after. 251

Other Neurological Symptoms During the acute phase of Covid-19, the most commonly reported neurological symptoms are loss of smell (60- 80%), myalgia of muscle pain (40%), encephalopathy (15-30%), and cerebrovascular events (1-5%), as well as peripheral nerve damage and encephalitis on occasion. Since these complications can significantly alter the structure of the brain, molecular brain imaging tools, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), are useful diagnostic tools. Encephalopathy, for example, is characterized by cognitive impairments, but these symptoms are also accompanied by significant hypometabolism in top layers of the frontal and parietal lobes, important for integrating sensory information into executive processing functions. Clinical studies show that hypometabolism in these areas negatively correlates with lower scores on cognitive tests. Also, when metabolic levels begin to stabilize following infection, most individuals previously hospitalized for Covid-19 show almost complete cognitive improvement. Researchers speculate that encephalopathy may partially manifest as microgliosis in white brain matter, during which the microglia cells that normally support brain tissue temporarily cluster to produce an immune response. The corresponding immune response activates several other proinflammatory cells, including cytokines, that injure healthy cells. Once the infection is eliminated, however, the microglia separate to restore normal brain function. In contrast, diagnosing long Covid can be more challenging. Long-lasting symptoms are often neuropsychiatric, such as fatigue, attention problems and memory loss and therefore cannot be seen using molecular imaging tools. This does not mean that people with these symptoms may not face significant challenges. Some studies suggest that adults and children with long Covid exhibit widespread hypometabolism, including in the orbitofrontal cortex, medial temporal lobes, thalamus, brainstem and cerebellum. Clinical investigations, however, have not found any correlations between cognitive or neuropsychiatric symptoms and PET imaging measurements. This makes it more difficult to determine the underlying mechanisms that contribute to long-lasting symptoms. Conclusion 252

Brain imaging tools are useful for identifying correlations between symptoms and brain activity, but they often do not prove causation. We are only just beginning to understand how SARSCoV-2 damages brain tissues during acute infection and long after. Although the full consequences may not be known for years to come, Meyer et al. seems confident that for a majority of people these neurological impairments will not be permanent or unresponsive to treatment. As Philipp T. Meyer, MD, PhD, head of the Department of Nuclear Medicine of the Medical Center– University of Freiburg in Germany, puts it, “To the best of our knowledge there are no convincing studies clearly demonstrating relevant and irreversible brain damage, except for disease complications like brain infarcts and bleedings.” Continuing clinical research on the consequences of Covid-19 in the brain will expedite the production of new treatments and vaccines bringing relief to millions. This article is featured on Forbes.org, and can be read online here: Imaging the Brain: What Brain Scans Reveal About the Consequences of Covid-19

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Innate Lymphoid Cells (ILCs): Guardians Against Infection Forbes | March 29, 2022 | Article

This is part of a series of stories on innate immune protection in the gut, focusing on SARS-CoV-2 infection. Read part one and part two. Previous articles in this series have discussed our immune responses —innate, adaptive, and trained— and given an overview of the particular immune environment of the human gastrointestinal tract. The gut is especially tricky for our immune system to manage since it’s forced to distinguish between things that should be there, like food and commensal bacteria, and things that shouldn’t be there. This not only means distinguishing self from other, but also distinguishing helpful other from harmful other. No easy feat. Here, we talk about a family of innate immune cells called innate lymphoid cells (ILCs). These cells play an active role in regulating barrier environments like the gut and the lungs. Subsequent articles in this series will zoom in on one particular subset of the ILC family, group 3 ILCs. Specifically, how ILC3s help regulate SARS-CoV-2 infection in the small intestine. Innate lymphoid cells are the most recently discovered innate immune cells. Hints of their existence emerged during the 1970s — by way of research on natural killer (NK) cells and lymphoid tissue inducer (LTi) cells— but a fuller characterization of the family didn’t arrive until the late 2000s. A lot of previous research on the ILC family has focused on their contribution to autoimmune diseases, especially rheumatic diseases and diseases of chronic inflammation in the gut, like Crohn’s disease. But, the more we learn about these cells, the more we realize they are a crucial part of the innate immune response to microbes across the body. We focus on these protective qualities of ILCs. In many ways, ILCs act as the innate counterpart to the T lymphocytes found in the adaptive immune response. Like T cells, they play an important role in the modulation of immune homeostasis, response to injury or infection, and repair of damaged 254

tissue. But they are set apart by one major difference: their lack of antigen specificity. Foreign substances or toxins that enter and have the potential to harm our body are called antigens. Antigens can stem from pathogens like viruses and bacteria all the way to pollen or chemicals. Luckily, our T cells come equipped to recognize basically any and all antigens. Through a process called gene recombination —a kind of random mix and matching of genetic code— T cells develop a surface protein called a T cell receptor. Each T cell has its own unique surface protein with its own unique shape. In theory, for each specific antigen we have a corresponding T cell whose receptor is shaped just right to allow it to bind to the antigen— think of it as a very elaborate and very large system of locks and keys. T cells that have at some point bound to an antigen proliferate and continue to circulate throughout the body for years or even decades after initial exposure. It is through their antigen specific receptors that the cells of the adaptive immune system can form lasting immunological memory, enabling a quicker and more efficient immune response the second time around. Instead, ILCs depend on cytokine receptors that allow them to pick up changes in their microenvironment in response to tissue damage. They also have an array of other receptors sensitive to microbial products, neuronal transmitters, and nutrient components. ILCs are first and foremost tissue cells, only very rarely found in peripheral blood, and localized mainly around the mucosa of the gut and the lungs. In total, the ILC family is made up of three groups: ILC1s, ILC2s and ILC3s (Figure 1). They are classified according to their functions. Broadly speaking, ILC1s are in charge of clearing pathogens that have managed to enter our cells. They are defined by their ability to produce interferon gamma (IFNγ). IFNγ is a small signaling protein that serves as the primary activator of macrophages—which recognize, engulf, and digest foreign substances. In general, IFNγ is a foundational component of both the innate and adaptive immune responses, helping to stimulate and modulate them in the face of infection. NK Cells are the prototypical group 1 ILCs.

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FIGURE 1. Schematic representation of the three groups of innate lymphoid cells (ILCs), including ... [+] FROM: “INNATE LYMPHOID CELLS — A PROPOSAL FOR UNIFORM NOMENCLATURE” HERGEN SPITS ET AL. 2013

Group 2 ILCs, on the other hand, are associated with immune activity in helminth infections and also response to allergen-induced airway inflammation. They are defined by their ability to produce interleukin-5 (IL-5) and interleukin-13 (IL-13). Interleukins are another kind of signaling molecule, primarily responsible for the growth and differentiation of different immune cells. Interleukins also have a hand in activating various immune cells, with different interleukins responsible for the activation of different immune cells. In the case of IL-5, it initiates and watches over B cell growth, which ultimately helps stimulate the production of antibodies. IL-13 seems to contribute to the regulation of the inflammatory response, inhibiting the production of inflammatory cytokines when and where needed.

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Finally, we have group 3 ILCs, which are predominantly involved in gut immunity. They act as sentinel cells, sending warning signals that trigger immune responses capable of killing not only host cells that have already been infected, but also the actual pathogen causing the infection. ILC3s do this by producing interleukin-17 and/or interleukin-22. Some also activate IFNγ. Figure 2 gives an overview of the role of ILC3s in intestinal homeostasis.

FIGURE 2. Intestinal ILC3 are a dominant source of interleukin (IL)-22 at steady state, produced in ... [+] FROM: “ORCHESTRATION OF INTESTINAL HOMEOSTASIS AND TOLERANCE BY GROUP 3 INNATE LYMPHOID CELLS.” PENNY ET AL. 2018

IL-17 stimulates a variety of different signaling cascades that lead to the production of chemokines— signaling proteins that help move important immune cells, like monocytes and neutrophils, to areas where they are most needed. Monocytes are a kind of white blood cell made in the bone marrow. They travel through the blood 257

to various tissues in our body, where they become either macrophages or dendritic cells. Macrophages, as discussed above, actively engulf and kill pathogens. Dendritic cells, in turn, strengthen the immune response by presenting antigens from the invading microorganism to other immune cells, getting them involved in the action. Neutrophils, another kind of white blood cell, are part of the first responders of the innate immune system. They travel to sites of infection and infiltrate damaged tissue. Once there, they engulf and destroy invading microbes. They also release antimicrobial enzymes and peptides, inhibiting the advance of pathogens. Neutrophils also help lay the groundwork for repair of damaged tissues, which is more fully managed by macrophages. In stimulating monocytes and neutrophils, IL-17 promotes inflammation at sites of injury or infection, helps clear invading pathogens, and also initiates the repair of damaged tissue. IL-22 is also closely tied to the inflammatory response, functioning to moderate cell survival and, importantly, stimulate antimicrobials including defensins and S100 proteins. S100 proteins help defend against pathogens by depriving them of the nutrient metals they depend on for growth and replication, a process known as “nutritional immunity”. They also act as signaling beacons released by damaged or dying cells, calling into action nearby immune cells and kickstarting inflammation. Defensins, in turn, are small peptides with direct antimicrobial abilities as well as immune signaling abilities. In cases of viral infection, they help reduce infectivity by interfering with and inhibiting fusion of virions with host receptors, or, once the virus has managed to enter the cell, by preventing the virus from exposing its genetic material to the host machinery it needs for replication. When confronted with bacterial infections, defensins help stymie the threat by binding to the bacterial membrane and breaking it down or by preventing the synthesis of the bacterial cell wall. They also help neutralize the toxins secreted by bacteria. Greater knowledge of this system, and how it works, is necessary to understand both SARS-CoV-2 pathogenesis and how to control it late in disease. This article is featured on Forbes.org, and can be read online here: Innate Lymphoid Cells (ILCs): Guardians Against Infection

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New And Effective Monoclonal Antibody Treatment For Ebola On The Horizon Forbes | March 29, 2022 | Article

Scientists at the Center for Infectious Disease and Vaccine Discovery in La Jolla, California have developed a remarkable pair of monoclonal antibodies that may be the long-sought answer for effective treatments of multiple ebolavirus strains. Ebola poses a threat to human health not only in Africa, where it originates but with the possibility that it may spark another pandemic. We can all recall the fears of Ebola as it spread from the African continent to international shores. Fortunately, these cases were isolated and a pandemic did not ensue. There are no guarantees that ebolaviruses could not be the cause of the next global pandemic. At present, our tools to confront Ebola are lacking and not fully effective. Current vaccines only treat one strain of ebolavirus, EBOV, and only with partial protection that fades over time. There are mRNA vaccines in development to treat ebolavirus, but it remains to be seen if those will have lasting durability against disease and death. Although many small molecule drugs have been tested for efficacy, none have yet been approved. The most effective drug against Ebola is Regeneron’s monoclonal antibody cocktail: REGNEB3. The drawback is that it only protects against one of the major strains of ebolavirus, leaving strains like Sudan virus (SUDV) untreated. Isolation Of Broadly Neutralizing Antibodies The target for neutralizing antibodies in ebolaviruses, much like SARS-CoV-2, is the surface protein that regulates binding cell surface and cell entry. The protein binds receptors on the cell, triggering fusion of the viral and cell membrane, providing an opening for the virus particle to enter the cell. Entry is mediated by the trimeric surface protein of Ebola. There are three glycoproteins to a trimer and each is divided into two separate proteins: GP1 and GP2. 259

GP1 binds to the receptor, forming a binding pocket while GP2 mediates the fusion of the virus and cell membranes. GP1 is noncovalently attached to GP2, which oversees fusion to the cell surface once receptor-binding is complete.

FIGURE 1: Structure of the EBOV glycoprotein (GP).MOLLER-TANK ET AL.

Search For Broadly Neutralizing Antibodies Milligan et al. isolated B-cell samples from patients within one year of their Ebola infection. They identified 36 mAb candidates. From these, they screened for binding strength to Ebola (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), all members of the ebolavirus genus. They also screened for antibodies that bound outside the ectodomain, meaning they were not restricted to the head and core regions. This screening process narrowed their search to nine, which they examined individually for their neutralization of the diverse ebolavirus strains. A subset of those that bound these viruses 260

neutralized the SUDV and BDBV strains. They separated the nine into two groups: those that bound to GP1 and those that bound to GP2. Their search resulted in the discovery of two new antibodies: 1C11 and 1C3. Both of these strongly neutralized all of EBOV, SUDV, and BDBV, with the exception of BDBV for 1C3. 1C11 targeted the base region of the ebolaviruses glycoprotein, whereas 1C3 targeted the head region. Milligan et al. note that 1C3 also induces antibody-dependent cytotoxicity, not only neutralizing the virus but also targeting and killing the infected cell. 1C3 Binding

FIGURE 2: Molecular surface of EBOV GP in gray, with a single bound 1C3 reaching down into the ... [+] MILLIGAN ET AL.

Figure 2 clearly shows how the Fab fragment of the 1C3 antibody binds to the chalice. Please note that the antibody makes asymmetric contacts, binding overlapping but distinctly different sets of amino acids of the three receptor binding interfaces. 1C3, therefore, has a dual function. The three-face asymmetric binding not only inhibits binding to the cell but also effectively locks GP1 into position preventing fusion. Please note the simultaneous asymmetric binding residues of 1C3 to the trimer below. 261

FIGURE 3: Overlapping asymmetric binding sites in the receptor-binding chalice for the 1C3 antibody. MILLIGAN ET AL.

1C11 Binding

FIGURE 4: Molecular surface with the 1C11 antibodies connecting the trimers at the base. MILLIGAN ET AL.

1C11 also has very unusual properties. Not only does it bind across two of the trimer surfaces, but it also binds an entirely different site down below 1C3 in the base of the glycoprotein, GP2, where the fusion domain lies. 1C11 contacts GP1 residues 34, 88–90, and 155 and the GP2 residues 523–524, 527–532, 534–536, and 563– 566. The net result is the locking of the entire structure in place, without the ability to release glycoproteins after infection. Combination Antibody The resultant combination therapy was more effective than both antibodies in isolation. In comparison to 1C3, the combination was 262

2.64-fold more effective and in comparison to 1C11, the combination was 3.93-fold more effective in vitro. There are two reasons this combination therapy is so broadly binding. First, the base region that is bound by 1C11 varies little across ebolavirus strains. Second, while the amino acids of the chalice differ, the 1C3 footprint touches so many residues that the commonalities may compensate for the variation. Protection From Disease And Death In Rodents And NonHuman Primates They also tested the combination therapy in vivo on both rodents and nonhuman primates. They first used a mouse-adapted virus. In pretreated EBOV-infected mice, 1C3 and 1C11 provided 90-100% protection from death as compared to the 10% survival rate of the control group. Similar results were found in BDBV-infected mice, in which the combination yielded 100% protection from death. In guinea pigs, neither antibody was effective individually, but the combination protected 80% of subjects for both EBOV and SUDV. In nonhuman primate models, SUDV and EBOV-infected primates treated with the combination therapy had a 100% survival rate with the viruses being completely cleared according to PCR test between two and three weeks post-infection. The viruses were undetectable only days after inoculation with the treatment. Milligan et al. also emphasize the restriction of low dosages throughout their testing process as these antibodies can be expensive and would primarily be used in low-income countries. In a piece of truly excellent work involving antibody isolation, characterization, and animal model work, Milligan et al. present a tour de force. This work provides a key not only for effective treatment for ebolaviruses, but we believe for many other viruses as well. The analog for SARS-CoV-2 would be antibodies that bind across the S1/S2 subunits in a similar fashion, locking the trimer into place. An equivalent may be the camelid antibodies that lock the SARS-CoV2 Spike protein in place in a similar way, which may be a class of antibodies worth exploring for future treatments. This article is featured on Forbes.org, and can be read online here: New And Effective Monoclonal Antibody Treatment For Ebola On The Horizon

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Group 3 Innate Lymphoid Cells (ILC3s) And Trained Immunity:

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Faster And Stronger.

Forbes | March 30, 2022 | Article

This is part of a series of stories on innate immune protection in the gut, focusing on SARS-CoV-2 infection. Read part one, part two, and part three. The past few decades have yielded a wealth of new research on innate lymphoid cells (ILCs), including work on the development of immunological memory in ILC1s and ILC2s. The ability of ILC3s to develop such “trained immunity,” however, has remained somewhat of a mystery. Scientists from the Institut Pasteur and Inserm have tackled the issue head on, discovering that ILC3s do, in fact, have the ability to learn from their previous encounters with pathogens, allowing them more quickly to recognize and engage should they reencounter them down the line. To check for functional changes to ILC3s in response to pathogens, Serafini et al. exposed mice to a bacteria called Citrobacter rodentium. Mice infected with C. rodentium display similar symptoms and a similar disease trajectory to humans infected with harmful Escherichia coli (E. coli), both producing severe inflammation of the gastrointestinal tract. Once in the gut, C. rodentium attaches itself tightly to the walls of the small intestine and colon. This causes innate dendritic cells to activate, and in turn, triggers ILC3s into action. However, dendritic cells also get the adaptive branch of the immune system involved. So to focus solely on the ILC3 portion of the immune response, the researchers gave the mice a short course of antibiotics, preventing the adaptive response from kicking in while still allowing ILC3related immune responses to function as normal. During the initial infection with C. rodentium, both the number of ILC3s and the number of T cells, a quintessential adaptive immune cell, remained fairly constant. One month after initial infection, Serafini et al. reinfected the mice with C. rodentium: this time, the number of intestinal ILC3s skyrocketed (Figure 1). There was a particularly strong response in two ILC3 subsets, NKp46+ and 265

CCR6+, which the researchers decided to name “trained ILC3s”. All the while there was little to no change in T cell populations.

FIGURE 1. Overview of T cell and ILC3 count during first exposure to C. Rodentium (CR) and ... [+] FROM: “TRAINED ILC3 RESPONSES PROMOTE INTESTINAL DEFENSE” SERAFINI ET AL. 2022

Along with the spike in ILC3s, there was no observable bacterial growth during reinfection (Figure 2). This suggests that ILC3s can spawn functional, specific and persistent subsets in response to even a short initial exposure to a bacterial pathogen. In a sense, a kind of immunological memory.

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FIGURE 3. Absolute numbers of total ILC3s in the lamina propria of the small intestine. SERAFINI ET AL. 2022

Dotting their i’s and crossing their t’s, the researchers checked to make sure that trained ILC3s could be generated upon reinfection even without an adaptive immune response. They tested this by exposing mice that had been mutated to produce no mature T or B cells, known as Rag2−/− mice, to the C. rodentium bacteria. Those Rag2−/− mice that had already been previously exposed to the C. rodentium, and thereby built up trained ILC3s, had significantly higher levels of ILC3s than did those Rag2−/− mice exposed for the first time. Again, bacterial growth was also substantially lower in the mice with trained ILC3s. This confirmed that trained ILC3s can function just as well during pathogen reinfection without T cells present as they do with T cells present. Curiously enough, these “memory-like” features of the trained ILC3s weren’t restricted to C. rodentium. For example, upon exposing C. rodentium-activated trained ILC3s to a different kind of bacteria, Listeria monocytogenes, Serafini et al. still saw an enhanced ILC3 response. That said, both classical and innate immunological memory responses are characterized by durable metabolic changes, and this was clearly the case in trained ILC3s. These subsets shifted from glycolysis and glutaminolysis to enhanced tricarboxylic acid (TCA) cycle, also known as the Krebs cycle. This shift was also accompanied by a change to fatty acid synthesis and oxidation-associated gene expression. Serafini et al.’s work offers an exciting new insight into the world of innate immunological memory, and confirms that ILC3s can, and 267

do, develop trained immunity that allows them to respond to pathogen reencounters with heightened sensitivity and heightened efficacy. This article is featured on Forbes.org, and can be read online here: Group 3 Innate Lymphoid Cells (ILC3s) And Trained Immunity: Faster And Stronger.

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Hide And Seek: How Group 3 Innate Lymphoid Cells Help Cloak ACE2 Receptors From SARSCoV-2 Forbes | March 31, 2022 | Article

This is part of a series of stories on the protective role of innate lymphoid cells (ILCs) in the gut and other tissues, focusing on SARS-CoV-2 infection. Read part one, part two, part three, and part four. Innate lymphoid cells (ILCs) are found throughout the mucosal surfaces of our body, but they are particularly well-represented in the lung and in the gastrointestinal tract. Here they help our innate immune system with the difficult task of sorting commensal microorganisms —which share our body without causing us harm— from those with a pathogenic inclination. Of the ILC family, group 3 ILCs are most closely associated with gastrointestinal immunity. Their main function in the immune response is the production of interleukin-17 (IL-17) and interleukin-22 (IL-22), more on this below. SARS-CoV-2, although mainly known for its impact on the lungs and the respiratory system, is also very much a virus of the gut. Covid-19 patients have displayed symptoms related to the gastrointestinal tract, including diarrhea, nausea, and vomiting. In some, gastrointestinal issues remain up to 6 months after initial infection, contributing to issues of malnutrition and inability to gain weight. Here we explore two different studies which, taken together, implicate ILC3s as an important part of the immune response against SARS-CoV-2 infection of the gut. IL-22, IL-17, and α-Defensins The first paper comes from a group of researchers based out of the University of Tokyo. Kamioka et al. describe the process by which ILC3s, in combination with commensal bacteria, help regulate Paneth cells.

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The epithelium of our small intestine —a single layer of cells that helps protect our gut from a variety of threats and stressors— is organized into crypts and villi (Figure 1). Villi are finger-like protrusions that extend towards the center of our small intestine, helping to absorb nutrients from our food and shuttle them into the dense system of capillaries directly underneath. Each villus is surrounded by a number of crypts, which is where new epithelial cells are formed to replace those that have been worn down by passing food. Paneth cells sit at the base of these crypts. From there, they secrete a host of antimicrobial peptides, such as α-defensins, and immunoregulatory proteins, both of which help keep our gut’s ecosystem in balance. By extension, they also contribute to the repair of the epithelial layer and modulation of intestinal inflammation.

FIGURE 1. (Left) Image of mouse small intestine, with villus, crypt, and lamina propria clearly ... [+] FROM: “THE PANETH CELL: THE CURATOR AND DEFENDER OF THE IMMATURE SMALL INTESTINE” LUESCHOW AND MCELROY 2020

Kamioka et al. noticed that, by virtue of metabolic differences, Paneth cells can actually be broken down into two subsets: Fut2− Paneth cells and Fut2+ Paneth cells. Compared to Fut2− Paneth cells, the Fut2+ cells were a lot more complex in structure and possessed a higher density of granules containing α-defensin, both of 270

which suggest that Fut2+ is the subset of Paneth cells more actively involved in host defense and immune response. Recognizing that IL-22 plays a role in the growth and repair of epithelial cells, the researchers next tested to see if IL-22 signaling worked to regulate Fut2+ Paneth cell development and maturation. Comparing mice that had been mutated to lack IL-22 with their unmutated counterparts, Kamioka et al. confirmed that the IL-22deficient mice had far fewer Paneth cells in their ileum —the last part of the small intestine before it joins the large intestine— than did the wildtype mice. This suggests IL-22 signaling is part of the regulatory process underlying Paneth cell development. Spurred on by this finding, the researchers wanted to see if IL22 also affects Paneth cell secretion of α-defensins. To test this, they exposed a mouse-derived intestinal organoid —a kind of miniature replica of the intestine grown from stem cells— to IL-22 for a period of two days. When compared to pre-exposure levels, the number of α-defensins in the organoid had increased significantly, confirming that IL-22 has a direct impact on secretion. Kamioka et al. also discovered that IL-17 contributes to αdefensin secretion. Areas of the small intestine rich in Fut2+ Paneth cells were found to express much higher levels of IL-17 than others. Further, IL-17-deficient mice had far lower levels of α-defensins in their feces compared to wildtype mice, again suggesting that IL-17 regulates the amount of α-defensins produced by Paneth cells. All in all, their work shows that the two main interleukins produced by ILC3s, IL-17 and IL-22, play a central role in the development of Paneth cells and, by extension, the secretion of αdefensins. But, do α-defensins offer any particular protection against SARS-CoV-2 infection? Here we turn to the second study. Human Defensin-5 (HD5), ACE2, and SARS-CoV-2 In September of 2020, a group of Chinese researchers based at The Army Medical University in Chongqing, China, were struck by a puzzle: how is it that the human gastrointestinal tract, despite spanning more than 2150 square feet and being covered in angiotensin-converting enzyme-2 (ACE2) receptors —the main receptor for SARS-CoV-2 entry into cells—, is less susceptible to infection than the lungs? To find out why this might be, Wang et al. made use of immunofluorescence microscopy (IFM). IFM is a microscopy 271

technique that can help visualize the presence of a specific protein or antigen in cells or tissue sections. This is done by binding a special antibody to the target cells or target tissue; namely, an antibody that has been chemically combined with a fluorescent dye. If the target protein or antigen is present, then the area will glow from the fluorescent dye, showing up clearly under the microscope. For this particular experiment, the researchers focused on human defensin-5 (HD5). This is the most common α-defensin secreted by intestinal Paneth cells and, as Wang et al. reported, is in close contact with the ACE2 receptors found in enterocytes, the most abundant epithelial cell of the intestines (Figure 2). The scientists discovered that HD5 can bind to the ACE2 receptors found on the surface of enterocytes. More specifically, they determined that HD5 attaches to and “cloaks” a part of the ACE2 receptors known as the ligandbinding domain (LBD) (Figure 3).

FIGURE 2. Immunofluorescence displaying the locations of ACE2 (red) and FITCHD5 (green) in human ... [+] FROM: “HUMAN INTESTINAL DEFENSIN 5 INHIBITS SARS-COV-2 INVASION BY CLOAKING ACE2” WANG ET AL. 2020

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FIGURE 3. Schematic showing the cloaking of the ligand-binding domain (pink) of the ACE2 receptor ... [+] WANG ET AL. 2020

Recognizing that the LBD plays an important role in SARSCoV-2 entry into cells, the researchers were curious to see if this relationship between HD5 and ACE2 receptors had any impact on SARS-CoV-2 infection. To test their hunch, they exposed Caco-2 cells —a type of cell often used to model the intestinal epithelial barrier— to SARS-CoV-2 Spike (S) pseudovirions. When compared to a control group, those Caco-2 cells pre-treated with HD5 for one hour before infection displayed a significant reduction in SARS-CoV-2 invasion (Figure 4). This was tested three times across three different days, and held true each time. Further, it was also shown to be the case in human renal proximal tubular epithelial cells, a kind of epithelial cell found in a different part of the gut.

FIGURE 4. Immunofluorescence revealing the inhibition of HD5 on SARS-CoV-2 S pseudovirions entry to ... [+] WANG ET AL. 2020

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Wang et al. conclude, “[t]o our knowledge this is the first study demonstrating the innate defense function of human intestine against SARS-CoV-2.” In closing, these two reports suggest that: a) ILC3s play a crucial role in the development and maturation of Paneth cells, including the production of α-defensins, and b) that human defensin-5, the αdefensin most abundantly produced by Paneth cells, can hide part of the ACE2 receptors found in epithelial cells across the human gastrointestinal tract, effectively blocking SARS-CoV-2 binding. It would also be interesting to know, as Wang et al. mention, whether in vivo supplementation of HD5 can help assuage the severity of SARS-CoV-2 infection— especially in those with HD5 deficiencies, such as patients suffering from Crohn’s disease or other inflammatory bowel diseases. This article is featured on Forbes.org, and can be read online here: Hide And Seek: How Group 3 Innate Lymphoid Cells Help Cloak ACE2 Receptors From SARS-CoV-2

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April 2022

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Coronaviruses Can Recombine With Cellular And Heterologous Viruses To Create Unexpected Variants Forbes | April 01, 2022 | Article

Recent concerns that recombinants may arise amongst SARSCoV-2 viruses are now a reality. Recombinants have been identified between Delta and Omicron and between variants of Omicron itself, namely BA.1 and BA.3. The following question arises: may other SARS-CoV-2 variants arise that incorporate genes from homologous or heterologous viruses or even cellular genes, and might such viruses have novel properties that increase the transmission, immune evasion, or virulence? To replicate, the RNA-dependent RNA polymerase of coronaviruses transcribes the positive strands of the genome. To produce protein-subgenomic 3’ messenger RNAs, the SARS-CoV2 RNA-dependent RNA polymerase must pause, dissociate from the genomic positive-strand RNA, and jump to a homologous sequence at the leading 5’ end of the virus to complete the messenger RNA. It is possible that when jumping, the polymerase may inadvertently connect to heterologous cellular messenger RNA or to the messenger RNA of a virus that is co-infecting the same cell. A second jump from the end of the heterologous message back to the genome would result in the insertion of the heterologous gene into a full length minus stand. When copied into a full-length positive-strand would result in the inclusion of the heterologous gene into newly formed genomes. If the newly acquired gene is tolerated, especially if it provides a survival advantage, the newly formed hybrid virus may spread through a population.

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FIGURE 1: (A) Insertion of a heterologous protein from a heterologous virus inserted into a ... [+] ACCESS HEALTH INTERNATIONAL

To examine whether or not such a scenario is plausible for SARS-CoV-2, we reviewed the recent literature. We came across a 2016 study by the laboratory of George Gao (Huang et al.)that describes just such an event, recombination between a bat betacoronavirus and a gene from a bat orthoreovirus. As part of a systematic study of bat betacoronavirus in the southern province of Yunnan, the team determined the full-length sequence of several independent bat isolates. One such sequence differed from all others by the inclusion of a novel sequence between the genes encoding the nucleocapsid protein and the accessory NS7a gene (Figure 2).

FIGURE 2: Genome organization of Ro-BatCoV GCCDC1. Nonstructural genes and putative mature ... [+] HUANG ET AL.

Origin Of The Heterologous Sequence 277

A search of gene databases revealed a match for the anomalous insertion, the P10 gene of a bat orthoreovirus. An analysis of the P10 TRS sequences indicates that they complement the TRS sequences of the N and NS7a proteins, resulting in their configuration between the two (Figure 3).

FIGURE 3: Identification of the recombinant p10 gene and its TRS. HUANG ET AL.

FIGURE 4: Phylogenetic analyses of P10 from representative reoviruses and RoBatCoV GCCDC1. HUANG ET AL.

The orthoreovirus P10 protein belongs to a family of fusionassociated small transmembrane proteins called FAST protein. The amino terminus of these proteins is typically myristoylated, followed by a hydrophobic extracellular domain. The proteins all have a single transmembrane domain followed by a polybasic, positively-charged, intracellular domain (Figure 5). The FAST proteins drive cell-to-cell fusion, also known as syncytia formation.

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FIGURE 5: FAST protein structure. DUNCAN ET AL.

Huang et al. compared the ability of the bat coronavirus-derived P10 protein to initiate cell to cell fusion with the P10 protein isolated in an authentic orthoreovirus (Palau). They cloned both into expression vectors. Both are active FAST proteins capable of triggering cell-to-cell fusion (Figure 6).

FIGURE 6: Syncytium formation and functional analyses of Ro-BatCoV GCCDC1 p10 gene. HUANG ET AL.

Unfortunately, Huang et al. could not isolate replicationcompetent virus from the bat with the bat P10 insertion. They were,

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however, able to detect the P10 protein shed into the bat feces by immuno-assay. The original work left unanswered whether or not the heterologous recombinant is viable. Our colleague Roberto Patarca searched the coronavirus database and revealed 13 additional identical independent isolates from bats between 2010 and 2016 in China (three isolates), Singapore (one isolate), and Cambodia (nine isolates), indicating the viability and transmission of this recombinant. We note that including a FAST protein within a coronavirus genome may confer a selective advantage. Syncytial forming viruses may spread from cell to cell within a host, avoiding recognition by antiviral antibodies. Others have noted the ability of coronaviruses to undergo nonhomologous recombination with other viruses, including toroviruses, influenza C/D, reoviruses, rotaviruses, and astroviruses. As is the case with the bat betacoronavirus described here, these all arise in the 3’ region of the virus that encodes the structural and accessory proteins. Similar heterologous events have been identified for toroviruses that, like coronaviruses, are members of the nidovirus family, which share this continuous synthesis of their messenger RNA. Previous works show that nidoviruses, including betacoronaviruses, can recombine with other viruses, and host cells' genes coproduce viable offspring. Although these events are maybe rare, such recombinants exist. Several billion people and innumerable animals have been infected by-y SARS-CoV-2. The appearance of such a variant with new and unexpected properties should not come as a surprise. Instead, we should increase genomic surveillance and immediately characterize such a SARS-CoV-2 variant should it arise. This article is featured on Forbes.org, and can be read online here: Coronaviruses Can Recombine With Cellular And Heterologous Viruses To Create Unexpected Variants

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The Artful Dodger SARS-CoV-2: Evading Immunity By Inhibition Of Interferon Forbes | April 01, 2022 | Article

The Covid-19 pandemic is characterized by successive waves of infection driven by variants that have evolved to become more transmissible and immune evasive. Much of the attention has focused on variations in the spike protein as the primary site for virus neutralization and the main driver of emerging variants. Less attention has been focused on the SARS-CoV-2 virus’s ability to dampen the innate immunity response, our frontline defense against the virus. A new preprint study by Guo et al. from the University of Colorado suggests the evolution of SARS-CoV-2 to evade innate immunity by developing resistance to interferons (IFN), may also contribute significantly to the increase in transmission and immune evasion. Our bodies are equipped with a number of alarms that trigger anti-microbial defenses against novel microbes collectively known as innate immunity. Interferons play a central role in innate immunity. Interferons are proteins that are made and released by host cells in response to the invading microbes. A virus-infected cell releases interferons that in turn stimulate the activation of literally hundreds of antimicrobial and antiviral genes by the infected cells themselves. It is these interferon-stimulated proteins that attack and eliminate the infecting micro-organisms. Figure 1 illustrates the activation pathway of both interferons and of interferon-stimulated genes.

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Figure 1: The two-panel defense pathway figure that includes SARS-CoV-2 viral proteins with immunosuppressive functions. The figure shows: the nonstructural proteins NSP1, NSP3, NSP5 (main protease), NSP6, NSP8, NSP9, NSP12 NSP13, NSP14, and NSP16; s ACCESS HEALTH INTERNATIONAL.

Generally, most healthy people suffer either no or only mild symptoms from SARS-CoV-2 infection and the virus has cleared the host well before adaptive immunity via B cell recognition kicks in. It is only when innate immunity fails to control the infection that the disease progresses raising the risk of severe disease or death. Guo et al. examine the potential of new SARS-CoV-2 variants to resist interferon. The results are remarkable. They report that each successive variant is more resistant to interferons than earlier isolates. Interferon sensitivity of SARS-COV-2 variants Guo et al. compared the potency of 17 different human interferons against several viral variants. The experiments were performed between December 2020 and March 2021. During this time period, the ancestral lineages were rapidly replaced by the variants of concern. The researchers selected isolates from prominent lineages during this phase of the pandemic. The isolates used included the original Wuhan isolate (B), the initial variant D614G (B.1), the Alpha variant (B.1.1.7), and the Beta variant ( B.1.351). To test the sensitivity of the virus to interferons, cells were preincubated with the recombinant interferons overnight, then infected 282

with each variant. The total amount of virus was measured 24 hours post-infection. Overall, IFNβ, IFNα8 and IFNω were found to be most potent, followed by INFα5, INFα17, and INFα14. The type III 113 (λ) interferons were found the be the least potent seen in Figure 2 below.

Figure 2: Sensitivity of SARS-CoV-2 variants to diverse Interferons. INTERFERON RESISTANCE OF EMERGING SARS-COV-2 VARIANTS, GUO ET AL, 2022.

The B.1 variant is significantly more resistant to interferons than is the parental Wuhan strain (B). Resistance of the Gamma variant (B1.351) is roughly similar to that of the B.1 variant. However, the Alpha variant is significantly more resistant to interferon treatment than are the b, B.1, and Gamma isolates. Alpha is approximately 100 fold more resistant to interferons than the earlier isolates. Mutations in viral genes that regulate the interferon pathway SARS-CoV-2 is a potent inhibit of the cellular innate immune response. Not only do many of the SARS-CoV-2 inhibit the synthesis if type 1 interferons, they also inhibit interferon-stimulated gene expression. Figure 1 summarizes both pathways and indicates the action of each gene in the pathway. The left side of the panel illustrates inhibition of the interferon induction pathway. The right 283

side of the figure describes the viral genes that inhibit the production of interferon-stimulated genes. It is important to note that viral resistance in these studies reflects only resistance to either induction of the interferon stimulates genes or resistance to the interferonstimulated genes resistant themselves. This is so as interferon is supplied exogenously in these experiments. Figure 3 illustrates the location of SARS-CoV-2 genes along the genome, including the location of the nonstructural replication enzymes (Orf1a and Orf 1b), the viral structural proteins S,E, M and N, and the accessory genes Orfs 3, 6, 7, 8, 9 and 10.

Figure 3: SARS-CoV-2 genomic map of mutations impacting proteins that influence viral immune evasion. Those in red are found in the Alpha variant; those in orange are found in the Beta variant; those in blue are found in the Gamma variant; those in p ACCESS HEALTH INTERNATIONAL.

The figure 3 and table 1 also indicate the location of mutations in each of the genes known to regulate interferon and interferonstimulated gene expression in a set of variants including Alpha, Beta, Gamma, and Delta variants. This observation raises the possibility that changes in these genes increase interferon resistance thereby increasing the increasing immune evasion and transmissibility.

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Table 1: SARS-CoV-2 proteins inhibit the induction of interferon-stimulated genes. ACCESS HEALTH INTERNATIONAL.

Current understanding stops well short of tracing interferon resistance to any of these mutations. Further work is required to examine the properties of viruses that differ only by single mutations in the genes that relate to the interferon pathway. It will be helpful to extend these studies to include all variants of concern including the Omicron family of variants. In summary, Guo et al. make a valuable contribution to our understanding of the sensitivity of viral variants to interferon stimulated genes. It will be equally important to understand the role of variants in triggering the production interferon itself. This article is featured on Forbes.org, and can be read online here: The Artful Dodger SARS-CoV-2: Evading Immunity By Inhibition Of Interferon

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Quantitative Markers for Covid-19 Brain Injury Forbes | April 04, 2022 | Article

This story is part of a larger mosaic of stories on the neurological consequences of Covid-19 and Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part one, two, three and four of this series. Here, we discuss a series of studies examining the objective measures of blood and cerebrospinal fluid (CSF) that may offer clues as to how Covid-19 injures the brain. Estimates show that half of those hospitalized for neurological complications with Covid-19 continue to experience cognitive impairments. Even more concerning are the increasing reports of long-lasting cognitive symptoms among individuals that experienced mild or asymptomatic infections. It is clear that SARS-CoV-2 can significantly damage brain cells without direct infection. Systematic efforts to detect brain injury have failed to provide explicit evidence of inflammatory damage to brain cells. Previously in this series, for example, we featured brain imaging studies suggesting that Covid-induced structural changes in the brain may uncover or accelerate neurodegeneration. More subtle brain damage also may be occurring that is not as obvious. Researchers are looking to identify quantitative biological markers in blood and cerebrospinal fluid that may reveal more widespread Covid-19-related injury. Although hypoxia likely accounts for some of the damage seen in the most severely affected individuals, it is unlikely that hypoxia is the primary mechanism for underlying brain injury, The current hypothesis suggests that structural changes in the brain and the longterm cognitive symptoms may be a consequence of hyperinflammation. Measuring inflammation, however, can be difficult. While some symptoms, such as memory loss, fever, and loss of smell, are more obvious, inflammation can also induce psychosomatic symptoms, including fatigue, insomnia and body aches, that are subjectively reported. Identifying neurological biomarkers for inflammation may provide an objective measure for

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the ongoing neurological symptoms that millions of people have experienced throughout the pandemic. Inflammatory Biomarkers in CSF Unlike blood, cerebrospinal fluid is exclusively found in the central nervous system. Measuring inflammatory biomarkers in CSF can, therefore, provide a direct marker for possible injuries in the brain and spinal cord. For instance, when brain tissues shrink as with old age, increased CSF fills the gaps to maintain the brain’s structural integrity. In the case of an infection, the protein contents of CSF may change as a result of inflammation in the brain and other peripheral tissues. According to a recent study from Switzerland, pro-inflammatory changes in CSF may be linked to structural changes in the brain generated by Covid-19. One of the major reasons Sanabria-Diaz et al. chose to investigate CSF in the first place is that it provides insight into the integrity of the blood-brain barrier. The CSF/bloodalbumin ratio, in particular, is useful for determining the level of albumin in CSF compared to blood. A high CSF/blood-albumin ratio indicates that the permeability of the blood-brain barrier has been altered, often seen in multiple sclerosis and other neurodegenerative conditions. For their investigation, Sanabria-Diaz et al. selected individuals hospitalized for Covid-19 related neurological complications. Compared to those not exposed to Covid-19, these individuals had significantly high CSF/blood-albumin ratios that positively correlated with widespread reductions in the gray matter of orbitofrontal, frontal and temporal brain regions. This suggests that structural changes caused by Covid-19 may correspond with disruption of the blood-brain barrier, but it is unclear whether one causes the other. To more directly measure inflammation, investigators also tested for three cytokines in the CSF: plasma-receptor for advanced glycation end-products binding protein (EN-RAGE), plasma-tumor necrosis factor-related activation-induced cytokine (TRANCE), and osteoprotegerin (OPG). The most significant negative correlations were seen between EN-RAGE cytokines, of which are known to activate inflammatory cascades, and cortical thickness in seventeen brain regions. Interestingly, enhanced EN-RAGE levels also correlated with increased volume in other parts of the brain. 287

Finally, Sanabria-Diaz et al. measured levels of lactate in CSF through a test often used to differentiate between viral and bacterial meningitis. Not only did individuals exhibit enhanced lactate levels corresponding with viral infection but the presence of lactate in CSF also negatively correlated with brain volume in orbitofrontal, frontal and temporal regions. Sanabria-Diaz et al. concluded that brain damage may be a consequence of a secondary parainfection complication likely arising from the robust inflammatory response to SARS-CoV-2 that alters the composition of CSF and impairs the blood-brain barrier. Whether these abnormalities may lead to permanent damage remains to be determined. Inflammatory Biomarkers in Blood Elevated levels of inflammatory biomarkers have also been detected in the blood. In a recent study from the NYU Grossman School of Medicine, individuals hospitalized with Covid-19 displayed significantly high levels of inflammatory neurodegenerative biomarkers in their blood. Despite no previous history of dementia or cognitive deficits, these individuals experienced worse outcomes associated with encephalopathy, or injury to the brain. When the body encounters SARS-CoV-2, a robust immune response launches to release various immune cells, such as cytokines, into the bloodstream. In addition to cytokines that can cross through the blood-brain barrier, the brain may also produce its own inflammatory proteins to protect brain cells from injury. Studies of chronic inflammation in the brain indicate that enhanced production of pro-inflammatory neurodegenerative biomarkers, particularly tau proteins, neurofilament light chain (NFL) and glial fibrillary pacific protein (GFAP), may serve as an indicator that neurons are not only being injured but also glial supportive cells. First, Frontera et al. asked if blood biomarkers for neuronal and glial injury correlated with the onset of new neurological complications among those hospitalized for Covid-19. Indeed, individuals with neurological symptoms displayed significant levels of NFL, GFAP, tau and several other neurodegenerative biomarkers, compared to pre-pandemic blood samples. Even when race, age, and pre-existing conditions were controlled, individuals with increased

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levels of NFL, GFAP, and tau, in particular, were less likely to be discharged home. Next, Frontera et al. found that higher levels of inflammatory biomarkers correlated with increased disease severity and worse outcomes. Especially high elevations were seen in those diagnosed with toxic-metabolic encephalopathy, a form of brain injury that can produce behavioral changes, induce seizures, and alter consciousness. These individuals were also more likely to require mechanical ventilation, suggesting a possible link to hypoxia in the brain. Interestingly, enhanced levels of NFL, GFAP and tau biomarkers have also been implicated in the development of Alzheimer’s Disease. As Alzheimer’s progresses, amyloid plaques and tau neurofibrillary tangles accumulate in the brain leading to worsening cognitive function. Build up of these proteins induces chronic inflammation in the brain, consequently damaging neurons and supportive glial cells. Animal models of Alzheimer’s disease also seem to suggest that hypoxia, resulting from extensive build of these proteins, can phosphorylate, or activate, tau proteins prompting greater build up in the brain and more inflammation. Therefore, investigators speculate that the combined consequences of hypoxia and hyperinflammation caused by Covid-19 may capitalize on the pathology of Alzheimer’s Disease to produce widespread damage in a short period of time. To their surprise, Frontera et al. found that some hospitalized individuals had more circulating inflammatory biomarkers than Alzheimer's patients not exposed to Covid-19. This may be linked to the robust immune response unique to SARS-CoV-2 infection. For example, inflammatory cytokines produced to fight the virus have been shown to injure endothelial cells lining blood vessels, including those that make up the blood-brain barrier. Not only does this disrupt cognitive function but animal studies have also shown that the activation of these cytokines can prompt the development of amyloid plaques and neurofibrillary tau tangles. The pathology of Covid-19, however, cannot be entirely conflated with Alzheimer’s Disease. Frontera et al. did not find any correlations between SARS-CoV-2 and the development of beta amyloid plaques, a significant hallmark for Alzheimer’s Disease. Also, blood tests alone cannot differentiate between an Alzheimer’s diagnosis or another form of dementia. Since age seems to correlate 289

with Covid-19 symptom severity, it is possible that infection may uncover or accelerate late-onset neurodegeneration in some individuals. Does this mechanism extend to neurological complications associated with long Covid? A recent study from researchers at Northwestern University Feinberg School of Medicine compared blood samples of individuals currently hospitalized for Covidinduced encephalopathy to those that were not hospitalized during infection, as well as those previously hospitalized for other Covid19 complications, but now are experiencing new neurological complications associated with long Covid. Among individuals 50 years and older, the encephalopathy group had significantly high levels of NFL and GFAP, consistent with the previous study. However, the other groups did not differ from the controls, suggesting that the Alzheimer-like consequences of Covid-19 may be unique to the development of encephalopathy during acute infection. Additional investigations are needed to determine whether these changes in the brain will progress into full Alzheimer’s Disease or other forms of dementia. Conclusion Inflammatory biomarkers provide an objective measure for the damage we know is occurring in the brain. Despite the overwhelming evidence from CSF and blood tests that hyperinflammation underlies much of the neurological damage caused by Covid-19, we still do not know why some develop more severe complications than others. Future studies comparing the biological markers found in both CSF and blood may provide greater insight. It is possible that infection uncovers or accelerates neurodegeneration that is already occurring, especially among older populations. Understanding the mechanisms underlying Covid-19 induced brain injury now will help us to better prepare for a growing class of people living with cognitive disabilities. This article is featured on Forbes.org, and can be read online here: Quantitative Markers for Covid-19 Brain Injury

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Two More Members Of The Omicron Family To Keep An Eye On Forbes | April 06, 2022 | Article

As countries worldwide relax Covid restrictions to pre-pandemic levels, the Omicron family of variants continues to prove that now is not the time to take off your mask and go to a crowded event. New variants of Omicron, the most transmissible virus in decades, appear week after week, each with a unique addition that may make the virus that much more infectious. Here we examine some of these emerging substrains and how they could impact viral characteristics like transmission, immune evasion, and virulence. Omicron XE In a previous publication for Forbes, we discussed Omicron and its ability to recombine with other viruses. To recount, recombination is the exchange of one portion of a virus's viral genome for another during the replication process. This is possible when a host is coinfected with two different viruses; the replicating polymerase of one virus replicates a portion of the other virus by mistake, incorporating it into its genome. The UK Health Security Agency has identified at least three of these recombinants worth mentioning within the past few weeks. The first two are Omicron XD and XF. Both of these recombinants join together portions of Omicron and Delta. We have previously discussed the implications of Deltacron recombinants in Forbes. Here we focus on Omicron XE, a recombinant of the BA.1 and BA.2 substrains of Omicron.

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FIGURE 1: Three new Omicron recombinants described by the UK Health Security Agency. TOM PEACOCK TWITTER

As the figure above illustrates, XE recombines in the Orf1a region of the virus. Orf1ab is the replication machinery of SARSCoV-2, meaning proteins in Orf1ab affect replication efficiency and virus load in a host. Omicron XE recombines at nucleotide position 11,537. This lies in nonstructural protein (NSP) 6. All nucleotides before this position resemble BA.1, and all those after resemble BA.2. In addition to recombining between the two Omicron substrains, Omicron XE includes three novel mutations. Two of these mutations lie in NSP3: synonymous nucleotide mutation C3241U and amino acid mutation valine to isoleucine at position 1069 (V1069I). The amino acid mutation V1069I is located in the papain-like protease, which oversees the processing of viral polyproteins. The mutation is present in several thousand Omicron family samples in the GISAID sequence database. The other lies in NSP12: synonymous nucleotide mutation C14599U. We also note the synonymous nucleotide mutations as they could impact cisacting regulatory sequences. This recombinant is particularly interesting because of its potential transmissibility. The UK Health Security Agency found that BA.1 was the most transmissible virus in several decades, with some estimates showing two to four times greater infectivity than 292

Delta. BA.2 pushed infectivity even further, yielding a 33% increase over BA.2 according to Charles Schmidt in Scientific American. Early indications from the United Kingdom suggest that indeed XE, may be more transmissible than prior variants. While less than 1,000 identified cases are confirmed to be the XE variant, genomic comparison between XE and its parental viruses shows that the new recombinant is 9.8% more transmissible than BA.2, a modest increase but a significant one. This was enough for the World Health Organization to issue a formal warning over Omicron as the most transmissible version of Covid to date. Yet Another Variant: BA.2 + L452R Recent reports highlight the potential danger of yet another variant: BA.2 + L452R. This variant is identical to the BA.2 genome with the addition of leucine to arginine at position 452 (L452R) in the receptor-binding domain of the Spike protein. The L452R mutation is present in Delta, Kappa, and Lambda variants of concern and interest. The mutation increases both the infectivity and virulence of SARS-CoV-2, triggering concern about the potential epidemiology of this newly detected variant.

FIGURE 2: BA.2 + L452R Genome and Spike protein. Mutations in blue are those shared with BA.2 and ... [+] ACCESS HEALTH INTERNATIONAL

Early reports of BA.2 + L452R have mostly been isolated to South Africa and are relatively limited. However, if the virus is more 293

severe and infectious, we expect cases caused by this subvariant to rise rapidly. Ding et al. conducted a study in which they developed an L452R-mutated BA.1 virus to analyze the mutational impact. They found that BA.1 + L452R increased fusogenicity and strengthened the high infectivity of Omicron by enhancing the cleavage of the Spike protein. Additionally, The mutated BA.1 enhanced Omicron infection of lung tissue in humanized ACE2 mice. We fully anticipate additional Omicron variants to appear over the coming weeks. It will be important to track the course of each variant to determine which, if any, poses the greatest threat. This article is featured on Forbes.org, and can be read online here: Next Article

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Innate Lymphoid Cells And Covid-19 Severity: Chicken Or The Egg? Forbes | April 07, 2022 | Article

This is part of a series of stories on the protective role of innate lymphoid cells (ILCs) in the gut and other tissues, focusing on SARS-CoV-2 infection. Read part one, part two, part three, part four, and part five. Recent discoveries point to the importance of innate lymphoid cells (ILCs) in the protection from disease, including Covid-19. New research by Silverstein et al. suggests that lower blood ILC levels may help predict disease outcome. In particular, the decrease of ILCs with age correlates strongly with an increased risk of severe Covid-19. They also show that the difference in concentrations of blood ILCs between males and females may contribute to the increased susceptibility of males to serious disease. Innate Lymphoid Cells and Disease Tolerance Innate lymphoid cells are generally found in “barrier environments” —areas of our body that are constantly exposed to the outside world, like our lungs and gastrointestinal tract— where they play an active role in regulating homeostasis. This includes responding to invasive and potentially harmful microorganisms. When faced with an intrusive pathogen, our body’s first priority is to contain it. This is where our immune system comes in, kickstarting inflammation and redirecting immune cells to areas where they are most needed. Both the infectious pathogen so well as our inflammatory response can cause serious tissue damage. So a secondary priority of the immune system is making sure that this damage is kept in check, known as “disease tolerance”. Although disease tolerance mechanisms do not directly engage or eliminate the

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pathogen, they work to sustain overall host health during infection, curbing disease severity. In the case of SARS-CoV-2, the amount of virus circulating through a patient’s body, known as viral load, doesn’t neatly correlate with Covid-19 severity; some people, children in particular, can have very high viral loads while remaining asymptomatic or only mildly symptomatic. This suggests disease severity in Covid-19 is determined more directly by age-dependent, disease tolerance mechanisms than by viral replication and viral loads. Prior studies have indicated that ILCs may contribute to disease tolerance, with one specific subset of the ILC family, group 2 innate lymphoid cells (ILC2s), having an especially active hand. In response to tissue damage, ILC2s can produce a protein called amphiregulin (AREG). By binding to epidermal growth factor receptors (EGFR), amphiregulin helps stimulate cell growth, cell survival, and cell migration. In doing so, ILC2-induced amphiregulin helps repair damaged tissue, maintaining the integrity of the epithelial lining in the lungs and in the intestine. But determining to what extent ILC levels contribute to Covid19 severity is trickier than it may initially seem. For one, there are multiple factors that increase the risk of progressing to severe Covid19. Two of the most common are age and sex: men are more likely than women to develop serious disease, and similarly, the older you are the higher the risk. Added to this is the fact that the levels of lymphocytes —a class of white blood cells made up of T and B cells, as well as ILCs— are already reduced during SARS-CoV-2 infection, a phenomenon known as lymphopenia. Finally, lymphocyte levels also vary according both to age and sex, decreasing with age and lower in men right off the bat. The challenge for Silverstein et al. was establishing whether blood ILC levels were altered in Covid-19, independently of the aforementioned factors— age, sex, and global lymphopenia. Additionally, whether ILC levels correlated with Covid-19 severity. To do this, the researchers compared blood samples from three groups: adults hospitalized for Covid-19, adults treated for Covid19 who didn’t require hospitalization, and a control group of adults who had not been infected with SARS-CoV-2. The researchers first sought to calculate a baseline for age- and sex-related lymphopenia, giving them a reference point with which 296

to compare the lymphopenia seen in Covid-19 patients. To do so, they collected peripheral blood mononuclear cell readings from 103 SARS-CoV-2-negative blood donors. Peripheral blood mononuclear cells are a large collection of immune cells, including lymphocytes, that circulate in the blood, acting as a first line of defense in response to invading pathogens. The donors ranged from 2 to 79 years of age, with a nearly equal number of males and females. Innate Lymphoid Cells Decrease Dramatically with Age Silverstein et al. zeroed in on the lymphocyte readings, focusing on the four major subsets: CD4+ T Cells, CD4+ B Cells, ILCs, and CD16+ natural killer cells (NK cells). Although all subsets were affected by age, only ILCs displayed consistently significant differences across all age groups. The median decrease for ILCs was 2-fold every 20 years, with a 7-fold absolute decrease from the youngest age group to the oldest. The age-dependent decrease in ILCs was mirrored closely by the aggressive, age-dependent increase in Covid-19 mortality (Figure 1).

FIGURE 1. Blood ILC levels by age (left) are closely correlated with Covid-19 mortality rate by age ... [+] FROM: “INNATE LYMPHOID CELLS AND COVID-19 SEVERITY IN SARS-COV-2 INFECTION” SILVERSTEIN ET AL. 2022

Men Have Fewer Innate Lymphoid Cells than Women The team of researchers also discovered that ILC levels in males were naturally lower than they were in females (Figure 2). Again, this correlates with the fact that males are more likely to suffer from severe Covid-19 than their female counterparts. No other 297

lymphocyte subset, with the exception of CD4+ T cells, displayed such a pronounced decrease on the basis of sex.

FIGURE 2. Blood ILC levels by sex (left) and blood CD4+ T cell levels by sex (right). SILVERSTEIN ET AL. 2022

The Lower the ILC Levels, the Higher the Risk of Hospitalization Next, the group of researchers set out to confirm if lymphocyte levels are actually lower in Covid-19 patients once the effects of age and sex are factored in. They determined that hospitalized Covid19 patients displayed a 1.33-fold reduction in total lymphocytes among PMBCs as compared to their control group. This included 1.8-fold fewer ILCs and 2.3-fold fewer CD16+ NK cells as compared to the control group (Figure 3). No such significant decrease could be seen in T and B cell levels. For the group of patients with less severe Covid-19, there was no decrease in ILCs and only a slight decrease in CD16+ NK cells.

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FIGURE 3. Comparison of ILC and CD16+ NK cell blood levels across control, hospitalized, and ... [+] SILVERSTEIN ET AL. 2022

Importantly, as ILCs decreased, both the risk of hospitalization and the duration of hospitalization increased considerably. Every 2fold decrease in ILCs meant a 55% higher chance of hospitalization and an extra 9.4 days of hospital stay. None of the other lymphocyte subsets influenced rate and duration of hospitalization in the same way. On top of this, lower levels of blood ILCs were accompanied by an increase in common inflammatory markers. Although this study establishes a clear correlation between ILCs and Covid-19 severity, the authors admit that the direction of the causal arrows remains a little unclear: which came first, the chicken or the egg? Do low blood ILC levels predispose us to severe Covid19, or does severe Covid-19 cause our blood ILC levels to drop? Silverstein et al. think it’s more likely for low ILC levels to be driving Covid-19 severity, not the other way around. First, unlike other lymphoid cells, ILCs decrease exponentially with age. This decrease is mirrored very closely by an age-dependent, exponential increase in Covid-19 mortality. Second, males naturally have lower levels of ILCs in their blood, including those that produce amphiregulin, and they also happen to have a greater risk of progressing to severe Covid-19. Finally, conditions which independently correlate with lower levels of blood ILCs, like HIV-

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1 and obesity, heighten the risk of being infected with, and getting severely ill from, SARS-CoV-2. This work adds to the ever-growing list of research labeling innate lymphoid cells an integral part of the innate immune response. Not only in challenging invading pathogens, but also in contributing to disease tolerance. Hopefully these insights inspire others to pay more attention to ILCs, their multifaceted impact on SARS-CoV-2 infection, and beyond. This article is featured on Forbes.org, and can be read online here: Innate Lymphoid Cells And Covid-19 Severity: Chicken Or The Egg?

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Head in the Clouds: Living with Covid-19 Forbes | April 07, 2022 | Article

Emerging studies have increasingly recognized Covid-19 as an inflammatory disease. Brain shrinkage, brain-blood barrier disruptions and neurodegeneration, as we previously reported in this series, seem to emerge as an inflammatory consequence of acute infection that for some progresses into Long Covid. Cognitive impairments are consistently reported as one of the most persistent and some of the more impairing symptoms of Long Covid. Brain fog, characterized by sluggish or slow executive function, and memory problems, for example, can severely interfere one’s ability to return to work and other daily activities. The broader implications of these symptoms could significantly disrupt the economy and society as a whole. Although the long term effects remain unclear, the immediate repercussions are just now being uncovered. In the journal Frontiers in Aging Neuroscience, researchers from the University of Cambridge recently published their first findings from their longitudinal online study: COVID and Cognition. As Dr. Lucky Checke, a professor at the University of Cambridge' Department of Psychology and senior author of the study, says, “"Long COVID has received very little attention politically or medically. It urgently needs to be taken more seriously, and cognitive issues are an important part of this. When politicians talk about 'Living with COVID' – that is, unmitigated infection, this is something they ignore. The impact on the working population could be huge," Here, we discuss the early findings from this study, which considers how cognitive impairments associated with Long Covid may develop from acute infection regardless of disease severity.

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Guo et al. reported their finding in two parts. The first asked 481 adult participants to complete questionnaires related to previous Covid-19 infections and the prevalence of ongoing symptoms. To provide a more objective measure of cognitive impairments, the second investigation employed a series of online cognitive tests to the same sample of participants. Since this data was collected between October 2020 and March 2021, infections were primarily linked to wild-type Covid-19 and alpha strain. Self-Reported Symptoms Although almost all the individuals sampled for this study were not hospitalized for Covid-19, Guo et al. found that more severe disease during the acute phase was associated with ongoing symptoms. In fact, ongoing symptoms were more severe among those who experienced more severe initial infections, compared to individuals who experienced mild or moderate infections. Those that reported limb weakness, in particular, were more likely to experience severe long-lasting symptoms following infection. The severity of the initial infection therefore may be a strong predictor for ongoing symptoms associated with Long Covid. More specifically, disease severity seems to correlate with selfreported cognitive impairments. This includes fatigue and related symptoms such as headache, brain fog and dizziness, in addition to neurological symptoms such as abnormal vision, altered consciousness, delirium and disorientation. Guo et al. speculate that the presence of these symptoms may involve neurological mechanisms related to inflammation and encephalitis, consistent with previous studies. To their surprise, investigators also found strong correlations between ongoing cognitive impairment and gastrointestinal and cardiopulmonary complications. Given that there is no evidence that SARS-CoV-2 directly infects the brain, this finding may suggest that brain injury is instead a consequence of greater systemic inflammation throughout the body. Both cardiopulmonary symptoms and fatigue, in particular, seemed to correlate with longer acute infection periods. It is possible that these symptoms manifest through similar mechanisms related to damage to blood vessels, or they may simply be two of the most commonly reported symptoms associated with long-lasting illness. Implications for Cognitive Performance 302

Part two of Guo et al.’s investigation assessed the same sample of participants on cognitive tests for memory, executive function, and language. Participants were asked to complete six tasks: the Word List Recognition Memory Test, Pictorial Associate Memory Test, Category Fluency Test, Mental Rotation Test, Wisconsin Card Sorting Test, Number Counting Test, and Relational Reasoning Test. Consistent with self-reported symptoms, individuals exposed to Covid-19 displayed significant deficits associated with memory. In fact, regardless of ongoing symptoms, those previously infected with the virus performed worse on memory tasks but not other cognitive domains. This was most evident during verbal memory tasks, during which these individuals had much slower reaction times. Once infection severity was taken into account, those with ongoing symptoms performed worse than recovered individuals, and memory deficits once again seem to be a significant factor. Worse verbal memory, in particular, was associated with greater severity of ongoing symptoms. Those with the most severe symptoms not only had slower reaction times but also recorded fewer correct answers. Although the results for non-verbal associative memory were less clear, Guo et al. reported that individuals that had not been exposed to Covid-19 displayed a clear advantage in their performance. A major limitation of conducting tests for cognition online is determining whether the findings actually vary with the hypothesis being tested or whether the particular participants selected for the study were simply having a “bad day.” This is made more difficult by the fact that Long Covid seems to be a cyclical illness. Although increasing reports show that the severity of symptoms change over time, we still do not know how or when cognitive symptoms may change during Long Covid. With this in mind, Guo et al. also asked participants to report if they were having a “bad day”, or the extent to which any ongoing symptoms were affecting their well-being at the time of the task. Indeed, they found that having a “bad day” did not predict cognitive performance on any tasks, thus strengthening the hypothesis that general severity of ongoing symptoms correlates with cognitive impairments. How did the subjective reports of impaired cognitive performance compare to actual performance? Interestingly, not every self-reported cognitive symptom was confirmed by cognitive 303

testing. While those that reported memory problems scored lower on memory tests, individuals that reported linguistic deficits did not perform any worse on language tasks. It is possible, however, that the tests selected were not sufficient to detect these deficits. Conclusion Investigators are hopeful that future publications from the COVID and Cognition Study will provide a greater understanding of how cognitive symptoms and performance change throughout Long Covid. Although these complications likely will not be permanent, it is important to recognize that “Living with Covid” will not look the same for everyone. Some people may experience more noticeable impairments in memory, executive function and attention than others. We urge lawmakers to not only consider the economic consequences but also the personal challenges associated with Long Covid. Guiding care for these individuals needs to be a political priority as this pandemic transitions into an endemic. This article is featured on Forbes.org, and can be read online here: Head in the Clouds: Living with Covid-19

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S100s: A Blunderbuss Approach To SARS-CoV2 Defense Forbes | April 09, 2022 | Article

One of the primary anomalies of SARS-CoV-2 infection lies in our gut. Our intestine spans hundreds of feet in surface area and is rich with ACE2 receptors—the key receptors by which SARS-CoV-2 infects cells. Yet, most of the identifying SARS-CoV-2 symptoms are isolated to respiratory illnesses. Why is it that even though the gut is an ideal entry point for SARS-CoV-2 infection, few patients report any intestinal symptoms? In previous installments of this series, we have discussed several different molecules that appear in the intestine’s natural defense system including type 3 innate lymphoid cells (ILC3s), interleukins, and defensins. Now, we introduce an unsung hero of the gut that offers even greater protection against the effects of infection— S100s located in the intestine. S100s are proteins characterized by their ability to bind to calcium. There are currently 25 known members of the S100 family, and they are typically found in neural cells such as dendritic cells and chondrocytes but can be found in other cells of the body as well. S100s are categorized into three subgroups: those that perform only intracellular functions, S100s that possess both intracellular and extracellular roles, and S100s that only have extracellular effects. Because the scope of the S100 family is so broad, S100s have been implicated in several key immune functions including apoptosis and inflammation.

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Figure 1: Structure of S100B.EMW, CC BY-SA 3.0 <HTTPS://CREATIVECOMMONS.ORG/LICENSES/BY-SA/3.0>, VIA WIKIMEDIA COMMONS

One of the key features of S100s is that they are damageassociated molecular pattern molecules (DAMPs). DAMPs are messenger molecules. When a cell is damaged by trauma or a pathogen, it will release DAMPs into the extracellular space. The DAMPs can then interact with other cells to communicate that there may be danger nearby by inducing an inflammatory response.

Figure 2: S100s can trigger inflammation and other defense mechanisms through multiple pathways.CHANG, X. ET AL. "S100 PROTEINS AS AN IMPORTANT REGULATOR OF MACROPHAGE INFLAMMATION". FRONTIERS IN IMMUNOLOGY. 2018. DOI: 10.3389/FIMMU.2017.01908

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S100s are particularly effective DAMPs because there are so many different forms of the molecule, each with the ability to bind to 1 or more receptor types (Figure 2). Since cells often have unique receptors on their cell membrane, the versatility of the S100 family allows them to communicate the danger of a nearby infection to several, if not all cells. Once an S100 binds to a receptor on the cell membrane, this triggers a chain of phosphorylation events within the cell, ultimately leading to the production of AP1 or NFkB. Both AP1 and NFkB are transcription factors that are crucial for triggering several cell defense mechanisms, including inflammation. How do S100s relate to the gut? While S100s are generally found in neural cells, they are also found in the gut and in the cytoplasm of white blood cells called neutrophils. When viruses invade the gut, they activate interleukin-3 (IL3) molecules. These IL3 molecules then activate interleukin-22 (IL-22). IL-22 stimulates the production of several antimicrobials including defensins and S100s. Once S100s have been produced by IL-22, they are free to trigger or amplify an inflammatory response in the gut against a virus. Inflammatory responses are built to protect the body against infection. However, much like a fire becomes deadly when it is out of control, overactive or late-stage inflammatory responses can often become lethal. Now, researchers at Peking University have found that not only are S100s a marker for severe SARS-CoV-2 infection, but they may be responsible for late-stage hyper inflammation and severe SARSCoV-2 symptoms. Guo et al. focused their project on exploring how S100s contribute to the pathogenesis of SARS-CoV-2. To investigate this relationship, the researchers infected monkeys (rhesus macaques) with SARS-CoV-2 and analyzed the RNA molecules in the monkeys’ lungs and blood after infection. A Unique Immune Response to Coronavirus Researchers found that within the first few days of infection, the presence of type-1 interferons, T cells, and B cells did not increase. Instead, they discovered several genes that were related to the activity of anti-bacterial pathways. They also found a significant presence of neutrophil marker genes. These results suggested that in the early stages of SARS-CoV-2 infection, the virus provokes an 307

anti-bacterial response and triggers an uptick in neutrophil activity or inflammation. To determine how SARS-CoV-2 infection triggers the activation of anti-bacterial pathways, the researchers then examined which genes were expressed after infection and compared them to the genes expressed before infection. Guo et al. found that there was robust expression of S100A8. The expression of S100A8 also increased dramatically in monkeys that had greater amounts of SARS-CoV-2 in their bodies. These results were consistent with data from SARS-CoV-2 patients. Guo et al. found that when they compared post-mortem lung samples from SARS-CoV-2 patients to healthy lung tissue from uninfected individuals, both S100A8 and neutrophil marker genes were significantly upregulated, confirming that hyper-inflammation had taken place. When these experiments were repeated with mice infected with influenza A virus, encephalomyocarditis virus, herpes simplex virus1, or coronavirus MHV-A59, only coronavirus MHV-A59 was linked to an upregulated expression of S100A8 and neutrophils. Guo et al. then examined the structures of the neutrophils in mice infected with SARS-CoV-2 and MHV-A59, and found that surprisingly, infected mice’s neutrophils were distorted. This suggests that coronaviruses induce an abnormal immune response that causes hyper inflammation and distorted neutrophil cells. Discovering the role of S100s From this, Guo et al. hypothesized that the distorted neutrophils may contribute to the lethality of coronaviruses, but how were the abnormal neutrophils formed? Since S100A8 is found in the cytoplasm of neutrophils, Guo et al. designed an experiment to investigate the role of S100A8 in the formation of abnormal neutrophils and how the small proteins might contribute to the lethality of coronavirus. An interesting aspect of S100A8 is that it binds to S100A9. The two molecules then act together as a single DAMP to activate immune responses in the body. Researchers used a drug called Paquinimod that suppresses S100A9’s ability to cell receptors. By blocking S100A9 activity in infected mice, Guo et al. hypothesized that they could prevent the S100A8/A9 from forming and activating an immune response. 308

When infected mice were treated with Paquinimod, the drug was highly successful at minimizing severe SARS-CoV-2 and MHV-A59 symptoms. Paquinimod significantly alleviated damage to the lungs, decreased the activity of neutrophils, and decreased the level of coronavirus in the mice. Most neutrophils in the Paquinimod-treated mice also returned to their normal structures. In other words, by suppressing S100 activity, researchers successfully rescued mice from fatal coronavirus infection. While Guo et al.’s research suggests that S100s in neutrophils might be more harmful than they are helpful, the discovery of S100A8/S100A9’s role in inflammation suggests that we might need to be paying closer attention to how S100s react to SARS-CoV-2 across the body. So far, we’ve seen that their inflammatory properties help the gut evade the effects of SARS-CoV-2, but their potential to induce hyper inflammation may cause severe symptoms in the lungs. Now that we’re seeing that SARS-CoV-2 causes inflammation-induced brain damage, it will be interesting to discover whether S100s are to blame. Overall, Guo et al.’s research provides significant insight into the intricacies of SARS-CoV-2 and offers a new avenue of research for the development of anti-SARS-CoV-2 medications. This article is featured on Forbes.org, and can be read online here: S100s: A Blunderbuss Approach To SARS-CoV-2 Defense

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In the Eye of the Storm: How Covid-19 Impacts the Eye Forbes | April 11, 2022 | Article

While Covid-19 is commonly associated with infection of the lungs, heart and other vital organs, a growing body of evidence suggests that infection can also impact the eye. Approximately 1 in 10 people exposed to Covid-19 experience at least one eye problem, such as dryness, redness, blurred vision and sensitivity to light. Conjunctivitis, or “pink eye,” may also appear in the early stages of infection, suggesting that it may be one the first markers of Covid19 infection. Given the direct connection between the eye and brain via the optic nerve, infection of the retina could open a window to the rest of the brain. Thanks to a study published by researchers at the Max Planck Institute for Molecular Biomedicine in Germany we may now have a better picture of how SARS-CoV-2 infects cells in the eye and the possible consequences to the rest of the nervous system. To track changes in the eye after exposure to Covid-19, Menuchin-Lasowski et al. used retinal organoids, small threedimensional tissues cultures resembling human retinas. These stemcell derived tissues contain most types of cells found in the retina, including photoreceptors and supporting amacrine and horizontal cells, correctly organized in a layered structure as shown in the figure below.

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FIGURE: ILLUSTRATION OF RETINAL ORGANOID (LEFT) AND ORGANIZATIONAL STRUCTURE OF RETINA (RIGHT). ON THE LEFT, CELL BODIES ARE SHOWN IN DISTINCT LAYERS: GANGLION CELL (GREEN), AMACRINE AND HORIZONTAL CELLS (WHITE), AND MÜLLER GLIAL CELLS (RED). THE SCHEMATIC ON THE RIGHT SHOWS ALL THE RESPECTIVE LAYERS OF RETINAL CELLS (FROM TOP TO BOTTOM): OUTER NUCLEAR LAYER (ONL), OUTER PLEXIFORM LAYER (OPL), INNER NUCLEAR LAYER (INL), INNER PLEXIFORM LAYER (IPL), AND GANGLION CELL LAYER (GCL). VISUAL INPUTS ARE PASSED FROM THE TOP TO THE BOTTOM LAYERS BEFORE RETINAL GANGLION CELLS (SHOWN IN GRAY), DELIVER THAT INFORMATION TO THE BRAIN. FROM: “SARS-COV-2 INFECTS AND REPLICATES IN PHOTORECEPTOR AND RETINAL GANGLION CELLS OF HUMAN RETINAL ORGANOIDS” MENUCHIN-LASOWSKI ET AL. 2022

As these cells grew and reached maturity, investigators exposed the retinal organoids to the SARS-CoV-2 virus. To their surprise, Menuchin-Lasowski et al. found that SARS-CoV-2 not only infected cells but also replicated. This was evident by the production of viral plaques in the cells that were exposed to the virus. The amount of viral plaque significantly increased within the first 24 hours and nearly doubled after another 24 hours. Interestingly, after 72 hours, the amount of plaque decreased back to the levels detected during the first 24 hour time point. Previous studies have observed a similar pattern of Covid-19 infection in human brain organoids. How are cells infected in the first place? Although viral transmission through the eye has not been well described, SARS311

CoV-2 can penetrate mucous membranes that line several organs including the respiratory system, as well as the eye’s surface and eyelids. When a droplet containing the virus lands on the surface of the eye, SARS-CoV-2 likely travels through the mucous membrane into the internal cavity of the eye before eventually reaching the retina found all the way in the back of the eye. Relatively low expression of the ACE-2 receptor used by SARS-CoV-2 to gain entry to cells has been detected in the eye. Strikingly, Menuchin-Lasowski et al. found that blocking this receptor could prevent infection in the retinal organoids, suggesting that ACE-2 may be the primary mechanism for infection in the eye despite low levels of expression. Which retinal structures were directly infected? Retinal ganglion cells seem to be the most vulnerable to Covid-19 infection. retinal ganglion cells are neural cells that span the inner surface of the retina. They receive visual information detected by photoreceptors and passed through horizontal and amacrine cells. Ganglion cells axons then bundle to form the optic nerve, which directly connects to visual areas of the brain Widespread damage to these cells can significantly impair vision. Emerging studies show that degeneration of retinal ganglion cells and/or photoreceptors by Covid-19 can lead to permanent virtual impairments or blindness. Approximately 40% of cells that were infected by SARS-CoV2 in this investigation were retinal ganglion cells, compared to a small percentage of photoreceptors and even fewer bipolar and amacrine cells. Despite being located in the inner layers of the retina, ganglion cells seem particularly susceptible to infection. Reports of pink eye and other eye problems with Covid-19 signal that inflammatory damage is occurring in the eye. It is possible that retinal injury is a consequence of vascular dysfunction around the eye related to the infection of epithelial cells lining blood vessels. Menuchin-Lasowski et al. also speculates that infected retinal ganglion cells may activate inflammatory signal pathways that damage nearby cells. In fact, common retinal injuries generated by Covid-19, such optic nerve swelling and lesions in the ganglion cell layer, seem to be linked to retinal ganglion cells damage. Menuchin-Lasowski et al., for example, reported that infection of the retinal organoids increased expression of several inflammatory genes, consistent with previous findings that SARS-CoV-2 induces 312

a robust immune response. Cytokine IL-33, in particular, was significantly upregulated. The prevalence of IL-33 in the blood of individuals exposed to Covid-19 has been shown to correlate with disease severity. Enhanced IL-33 was also accompanied with increased expression of NLRP1, an inflammatory protein implicated in degenerative eye diseases that cause irreversible blindness. Interestingly, genes associated with DNA repair, recombination and metabolism were significantly downregulated, which could explain why symptoms related to Covid-19 infection continue to linger long after the initial infection. These findings only add to the increasing body of evidence that SARS-CoV-2 is largely an inflammatory disease. Previously the impact of Covid-19 on the eye had not been well understood. "However, our current retina-organoid study shows that infection with Sars-CoV-2 can have direct pathological consequences for retinal ganglion cells, even though visual impairment is not common in patients with Covid-19," said Dr. Thomas Rauen, co-leading author of this study and head of the White Paper Project Group "Brain Organoids: Alternatives to Animal Testing.” These findings may help us to better understand the pathology of Covid-19 not only the eye but also the rest of the nervous system. This article is featured on Forbes.org, and can be read online here: In the Eye of the Storm: How Covid-19 Impacts the Eye

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Two Emerging Viral Adversaries—Nipah And Hendra Virus—May Soon Meet Their Match Forbes | April 11, 2022 | Article

Two emerging viral adversaries—Nipah and Hendra Virus— may soon meet their match. Our recent experience with Covid-19 has taught us to be aware of new and emerging viruses that have pandemic potential. Amongst these viruses, we draw attention to two members of the zoonotic henipavirus genus: Nipah virus and Hendra virus. These two viruses are responsible for outbreaks of encephalitis, or the inflammation of brain tissue, and respiratory illness, with a staggering 50-100% fatality rate. To date, Nipah Virus and Hendra Virus outbreaks have been localized in poorer communities within Bangladesh, India, the Philippines, and Africa. Exciting new work underway may pave a path for preventing these viruses from growing out of hand in the coming years. People have long noted the potential of these viruses to cause much wider outbreaks. The viruses are transmitted both from infected animals to humans via direct contact or contaminated food supplies, as well as from infected human-to-human via direct contact. Person-to-person transmission is most commonly noted in families and healthcare settings. The virus is not airborne but can spread via droplets, such as sneezing or coughing. Nipah Virus Glycoprotein Structure Monoclonal antibodies have proven to be a mainstay for postexposure prophylaxis and treatment of Covid-19. It is therefore of great interest to understand how monoclonal antibodies may work for other viruses and in turn, such knowledge may inform antiCovid monoclonal strategies as well. The following paper by Veesler et al. is a welcome insight. It elucidates both the structure of the Nipah virus external protein, as well as describes a neutralizing antibody combination that could overcome both Nipah Virus and Hendra Virus. This information may provide a pathway for future treatments and vaccines to follow. 314

The Nipah Virus binds and fuses to a host cell via the glycoprotein. This protein comes in sets of four, or as a tetramer, like a claw in an arcade crane game with four arms. It is an asymmetrical tetramer consisting of a stalk, neck, linker, and four heads. The Nipah tetramer binds ephrin-B2 or ephrin-B3, which are transmembrane protein tyrosine kinases on the host cell surface.

FIGURE 1: Architecture of the NiV G homotetramer. (A) Linear representation of the NiV G ectodomain ... [+] VEESLER ET AL.

The figure above is a beautiful depiction of the tetramer. The amino terminus of the glycoprotein is a stalk, which descends from the protein as an alpha helix to the membrane. Next is the neck 315

region which connects the stalk to the large head region comprising over two-thirds of the glycoproteins amino acids. We emphasize the asymmetric nature of the tetramer. Many viruses, including HIV and coronaviruses, form symmetric trimers or sets of three, but Nipah Virus is an asymmetric tetramer, wherein only one head binds. A detailed understanding of virus Spike proteins allows for a more intricate analysis of potential antibody treatments, such as the following. Three Monoclonal Antibodies For Nipah Virus And Hendra Virus After modeling the Nipah Virus glycoprotein in clear detail using cryo-electron microscopy, Veesler et al. then began testing a number of potential antibody candidates in hopes of finding a treatment for the disease. Blocking The Receptor-Binding Site: m102.4 One antibody, m102.4 targets the receptor-binding domains of the tetramer’s individual monomers, essentially mimicking the binding surface of the host cell and preventing transmission. It directly competes with ephrin binding sites, blocking any chance of the virus binding to the cell surface. This antibody recently completed a phase 1 clinical trial in Australia against Hendra Virus or Nipah Virus infection. With IC50 values ranging between 17-58 ng/mL, the neutralization potency was relatively solid against both Nipah Virus and Hendra Virus. Interfering With Fusion-Triggering Mechanism: nAH1.3 The second antibody, nAH1.3, was isolated from a mouseadapted Nipah Virus infection back in 2016. The nAH1.3 antibody targets each Nipah Virus head domain, interacting with antigenic sites away from the receptor-binding domain. Unlike m102.4, nAH1.3 does not impact binding but rather inhibits HNV entry into cells by interfering with fusion triggering mechanisms. If the virus is unable to fuse, it cannot transmit its RNA into the host cell and continue to reproduce within the host. The IC50 values range between 33 and 32 ng/mL for the Nipah Virus and Hendra Virus viruses, which the authors note is a similar neutralizing capability to that of m102.4.

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FIGURE 2: Superimposition of the NiV G head domain (blue surface) bound to nAH1.3 (heavy and light ... [+] VEESLER ET AL.

Antibody Synergy Against SARS-CoV-2 and other viruses, combination antibody treatments have been used to give antibodies an edge against escape viruses by targeting multiple nonconflicting sites. Both m102.3 and nAH1.3 binding to the Nipah Virus and Hendra Virus tetrameric glycoproteins in a combination treatment resulted in a highly synergistic neutralization. Synergy is determined by a ZIP score, of which greater than ten indicates a stronger combination together than separate. For Nipah Virus, the ZIP score of the two antibodies was 10.628 and for Hendra Virus, the score was 17.248.

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FIGURE 3: Superimposition of the NiV G head domain (blue surface) bound to nAH1.3 (heavy and light ... [+] VEESLER ET AL.

Yet Another Arrow In The Quiver: HENV-32 They also note a third antibody, HENV-32, which binds the neck and stalk regions of the viruses. These regions are typically more conserved among the henipavirus genus, indicating potential cross-reactivity. Based on its target region, HENV-32 would be very unlikely to negatively interfere with nAH1.3, m102.3, or their synergistic effects. A triple antibody combination of the three could potentially be our most effective strategy for treating Nipah Virus and Hendra Virus in the near future.

FIGURE 4: Surface representation of the NiV G tetramer with three head domainspecific Fabs bound ... [+] VEESLER ET AL.

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This paper is a dramatic understanding of the structure and function of the Nipah Virus that should lead to a strong step forward in attempts to both prevent and control infections. We urge continued work by researchers and pharmaceutical companies to develop vaccines, small molecule drugs, and monoclonal antibodies. These medical advances could treat current epidemics of viruses such as these and prevent the potential for future pandemic spread of a deadly pathogen. This article is featured on Forbes.org, and can be read online here: Two Emerging Viral Adversaries—Nipah And Hendra Virus—May Soon Meet Their Match

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Remdesivir Resistance Reported Upon Treatment Of An Immunocompromised Patient Forbes | April 12, 2022 | Article

Remdesivir has been approved as a stand-alone therapy for the treatment and prevention of COVID-19. One of the questions that arises with any single drug treatment of an RNA virus is the level of resistance. Resistance is documented for remdesivir in vitro, however, to date no example of remdesivir resistance has been documented. Here we describe research from a team at Yale University, that documents SARS-CoV-2 remdesivir resistance in a patient with an unusually prolonged infection. Clinical description Gandhi et al. treated a 70 year old female, immunocompromised patient who contracted a prolonged SARS-CoV-2 infection. Figure 1A shows the patient’s time course and symptoms. During the initial period, she suffered from persistent fever, anosmia with intermittent cough and rhinorrhea. The viral load of the patient was followed throughout the course of her infection, a spike was detected on day 148. At this point, it was decided to treat the patient intravenously with a ten day course of remdesivir. The treatment resulted in the termination of the patient’s fever, normal CRP, and improvement on opacities of her chest computed tomography (CT). However, by day 160 there was a steady increase in the amount of virus produced as judged by PCR. The patient was treated with antibodies on day 163 for a week, specifically casirivimab-indevimab, until symptoms and tests came back normal. Following antibody treatment her recovery was carefully monitored for the remainder of her stay.

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Figure 1. Symptoms and clinical discourse of immunocompromised patient with SARSCoV-2. Patient (a) ... [+] GANDHI, SHIV, ET AL. “DE NOVO EMERGENCE OF A REMDESIVIR RESISTANCE MUTATION DURING TREATMENT OF PERSISTENT SARS-COV-2 INFECTION IN AN IMMUNOCOMPROMISED PATIENT: A CASE REPORT.” NATURE COMMUNICATIONS, VOL. 13, NO. 1, 2022, HTTPS://DOI.ORG/10.1038/S41467-022-29104-Y.

Isolation of remdesivir resistant the virus Although the antiviral drug alleviated symptoms, there was a recurrence of viral shedding which suggested resistance had occurred. To confirm this suspicion the virus was isolated from infected patient nasopharyngeal tissue, saliva, stool, and blood samples to clone the viral genome. Upon sequencing of the virus, Gandhi et al. noted the appearance of a mutation at E802D seven days after the initial dose of remdesivir. The mutation E802A had been previously associated with remdesivir resistance in vitro. In addition to E802D, mutations A504V in nsp14 (exonuclease) and I115L in nsp14 (endoRNAse), were also found in the isolated virus. Resistance to remdesivir decreases viral fitness To determine if E802D did in fact confer resistance, the mutation was cloned into an isogenic SARS-CoV-2 strain and resistance to remdesivir was measured. Figure 2 shows that both the E802D/A mutations are resistant to remdesivir. E802D causes an overall 4.2 fold increase in drug resistance while E802A has a 2.7 fold resistance. As Gandhi et al. noted, the fitness disadvantage is only noticed in the absence of remdesivir. In the presence of remdesivir the E802D variant has a marked growth advantage. The implication is that it is unlikely that this particular mutation will spread through the population.

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Figure 2. Inhibition of SARS-CoV-2 by remdesivir at 48hrs post-infection. GANDHI, SHIV, ET AL. “DE NOVO EMERGENCE OF A REMDESIVIR RESISTANCE MUTATION DURING TREATMENT OF PERSISTENT SARS-COV-2 INFECTION IN AN IMMUNOCOMPROMISED PATIENT: A CASE REPORT.” NATURE COMMUNICATIONS, VOL. 13, NO. 1, 2022, HTTPS://DOI.ORG/10.1038/S41467-022-29104-Y.

Structural analysis Figure 3 illustrates the 3-dimensional diagram of the location of the E802D mutation within the nsp12 RNA-dependent RNA polymerase as determined by cryo-electron microscopy. The E802D mutation occurs in the active site. The substitution at E802D is a substitution of aspartic acid for glutamic acid in the active site of nsp12 polymerase. Note that the two amino acids that lead to a mutation, alanine and aspartic acid, are shorter than glutamic acid. The mechanism by which this leads to remdesivir resistance is not immediately evident. The authors speculate it may either permit elongation at the site of insertion of remdesivir or exclude remdesivir from binding. Of the two it seems more likely that a more open configuration allows for elongation of the growing chain past the remdesivir insertion.

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Figure 3. Structure of remdesivir resistance mutation (left) crystal structure depicting interaction ... [+] GANDHI, SHIV, ET AL. “DE NOVO EMERGENCE OF A REMDESIVIR RESISTANCE MUTATION DURING TREATMENT OF PERSISTENT SARS-COV-2 INFECTION IN AN IMMUNOCOMPROMISED PATIENT: A CASE REPORT.” NATURE COMMUNICATIONS, VOL. 13, NO. 1, 2022, HTTPS://DOI.ORG/10.1038/S41467-022-29104-Y. CHEMDRAW (RRID:SCR_016768).

As mentioned, other mutations associated with RDV therapy have been found: A504V in nsp14 (exonuclease) and I115L in nsp14 (endoRNAse) as well as four other more low-grade mutations. Both have demonstrated an increase in allele frequencies from the remdesivir treatment but have yet to be further tested. This study notes the idle characteristic of the virus under remdesivir therapy despite the increase in viral shedding for immunocompromised patients. As we’ve known for some time with viruses, single drug treatment is not optimal as very often mutations occur, remdesivir being no exception. As it’s often the case, the resistance mutation leads to a fitness cost of which the implication is unlikely the mutation will spread across the population. For now, this means monitoring the effects of Remdesivir as an antiviral drug for SARSCoV-2 and considering multi-antibody therapies for immunocompromised patients. Conclusion A take home message from this study is that the use of single drugs to treat RNA viruses often lead to resistance. The future utility of remdesivir as a first line therapy for SARS-CoV-2, should include pairing with another active antiviral drug, as well as attempts to ensure that the drug can be administered intramuscularly or subcutaneously. This article is featured on Forbes.org, and can be read online here: Remdesivir Resistance Reported Upon Treatment Of An Immunocompromised Patient

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New Analysis Shows Fluvoxamine Has The Potential To Reduce Covid-19 Hospitalizations By More Than 90% Forbes | April 13, 2022 | Article

The United States has launched a new and innovative program called Test And Treat. This program intends to solve one of the most immediate problems with Covid-19 cases: the prevention of symptomatic progression by taking drugs in a timely manner. Antiviral drugs must be used early in an infection to reduce disease outcomes. At present, to receive Covid-19 treating drugs, a patient must test positive, go to a doctor, receive a prescription, and fill the prescription at a pharmacy, all of which could take days or even weeks. Test And Treat aims to shorten this process, by having a patient test for Covid-19, receive a prescription, and fill that prescription all in one location, for the most part in pharmacy-based clinics. This reduces the several day-long process of receiving antivirals to a single trip to the pharmacy. A current roadblock to the nationwide implementation of Test And Treat is the accessibility and convenience of antiviral drugs. One drug, remdesivir, must be administered intravenously at an infusion center. Another treatment is paxlovid, which is orally administered but is limited principally by the long list of exclusions for other medications, including widely prescribed statin drugs for lowering blood pressure. The third treatment, molnupiravir, has not shown high efficacy and has the additional drawback, as a mutagen for SARS-CoV-2, of speeding the creation of new and more viral forms of the virus. 324

The drug fluvoxamine may address many of these issues. Fluvoxamine is inexpensive, orally-administered, FDA-approved drug, with a strong safety profile, and widely available worldwide. A recent meta-analysis of fluvoxamine clinical trials suggests that it may have a powerful effect on reducing hospitalization. Collectively, trials found that fluvoxamine’s probability of reducing the risk of hospitalization is between 94.1 and 98.6%, which would be remarkable if reproduced at a large scale. Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI), originally designed to treat depression. A recent meta-analysis by Lee et al. reviews three clinical trials of fluvoxamine. Of the 19 trials Lee et al. found searching the World Health Organization trial database, ten were retained after filtering for duplicates. Of those ten, seven were excluded because they were either still recruiting, focusing on inpatient, suspended, or had not begun. The process left three trails of 2,196 outpatients. All three trials were placebo-controlled randomized trials with unvaccinated, symptomatic adults PCR-test confirmed to be infected with SARS-CoV-2. We note that all trials predate the Delta and Omicron variants. Between the three trials and over 2,000 outpatients, Lee et al. found that the overall probability of association with reduced hospitalization ranged from 94.1% to 98.6%. We would be remiss not to mention the limitations of this metaanalysis. First, the STOP COVID 1 and STOP COVID 2 trials only account for 699 patients. The third trial, TOGETHER, accounts for 1497, which yields a definite skew towards their results. Second, The STOP COVID 2 trial was terminated before its intended close date due to lower than expected recruitment. Third, these trials did not examine partially, fully, or booster-vaccinated outpatients, who have a lower risk of hospitalization. Further trials to address these concerns would be welcome. There are also drawbacks to fluvoxamine itself. As an SSRI, there are several potential side effects from regular use, but none have precluded the widespread use of fluvoxamine as an antidepressant. Despite any shortcomings of the drug or this analysis, it is ultimately worth it to examine new tools in our fight against Covid19. Infections will continue and new variants will arise, likely causing swells of cases for at least the months to come. If we can prevent

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hospitalizations with a drug that is cheap, readily available, and already in existence, that is an avenue we should heavily consider. This article is featured on Forbes.org, and can be read online here: New Analysis Shows Fluvoxamine Has The Potential To Reduce Covid19 Hospitalizations By More Than 90%

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‘Test-to-treat’ could transform the pandemic The Hill | April 13, 2022 | Article

The Biden administration’s “test-to-treat” initiative has the potential to be one of the most significant advances in health care delivery in recent years. It is also a positive example of how the pandemic is encouraging us to reinvent infectious disease protocols. Early testing and rapid treatment are critical for the best individual health outcomes and to control the spread of the virus. Yet we must ensure that logistical challenges and issues of equitable access don’t hamper the potential of this groundbreaking initiative. As we move into the third year of the pandemic, fatigue around COVID-19 protocols and interventions, coupled with ongoing surges, means that our public health strategies must focus on local and nimble responses. The strategy behind the test-to-treat initiative is to use pharmacies to quickly identify those who are positive for COVID-19 through free rapid testing and then provide free oral antiviral treatments in the early days of illness when they are most effective. This could reduce the whole process to less than 30 minutes, reducing barriers to care and encouraging more rigorous uptake of testing and treatment among the broader population. We now know that the vaccines reduce the risk of severe disease and hospitalization, but they only provide temporary protection, at best, against infection from emerging variants. We need the additional layers of protection that test-to-treat can provide, especially for vulnerable populations. We now have a range of COVID drugs that are powerful tools to improve health outcomes and reduce the burden on hospital systems. A new meta-analysis of oral antiviral therapies found they reduced the risk of hospitalization and death from COVID by nearly 67 percent. The current oral anti-viral drugs included in test-to-treat are Paxlovid and Molnupiravir, which have an advantage over Remdesivir as they don’t require infusion in a specialized facility over several hours.

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But they are most effective only if accessed in the early days of infection. In clinical studies, Paxlovid was found to reduce the proportion of people with COVID-19 related hospital admission or death by about 88 percent compared to the placebo treatment arm only if they received the treatment within five days of the onset of symptoms. There could also be even more help on the way. New data suggest early use of the antidepressant fluvoxamine reduces the risk of all-cause hospitalization in symptomatic adult outpatients. A second drug, proxalutamide, has also shown promising trial results, lowering hospitalization or mortality in outpatients with mild-tomoderate COVID-19. In March, the Department of Health and Human Services began distributing Pfizer’s Paxlovid and Merck’s Molnupiravir pills directly to participating test-to-treat pharmacy-based clinics. The Office of the Assistant Secretary for Preparedness and Response has also launched a program for long-term care pharmacies that will facilitate increased access for long-term care residents who are at increased risk for developing severe COVID-19. Test-to-treat is intended to eliminate any barriers to accessing testing and treatment in the early days of infection. The intended scenario is for patients to walk into a pharmacy that has a health clinic staffed by nurse practitioners, physician assistants or physicians where they can be tested for COVID-19 at no cost, and if treatment is deemed appropriate be prescribed oral antiviral therapy at no cost. Unfortunately, there are some steep disparities with the pharmacies that can offer this scenario. Only 2,800 of the approximately 50,000 retail pharmacies in the U.S. have health clinics staffed by nurse practitioners, physician assistants or physicians, and about a third of them are located in California, Florida, Illinois, Minnesota and Texas. Additionally, just 12.5 percent of these pharmacies with health clinics are located in medically underserved areas. Not to mention the shortages of doctors and nurses that health systems are already facing in more traditional settings such as hospitals. This leaves much of the country, and especially populations who may be particularly vulnerable to COVID-19, without access to the test-to-treat initiative.

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One approach to resolving these disparities is to authorize pharmacists to prescribe oral antiviral drugs to patients with positive COVID-19 tests who meet the criteria. Not dissimilar to how pharmacists were given broad, national authorization to administer COVID-19 vaccines. Pharmacies are often used to alleviate the problem of health care deserts. However, this poses problems of potential liability and fear of litigation for pharmacists. The American Medical Association (AMA) has rejected the concept of test-to-treat in pharmacies, whether or not they have a health clinic with nurse practitioners and physician assistants in them. The AMA cited concerns over the complexity of treating COVID and the high number of drug reactions, suggesting they seek care from their primary care physician instead. But this position seems to neglect the medical conditions pharmacists already successfully manage, such as heart failure and blood clotting, and the large number of Americans who don’t have access to a primary care physician. There also needs to be clear messaging and transparent communication about the effectiveness of oral antiviral drugs and the accessibility through the test-to-treat initiative. The recent launch of COVID.gov provides important tools for the public such as a Centers for Disease Control and Infection (CDC) community risk lookup allowing for situational awareness to determine infection risk. But more work needs to be done to restore trust in the public and encourage them to take advantage. If we can resolve these disparities, the test-to-treat initiative could have huge potential to mitigate the consequences of future COVID-19 surges, and we could begin to introduce this model to other diseases. This article is featured on The Hill, and can be read online here: ‘Testto-treat’ could transform the pandemic

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SARS-CoV-2 Actively Infects And Kills Lymphoid Cells Forbes | April 14, 2022 | Article

Much of the public discussion surrounding SARS-CoV-2 infection has focused on the interactions between the SARS-CoV2 spike protein and cellular ACE2 receptors. Now, researchers at the University of the Chinese Academy of Sciences have found that this may only be part of the story. In a recent paper published in the journal of Nature, scientists describe a new target for the SARSCoV-2 virus that is independent of ACE2 receptors—our T cells. One of the critical symptoms of SARS-CoV-2 infection is lymphopenia. Lymphopenia is a condition in which patients exhibit reduced levels of white blood cells called lymphocytes. Lymphocytes are the fundamental defense cells of our adaptive immune system. They consist of natural killer cells, T cells, and B cells. When any of these cells are reduced, it can inhibit our body’s ability to protect itself from viruses. Recent reports have shown that marked lymphopenia is observed in 83.2% of SARS-CoV-2 patients, but little is known about how SARS-CoV-2 effectively dismantles one of our primary tools of defense. Shen et al. explored this question by first determining which lymphocytes SARS-CoV-2 targeted. Researchers collected blood cell samples from 22 patients with severe SARS-CoV-2 and from 15 healthy donors. They then tested each sample for three major lymphocyte cell types to determine any differences in lymphocyte counts between the healthy and infected donors. To their surprise, patients with severe SARS-CoV-2 exhibited a significant decline in T cells only. This suggested that SARS-CoV-2-induced lymphopenia is due to a decline in T cells and that T cells are the primary lymphocyte targets of the SARS-CoV-2 virus. How does SARS-CoV-2 attack T cells? Shen et al. hypothesized that the virus might directly infect T cells, killing them in the process. To test this theory, they analyzed the T lymphocytes of infected patients for the presence of viral antigens. This analysis 330

confirmed that SARS-CoV-2 antigens were present inside the T lymphocytes and that the virus could directly infect T cells. To confirm this result, Shen et al. examined lung sections from patients who had suffered from fatal SARS-CoV-2 infections. They found that the lung tissue contained high levels of T lymphocytes and that many T lymphocytes tested positive for SARS-CoV-2 proteins, indicating infection of those T cells (Figure 1).

Figure 1: Patients with fatal SARS-CoV-2 infections displayed substantial levels of T lymphocytes in their lungs (left, green) along with evidence of SARS-CoV-2 infection (middle, red). Many T cells also exhibited SARS-CoV-2 infection (right, T cellsSHEN, XR., ET AL. ACE2-INDEPENDENT INFECTION OF T LYMPHOCYTES BY SARS-COV-2. SIG TRANSDUCT TARGET THER 7, 83 (2022). HTTPS://DOI.ORG/10.1038/S41392-022-00919-X

These results were further corroborated by in vitro experiments. Shen et al. infected three model T cell types with SARS-CoV-2— Jurkat cells, MT4 cells, and T cells isolated from healthy donors. After initial infection, Shen et al. tested the cells for viral subgenomic mRNA. Subgenomic mRNA is an ideal marker for SARS-CoV-2 because it is only produced in infected cells during viral replication. If the researchers found SARS-CoV-2’s subgenomic mRNA in the cells, this would indicate that those cells were actively infected and that the virus was replicating.

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Figure 2: SARS-CoV-2 subgenomic mRNA and proteins were discovered in infected Jurkat and MT4 cells (indicated by asterisks).SHEN, XR., ET AL. ACE2INDEPENDENT INFECTION OF T LYMPHOCYTES BY SARS-COV-2. SIG TRANSDUCT TARGET THER 7, 83 (2022). HTTPS://DOI.ORG/10.1038/S41392-022-00919-X

After the experiment, not only did Shen et al. find substantial levels of subgenomic mRNA in the infected cells (Figure 2), but subgenomic mRNA levels continued to grow significantly after 24 hours. These results were consistent across all three cell types. Next, the researchers investigated whether SARS-CoV-2 infection could induce T cell death. To do so, Shen et al. analyzed infected cells for markers of apoptosis. Apoptosis is a natural consequence of infection and occurs when an infected cell selfdestructs to prevent a virus from replicating and spreading to other healthy cells. Surprisingly, T lymphocytes from SARS-CoV-2 patients displayed nearly ten times the number of apoptotic cells than healthy individuals. In many of the patients, the apoptotic cells also contained viral antigens, further indicating that apoptosis was a direct consequence of SARS-CoV-2 infection. It is common knowledge that SARS-CoV-2 primarily infects cells by interacting with ACE2 receptors expressed on the cell membrane. However, lymphocytes are known to contain very few, if any, ACE2 receptors. How then, could SARS-CoV-2 have such a profound effect on T cells? Shen et al. conducted an experiment using model T cells (Jurkat cells) and non-lymphocyte cells (Caco2 cells) as control cells. To test 332

whether SARS-CoV-2 infected the T cells through the ACE2 receptors, the researchers inactivated the genes responsible for producing ACE2 receptors in both the Jurkat and Caco2 cells. They then exposed both cell types to SARS-CoV-2. Surprisingly, a lack of ACE2 receptors decreased SARS-CoV-2 infection in the Caco2 control cells but had no effect on infection in the Jurkat cells. This finding demonstrated that SARS-CoV-2 infection of T cells occurs independently from the ACE2 receptors. Motivated by this finding, Shen et al. sought to discover which T cell receptors were involved in SARS-CoV-2 infection. Two of the most common receptors found on T cell membranes are AXL and LFA-1. These two receptors are also highly expressed in the model Jurkat cells. Once again, the researchers inactivated the genes for these receptors in Jurkat cells. They then infected Jurkat cells with SARS-CoV-2. After running this experiment for 24 hours, they found that an absence of AXL receptors did not block SARSCoV-2 infection. However, when AXL was overexpressed on the membrane, it seemed to increase SARS-CoV-2 infection. An absence of LFA-1 receptors significantly decreased viral infection. When LFA-1 was overexpressed, it also promoted SARSCoV-2 infection. This indicated that while AXL may play a small role in SARS-CoV-2 infection of T cells, LFA-1 is a far more convincing candidate and may be the key to developing medications that limit SARS-CoV-2 induced lymphopenia. Currently, there are several theories surrounding SARS-CoV-2induced lymphopenia and how the virus can attack lymphocytes in the absence of ACE2 receptors. However, this paper represents real progress in our understanding of the scope of SARS-CoV-2 infections and provides an interesting theory about how SARSCoV-2-induced lymphopenia occurs. As research continues to develop, it will be interesting to discover exactly how this virus attacks and dismantles one of our key defense systems. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Actively Infects And Kills Lymphoid Cells

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Six Ways Fluvoxamine May Act To Prevent Severe Covid-19 Forbes | April 18, 2022 | Article

Portrait of young athlete jumping over a hurdle during training on race track. GETTY A recent meta-analysis of a small number of fluvoxamine efficacy trials enrolling just over 2,000 patients found that fluvoxamine reduces the chances of severe hospitalization by up to 95%. If this efficacy is maintained in larger studies, fluvoxamine could be an important component of the Test-to-Treat strategy that the US is in the process of rolling out. Fluvoxamine was not developed as an anti-COVID drug. It has long been used as a mood-stabilizing selective serotonin reuptake inhibitor (SSRI) and is now available as an inexpensive generic drug worldwide. Hallmarks of Severe Covid The apparent effectiveness of fluvoxamine raises the question of how it might act independently of its use as a mood stabilizer. The following review by Sukhatme et al. provides details of the potential mechanisms by which fluvoxamine has the potential to interfere with the most severe consequences of COVID 19 infection, including cytokine storms, coagulation, and hyperinflammation.

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FIGURE 1: Fluvoxamine acts independently to inhibit several of the pathways that are characteristic ... [+] ACCESS HEALTH INTERNATIONAL

There are six known mechanisms, potentially more, by which fluvoxamine may act to treat late-stage Covid and prevent late-stage sequela: reduction In platelet aggregation, decreased mast cell degranulation, interference with endolysosomal viral trafficking and membrane binding, sigma-1 receptor activity, and increased melatonin levels.

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FIGURE 2: Potential anti-COVID-19 mechanisms of action of fluvoxamine, including reduction of ... [+] SUKHATME ET AL.

Inhibition of Cytokine Storm: Decreased Mast Cell Degranulation The cytokine storm is a life-threatening systemic inflammatory syndrome involving elevated levels of circulating cytokines and immune-cell hyperactivation. Essentially, this is the alarm bell in your immune system once a dangerous pathogen is detected, sending immune defenses to fight off the invader and resulting in severe symptoms. 336

One of the primary triggers of the cytokine storm is the interaction between SARS-CoV-2 and human mast cells, which are often viral reservoirs for RNA viruses. Pulmonary tissues in the lungs of deceased Covid patients are often linked to activated mast cells, which are degranulated by viruses upon infection. Fluvoxamine also interacts with mast cells, decreasing mRNA levels of protease-1, which promotes mucosal permeability in intestinal allergic hypersensitivity reactions. This likely impacts the efficiency of interaction between the virus and mast cells, leading to less severe cytokine storm responses and symptoms. Therefore, fluvoxamine has the potential to reduce the cytokine storm pathway of severe SARS-CoV-2 infection. Inhibition of Coagulation: Reduction In Platelet Aggregation Coagulation is the thickening of the blood as a result of blood platelets aggregating. This may lead to bloodstream issues like hypertension. Although platelets do not produce serotonin, they do absorb and contain high concentrations of the chemical. When aggregating in the bloodstream, platelets release serotonin to facilitate hemostasis. Fluvoxamine inhibits the uptake of serotonin from platelets and therefore inhibits platelet aggregation. Fluvoxamine is a serotonin uptake inhibitor and inhibits serotonin concentration in platelets prior to clot formation. A 2011 study showed that patients taking SSRIs measured lower levels of coagulation than those that were not taking the drugs. Therefore, fluvoxamine has the potential to reduce the coagulation pathway of severe SARS-CoV-2 infection. Inhibition of Hyperinflammation: Sigma-1 Receptor Activity Hyperinflammation following coagulation causes a number of health issues, including joint pain, gastrointestinal issues, and in some cases, brain injury. One player in inflammation is the sigma-1 receptor, which regulates endoplasmic reticulum mitochondrial calcium ion signaling, resulting in pro-inflammatory responses in higher concentrations. Fluvoxamine upregulates interleukin-10 (IL-10), which is an anti-inflammatory protein. The sigma-1 receptor downregulates other interleukins, such as IL-6 and IL-8, but not IL-10. Therefore high levels of IL-10 can inhibit the sigma-1 receptor and its hyperinflammatory processes. In other words, fluvoxamine may reduce Covid-related hyperinflammation. 337

Indirect Mechanisms: Endolysosomal Viral Trafficking And Membrane Binding SARS-CoV-2, along with other beta-coronaviruses, use lysosomal trafficking to escape infected cells. Lysosomes break down worn-out cell structures in a process called apoptosis, which the virus uses as a means of leaving one infected cell to find another. Basic SSRIs like fluvoxamine actually hinder lysosomes by interfering with their charge and reducing their membrane-crossing efficiency. This may not impact the virus itself, but it impedes one of its major routes of escape, resulting in antiviral effects that could make extended infections more difficult. We also note that lysosomotropic drugs also impact viral entry of the SARS-CoV-2. While fluvoxamine has not been tested specifically for this feature, it has an anti-lysosome function, meaning it may also impact viral entry. Inhibition of Hyperinflammation: Increased Melatonin Levels Fluvoxamine elevates melatonin levels by the inhibition of the enzyme CYP1A2, which is involved in the metabolization of polyunsaturated fatty acids. Some CYP1A2 reactions result in elevated blood pressure and promoted inflammation responses. Melatonin mitigates inflammation from the NLRP3 pathway, a common tool for SARS-CoV-2. Fluvoxamine, therefore, may reduce Covid-related hyperinflammation. Although it is still early days, given the relatively limited trials fluvoxamine has been shown prevention of serious COVID, the results already look promising. Moreover, the study by Sukhatme et al. finds that there are many independent biochemical pathways that may explain how these drugs, given earlier in infection, may prevent serious disease. Although fluvoxamine does not seem to have a direct antiviral effect other than effective endosomal viral entry into the cell, it does have a number of activities that fortuitously inhibit precisely those pathways that are characteristic of the most serious forms of COVID-19. We can only hope that these promising results hold up against further investigation. This article is featured on Forbes.org, and can be read online here: Six Ways Fluvoxamine May Act To Prevent Severe Covid-19

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New Members Of The Omicron Family Of Viruses: BA.2.12.1, BA.4, And BA.5 Forbes | April 20, 2022 | Article

Emerging variants as genomic prevalence in South Africa over time. DR. TULIO DE OLIVEIRA SARS-CoV-2 variants continue to be a topic of great concern. As weeks go by, it is evident that the Omicron family of viruses continue to grow in complexity. Figures one and two show the extent of variation from the beginning of the pandemic up to the present.

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FIGURE 1: Relative growth rate of SARS-CoV-2 variants compared to confirmed cases per day. OBERMEYER ET AL.

Figure 1 roughly illustrates the complexity of SARS-CoV-2 variants, with each strain labeled by its PANGO lineage designation. The vertical axis illustrates the number of people infected, and the horizontal axis displays the rate of spread of that particular variant in the population. Both axes are on a logarithmic scale. The relative growth rate is described as how many additional people are infected by one infected person, for instance, BA.2 exceeding 12. Reading the figure from left to right, the original set of variants before Delta in late 2020 and early 2021 is shown as a cloud of variants, distinguished by unique mutations around the common blueprint D614G Triad variant. Figure 2 illustrates the emergence of new variants by date and relative growth rate. As we move from left to right across, we note that the rate of spread in populations increases. Notably, this occurs in many populations that have been either heavily infected, highly vaccinated, or both. The second cloud of variants comes from the Delta variant. These variants were more pathogenic and spread through the population more rapidly than the previous isolates. There is then a gap as the emergence of new variants slowed. Over the past few months, the Delta cloud was displaced by two distinct families. The earlier was the BA.1 family of variants, followed by the current BA.2 family. Within the BA.2 family, many different variants have been identified, including some that fuel more

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infections than others, for example, the BA.2.12.1 variant spreading widely in the Northeast United States.

FIGURE 2: Relative growth rate of SARS-CoV-2 variants compared to their date of lineage emergence. OBERMEYER ET AL.

Outside of the United States, two new variants are gaining attention from the World Health Organization as they grow in their sequence and infection count: BA.4 ad BA.5, which are offshoots of the BA.2 family. As the thumbnail of this article makes clear, BA.4 and BA.5 are growing rapidly to dominate the South African population. We note that just because instances of BA.4 and BA.5 are increasing as a relative proportion. That does not mean that the total number of cases is increasing in South Africa. Case rates are low and stable at the time of writing. The Omicron subvariants BA.4 and BA.5 share most mutations with the original BA.2 Omicron variant, but each has its distinct mutations from BA.2 and one another. Here we examine these two subvariants and the new modifications they possess. For reference, the BA.2 Spike protein contains 28 amino acid mutations compared to the original Wuhan strain. Fifteen of these are concentrated in the receptor-binding domain. In addition to the mutations in the receptor-binding domain, there are also a number of mutations in the N-terminal domain and the membraneassociated S2 region in the Spike.

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The BA.4 and BA.5 subvariants share an identical Spike protein with four modifications relative to BA.2. Receptor-Binding And N-Terminal Domain Mutations The first of these four is a reversion of a mutation back to its original amino acid. The mutation of glutamine to arginine at position 493 (Q493R) in BA.2 returns to glutamine in BA.4 and BA.5. There are studies indicating that Q493R is an escape mutation for some monoclonal antibody treatments, such as bamlanivimab and etesevimab. A reversion to Q493 may suggest that BA.4 and BA.5 are more susceptible to neutralization by monoclonal antibodies, though further investigation would be needed.

FIGURE 3: Spike protein mutation profile for BA.4 and BA.5

DR. TULIO DE OLIVEIRA The next mutation, leucine to arginine at position 452 (L452R), is common in existing variants. The mutation induces a change in charge from positive to neutral. The mutation is cited for contributing to immune escape from antibody binding and may also contribute to stronger cell attachment to the virus, leading to increased transmissibility and pathogenicity. The least common of the three amino acid changes is phenylalanine to valine at position 486 (F486V). This mutation does not induce a polar or charge shift. Dr. Jesse Bloom suggests that F486V could “lead to more antibody escape from serum elicited by current vaccines / early infections,” though further research to confirm this hypothesis would be useful.

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There is also a deletion of positions 69 and 70 in the N-terminal domain. This is a common mutation found in natural variants and likely knocks out an antibody binding site, meaning this specific mutation could impact overall immune evasion. The rate at which the virus spreads through a population that has a large percentage of Covid-19 recoverees is determined both by the number of people who have been previously infected as well as the number of those vaccinated. A new variant must have the viral fitness to overcome immune defenses developed during previous infections and vaccinations to spread in such a population. One way of understanding escape from a waning immunity is to look at the sensitivity of variants to monoclonal antibodies based on certain escape mutations. Despite the mutation at position 493 reverting to its original state, the inclusion of the N-terminal domain deletion, F486V, and L452R may yield a more immune evasive virus overall. Both receptor-binding domain mutations were cited by Dr. Tulio de Oliveira as being associated with escape neutralization from all classes of monoclonal antibodies.

FIGURE 4: Escape mutations for BA.4 and BA.5 from SARS-CoV-2 monoclonal antibody therapies.

DR. TULIO DE OLIVEIRA The BA.4 and BA.5 subvariants also come with a few modifications to the genomic profile of the virus outside the Spike protein.

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FIGURE 5: Non-Spike protein mutations in BA.4 and BA.5

DR. TULIO DE OLIVEIRA Nonstructural, Structural, and Accessory Protein Mutations Both BA.4 and BA.5 revert two mutations back to their wild type: NSP4 L438 and Orf6 D61. The exact functions of these mutations are unknown, though we can speculate based on the function of their resident protein. NSP4 is involved in forming the double-membrane vesicle, which is involved in replication. Orf6 inhibits the innate immune response, downregulating various signals, proteins, and enzymes that impede virus replication. BA.4 adds two mutations closer to the 3’ end of the genome: leucine to phenylalanine at position 11 (L11F) in Orf7b and proline to serine at position 151 (P151S) in the N protein. Both Orf7b and N are involved in the evasion of innate immunity, and these mutations may contribute to evasion efficiency. The N protein mutations may also affect the packaging and stability of the virus particle. BA.5 adds aspartic acid to asparagine at position 3 (D3N) in the M protein. The Membrane protein surrounds the virus particle and is involved in viral entry, and this mutation may increase functional efficiency. The M protein also plays a role in the suppression of innate immunity. The D3N mutation is relatively uncommon, but D3G is found in some Omicron lineages. These viruses should not be underestimated. For every person infected with the Wuhan strain, these viruses may infect roughly 12. Previous mitigation methods have spared China the level of infections rampant elsewhere. However, mild increases in cases in 344

comparison seem to show it is more difficult to contain the spread of the Omicron variants. Based on observations thus far, I doubt that SARS-CoV-2 has reached the upper limits of its rate of spread in either vaccinated or unvaccinated populations. I think new variants may emerge with substantially increased virulence. We should remember that a closely related virus to SARS-CoV-2—MERS-CoV—kills roughly 50% of those it infects. The story of the omicron family of variants is still emerging and one that the world is watching closely. It is unfortunate that at a time when surveillance of infection rates and strain identification is crucial, those efforts are declining rapidly worldwide. This strikes as a grave mistake, as surveillance is the best early warning signal for new and perhaps more dangerous variants. This article is featured on Forbes.org, and can be read online here: New Members Of The Omicron Family Of Viruses: BA.2.12.1, BA.4, And BA.5

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Moral Injury Is Similar in Healthcare Workers and Veterans Psychology Today | April 20, 2022 | Article

A new study demonstrates that potential moral injury is similarly high among Covid-19 healthcare workers and combat veterans who served post-9/11. Moral injury is defined as the strong cognitive and emotional response that can occur following events that violate a person's moral or ethical code or when they feel betrayed by authority figures. It should be distinguished from the term ‘burnout’ that is often used to describe the effects of ongoing stress in the workplace, but the two conditions can overlap. While moral injury is not classified as a mental illness, it is associated with post-traumatic stress disorder, depression, social isolation, and suicidality and should be treated seriously. The study published in the Journal of General Internal Medicine was a collaboration between Duke University, Vanderbilt University, and the Department of Veterans Affairs. The researchers relied on data from a study of 618 post-9/11 combat veterans and a separate survey of 2,099 people working in health care during the Covid-19 pandemic. The study builds on a decade of research into moral injury among veterans by comparing data from the Post-Deployment Mental Health Moral Injury (PDMH-MI) study with findings from the Healthcare Worker Exposure Response and Outcomes (HERO) registry. The term potential moral injury is used as there is currently no threshold for when an event meets the criteria for moral injury and uncertainty exists about how to measure the syndrome in a clinical setting. Most research to understand moral injury has been associated with combat veterans and military service members. This is likely due to the fact that the nature of war and combat create situations where people may have experiences that contradict their values. Clinical psychiatrist Jonathan Shay first coined the term when he noted that some American soldiers who had experienced traumatic 346

events in Vietnam returned with profound changes to their character. Their symptoms shared similarities but were different from what would normally be recognized as PTSD. After further research, he discovered that their symptoms were predominantly triggered by events that violated a soldier’s moral code rather than the experience of narrowly escaping death. I have written extensively about this in Covid Related Post Traumatic Stress Disorder (CV-PTSD): What It Is and What To Do About It. Further research into how moral injury affects other professions and the general population, as well as how it can be measured in a clinical setting is sorely needed. The current definition of moral injury is limited to the experiencing of a violation of morals and ethics in one’s profession. Yet many members of the public feel a deep sense of betrayal by the governments and public health authorities who failed to protect them from the worst effects of the virus. In particular, those who are immunocompromised or at high risk for severe Covid-19 feel betrayed and left behind by the continued shedding of policies such as mask mandates that are occurring globally and the false characterization of the Omicron as mild. Long Covid sufferers may not feel the risks of the virus were adequately communicated. For this study, veterans were asked about moral experiences in the context of their military service, and health care workers were asked about their experiences during Covid-19. The potential moral injury was assessed in two categories: other-induced and selfinduced. The overall prevalence rates of potential moral injury were similar across samples, with veterans endorsing other-induced potential moral injury slightly less often than HCWs and selfinduced PMI slightly more often. The findings show that 46 percent of veterans and 51 percent of healthcare workers indicated being troubled by others’ immoral behavior, whereas 24 percent of veterans and 18 percent of healthcare workers indicated being troubled by violating their own morals and values. Both healthcare workers and veterans selfreported significantly higher levels of depression and a lower quality of life. Health care workers cited a number of experiences that conflicted with their morals. These included witnessing the public’s disregard for preventing Covid-19 transmission, witnessing mass 347

casualties and deaths, enduring staffing shortages, rationing of care and personal protective equipment, and policies barring visitors from seeing dying patients. Doctors and nurses have felt a deep sense of betrayal and hypocrisy from the public during the pandemic. Public tributes such as the nightly cheering and clapping and other signals of heroworship were juxtaposed with a refusal to wear masks, get vaccinated to relieve the burden on health systems, and take other key public health precautions. Lead study author Jason Nieuwsma, a researcher with the VA and the Department of Psychiatry and Behavioral Sciences at Duke University School of Medicine, stated: “It is sobering to see how many health care workers are troubled at a moral level because of their work experiences during the pandemic. This may help us to understand some of the current challenges facing health care systems across the country,” Senior study author Keith Meador, a professor in the departments of Psychiatry, Religion, and Health Policy at Vanderbilt, stated that: “Examples we saw most were individuals being expected to do things that made them feel questionable about their participation. In the health care context, that may look like not being able to provide the level of care one would like to provide due to the complexities of the ongoing pandemic. As a result, HCWs were vulnerable to the consequences of potential moral injury and reduced quality of mental health, similarly to what we’ve seen in post 9/11 veterans.” Meador has identified collaboration between chaplains and mental health providers as a potential opportunity to improve the mental health of healthcare workers. While additional and alternative forms of mental health support are surely welcomed by healthcare workers, systematic and institutional change within healthcare systems is also required to ensure that workers are not placed in positions that cause conflicts with their morals and ethics. Pandemic preparedness funding needs to be consistent so that hospitals can be well resourced and fully equipped and so that staff receives appropriate ongoing training to deal with crises. Physician shortages need to be addressed through creative solutions. Federal and local public health policy needs to account for the burden on health systems when cases remain steady but hospitalizations rise. 348

The American Psychiatry Association recommends putting systems in place like an anonymous hotline that empowers and encourages healthcare workers to speak freely about the stressors they face and to advocate for their own health as well as that of their patients. This could be a useful strategy for addressing moral injury, but only if executive leadership is willing and ready to implement the feedback received. Finally, there is a dire need for more legislation like the recently passed Lorna Breen Act, which provides funding for training healthcare providers on suicide prevention and behavioral health. Healthcare workers were already under immense strain prior to the pandemic. As we enter its third year, we must make the mental health of healthcare workers a priority. This article is featured on Forbes.org, and can be read online here: Next Article

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SARS-CoV-2 Infection Of Monocytes Triggers Inflammation Forbes | April 21, 2022 | Article

This is part of a series of stories on inflammation triggered by SARSCoV-2 infection. Other articles in the series include: lectins, Covid-19 and brain injury, and long Covid. They may also be found on my website, www.williamhaseltine.com/. Inflammation is a key feature of SARS-CoV-2 infection, both during the acute phase of Covid-19 and also as a contributor to postacute sequelae SARS-CoV-2 infection (PASC), otherwise known as long Covid. Inflammation in reaction to any microbe is Janus-faced. On the one hand, it can serve as a barrier to prevent the spread of, and also destroy, invading pathogens. On the other hand, if uncontrolled, inflammation can lead to serious multi-organ damage and even death. New research from Harvard Medical School and Boston Children’s Hospital provides insights into the multiple ways SARS-CoV-2 can trigger inflammation. Junqueira et al. found that non-productive infection of monocytes and macrophages stimulates the release of potent inflammatory signals, explaining at least in part the inflammation associated with Covid-19. The study was based on a comparison of blood and lung tissues from a matched-set of 22 Covi-19 patients and 19 healthy donors. The researchers searched for markers of pyroptosis, a form of programmed cell death. Cells are equipped with multiple alarms that signal the presence of an invading microorganism or inappropriate cell damage. These alarms are set of either by pathogen-associated molecular patterns (PAMPs) — molecular “motifs” that are preserved across most pathogens, be they viral, bacterial, or fungal— or by damage-associated molecular patterns (DAMPs), released by damaged or dying host cells. Some of these alarms stimulate the inflammatory response, mediated by a protein signaling complex dubbed the inflammasome. Once assembled, the inflammasome activates a protease called caspase-1. This, in turn, stimulates the production of proinflammatory cytokines, like interleukin-18, and a 350

pore-forming protein called Gasdermin-D. Gasdermin-D punches holes in the cell’s membrane, eventually causing it to explode and spill all of its contents. This helps call other immune cells to the area, increasing inflammation and assisting with host defense. It also prevents the host cell from having its cellular machinery hijacked by the pathogen for purposes of self-replication. The full pathway leading to pyroptotic cell death is depicted in Figure 1.

FIGURE 1. Induction of pyroptotic cell death: Detection of cell stress, toxic molecules or pathogens ... [+] SOURCE: PYROPTOTIC CELL DEATH: CONTROLLED SELF-DESTRUCT THAT CAUSES INFLAMMATION AND DISEASE

But sometimes this process goes haywire and fails to reel itself in, causing extreme inflammation that actually furthers tissue and organ damage. The researchers discovered that the cells showing the most signs of pyroptosis —as determined by cell membrane damage and inflammasome activation— were monocytes. These are immune cells that act as lookouts, keeping an eye out for any microbes in the bloodstream, and also serve as early responders to infection. Lung macrophages, a similar immune cell found in tissue instead of the blood, also showed signs of pyroptosis. “In the infected patients, about 6 percent of blood monocytes were dying an inflammatory death,” said Judy Lieberman, senior author of the study. “That’s a large number to find, because dying cells are rapidly eliminated from the body.” Junqueira et al. then investigated the possibility that the monocytes and macrophages themselves were being infected by SARS-CoV-2. To do so, they engineered a SARS-CoV-2 clone that encoded a fluorescent reporter of viral replication. They found that about 10% of monocytes and 8% of lung macrophages displayed 351

clear signs of infection. This included the presence of viral RNA and viral proteins. No such monocyte or macrophage infection was seen in the control group of healthy blood donors. Curiously, the researchers noticed that lung epithelial and endothelial cells, whose surfaces are covered with angiotensin converting enzyme 2 (ACE2) receptors, the primary portal of entry for SARS-CoV-2 infection of cells, showed neither signs of inflammasome activation nor signs of pyroptosis. Monocyte infection was unexpected as the well-known ACE2 receptor is absent from the cell's surface, and found only in very low concentrations on the surface of macrophages. But they were clearly being infected. So, how is SARS-CoV-2 binding to and entering these cells? The team of researchers suspected that infection may be occurring via antibody-dependent enhancement (ADE). Antibodies are produced by our adaptive immune system in response to microbial threat. Although there are a variety of different antibodies, they generally fall into one of three functional categories: those that bind to the surface of a pathogen to prevent it from being able to enter its target cell (neutralizing antibodies); those that cover the surface of a pathogen and mark it for removal by monocytes, macrophages, and other immune cells (opsonins); and finally, those that trigger other immune responses, including the complement pathway, to help eliminate pathogens. Put simply, when nonneutralizing antibodies bind to a pathogen they sometimes facilitate the pathogen’s entry into cells that carry antibody receptors (Figure 2).

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FIGURE 2. In antibody-dependent enhancement, sub-optimal antibodies (the blue Yshaped structures in ... [+] SOURCE: WIKIPEDIA

Junqueira et al. discovered that a particular subset of monocytes were especially sensitive to SARS-CoV-2 infection: those that express the CD16 receptor. This is one of three receptors monocytes use to bind with antibodies. Although CD16 monocytes usually comprise only 10% of the total monocyte population, they are noticeably increased in Covid-19 patients. Confirming the team’s suspicions, CD16 receptors were able to recognize viral particles tagged by antibodies, allowing them access into the cell. This did not happen with monocytes from the healthy donor group, unless mixed with anti-Spike antibodies or antibodycontaining plasma from Covid-19 patients. Crucially, vaccine-derived antibodies did not facilitate viral entry into monocytes. Although not entirely sure why this is, Junqueira et al. hypothesize it may come down to differences in the properties of the antibodies, with those developed post-vaccination less prone to binding to CD16. This work provides clear evidence that SARS-CoV-2 infection of monocytes and macrophages does occur and that, when it occurs, it triggers an inflammatory response. However, the researchers noted that infection of the monocytes and macrophages does not lead to the production of infectious virus particles. Evidently the full

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replication cycle is blocked, possibly by the inflammatory response itself. “In some ways, uptake of the virus by these ‘sentinel’ cells is protective: it sops up the virus and recruits more immune cells,” said Lieberman. “But the bad news is that all these inflammatory molecules get released. In people who are more prone to inflammation, such as the elderly, this can get out of control.” Junqueira et al.’s research goes partway to explain the inflammatory response of those infected with SARS-CoV-2. Other contributors include lectins, which distort our neutrophils, and innate lymphoid cells (ILCs), which produce proinflammatory chemokines like interleukin-17. Clearly, unless infection is controlled by the initial innate immune response, or high-levels of neutralizing antibodies, virus replication can trigger a violent cycle of hyper-inflammation leading to severe disease and even death. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Infection Of Monocytes Triggers Inflammation

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STING: It Takes Two To Tango Forbes | April 21, 2022 | Article

This is part of a series on Covid-19 and inflammation related to severe disease and Long Covid. Although innate immunity is the body’s first defense against an infection, SARS-CoV-2 cleverly avoids activating much of the innate immune system when it first infects the body. How effectively the body re-engages these mechanisms could mean the difference between mild and severe infection. Delaying activation of the innate immune system gives the virus more time to replicate, which contributes to increased inflammation throughout the body. Even in tissues that SARS-CoV-2 does not directly infect like the brain, the inflammatory consequences can induce a range of symptoms that for some continue to linger long past the initial infection. Now, the critical question for researchers is how can we activate the innate immune system early to prevent more severe infection later? Can we introduce a drug that controls inflammation to our advantage? One major therapeutic target that has emerged is STING (stimulator of interferon genes). When abnormal DNA is detected in the cell, STING activates to mount a defense. In healthy cells, DNA is enclosed within the nucleus. DNA in the cytosol, however, signals that a pathogen or tumor has invaded the cell. Infected cells will also release mitochondrial DNA that can trigger the STING pathway. Capitalizing on STING-mediated immunity may help to fight Covid-19, but the story of STING is far more complicated. Originally, it was thought that this protein could only be activated by cyclic GMP-AMP (cGAMP), or similar cyclic dinucleotide molecules. Now, a recent study from the University of Texas Southwestern has uncovered a previously hidden binding site on the STING protein. On one hand, the discovery of this “cryptic pocket” may be a useful target for enhancing activation. One the other hand, previous studies have also found that when some molecules bind to STING, they inhibit its activation. Considering that heightened STING activation seems to underlie some 355

autoimmune disorders, knowing when to activate or inhibit this protein may be the key to controlling the robust inflammatory consequences of Covid-19. Here, we will discuss the discovery of the new binding site on STING as a promising therapeutic target for activating the immune system. Before we begin, we must consider the mechanisms involved in STING-mediated immunity beginning with the detention of DNA by an enzyme called cGAMP synthase (cGAS). Upon binding to DNA, cGAS triggers a reaction that produces the second messenger cyclic GMP-AMP (cGAMP). cGAMP then binds to STING, located in the endoplasmic reticulum around the cell's nucleus. This triggers profound changes in the STING protein that allow it to move around the cell and mount an immune defense against the invading pathogen. The cGAMP- bound STING protein then travels to the golgi apparatus, where it recruits a protein called TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). TBK1 phosphorylates, or “activates”, both STING and the interferon regulatory factor. Now in its final active form, STING mediates the removal of the pathogen through autophagy, while the activated interferon regulatory factor enters the nucleus to stimulate increased expression of inflammatory genes to fight the infection, including a group of cytokines known as type 1 interferons. Type 1 interferons have a dual effect that not only fights infection in the cell they were generated in but also protects nearby healthy cells. The entirety of this pathway is illustrated below.

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FIGURE: ILLUSTRATION OF THE CGAS-CGAMP PATHWAY. ON THE RIGHT HAND SIDE, CGAS FIRST DETECTS THE PRESENCE OF FOREIGN DNA IN THE CYTOPLASM AND THEN TRIGGERS A REACTION THAT PRODUCES THE SECOND MESSENGER CYCLIC GMP-AMP (CGAMP). CGAMP BINDS TO A PROTEIN C

FROM: “TBK1, A CENTRAL KINASE IN INNATE IMMUNE SENSING OF NUCLEIC ACIDS AND BEYOND” RUYUAN ET AL. 2020 As a transmembrane protein, STING has two main domains: a transmembrane domain and cytoplasmic ligand-binding domain. Although cGAMP and other cyclic dinucleotides bind strongly to the center of the butterfly-shaped ligand-binding domain, the challenge with treating Covid-19 with cyclic dinucleotides is that these molecules are not only involved in immune regulation. They have multiple functions throughout the body. cGMP, for example, also plays an important role in detecting light in the visual system, forming long-term memories, and the process of contracting and relaxing muscles. Administering high amounts of these molecules can significantly disrupt several of these systems.

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FIGURE: SCHEMATIC DIAGRAM OF CYCLIC GMP-AMP (CGAMP). WIKIMEDIA COMMONS

Activating STING causes the entire structure of the protein to shift and facilitates the formation of a larger structure called an oligomer. During oligomerization, multiple individual STING proteins come together in a line to form side-by-side connections. Forming an oligomer not only allows STING to travel around the cell but this step is also important for recruiting the TBK1 enzyme needed for phosphorylation. Despite their high affinity for STING, however, Lu et al. were surprised to find that cGAMP alone could not activate STING in a test tube. Little to no oligomers were formed and there was no evidence of phosphorylation. Since cGAMP is known to STING, researchers speculated whether adding another agonist could promote activation. Fortunately, a previous discovery published in the European Journal of Medicinal Chemistry revealed that non-cyclic dinucleotides can also activate the immune properties of STING. This study identified a group of small compounds from the benzothiazinone family that bind to STING with varying degrees. Pryde et al. found that one molecule in particular, compound 53 (C53) shown in the figure below, robustly and consistently activated STING. However, considering how chemically and structurally different C53 is from cGAMP, where this molecule was binding to on the STING protein was a mystery. This is when researchers initially began speculating whether there may be an additional binding location somewhere else on the protein.

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FIGURE: SCHEMATIC DIAGRAM OF COMPOUND 54 (C53).FROM: “THE DISCOVERY OF POTENT SMALL MOLECULE ACTIVATORS OF HUMAN STING” PRYDE ET AL. 202

Using the STING agonists identified from the previous study, Lu et al. found that adding both cGAMP and C53 consistently generated high amounts of the STING oligomer. However, this effect could not be replicated when either of these compounds were mixed STING alone. Using cryo-electron microscopy, a technique that freezes proteins in place, Lu et al. confirmed that both cGAMP and C53 could simultaneously bind to the STING oligomers. Shown in the figure below, cGAMP sat in the ligand-binding domain, while C53 fit within a deep pocket within the transmembrane domain.

FIGURE: REPRESENTATION OF STING OLIGOMER WITH BOTH C53 (LABELED IN PINK) AND CGAMP (LABELED IN CYAN) BOUND TO THE TRANSMEMBRANE DOMAIN AND LIGAND-BINDING DOMAIN,

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RESPECTIVELY. FROM: “ACTIVATION OF STING BY TARGETING A POCKET IN THE TRANSMEMBRANE DOMAIN.” LU ET AL. 2022

What role does C53 have in activating STING? To answer this question, Lu et al. further examined the structure of the STING oligomer. What they found was that these oligomers consisted of two smaller STING proteins, stabilized by interactions between the two ligand binding sites, as well as the two transmembrane domains. While the ligand-binding connections were rather weak, interactions between the transmembrane domains seemed to provide most of the structure’s stability. In fact, when investigators genetically mutated regions of the protein that facilitate interactions across transmembrane domains, the number of STING oligomers generated significantly reduced despite the fact that both C53 and cGAMP were bound to the protein. Lu et al. therefore speculated that when C53 binds to STING, it significantly changes the transmembrane domain that promotes stronger interactions. This likely explains why cGAMP alone did not produce stable STING oligomers, but adding C53 did. Previously, it was unclear whether the transmembrane domain even had the ability to bind agonists. Now it seems like this “cryptic pocket” may be critically important for mediating innate immunity. When researchers introduced mutations in this binding pocket, for instance, the number of STING oligomers dramatically reduced, even when exposed to both C53 and cGAMP. These mutations also prevented the phosphorylation of STING later on, suggesting that this binding pocket within the transmembrane domain may mediate the effectiveness of STING after it has been activated by cGAMP. The discovery of this hidden binding site may provide a new target for drugs and vaccines against SARS-CoV-2. Compounds, like C53, that can bind to the transmembrane domain in fact may be better therapeutic agonists for activating STING than those mimicking cGAMP. However, the specifics of how STING is innately activated in the body, not to mention how different molecules activate or inhibit its activity, remain unclear. Researchers speculate that cells have an unknown molecule similar to C53 that binds to STING’s transmembrane domain to activate the protein as needed. Identifying this molecule and understanding its mechanism of action may provide better insight into how we can capitalize on 360

innate immunity to fight SAR-CoV-2. Next, in this series, we will take a closer look at the structure of C53 to understand which components may be the most important for STING activation. This article is featured on Forbes.org, and can be read online here: STING: It Takes Two To Tango

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Survival Of The Fittest: The Rise Of BA.2.12.1 Forbes | April 21, 2022 | Article

Contrary to the popular belief that Covid infections are receding universally, recent reports show that several countries are in the midst of accelerating Covid rates. This draws cause for concern with the Summer fast approaching. Recall that Summer 2021 was dominated by Delta variant infections. This Summer may be in for something similar. The current increase is almost certainly due to new members of the Omicron family of viruses.

FIGURE 1: Omicron family tree as BA.1, BA.2, and BA.3 first emerged.

ACCESS HEALTH INTERNATIONAL Current estimates suggest that BA.2 variants have replication rates at least 30% greater than BA.1 variants. Two variants, BA.4 and BA.5 in Europe, were recently identified as strains to monitor by the World Health Organization. Figure one illustrates how rapidly descendants of Omicron have diversified and established themselves not only in the United States, but around the world.

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FIGURE 2: Extension of the Omicron family tree focusing on the BA.2 sub variants. Horizontal distance ... [+] NEW YORK DEPARTMENT OF HEALTH

One such subvariant is already causing significant case numbers in the American Northeast. In the GISAID SARS-CoV-2 database, BA.2.12.1 is attributed to roughly 2,000 sequenced infections, which suggests that the actual number of BA.2.12.1 cases is at least in the tens of thousands. A recent analysis by the CDC suggests that BA.2.12.1 comprises at least 19% of new cases while BA.2 lineages overall comprise over 90%. As its lineage name suggests, BA.2.12.1 is a descendant of the BA.2 virus. BA.2 was the main driver of the winter wave of Omicron infections that peaked in the United States on January 14th, 2022, when the daily average was over 800,000 new infections. Part of BA.2’s viral fitness was its remarkably high transmissibility, and another was its expert evasion of the immune system, including antibodies developed from previous infections and vaccinations. The BA.2 virus’s viral fitness is attributed to its wealth of mutations within and external to the Spike protein. The BA.2 genome contains 53 amino acid mutations, 29 of which are in the Spike protein, far outnumbering the Spike mutations of previous variants of concern and interest.

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FIGURE 3: BA.2 genomic and Spike mutations. Those in blue indicate a shared mutation with other ... [+] ACCESS HEALTH INTERNATIONAL

One mutation lies in the receptor-binding domain. This is a substitution of lysine for glutamine at position 452 (L452Q). This mutation is notable for two reasons. First, L452Q has been previously identified in several variants of concern, like Lambda, Delta, Delta plus, and Epsilon. Second, unlike many mutations where we must only speculate on their impact on viral fitness, L452Q was studied and found to increase escape immunity, allowing the virus to attach more strongly to human cells and avoid neutralizing antibodies. The second mutation is more mysterious. It is a substitution from serine to leucine at position 704 (S704L). Unlike L452Q, S704L is a relatively rare mutation by way of major variants, and its exact impact on the virus is unstudied. Based on its position in the S2 region of the Spike protein, we can speculate that the mutation may affect either viral fusion or furin cleavage efficiency, but further study would be welcome. We can say with near certainty that BA.2.12.1 is at least as transmissible and immune evasive as BA.2. It is unlikely that the two Spike mutations would decrease either of these facets of the virus strain. However, due to the wealth of BA.2 cases in previous months, the BA.2.12.1 virus may be impeded by antibodies developed from infections during that wave, which could explain why cases are low relative to the Omicron wave. However, it is critical to remain vigilant in our surveillance of these subvariants. Recent reports from Hong Kong note that Omicron BA.2 subvariants deliver a similar fatality risk to those infected relative to earlier strains of SARS-CoV-2. This is contrary to many assumptions that Omicron is less severe than previous strains. Additionally, there are indications that Omicron is as

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dangerous in terms of long-term complications in children as the Delta variant of last summer. We also note the growing danger of long Covid. As many as 3050% of Omicron recoveries describe at least some lingering issue post-infection. This is independent of vaccination status and severity of their given infection—the rate increases for those hospitalized. A new wave of Omicron with BA.2.12.1 or another variant yet to be identified could be as catastrophic as the wave from December to February. At the time of writing, 988,610 people died due to Covid-related complications. We should make every effort to keep that number from continuing to increase. One such avenue is to continue surveilling new variants to inform public health measures and aid the development of Covid-19 treatments. This article is featured on Forbes.org, and can be read online here: Survival Of The Fittest: The Rise Of BA.2.12.1

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SARS-CoV-2 Mimics Inflammatory Proteins In Our Body Forbes | April 25, 2022 | Article

This is part of a series of stories on inflammation triggered by SARSCoV-2 infection. Other articles in the series include lectins, Covid-19 and brain injury, Covid-19 infection of monocytes, and long Covid. They may also be found on my website, www.williamhaseltine.com/. One of the paradoxes of SARS-CoV-2 is that while it represses interferons in the innate immune system, it ignores and may even exacerbate our innate immune system’s most violent form of defense: the inflammatory response. Inflammation is one of the most serious consequences of viral infection and is a hallmark of severe SARS-CoV-2. When SARS-CoV-2 replication and inflammation are left unchecked, they not only lead to severe immediate consequences but long-term consequences. Now, recent studies show that SARS-CoV-2 activation of the inflammatory response is not a passive process, but an active one. Two studies published in iScience and mBio indicate that SARSCoV-2 actively produces a protein called ORF8 which mimics one of the most potent inflammatory response triggers, interleukin-17.

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Interleukin-17 (IL-17) proteins bind to IL-17 receptors to activate an inflammatory pathway. GROEN, S, ET AL. EXPLORING IL-17 IN SPONDYLOARTHRITIS FOR DEVELOPMENT OF NOVEL TREATMENTS AND BIOMARKERS. AUTOIMMUNITY REVIEWS. 2021:20:3. DOI: HTTPS://DOI.ORG/10.1016/J.AUTREV.2021.102760 Interleukin-17 is a family of proteins that are produced by Thelper immune cells. They are one of the principal molecules tasked with triggering the inflammatory response in the body. When interleukin-17 proteins interact with their corresponding interleukin-17 receptors on the cell membrane, a cascade of reactions is induced within the cell. This cascade of reactions leads to the activation of the transcription factor NFkB. NFkB induces several cell defenses, including inflammation. Clinical data has shown that patients with severe SARS-CoV-2 display high levels of inflammation, leading to acute respiratory distress syndrome and multiple organ failure. While antibodies that target interleukin-6 are commonly used to inhibit inflammation, they did not seem to have the same effect in SARS-CoV-2 patients.

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The question that remained was how does SARS-CoV-2 cause inflammation and what treatments could be used to diminish inflammation? Lin et al. hypothesized that inflammation in SARS-CoV-2 patients may be due to interleukin-17 rather than interleukin-6. To test their hypothesis, the researchers examined three different SARSCoV-2 proteins: NSP2, ORF7a, and ORF8, and tested their interactions with interleukin-17 receptors. To their surprise, only ORF8 exhibited interactions with the interleukin-17 receptors. These results were confirmed by in vitro experimentation and are in line with clinical data. Patients infected with SARS-CoV-2 containing mutated ORF8 proteins displayed lower levels of inflammation. While this determined that ORF8 interacted with the interleukin-17 receptors, it was unclear whether ORF8 directly triggered inflammation or if it promoted inflammation by increasing the expression of interleukin-17. To test this, Lin et al. engineered cells that did not contain any interleukin-17. They then exposed the cells to ORF8. Researchers found that the inflammatory pathway was triggered even in the absence of interleukin-17. This suggested that ORF8 could mimic interleukin-17 and interact with its receptors to directly induce an inflammatory response. Lin et al. successfully demonstrated that the interactions between ORF8 and interleukin-17 receptors caused inflammation, but how could those interactions be prevented? The researchers began by testing an interleukin-17 receptor antibody treatment. By using an antibody treatment, researchers believed that the antibodies would block the interleukin-17 receptors so that the ORF8 protein could not bind to the receptors.

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Antibody treatments can be used to block receptors from interacting with their corresponding ligands. SIMON CAULTON, OWN WORK. WIKIMEDIA COMMONS To test this theory, Lin et al. engineered a pseudovirus that expressed the ORF8 protein. They then genetically modified mice so that the mice would no longer contain interleukin-17. Lin et al. infected interleukin-17 deficient mice with the pseudovirus and compared inflammation levels in mice treated with receptor antibodies and mice who were left untreated. While inflammation occurred in both the treated and untreated mice, the treated mice exhibited much lower levels of inflammation than untreated mice. This determined that interleukin-17 antibodies were an effective treatment to reduce SARS-CoV-2-induced inflammation. In a second, related story, scientists from the Lerner Research Institute found similar results. When analyzing the interactions between ORF8 and interleukin-17 receptors, Wu et al., found that inflammation was a direct result of these interactions. A puzzling aspect of the ORF8/interleukin-17 receptor interaction, however, is that ORF8 and interleukin-17 are not very structurally similar. So, how effective could ORF8 be at actually mimicking the effects of interleukin-17? Wu et al. isolated blood cells containing interleukin-17 receptors and treated each sample with either ORF8 or interleukin-17 proteins. After these treatments, researchers analyzed the RNA of each blood cell sample to determine how gene expression differed 369

between the samples. These results would indicate how well ORF8 could mimic the effects of interleukin-17 inside the cell. Interestingly, they found that among the upregulated genes, 81.7% of genes were shared between the two sample groups. Among the downregulated genes, 64% of genes were similar between the two groups. These results confirmed that while ORF8 mimics interleukin-17 to a high degree by initiating similar inflammatory pathways, there are still some distinctions between the pathways they activate through the interleukin-17 receptor. To further investigate the relationship between ORF8 and the interleukin-17 receptors, Wu et al. examined the interactions between three common ORF8 variants and the interleukin-17 receptors. Through these experiments, researchers found that variants of ORF8 displayed decreased ability to bind to interleukin17 receptors. Considering that ORF8 is one of the most frequently mutated proteins of SARS-CoV-2, these interactions may provide an explanation for why some variants are more or less likely to cause severe SARS-CoV-2 symptoms. Both of these studies represent real progress in understanding the inflammation that occurs in patients with severe SARS-CoV-2 and reveal potential treatments against inflammation that could significantly reduce the danger of SARS-CoV-2 infection. It is still a mystery as to why coronaviruses developed a protein that could induce an inflammatory reaction. However, these papers reveal that protein mimics of interleukin-17 and their ability to bind to the interleukin-17 receptor may be the key to why some variants of SARS-CoV-2 are more deadly than others. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Mimics Inflammatory Proteins In Our Bod

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STING-Mediated Innate Immunity: How One Discovery Unlocked New Possibilities Forbes | April 26, 2022 | Article

This is part of a series on Covid-19 and inflammation related to severe disease and Long Covid. When an individual is first exposed to Covid-19, STING (stimulator of interferon genes) proteins play an important role in generating a rapid innate immune response. Studies increasingly show that these proteins may be useful therapeutic targets not only against viral infection but also other medical conditions including cancers that damage healthy cells. Activating STING, however, requires second messenger molecules to bind onto the protein. In the body, cells have DNA sensors called cyclic GMP-AMP synthase (cGAS), which recognize foreign DNA in the cytoplasm and trigger the production of cyclic dinucleotides (CDNs). These cyclic dinucleotides are the second messengers that activate STING. Several clinical trials treating cancer have already been successful using molecules derived from the hybrid cyclo-(AMP-GMP) (cGAMP), one of the most potent STING agonists. Considering the limitations and possible side effects of administering cyclic dinucleotides, only a few useful agonists have been identified. However, as we previously reported in this series, cyclic dinucleotides are not the only molecules that activate STING. Small molecules known as benzothiazinones also seem to bind strongly to STING and activate its immune properties. In this part of the series, we will delve deeper into the structure of these molecules and their role in mediating innate immunity. The initial discovery of these potent STING activators came from a study published in the European Journal of Medicine. Pyrde et al. began their investigation with a series of benzothiazinones compounds that had previously been identified as potent antibacterial candidates for treating severe bacterial infections such as tuberculosis. Initial forms of these molecules, however, only weakly activated STING. Rather than looking elsewhere, investigators 371

speculated whether these compounds could be modified to bind better to STING. The biggest challenge with designing drugs that activate STING is that the structure of this protein not only varies between different species but also within species. Humans, for example, can have one of several naturally occurring variations of STING categorized by haplotype. 99% of the population has one of the five major STING haplotypes: H232, R232, HAQ, Q and AQ. Any drug targeted towards STING would need to be highly effective against all five of these haplotypes. In a previous investigation, Pryde et al. discovered that introducing simple substitutions on a benzothiazinone molecule like compound 4 (not shown) could transform it from being a weak activator of the HAQ STING haplotype to one that was highly potent. Investigators, therefore, used compound 4 as a template for identifying other STING agonist candidates. Starting from this template (shown below), they modified both R groups (R4 and R3), as well as the core. Fourteen different compounds were identified from this first set of experiments, ranging from -1% to 116% activation of STING compared to the template molecule. Those with reported -1% activation somehow performed worse than the initially weak compound 4 agonist.

FIGURE: SCHEMATIC DIAGRAM OF THE BENZOTHIAZINONE TEMPLATE DERIVED FROM THE STRUCTURE OF COMPOUND 4. R GROUPS (R3 AND R4) AND CORE WERE CHANGED TO CREATE DIFFERENT COMPOUNDS. FROM: “THE DISCOVERY OF POTENT SMALL MOLECULE ACTIVATORS OF HUMAN STING” PRYDE ET AL. 2021

Pryde et al. realized that they could optimize some of these compounds by keeping the most effective R groups and only changing the core. These experiments produced another 13 compounds, with activation strengths ranging from 5% to 102%. All these compounds successfully activated the three most common 372

STING haplotypes, H232, HAW and R232, suggesting that they may be useful drug candidates for activating STING-mediated innate immunity.

FIGURE: TABLE ILLUSTRATING SOME OF THE FEATURED CORE SUBSTITUTIONS THAT PRODUCED POTENT STING AGONISTS, INCLUDING COMPOUND 53 (C53). FROM: “THE DISCOVERY OF POTENT SMALL MOLECULE ACTIVATORS OF HUMAN STING” PRYDE ET AL. 2021

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To determine how effective these agonists could be in regulating immunity, investigators selected compound 53 (C53) from the previous table for further testing. This small compound showed high potency across all five main human STING haplotypes, as well as those found in monkeys. Interestingly, C53 did not seem to activate mouse STING. Still, given the 81% similarity to human STING, Pryde et al. used rodent models to evaluate how C53 reacted in the body. These results confirmed that this compound could remain stable as it is absorbed in the body. Most importantly, they found that the C53 was particularly selective for STING, making it a good candidate for further research. How does this compare to human STING? Since it is too early to perform human clinical trials using these newly discovered compounds, Pryde et al. instead exposed C53 to human embryonic kidney cell samples generated in a lab. The effectiveness of the compound was determined by how well C53 could trigger phosphorylation, or activation, of STING proteins within the cell samples. Based on the pre-established pathway of STING-mediated immunity, we know that the activation of STING begins with the binding of cGMAP. As it further activates, individuals STING dimers come together to form a tetramer structure, important for recruiting a specialized protein kinase called TBK1. TBK1 importantly phosphorylates STING, as well as interferon regulatory factor proteins that then enter the nucleus to enhance immune regulation. In this experiment, Pryde et al. observed the phosphorylation of both STING and interferon regulatory factors across all the STING variants tested. The activation of STING also correlated with the recruitment of several cytokines, confirming that C53 can in fact stimulate the immune system. In particular, this included the production of several type 1 interferon cytokines important for not only fighting infection in damaged cells but also protecting nearbering healthy cells. Cytokine activity remained high even after 24 hours. C53 is only one of several potent STING agonists identified in this study. Understanding the structure of these compounds may be the key to uncovering more candidates for treatment. These compounds, however, are not a replacement for the second messenger cGAMP. Instead, it seems that both are needed to fully 374

activate STING. cGAMP first binds to the ligand-binding domain on individual STING dimers, which then prompts the binding of C53-like compounds to a second binding site within the transmembrane domain. A recent study from UT Southwestern discovered that binding to STING’s transmembrane domain in this second step is critical for forming STING tetramers and triggering the rest of the inflammatory pathway. In the figure below, Lu et al. show where cGAMP and C53 respectively bind to fully activate STING.

FIGURE: REPRESENTATION OF STING OLIGOMER WITH BOTH C53 (LABELED IN PINK) AND CGAMP (LABELED IN CYAN) BOUND TO THE TRANSMEMBRANE DOMAIN AND LIGAND-BINDING DOMAIN, RESPECTIVELY.

FROM: “ACTIVATION OF STING BY TARGETING A POCKET IN THE TRANSMEMBRANE DOMAIN.” LU ET AL. 2022 In addition to fighting Covid-19 infection, synthesizing drugs that activate STING-mediated innate immunity could also be useful against some cancers. Now, the next question is how can we capitalize on these findings to create targeted, fast-activating drugs? Although we are still in the early phases of identifying effective STING-mediated treatments, researchers are hopeful that future discoveries will save millions of lives. 375

This article is featured on Forbes.org, and can be read online here: STING-Mediated Innate Immunity: How One Discovery Unlocked New Possibilities

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SARS-CoV-2 Infection Causes Cell Fusion In The Lungs, Triggering Inflammation. Forbes | April 26, 2022 | Article

This is part of a series of stories on inflammation triggered by SARSCoV-2 infection. Other articles in the series include:lectins, Covid-19 and brain injury, long Covid, STING, monocytes, and ORF8. They may also be found on my website, www.williamhaseltine.com. When it comes to SARS-CoV-2 infection, our very own immune system can end up contributing to the damage. Inflammation, though an important part of defense against microbes, can sometimes get out of hand, leading to multiorgan injury and even death. This is evidenced by the hyper-inflammation commonly associated with severe Covid-19. The mechanisms by which SARSCoV-2 triggers such an intense inflammatory response are still being uncovered. New work by a group of researchers at Peking Union Medical College adds another piece to the mosaic of causes. Published in Science Signaling, Liu et al. discovered that SARS-CoV2 spike protein can prompt cells in the lungs to fuse together, causing damage to their DNA and triggering an immune response that culminates in excessive inflammation. Although the impacts of SARS-CoV-2 infection have been recorded across the board —from the liver to the heart to the brain, and beyond— nowhere is the damage quite as intense as in the lungs. The presence of syncytia, along with alveolar destruction and massive blood clotting, are all characteristic of severe Covid-19. Syncytia are large, cell-like structures that form when two or more cells join together; essentially, an amorphous mass of cytoplasm and nuclei (Figure 1). Even though syncytia are crucial to the formation of our muscle tissue, when induced in response to viral infection they are pathological. Importantly, the syncytia formed in response to SARSCoV-2 infection do not seem to heal or regenerate with time, as they do in response to the flu.

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FIGURE 1. Syncytia formation. FROM: “MULTISCALE MOVING BOUNDARY MODELLING OF CANCER INTERACTIONS WITH A FUSOGENIC ONCOLYTIC VIRUS: THE IMPACT OF SYNCYTIA DYNAMICS” ALZAHRANI ET AL. 2020

To study the formation of syncytia and its knock-on effects, Liu et al. co-cultured embryonic kidney cells containing SARS-CoV-2 spike protein and enhanced green fluorescent protein (EGFP) — which makes the S protein-containing cells show up green under a microscope— with a range of ACE2-containing human endothelial cells. ACE2 acts as the primary portal of entry for SARS-CoV-2, mediated by the spike protein. As expected, the researchers witnessed cell fusion between the spike protein-containing cells and the endothelial cells. This had already been demonstrated in prior studies. But Liu and colleagues noticed that cell fusion depends greatly on host proteases used to break down the spike protein, such as transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 spike undergoes cleavage at two sites: S1/S2 and S2’ (Figure 2). When the researchers generated spike protein mutants with alterations to these sites —rendering them resistant to cleavage— they witnessed only modest, if any, syncytia formation. The unmutated spike protein, in contrast, induced extensive cell fusion.

FIGURE 2. Schematic representation of the S1/S2 and S2’ cleavage sites along the SARS-CoV-2 spike ... [+] FROM: “SARS-COV-2 SPIKE PROTEIN–

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INDUCED CELL FUSION ACTIVATES THE CGAS-STING PATHWAY AND THE INTERFERON RESPONSE” LIU ET AL. 2022

Following cell fusion and the formation of syncytia, Liu et al. witnessed upregulation of interferon-beta (IFN-β). Interferons are a class of signaling proteins produced and released by host cells in response to viral infection, warning other nearby cells of danger. IFN-β is particularly closely linked to the innate immune response and, crucially, the activation of inflammation. Although helpful in containing viral spread across the body, prolonged and misregulated production of interferons can impair lung cell regeneration and worsen disease outcome. Curious to determine how exactly cell fusion triggers IFN-β activation, the team of scientists turned to one of the central signaling pathways of the innate immune system, the cGAS–STING pathway (Figure 3). This pathway kicks off with the detection of misplaced DNA by an enzyme called cGAMP synthase (cGAS). DNA is usually found in the nucleus of a cell, so its presence in the cytoplasm indicates either cellular damage or microbial infection. cGAS binds to the misplaced DNA, triggering the formation of cyclic GMPAMP (cGAMP). This, in turn, goes on to bind to Stimulator of Interferon Genes (STING), a protein in charge of inducing the production of type 1 interferons such as IFN-β. With STING in the game, interferon regulatory factor 3 (IFR3) undergoes phosphorylation. Once phosphorylated, IFR3 can travel to the nucleus of the cell in order to upregulate inflammatory genes.

FIGURE 3. A simple diagram illustrating the various steps involved in the cGAS– STING pathway, ... [+] SOURCE: BELLBROOK LABS

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Liu et al. returned to their co-cultured cells and looked for signs of cGAS–STING pathway activation. Substantiating their hunch, they found traces of phosphorylated IRF3 in the cell culture. To further confirm their hypothesis, they co-cultured cells engineered to lack cGAS and STING with spike-protein containing cells. The result? A marked decrease in IFN-β activation. Interestingly, the researchers found that stimulation of IFN-β also depended on spike cleavage. Returning to the cleavage-resistant spike protein they had engineered, Liu et al. compared the activation of the cGAS–STING pathway between the mutated spike and the original SARS-CoV-2 spike. Only the unmutated spike protein induced the expression of IFN-β. They also found that cells overexpressing TMPRSS2 displayed higher levels of IFN-β expression. So the cGAS–STING pathway is responsible for triggering IFNβ upregulation in response to SARS-CoV-2-induced cell fusion. But, how is cell fusion triggering the pathway? Genetic analysis of the cell cultures that triggered the cGAS– STING pathway revealed an increased induction of DNA damage responses (DDR) — an elaborate network of genes in charge of recognizing and tending to DNA damage. Liu et al. turned to the microscope; they observed that spike protein-induced syncytia were peppered with ruptured nuclei and with micronuclei. This was not seen in co-cultures formed using the cleavage-resistant, mutated spike protein. Finally, the researchers sought to confirm whether the same series of events unfold during viral infection. Instead of the spike protein-containing kidney cells they used for their initial co-cultures, the team directly infected a range of human endothelial cell lines with a vesicular stomatitis virus (VSV) that had been engineered to express the SARS-CoV-2 spike protein. Again they witnessed the formation of syncytia, ruptured nuclei, the activation of the cGAS– STING pathway, and ultimately, the upregulation of IFN-β. The work by Liu et al. offers us insight into another mechanism through which SARS-CoV-2 induces inflammation. Covid-19 patients initially display a weakened type 1 interferon response, in large part orchestrated by the virus’ immunosuppressive tactics. This isn’t the case with patients that progress to severe disease, who often display signs of robust interferon response. More research needs to 380

be done to understand the contribution of type 1 interferon to hyper-inflammation and severe Covid-19. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Infection Causes Cell Fusion In The Lungs, Triggering Inflammation.

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The Role Of N Protein Mutant Variants As Determinants Of SARS-CoV-2 Replication And Pathogenesis Forbes | April 26, 2022 | Article

An often overlooked aspect of SARS-CoV-2 variation is the potential impact single mutations can have on characteristics like transmission and virulence. One variant may contain a single change over another enabling it to be far more dangerous to the global population. Among the first major mutations that have manifested widely two years into the Covid-19 pandemic are the N protein mutations R203K and G204R. These mutations were first assumed to have emerged with the Alpha variant, originally denoted B.1.1.7 in late 2020. However, analysis of sequenced viruses in the GISAID SARS-CoV-2 database shows that R203K and G204R were first observed as early as Spring 2020. Today, these two mutations are found in 4.8 million of the 10.4 million viruses in the GISAID database, indicating that about half of SARS-CoV-2 infections involve these mutations. N Protein Mutations In Saudi Arabia A recent study by Mourier et al. delved deeper into why these two N protein mutations are so omnipresent. Their observations centered on the kingdom of Saudi Arabia, hypothesizing that the relatively high level of population movement, specifically in reference to religious mass gatherings, could be a breeding ground for SARS-CoV-2. Their observations were conducted from March to August 2020, meaning little was known about the virus or the variants that would follow. Mourier et al. collected 892 SARS-CoV-2 samples via nasopharyngeal swabs from patients in Jeddah, Makkah, Madinah, Riyadh, and the east coast of Saudi Arabia. They then performed phylogenetic analysis on the samples to ascertain the genetic diversity of their samples. To my knowledge, this is one of the earliest large-

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scale examinations of genetic diversity in collected samples during the Covid-19 pandemic.

FIGURE 1: Regions where samples were collected over the span of six months. MOURIER ET AL.

Of the 892 samples, Mourier et al. noted 836 nucleotide modifications as compared to the Wuhan wild-type. A vast majority of these nucleotide mutations were isolated to a single sample and were generally disregarded, however, some nucleotide mutations were more frequent than others. Specifically, the researchers noted a high volume of A23403G, resulting in a mutation from aspartic acid to glycine at position 614 (D614G) in the Spike protein, and three consecutive nucleotide mutations of G28881A, G28882A, and G28883C, resulting in mutations from arginine to lysine at position 203 (R203K) and glycine to arginine at position 204 (G204R) in the N protein. N Protein Functional Characteristics The high prevalence of R203K and G204R initiated a closer examination of the mutations in reference to functional analysis and impact on virological characteristics. Mourier et al. first examined the viral load implications of the N protein mutations. Using cycle threshold values obtained through PCR analysis of the samples, they found that samples including the two N protein mutations displayed a 33% increase in viral load over samples excluding the mutations. They also note that patients with severe symptoms were more often associated with viruses including R203K and G204R, implicating higher viral load as one reason for more intense Covid outcomes. 383

FIGURE 2: Density distributions of virus copy numbers derived from Ct measurements. MOURIER ET AL.

They next examined the oligomerization potential and RNAbinding affinity. Noting that the mutations were in the N protein linker region, which is involved in N protein oligomerization. Oligomerization is a key function of the N protein, binding and encoding the full-length viral RNA. Mourier et al. found samples with the mutant N protein had a higher oligomerization potential than others. As oligomerization is involved in viral RNA interactions, they used an in vitro assay to examine binding affinity. They note a significantly stronger binding affinity than non-mutant viruses, suggesting increased efficiency of N protein functions.

FIGURE 3: A schematic diagram showing the SARS-CoV-2 N protein's different domains (Upper: control, ... [+] MOURIER ET AL.

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The N protein is multifunctional and pleiotropic. Among its many functions are import and export packaging of RNA from the nucleus, synthesis of genomic RNA, cell cycle manipulation of the host cell, and suppression of interferon responses to name a few. To begin to explore the effect of the N protein mutations on non-packaging functions, Mourier et al. then examined the mutant N protein interactions with host proteins. Using a mass spectrometry analysis, they found that of the 43 proteins that displayed significant differential interactions with the SARS-CoV-2 N protein, 42 showed increased interaction with the mutant protein. They found that many of the 42 host proteins are involved in significant immune processes, including viral processing, regulation of RNA nuclear export, apoptosis, and immune regulation. One characteristic that sets the N protein apart from most others is that it is phosphorylated and the degree of phosphorylation can dramatically affect its function. For this reason, Mourier et al. examine the phosphorylation of the mutant protein. They note that S206 is highly phosphorylated in the mutant N protein as compared to the wild-type N protein. We emphasize that S206 is unmutated and is only adjacent to the mutant amino acids. The authors speculate that the changes at positions 203 and 204 increase phosphorylase affinity, leading to greater N protein phosphorylation, which is positively correlated to the efficiency of viral genome processing and nucleocapsid assembly. A highly phosphorylated S206, which lies in the critical linker region, likely improves the mutants' viral fitness. Finally, Mourier et al. examine the relationship between the N protein mutations and pathogenesis. They found that the mutant N protein upregulates over 100 interferon-related genes in infected host cells. Interferon genes allow for communication between cells to trigger protective defenses by the immune system to eradicate pathogens. Among these defenses are events like cytokine storms, which are floods of circulating cytokines and activated immune cells, leading to severe disease symptoms. The mutant N protein is more likely to induce severe disease as a result of this upregulation.

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FIGURE 4: Upregulation of mutant and control transfected cells. MOURIER ET AL.

These results align with a study from early November 2021 by Syed et al. that examined N protein mutations in the Delta virus. Using virus-like particles in a lab setting, Syed et al. analyzed dozens of N protein mutations for impacts on transmissibility and virulence. Their examination also highlighted the linker region, wherein they noted that all natural variants of concern or interest contained at least one amino acid mutation between positions 199 and 205. One such mutation, S202R, resulted in 166-fold higher infectious titers as compared to the wild-type. While R203K and G204R do not impact viral efficiency this extremely, the congruence between these two studies is notable. The N protein mutations are but one example among many. The SARS-CoV-2 genome is roughly 30,000 amino acids long and any one of those can be mutated in dozens of different ways. While most mutations will not make much of an impact, many can and do. Using the sort of analyses that Mourier et al. conduct, we can monitor emerging variants for dangerous mutations to estimate their transmissibility, virulence, and pathogenesis before they begin to impact the population, and then we may adjust our Covid counterstrategies accordingly. 386

This article is featured on Forbes.org, and can be read online here: The Role Of N Protein Mutant Variants As Determinants Of SARSCoV-2 Replication And Pathogenesis

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The Center For Forecasting And Outbreak Analytics Could Represent A New Era In Infectious Disease Control, But Challenges Lie Ahead Forbes | April 28, 2022 | Article

Leaders have relied on data-driven weather forecasts to understand when to deploy resources in response to natural disasters and advise the general public on the level of risk so they understand whether to shelter at home, evacuate, or simply bring an umbrella when leaving the house. The CDC's new Center for Forecasting and Outbreak Analytics (CFA), aims to act in a similar way to guide decisions about broad public health needs like deploying antivirals or additional testing capacity and advising the general public on the level of risk for different behaviors like wearing a mask or attending large events. This is a long-overdue element of infectious disease control that replaces a patchwork of mostly volunteer academic experts and institutions. Designing and implementing infectious disease policies and guidelines without a centralized reliable source for forecasting is challenging. The CFA could have a profound impact on how we approach and control this pandemic and inevitable future infectious outbreaks. The CFA is intended to support federal, state, and local decisionmakers with analyses at every stage of a health threat. The work will be focused on three main areas; predicting and modeling, informing, educating and communicating and innovation. This will allow the center to determine the outbreak risk and its potential to reach epidemic status, early on. There will be a strong focus on effective communication and how to translate the data into clear decisions, interventions, and resource allocation. However, the system will only be as effective as the data it receives. There needs to be a clear, real-time, pipeline of anonymized infectious disease data identified via zipcodes from 388

hospitals and primary care clinics to the CFA. We can not also ignore the use of open-source intelligence. Analyzing the data is just the first step. A lack of general testing capacity or general ambivalence about testing amongst the public as Covid fatigue sets in could threaten the accuracy of the data. Recent cuts to federally subsidized testing programs will undercut the expansive data needed for this program to be successful. Antiviral drugs like Paxlovoid need to be stockpiled so they can be swiftly distributed free of cost to communities through programs like test and treat. Egypt’s 100 Million Healthy Lives initiative provides a recent example of how universal disease surveillance, treatment, and eradication is possible. Egypt previously had the highest rate of Hepatitis C in the world. In 2018, Egypt launched the 100 Million Healthy Lives program. The goal was to screen all Egyptians over the age of 12 for active hepatitis C virus replication along with other chronic conditions such as hypertension, diabetes, and obesity. Treatment was offered for free in government clinics for those who tested positive for hepatitis C, hypertension, and diabetes; free counseling was available for those considered obese. Approximately 4 million people with active hepatitis C were identified and treated with the antiviral medication Sovaldi (sofosbuvir), a nucleotide analog that inhibits the polymerase enzyme of hepatitis C and blocks its replication, effectively eliminating hepatitis C from Egypt. Once initiated, the program was completed in 18 months. Implementing this kind of program will not only improve our disease surveillance but allow us to work towards universal healthcare goals and emerge as a healthier society. The CFA will hopefully begin to rebuild trust in the general public which has been eroded by conflicting and confusing guidelines that have been issued by various federal agencies. A centralized, data-driven source of guidelines will relieve the burden of risk assessment that has fallen heavily on the shoulders of the public and business owners for years. The launch of the CFA puts us in a better position to assess the early risk of emerging Omicron subvariants and any new variants that are likely to occur as the virus evolves and responds more swiftly than we did with past devastating variants. But the center’s work needs to be funded sustainably for the long term and state and public 389

health departments need a skilled workforce that understands methods for modeling diseases. Funding for public health programs has always been fraught with difficulties. But we should look to the substantial investments made in HIV/AIDS as an example to follow. Within the US, we now have the tools to treat and significantly improve the quality and longevity of those living with HIV. It is no longer viewed as a death sentence and should be the standard we aspire to for treating all diseases. This article is featured on Forbes.org, and can be read online here: The Center For Forecasting And Outbreak Analytics Could Represent A New Era In Infectious Disease Control, But Challenges Lie Ahead

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Additional Support Arrives For The Covid-19 Test-To-Treat Program Forbes | April 29, 2022 | Article

The Biden Administration's Test-to-treat program has the potential to transform the pandemic, especially for those at high risk of severe disease. The program represents a great advance in healthcare delivery. Test-to-treat is intended to eliminate any barriers to accessing testing and treatment in the early days of infection. The ideal scenario is for patients to walk into a pharmacy that has a health clinic staffed by nurse practitioners, physician assistants, or physicians where they can be tested for Covid-19 at no cost, and if treatment is deemed appropriate be prescribed oral antiviral therapy at no cost. Currently, only 2,800 of the approximately 50,000 retail pharmacies in the U.S. have health clinics staffed by nurse practitioners, physician assistants, or physicians causing issues of access and equity. But the Biden Administration is working to address these issues and I hope that there continue to be many incremental improvements. Oral antivirals are currently available in 20,000 locations across the US including pharmacies, community health centers, hospitals, urgent care centers, Veterans Affairs clinics, and Department of Defense Medical Treatment Facilities. The Biden administration intends to increase that number to 30,000 in the coming weeks. The drugs are intended for those with mild or moderate COVID-19 who are at risk of severe disease. This can include older people and those who are immunosuppressed or have conditions such as heart disease, cancer, or diabetes. The drug Paxlovid has a distinct advantage, as it has a higher efficacy and is also authorized for children ages 12 and older. The U.S. has also committed to the purchase of 20 million treatment courses of Paxlovid. But despite the abundance of medication, state health officials have reported that many Americans who are good candidates for Paxlovid do not seek it out. Pharmacists 391

have also reported that they have struggled to use up the drugs despite recent increases in cases. While the cost of the pills is covered by the federal government, obtaining a prescription can be expensive for those who are not insured. CVS pharmacies is one of the largest participants in the Test-to-treat program and primarily where those who don’t have a primary care physician or who live in a healthcare desert would seek out Test-and-treat. CVS doesn’t charge uninsured patients for Covid testing, however, an appointment at a MinuteClinic which is required to prescribe an antiviral can cost upwards of $100. Those without insurance or whose health plans don't cover visits to the clinics are responsible for the cost of the appointment. The fees associated with getting a prescription from an urgent care clinic or hospital would be significantly higher than a Minute Clinic for the uninsured. One way I suggest the cost barrier could be resolved is to have the appointments made free of charge through federal funds or to authorize pharmacists to dispense oral antiviral drugs to patients with positive Covid-19 tests who meet the criteria. Not dissimilar to how pharmacists were given broad, national authorization to administer Covid-19 vaccines. However, this poses problems of potential liability and fear of litigation for pharmacists. The American Medical Association (AMA) has rejected the concept of Test-to-treat in pharmacies, citing concerns over the complexity of treating Covid19 and the high number of drug reactions, suggesting they seek care from their primary care physician instead. Other reasons Paxlovid may be underutilized are due to poor communication and messaging. Members of the general public may be unaware they qualify for it, hesitant about taking a new medication or confused by healthcare providers who interpret the eligibility guidelines more narrowly than others. The Biden Administration intends to address these issues by making sure that health care providers have the latest information on Covid treatments and launching an awareness campaign for the general public. This will include an online test-to-treat locator and a call center (1-800-232-0233) to provide help in more than 150 languages. However, they will need to address the same trust issues that have contributed to lagging vaccination rates.

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However, Paxlovid does have the disadvantage of several drug interactions with common medications such as statins because of the use of the drug Ritonavir. Hopefully, we will soon have a range of antiviral drugs that match or exceed Paxlovid’s efficacy with fewer interactions. Recent data for Shinogi's new protease inhibitor drug S-217622, show promising results and does not use Ritonavir. On day four of treatment, the proportion of patients with positive viral titer decreased by approximately 90% versus placebo. At this stage in the pandemic, we have some highly effective tools to reduce the severity of the Covid-19 for individual health outcomes and then reduce its spread within the community. If we can remove the barriers of cost, poor communication, and access we can make enormous progress in our fight against the virus. This article is featured on Forbes.org, and can be read online here: Additional Support Arrives For The Covid-19 Test-To-Treat Program

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35B5: A Potent, Broadly-Neutralizing Monoclonal Antibody Effective Against All Known Variants Forbes | April 29, 2022 | Article

Monoclonal antibodies have proven to be a potent tool in our ability to prevent and treat SARSCoV-2 infections. The current epidemic is driven in part by naturally occurring variants that elude vaccine-induced and monoclonal antibodies. The search is on broadly neutralizing antibodies. Here we describe one such antibody recently reported by Wang et al. in Japan. Discovering 35B5 In a previous study from earlier this year, Wang et al. attempted to discover broadly neutralizing monoclonal antibodies that would be effective against both existing and future variants. Their experiments took place in the latter stages of the Delta surge of infections. Sorting the B cells for immune memory of the SARSCoV-2 receptor-binding domain, they found a match. Wang et al. cloned the monoclonal antibody 35B5. They then analyzed the neutralizing capacity, finding that 35B5 potently neutralized not only the SARS-CoV-2 wild-type but also a broad spectrum of variants, including Beta and Delta, leading the researchers to investigate the structural-functional mechanisms of 35B5 in greater detail.

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FIGURE 1: Neutralizing activity and virus culture analysis of 35B5 against authentic SARS-CoV-2 ... [+] WANG ET AL.

35B5 Also Neutralizes Omicron After the rise of the Omicron variant, Wang et al. repeated their experiments in the context of the new variant. They observed 35B5 exhibiting binding efficiency to Omicron comparable to Delta and the SARS-CoV-2 wild-type, as well as trimer dissociation. The authors note that Omicron’s binding efficiency with 35B5 is slightly weaker than the wild-type likely because of a hydrogen bond between the antibody and N481 in the receptor-binding domain, which is impaired in the Omicron Fab interface. In a neutralization assay, they found that both pseudotype and authentic Omicron viruses were potently neutralized by 35B5. While Omicron was not neutralized as effectively as Delta or the wild-type, it was still efficient enough to disable the virus.

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FIGURE 2: Neutralizing activity of 35B5 against SARS-CoV-2 variants. (A) Binding capacities and ... [+]WANG ET AL.

35B5's Residue Target Despite dozens of mutations in the Omicron Spike receptorbinding domain and N-terminal domain, 35B5 still binds tightly over a footprint of 29 interacting residues. These residues interact via salt bridges and hydrogen bonds to allow for interaction between the antibody and the Spike.

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FIGURE 3: Interface between the N165-glycans and the down-RBD in Omicron SECD. The down-RBD and ... [+] WANG ET AL.

One crucial glycan that 35B5 interacts with is N165. One of the functions of glycans is to shield the virus particle beneath, but N165, along with N234, also possesses a unique responsibility. These two act together as a molecular switch to control the Spike conformational transition. The Spike alternates between up and down conformation based on whether it is currently infecting a host cell. The two glycans clamp the two sides of the receptor-binding domain and enable the trimer to alter between the up and down configurations, acting as a switch. The 35B5 antibody displaces N165 from its binding pocket, disabling the switch mechanism, like breaking a light switch lever.

FIGURE 4: Location of the N165- and N234-glycans from the NTD at the interface between the NTD and ... [+] WANG ET AL.

Were the Spike in the up configuration, Wang et al. found that the 35B5 antibody would continue to take a toll on the virus. When superimposed into the up configuration, the researchers observed the N165 and N234 glycans are displaced from their native binding pocket. This displacement results in significant dysfunction of the glycan switch necessary for transition between up and down configurations, suggesting that the 35B5 antibody forces the trimer in the up configuration, thereby destabilizing the Spike. 397

What makes this antibody so exciting is the conservation of the N glycans across SARS-CoV-2 genomes. Of the 10.5 million SARS-CoV-2 genomes in the GISAID database, only 3,129 contain a mutation at position N165 or less than .03%. The same can be said about N234, which only displays a mutation in 1,723 viruses or less than .02%. This suggests that 35B5 could have neutralized over 99% of the viruses circulated since the start of the pandemic, meaning they could be a great tool for variants yet to come. Additionally, the mutations in Omicron are far from the 35B5 epitope, suggesting an additional advantage to 35B5. Because the 35B5 epitope residues are highly conserved, it is unlikely that Omicron RBD mutations interfere with 35B5’s virus neutralization.

FIGURE 5: Interactions between 35B5 Fab and the Omicron RBD; Surface distributions of the Omicron ... [+] WANG ET AL.

This is not the only antibody that recognizes highly conserved sequences in the Spike. We note that Zhou et al. describe two antibodies that target conserved sequences in the Omicron Spike: Ly-CoV1404 and S2E12. Additionally, Li et al. describe the CV325 antibody, which binds a linear epitope in the S2. We suggest that a combination of 35B5 and CV3-25 might be ideal for the prevention and treatment of Covid-19. Similar combination antibodies that bind both the membrane-associated protein and the receptor-binding protein display broad neutralizing activity against most of the existing strains of Ebola. Such a combination antibody therapy for Covid-19 could be a successful path for treatment and prophylaxis against current and future strains of the virus.

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This article is featured on Forbes.org, and can be read online here: 35B5: A Potent, Broadly-Neutralizing Monoclonal Antibody Effective Against All Known Variants

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May 2022

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Asthma Medication Points The Way To Drugs To Prevent And Treat Covid-19: Nsp1, A Novel Target Forbes | May 02, 2022 | Article

Targeting Nsp1 with montelukast blocks a critical early step in SARS-CoV-2 replication. CREDIT: MOHAMMAD AFSAR To survive and replicate inside our cells, a virus has to be able to adapt them to its own advantage. This means altering the cells’ metabolism, physiology, and gene expression. Often, these adaptations are part of a strategy to bypass and thwart our innate immune system — a virus can’t get very far if our immune system manages to recognize it early on. So, suppressing our immune response is one of a virus’ top priorities. SARS-CoV-2 is particularly crafty in this regard, possessing many different tactics that help keep it under the radar. But there’s a flipside to this craftiness, it is highly complex. A great deal of different processes and pieces need to come together for it to work. Block one, and the whole thing can come crashing down. This makes it an excellent avenue for therapeutic interventions. A group of researchers based at the Indian Institute of 401

Science (IISc) have uncovered a new target: SARS-CoV-2 nonstructural protein 1 (NSP1). Published in eLife, their work shows that binding to and blocking NSP1 leads to a decrease in viral replication in human immune cells. There are two major categories of viral proteins: structural and nonstructural. Structural proteins are the components of the virus particle itself. They provide the essential elements the virus needs to transmit from one person to another. Nonstructural proteins, on the other hand, are not a component of the virion particle itself. Instead, they are produced by the virus once inside host cells. Here, the nonstructural proteins help with viral replication by regulating transcription and impairing host defenses. NSP1 is an especially versatile nonstructural protein, with a variety of different functions related to both immunosuppression and viral replication. It is one of the first viral proteins released once inside the cell. The primary function of NSP1 is to inhibit the translation of host messenger RNA (mRNA). To replicate, viruses need to suppress the genes of the host cell and instead favor synthesis of their own. NSP1 contributes to this by binding to ribosomes, which act as the “cellular machinery” via which proteins are made. More specifically, NSP1 is made up of two sections, an N terminal and a C terminal. It is the C terminal that binds deep into the mRNA entry tunnel of the small ribosomal subunit (40S). Once bound, NSP1 prevents host messenger mRNA from being translated, but in an act of delicate trickery, still allows for viral mRNA to be translated (Figures 1 & 2).

FIGURE 1. NSP1 blocks ribosome entry of cellular messenger RNA. NSP1 permits ribosome entry of viral ... [+] CREDIT: ACCESS HEALTH INTERNATIONAL

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FIGURE 2. NSP1 binds to the mRNA entry channel of the 40S Ribosome, inhibiting host mRNA ... [+] FROM: “SARS-COV-2 DISRUPTS SPLICING, TRANSLATION, AND PROTEIN TRAFFICKING TO SUPPRESS HOST DEFENSES” BANERJEE ET AL. 2020

Downstream, the sabotaging of host mRNA translation means a variety of important antiviral proteins don’t get produced. This weakens the immune response our bodies can muster, and in a dangerous feedback loop, makes it even easier for the virus to replicate and spread unchallenged. Aside from its pivotal role in viral replication and host immunosuppression, Asafar et al. focused on NSP1 because it is one of the SARS-CoV-2 proteins with the lowest mutational frequency. That is, it changes very little over time and across variants. Any drug that wishes to remain effective against SARS-CoV-2 has to contend with the rise of new variants — zeroing in on a highly conserved, functionally important protein like NSP1 improves the odds. Once they’d chosen their intended target, the team of scientists combed through a database of around 1,600 FDA-approved drugs. Turning to computational modeling, they isolated drugs with the 403

potential to bind the NSP1 C-terminal. Of a final list of a dozen or so candidates, Asfar et al. narrowed it down to one, montelukast. Montelukast is usually used to treat asthma and hayfever. How might it work to curb SARS-CoV-2 infection? In a nutshell, by binding to the C terminal of NSP1 before NSP1 has a chance to bind to our ribosomes. This clogs up NSP1’s binding site and prevents it from inhibiting our cell’s mRNA translation. At least, that’s how it should work in theory. To find out whether it worked in practice, the researchers engineered human kidney cells that express NSP1. They observed a noticeable drop in mRNA translation and protein synthesis. Next, Asafar et al. exposed these cells to montelukast. The drug restored protein synthesis to normal levels, successfully counteracting the effects of NSP1. Asafar and his colleagues also exposed human cells to live SARSCoV-2 virus and then treated those cells with montelukast. They witnessed a significant drop in the expression of SARS-CoV-2 spike protein, suggesting reduced viral replication. The results indicate that montelukast managed to prevent NSP1-mediated inhibition of host mRNA translation, allowing for normal production of antiviral proteins and a reduced capacity for viral replication (Figure 3).

FIGURE 3. Bar graphs showing the effects of montelukast on SARS-CoV-2 RNA copy number (left) and ... [+] FROM: “DRUG TARGETING NSP1RIBOSOMAL COMPLEX SHOWS ANTIVIRAL ACTIVITY AGAINST SARS-COV-2” ASAFAR ET AL. 2022

Despite the positive results, the strength with which montelukast binds to NSP1 —known as “binding affinity”— is still relatively low. Asafar et al. admit that montelukast on its own likely won’t be up to 404

the task. But its mechanism of action, the way in which it prevents SARS-CoV-2 NSP1 from inhibiting host mRNA translation, is a viable one, and a good starting point for future drug design. Importantly, it hints at an overall strategy for successful therapeutic intervention: isolating the specific processes by which SARS-CoV2 manages to suppress and evade our immune response and discovering drugs that inhibit each one. The book, Natural Immunity and Covid-19: What it is and How it Can Save Your Life, provides a detailed overview of these processes. Figure 4 highlights the virus' multiple points of sensitivity. I foresee a day when there will be twenty or more different classes of drugs (with multiple drugs in each class) each targeting a different viral protein. When used in combination, these drugs will be safe and effective in preventing and treating Covid-19, no matter what variant may arise.

FIGURE 4. A diagram of the SARS-CoV-2 viral genome mapping out potential drug targets. Viral ... [+]CREDIT: ACCESS HEALTH INTERNATIONAL

This article is featured on Forbes.org, and can be read online here: Asthma Medication Points The Way To Drugs To Prevent And Treat Covid-19: Nsp1, A Novel Target

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Structural Analysis Of The Omicron Spike Unveils Houdini-Like Immune Escape Forbes | May 03, 2022 | Article

Omicron is the ultimate escape variant. Not only does it escape from natural and vaccine-acquired immunity, but it also escapes most, but not all neutralization from monoclonal antibodies. A recent paper by Wang et al. not only describes a new broadly neutralizing antibody but also provides a deep understanding of the structural properties of the Omicron Spike protein uses to facilitate immune evasion while remaining sensitive to neutralization by some monoclonal antibody treatments. Here we describe some of Wang et al.’s most striking observations. As has been well established, the Omicron BA.1 Spike and all Omicron family variants that followed have the most mutated Spike protein of any natural variant to this point in the pandemic. In the Omicron BA.1 receptor-binding domain alone, there are 15 mutated sites. Monoclonal antibodies are described by where they bind, divided into four groups: classes 1, 2, 3, and 4. The mutations in the Omicron receptor-binding domain are located in such a way that they interfere with the binding of all four classes of antibodies. In their investigation of the 35B5 antibody, Wang et al. note that “most of the 15 mutation sites, including N501Y, G496S, K417N, Q493R, and G446S…severely change the epitopic residues of class 1 and 2 mAbs. The G339D and N440K mutations are located in the epitopes for class 3 mAbs…whereas S371L, S373P, and S375F are located at the RBD interface with class 4 mAbs.”

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FIGURE 1: Mutation sites in the Omicron RBD. The mutation residues are shown as red sticks. Classes ... [+] WANG ET AL.

In addition to amino acids changing the structural binding dynamics of the Spike, the mutations also induce electrostatic changes to the spike surface. Altering the charge or polarity of a binding site further inhibits the ability of an antibody to bind, acting as a shield of sorts. In particular, Wang et al. note increased positive charges for regions impacting class 1 and 2 antibodies, as well as increased hydrophobic characteristics at the class 4 interface.

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FIGURE 2: Surface electrostatic distribution of Omicron (left) and G614 (right) RBDs colored by ... [+]

WANG ET AL. The Omicron Spike also displays eight mutations in the Nterminal domain, which plays a role in Spike stability. Mutations like T95I and A67V increase hydrophobic interactions in the N-terminal domain, while other deletions like del69-70 and insertions like ins214EPE create disorder and antigenic shifts. The Omicron S2 subregion contains six further mutations. The mutations of N764K and N856K create further hydrogen bonding and strengthen the interaction between the different domains of the Spike. Other mutations, like D796Y and L981F, increase hydrophobic interactions in the S2 structures, yielding tighter packing and interaction of the Spike trimer, which we will explore in a later article. However, there are hidden workarounds for monoclonal antibodies that do not involve mutated Omicron residues. These are conserved amino acids that are crucial for basic SARS-CoV-2 functions.

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FIGURE 3: Interactions between 35B5 Fab and the Omicron RBD (shown as a yellow surface). The epitope ... [+] WANG ET AL.

The 35B5 antibody that Wang et al. investigated specifically avoids mutated residues in the receptor-binding domain and attacks conserved regions. This makes 35B5 and conserved-residuetargeting antibodies like it a major threat to Omicron replication and furthermore, any variant of SARS-CoV-2 with these conserved sequences. The SARS-CoV-2 Spike protein is comprised of a number of sheets and loops, which are structures made of connected amino acids. The stability of one such sheet, beta 5/6, is directly linked with ACE2 binding efficiency. In their investigation of 35B5, Wang et al. found that residues R346, S349, and Y351 are located in the L2 loop, which interacts directly with amino acids in beta 5/6 and stabilizes the conformation of that sheet. An additional residue in the L2 loop, V350, inserts a hydrophobic pocket under beta 5, providing further support for the sheet. The researchers conclude that the conserved amino acids in L2 are crucial for beta 5/6 and ACE2 binding in general.

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FIGURE 4: Interactions between the L2 loop and β5 and β6 in the core region of the Omicron RBD. WANG ET AL.

We note that there are mutations in the Omicron Spike protein that are known to decrease ACE2 binding affinity. McCallum et al. analyzed the receptor-binding domain mutations in great detail, noting that some mutations, such as lysine to asparagine at position 417 (K417N) and glutamine to arginine at position 493 (Q493R), individually reduce ACE2 binding affinity, while others like asparagine to tyrosine at position 501 (N501Y) and serine to asparagine at position 477 (S477N) increase affinity. Though despite competing mutations in terms of ACE2 binding efficiency, the Omicron BA.1 variant still binds 2.4-fold tighter as compared to the wild-type. In addition to the mutations that increase binding affinity, it is possible that the L2 residues from positions 344 to 354 are conserved to compensate for decreased affinity mutations. The required conservation of these residues is therefore a glaring target for the 35B5 antibody or similar antibodies that target the same region. This is one of many structural intricacies in Omicron and SARSCoV-2 in general. This analysis will be followed by further discussion of how Omicron differentiates itself from all previous variants of concern and interest, informing monoclonal antibody treatment moving forward.

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This article is featured on Forbes.org, and can be read online here: Structural Analysis Of The Omicron Spike Unveils Houdini-Like Immune Escape

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New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part II Forbes | May 03, 2022 | Article

This story is Part II of a series on respiratory syncytial virus vaccines. Here we describe the groundbreaking research that is both answering questions about the past vaccine failure and mapping a path towards the production of successful vaccines.This story is Part II of a series on respiratory syncytial virus vaccines. Here we describe the groundbreaking research that is both answering questions about the past vaccine failure and mapping a path towards the production of successful vaccines. Respiratory syncytial virus is one of the leading causes of death for infants globally, and a safe vaccine has yet to be approved for use. However, the long hunt for a vaccine may soon be over. The new structure-based vaccine technology behind the recent development of such vaccines holds great promise. In Part I of this series we discussed the magnitude of the disease and severity of its consequences, and here in Part II we discuss the science that underlies the recent advances in search for an RSV vaccine. Until recently, respiratory syncytial virus has posed obstacles to the technology that has been successful in producing functional vaccines that have succeeded in mitigating the spread and severity of many other infectious diseases. While the search for a vaccine for respiratory syncytial virus has eluded scientists and vaccine manufacturers for decades, just as of recently there are finally some promising vaccine candidates in phase 3 trials. The new structurebased vaccine design technology behind their development will have lifesaving consequences, as respiratory syncytial virus is the leading cause of hospitalizations in children under five, and infants and the elderly are particularly susceptible. Just over 55 years ago in 1965, a small vaccine trial for respiratory syncytial virus resulted in increased rates of hospitalization and the deaths of two infants from a treatment group of only 23. The 412

disastrous consequences of the trial stunted any further development of an RSV vaccine through the turn of the century. Recent advances in structural vaccine technology mean that vaccines may soon be available, even for young children.

One avenue of structure-based vaccine design technology consists of focusing on the pathogen’s physical form, down to the individual atom. By identifying the structures to which antibodies bind, scientists can then synthesize a replicate of the antigen structure. Formulating vaccines with the engineered antigens will prompt a response of antibody production in the body, priming the immune system to respond swiftly to future exposures. What follows is drawn largely from the research published by Graham, Modjarrad, and McLellan in 2015.

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Figure 2. Schematic drawing of RSV particlesThe F glycoprotein is known to exist on the virion membrane in both pre-F (functional) and post-F ... [+] GRAHAM ET AL.

The fusion (F) protein was identified to be the key viral structure to target for RSV. However, we now know that the F protein has two forms, converting from the prefusion state (pre-F) into the postfusion state (post-F). Antibodies target both forms of protein F, but those that bind to the prefusion form are much more effective at neutralizing the virus. Furthermore, the negative effects of the virus can be in part attributed to the non-neutralizing binding of antibodies to the postfusion form. Knowing this, it became clear to scientists that to engineer a vaccine to produce the maximum defense, they should focus on the prefusion antigen structure. The problem with this is that the prefusion F protein is highly unstable. The prefusion state is spring loaded and readily assumes the postfusion state. To incorporate the pre-F form into the vaccine for the purpose of stimulating more impactful antibody production, the challenge is to stabilize the F protein in its prefusion state. In 2013, researchers at the National Institute of Health’s Vaccine Research Center utilized x-ray crystallography to understand the precise molecular structure of pre-F and post-F forms while bound to one of the recently discovered potent antibodies. Building off previously established research that a virus could be stabilized in its prefusion form and the additional structural information provided by the x-ray crystallography, researchers Dr. Jason McLellan and Dr. 414

Barney Graham with the NIH’s Vaccine Research Center reported that they successfully genetically modified the protein to preserve its prefusion state. To stabilize the pre-F conformation, McLellan and his team analyzed the structure of pre-F for mutations that would maintain the structure maximizing antibody neutralization. From more than 100 variants, three were found to retain binding capacity to the pre-F-specific antibodies. The first of these mutation variants, named DS, was formed by substituting amino acids Ser155 and Ser290 for pairs of cysteines which formed stable disulfide bonds. These served as covalent bridges to lock the F protein in its prefusion configuration. The next, termed Cav1, contained the mutations S190F and V207L. Changing a serine to a phenylalanine and a valine to a leucine filled atomic level cavities to increase hydrophobic packing and maintain the structure of the most powerful antigenic site. The third variant, termed TriC, involved an F488W mutation, stabilizing the hydrophobic fusion peptide. However, after testing different combinations of the three mutations, it was clear that the DS-Cav1 combination was superior in maintaining stability under extreme conditions such as temperature, pH, osmolality, and freeze-thaw, all of which are important for vaccine manufacturing. When put to the test, the DS-Cav1 model of the stabilized pre-F form induced extremely high antibody levels as compared to the post-F form in an animal trial, confirming the research and paving the way for new RSV vaccine options.

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Figure 3. Design of soluble site Ø-stabilized RSV F trimers. The structure of the RSV F trimer in its D25-bound. Insets show enlargements of stabilizing ... [+] MCLELLAN ET AL.

Fig. 4. Crystal structures of RSV F trimers, engineered to preserve antigenic site Ø (B) Antigenic site Ø of a RSV F protomer is displayed in ribbon diagram, with the structure of ... [+] MCLELLAN ET AL.

The stabilization of the prefusion form is an essential development because this pre-F form of the protein contains more effective antibody neutralization target sites. As shown above in Figure 3, the red targets, labeled Site Ø, are rated “outstanding” in 416

value as a neutralization site. However, these are only present in the prefusion state. In the postfusion state, which occurs after the viral cell undergoes a conformational change and makes physical contact with the host cell, these essential binding sites are no longer present. Prefusion F also has additional neutralization target sites which are not yet named but shown in dark orange in Figure 3. These “excellent” neutralization value sites are also only present on the prefusion form, demonstrating the importance of the protein modifications that succeeded in stabilizing protein F for vaccine development.

Figure 5. Surface representation of RSV F glycoprotein

The RSV F glycoprotein exists in a prefusion (pre-F) conformation prior to an extensive ... [+] GRAHAM ET AL. It has been over half a century since the initial RSV vaccine candidate performed so poorly, resulting in higher levels of hospitalizations and mortality for the children vaccinated over the control group. With the recent developments in structure-based vaccinology, we have an explanation of what went wrong. The evidence now suggests that the 1965 RSV vaccine induced high levels of antibodies binding just to the post-F state, not the pre-F state. Since only the prefusion form contains the most neutralizationsensitive binding sites, the vaccine was not effective. Scientists at the time were unaware of protein F’s different forms, and this led to an immune response of hyper production of antibodies that bound to 417

the virus without actually neutralizing it. These non-neutralizing antibodies primed the young children who had not yet been exposed to RSV naturally to have an overly strong inflammatory immune response once they were exposed to the virus after vaccination. In some cases, this resulted in severe respiratory symptoms and serious illness requiring hospitalization. This tragic first vaccine trial in conjunction with the recent groundbreaking structural based research emphasizes the importance of having a fundamental understanding of the molecular structure. The recent advances in development of an RSV vaccine would not be possible without the current deep understanding of the RSV virus at an atomic level as this fundamental research is essential for successful vaccine production. In Part III of this series, we will describe the strategies behind vaccine development, as well as the current respiratory syncytial virus vaccines currently in late-stage clinical trials with the potential to save many lives. This article is featured on Forbes.org, and can be read online here: New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part II

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Far-Ultraviolet Light Is Another Way To Keep Our Public Spaces Safe Forbes | May 04, 2022 | Article

For some time now, it’s been established that the primary root of SARS-CoV-2 transmission is airborne. Research suggests that contagious particles are released into the air and linger for several minutes to hours. Inhaling these particles is what leads to infection. People have experimented with ways to sterilize the air from infectious particles. Here we describe work that shows, in addition to ventilation and filtration, how Far-UVC light inactivates the virus in a way that is harmless to us. Past uses of ultraviolet light Germicidal ultraviolet light has been demonstrated to work in reducing the transmission of several bacterial and viral infections. Some which include lessening the transmission of measles, mumps, and has shown to inhibit the spread of tuberculosis by 70% after being tested on guinea pigs in an experiment. A major challenge, however, has been its long wavelength of 254 nm which is detrimental to humans, causing skin cancer and scarring the eyes. Even with caution, accidental exposure of germicidal ultraviolet light can occur, leaving people at risk for painful burns and scarring. Far-UVC is harmless to humans To circumvent the effects of germicidal ultraviolet light, a group of scientists at Columbia University Irving Medical Center experimented with a different form of ultraviolet light known as FarUVC. This light has a shorter wavelength (200-230nm), inhibiting its ability to penetrate human skin while still being able to attack small viral air particles efficiently. Measuring the efficacy of Far-UVC viral inactivation To measure the rate of airborne viral inactivation, Eadie et al. used a room-sized chamber that simulated an office work-space. The ventilation simulated a normal work environment with 3-airchanges per hour. At the same time, Staphylococcus aureus aerosols were deployed into the room until a steady concentration was 419

reached. This process lasted around 60 minutes. Five Far-UVC lamps were then turned on from overhead lights with their emission directed towards the ground. In the span of five minutes, the inactivation of bacterial particles in the air was reduced by 98%. The experiment was also repeated with different levels of Far-UVC (i.e. high, medium, low), with aerosol particles being continuously released in the environment to test the efficacy of the levels. The best results were obtained when Far-UVC was maintained at high or medium level.

Figure 1. 3D schematics of the bioaerosol chamber configuration showing room dimensions, the ... [+] EADIE, E.; ET AL (222 NM) EFFICIENTLY INACTIVATES AN AIRBORNE PATHOGEN IN A ROOM-SIZED CHAMBER. HTTPS://WWW.NATURE.COM/ARTICLES/S41598-022-08462-Z.

Advantages of Far-UVC A key advantage of using this light technology is that viruses are not able to mutate under Far-UVC as they generally would if introduced into the human body first. Eadie et al. also confirm that current and future variants of SARS-CoV-2 are no exception to 420

inactivation. Another advantage of Far-UVC is that it may not require mixing of “good air” to work as you normally would with an air cleaners or GUV.

Figure 2. Percentage of viable airborne S. aureus remaining plotted on a linear y-axis

EADIE, E.; ET AL (222 NM) EFFICIENTLY INACTIVATES AN AIRBORNE PATHOGEN IN A ROOM-SIZED CHAMBER. HTTPS://WWW.NATURE.COM/ARTICLES/S41598-02208462-Z. While Far-UVC has been shown to work in real-life environments, precautions such as light intensity and exposure time are equally as important when considering this light technology on a large scale. Conclusion Although SARS-CoV-2 has prevented most of us from being in large indoor spaces, Far-UVC gives us confidence moving forward with the pandemic for safer environments. It is vital that we take air quality seriously especially in our public spaces including: schools, concert halls, bars, restaurants, etc. While Far-UVC works best when accompanied with ventilation/filtration of air, wearing a mask, washing our hands, and getting vaccinated cannot be forgotten. This article is featured on Forbes.org, and can be read online here: FarUltraviolet Light Is Another Way To Keep Our Public Spaces Safe 421

Study Shows Unvaccinated People Are At Increased Risk Of Infecting The Vaccinated Forbes | May 06, 2022 | Article

A new modeling study published in the Canadian Medical Association Journal demonstrates that unvaccinated people threaten the safety of the vaccinated even when SARS-CoV-2 vaccination rates are high. Researchers used a simple compartmental model of respiratory viral disease to explore the effect of mixing between unvaccinated and vaccinated people. People were represented as residing in 3 possible compartments including susceptible to infection, infected and infectious, and recovered from infection with immunity. Those compartments were divided to reflect the two connected subpopulations: vaccinated and unvaccinated people. The researchers simulated mixing of like-with-like populations in which people have exclusive contact with others of the same vaccination status as well as random mixing between different groups. When unvaccinated mixed with unvaccinated, the risk to vaccinated people was lower. When vaccinated and unvaccinated people mixed, a substantial number of new infections would occur in vaccinated people, even in scenarios where vaccination rates were high. Cumulative infection rates among vaccinated people were highest (15%) with random mixing. Unvaccinated participants exhibited a disproportionate contribution to infection risk following contact count adjustment.

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Impact of mixing between vaccinated and unvaccinated subpopulations on contribution to risk and final epidemic size for (A) varying reproduction numbers and (B) vaccine effectiveness. Both panels show the impact of increasing like-with-like mixing onIMPACT OF POPULATION MIXING BETWEEN VACCINATED AND UNVACCINATED SUBPOPULATIONS ON INFECTIOUS DISEASE DYNAMICS: IMPLICATIONS FOR SARS-COV-2 TRANSMISSION. FISMAN ET AL. 2022

The findings remained stable even when they modeled lower levels of vaccine effectiveness for the prevention of infection, such as in those who have not received a booster dose or with new SARSCoV-2 variants. The lower-bound estimate for vaccine effectiveness (40%) reflected uncertainty about the emerging Omicron variant at the time. In contrast, the upper-bound estimate for vaccine effectiveness (80%) reflected the higher effectiveness seen with the Delta variant. The authors acknowledge that there are some limitations to the simplicity of their model. Vaccine effectiveness against infection was modeled but not the additional benefits of vaccination for preventing severe illness and the impact of vaccines on prevention of forward transmission by vaccinated but infected individuals. However, the 423

simplicity of the modeling does unequivocally demonstrate the infection risk of the unvaccinated. Those opposed to vaccinations and vaccine mandates often argue that it is a matter of personal choice or individual rights. However, this study reinforces the fact that an individualistic or personal responsibility approach to Covid-19 will result in poor pandemic control. The actions of the unvaccinated impact the broader population. A report from Frontiers in Public Health found that the more “individualistic” a country was, the higher the instance of Covid cases and mortalities. The report also found that the more individualistic participants were, the higher the chances they would not adhere to epidemic prevention measures. According to the CDC, only 66.1% of the U.S. population has received two doses of the Covid-19 vaccine and even fewer have received boosters. We need better more nuanced messaging on why vaccination against Covid-19 is so critical. The original messaging that Covid-19 vaccines would prevent all instances of infection was unrealistic and not in keeping with results from other vaccines. But vaccines do not need to prevent infection to be highly effective. The Salk inactivated virus Polio vaccine for example does not prevent infection or transmission but has been responsible for the eradication of Polio in the US and in many other countries worldwide. Vaccination also radically reduces the chance of severe disease, death, and hospitalization. This reduces the burden on our healthcare systems. If hospitals are overwhelmed with Covid cases it affects the standard of care for all health issues. No person or population is safe from the scourges of Covid-19 until we all are. Public health policy and regulation in the past have been developed around behaviors that create health risks for the community, as well as individuals. For example, if we have public health statutes that limit indoor smoking and driving under the influence of alcohol and drugs, it may be time to consider vaccination against Covid-19 in the same regard. This article is featured on Forbes.org, and can be read online here: Study Shows Unvaccinated People Are At Increased Risk Of Infecting The Vaccinated

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Omicron Transmission And Immune Evasion Explained By The Omicron Spike’s Unique Structure Forbes | May 06, 2022 | Article

The SARS-CoV-2 Omicron family of variants differs from the original isolates as well as other variants. Omicron viruses contain a minimum of 30 mutations in the Spike protein and another 23 elsewhere in the genome. Figure 1 illustrates how significantly mutated one of the currently circulating Omicron strains, BA.2.12.1, compares to the wildtype Wuhan virus. There are also silent mutations that change the nucleic acid sequence without changing the protein-coding capacity.

FIGURE 1: BA.2.12.1 genomic and Spike mutations in black. Mutations in red are those found in other ... [+] ACCESS HEALTH INTERNATIONAL

In addition to looking different in terms of amino acids, Omicron variants behave differently as well. Omicron is by far the most transmissible variant, including both rate of infection globally and infectivity between individual hosts. The family is diverse and at least three of the variants are at epidemic proportions: BA.4 and BA.5 in South Africa, as well as BA.2.12.1 in the US. While the disease spectrum still requires further examination, it is clear that Omicron can cause severe disease at high rates in children and the elderly, even in fully vaccinated individuals.

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FIGURE 2: Proposed Omicron family tree. ACCESS HEALTH INTERNATIONAL

One of Omicron’s most salient features is how resistant the variant family is to vaccines and most monoclonal antibodies. Resistance contributes, at least in part, to the rapid spread of the Omicron variants. Previously, we described how amino acid changes in the Spike protein alter immune recognition by patient sera and monoclonal antibodies. Those infected with BA.1 can be reinfected with BA.2 and those infected with BA.2 can be reinfected by BA.4, BA.5, or BA.2.12.1. More Tightly Packed Omicron Spike Protein Wang et al. detail how multiple amino acid changes in the Omicron Spike protein not only eliminate antibody binding sites but also alter shape and function The Omicron Spike protein is significantly more compact than that of the original Wuhan (D614G) variant and the full range of variants that followed. Figure 3 compares the structure of the Omicron Spike with that of the Wuhan (D614G) wildtype. The Omicron protein is illustrated in green overlaid with the D614G variant in grey. The view is from the apex of the Spike looking toward the base. Major structural rearrangements are evident throughout the structure. I speculate that some antibodies which bind the looser structures fail to bind the Omicron Spike not only because of single amino acid changes but also as a consequence of the macro differences in structure. I also speculate that tighter packing implies that it has a more entropic structure, representing a higher energy state. That additional energy may be released during membrane fusion, increasing the efficiency of viral entry.

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FIGURE 3: Structural comparison of the Omicron S-ECD with the G614 S-ECD (PDB: 7BNO). The S-ECD ... [+] WANG ET AL.

I also speculate that tighter packing implies a more entropic structure, representing a higher energy state. That additional energy may be released during membrane fusion, increasing the efficiency of viral entry. Wang et al. attribute tighter packing of the Omicron Spike to specific amino acid substitutions. The H655Y mutation induces a tighter association of the monomers that comprise the trimer. Additionally, five mutations in the central helical region, N764K, D796Y, N856K, L981F, and N969K introduce and facilitate additional hydrogen bond and hydrophobic interactions between the S2 trimers (Figure 4).

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FIGURE 4: Mutation sites in Omicron SD1 and SD2 and the S2 region. The mutant residues are ... [+] WANG ET AL.

Alterations In Omicron Fusion The second major structural change involves one of the hallmarks of SARS-CoV-2 as compared to SARS-CoV-1: initial scission at the S1 furin cleavage site. For most variants, furin cleaves the SARS-CoV-2 Spike protein as the virus buds from the cell surface. No such cleavage occurs for the Omicron variants, or if it does, the efficiency is greatly reduced (Figure 5 by Yamasoba et al.). The furin cleaved S1/S2 complex is inherently less stable than the uncleaved monomer. Post-cleavage S1 readily disassociates from S2. In fact, one advantage the D614G mutant virus has over the original Wuhan variant is that it stabilizes S1/S2 association. Increased transmission of Omicron as compared to other variants may be partially attributed to increased retention of S1 on the mature virus particle.

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FIGURE 5: Western blot. Left, representative blots of S-expressing cells. ACTB is internal control. ... [+] YAMASOBA ET AL.

Wang et al. note that one mutation in the Omicron Spike, H655Y, may account for reduced furin cleavage at the furin cleavage site. The mutation increases the stability of the 630 loop in the region by interacting with residue F643. Wang et al. and Bing Chen speculate that increased rigidity of the loop containing the furin cleavage site reduces proteolysis.

FIGURE 6: Structural comparison of the closed promoters from the Omicron (green) and WT (light pink; ... [+] WANG ET AL.

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The Wuhan strain and all previous variants enter via membraneto-membrane fusion. The Omicron family by contrast enters via an endosomal entry route. This is the strategy employed by SARSCoV-1. Endosomal entry is very likely required in the absence of efficient furin cleavage. The S1/S2 cleavage site is at positions 685/686 and the S2 site is at positions 815/816.

FIGURE 7: Schematic representation of SARS-CoV-2 S protein and the location of S1/S2 and S2′ ... [+]XIA ET AL.

This observation prompts a paradox. The presence of the furin cleavage site was hypothesized to be a key event in the acquisition of efficient human-to-human transmission. In fact, such speculation was so prominent that some suggested the furin cleavage site was artificially inserted to increase the infectivity of the virus as part of a laboratory gain-of-function experiment. Many who made that speculation appear to have been ignorant to the fact that many naturally circulating alpha and beta coronaviruses contain furin cleavage sites It is notable that even though the Omicron family of viruses contain furin cleavage sites, they are poorly active at best. The paradox: The Omicron family of viruses are far more infectious than previous variants. It is evident that efficient cleavage is not necessary, either for infection or efficient transmission in the human population. This article is featured on Forbes.org, and can be read online here: Omicron Transmission And Immune Evasion Explained By The Omicron Spike’s Unique Structure

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Scientists Discover Genetic Cause of Lupus, Findings May Help Research on Long Covid. Forbes | May 09, 2022 | Article

Molecular structure of toll-like receptor 7 (TLR7) FROM: "STRUCTURE BASED MODELING OF SMALL MOLECULES BINDING TO THE TLR7 BY ATOMISTIC LEVEL SIMULATIONS" GENTILE ET AL. 2015 Systemic lupus erythematosus (SLE), more commonly known as simply lupus, is a chronic autoimmune disease that provokes symptoms from skin rashes and fevers all the way to chronic fatigue and inflammation of joints and organs. Severe cases of lupus can be fatal. Autoimmune diseases occur, in the broadest sense, when our body’s natural defense, the immune system, mistakes our own cells or tissues for foreign substances, accidentally turning on us and attacking otherwise healthy cells and tissues — in essence, a lapse in the ability to tell self from other. Even so, at a more granular level, the exact causes and mechanisms of the self-targeting seen in autoimmune diseases remain poorly understood. It is likely that a complex mix of genetic and environmental factors come together to trigger disease onset. A new study by researchers at the Australian 431

National University, Canberra heralds a major breakthrough. Published in Nature, the work by Brown et al. pinpoints a mutation in the gene of a protein that senses single-stranded RNA as one direct cause of systemic lupus erythematosus. The same protein, tolllike receptor 7 (TLR7), is activated upon infection by singlestranded RNA viruses like SARS-CoV-2. Toll-like Receptors (TLRs): What are they and what do they do? Toll-like receptors are a family of proteins closely involved in the innate immune response. They are usually found in the membrane of sentinel cells —macrophages and dendritic cells— that circulate throughout our body while keeping an eye out for pathogens. These cells are often the first to respond to infection, and it’s the toll-like receptors in their membrane that help them to spot any possible microbial threats. As pattern recognition receptors, tolllike receptors can detect molecular “motifs” that are highly conserved across pathogens, from bacteria and viruses to fungi and parasites. These motifs are known as pathogen-associated molecular patterns (PAMPs). When toll-like receptors uncover traces of PAMPs in places they shouldn’t be, they quickly activate and mobilize different immune cells. Toll-like receptor 7, one of the 10 different types found in humans, is in charge of detecting single-stranded RNA in the endosomes of cells — endosomes are “sorting stations”, located within the cytoplasm of cells, that help send important proteins to various destinations within the cell. Single-stranded RNA is a hallmark of certain viruses, including hepatitis C virus, human immunodeficiency virus (HIV), and coronaviruses such as SARSCoV-2. TLR7 is particularly responsive to guanosine and uridine, two nucleotides derived from single-stranded RNA (Figure 1).

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FIGURE 1. TLR7 can detect both uridine-containing single-stranded RNA as well as guanosine ligands. FROM: “STRUCTURAL ANALYSIS REVEALS THAT TOLL-LIKE RECEPTOR 7 IS A DUAL RECEPTOR FOR GUANOSINE AND SINGLE-STRANDED RNA” ZHANG ET AL. 2016

Once TLR7 detects viral single-stranded RNA, it stimulates a series of signaling cascades that ultimately activate nuclear factorkappaB (NF-κB). NF-κB is a protein complex that regulates a host of important cellular behaviors, including: the induction of type I interferon (INF1) and other inflammatory proteins, like cytokines; cellular growth; and, finally, the programmed self-destruction of cells, known as apoptosis. NF-κB is especially closely involved in the regulation of B lymphocyte survival, preventing apoptosis and promoting activation whenever possible. TLR7, Autoimmunity, and Lupus As mentioned, autoimmune diseases are a product of our immune system mistakenly attacking our own healthy cells and tissues, rather than a foreign threat. For the most part, the things that do the attacking are antibodies, which are secreted by B cells. Since they end up directed at us instead of microbial threats, they are called 433

autoantibodies. The cells and tissue they end up attacking are called autoantigens. Although an important component of the antiviral immune response, TLR7 has also been associated with autoimmune disorders. This is in large part because of its role in regulating inflammation and B cell survival via NF-κB. In the case of lupus, it has long been suspected that increased TLR7 signaling may be a major contributor. This new research by Brown et al. confirms the suspicion. To study the relationship between lupus and TLR7, the team of scientists performed a full genome analysis of a young Spanish woman who was diagnosed with systemic lupus erythematosus at the age of 7. Such early onset is quite unusual, and hints at a distinct genetic cause. Indeed, the genetic analysis revealed that she had a mutation to the TLR7 gene, a switch from tyrosine to histidine, giving rise to a new variant: TLR7Y264H. The researchers crossreferenced the mutation against databases of normal human genome variation, but didn't get any results. Next, Brown et al. sought to check if this variation enhances TLR7 signaling and the related downstream effects, like NF-κB activation. To do so, they engineered different cells to express the three TLR7 mutants they had discovered. They then activated the variants and measured the level of TLR7 expression. Compared to the unmutated TLR7 gene, TLR7Y264H expressed higher levels of the TLR7 protein than usual and induced enhanced NF-κB activation. The TLR7Y264H gene variant also increases the protein’s affinity to the activator guanosine, which dimerizes and activates the receptor (Figure 1). This increased sensitivity of TLR7 makes it more likely for our immune cells to mischaracterize our own healthy tissues as either damaged or as a foreign substance, causing them to go on the offensive. To see what this meant in the context of autoimmunity, the team of scientists introduced the Y264H mutation into mice. Both male and female mice carrying the TLR7 variant began displaying signs of autoimmune symptoms within 12 weeks. This included tissue damage at various different sites and an overall decreased survival compared to unmutated mice (Figure 2). The symptoms were accompanied by the development of autoantibodies towards single434

stranded RNA and Smith proteins (Sm), a family of RNA-binding proteins that help splice protein-producing sections of RNA. Presence of autoantibodies against Smith proteins has been used as a diagnostic criteria for lupus. So, along with the symptoms, the presence of these autoantibodies suggested that the TLR7Y264H variant was directly responsible for the onset of lupus in the mice.

FIGURE 2. Kidney damage in mice with the TLR7/Y264H variant (right) compared to a healthy kidney in unmutated mice (left). FROM: “TLR7 GAIN-OFFUNCTION GENETIC VARIATION CAUSES HUMAN LUPUS” BROWN ET AL. 2022

The researchers then focused on the impact the Y264H variant of TLR7 had on B cell production and survival. Analysis of the spleens of mice with the TLR7Y264H variant showed an increase in total B cells as compared to unmutated mice. B cells derived from the mutated mice also survived much longer, with a two-fold increase in the activation of genes related to cell survival. Their results suggest that the hypersensitive TLR7Y264H variant promotes the survival of autoantibody-producing B cells. Importantly, Brown et al. were able to confirm that the effects of the TLR7Y264H variant were independent of germinal centers (GCs). When B cells encounter an antigen they travel to germinal centers, where they start developing specific antibodies to neutralize the substance. One critical function of the germinal centers is to filter out those B cells that fail to differentiate self from other. B cells that have never been filtered through a germinal center are much more 435

likely to accidentally start targeting our own healthy cells and tissues. Introducing the Y264H mutation into mice that cannot form germinal centers still causes lupus, suggesting that the autoimmunity is being driven by TLR7 independently of germinal centers. Implications for Long Covid? There appear to be some parallels between the TLR7-driven autoimmunity seen in lupus and the autoimmune aspects of postacute sequelae SARS-CoV-2 infection (PASC), also known as long Covid. First, being a single-stranded RNA virus, SARS-CoV-2 also induces TLR7 activation. This has been confirmed by studies showing that individuals with TLR7 deficiencies are more likely to develop severe Covid-19. Second, like lupus, SARS-CoV-2 infection is accompanied by a proliferation of B cells that have not passed through germinal centers, and therefore maintain the capacity for self-recognition. Both lupus and long Covid are characterized by the persistence of such auto/antibodies. Also, both lupus and long Covid are more prevalent in women than in men. Given the results of their latest study, Brown et al. suggest that, in lupus, this may be because the TLR7 gene is found on the X chromosome, and females have two X chromosomes where males have one X and one Y chromosome. Although one X chromosome is usually inactive in women, silencing of the section of the chromosome on which the TLR7 gene sits is often incomplete. Dr. Carola Vinuesa, senior author of the study, mentions that, “There are other systemic autoimmune diseases, like rheumatoid arthritis and dermatomyositis, which fit within the same broad family as lupus. TLR7 may also play a role in these conditions.” Perhaps TLR7 may also be to blame for the autoimmune aspects of long Covid? At the very least, the many parallels between the two suggest it may be worth taking a closer look. This article is featured on Forbes.org, and can be read online here: Scientists Discover Genetic Cause of Lupus, Findings May Help Research on Long Covid.

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Targets for Infection: How SARS-CoV-2 Damages the Kidneys Forbes | May 09, 2022 | Article

Kidney complications are some of the most common and severe symptoms associated with Covid-19. Estimates show that 30% of people hospitalized for severe infection and 50% of those that are later admitted to intensive care units experience some injury to the kidneys. Even previously healthy people with mild infections can later show signs of kidney damage, evident by elevated levels of protein in urine, as well as in blood. Despite an increased prevalence of kidney complications, the rate of organ transplant operations significantly decreased in the early months of the pandemic due to increased precautions to ensure the health of the kidneys being donated. Now as transplant rates have rebounded, however, there is still a need to consider the long-lasting consequences of Covid-19induced kidney damage and the possible strain that it will put on our already weakened health systems. Thanks to a recent study from Duke University we are now closer to understanding how and why SARS-CoV-2 disrupts kidney function. Using lab-synthesized kidney cells derived from human stem cells, Kalejaiye et al. found that a particular group of cells within the kidneys, called podocytes, are especially vulnerable to infection. Injuring these cells can lead to considerable organ damage. This resembles a similar pattern of damage that has already been identified in another critical supportive cell: heart pericytes. As we discussed in a previous story, exposure to SARS-CoV-2 can make these endothelial heart cells “sticky” and vulnerable to clotting, putting the entire cardiovascular system at risk of severe complications. Similarly, viral damage to podocytes disrupts the kidney’s ability to remove waste and toxins from the body, which, left untreated, could put the entire body at risk. In this first installment of a two-part series, we will explore the consequences of viral infection in kidneys. Normal Function of the Kidneys

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Within each of the kidneys, there is an extensive network of capillaries, referred to as the glomerulus. This is the primary site for filtering toxins and waste from the blood, which are later released through urine. As illustrated in the figure below, a layer of capillary blood cells creates a barrier between circulating blood cells and the internal structures of the kidney. Small pores through these blood vessels allow particles from the blood to pass through the glomerular barrier and into the kidney’s cavities.

Figure 1: Illustration of a kidney glomerulus. Blood enters through the afferent arteriole, and as it circulates through the capillaries of the glomerulus, water containing dissolved solvents is filtered through the endothelium, or barrier, of the glomerulus. Podocytes and their pedicels (foot projections) help to further filter waste and toxins before it is transported through the proximal convoluted tubule to be removed from the body as urine. CREDIT: Cenveo

Only small particles dissolved in water can filter through the kidney’s porous blood vessels and through the three-layered glomerular barrier. A line of podocyte cells on the other side of the barrier further filters the water-based solution. Given by their name (“podo” meaning foot), these cells have long foot-like projections that wrap around the glomerular barrier. Small proteins, called nephrins, separate individual foot-like projections to allow the filtered water to pass through.

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Figure 2: Schematic diagram of podocytes attached to the glomerular basement of the kidney. When podocytes are injured, the foot processes retract and eventually the cell begins so large that it detaches and dies. From: “MicroRNAs in Podocyte Injury in Diabetic Nephropathy” Ishii et al. 2020

When the kidneys experience a bacterial or viral attack, podocytes begin retracting their foot projections. This allows increased levels of proteins to leak into the urine and signals that vital nutrients are being lost. Prolonged infections can increasingly injure podocyte cells, causing them to detach from the glomerular barrier. The loss of these supportive cells dysregulates the kidneys’ ability to filter blood, which could lead to organ failure. As a protective measure, however, podocytes can be regenerated to prevent further kidney damage. Stem cells, particularly those found in bone marrow, supply a virtually unlimited source of regenerative cells. When individual cells are injured, stem cells can transform into almost any cell type, including kidney podocytes. This allows the kidneys to resist severe damage, especially when exposed to toxins from alcohol and drugs. Impact of Covid-19 on Kidney Cells Exposure to SARS-CoV-2 can overwhelm the kidneys as a consequence of robust inflammatory responses to infection in other parts of the body, as well as through direct infection. The kidneys in 439

fact are a direct target for viral infection. The damage can be so severe in some cases that it disrupts the regeneration of new podocytes. How can SARS-CoV-2-induced kidney damage be so severe? Given how vulnerable they are to infection, it is likely that podocyte injuries underlie much of the damage to the kidneys. Yet to answer this question, Kalejaiye et al. needed to find a cell model that closely resembled the appearance and function of kidney podocytes. Rather than using animal models, they turned their attention to human stem cells. Although human stem cells have been successfully used to model different diseases for years, researchers only recently discovered how to create podocyte models to study kidney damage. The process of generating these cells capitalizes on the natural ability of stem cells to transform into any type of cell. For this investigation, Kalejaiye et al. obtained human induced pluripotent stem cells (hiPS) derived from skin and blood cell samples that were reprogrammed to look and function like embryonic stem cells. These cells were then exposed to growth factors that eased their development into mature cells. After a few days, the stem cells entered the mesoderm and intermediate mesoderm stages of development. At this stage, cells can develop into any type of internal tissue. To nudge them towards becoming kidney podocytes, Kalejaiye et al. added other podocytespecific growth factors when the stem cells reached the intermediate mesoderm stage. A figure of this process is shown below. Once these lab-synthesized podocytes reached maturity, the investigators were ready to begin their experiments

Figure 3: Schematic overview of the process of generating mature podocytes from human iPS cells; adapted from (Musah et al., 2017). Human stem cell samples (hiPS) are exposed to growth factors that promote their development into the mesoderm and intermediate mesoderm stages. Once they reach the intermediate mesoderm stage, the cells are exposed to podocyte-specific growth factors. From: “SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes” CREDIT: Kalejaiye et al. 2022

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First, Kalejaiye et al. wanted to determine how vulnerable kidney podocytes are to the SARS-CoV-2 spike surface protein used to gain viral entry. Rather than exposing cells to the live virus, they instead employed a pseudo virus that highly expressed the viral S protein. Each day that the podocytes were exposed to this pseudovirus they experienced an exponential increase in the number of viral RNA, confirming that the viral S protein had gained entry to these cells. How does this compare to the live SARS-CoV-2 virus? The podocytes were just as vulnerable to infection when they were exposed to the live virus, as they were with the pseudovirus. Even when exposed to very low amounts of the virus, Kalejaiye et al. measured a high amount of intracellular and extracellular viral RNA. They found that the longer the cells were exposed to the virus, the less viral RNA was detected. This did not necessarily mean that the infection had cleared. Rather, Kalejaiye et al. speculated that the severity of the infection triggered apoptosis and necroptosis, both of which are inflammatory responses that mediate cell death. Earlier studies have shown that a similar effect occurs when lung cells are infected with SARS-CoV-2. Before inducing their death, SARS-CoV-2 alters the expression of specific genes associated with severe podocyte damage. Kalejaiye et al., for instance, observed a significant increase in compensatory genes that normally help to restore and support podocyte function. Although the upregulation of these genes enables podocytes to further extend their foot-like projections, this maladaptive mechanism also exposes more of the cell’s surface to SARS-CoV-2, making the cell more susceptible to infection. Conclusion Now that the kidneys, and particularly podocyte cells, have been identified as vulnerable targets for Covid-19, there is a critical need to develop novel vaccines and antiviral treatments that prevent the progression of kidney complications and other related injuries. The next part of this series will continue discussing the findings from Kalejaiye et al.’s investigation, which takes a deeper look at how the expression of specific proteins and enzymes makes podocytes particularly vulnerable to infection. This article is featured on Forbes.org, and can be read online here: Targets for Infection: How SARS-CoV-2 Damages the Kidneys 441

New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part III Forbes | May 10, 2022 | Article

This story is Part III of a series on respiratory syncytial virus vaccines. Here we discuss the complex strategy of vaccine development and the RSV vaccine candidates currently primed to make history. Respiratory syncytial virus is one of the leading causes of death for infants globally, and a safe vaccine has yet to be approved for use. In Parts I and II of this series we discussed the magnitude of the disease and the science that underlies the recent advances in search for an RSV vaccine. In a triumph over the tragic history, many companies have recently taken on the challenge of producing an RSV vaccine, most of them using the new protein stabilization technology detailed in Part II of this series. By genetically modifying the key antibody target for effective neutralization as pre-fusion state glycoprotein F, it became possible to synthesize a replicate of the antigen structure for vaccine production.

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Structural vaccinology for vaccine design (A) Basic methods and tools for structural vaccinology. (B) Irreversible conformational change of viral antigens in RSV F. The three-dimensional structure of the antigen (II) will be determined using structural biology techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), and nuclear magnetic resonance (NMR). The antigen will then be re-engineered, adopting a specific conformation (I). Next, the re-engineered antigen could be incorporated into one of the vaccine platforms such as the recombinant protein vaccine platform. The safety and efficacy of the candidate vaccine should then be tested in animal models. ANASIR AND POH, 2019

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Part of the difficulty in developing a vaccine against respiratory syncytial virus available for young children is due to the complexity of vaccine development. There are strict stages of testing for safety and efficacy that must be observed. As a vaccine candidate progresses through clinical trials, it begins in Phase 1 with generally around 100 volunteers. This trial is predominantly focused on confirming the safety suggested by animal studies. It usually only includes healthy adults from a narrow age range to minimize confounding variables or adverse reactions. With a few thousand subjects, Phase 2 is larger and intended to confirm safety data and begin to measure efficacy. In Phase 3, often tens of thousands of volunteers receive the vaccine in order to provide a large enough data set to confirm that the vaccine confers significant protection against the pathogen. Phase 4 consists of ongoing studies post approval. Vaccine clinical trials are often slow-moving because significant time and resources go into analyzing the data before the money necessary to progress to the next phase is invested.

Vaccine Human Trial Phases ABC NEWS, WHO Due to the tragic history of vaccine trials with RSV specifically, an abundance of caution is being taken in the production of a vaccine for young children. Only once profuse safety data is available from vaccine trials with adults will further testing with children 444

commence. This is why it is expected that a vaccine will soon be available to adults before it is available to infants. As of September 2021, there were 20 proposed or active vaccine clinical trials, some of which are designed for at-risk adults, some for pregnant women, and some for children. Companies in the spotlight recently for the production of a COVID-19 vaccine such as Pfizer, Moderna, Johnson & Johnson, and AstraZeneca are leading the charge along with others such as GSK and Janssen, most of which had global phase III trials underway by the end of 2021.

Summary of RSV Vaccine Products in Late-Sate Development CCDR GOVERNMENT OF CANADA Janssen’s vaccine is meant for use only in older adults, while the GSK and Pfizer vaccines, the latter of which recently reported 85% efficacy in a phase 2b trial, are for use in pregnant women. The intention is to provide protection during pregnancy that will persist in newborns throughout their first six months of life, the most vulnerable period. In March 2022, Pfizer’s vaccine RSVpreF received Breakthrough Therapy Designation from the US Food and Drug Administration (FDA), which should expedite the process of further review and approval of the vaccine.

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With results published in the New England Journal of Medicine in April 2020, 406 pregnant women participated in a phase 2b trial, with 80.5% receiving the Pfizer RSVpreF vaccine. It was documented that most post-vaccine reactions were mild to moderate, with women receiving the vaccine version without aluminum hydroxide reporting lower incidence of local site reactions. For both the pregnant women and their post-vaccination born infants, incidences of adverse events were comparable in the vaccine and placebo groups, supporting the safety of the vaccine. Promising efficacy results showed significantly higher ratios of 50% neutralizing antibodies in infants of vaccinated mothers as compared to those receiving just placebo.

Pregnant Woman Receives a Vaccine GETTY IMAGES The earliest vaccines will likely be approved for use only in adults, even though infants in their first year of life of the most vulnerable to RSV infection. The specific time period is between 3 to 8 months, because it is during this timeframe that maternal antibodies resulting from previous natural infection with RSV of the mother are no longer very effective. However, vaccination of infants during this stage is still a complicated and precarious process, in part due to the presence of circulating RSV maternal antibodies, even if levels have decreased. This is why maternal vaccination is a competitive alternative route until a safe vaccine for infants is 446

approved for widespread use. Maternal vaccination will improve the efficacy of the RSV antibodies naturally received by newborns, granting them additional protection starting at birth. Despite the increased difficulties, promising late-stage clinical trial data provide hope that there will be a respiratory syncytial virus vaccine option available to young children in the near future. As a direct prophylactic option, AstraZeneca and Sanofi developed a single dose monoclonal antibody treatment with an extended halflife of efficacy compared to the current monoclonal antibody treatment in use, Palivizumab. Termed Nirsevimab, this preventative treatment is designed for all infants in their first year of life experiencing their first RSV reason, as well as those in additional years who are at higher risk of serious disease. The results of a Phase 3 clinical trial published in the New England Journal of Medicine demonstrated that a single injection of Nirsevimab prior to RSV season was able to protect healthy infants, both term and late preterm, from serious lower respiratory tract infection as a result of RSV infection by 74.5%, a significant efficacy rate. While the search for a vaccine for respiratory syncytial virus has eluded scientists and vaccine manufacturers for decades, there are finally some promising vaccine candidates and other preventative treatments in phase 3 trials. It looks as if there may be a vaccine in the foreseeable future, within the next few years. The next question is what availability will look like in high, medium, and low income countries. Childhood vaccines have been one of the singular successes of global health and this affirms hope that the success will be extended to new safe and effective respiratory syncytial virus vaccines. This article is featured on Forbes.org, and can be read online here: New Research Provides Hope In The Search For A Respiratory Syncytial Virus Vaccine- Part III

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A Dynamic Duo: How ACE-2 And CD147 Mediate Covid-19 Infection In The Kidneys Forbes | May 17, 2022 | Article

3D illustration of human kidneys with cross-section GETTY As one of the major targets of Covid-19, infection in the kidneys can lead to significant complications associated with kidney disease, as well as kidney failure. Kidney damage can occur even in those that experience mild infection. The first installment of this two-part series identified a group of supportive cells within the kidneys, called podocytes, which are particularly vulnerable to infection and subsequent injury associated with SARS-CoV-2. To answer why stem cell-derived podocytes are so susceptible to infection, we must consider how the virus is able to bind and invade these host cells. Rather than ACE-2 receptors, Kalejaiye et al. found that binding to CD147 receptors on stem cell-derived kidney podocytes is the preferred mechanism through which SARS-CoV-2 infects these cells. Here, we will continue discussing the significant findings from Kalejaiye et al., which may provide insight into how to protect not only the kidneys against infection but also other vulnerable cells and tissues throughout the body. 448

Early in the pandemic, angiotensin-converting enzyme 2, or ACE-2, was identified as a key binding site for SARS-CoV-2. Recent studies, however, have reported relatively low expression of ACE-2 receptors in the various tissues and organs that Covid-19 primarily infects, especially the lungs. Kalejaiye et al. were surprised to find that kidney podocytes express even lower levels of ACE-2 receptors. Compared to lung cells ACE-2 expression is 10 times lower in stem cell-derived kidney podocytes. These cells also expressed less of the TMPRSS2 enzyme, which normally works in tandem with ACE-2, than lung epithelial cells. Given the low level of ACE-2 expression in many of these cells, CD147, also known as basigin or EMMPRIN, has been increasingly recognized as an alternative mechanism for viral infection. This transmembrane glycoprotein is a member of the immunoglobulin superfamily that promotes the recognition, binding, and adhesion of extracellular substances to the cell’s surface. CD147 receptors play a critical role in facilitating cell metabolism and communication, as well as regulating the responsiveness of lymphocytes. Studies indicate that these receptors are implicated in several infectious diseases, including Hepatitis B and C viruses and HIV. Kalejaiye et al., therefore, speculated whether CD147 receptors may be the primary mechanism through which kidney podocytes are infected. They observed greater expression of these receptors in kidney podocytes, compared to both lung and colon cells. The degree of CD147 expression in kidney podocytes also correlated with the severity of infection, measured by how much viral content was detected in cells. Using antibodies to block these receptors reduced the rate of infection, suggesting that CD147 is a key receptor for SARS-CoV-2 binding and infection. This does not mean that SARS-CoV-2 does not also act on ACE-2 receptors when infecting kidney podocytes. Blocking these ACE-2 receptors alone also reduced the severity of Covid-19 infection in these cells. Kalejaiye et al. in fact observed the lowest rates of infection when ACE-2, as well as CD147 receptors, were blocked. Therefore, it is likely that both ACE-2 and CD147 are involved in Covid-19 infection of kidney podocytes. SARS-CoV-2 binding to ACE—2 and CD147 receptors may also be coregulated, in such a way that activity in one affects the expression of another. Kalejaiye et al. found that treating kidney 449

podocytes with ACE-2 antibodies induced the downregulation of CD147, to a similar effect that CD147 antibodies decreased the expression of ACE-2 receptors. Nonetheless, blocking these receptors did not completely block infection, suggesting that more research is needed to identify other types of receptors that may facilitate SARS-CoV-2 binding and entry into the cell. Understanding how the virus infects and enters cells can be a useful tool for identifying anti-viral targets for Covid-19. Although ACE-2 is widely recognized as a key receptor for SARS-CoV-2 binding, drugs that block these receptors may interfere with its various protective functions, including maintaining blood pressure and protecting against heart and kidney damage. Blocking CD147 proteins, on the other hand, may be able to reduce viral uptake, particularly in kidney podocytes, with relatively fewer side effects. However, we are just beginning to understand the complex role that CD147 plays in Covid-19. Future studies need to identify ways that drugs targeting both CD147 and ACE-2 can safely and effectively prevent SARS-CoV-2 infection. This article is featured on Forbes.org, and can be read online here: A Dynamic Duo: How ACE-2 And CD147 Mediate Covid-19 Infection In The Kidneys

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FDA Turns Down Fluvoxamine Emergency Use Authorization Request Forbes | May 19, 2022 | Article

The Food and Drug Administration denied an emergency use authorization for fluvoxamine as a SARS-CoV-2 antiviral. Fluvoxamine was proposed as a possible antiviral in 2021 due to its mechanisms of action for treating obsessive-compulsive disorder. These same mechanisms were suspected to inhibit SARS-CoV-2, leading to four trials testing the efficacy of the drug. In December 2021, Dr. David R Boulware requested an emergency use authorization for the outpatient treatment of adults testing positive for SARS-CoV-2 to prevent progression to severe symptoms based on early efficacy data from the fluvoxamine trials. The Food and Drug Administration denied his request, listing concerns such as insufficient data. Here we will discuss the FDA’s decision and reasoning. Trials The FDA’s main concern was the lack of conclusive evidence from four trials that were below emergency use authorization standards. The largest of the four trials, the TOGETHER trial, was a randomized, double-blind, placebo-controlled trial of high-risk patients in Brazil. The primary endpoints of the trial were (1) emergency room visits lasting greater than six hours and (2) hospitalization due to progression of Covid-19. The FDA found that the success of the trial was fueled by the six-hour threshold, which they found to be arbitrary. A patient remaining in the emergency room for 5.9 hours after taking fluvoxamine was considered a positive data point, whereas 6.1 hours was considered a negative. In the FDA’s words, “there are uncertainties about…whether the 6-hour timepoint represents a clinically meaningful threshold.” The other three trials were found to be inconclusive as well. The FDA noted that the STOP COVID trial had several damaging design flaws, including a lack of randomization, a small sample size, and 451

only a single testing center. The STOP COVID 2 trial and the COVID-OUT trial both failed to demonstrate a positive efficacy for fluvoxamine and were terminated early for futility. While the TOGETHER trial did achieve a positive efficacy based on its trial design, the FDA ultimately concluded that the four trials failed to provide sufficient data to conclude that fluvoxamine may be an effective treatment for nonhospitalized Covid-19 patients. Mechanisms of Action Among fluvoxamine’s mechanisms of action, which we discussed in a previous article, are binding to sigma-1 receptors on immune cells, reduced expression of inflammatory genes in human endothelial cells, and mediation of lysosomotropic properties. These factors were theorized to reduce the immune impacts following SARS-CoV-2 infection, including cytokine storms, hyperinflation, and coagulation. While some in-vitro data supported the claim that fluvoxamine’s mechanisms of action could work against SARS-CoV-2, no in vivo animal or human studies have been conducted to confirm this hypothesis. The FDA notes that the lacking evidence and lack of detailed characterization for fluvoxamine in a Covid-context is a primary data point for their rejection of the emergency use. Alternate Treatments The FDA’s notice of emergency use rejection for fluvoxamine was concluded with currently available and approved treatments for Covid-19. We have detailed each of these and encourage their use whenever applicable. These include Paxlovid, Remdesivir, Bebtelovimab, and convalescent plasma. We note that Molnupiravir is listed among these as well, but we discourage the use of this drug due to concerns about mutagenesis and cytotoxicity. Many further treatments are currently in development, including a number of monoclonal antibody treatments that aim to broadly neutralize all variants of SARS-CoV-2, which is an ongoing concern two years into the pandemic. This article is featured on Forbes.org, and can be read online here: FDA Turns Down Fluvoxamine Emergency Use Authorization Request

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The Growing Threat Of Tick-Borne Disease Part I: Powassan Virus Forbes | May 23, 2022 | Article

Ixodes sp. Hypostome and Palps. The hypostome of the tick is used to anchor itself into the skin of the host while feeding on blood. This barbed structure is covered in backwards-facing “teeth” to make removal difficult. According to the CDC, the best method of tick removal is to grasp the tick as close as possible to the skin’s surface using a pair of clean tweezers and pull straight upwards with steady pressure. Afterwards, clean and sanitize the area and dispose of the tick by putting it in alcohol, tape, or flushing down the toilet. ANDREA HALLGASS- FLICKR This story is Part I of a series on tick-borne disease. Here we provide background information on the current state of tick-borne illness in the United States and then focus on the swiftly growing threat of Powassan virus. This spring, when everything is in full bloom and you’re looking forward to spending time outside, you may have unwanted company. Although small, ticks carry many different potentially dangerous pathogens, and Powassan virus is one of growing concern.

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This month, the Connecticut Department of Public Health reported the first case of Powassan virus in the state for 2022. The victim was hospitalized with neurological symptoms but recovered. Unfortunately, a person who likely acquired the pathogen in Maine was not so lucky, and died in the hospital this past April. Though the northeastern states are no stranger to tick-borne disease, Powassan virus, which carries a mortality rate of 1 in 10, is especially threatening.

Blacklegged or Deer Tick Crawling on Skin STEVE ELLINGSON- FLICKR Carried and transmitted by both the Woodchuck tick and the vector better known for Lyme disease, the blacklegged or deer tick (Ixodes scapularis), Powassan virus can be transmitted from tick to human in a mere 15 minutes. Due to the combination of an incredibly short transmission time and the severity of the neurological symptoms of the resulting illness, Powassan virus is a growing threat to public health and safety throughout the range of the blacklegged tick, primarily across the Northeast and Great Lakes regions.

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Total Reported Cases of Tick-Borne Disease in the US, 20042019 CDC Although Powassan virus is very rare, the infection is serious, and case numbers have approximately tripled in the last decade. They hit an all-time high in 2019 with 39 cases over the course of the year in the US. Human Powassan virus cases are not limited to the US, however, as they have been reported in the United States, Canada, and Russia. Cases occur most frequently in late spring, early summer, and mid fall, which corresponds to high activity levels within the life cycle of the blacklegged tick. Researchers are not yet certain which animal species serves as the reservoir host for Powassan virus, or the population of animals in which the pathogen lives and reproduces, providing opportunity for other organisms to become infected. However, it is suspected that it may be white-footed mice, shrews, or even voles, as blacklegged ticks are known to feed on all of these species of small mammals.

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Number of Powassan virus Cases Reported Each Year in the United States. From 2011 through 2020, an average of 18 cases were reported annually CDC Powassan virus (POWV) is the only member of the tick-borne encephalitis serogroup of flaviviruses in North America. It is a type of positive-sense, single-strand RNA viruses from the family Flaviviridae which are often spread through arthropod vectors. Some other viruses in the flavivirus genus include West Nile virus, Dengue virus, Yellow Fever virus, and Zika virus, all additional examples of vector-borne diseases, spread to humans by mosquitoes. Generally, the flavivirus genomes are non-segmented and around 10-11 kbp in length. The genome encodes three structural proteins (Capsid, prM, and Envelope) and seven non-structural proteins.

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Phylogenetic Tree for Genus Flavivirus. Complete polyprotein amino acid sequences were aligned using MUSCLE. Species names are color-coded as follows: classical insect-specific- blue; dual-host insect-specific- green; NKV (no known vector)- red, mosquito/vertebrate- purple; tick/vertebrate- black. BLITVICH AND FIRTH, 2017 There are two genetically distinct lineages recognized, POWV (lineage I) and deer tick virus (lineage II). However, they are not serologically distinct, and either lineage could be responsible for a positive test for Powassan virus. POWV was first identified in humans in 1958 when a child died of encephalitis in the town of Powassan, Ontario, and the pathogen was isolated from his brain.

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Computer Illustration of Powassan Virus Particles KATERYNA KON- SCIENCE PHOTO LIBRARY ART As Hermance and Thangamani report in Vector Borne and Zoonotic Diseases, Powassan virus is the cause of a severe neuroinvasive disease with a 10% mortality rate. While the initial symptoms are generic and mild, such as a sore throat, drowsiness, and headache, serious cases progress to neurological involvement. This often consists of encephalitis and meningitis, swelling of the brain and inflammation of the fluids and membranes surrounding the brain and spinal cord, respectively. Symptoms present as persistent fever, vomiting, difficulty speaking, and seizures. Even in the cases that are not fatal, 50% of survivors experience serious and longlasting neurological symptoms such as recurring headaches and memory problems. Because there is no treatment other than basic supportive care, prevention and early diagnosis are key to recovery, making wide-spread awareness of Powassan virus vital.

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Timeline of POWV Infection, Symptoms, and Diagnostic Testing. Shown is the chronology of POWV infection in relation to the clinical signs and symptoms of disease. During the early, viremic phase of disease, POWV can be diagnosed by virus isolation or by detection of specific nucleic acid/viral antigen. At later stages of disease, POWV-specific IgM and IgG antibodies can be detected. HERMANCE AND THANGAMANI, 2017 Cases of tick-borne disease including Powassan virus are on the rise due to a number of factors. As mentioned previously, tick-borne diseases are significantly underdiagnosed and underreported, partially due to their often generic, flu-like symptoms, in part because of the failure to consider or test for tick-borne diseases by doctors, and finally due to the lack of a nation-wide standardized reporting system. The recent increase in awareness of tick-borne diseases by both medical professionals and the general public has led to increased diagnosis and reporting, but this is not the only factor. As will be discussed further in this series, the combination of climate change, globalization, and habitat fragmentation is resulting in the range expansion of both native and invasive ticks. As a result, we will likely experience greater exposure to both familiar and emerging pathogens in the US and globally. This article is featured on Forbes.org, and can be read online here: The Growing Threat Of Tick-Borne Disease Part I: Powassan Virus 459

Expelling COVID: It Takes Longer Than You May Think Forbes | May 31, 2022 | Article

The answer to the question, "How long can SARS-CoV-2 remain in the body of an otherwise healthy person?" is slowly coming into focus. The answer—Longer than you may think! The lungs and the intestine seem to be the major two organs infected by SARS-CoV-2. The lungs as the point of entry—the intestine, as the largest and richest site of the ACE 2 virus receptor. The latter may surprise you. Recent studies suggest that the virus harbors in the lungs and intestines for many, if not most, of us. One giveaway is the now frequent references to the detection of SARS-C0V-2 RNA in what are called sewer sheds. A sharp increase in the concentration of viral RNA in our sewers is a dead giveaway of yet another wave of infection in our community. Whence the RNA? Expelled with our stool from the infected intestine. The data emerged from a study by Natarajan et al. to determine whether or not a long-acting form of interferon-gamma (pegylated interferon) was useful in the treatment of Covid-19. Alas, the data show no effect of interferon treatment on the progress of the disease. However, the study did turn up interesting data on intestinal infection. The investigators measured viral RNA in the stool as a marker for infection. To their surprise, they detected virus in the stools of most participants. How did they do it? Natarajan et al. collected stool and respiratory samples from patients with mild to moderate symptoms and tested for viral RNA. The participants were randomly assigned into one of two groups, those that were administered the pegylated interferon or those that were administered a placebo. Of 120 total participants, 113 provided a stool sample at least one of the six defined time points, with 86 participants providing a stool sample thrice. Over the course of 10 460

months, 673 stool samples were collected and underwent an assay of the SARS-CoV-2 genomic RNA for the envelope protein, nucleocapsid protein, and RNA-dependent RNA polymerase (RdRp).

FIGURE 1: Schematic representation of study (top), graphic representation of detection of SARS-CoV-2 ... [+] FROM: "GASTROINTESTINAL SYMPTOMS AND FECAL SHEDDING OF SARS-COV-2 RNA SUGGEST PROLONGED GASTROINTESTINAL INFECTION" NATARAJAN ET AL. 2022

What did they find? The study reports a decline in the concentration of viral genomic RNA in fecal matter over the course of a month. 49% of participants tested positive for SARS-Co-V-2 fecal matter after the third day of infection, 40% of participants tested positive on day 14, and 11% of participants tested positive on day 28 (figure 1). At 120 days, or 4 months following infection, all participants who provided an oropharyngeal swap and stool samples tested negative for SARS-CoV-2 in their oropharyngeal (OP) sample but 12.7% tested positive in their stool sample. Natarajan et al. did not test OP samples after 4 months, but they did find that 3.8% of participants' stool samples tested for SARS-CoV-2 after 7 months (210 days). Zero participants tested positive after 300 days, or 10 461

months, following infection of SARS-CoV-2 in their RNA (figure 1). These findings suggest prolonged infection of Covid-19 and viral shedding. The study does not rule out the possibility that some of the longest positive samples are the result of reinfections. Natarajan et al. found that those who tested positive for genomic RNA in the stool were more likely to exhibit gastrointestinal symptoms. Within the first month of the study, participants who tested positive for Covid-19 RNA were statistically more likely to report nausea, vomiting, and abnormal pain. Natarajan et al. investigated the association between fecal viral shedding and observed symptoms and respiratory shedding. They found that participants who experienced shedding viral RNA from their oropharynx had higher rates of most Covid-19-related symptoms. However, there was no meaningful relationship between the observed symptoms and fecal shedding.

FIGURE 2: Percentage Breakdown of participants tested positive for SARS-CoV-2 in stool. NATARAJAN ET AL. 2022

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Summary Pegylated interferon treatment had no meaningful effect on the presence of viral shedding in fecal matter and observed gastrointestinal symptoms (figure 2). Patients with positive respiratory symptoms may experience a spectrum of Covid-19related symptoms independent of whether or not viral RNA is present in their stool. Natarajan et al. comment that fecal shedding may indicate the presence of an ongoing infection and that such infections can be accompanied by gastrointestinal systemic symptoms. These results should come as no surprise. Coronaviruses are well known to infect the gastrointestinal tracts of many species, including bats, the home species of SARS-CoV-2. The implications for longterm health, treatments, and pandemic control are subject to intense research. This article is featured on Forbes.org, and can be read online here: Expelling COVID: It Takes Longer Than You May Think

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The Vaccinia Virus That Hopped From Rabbits to Hares Forbes | June 01, 2022 | Article

Poxviruses are back, and it is no surprise. When the World Health Organization announced the eradication of smallpox over forty years ago, they also halted vaccinations against this lethal infectious disease. Consequently, much of the world population now has no protection against smallpox or the wide array of other poxviruses, including monkeypox, deerpox, rabbitpox and other zoonotic diseases. Researchers have long predicted that halting smallpox vaccinations would enable the emergence of new virulent strains of smallpox and other poxviruses. Increasing reports of monkeypox infections in humans has confirmed these concerns. Although the origin of monkeypox is not clear, usual properties in a newly detected strain has allowed this virus to spread more rapidly from indigenous regions of western Africa. At this stage, it is unlikely that these infections will lead to a major pandemic, but this will not always be the case. As viruses jump from one host to another, different molecular interactions influence the genes of both the host and virus. This fuels an arms race between a viral pathogen and its hosts. The goal of any virus is to infect as many hosts as possible but killing to much of the population would mean the virus has nowhere else to jump. Simultaneously, animal species over time will naturally develop mechanisms to reduce fatalities and weaken the severity of symptoms associated with viral infection. Through natural selection, individuals with certain genes are more likely to survive infection. This hostvirus arms race is what allows viruses to be contained within animal populations for multiple generations. Enough changes to the virus’s genome can enable a pathogen to cross to and infect other animal populations. Referred to as a spillover event, exposure to newly mutated viruses can have significant consequences as the virus further replicates and mutates

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in new hosts. When this happens, the critical question is whether the new viral strain is more or less virulent than the original virus. One of the most documented examples of this is the coevolution of the myxoma virus in European Rabbits. Initially detected in South American rabbits, the myxoma virus that causes rabbitpox was intentionally released in Australia to control the population of European rabbits in 1950. Since then, scientists have not only tracked how the rabbit population has changed but also variations in the viral genome. To their surprise, the myxoma virus that originally had a nearly 100% fatality rate was replaced by less fatal strains that only killed 7085% of its hosts. Some strains of the myxoma virus reportedly had less than a 50% fatality rate. How is it possible that a virus becomes less dangerous the more it spreads? Australian researcher Frank Fenner and his colleagues were the first to show that natural selection favored less virulent viruses. A highly virulent virus that rapidly infects and kills hosts has a much shorter infectious period, limiting its window to infect others. Decreased virulence, however, does not explain why different populations of rabbits experience varied fatality rates when exposed to the same myxoma virus. For example, within a seven-year period, a myxoma strain that once had a 90% fatality rate in rabbits living in Lake Urana only killed 26% of rabbits in the same area. These rabbits appeared to have developed a genetic resistance to the myxoma virus, in which innate and adaptive immunity could control the severity of infection even in response to the most virulent viral strains. While a strong immune response works to keep the animal alive, a particularly dangerous viral strain can spread more during the increased infectious period. This is why more virulent viruses never completely disappear. In this arms race, changes to the viral genome also enables new strains to suppress the increasingly resistant host immune response. Like other poxviruses, the myxoma virus encodes several proteins called host range factors that enhance infection. These proteins manipulate and suppress the host’s immune system to prolong the infectious period. One study from Pennsylvania State University found that increased infectability between different animal populations may be linked to single mutations, or multiple mutations 466

over time, that facilitate the expression of new host range factors. Therefore, despite how much hosts evolve to resist viral infection, the rabbitpox virus continues to find new ways to bypass these mechanisms. Since a virus’s host range factors are specific to the type of hosts they infect, other species are usually not affected by new viral strains. Occasionally, key mutations may enable poxviruses to cross the species barrier. When hundreds of hares from the Iberian Peninsula suddenly died from rabbitpox-like infections in fall 2018, it was suspected that such an event had occurred. Researchers at Arizona University recently published a report that identified the key mutation that allowed the rabbitpox virus to lethally cross into Iberian hares. These hares have lived alongside European rabbits since the 1990’s, but they only recently have been susceptible to a novel strain of the rabbitpox myxoma virus. Although rabbits and hares look alike, they are entirely different species. Physical, behavioral and lifestyle differences between rabbits and hares are mediated by genetic evolutionary variations from their common ancestor. As a result, these two species are not equally susceptible to the same diseases. When poxviruses jump from one species to another, there may be profound implications for not only animal but also human health. Understanding how this virus could cross from one species to another may provide insight to preventing further viral strains that could target humans. It is critical now more than ever to identify spillover events as they occur and isolate viruses before they have a chance to spread. In the next part of this series, we will examine the findings from this study to determine how this poxvirus hopped from one species to another. The take-home message here is that poxviruses, like other viruses, are not stable. They adapt and mutate with their environment. The SARS-CoV-2 virus was no exception. This virus thrived in bats that have genetically evolved to avoid getting sick. A recombination change in the viral genome, however, allowed the SARS-CoV-2 virus to become more lethal, eventually spreading to humans. Climate change and increased globalization has enabled viruses to mutate and spread at unprecedented rates. There are steps that we can take now to delay the next major pandemic: 467

(1) Reinstate smallpox vaccinations to target emerging poxvirus strains. (2) Increase testing for antiviral treatments by supporting academic and pharmacological research. (3) Develop a multidimensional therapeutic approach that includes vaccinations and antivirals to not only prevent infections but also effectively respond to outbreaks as they occur. This article is featured on Forbes.org, and can be read online here: The Vaccinia Virus That Hopped From Rabbits to Hares

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Tick-Borne Disease Part II: Crimean-Congo Hemorrhagic Fever Forbes | June 03, 2022 | Article

This story is Part II of a series on tick-borne disease. Here we discuss the severity and spread of Crimean-Congo Hemorrhagic Fever, the only hemorrhagic fever transmitted by ticks. At present there is a serious outbreak in Iraq of Crimean-Congo Hemorrhagic Fever, a rare but deadly tick-borne disease. This is not the first time, as it is endemic to many parts of Africa and the Middle East, but this outbreak is more severe than is usually seen. The World Health Organization received reports from Iraq of 212 cases, 46% of which were laboratory confirmed. There have been 27 fatalities so far, and the death toll is expected to continue to rise. As of 2020 there were at least 27 tick-borne diseases recognized around the world. Nicknamed the “Nose-Bleed Fever,” CrimeanCongo Hemorrhagic Fever has been around for a long time but is now spreading on multiple continents, including through Europe. Carried and transmitted most frequently by ticks of the genus Hyalomma, Crimean-Congo Hemorrhagic Fever (CCHF) carries a mortality rate often as high as 40%. There is currently no known specific vaccine or treatment. Cases have been documented in many different parts of the world, including Africa, Asia, the Middle East, Russia, and the Balkans. Recent and more frequent outbreaks have occurred in Western Europe, leading the World Health Organization to categorize CCHF as a priority pathogen.

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Crimean-Congo Hemorrhagic Fever Distribution Map as of 2014 CDC From the family Nairoviridae and order Bunyavirales, CrimeanCongo Hemorrhagic Fever virus has a circular, negative-sense, single-stranded RNA genome. There are three segments, Small (S), Medium (M), and Large (L), each of which encodes different aspects of the virion. The L segment, the biggest, encodes the RNA polymerase, the M segment encodes the glycoproteins, and the S segment encodes the nucleocapsid protein.

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Crimean-Congo hemorrhagic fever virus (CCHFV) virion and replication cycle. CCHFV attaches to an ... [+] ZIVCEC ET AL., 2016 While Crimean-Congo Hemorrhagic Fever is only carried and transmitted by ticks, there is diversity in the vectors and targets of other Bunyaviruses. Members of this order can infect arthropods other than ticks, plants, protozoans, and vertebrates. However, the majority are vector-borne, and excluding only Hantaviruses and Arenavirus, all other viruses in the Bunyavirales order are transmitted by arthropods such as ticks, mosquitos, midges, and sandflies. Due to their segmented genomes, Bunyaviruses are capable of swift recombination, increasing the risk of outbreaks. It is possible for Crimean-Congo Hemorrhagic Fever specifically to undergo recombination through the reassortment of genome segments as well as intragenic homologous recombination.

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Schematic representation of different types of nairovirus genome organization. CCHFV has a similar ... [+] GARRISON AND ALKHOVSKY ET AL., 2020 It is thought that Crimean-Congo Hemorrhagic Fever Virus may have evolved around 1500 – 1100 BC. In the 12th century, a reported instance of hemorrhagic disease in what is now Tajikistan is thought to be the first known case of CCHF. During the Crimean War in the 1850s, CCHF was common and at the time known as Crimean Fever. It infected many during the war and according to some reports, this even included Florence Nightingale, who was working as a nurse. Then in 1944, scientists in Soviet Russia identified a disease in Crimea they named Crimean Hemorrhagic Fever. Although scientists were not aware at the time, a virus from the Congo was isolated in 1956, and in 1969 it was determined that these two strands were identical. The hemorrhagic virus was officially renamed the Crimean-Congo Hemorrhagic Fever Virus a few years later. CCHF is a type of hemorrhagic fever, meaning it interferes with the blood’s ability to clot. Early symptoms are fairly general, consisting of fever, muscle aches, headaches, and dizziness. Nausea, diarrhea, and vomiting can follow, along with agitation and confusion. After a few days as the disease progresses, severe bruising and nosebleeds are common, as well as a rapid heart rate and petechiae, a rash caused by bleeding into the skin. Severely ill patients may experience kidney, liver, or pulmonary failure after the fifth day of illness, and fatalities mostly occur in the second week after onset of symptoms. In patients who recover, symptoms usually 472

begin to improve early into the second week, although recovery is slow.

Crimean-Congo Hemorrhagic Fever Transmission Schematic HAWMAN AND FELDMANN, 2018 There is currently no vaccine authorized for preventative use against CCHF. Although there have been several vaccine trials around the world, most have been terminated due to high toxicity and lack of safety. There is also no specific treatment, and care for those infected is primarily supportive. Ribavirin, a nucleoside producing broad-spectrum activity against several RNA and DNA viruses, is an antiviral agent most commonly used to treat Hepatitis C. It has been used to treat CCHF during outbreaks, but the evidence supporting its efficacy is unclear, and it can cause serious side effects such as anemia and liver damage. Due to the lack of effective vaccines and treatments, the World Health Organization named CCHF as a top priority on a list of emerging pathogens with epidemic potential for which there are no medical treatments. There is great variation in the symptoms and outcome of infection between cases, and the cause for this has not been very well understood until recently. There has been no suitable animal model for studying the disease until recently, adding to the challenges in understanding the virus. However, a study just published in May 2022 has shown important development in the understanding of factors leading to more severe manifestation of disease. In this study, Golden et al. demonstrated both in cell culture and in mice that mitochondrial antiviral signaling protein (MAVS) activation along with cytokine production contribute to the pathogenesis of

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Crimean-Congo Hemorrhagic Fever Virus, identifying key new targets for potential treatments. It is mostly people who experience greater exposure to ticks in areas where CCHF is common that are at a higher risk for contracting the disease. This primarily includes agricultural workers, but another common route of exposure is not directly from a tick bite itself, but rather from exposure to the blood of an infected animal. Slaughterhouse workers and those with other frequent exposure to livestock are therefore also susceptible. Healthcare workers in areas where CCHF is endemic who are exposed to blood or bodily fluids without sufficient protections are also at risk for catching the blood-borne virus. However, as climate change results in the expansion of the range for the ticks that carry CCHF and outbreaks become more common, the risk of contracting CrimeanCongo Hemorrhagic Fever increases, as does the importance of developing effective therapies. This article is featured on Forbes.org, and can be read online here: TickBorne Disease Part II: Crimean-Congo Hemorrhagic Fever

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Rabbitpox: the Story of a Specialized Killer Forbes | June 06, 2022 | Article

With poxviruses now back on the radar, we must consider the outstanding threat of possible human pathogens in much greater detail. It is critically important to understand not only how poxviruses jump from one species to another but also how they evolve across generations. As we reported in part one of this series, the “arms race” between the myxoma virus and its primary hosts, rabbits, has been one of the most well studied examples of how viruses mutate and coevolve with their environment. Scientists initially introduced the myxoma virus to European rabbits to control their growing populations in parts of Europe and Australia. This virus that was naturally found in South American sylvilagus rabbits killed 99% of the European rabbits it infected. Over time, surviving generations developed mechanisms to resist viral infection, which reduced the virus’ fatality rate by nearly 50%. Just as European rabbit populations were evolving, however, so was the virus. Newer strains of the myxoma virus, in fact, increasingly suppress the immune systems of their rabbit hosts. Like any other virus, the myxoma virus must overcome the host’s immunity barriers to replicate within cells successfully. As the cell’s first line of defense, innate immune responses block viral replication by inducing the expression of interferon-stimulated genes. Suppressing these innate immune responses, as well as later immune mechanisms, determines how well the virus can replicate and infect others. Expressing host range factors is one way that viruses can evade immune detection. The function and prevalence of these virusencoded proteins varies across different cell types, hosts and species. For example, poxviruses are the only viruses that express host range factors from the C7L family. The C7 sequence was originally identified in the vaccinia virus that causes smallpox. Deleting the C7L gene from the smallpox virus resulted in a mutated virus that could no longer replicate in human and other animal cells. Without 475

the C7L protein, experimentally enhancing the expression of interferon-stimulated genes could be a potent treatment against smallpox, but reintroducing C7L blunted this effect. This finding revealed that C7L and other similar host range factors may interfere the effectiveness of interferon-stimulated genes recruited during innate immunity. C7L and its homologs are expressed in nearly every poxvirus that infects mammals, including the myxoma rabbitpox virus. The C7L family consists of all the protein homologs across different poxviruses that exhibit a similar genetic sequence to the original smallpox C7L protein. Despite the similarity of their genetic sequences, this family of host range factors can be divided into two major groups based on their role in viral replication. The first group, which includes most members of this family, determines how well the virus can replicate and therefore are not functionally different from the C7L smallpox protein. The second group does not play a critical role in replication in most cells. Within this group, however, a member of the C7L family may enhance replication in one species but not others. The Crossover In fall 2018, hundreds of hares from the Iberian Peninsula seemingly died overnight from a mysterious virus. A genetic analysis found that this novel virus, which researchers called MYXV-Tol, was very similar to the original South American rabbitpox virus, suggesting that this virus was a novel strain of the myxoma virus. Given that these hares were previously resistant to rabbitpox, what changed? The key difference was that the new MYXV-Tol virus acquired a unique “gene cassette” through DNA recombination. Gaining approximately 2,800 base pairs from some unknown poxvirus, this hybrid strain expresses several new genes compared to the original virus. The placement of these new protein-encoding genes is shown below in Figure 1.

FIGURE 1: A REPRESENTATION OF MXYV-TOL VIRAL GENOME. NEW GENES THAT WERE ACQUIRED FROM DNA

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RECOMBINATION ARE SHOWN IN BLUE AND PURPLE, INCLUDING M159.

FROM: “IDENTIFICATION OF A NOVEL MYXOMA VIRUS C7-LIKE HOST RANGE FACTOR THAT ENABLED A SPECIES LEAP FROM RABBITS TO HARES” ÁGUEDAPINTO ET AL. 2022 Now, in a recent report, Águeda-Pinto et al. from Arizona State University singled out a key gene from the novel recombinant virus, called M159, that may have enabled the myxoma virus to jump from rabbits to hares. Characterized as a host range factor, understanding the role of this protein may provide insight into how poxviruses bypass innate immunity to successfully infect hosts. To investigate the role of this protein, Águeda-Pinto et al. knocked out, or removed, the M159 sequence from the myxoma virus they detected in the hares. How well this knockout virus could replicate within the extracted hare cells was then compared to unchanged versions of the virus. Subsequent findings from this experiment confirmed that the replication of the myxoma-like virus within hare cells depended on the expression of M159 proteins. Removing this gene prevented the virus from infecting or replicating within host hare cells. Exposure to the unchanged virus, however, did lead to infection. When researchers inserted the M159 protein sequence into an alternate South American strain of the myxoma virus, the virus increasingly replicated within the cell samples, even though Iberian hares are not normally susceptible to this viral strain. Águeda-Pinto et al. reported that the specific backbone of the virus, in addition to the presence of M159, is critical for infecting these hare cells. Investigators also found that the addition of M159 enabled the virus to infect human cancer cells. Humans are not normally susceptible to rabbitpox. However, when Águeda-Pinto et al. exposed pancreatic and melanoma cancer cells to M159-enhanced strains of the myxoma virus, they were able to engineer a tumorfighting virus that successfully replicated within cells. The ability for M159 to enhance viral replication may provide a useful target for fighting cancers in humans. How is M159 enhancing viral replication? In vaccinia viruses, these proteins have been shown to suppress early innate immune 477

responses to promote viral replication. How this happens has yet to be well understood. What we do know is that without this immunosuppressive gene, a cell can more easily shut down the invading virus and block further replication. Investigators speculate that M159 likely binds to and interferes with interferon-stimulated gene products that are recruited for innate immune responses to viral pathogens. Like other members of the C7 family, M159 folds into a protein scaffold that can bind to specific host-generated antiviral proteins. The structure of this protein looks like a hand folded upwards. Despite being a member of the C7 host range factor family, M159’s genetic sequence only slightly resembles that of C7L. In fact, this protein only shares five amino acids with C7L. Like a hand with many fingers, only the fingertips are conserved. The rest of the hand has an entirely different protein sequence. However, it is the conserved fingertips of this protein that are reportedly critical for binding host-generated antiviral proteins.

FIGURE 2: AN ILLUSTRATION OF THE M159 PROTEIN GENE IDENTIFIED IN THE MYXV-TOL RABBITPOX VARIANT THAT JUMPED FROM EUROPEAN RABBITS TO IBERIAN HARES. (A) A COMPARISON OF THE AMINO ACID SEQUENCE FROM DIFFERENT HOST RANGE FACTORS FROM THE C7 FAMILY. IN ADDITION TO

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M159, THIS LIST INCLUDES C7 FROM THE VACV SMALLPOX VIRUS, SPPV-63 FROM SHEEPPOX, SWPV-64 FROM SWINEPOX, YLDV-67 FROM YABBA-LIKE MONKEY VIRUS, AS WELL AS M062, M063, AND M064 FROM THE INITIAL SOUTH AMERICAN RABBITPOX VIRUS. ONLY FIVE AMINO ACIDS ARE CONSERVED FROM THE SMALLPOX C7 PROTEIN SEQUENCES ACROSS THESE VARIOUS HOST RANGE FACTORS, SHOWN IN RED. THE SPECIFIC BETA AND ALPHA PROTEIN FRAMEWORK AS HIGHLIGHTED IN BLUE AND GREEN, RESPECTIVELY, FOR M159. (B) THE MODELED STRUCTURE OF M159, COMPLETED WITH BETA AND ALPHA STRUCTURES IN LIGHT GREEN. THE AMINO ACIDS THAT WERE CONSIDERED FROM C7 ARE SHOWN IN RED. FROM: “IDENTIFICATION OF A NOVEL MYXOMA VIRUS C7-LIKE HOST RANGE FACTOR THAT ENABLED A SPECIES LEAP FROM RABBITS TO HARES” ÁGUEDAPINTO ET AL. 2022

Unlike other DNA viruses that need to enter the nucleus to replicate, poxviruses can operate directly from the cell’s cytoplasm. At early, intermediate and late stages of infection, these viruses capitalize on different gene regulatory mechanisms to enhance replication. Once the virus binds to and invades a host cell, a viral inner core is released into the cytoplasm. This prompts the transcription of early viral mRNA proteins, including host range factors that can evade the immune system and facilitate the synthesis of viral RNA products. Intermediate and late mRNAs, then, synthesize products needed to assemble new versions of the virus. To determine which stage M159 is implicated, Águeda-Pinto et al. examined the expression of known early and late viral proteins in hare cells following exposure to both changed and uncharged versions of the myxoma-like virus. Gene expression of early viral proteins was unaltered with exposure to various forms of this virus. Deletion of M159, however, prevented the formation of new viruses, suggesting that this viral protein is involved somewhere between early and mid-to-late stages of infection. Yet, there is still another pressing question: did the addition of M159 make European rabbits more vulnerable to severe infection? To their surprise, Águeda-Pinto et al. found that the presence of M159 did not enhance viral infection and replication in rabbit cells. Although it has been reported that European rabbits can contract the myxoma-like virus, the rate that they are infected is much less

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frequent than Iberian hares. M159 is likely a host range factor that is specific to hare species. We should not rush to assume that the addition of M159 in the novel myxoma strain was the only change that enabled the virus to cross the species barrier, however. This recombinant virus expresses several new genes, in addition to M159, that may have enhanced the virulence of rabbitpox in hares. Since the full nucleotide sequence of the myxoma-like virus has yet to be elucidated, there may have been several factors that contributed to the enhanced infectability of the Iberian hares. As Grant McFadden, director of the Biodesign Center for Immunotherapy, Vaccines and Virotherapy at ASU and coauthor of this study, says, "Every time a virus leaps from one host species into another, we learn something new about Mother Nature." Sometimes the things we learn, however, only lead to more questions. What does this new myxoma strain mean for other animal species that live alongside these hares and rabbits? Can we predict how the virus will evolve in the future to prevent other species from being infected? These and many other questions exemplify why it is important to identify viral spillover events as they occur. The next pandemic may likely be a zoonotic poxvirus. This article is featured on Forbes.org, and can be read online here: Rabbitpox: the Story of a Specialized Killer

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Study Finds Previous Covid-19 Infection Doesn’t Protect Children From Omicron Forbes | June 09, 2022 | Article

A new study found that less than 10 percent of children who contracted Covid-19 in 2020 or early 2021 developed neutralizing antibody titers against the Omicron variant of SARS-CoV-2. This finding reinforces the need to vaccinate children and teens against SARS-CoV-2 even with a prior infection. Not only are unvaccinated children significantly more likely to develop severe or fatal Covid-19 disease, but they can transmit Omicron to other highly vulnerable populations. The study published in Nature was led by researchers at Harvard Medical School, Boston Children’s Hospital, and the U.S. Food and Drug Administration. It is now well established that prior infection does not protect adults from re-infection, but there is less research on children. The study assessed children’s ability to neutralize the Alpha, Beta, Gamma, Delta, and Omicron Covid-19 variants. Serum and plasma samples were collected from 3 independent pediatric disease cohorts: children younger than 5, children 5-11 years, and adolescents 12-21 years. None of the children and adolescents were vaccinated. The researchers performed antibody profiling on the samples from 177 children and adolescents who were hospitalized with acute Covid-19 or MIS-C, or outpatient mild convalescent Covid-19. By using pseudovirion (a pseudovirus derived from SARS-CoV-2 but stripped of its virulence) neutralization assays, the researchers determined the antibody neutralization activity of the samples against the predominant SARS-CoV-2 WA1 strain and the 5 variants of concern. For the control group, the researchers used samples from 10 critically ill children who tested positive for seasonal coronaviruses before 2019. None of these controls demonstrated neutralization titers against Covid-19. Children under the age of 5 who were hospitalized with severe acute Covid-19 were found to have lower neutralizing antibodies to 481

SARS-CoV-2 variants compared with children older than 5. Convalescent pediatric Covid-19 and MIS-C cohorts showed higher neutralization titers than hospitalized acute Covid-19 patients. The researchers acknowledge that a limitation of the study is that it was able to test only for antibody responses, not other measures of immunity such as the production of T-cells. All cohorts showed some loss of cross-neutralization against all variants, with the most pronounced loss against Omicron. In contrast to prior SARS-CoV-2 infection, children who were vaccinated twice demonstrated higher titers against Alpha, Beta, Gamma, Delta, and Omicron. Only 29 percent of 5 to 11-year-olds and 59 percent of 12 to 17-year-olds in the US have currently received two vaccine doses. I am hopeful that these findings dispel the misunderstanding that prior infection provides protection and encourages any vaccine-hesitant parents to immediately vaccinate their children. As the virus continues to evolve, we sit on a knife’s edge with some SARS-CoV2 strains only a mutation or two away from far greater lethality. This article is featured on Forbes.org, and can be read online here: Study Finds Previous Covid-19 Infection Doesn’t Protect Children From Omicron

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Unraveling Hope For Prion Disease And Other Progressive Neurodegenerative Diseases Forbes | June 09, 2022 | Article

Prion diseases are rare but deadly neurodegenerative brain diseases that result from misfolding protein. Impacting both animals and humans, such rapidly progressive diseases result in abnormal physical and impaired mental functioning within months of diagnosis until death. Examples include mad cow disease, scrapie, Kuru, and Creutzfeldt-Jakob disease.

Figure 1: Conversion of a normal PrPc protein into the disease-causing prion PrPSc.

DU PLESSIS ET AL 2008. The normal cellular host protein PrPC holds an intrinsic property to fold. When it folds differently, the protein undergoes a conversion to the pathogenic PrPSc conformation (Figure 1). Specifically, the protein loses some of its alpha helical nature and becomes greatly composed of beta-pleated structures, which are prone to 483

aggregation. The infectious particle can consequently accumulate in the brain and have detrimental impacts in cognition. PrPSc also replicates like an inanimate crystal; devoid of genetic material, PrPSc auto-catalyzes and triggers a chain-like reaction to produce more of itself. PrPSc is also known to ravage through the brain, creating a sponge-like (spongiform) appearance. At present, there are no cures available to treat prion disease. Novel work done by Mead et al. however provides a glimmer of hope regarding an approach to treat patients with Creutzfeldt-Jakob disease. They designed a monoclonal antibody as a potential treatment for Creutzfeldt-Jakob disease. However, unlike most others who create antibodies that bind to aberrant protein, Mead and colleagues designed an antibody that stabilizes the normal protein precursor. If successful, this may prove to be a valuable approach for treatment for a number of similar diseases, including Alzheimer's. What is Creutzfeldt-Jakob disease? Creutzfeldt-Jakob disease is a rapidly progressive neurodegenerative disease that causes dementia and ultimately death. This devastating disease affects one person per million per year. In the United States, that is roughly 320 cases annually. However, because it is very difficult to diagnose, researchers and health care professionals speculate that its prevalence may be inaccurate. There are four types of Creutzfeldt-Jakob disease: sporadic Creutzfeldt-Jakob disease, genetic Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, and iatrogenic Creutzfeldt-Jakob disease. Sporadic Creutzfeldt-Jakob disease is the most common form of Creutzfeldt-Jakob disease, accounting for 85% of cases, but its causes are unknown. Whereas genetic Creutzfeldt-Jakob disease results from an inherited mutation from one or both parents. This form represents 10% to 15% of patients. Variant Creutzfeldt-Jakob disease comes from exposure to bovine spongiform encephalopathy (BSE), most often through consumption of infected cattle. Iatrogenic Creutzfeldt-Jakob disease is reported to be the least common type of Creutzfeldt-Jakob disease, representing less than 1% of cases. Patients contract the disease through exposure to contaminated medical equipment or medical procedures.

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Figure X: A FLAIR and Diffusion MRI scans of the horizontal sections of the brain of a 33- year old patient with sporadic Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease). Top row are MRI scans of the patient’s brain taken in April 2016. Bottom row are MRI scans of the patient’s brain taken in May 2016.

WIETSE ET AL. 2018. In their study that spanned from mid-October 2018 to July 2019, Mead and colleagues investigated the effect of PRN100, an antibody to PrPC, on Creutzfeldt-Jakob disease progression. The study consisted of six patients: five patients were clinically diagnosed with probable sporadic Creutzfeldt-Jakob and one patient was clinically diagnosed with iatrogenic Creutzfeldt-Jakob disease. Patients followed a gradual dosing schedule of PRN100 and were assessed using the MRC Disease Rating Scales. Beginning at 1 mg/kg, PRN100 doses were administered in 2 to 6 day increments with the maximum dosage of 80 to 120 mg/kg administered every two weeks. Their scores were then compared to those obtained from the National Prion Monitoring Cohort observational study that served as controls. Based on literature precedent, researchers aimed for a 50nM concentration of PRN100 in the cerebrospinal fluid.

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Figure 2: Patients’ MRC Prion Rating Scale scores over time (days) since enrollment. Panel (A) represents scores of patient 1(red) compared to 18 controls (gray) with sporadic Creutzfeldt-Jakob with a heterozygous genotype codon on the PRNP gene and baseline score on the MRC Prion Disease Rating Scale within 16-18. Panel (B) represents scores of patient 2 (red) to 17 (gray) controls with iatrogenic Creutzfeldt-Jakob, any genotype codon on the PRNP gene and any score on the MRC Prion Disease Rating Scale. Panel (C) represents scores of patient 3 (red) compared to 17 controls (gray) with sporadic Creutzfeldt-Jakob, a baseline score of 10-12 on the MRC Prion Disease Rating Scale and a genotype at codon 129 in the PRNP gene. Panel (D) represents scores of patient 4 (red) compared to 18 controls (gray) with sporadic Creutzfeldt-Jakob with a score of 16-18 on the MRC Prion Disease Rating Scale and a heterozygous genotype at codon 129 on the PRNP gene. Panel (E) represents scores of patient 5 (red) compared to 10 controls (gray) with sporadic Creutzfeldt-Jakob, a baseline score of 12-15 on the MRC Prion

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Disease Rating Scale, and a methionine homozygous genotype at codon 129 in the PRNP gene. Panel (F) represents scores of patient 6 (red) to 10 controls (gray) with sporadic Creutzfeldt-Jakob, a baseline score of 13-15 on the MRC Prion Disease Rating Scale, and a heterozygous PRNP genotype at codon 129 in the PRNP gene. The orange bar represents a concentration lower than 50nM of the PRN100 antibody in the cerebrospinal fluid. The orange bar represents a concentration equal to or greater than 50nM of the PRN100 antibody in the cerebrospinal fluid.

With a small sample size and limited stock of drug availability in mind, researchers saw promise in the preliminary data. Two patients passed away before 50nM of PRN100 was reached (patients 3 and 5, both of whom were diagnosed with sporadic Creutzfeldt-Jakob disease); however, the scores from the MRC Prion Disease Scales from three patients did stabilize when there was a 50nM or higher concentration of PRN100 (figure 2). Their careful gradual dosing approach contributed to the average of 47 days to reach 50nM of PRN100 in the cerebrospinal fluid and clinically significant neurological decline among patients with sporadic Creutzfeldt-Jakob disease during that period. For additional context, the median survival for patients with sporadic Creutzfeldt-Jakob disease in the National Prion Monitoring Cohort observational study was 25 days from their respective enrollment into the study.

Figure 3: The impact of PRN100 on the removal of PrPSc from the brain. Panels A-D come from PrP labeling in the brain of PRN100 treated patient 2. Panels E-H are PrP

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labeling of the brain of an untreated patient with iatrogenic Creutzfeldt-Jakob. Panels I-L compare the optical region of the PRN100-treated brain of patient 2. Panels M-P compare the optical region of the non-treated brain of a patient with iatrogenic CreutzfeldtJakob.

THE LANCET Based on the autopsy of patients 2 and 3 (diagnosed with iatrogenic and sporadic Creutzfeldt-Jakob disease, respectively) reported no evidence of cytotoxicity, lymphocytic inflammation, or increased formation of vacuoles. There was a striking decrease in PrPSc immunoreactivity in the parietal and occipital cortex, change in PRPSc distribution in the subventricular areas, and an accumulation of amyloid in the arteries in the brain (cerebral amyloid angiopathy) in patient 2 compared to the untreated patients with iatrogenic Creutzfeldt-Jakob disease (figure 3). Mead and colleagues attributed the cerebral beta amyloid angiopathy to be the result of treatment, however, they report no amyloid-related imaging abnormalities in any patient. The autopsy of patient 3 also had different PrP labeling in the periventricular regions in comparison to the historical controls. Mead and researchers also observed PRN100 in the patient’s brain tissue to be similar in concentration observed in the cerebrospinal fluid (well above concentrations shown to clear cells of PRPSc infection). Although their results are highly preliminary, it does seem that the alternative approach of Mead et al stabilizing the normal protein that is the pathogenic form may be promising for a large number of neurological diseases. This article is featured on Forbes.org, and can be read online here: Unraveling Hope For Prion Disease And Other Progressive Neurodegenerative Diseases

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Paxlovid Resistance: Challenges And Opportunities Forbes | June 09, 2022 | Article

We find ourselves in a challenging scenario with the current state of the pandemic. While proving to be effective at reducing hospitalizations, and severe disease, our best vaccines have failed to protect against infection. Tolerance for public health measures is waning or even nonexistent around the world. So how might protect ourselves against this ever-evolving virus? Paxlovid has been our first line of defense in treating breakthrough Covid-19 infections that occur despite vaccination. Public health officials have attributed lower hospitalization rates in the most recent surge to the use of Paxlovid. The seven-day average of new Covid hospitalizations is approximately 28,000 this week, down from nearly 160,000. However, two recent preprint studies demonstrate evidence of potential Paxlovid resistance. These findings present both challenges and opportunities for the future of the pandemic. Paxlovid combines nirmatrelvir, an inhibitor of the SARS-CoV2 main protease (MPro, also known as 3CL or nsp5), and ritonavir, an HIV-1 protease inhibitor and CYP3A inhibitor. Two recent preprint studies in which SARS-CoV-2 was passaged in cells exposed in culture to nirmatrelvir documented the selection of a combination of two and three amino acid substitutions in the main protein protease of SARS-CoV-2 that confer resistance to the nirmatrelvir. Three substitutions were identified: L50F (in which leucine at position 50 is substituted by phenylalanine), E166A or E166V (in which glutamic acid at position 166 is substituted by alanine or valine) and L167F (in which leucine at position 167 is substituted by phenylalanine). In the study by Jochmans and collaborators, E166A and L167F individually provided low-level resistance (10- and 4-fold) in a biochemical assay, while the triple mutant L50F+E166A+L167F resulted in the highest levels of resistance (72-fold). All substitutions 489

were associated with a significant loss of protease activity, suggesting a reduction in viral fitness. Consistently, in structural biology analyses, the different substitutions reduced the number of interactions between the inhibitor and the enzyme. In the study by Zhou and collaborators, L50F+E166V provided up to 80-fold resistance, with resistance being conferred by E166V (the double mutant L50F+A173V showed no-to-little resistance) in reverse genetic studies in a homologous cell culture system. Moreover, engineered double mutants showed high fitness in transfection and passage cultures with infectivity titers comparable to those of the original virus. The fitness cost of single substitutions E166V and A173V was compensated by L50F. In molecular dynamics simulations, E166V and L50F+166V weakened while L50F improved nirmatrelvir binding. Both studies are consistent in that the triple mutant L50F+E166A+L167F displays a similar fold increase in resistance of 72-fold relative to the unmutated virus than the double mutant L50F+E166V. Jochams and collaborators also describe that the appearance of the double mutant L50F+E166A preceded that of the triple mutant L50F+E166A+L167F. Although it is often the case that the resistance mutation(s) carry a viral fitness cost which renders it unlikely for the mutation to spread across the population, the two publications differ in their findings on the fitness phenotype of the variants, which warrants further study. Presently, the only treatment alternatives we have for Paxlovid are Remdesivir and Monoclonal Antibodies. We have learned that SARS-CoV-2 has developed resistance to Remdesivir and the Omicron variants have developed resistance to most monoclonal antibodies treatments with the exception of Sotrovimab. However, as the virus continues to evolve we can expect that it will develop resistance to Sotrovimab in the near future. These observations suggest that the way forward is to take a lesson from HIV research, another virus that we have failed to produce an infection-blocking vaccine. We should be focusing on developing combinations of small-molecule antiviral drugs that have proved effective at preventing and treating HIV. The goal of the biotechnology and pharmaceutical industries should be to develop an array of highly potent and specific drugs, each of which targets a different function of the virus. 490

This will require accelerated research and global collaboration that not only breaks down geographic borders but also the silos that exist between academia and the pharmaceutical biotechnology industry. It will require similar levels of investment and resources that we saw used by The National Institutes of Health for an HIV/AIDS research budget, approximately $2-3 billion a year. With this plan in place, we can hope to have highly effective drugs that both treat and prevent SARS-CoV-2 and put an end to repeated assaults of this pandemic. This article is featured on Forbes.org, and can be read online here: Paxlovid Resistance: Challenges And Opportunities

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The Virus Is Still Winning

Project Syndicate | June 10, 2022 | Opinion

BOSTON – Once again, COVID-19 is rampaging unchecked through the US population, because there are no longer any publichealth measures in place to protect adequately against infection. I myself have taken extraordinary precautions to avoid infection for the last two and a half years, because I met three of the qualifications for severe COVID-19: I am 77 years old; I am a cancer survivor; and for the past 40 years I have been treated for a chronic inflammatory condition that leaves me moderately immunocompromised. Luckily, I had the privilege of being able to sequester myself in my country home, where I could control my personal interactions. I was vaccinated within weeks of vaccines becoming available, and I have received boosters every three to four months since. Moreover, I was given Evusheld, a monoclonal antibody treatment, four weeks ago. Yet, toward the end of May, after many months of seclusion, I permitted myself one social occasion – a fundraising event with about 100 people in attendance. Four days later, in bed with a fever, cough, and malaise, I tested positive for COVID-19. Even with multiple vaccinations and Evusheld, I was not protected. My experience has underscored a sad truth. In the race to return to some semblance of normalcy, the needs of the vulnerable have been forgotten. Given that infection by SARS-CoV-2, the virus that causes COVID-19, has now become almost inevitable for most people, the least we could do is develop drug treatments to alleviate disease symptoms and prevent death and long-term consequences. But we have not done so. Pharmaceutical companies designed and manufactured COVID-19 vaccines on an unprecedentedly accelerated timeline, but drug development has not proceeded apace.

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There are three main treatment options for those who develop COVID-19 and are at risk for long-term effects and disability. All three have limitations that prevent wider use. The first is remdesivir, which requires an IV-administered infusion and is thus available only in clinical settings. The second is Evusheld, which its developer, AstraZeneca, claims is effective at preventing infection from the latest Omicron subvariants. However, in my case at least, the treatment failed to work as it should. The third is Paxlovid, an antiviral drug developed by Pfizer that targets the SARS-CoV-2 protease. Paxlovid gained popularity as a COVID-19 treatment when studies showed that it reduced the risk of serious illness and death by up to 90%. But research into its prophylactic potential has turned up empty, and new evidence suggests that some who take it experience rebound infections, meaning they might unwittingly transmit the virus to others. What is to be done? Since the start of the pandemic, I have argued that vaccination alone will not be sufficient to prevent repeated COVID-19 infections. The literature on cold-causing coronaviruses demonstrates that they can reappear like clockwork. And with SARS-CoV-2, waves of infection occur not annually, as I would have expected based on previous data, but rather at much shorter intervals – every four to six months. In addition to recognizing the limitations of vaccination as a form of pandemic control, we are becoming more aware of the seriousness of “long COVID,” which describes a broad range of symptoms – respiratory, gastrointestinal, neurological – that persist after an initial COVID-19 infection has cleared. While more than one-third of COVID-19 patients will develop long-term symptoms of some kind, 2-4% will experience consequences severe enough to be disabling for many months, if not years. These include intense fatigue, persistent pain, and damage to vital organs, such as the heart, lungs, pancreas, and possibly others. Millions of people around the world have long COVID, and millions more will end up with it. Recent data suggest that the vaccines reduce its impact by a mere 15%. While our path out of the pandemic is not as straightforward as when I was working as an HIV researcher, the overarching direction of travel is still clear. We must develop strong antiviral drugs targeted at SARS-CoV-2 specifically. Though only six drugs targeting HIV 493

have been identified, we have several treatments that are effective at clearing the virus. Given the technological advances made in the last 30-35 years, and the speed with which these advances led to a COVID-19 vaccine, I had expected that we would have numerous antivirals for COVID-19 by now. And yet, of the treatments that have been approved, all are retreads or drugs developed for other viruses, not SARS-CoV-2. What we need now is to repeat the approach that worked during the HIV/AIDS epidemic. That effort involved a multiyear drugdevelopment program to guide government- and industrysponsored research. As a member of the National Institute of Allergy and Infectious Diseases Council, I proposed an intensive, cooperative program whereby universities with industrial partners would receive large grants to fund basic research and practical drug development. The potential for a payoff attracted the interest of large pharmaceutical and biotechnology companies and spurred the formation of several public-private partnerships. The program was a resounding success, but it didn’t come cheap. Each year, the National Institutes of Health set aside $2-3 billion in a special HIV/AIDS research budget. The same level of investment is needed again. My colleagues around the world who have the skills required for intensive COVID-19 research are desperate for additional funding. To its credit, the NIH recently awarded $108 million in grants to the Metropolitan AntiViral Drug Accelerator, a collaborative initiative by world-class research institutions in New Jersey and New York that will investigate the potential for small-molecule drugs to treat and prevent coronaviruses. Combinations of small-molecule antiviral drugs have proved effective at preventing and treating HIV, and can be manufactured and sold cost-effectively. A year’s treatment with HIV drugs now costs well under $100 in many places around the world. A protocol to prevent and treat COVID-19, lasting two to three weeks at most, should cost far less. Unless we find powerful ways to eliminate the SARS-CoV-2 virus, it will remain a scourge. Some strains are only a mutation or two away from a leap in lethality. To prevent that, we must learn from pandemics past. 494

This article is featured on Project Syndicate, and can be read online here: The Virus Is Still Winning

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This is how we should be diagnosing long COVID The Hill | June 10, 2022 | Opinion

For those who have struggled to obtain a diagnosis for chronic illnesses such as chronic fatigue syndrome/myalgic encephalomyelitis or endometriosis, a familiar process is now playing out for a large portion of the population who are struggling to get a long COVID-19 diagnosis. Chronic fatigue syndrome/myalgic encephalomyelitis patients often wait up to five years for a diagnosis. The condition remains a diagnosis of exclusion, which often means patients are subjected to many time-consuming and costly tests. Endometriosis has an average diagnostic delay of seven to 10 years from the onset of symptoms. These diagnostic delays are due to a variety of factors, from outdated clinical definitions and a lack of health care provider education to complicated and prohibitive coding and referral systems. We must heed these lessons and not repeat the same mistakes when diagnosing long COVID patients. A correct diagnosis doesn’t just give patients a name for their ailments. A correct diagnosis unlocks treatment options, eligibility for clinical trials and financial coverage through insurance. A diagnosis also allows some to seek necessary disability benefits or work accommodations from the agencies that determine benefit eligibility. A meta-analysis by the Brookings Institution suggested that long COVID may be responsible for over 1 million workers being out of the labor force at any given time. Misdiagnosis or delayed diagnosis of chronic illness is a critical public health issue. The first challenge is to create a universal and inclusive clinical definition of long COVID and subsequent medical coding. Long COVID is presently known by several other names, including longhaul COVID, chronic COVID and post-acute sequelae of COVID19. Yet using terms such as “post,” “chronic” or “syndrome” can cause assumptions about the underlying physiological processes associated with the condition, which still largely remain unclear. 496

The lack of a single name and clinical definition causes problems for epidemiological data reporting, research, policy and treatment. But this must also be balanced against the need to provide an inclusive definition that ensures that no long COVID patients are left behind. In October 2021, long COVID was recognized with a diagnostic ICD-10 code known as U09.9 Post Covid Condition, an important step for patients who need their care and treatment to be reimbursed by insurance providers. Guidelines for use of the code state that it should be used for patients who experienced a probable or confirmed COVID-19 infection. This is an important distinction as unequal access to testing at different stages of the pandemic means that many patients may not have serological proof of SARS-CoV-2 infection. Unfortunately, this code also excludes any potential long COVID patients who experienced an asymptomatic COVID-19 infection. It is now well understood that the severity of the initial infection has no bearing on the severity of long COVID symptoms. A recent analysis of medical records in California found that 32 percent of patients with long COVID symptoms had asymptomatic infections. This unnecessary criterion could prevent thousands of long COVID patients from accessing treatment and care. The code also needs to better represent the broad range of symptoms associated with long COVID. The next challenge is developing screening systems and educating health care providers on the full spectrum of symptoms and experiences. The symptoms associated with long COVID continue to evolve and grow. One Lancet study lists up to 200 symptoms. In addition to collaborating with researchers and physicians to develop guidelines for diagnosis, treatment and care, the Centers for Disease Control and Prevention (CDC) and other physician groups should be working with patient advocacy groups to fully understand the barriers to care and the breadth of the patient experience living with this condition. Health care provider education should be ongoing. Since long COVID spans so many medical specialties, we also need to break down silos that exist between different physician groups to share resources and collaborate. Only then can we create comprehensive guidance and pathways for physicians to make long COVID diagnoses. 497

Between 10 percent to 30 percent of COVID-19 cases are now resulting in long COVID symptoms. This growing population means that screenings for long COVID symptoms should be integrated into annual primary care checkups. In particular, cardiovascular complications from COVID-19 are becoming increasingly common. This is true even for patients without previous cardiovascular disease, comorbidities and an otherwise low risk of cardiovascular disease. We need to ensure that cardiovascular issues are picked up at the earliest possible stage to avoid severe disease. Another complex challenge is the institutional change required for doctors to work most effectively with long COVID patients. Many physicians are heavily restricted by the amount of time they can spend with patients due to insurance reimbursements and a feefor-service payment model. The average 15-minute primary care doctor appointment mandated by many insurance companies is barely enough time to take a medical history for a condition as complex as long COVID let alone conduct an exam and order tests or make referrals. Long COVID care should be integrated and patient-centered and utilize a multidisciplinary team. Primary care doctors who may not have the expertise required to diagnose or care for certain long COVID patients should have a network of local specialists and treatment clinics available for referrals. Only by addressing these challenges can we begin to move forward toward an inclusive, swift, and accurate diagnosis process for long COVID patients that leaves no one behind. This article is featured on Forbes.org, and can be read online here: This is how we should be diagnosing long COVID

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There May Be A New Polio Epidemic On Its Way- If So, What We Can Do Forbes | June 10, 2022 | Article

This is Part I in a series on the enteroviruses that appear to cause a poliolike neurological disease, Acute Flaccid Myelitis. Reports of this lifethreatening condition have increased within the US and elsewhere, and viruses from the same family as the poliovirus are implicated. Most people have either lived through or heard horror stories of polio, a severe disease caused by the poliovirus which can cause paralysis or even death through infection of the spinal cord. Thanks to vaccines, the United States has been polio-free since 1979. However, there are other lesser known viruses that can cause similar life-threatening neurological conditions. Acute Flaccid Myelitis is one of these conditions, and there have been increased reports of cases in the US in the past 10 years. Acute Flaccid Myelitis (AFM) affects the nervous system, specifically the area of the spinal cord referred to as gray matter, and results in a weakening of the body’s muscles and reflexes. In serious cases this can lead to respiratory failure, as the patient’s muscles required for breathing are too weak and lifesaving mechanical support is required. Up to 90% of cases of AFM are seen in children, and there is no specific treatment or cure.

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Magnetic Resonance Imaging (MRI) of the Spinal Cord in a Case of AFM. Visible swelling is apparent in (D) when compared to (E) where the swelling has resolved. ESPOSITO ET AL., 2017 As reported by the CDC, there have been sharp spikes in the number of cases of AFM in the years 2014, 2016, and 2018. It is unclear how the Covid-19 pandemic may have impacted diagnosis and reporting of AFM in the previous few years, potentially explaining why the trend of uptick in cases is not observed after 2018. The observed increase every other year may suggest that immunity from natural infection only lasts one year, or there may be some other underlying cause for this new pattern of alternating increase in case rates. Within each year, the number of cases in the US are concentrated during the months between August and November, which is the same time of year that many viruses circulate. This includes enteroviruses, which are thought to be the culprit behind Acute Flaccid Myelitis.

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Confirmed AFM Cases by Month 2014 - 2022 CDC Enteroviruses are a genus of positive-sense single-stranded RNA viruses characterized by their transmission route through the intestines. They usually occur in the gastrointestinal tract, sometimes spreading to the central nervous system. Poliovirus is one of the many enteroviruses, and there are other non-polioviruses within the genus that are hypothesized to cause AFM. More than 90% of patients who developed AFM had mild respiratory symptoms or a fever consistent with viral infection prior. The most common initial symptoms of AFM are sudden onset of arm or leg weakness, loss of muscle tone, and loss of reflexes. Some will have drooping of eyelids or facial muscles, difficulty swallowing and slurred speech, and pains in arms, legs, neck, or back. In some patients with diagnosed Acute Flaccid Myelitis, coxsackievirus A16, EV-A71, and EV-D68 have been detected in the spinal fluid. These are all types of enteroviruses, and it is likely that there are multiple capable of causing AFM. Additionally, the stool samples of patients with AFM tested negative for poliovirus, so this is confirmed not to be polio. Sometimes, no pathogen is detected in the spinal fluid of patients with AFM. Some possible explanations behind this may be that the body has already cleared the pathogen or that the pathogen is hiding in tissues, making detection difficult. It is also possible that a now-cleared pathogen 501

triggered an immune response which then caused damage to the spinal cord.

Life Cycle and Genome of EV-D68. The mature EV-D68 virion attaches to the plasma membrane of the host cell and then undergoes receptor-mediated endocytosis. The capsid then undergoes uncoating allowing viral (+)-ssRNA to enter the cytoplasm. Next, the viral genome is translated into a polypeptide that undergoes further proteolytic processing to generate structural and nonstructural proteins. In addition, (−)-ssRNA is generated by RNA replication, which occurs on replication organelles. These RNAs become the template for new copies of the (+)-RNA genome. Virions assemble from structural proteins and VPg-linked RNA. These immature viral particles are largely taken up by autophagosomes, within which the acidic environment stimulates maturation of the capsid. Mature virions are released either by exocytosis of these autophagic vesicles or by cell lysis and release of nonenveloped viral particles. ELRICK, PEKOSZ AND DUGGAL, 2021 Recently, enterovirus D68 (EV-D68) has been targeted as the likely primary cause for AFM. This suspicion was just recently confirmed by researchers who published a case report in the New England Journal of Medicine in May 2022. They provided evidence that EV-D68 RNA and protein were found in the spinal cord of preserved tissue from the autopsy of a child who died of AFM. First identified in California in 1962, EV-D68 is primarily transmitted as a respiratory infection and often causes only mild respiratory illness. 502

It is suspected to be spread when an infected person coughs, sneezes, or touches a common surface in the close proximity of another person. There are no symptoms specific to EV-D68, and most people infected with it will never know unless it progresses to AFM.

Surface View of EV-D68 Viral Particle LIU ET AL.

Hypothesized Mechanisms of Neuroinvasion of EV-D68 ELRICK PEKOSZ AND DUGGAL, 2021 There is currently no specific vaccine or treatment of EV-D68 or AFM, but the best strategy likely consists of a multifaceted approach focused both on anti-viral and anti-inflammatory 503

strategies. Dr. Vogt at the University of North Carolina School of Medicine who published the evidence confirming that EV-D68 causes AFM suggests that there needs to be a balance between limiting the immune-mediated damage without restricting the immune system so much that the virus is able to thrive. Despite the lack of controlled clinical trials, immunoglobulin therapy (IGIV) has been used as treatment for EV-D68 infection and mitigation of the progression of AFM. It has been observed to produce high levels of neutralizing antibodies against EV-D68, as well as anti-inflammatory effects which may aid in the management of AFM. Monoclonal antibodies are a potential future treatment, and there is a push for rapid development of a vaccine. In the next piece in this series, we will discuss the production of vaccines in Asia for EV-A71, a similar enterovirus also capable of causing Acute Flaccid Myelitis. This article is featured on Forbes.org, and can be read online here: There May Be A New Polio Epidemic On Its Way- If So, What We Can Do

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Attack and Counterattack: How SARS-CoV-2 Blocks Our Natural Immune Defenses Forbes | June 10, 2022 | Article

This article is an extension of our series on immune suppression by SARS-CoV-2. The series has since been published as a book, Natural Immunity and Covid-19: What it is and How it Can Save Your Life. It is also available to read on my website. Here, we discuss new data regarding the ORF7a viral protein. During the initial stages of cellular infection, SARS-CoV-2 releases a number of accessory proteins to help suppress and evade our immune system. ORF7a is one such protein. Suppression of the innate immune system is necessary for the virus to establish infection. The several-day suppression of the immune system by the virus also contributes to the asymptomatic incubation period. During this period the virus can be transmitted from person-to-person before anyone is even aware they have been infected. ORF7a is a key player in preventing the phosphorylation of a host protein called signal transducer and activator of transcription 2 (STAT2), which is critical for the induction of antiviral interferon-stimulated genes. Novel research highlights additional means by which ORF7a contributes to the infectivity, pathogenesis, and transmission of SARS-CoV-2. Published in Nature Communications, the work by Timilsina et al. details how ORF7a counteracts the protective effects of serine incorporator 5 (SERINC5), a host protein that blocks viral entry into cells. On the Offensive: SERINC5 The antiviral functions of SERINC5 against retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), are well-documented. Yet little is known about its role in coronavirus infections. Timilsina and his colleagues set out to fill this gap in our knowledge. They first examined the expression levels of each member of the SERINC gene family —one through five— in lung tissue and Calu3 lung cells. All except SERINC4 were abundantly expressed. 505

Infection with SARS-CoV-2 did not influence the expression of SERINC1,2,3, and 5 in the lung tissue or the Calu-3 lung cells. Next, the researchers examined if any of the SERINC genes expressed in the lung tissue exhibit protective effects similar to those seen with SERINC5 in HIV-1. To do so, they produced pseudoviruses that replaced the HIV-1 exterior protein with the SARS-CoV-2 Spike protein —which the virus uses to bind to host receptors and enter into cells. All SERINC proteins were absorbed into the SARS-CoV-2 Spike pseudovirions. When exposed to these SERINC-containing pseudovirions, lung cells and kidney cells were significantly less likely to become infected. SERINC5 proved to be especially effective at reducing infectivity in both cell types (Figure 1). SERINC3 modestly reduced viral infectivity. SERINC1 and SERINC2 had no effect.

FIGURE 1. Relative infectivity of lung cells (Calu-3) and kidney cells (293T-hACE2) when exposed to ... [+] FROM: TIMILSINA ET AL. 2022

But retroviruses and coronaviruses are quite different from one another. For one, they assemble in different parts of the host cell; retroviruses in the plasma membrane and coronaviruses in the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC). The pseudoviruses the researchers generated may not accurately represent what happens with coronaviruses. To remedy this, they performed the same experiments on both SARS-CoV-2 virus-like particles (VLPs) —non-infectious replicas of the virus— and infectious SARS-CoV-2. As before, SERINC5 successfully incorporated itself into the Spike protein and successfully reduced 506

viral infectivity (Figure 2). SERINC3 was incorporated into the infectious SARS-CoV-2, but had a much smaller impact on viral infectivity.

FIGURE 2. Relative infectivity of lung cells (Calu-3) and kidney cells (293T-hACE2) when exposed to ... [+] FROM: TIMILSINA ET AL. 2022

The researchers also tested for SERINC5 incorporation into the Spike protein of different SARS-CoV-2 variants —Alpha (B.1.1.7), Beta (B.1.351), Gamma (P1), and Delta (B.1.617)— to make sure any mutations to the Spike protein did not negate the protective effects to SERINC5. Across all variants, SERINC5 continued to restrict viral infectivity (Figure 3).

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FIGURE 3. Relative infectivity of kidney cells (293T-hACE2) when exposed to different variants of ... [+]FROM: TIMILSINA ET AL. 2022

So SERINC5 gets incorporated into the Spike protein of SARSCoV-2 and, from there, manages to reduce infection. But, how exactly does it accomplish this? Timilsina et al. initially suspected that SERINC5 interferes with receptor binding, the first step of the process through which SARSCoV-2 gains entry into our cells. This happens when SARS-CoV2 uses its Spike protein to bind to angiotensin converting enzyme 2 (ACE2) receptors on the outside of our cells. Blocking this interaction would block the possibility of infection. To the surprise of the researchers, SERINC5 had no impact on the interaction between the Spike protein and our cells’ ACE2 receptors. Nor did SERINC5 interfere with the next stage of cellular infection — cleavage of the Spike protein into two parts. Once the SARS-CoV-2 Spike protein has bound to ACE2 and has been cleaved, the remaining section of the Spike protein inserts itself into the host cell membrane and pulls itself in, fusing the two together. Timilsina et al. discovered that, in the presence of SERINC5, the Spike-mediated fusion to the host cell membrane was noticeably reduced. This suggests SERINC5 blocks viral entry

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by interfering with the fusion stage of infection. The precise mechanism by which it does this remains to be determined. Viral Counterattack: ORF7a HIV-1 has developed a way of parrying the blow dealt by SERINC5; it encodes a protein called Nef that prevents the antiviral protein from being incorporated into the budding virions. What about SARS-CoV-2, does it wield any counterattacks of its own? The group of researchers turned to SARS-CoV-2 accessory proteins, known to block host antiviral genes and suppress the immune response. They honed in on ORF7a. During infection with SARS-CoV-2, ORF7a moves to the endoplasmic reticulum and Golgi apparatus of the host cell. This is the area where new viral particles are assembled and, by extension, where SERINC5 may incorporate itself into the nascent Spike protein. Timilsina et al. tested their hypothesis by exposing lung and kidney cells to a knock-out strain of SARS-CoV-2 that does not contain ORF7a. They compared the results to infection with unmutated wild-type SARS-CoV-2. The amount of SERINC5 packaged into the budding virions was significantly increased in the ORF7a knock-out strain. Higher levels of SERINC5 in the knockout strain were reflected by poorer viral infectivity overall. Reintroducing ORF7a to the knock-out strain salvaged infectivity. These findings confirm that, in the context of SARS-CoV-2 infection, ORF7a works to prevent the incorporation of SERINC5 into nascent viral particles. Timilsina and his colleagues propose two methods through which ORF7a inhibits SERINC5. One method is by preventing SERINC5 from being packaged into the nascent virion particles in the first place. The second method happens within the viral particles. The researchers suggest ORF7a forms a complex with the Spike protein and with SERINC5 that ultimately blocks SERINC5 from restricting viral entry. They were able to confirm that the SARSCoV-2 Spike protein, ORF7a, and SERINC5 all come together and interact at the endoplasmic-reticulum–Golgi intermediate compartment (ERGIC) (Figure 4). The nuances of how this complex undermines SERINC5 remain unknown. By analogy to HIV-1, the authors hypothesize that SERINC5 may alter the structure of the Spike protein and that ORF7a binds to the Spike

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protein to prevent any such changes. Future research should aim to resolve this unknown through in-depth structural analysis.

FIGURE 4. Co-location of SERINC5, ORF7a, and SARS-CoV-2 Spike protein at the ERGIC.

FROM: TIMILSINA ET AL. 2022 Mutations to accessory proteins are not rare and a number of naturally occurring deletions have been detected in ORF7a; do any of them undermine its ability to mount a counterattack against SERINC5? Timilsina et al. tested four naturally occurring deletions of ORF7a — Δ9nt, Δ18nt, Δ57nt, and Δ96nt— isolated from clinical samples of infected patients. The ability to block SERINC5 was preserved across all four of the SARS-CoV-2 variants with naturally occurring ORF7a mutations. There was little to no difference in the extent of SERINC5 restriction between the ORF7a mutations and the wild-type ORF7a. Implications Of more general importance is that inhibition of ORF7a will weaken SARS-CoV-2 replication, allowing our natural cellular defenses to be more effective in warding off the virus. This work by Timilsina et al. adds another reason to consider ORF7a an important antiviral target for future drug development. This article is featured on Forbes.org, and can be read online here: Attack and Counterattack: How SARS-CoV-2 Blocks Our Natural Immune Defenses

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Is It Safe To Fly? The National Academy Of Sciences, Engineering, And Medicine Weighs In Forbes | June 14, 2022 | Article

For many years, many of us have flown for business, pleasure, or to see relatives and friends. For the past two and half years, Covid19 introduced a significant risk to travel. Many wonder how to mitigate this risk. This is the first in a series discussing the airline industry in the Covid era, the risks involved, and how to mitigate them. To inform this discussion, we will use two recent reports from the National Academy of Sciences, Engineering, and Medicine that detail aviation in the Covid era. Over 4.5 billion passengers flew on commercial airlines in 2019. The Covid-19 pandemic grounded thousands of planes and delayed travel plans for all nonessential travelers. Only 1.8 billion flew in 2020 and 2.2 billion in 2021. Halfway through 2022, current estimates anticipate 3.4 billion by the end of the year, which is more than 2015 and all preceding years. In short, the airline industry is back, but that does not mean that Covid-19 is no longer a threat on airplanes and in airports. During the height of the Omicron variant wave, Dr. Dave Powell, physician and medical adviser to the International Air Transport Association, stated that “aircraft passengers were two or even three times more likely to catch Covid-19 during a flight.” Close contact with dozens or even hundreds of people for several hours in a closed metal vessel is ideal for transmitting a respiratory virus. More recently, a swath of airline policy relaxations put those flying in even more danger of Covid-19. In April, US District Judge Kathryn Kimball Mielle of Florida ruled against the federal mask mandate on public transportation. Within hours, all major US airlines, including United, American, Delta, Southwest, and others, stated masks were now optional on their aircraft. Commercial aircraft heavily ventilate the air in the cabin. One Covid-19 patient on a plane does not guarantee that all on board 511

will be infected. However, those within a few rows of the infected person are at much greater risk. Removing mask mandates will steepen those odds further. A policy failure that has gone from bad to worse recently is negative Covid test requirements. At no point in the Covid-19 pandemic did the federal government require a negative test for domestic travel. A passenger could be at peak infectivity and board a plane without restriction. For international travel, the United States did require a negative antigen or PCR test to enter the country to prevent the spread of international variants. This mandate has now come to an end. As of June 12th, “international travelers will no longer need to show proof of a negative Covid test before boarding flights to the United States, ending one f the nation’s last pandemic-related travel requirements.” The decision will be reassessed in 90 days, according to the CDC. All non-citizens, nationals, or permanent residents must still show proof of vaccination to enter the country. These policy retractions coincide with the busy summer season of air travel. The US Travel Association expects the most significant travel season since 2019, as 6 in 10 Americans will take at least one summer trip, and roughly $100 billion will be spent on travel nationwide. While infection counts may be down compared to the Omicron wave due to vast reductions in national testing, rest assured, Covid19 will thrive in the coming months. Descendants of the Omicron strain of viruses, namely BA.4 and BA.5, are more infectious than their parental strains and may reinfect those infected with earlier versions of Omicron. Hundreds of millions traveling to highly packed travel destinations on board aircraft with no Covid restrictions will yield millions of infections. Not to mention, other viruses and diseases will circulate efficiently in crowded social settings as well, such as influenza or even the evermore concerning monkeypox. There are some precautions to take if traveling. Of course, the greatest protection from Covid is to avoid traveling altogether. However, this is becoming more difficult in what many claim is a post-pandemic world. First, be fully vaccinated, meaning two initial doses, plus one or even two booster doses, if available. Second, keep your mask 512

(preferably an N95 mask) on as much as possible in the airport and aboard an aircraft. While masks primarily protect those around you from your respiration, they also protect the wearer to a lesser extent. Third, if seat selection is available, try to have an empty seat between you and another passenger. Also, forego the views of the outside world for an aisle seat as those receive ventilated air first. Fourth and finally, be thoughtful about social interactions. Avoid sharing drinks with strangers, large gatherings in enclosed spaces, etc. Covid-19 remains prevalent, but the airline industry is pushing the narrative that this is a post-pandemic world. It is up to those that do not want this disease to take the measures they can to avoid infection. This article is featured on Forbes.org, and can be read online here: Is It Safe To Fly? The National Academy Of Sciences, Engineering, And Medicine Weighs In

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Don’t You Wish You Didn’t Need To Breathe Someone Else’s Exhaled Air While Flying? Forbes | June 16, 2022 | Article

The Covid era restrictions on passenger aircraft, including mask mandates and negative tests, are officially ended. From now on, any American traveling domestically or returning to the United States from a foreign nation does not have to wear a mask, does not have to provide a negative Covid test, and does need to present proof of vaccination. We have returned to a pre-Covid aviation industry, which offers several dangers as Covid continues to loom large. As more people resume standard travel practices, Covid transmission on airplanes will increase. As the government and airlines roll back passenger Covid protections, it is now up to individuals to take every measure to protect themselves from infection. Continuing our series on air travel during Covid, here we will discuss the intricacies of Covid transmission on aircraft and how individuals can protect themselves from Covid on upcoming flights. Recently, the National Academy of Sciences, Engineering, and Medicine released a report detailing aviation in the Covid-19 era. This report presents science-based risks and mitigation for passengers flying commercial aircraft, which we will use to guide our discussion. Being in the same plane as another passenger infected with Covid-19 does not guarantee transmission to everyone on board. The likelihood of infection is low unless you are sitting within two or three rows of an infected person. Air in airplanes does have a higher CO2 count than outside or indoors, which means your chances of contracting Covid-19 in a plane are higher than at home or the park, but still not guaranteed. CO2 in enclosed spaces such as an airplane is generally the result of the gas, as well as infectious particles, in other people’s exhalations. The air you breathe is ventilated, reoxygenated, filtered, and sent back into the cabin to be breathed again. However, the air is circulated along the circumference of the cylindrical plane, not 514

lengthwise. This means that the air you breathe is mainly shared between those in your row and those directly near you. The process is far from perfect. We recently monitored the CO2 concentration several hours into a flight on a domestic airline and found it to be four times that of the open air. A study of a Vietnam Airlines flight from London to Vietnam examined the importance of proximity. Of the 217 passengers and crew aboard, one passenger displayed symptoms of Covid-19 midflight. Later on, 14 passengers and one crew member tested positive. Notably, 11 of the 14 passengers were sitting within two meters of the symptomatic carrier. These results are mirrored in several studies in the National Academy of Sciences, Engineering, and Medicine report. One simulation found that the general odds of Covid infection for a passenger on a full flight is one in 3,900. These odds increase drastically, as high as 80% if sitting in direct proximity to an infected person without a mask. Notably, the same simulation found that rigorously enforced mask-wearing drops transmission odds significantly; however, no US-based airlines now enforce maskwearing. The SARS-CoV-2 virus transmits in three ways. First, via droplets such as sneezing and coughing. Masks would essentially erase the risks of droplet transmission. Still, US District Judge Kathryn Kimball Mielle of Florida saw fit to overrule the federal mask mandate, meaning mask usage on domestic airlines is optional. The second transmission method is aerosols, which are like droplets but much smaller produced by breathing. Aerosol transmission is how the typical asymptomatic carrier passes on Covid-19. Again, aerosol risks would be mitigated by masks. The third transmission method is via fomites, or surfaces that infectious particles may latch. An infected person may have trace virus on their hands from touching their eyes, nose, or mouth, and by touching an object, they leave trace virus for another potential carrier to pick up. This could manifest in an airplane as handing something to the flight attendant, touching the bathroom handle, etc. This transmission method is easily mitigated by standard hand sanitizer and general health practices like avoiding touching your face.

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Depending on the level of community transmission, the chance that one person infected with Covid-19 boards a plane is relatively high. For instance, the report notes that in Texas, which at the time of writing had a Covid rate of 73 cases per 100,000 people, there is a 46% chance that one passenger on a 100-passenger plane has Covid. This number skyrockets to 96% when considering many cases are undiagnosed. As the federal government and airlines have rolled back nearly all Covid-19 restrictions, it is up to the individual to protect themselves against these chances. The report recommends nonpharmaceutical interventions like N95 masks, keeping as socially distant from others as possible, moving about the plane as little as possible, and using hand sanitizer regularly for personal use and on surfaces. Additionally, strategic flight bookings could reduce the chances of transmission as well. For instance, flights departing on Tuesdays, Wednesdays, and Thursdays are less crowded than those leaving on Friday through Monday. Try to book midweek flights and select a seat beside an empty middle seat, if possible. Covid remains prevalent, and the risk of infection mid-flight is significant, but maintaining safe Covid practices could prevent a case of Covid that would have otherwise derailed your trip and potentially caused long-term issues for months and years. This article is featured on Forbes.org, and can be read online here: Don’t You Wish You Didn’t Need To Breathe Someone Else’s Exhaled Air While Flying?

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Flying Under The Radar: How SARS-CoV-2 ORF7a Contributes To Immune Evasion And Inflammation Forbes | June 16, 2022 | Article

This article is an extension of our series on immune suppression by SARS-CoV-2. The series has since been published as a book, Natural Immunity and Covid-19: What it is and How it Can Save Your Life. It is also available to read on my website. Here, we discuss new data regarding the ORF7a viral protein. Click for part 1. The success of SARS-CoV-2 as a virus hinges in large part on its ability to suppress and evade our immune system. Usually, our immune system encounters an invading microbe and immediately springs into action. But SARS-CoV-2 is a master of flying underneath the radar, staying hidden from our innate immune response. It also evolves rapidly to evade our adaptive immune response. Interferons —a group of signaling proteins produced by cells in response to microbial threat— are key to both the innate and adaptive immune response. In a previous article, we outlined how ORF7a inhibits the induction of interferon-stimulated genes by preventing the phosphorylation of STAT2. This obstructs our interferon response, making it more difficult for our immune system to mount a successful counteroffensive. More recently, we described the discovery that ORF7a also blocks the antiviral function of a host protein called SERINC5, which would otherwise help prevent viral entry into cells. A study by a group of researchers based at The Fifth Affiliated Hospital of Sun Yat-sen University highlights yet another immune evasion capability of ORF7a: the suppression of CD14+ monocytes. In addition, Zhou et al. suggest that ORF7a contributes to one of the most troubling aspects of Covid-19, an overly aggressive release of pro-inflammatory molecules. These molecules are called

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cytokines, and their overexpression can lead to the life-threatening systemic inflammatory syndrome known as ‘cytokine storm’. Covid-19 Severity: Monocytes and Inflammation Most people who contract Covid-19 will suffer only mild, if any, symptoms. But there is a subset of patients who go on to develop severe disease — with upwards of half a billion confirmed cases, this subset quickly becomes very large. Severe cases are generally characterized by an overactive inflammatory response associated with elevated cytokine levels, a drop in the number of white blood cells, and infiltration of macrophages and monocytes into different tissues. Monocytes, in particular, appear to hold an important role in the hyperinflammation seen in severe Covid-19 cases. Monocytes are large white blood cells that circulate the body through the bloodstream, keeping an eye out for any microbial threats. They recognize microbes via pattern recognition receptors (PRRs) that line their surface. These receptors pick up on age-old molecular patterns typical of pathogens — be they viral, bacterial, fungal, or parasitic. Once they have spotted a pathogen, they migrate to the affected area and help stimulate the inflammatory response by producing cytokines. Monocytes can also differentiate into two other types of immune cells: phagocytes, which engulf and destroy microbes, and dendritic cells, which present T cells with antigens to help stimulate a more specific immune response. As such, suppression of monocytes can lead to all kinds of knock-on immune dysregulation. Although aberrant inflammation and upregulation of monocytes had been associated with worse Covid-19 outcomes, the viral proteins responsible for this excessive inflammation remained largely unknown. ORF7a: New Structural Insights Certain proteins contain immunoglobulin-like (Ig-like) molecular structures. These play a critical role in modulating interactions in the immune system. Usually these are host proteins, such antibodies. Certain viruses have developed proteins with similar structures to help them hijack the host immune system. Zhou et al. scanned SARS-CoV-2 proteins for Ig-like structures and noticed that ORF7a contains an Ig-like ectodomain — the section of a protein that extends out from the surface and initiates contact with other proteins and cells. The ORF7a ectodomain is 518

made up of seven beta strands (β-strands) that form two connected beta sheets (β-sheets) (Figure 1). The resultant structure looks like a hand, palm facing inwards. It is the “fingers” of this hand that end up reaching out and binding to other proteins.

FIGURE 1. (LEFT) Ribbon representation of a SARS-CoV-2 ORF7a molecule. Disulfide bonds are ...[+] FROM: (LEFT) ZHOU ET AL. 2021 (RIGHT) GETTY IMAGES

ORF7a contains an Ig-like ectodomain, but does it actually interact with host immune cells? To find out, the researchers exposed human peripheral blood mononuclear cells (PBMCs) — lymphocytes and monocytes— from healthy donors to SARS-CoV2 ORF7a. They discovered that ORF7a binds to CD14+ monocytes with a high degree of efficiency. It also binds to lymphocytes, but much more weakly. SARS-CoV-1, the virus responsible for the 2003 SARS outbreak, also has an ORF7a protein. The sequence similarity between the two is 87% and they share a very similar structure as well. Still, the ORF7a protein of SARS-CoV-1 binds only weakly to monocytes. The researchers used this difference in binding affinity to identify the precise structural features of SARS-CoV-2 ORF7a that allow it to bind so efficiently. They discovered that the two ORF7a proteins differ from one another in the distribution of the binding site residues. Compared to SARS-CoV-1 ORF7a, the binding residues of SARS-CoV-2 ORF7a were all located on the 519

larger beta sheet (strands A, G, F, and C). None were located on the smaller, three-strand beta sheet (strands D, E, and B). This implies the key binding residues are located on the larger beta sheet (Figure 2).

FIGURE 2. Structural alignment between SARS-CoV-2 ORF7a (this study) and SARS-CoV ORF7a (PDB: 1XAK)[+] FROM: ZHOU ET AL. 2021

ORF7a Suppresses Monocytes Having established that ORF7a can bind to monocytes and lymphocytes, the researchers looked into the effects of this interaction. They co-incubated SARS-CoV-2 ORF7a with human monocytes and lymphocytes for a 24 hour period. Zhou et al. then measured the expression levels of human leukocyte antigen (HLA) surface molecules on the immune cells. The HLA system is used to present antigens to other cells, and is a critical component of the adaptive immune response. It can be broken down into two classes. First, those proteins that help to move antigen snippets from the inside of a cell to the cell surface. This signals to the immune system that a cell has been infected and needs to be destroyed, thereby slowing the spread of the pathogen. This 520

class is made up of HLA-A, HLA-B, and HLA-C. The second class is composed of HLAs that present antigens found outside of the cell to T-helper cells. The T-helper cells then go on to stimulate the production of B-cells, which secrete antibodies specific to the microbial threat at hand. This class includes HLA-DR, HLA-DP, and HLA-DQ. The researchers noticed a significant decrease in the expression of the second kind of HLAs on the surface of CD14+ monocytes — roughly 30% lower than normal. There was no difference in the expression of HLA-A/B/C. The expression of HLAs on the surface of lymphocytes remained unchanged. So ORF7a decreases the amount of HLA receptors on the surface of CD14+ monocytes, hindering their ability to signal for additional support from other immune cells. Zhou et al. conclude that this is likely an immune escape tactic on behalf of SARS-CoV2 — essentially, buying it more time to spread unnoticed. The precise mechanism through which the ORF7a ectodomain modulates the antigen-presenting ability of CD14+ monocytes remains to be determined. ORF7a Triggers Inflammation As mentioned, hyperinflammation is a hallmark of Covid-19. In a previous article we discussed how SARS-CoV-2 can infect monocytes, leading to a form of cell death called pyroptosis and the sudden release of a mass of pro-inflammatory cytokines. Findings by Zhou et al. implicate ORF7a as an additional source of monocytemediated inflammation. To study the effects of ORF7a on inflammation, the team of scientists tested blood samples for markers of inflammation. They discovered that co-incubation with ORF7a triggered a sharp increase in the production of proinflammatory cytokines. More specifically, they noticed that co-incubation led to the upregulation of those cytokines most closely associated with cytokine storms — IL-6, IL-1β, IL-8, and TNF-α. The spike in cytokine production implies that ORF7a immune modulation of monocytes may be a contributing factor to the hyperinflammation seen in severe Covid19 cases. Interestingly, the cytokine profiles differed depending on the donor. Some blood samples had a more intense cytokine response than others. This indicates there may be genetic differences 521

governing ORF7a interaction and, by extension, disease outcome. Future research should aim to pinpoint the factors responsible for the variation in cytokine response. Why Inflammation? ORF8, another SARS-CoV-2 accessory protein, is also known to trigger inflammation. In this case, the process is very targeted, with ORF8 mimicking one of our immune system’s most powerful inflammatory response triggers, interleukin-17. The question arises, is the inflammation triggered by SARSCoV-2 infection simply an unfortunate side effect, or might it be to the benefit of viral replication? One observation that supports the hypothesis that inflammation may contribute to the success of the virus is that the receptor for viral entry, angiotensin converting enzyme 2 (ACE2), is induced as part of the inflammatory response in endothelial and other cells. A release of inflammatory substances may therefore be advantageous, and not entirely adventitious. The ability of ORF8 and ORF7a to, in and of themselves, induce an inflammatory response favors the advantageous hypothesis. Take-home Message This work by Zhou et al. opens the door to a new target for drug development, ORF7a. It also adds to the growing list of reasons to shift our focus onto the entirety of the SARS-CoV-2 genome, not just the Spike protein. There are many accessory proteins that contribute to the virus’ ability to evade and suppress our immune system; the sooner we learn about their role in infection, the sooner we can begin creating effective anti-Covid drugs. This article is featured on Forbes.org, and can be read online here: Flying Under The Radar: How SARS-CoV-2 ORF7a Contributes To Immune Evasion And Inflammation

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Medical Science Must Go Hand In Hand With Social Science For Effective Control Of Covid19 And Other Pandemics Forbes | June 17, 2022 | Article

We find ourselves in a particularly challenging stage of the pandemic. While proving to be effective at reducing hospitalizations, and severe disease, our best vaccines have failed to protect against infection. While Paxlovid has been an effective line of defense in treating breakthrough Covid-19 infections of those most critically at risk, we are yet to explore the potential of prophylactic drugs. Prevention is always better than a cure. Especially when more than one-third of Covid-19 patients will develop long-term symptoms and some will experience symptoms severe enough to be disabling for many months, if not years. HIV is another infectious disease that has eluded an effective vaccine, but after many years of research and investment in drug development, it is no longer a death sentence. I have long advocated that drug developers should take a lesson from HIV research and focus on developing combinations of small-molecule antiviral drugs to prevent and treat Covid-19. However, it is not enough to just to focus on the development of prophylactic drugs. The drugs will be rendered ineffective if there is hesitancy around taking the drugs or low levels of adherence to medication regimens. With each global health crisis it becomes clear that we need both medical solutions and the public health-orientated behavioral and social change programs to implement them. Policy makers and public health officials need to work with social scientists to create education and outreach initiatives and restore trust in institutions. We also need to ensure equity and access to preventative treatments, particularly in healthcare deserts and low socioeconomic areas. No population is safe until we all are. A recent study, on the real-world effectiveness of pre-exposure prophylaxis in men at high risk of HIV infection in France 523

demonstrates how even a highly effective drug such as PrEP can be weakened by a lack of medication regime adherence. Pre-exposure prophylaxis (PrEP) for HIV has shown to be highly effective in clinical trials, but there is less research on the effectiveness when prescribed in real life to diverse populations. The authors of this study used a matched, nested case-control study among adult men at high risk of HIV infection between Jan 1, 2016, and June 30, 2020, with data from the French national health data system. Men who were newly diagnosed with HIV infection before Dec 31, 2020, were individually matched with up to five controls for age, socioeconomic status, place of residence, calendar year, and follow-up duration. Among a total of 46 706 individuals, 256 patients with HIV infection were identified and matched with 1213 controls. PrEP users accounted for 29% of cases and 49% of controls. Real-world PrEP effectiveness was found to be 60% overall, expanding to 93% for a high amount of PrEP consumption, and 86% if excluding periods after PrEP discontinuation. PrEP effectiveness was significantly reduced in people younger than 30 years and in those with low socioeconomic circumstances. Both groups showed low amounts of PrEP consumption and high rates of PrEP discontinuation. While this study demonstrates that PrEP effectiveness appears to be lower in real-world conditions than is reported in clinical trials. Lower effectiveness appears to be linked to compliance. Strengthening efforts to improve the monitoring of PrEP compliance is essential to ensure PrEP higher effectiveness in a realworld setting. This article is featured on Forbes.org, and can be read online here: Medical Science Must Go Hand In Hand With Social Science For Effective Control Of Covid-19 And Other Pandemics

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There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part II Forbes | June 20, 2022 | Article

This is Part II in a series on the enteroviruses that appear to cause a polio-like neurological disease, Acute Flaccid Myelitis. The US still has no vaccine against the primary causative agent while China has three, with more in Asia on the way. As case reports rise, Acute Flaccid Myelitis (AFM) is a growing concern in the United States, heightened by the fact that we have yet to develop any vaccines or specifically targeted antiviral medications. Caused by an enterovirus in the poliovirus family, this neurological condition often results in weakening of the muscles and paralysis. However, the lack of success in vaccine production exhibited in the US is not mirrored in other parts of the world. In Asia, there are currently four vaccines in late stage clinical trials. These vaccines protect against EV-A71, an enterovirus very similar to EV-D68, the main culprit of Acute Flaccid Myelitis in the US. Three of these vaccines are currently licensed for use in children in China, and a fourth targeting a different subgenotype performed very well in a phase 3 clinical trial in Taiwan and Vietnam. Successful prevention of this enterovirus across the world is very hopeful for our prevention efforts here in the United States. Polio, caused by an enterovirus in the same family, has shown us that prevention and eradication is possible. Hopefully we will be able to replicate the successful development of vaccines in Asia in the United States very soon. EV-A71 infections often manifest as hand, foot, and mouth disease (HFMD), primarily in children. Symptoms generally consist of fever, mouth sores, and a rash found on hands, feet, and/or the mouth. Infections are often mild and many clear up within a week. However, some infections of EV-A71 have more serious neurological complications such as meningitis or Acute Flaccid Myelitis, leading to potential paralysis or loss of life.

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Child with Hand, Foot, and Mouth Disease with a Visible Rash GETTY IMAGES Over the past 25 years, the Asia-Pacific region has experienced cyclical outbreaks of EV-A71, some leading to hundreds or even thousands of deaths, mostly in infants and children. Therefore, access to effective vaccines is essential. The three vaccines currently authorized for use in China are inactivated whole virus C4 subgenotype-based vaccines and are manufactured by Beijing Vigoo Biological, Chinese Academy of Medical Sciences, and Sinovac Biotech in China, all three of which are based in Beijing. In phase 3 trials, a two-dose series showed high efficacy rates (90.0% - 97.4%) in children aged 6 - 35 months. While the vaccines have shown cross-reaction against genotypes other than C4, there is no hard efficacy data with other strains. The fourth vaccine, EV71vac, is based on the B4 genotype and included participants as young as two months old in the phase 2 clinical trial. This is valuable because the 2 - 5 month age group carries the highest risk of case severity and fatality, and the three Chinese vaccines are not authorized for use in infants of this age. This inactivated aluminum phosphate-adjuvanted B4 subgenotype based vaccine has shown promising results thus far, with no adverse events in clinical trial phases 1 and 2. Published April 2022 by Dr. Nguyen and collaborators in The Lancet, the results of a double-blind, placebo-controlled phase 3 clinical trial demonstrate a 96.8% efficacy rate. Of the 1,426 total participants that received two doses of EV71vac, there were zero reports of hand, foot, and 526

mouth disease (HFMD). In the placebo group of 1,483, there were 22 cases of both laboratory and clinically confirmed HFMD. Furthermore, seroprotection rates were close to 100% at all time points monitored after the second vaccination, up to 10 months after the second dose.

Clinical Efficacy of the Total Cohort Vaccinated with EV71vac Against EV-A71-associated Diseases NGUYEN ET AL., 2022

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Table of Clinical Efficacy of EV71vac Against EV-A71 Associated Diseases NGUYEN ET AL., 2022 Although EV71vac was based on B4, a different subgenotype than the Chinese vaccines which were based on C4, the vaccine still showed both in-vitro and real-world cross-protective efficacy against B4, B5, and C4 subgenotypes. The safety data and impressive efficacy rates in infants carry great potential in preventing severe disease and mortality amongst the most vulnerable population. Pending approval in Vietnam and Taiwan of the recently submitted results of the phase 3 clinical trial, EV71vac will be ready for wide distribution, adding to the arsenal of protection against diseases associated with enterovirus A71 like Acute Flaccid Myelitis. The success of vaccine development in Asia lends hope to the fight against the growing concern of non-polio enteroviruses. However, with zero vaccines in the United States against EV-D68, the primary cause of Acute Flaccid Myelitis in the country, our work is clearly cut out for us. Coronaviruses have taught us a lesson of the potential for an often insignificant viral infection to quickly turn catastrophic. Coronaviruses cause colds around the world, just as many enteroviruses cause mainly mild symptoms, without threat of serious illness. However, as Covid-19 has shown us, there are new and dangerous strains within the coronavirus family. The same is true for enteroviruses. While most are common and relatively mild, there is the potential for some to be much deadlier, just as was polio. This article is featured on Forbes.org, and can be read online here: There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part II

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Biggest Risk Factor for Severe Covid-19 Other than Age? Autoantibodies Forbes | June 21, 2022 | Article

The vast majority of people suffer only moderate to mild symptoms during SARS-CoV-2 infection — sore throat, fever, fatigue, and so on. But a portion of those infected go on to develop severe disease. This includes hyperinflammation, immune dysregulation, and serious organ damage. Such cases often require hospitalization and even intubation. A group of international researchers has discovered a new metric to help predict Covid-19 disease outcome: autoantibodies against type 1 interferons (IFNs). Published in Proceedings of the National Academy of Sciences (PNAS), the work by Manry et al. will allow for a more efficient distribution of care, affording the most at-risk patients priority access to limited resources including beds in intensive care units (ICU) and ventilators. What are Interferons and Autoantibodies? Our immune system helps protect us from microbial threats. It can roughly be broken down into two main branches: innate and adaptive. The innate immune system is the first to respond and kicks in immediately following infection. It is generalized and reacts to any and all invading microbes. The adaptive immune system, in turn, takes around seven to eight days to develop specificity towards a pathogen and reaches its peak around 15 days after initial infection. Once this specificity has been established, it can spring into action right away the next time it encounters the pathogen. A key feature of innate immunity is the interferon response. When cells sense an invader, they produce the signaling protein interferon. The production of interferon releases a storm of different antimicrobial substances, genes, and proteins which attack foreign organisms and create an unfriendly environment for the organisms’ proliferation. Interferons come in three types. Type I interferons bind to surface receptors, called Interferon-alpha/beta receptors (IFNARs), 529

found on nearly all cell types. This stimulates the production of proteins that interfere with a virus’ ability to replicate their genome. Humans make five different type I interferons— IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. Some people develop autoantibodies against type I interferons. Antibodies are antimicrobial proteins produced by B cells as part of the adaptive immune response. Usually they defend us against foreign organisms. Sometimes our immune system may accidentally label our own healthy tissues, cells, and proteins as a threat, leading to the production of antibodies that attack these healthy tissues. Such antibodies are called autoantibodies. Autoantibodies against type I interferons may interfere with the functioning of our interferon response, leading to a weakened innate immune system. Type I IFN Autoantibodies and Covid-19 Mortality To investigate the relationship between Covid-19 severity and the presence of type I IFN autoantibodies, Manry et al. turned to blood samples of 1,261 unvaccinated patients who had died from Covid-19 and blood samples of 34,159 individuals gathered before the pandemic. Across age groups, 20% of the deceased Covid-19 patients had autoantibodies against IFN-α, IFN-ω, and/or IFN-β circulating in their blood. This is compared to 1% in the general population younger than 70 and 4% in the general population older than 70. The researchers used these samples to estimate the infection fatality rate (IFR) and relative risk of death (RRD) across age groups for those carrying type I interferon autoantibodies relative to those not carrying the autoantibodies. First, they estimated the relative risk of death for individuals who carried only low concentrations of type I IFN autoantibodies (100 pg/mL). They focused primarily on IFN-α2 and IFN-ω. As compared to noncarriers, the relative risk of death was noticeably higher. Especially in those younger than 70, presence of autoantibodies strongly correlated with an increased relative risk of death compared to their noncarrier counterparts. The same result held for other combinations of type I IFN autoantibodies as well (Figure 1).

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FIGURE 1. Relative risks of death (RRDs) for individuals with auto-Abs neutralizing different ... [+] FROM: “THE RISK OF COVID-19 DEATH IS MUCH GREATER AND AGE DEPENDENT WITH TYPE I IFN AUTOANTIBODIES” MANRY ET AL. 2022

Although still higher than in noncarriers of the same age, those with type I IFN autoantibodies over the age of 70 had a lower relative risk of death than carriers under 70. This may seem counterintuitive, but the authors suggest it can be explained epidemiologically by a larger contribution of other mortality risk factors that correlate with old age, including comorbidities such as hypertension. At the cellular and molecular levels, old age is accompanied by a general weakening of both the innate and adaptive branches of the immune system as well as a specific decline in type I IFN immunity in the blood and respiratory tract. This means that autoantibodies against type 1 IFNs play a smaller role in Covid-19 mortality with age.

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The results when selecting for higher concentrations of type I IFN autoantibodies (10 ng/mL) were similar, albeit with an even more extreme increase in the relative risk of death for carriers. Across concentrations, the relative risk of death was highest in those who carried autoantibodies neutralizing both IFN-α2 and IFNω (Figure 1). Manry et al. also estimated the infection fatality rate (IFR) —the proportion of fatal outcomes upon infection— across sexes and across age groups in unvaccinated individuals carrying autoantibodies against type I IFNs. They compared this to the general infection fatality rate amongst the unvaccinated population. Again, the infection fatality rate was significantly higher in carriers (Figure 2). Even in those who carried only low concentrations of autoantibodies, the infection fatality rate was worrying; up to 10% in those aged 60, and a staggering 40.5% in those aged 80 or older. Those who carried higher concentrations of autoantibodies were even more likely to pass away during infection. For reference, the global infection fatality rate for SARS-CoV-2 hovers somewhere around 1%. Unlike the relative risk of death, the infection fatality rate increases steadily with age.

FIGURE 2. SARS-CoV-2 IFRs by age. IFRs are provided for the general population for both sexes (gray) ... [+] FROM: MANRY ET AL. 2022

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Male carriers were particularly prone to fatal outcomes. Those who carried high concentrations of autoantibodies were 5 times as likely to pass away from SARS-CoV-2 infection than female carriers at the same concentration, regardless of autoantibody combination and of age group. This reflects Covid-19 outcomes more generally, where men are at an increased risk of developing severe disease when compared to women. Implications Although only between 1% and 4% of individuals carry autoantibodies that neutralize type I IFNs, they consistently make up around 20% of Covid-19 fatalities across age categories. Other than age, presence of type I interferon autoantibodies is the strongest predictor of severe Covid-19 — more so than sex, common comorbidities, and most genetic variants. Future research should focus on how and why people develop autoantibodies against type I IFNs, and whether or not this is preventable. This article is featured on Forbes.org, and can be read online here: Biggest Risk Factor for Severe Covid-19 Other than Age? Autoantibodies

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The Future Of Aviation In A Covid And PostCovid World Forbes | June 21, 2022 | Article

As the Omicron subvariants continue to spread, world governments and airlines are advancing towards a post-Covid aviation industry. Despite continued infections and concerns about Long Covid health implications, Covid restrictions for flights are all but over. The Covid-19 pandemic nearly destroyed the entire aviation industry. Likely, Covid is not the last dangerous pathogen to befall modern society. How can we adapt such that air travel is safe and adaptable if another pathogen comes to pass? Here we discuss the future of aviation in a post-Covid world. The National Academy of Sciences, Engineering, and Medicine’s recent report “Flying in the COVID-19 Era” once again provides a detailed assessment of airlines' current state and future in the pandemic context. The authors analyze likely upcoming challenges and how we may face them. Most models project air traffic volume to match pre-pandemic levels by early 2024 or even late 2023, roughly two years after the Omicron peak. Prices will steadily rise as demand outpaces supply, particularly for international travel. The removal of adverse test requirements on US-based airlines will lead to an acceleration of demand. A significant concern for some travelers is the fear of going on an international trip and being infected with Covid-19 while abroad. Restrictions easing will open this customer base to travel sooner, perhaps as early as the Summer 2022 season. As hundreds of millions return to the skies with lower mask usage, there are two factors that airlines must control to keep up with demand while Covid is still prevalent. First is the health and safety of airline crew members. A plane cannot fly without two pilots and the entire attendant staff. There is already a significant labor shortage for pilots as the rigorous job is often underpaid. Losing pilots or staff to Covid for a week or two could cause rippling effects in terms of 534

delays and cancellations. While most airlines have adopted the optional approach to masks and vaccinations, we highly recommend that all staff continue to use N95 masks, plastic gloves, and complete vaccinations if they have not already. Another factor to consider is the efficiency of airline parts manufacturers. The confluence of a Covid-19 and a declining economy, among other factors, yielded significant supply chain shortages worldwide. The average age of domestic airline fleets is roughly two to three decades. Airplanes will need repair and maintenance like any vehicle. Any sort of system-wide mechanical failure could be particularly devastating at this time. We also emphasize the continued importance of airplane CO2 ventilation. Advances in ventilation should be one of the highest priorities for the aviation industry. Lingering CO2 contains droplets and aerosols that propagate the spread of Covid-19 and other pathogens. Decreasing mask usage will likely increase baseline CO2 levels, allowing more droplets and aerosols to spread about the cabin. We recently collected CO2 data on a domestic flight from New York to Tampa on an Airbus A319. While CO2 levels were not as high as the enclosed space of a cab to or from the airport, levels ranged from 2.5 to four times that of the New York outdoors prior to departure. The average A319 is almost 20 years old. Perhaps upgrades to ventilation systems on older aircraft should be a policy priority in the coming months and years.

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CO2 levels for domestic air travel. ACCESS HEALTH INTERNATIONAL Optimal indoor air quality is below 1,000 ppm of CO2, according to a 2015 Harvard air quality study. At 1,000 ppm, brain cognitive function decreases by 15% on average over the course of a day. At 1,400 ppm, cognitive function decreases by 50%. Extended exposure to CO2 on aircraft or elsewhere can damage brain function in the long term, in addition to the dangers posed by Covid-19. CO2 levels in the air are a proxy for breathing the air of others. The higher the CO2 ppm, the more air you inhale that was previously inhaled by others without ventilation. Many airlines may use modernized high-efficiency particulate air (HEPA) filters, even in older aircraft, to remove exhaled airborne particles onboard flights. These filters remove air debris as small as 0.3 microns, however, SARS-CoV-2 particles are as small as 0.1 microns, meaning some virus particles may escape. Another option may be ultraviolet germicidal irradiation. Extended exposure to UV light inactivated viruses on surfaces. UV light is not visible to the human eye. Installing UV lights within airplanes may serve to reduce viral infection via surface contamination. The National Academy report indicates several other trends that you may notice become more regular in the coming years. These build passenger confidence, speed up processing times, reduce touch points, and improve the airport experience. The first is at check-in. Airlines are trying to move away from long check-in lines at the front end of the airport. This manifests in emphasis on early online check-in via website or app and self-bag check machines that produce a tag you put on your bag yourself. The aim is to reduce the number of people at the check-in desk to below 50% of travelers, mainly those who had issues checking in or had exceptional circumstances. Another trend is the advances in TSA security screening. TSA Precheck and Clear are two paid services that streamline the security process, but general screening technologies are advancing rapidly, particularly with baggage screening. Perhaps most notably in the Covid era are airport and airplane cleanliness progressions. Since the pandemic's start, a major avenue to regain customer confidence was assurances of disinfection and 536

hygiene. A passenger does not want to sit in a seat occupied by someone with Covid. Sanitizer is more regularly available throughout airports, and general cleaning practices are more stringent. Expect these practices to stick around well into the future. Until Covid-19 infections are at a much more manageable rate, as we have emphasized in all our airline articles, personal protection is now on the individual. Continue to wear N95 masks on aircraft, receive as many vaccinations as you qualify, try to book a seat with an empty seat next to it, and reduce unnecessary risks like touching your eyes or mouth mid-flight. We all want to return to safe travel as soon as possible, and these suggestions are the safest way to do so. This article is featured on Forbes.org, and can be read online here: The Future Of Aviation In A Covid And Post-Covid World

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What We Can Learn From Other Poxviruses About Monkeypox Forbes | June 21, 2022 | Article

Since the eradication of smallpox in 1980, there have been growing concerns that we are not prepared for the next poxvirus pandemic. It is important to remember that smallpox killed millions of people throughout human history. The variola virus that caused smallpox was not only lethal but also highly contagious. Inhaling droplets from an infected person was the primary route that monkeypox was transmitted, albeit the virus could also spread through direct contact with contaminated objects or surfaces. Symptoms usually began with flu-like symptoms, such as muscle aches, headaches, and a fever. As the infection spread, the digestive tract was commonly implicated, causing nausea, vomiting, and severe backaches. Like other viral infections, most of these symptoms lasted two to four days. Around the twentieth day of infection, visible lesions appeared on the skin. The rash first appeared on the mucous membranes of the throat, mouth, and tongue. When enlarged lesions ruptured, they released large amounts of the virus, which increasingly infected other skin cells. Depending on which strain of the smallpox virus one was infected with, the fatality rate was anywhere between 1% and 75%. Now, we are seeing a resurgence of poxvirus infections in humans. Given that we no longer vaccinate against smallpox, much of the world population is not protected against the outstanding threat of other orthopoxviruses, such rabbitpox, deerpox, and not to mention, monkeypox. While smallpox only infected humans, many of these poxviruses are zoonotic in nature. A human case of monkeypox, for example, was first detected in 1970 in what is now the Democratic Republic of Congo. Initial studies reported that animal-to-human transmission of the monkeypox virus was rare and sporadic. However, as humans have increasingly come into contact

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with rainforests and jungles, human monkeypox has become endemic in several central and west African regions. How big of a threat is monkeypox? Although there is currently no cause for alarm, this will not always be the case. There are already more than 1,000 confirmed cases of human monkeypox across almost 30 countries that do not normally experience outbreaks, including the United States, United Kingdom, and Canada. Using a strategy called “ring vaccination”, several countries have begun administering smallpox vaccines to individuals that have been exposed to someone with monkeypox. Although smallpox vaccines are known to be effective against various poxviruses, we do not know exactly how effective they are against monkeypox. At this stage, however, this remains one of the most powerful tools we have to keep case numbers low. The recent surge of human infections has been linked to two predominant strains of the monkeypox virus, one indigenous to West Africa and the other to Congo Basin. These outbreaks, particularly the West African strain, have produced less severe infections than previous variants. So far, no deaths associated with monkeypox have been reported in the United States or Europe, but this may likely change if the virus reaches more vulnerable populations like immunocompromised individuals or young children. We should remember that poxviruses are not stable; they evolve with their environment. I have previously written about the coevolution of the myxoma rabbitpox virus and European rabbits exposed to a South American viral strain. While natural selection favored rabbits that could resist infection, this virus increasingly evolved to suppress the immune system of its hosts. Given the rate that rabbits multiply, this host-virus “arms race” continues to change the genome of the myxoma virus. Over time, genome analyses of newer strains have not only identified several mutations from the original South American myxoma viral strain but also the addition of entirely new gene sequences. These sequences have enabled the expression of novel host range factors, known to enhance viral infection and replication. Across different poxviruses, the role of host range factors is complex and not well understood. It has been suggested that the expression of specific host range factors can allow viruses to cross 539

over and infect other species. This is what researchers speculate occurred in fall of 2018, when hundreds of Iberian hares suddenly died from rabbitpox. The myxoma-like virus detected in these hares had gained approximately 2,800 new base pairs, compared to a South American rabbitpox strain. How does a poxvirus gain that much new genetic information? These new genes were likely acquired through a process called DNA recombination. DNA recombination occurs when two closely related viruses simultaneously infect the same cell. During replication, these viruses may swap genetic information, thus creating a new hybrid virus. It is unclear what other poxvirus the myxoma rabbitpox virus interacted with to cause the mutation that led to the infection and subsequent death of the Iberian hares. For a virus, recombination promotes its survival by creating new genetic traits. However, there is no telling what the consequences of future hybrid poxviruses may be to animal and human health. We must prepare for the possibility that a zoonotic poxvirus could become a highly infectious human pathogen. Our alreadyfragile health systems cannot afford to be caught in another global pandemic that we are not prepared for. First, we need to invest in and expand research on smallpox vaccinations. Smallpox may be gone, but other poxviruses continue to threaten human health. Before cases get too high, we must develop a new generation of vaccines that specifically target emerging strains of monkeypox and widely distribute them not only in countries that are experiencing new outbreaks but also in regions where infections are endemic. Second, we need to produce antiviral treatments that fight infections early and robustly. As we have learned through the Covid19 pandemic, it is not enough to simply prevent infections from occurring in the first place; we must also stop the progression of severe infections that could overwhelm hospitals and kill vulnerable populations. We must heed lessons from pandemics' past and prioritize pandemic and epidemic preparedness, otherwise, we will continue to suffer constant disruption to our lives and needless deaths. This article is featured on Forbes.org, and can be read online here: What We Can Learn From Other Poxviruses About Monkeypox

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Study Uncovers A New Way To Think About Alzheimer’s Disease Forbes | June 23, 2022 | Article

Cells throughout the body naturally accumulate DNA mutations as we age. With Alzheimer’s disease, mutations occur in brain cells at a much faster rate than normal. Thanks to a recent study from researchers at Brigham Women’s Hospital and Boston Children’s Hospital we may be one step closer to understanding why this happens. Whole-genome sequencing of more than 300 brain cells uncovered significant oxidative DNA damage in the hippocampus and prefrontal cortex, two of the primary regions Alzheimer’s affects. Widespread mutations to the genome appear to be related to increased exposure to reactive oxidative species, produced in response to the accumulation of tau and amyloid-β proteins during Alzheimer’s. This study by Miller et al. not only sheds light on underlying mechanisms of Alzheimer’s disease but also the natural consequences of aging. Oxidative DNA damage comes in different forms from both external and internal sources. Even normal cellular metabolic processes can produce superoxide byproducts, a molecule known to be a precursor for other reactive oxygen species. At low levels, reactive oxygen species have been shown to play a role in cell signaling and maintaining homeostasis. Allowing these molecules to accumulate in a cell, however, can disrupt cellular function, not to mention destabilize DNA. Although cells have developed ways to minimize the impact of reactive oxygen species, these mechanisms are not perfect. Repairing DNA regions with oxidative damage can also come at the risk of further destabilizing the genome and producing more mutations. When a region of DNA undergoes oxidative damage, the cell must make a delicate decision of whether to repair the damage or leave it unrepaired. DNA mutations are passed down each time a cell is regenerated and as a result, accumulate over time. Studies suggest that such 541

mutations not only contribute to the aging process but also the development of some age-related diseases. Alzheimer’s disease, for example, is associated with extensive oxidative stress marked by the increased production of reactive oxygen species and oxidative damage to both DNA and RNA. To determine the extent of such damage, this study is the first to sequence the entire genome of individual neurons located in the prefrontal cortex and hippocampus from the post-mortem brain samples of those with and without Alzheimer’s. Compared to neurotypical adults, Miller et al.’s first investigation uncovered significantly more DNA mutations among those diagnosed with Alzheimer’s disease. As Dr. Michael B Miller, the lead author and professor of Pathology at Brigham, said, these “results suggest that AD neurons experience genomic damage that causes immense stress on cells and creates dysfunction among them. These findings may explain why many brain cells die during AD.” DNA mutations can have significant consequences on the transcription, as well as expression, of genes. Transcription of an altered nucleotide may prevent the correct amino acid from being attached to a protein sequence and completely alter the function of the protein. As these mutations accumulate over time, an entire gene may stop being expressed permanently. In fact, investigators found a greater prevalence of dysfunctional neurons with important genes that were no longer being expressed in those with Alzheimer’s compared to the neurotypical control group. The DNA damage observed in individuals diagnosed with Alzheimer’s was beyond the pattern of damage associated with normal age-related mutations. Also, a greater portion of mutations among this group more often impacted genes that are important for neuron function, as well as survival. Investigators concluded that there are likely several mechanisms contributing to increased DNA mutations that may be specific to Alzheimer’s disease. Although there was some evidence of increased age-related DNA changes, most of the damage investigators observed appeared to be a result of oxidative damage to nucleotides. In particular, DNA mutations commonly affect guanine nucleotides. When exposed to reactive oxygen species, these nucleotides may mutate into 8oxoguanine. Given that the prevalence of this altered nucleotide is often used as a biomarker for oxidative DNA damage, investigators 542

were surprised to find significantly high levels of 8-oxoguanine in the DNA of neurons from those with Alzheimer’s, How did these cells acquire so much oxidative damage? Several factors likely contributed to these mutations. One of the leading theories suggests that increased inflammation in the brain during Alzheimer’s exposes brain cells to high levels of oxygen reactive species. In addition to the buildup of -β and neurofibrillary tau proteins, repeated activation of the brain’s primary immune defense mechanism, microglia, has been shown to correlate with cognitive decline during Alzheimer’s disease. The presence of amyloid-β proteins reportedly triggers microglia to not only release cytokines but also reactive oxygen species in an attempt to clear the extracellular space. As the disease progresses and proteins continue to build up, microglial cells never cease producing cytokines and reactive oxygen species, which consequently damages cells. One major piece of the puzzle remains: what causes amyloid-β and tau to build up in the first place? Previous studies have found that amyloid-β plaques can accumulate in the brain for up to 10 years before one ever experiences any symptoms. Yet, there are several critical aspects of Alzheimer’s disease that we still do not understand, including the mechanism through which the presence of amyloid-β and tau proteins induce inflammation and oxidative stress. The findings from this study do bring us one step closer to uncovering these mysteries. More than six million Americans currently have Alzheimer’s, albeit current projections warn that this neurodegenerative disease will become increasingly common as more of the general population gets older and lives longer. Even if we cannot prevent amyloid-β and tau proteins from building up in the first place, we may at least be able to develop treatments that reduce the level of oxidative damage in the brain and prolong the life expectancy of those diagnosed with this and other neurodegenerative disorders. This article is featured on Forbes.org, and can be read online here: Study Uncovers A New Way To Think About Alzheimer’s Disease

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Carbon Dioxide Levels May Predict Covid Risk In Your Immediate Surroundings Forbes | June 23, 2022 | Article

As mask usage decreases in the United States and worldwide, the risk of aerosol transmission of SARS-CoV-2 increases inversely. A maskless gym, grocery store, or gas station is much more dangerous than those settings in a masked context. We ought to be acutely aware of the levels of risk maskless settings pose as world governments roll back Covid policies while Covid continues to thrive. In our recent series on airline travel, we discussed exhaled CO2 as a Covid-19 risk proxy aboard aircraft. The concept is that the higher your surrounding CO2 levels, the more significant the proportion of air exhaled from nearby people, and thus the greater the risk of SARS-CoV-2 particles in your immediate vicinity. This concept can be extended past airplanes into our daily lives. In the initial months of the pandemic, we were in fewer situations with high CO2 levels, reducing the risk of infection. More recently, many are resuming daily activities with office jobs, eating at restaurants, and going on vacations. Monitoring CO2 levels, either personally or by the business or company hosting a social gathering, may grant us a better sense of Covid risk in the coming months. In the first year of the pandemic, many suggested that SARSCoV-2 could not be transmitted via aerosols, i.e., exhaling virus particles. Their view restricted virus transmission to larger droplets, such as with sneezing or coughing, as well as fomites, which are object surfaces to which the virus may cling, for example, door handles or countertops. This dissenting view yielded skepticism about mask usage and is one reason behind a lack of true mask mandates in the United States as many other countries implemented them. This theory was eventually dispelled, in part, due to the examination of CO2 in social settings. Studies such as that of Peng and Jimenez analyzed CO2 in closed indoor environments and 544

quantified CO2 levels in terms of Covid-19 risk. As expected, closed environments with higher CO2 levels are much more likely to harbor transmission if an infected person is within the system. In unventilated areas, gases move rapidly about space to fill the volume of the contained environment. Much like how liquids take the shape of whatever container they inhabit, gases do the same at a much higher rate. This is why being in the same room as an infected person may get you sick even if you do not touch them. The gas carries virus particles across the room towards you. That is why CO2 levels are an adequate proxy for Covid risk. The more CO2 that is in the air, the more likely that CO2 is transporting virus particles around the room if an infected person is there as well. With this in mind, we can introduce three tools to help reduce aerosol-based infection. he first tool is the CO2 monitor. The CO2 levels of an enclosed space correlate to the risk of Covid infection, assuming there is someone infected in the room. Personal monitors can be purchased for as low as $50 and could be a strong investment to prevent Covid infection and potential long Covid. Additionally, businesses and companies could provide CO2 level monitoring in their stores to assure customers of their safety. We recently measured the CO2 levels of various locations on a typical day in New York City. CO2 levels varied by location and density of people, ranging from 458 ppm in outdoor spaces to 2366 ppm on a very crowded subway. We cannot avoid many aspects of life, such as subway transport in New York, but being aware of CO2 levels to some degree may help mitigate risk to some extent. This article is featured on Forbes.org, and can be read online here: Carbon Dioxide Levels May Predict Covid Risk In Your Immediate Surroundings

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SARS-CoV-2 Spike: A Potential Biomarker For Long Covid Forbes | June 24, 2022 | Article

Ten to thirty percent of COVID-19 survivors face Long Covid, another battle after initial recovery. PASC, (post-acute sequelae of COVID-19) or PCC (Post-Covid Conditions) encompasses a wide range of symptoms which appear after the acute phase of COVID19. These symptoms vary widely—from fatigue, chest pain, diarrhea and more—and the mechanisms are not well understood. At present, there is no clinical test to distinguish Long Covid from several other diseases. And because Long Covid can develop months after mild or asymptomatic COVID-19 cases, even the initial SARS-CoV-2 infection may not be confirmed. This leaves physicians and patients alike at a loss of what to do. Here inlies the urgent need for a reliable Long Covid biomarker. A biomarker, a measurable indication of a condition or disease, would remove much of the mystery surrounding the complex condition. The ability to confirm a diagnosis would provide wellneeded clarity to people who suspect they have Long Covid and to medical professionals trying to provide accurate and helpful patient care. Potential treatment strategies would also be easier to evaluate if the mechanism was more understood. A New Biomarker Study Swank et al. discuss a potential biomarker for identifying Long Covid in their preprint, Persistent circulating SARS-CoV-2 spike is associated with post-acute COVID-19 sequelae. The researchers collected blood samples from confirmed COVID-19 patients—37 with a PASC diagnosis, 26 without. They analyzed the plasma for concentrations of SARS-CoV-2 antigens using an ultra-sensitive single molecule assay (SIMOA) pioneered at the Walt lab. The SIMOA technique allowed the team to detect full length spike (S), S1 subunit of spike and nucleocapsid (N) at around 1000x higher sensitivity than typical ELISA assays.

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FIGURE 1. SARS-COV-2 ANTIGEN LEVELS VERSUS TIME. THE CONCENTRATION OF S1 (A), SPIKE (B), AND N (C) MEASURED IN THE PLASMA OF INDIVIDUALS OVER TIME AFTER DIAGNOSIS WITH PASC OR COVID-19 FOLLOWING SARS-COV-2 INFECTION. MULTIPLE DATA POINTS MAY…

SWANK ET AL Of the three antigens, the study’s findings attribute the full SARS-CoV-2 spike antigen as the best biomarker candidate. The full length spike (S protein) was detected in 60% of PASC patients, with many displaying sustained spike levels over a period of several months. The full length spike was not detected in COVID-19 only patients during the acute phase of infection. The results highlight the S protein’s potential as a biomarker of Long Covid. These findings, if reproduced in a larger study, could significantly improve the study and treatment of Long Covid. Long Covid and Possible Viral Reservoirs Intriguingly, Swank et al. detected free floating S1 in only 20% of their Long Covid cohort. It’s unusual to see full length spike circulating the serum in such an absence of S1. The S protein is composed of two subunits: S2, which is anchored in the SARSCoV-2 transmembrane; and S1, which sits on top and contains the receptor binding domain. For most strains of SARS-CoV-2, the complete spike precursor protein is cleaved upon viral exit, freeing S1 to float and leaving the S2 attached to the transmembrane. Why, then, was full S protein detected more than S1? A possible explanation has been observed by researchers who found that uncleaved, full length spike protein circulates while attached to exosomes. Maybe these small extracellular membrane vesicles, equipped with the entire S protein, are present in Long Covid patients. Swank et al.’s observations raise another puzzling question: why is the S protein still circulating despite its short half-life in the blood? 547

It seems the spike protein is being made, although how is uncertain. One hypothesis argues for the existence of a persistent reservoir of active virus. This reservoir could be replicating SARS-CoV-2 virus at low levels. An earlier study found a reservoir in the gastrointestinal tract of children who developed post-Covid related MIS-C, but post-mortem tissue analyses revealed SARS-CoV-2 RNA and protein expression in several other tissues, suggesting reservoirs may also be found elsewhere in the body. Another possibility is that subgenomic RNA capable of producing spike protein persists in the absence of full viral replication. In this regard, it’s worth noting some coronaviruses produce defective interfering viruses which account for persistence of subgenomic RNA fragments in infected subcultures. Other Markers of Inflammation A previous study also used SIMOA technology to search for potential biomarkers of COVID-19 related damage. In this study from the NYU Grossman School of Medicine, Frontera et al. found that individuals hospitalized for COVID-19 displayed elevated levels of inflammatory neurodegenerative biomarkers in their blood—total tau, Neuro-filament light, Glial fibrillary acidic protein and others. Those hospitalized with new neurological symptoms displayed higher Ubiquitin carboxy-terminal hydrolase L1 and ptau-181. These markers are also characteristic of late-stage Alzheimer's disease. Both studies observed typical cytokine IL-6 levels in their populations, suggesting it may be best to prioritize other inflammatory biomarkers. It would be interesting to test for these neurodegenerative markers in Swank et al.’s Long Covid patients. The Long Covid cohort was much younger (median age of 46 compared to 71) and was not specifically selected for neurological symptoms. Another difference is severity: only 21 of the 63 total participants were hospitalized for COVID-19. Considering brain fog and memory loss are common complaints for Long Covid, these biomarkers may warrant additional investigation. A Hopeful Beginning Swank et al. bring promise to Long Covid biomarker research. Although the cohort was small, the study provides strong support for full spike as a Long Covid-specific indicator. If clinically adapted, physicians would finally have a conclusive means to diagnose the 548

condition; people with Long Covid would no longer be told their symptoms originate from psychosomatic disease; and effective treatments could be tailored to target the possible viral reservoirs in Long Covid patients. Additional studies are needed to validate these preliminary Long Covid associated biomarkers. Future efforts should follow large patient populations for a long time. The SIMOA methods may offer a convenient tool for such studies. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Spike: A Potential Biomarker For Long Covid

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Covid-19 During Pregnancy: Increased Risk Of Preterm Delivery And Infant Neurodevelopmental Issues Forbes | June 27, 2022 | Article

During pregnancy, a mother’s body suppresses parts of the immune system to help tolerate the growing fetus. This means pregnant women are at an especially high risk of contracting disease, including Covid-19. The risk they face is two-fold: first, towards their own health, and second, towards the health of their unborn child. Still, not much is known about the impacts of SARS-CoV-2 infection during pregnancy. In a previous article, I summarized some of the key findings thus far. Since then, two new studies have been published, offering additional insights into the dangers of Covid-19 exposure during pregnancy. The first findings come from researchers at the University College London. Their work suggests that mothers who catch Covid-19 during their first trimester are more likely to have an early miscarriage. The second study, led by a group at Harvard Medical School, found that infants born to mothers who had Covid-19 during pregnancy are more likely to receive a neurodevelopmental diagnosis in the first 12 months following delivery. The good news? Maternal vaccination can help cut down many of the risks associated with SARS-CoV-2 infection during pregnancy. Covid-19 Raises the Risk of Early Miscarriage Between May and December of 2020, Balachandren et al. recruited 3500 women via social media to help participate in the COVID-19 Contraception and Pregnancy Study (CAP-COVID). All women had conceived during the pandemic and were within the first 12 weeks of pregnancy. The team of researchers asked the women to report on any current or prior pregnancy complications and their medical history. The participants were also asked every trimester whether they or anyone in their household had been diagnosed with SARS-CoV-2 infection. Depending on their 550

answer, the women were filtered into one of three groups: those who self-reported a SARS-CoV-2 infection within the first trimester (“presumed infected”), those who had symptoms or were exposed to household contacts with symptoms but had no official diagnosis (“uncertain”), and those who had neither symptoms nor household contacts with symptoms (“presumed uninfected”). After accounting for age, body mass index (BMI), number of previous miscarriages, ethnicity, and smoking status, Baachandren and his colleagues discovered that the risk of early miscarriage was around 1.7 times higher in the “presumed infected” group — a rate of 14%, compared to 8% in the “presumed uninfected” group and 5% in the “uncertain” group. How SARS-CoV-2 infection during the first trimester contributes to early miscarriage is not entriely clear. Dimitrios Mavrelos, co-lead author of the study and Honorary Associate Professor, proposed that cytokines may be involved: “Coronaviruses have also been shown to induce a pro-inflammatory cytokine storm in the body. Cytokines are thought the play a role in the development of recurrent pregnancy loss, which could explain the increased risk of early miscarriage in women infected with COVID19.” The researchers stress that they have not established a strict causal relationship between Covid-19 and early miscarriage, but rather a correlation between the two. Further research will be needed to prove that SARS-CoV-2 is directly at fault. It is worth noting that the study suffers from a few limitations. For one, the sample size was too small to establish statistical significance, the standard by which results are deemed “real” rather than simply a byproduct of chance or sample bias. Second, Balachandren et al. mention that the use of self-reported data always runs the risk of inaccuracy, since there’s no way of guaranteeing validity. Some of the participants in the “presumed uninfected” group may have simply been asymptomatic, for example. Still, the results are concrete enough to warrant the recommendation that women in their first 12 weeks of pregnancy take extra precautions to avoid SARS-CoV-2 infection. Covid-19 Raises the Risk of Neurodevelopmental Issues Andrea et al., based at the Massachusetts General Hospital in Boston, set out to study whether infants born to mothers who caught 551

Covid-19 during pregnancy are at a higher risk for neurodevelopmental disorders within their first 12 months of life. They followed a cohort of 7800 infants born at the height of the early pandemic, between March and September of 2020. Of these, 222 infants were born to mothers who had caught Covid-19 at some point during their pregnancy. Maternal SARS-CoV-2 infection was confirmed via polymerase chain reaction (PCR) test, the gold standard for diagnosis. Their findings confirmed prior research: Covid-19 during pregnancy increases the risk of preterm delivery. In this particular case, 14.4% of mothers who suffered from Covid-19 delivered early. The likelihood of preterm delivery in the control group, on the other hand, stood at around 8%. Andrea and colleagues also received an answer to their primary question. The odds of suffering from neurodevelopmental disorders are twice as high for infants born to mothers who tested positive for SARS-CoV-2 during pregnancy. Of the 222 exposed infants, 14 went on to receive a neurodevelopmental diagnosis within their first year of life — around 6%. In comparison, only 227 of the 7550 — roughly 3%— of the exposed infants went on to receive a diagnosis. The same trend holds true when adjusting the model for race, ethnicity, insurance status, offspring sex, maternal age, and preterm status. Under the adjusted model, maternal SARS-CoV-2 positivity still correlates with an 80% increase in the odds of neurodevelopmental issues in infants. Maternal Covid-19 during the third trimester of pregnancy was associated with an especially steep increase in the odds of infant neurodevelopmental disorders — almost two and a half times the odds. Most of the diagnosed neurodevelopmental disorders reflected issues with motor function or of speech and language. Take-home Message As much of the world seems to want to forget the pandemic, these two studies stress the importance of continued caution on behalf of expecting mothers. Measures such as social distancing, avoidance of crowded and poorly ventilated spaces, and masking with N95/KN95 masks are still the best strategies for the prevention of Covid-19. At this point, there is also a wealth of safety data on vaccination during pregnancy; all of it suggests that vaccination against Covid552

19 significantly reduces risks for both the mother and the child. For one, the risk of developing Covid-19 in the first place is lower in vaccinated mothers. The likelihood of stillbirth is markedly lower in vaccinated mothers as well — 15% lower, compared to unvaccinated mothers. The rate of adverse effects in vaccinated mothers — including the risk of miscarriage— mirrors that of mothers who gave birth before the pandemic, confirming the safety of vaccination. Maternal vaccination even protects the baby. Antibodies developed by the mother in response to vaccination can pass through the placenta to the fetus, transferring on immunity against SARSCoV-2. These elevated antibody levels can persist up to 6 months after birth. The result? Infants born to vaccinated mothers are much less likely to require hospitalization for Covid-19. mRNA vaccines are 52% effective at preventing Covid-19-related hospitalization in infants. This number jumps up to 70% when looking at effectiveness against admission to an intensive care unit. Spreading out vaccine delivery so that the second dose is administered at some point after the first 20 months of pregnancy is particularly effective at preventing infant hospitalization. This article is featured on Forbes.org, and can be read online here: Covid-19 During Pregnancy: Increased Risk Of Preterm Delivery And Infant Neurodevelopmental Issues

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There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part III Forbes | June 28, 2022 | Article

This is Part III in a series on the enteroviruses that appear to cause a polio-like neurological disease, Acute Flaccid Myelitis. Hopefully we can use what we have learned from both the success and challenges of poliovirus vaccines to mount a formidable defense against emerging enteroviruses such as those causing Acute Flaccid Myelitis. Poliomyelitis, more commonly known as polio, has nearly been eradicated worldwide. This is mostly thanks to effective vaccines and the expansive vaccination efforts reaching virtually all ends of the world. Afghanistan and Pakistan are now the only countries left in the world where polio is endemic. However, there have already been multiple cases of paralysis due to polio reported across the globe this year, from Malawi to Ukraine, and poliovirus was very recently detected in wastewater in London. These outbreaks are evidence that the two polio vaccines currently in use are not sufficient to reach complete eradication, and the development of an even safer and more effective vaccine is essential. Despite the overarching success of vaccination against poliovirus, the push for complete eradication has faced considerable setbacks, with some recent discouraging ones. The entire continent of Africa was declared free of the wild poliovirus in 2020, but at least two new cases were reported in Malawi and Mozambique this year. They were discovered in young children who were both paralyzed as a result of the infection, meaning there are likely other unidentified cases as well. In 95% of cases, the poliovirus is only present in the bloodstream and the infection is asymptomatic. In 5% of cases, the virus spreads and replicates in other areas of the body outside of the bloodstream. The continued viral replication causes sustained secondary viremia and leads to the development of mild symptoms such as fever, headache, and sore throat. Paralytic poliomyelitis, causing the paralysis polio is infamously known for, occurs in less than 1% of all poliovirus infections. This 554

happens when the virus spreads into the central nervous system and replicates in motor neurons, leading to the selective destruction of said motor neurons in the spinal cord, brain stem, or motor cortex and resulting in temporary or permanent paralysis. We still do not really understand why the virus has very severe effects on some people but spares most others, and the frequency of asymptomatic cases means that there is a high likelihood of additional unidentified resurgent polio cases.

Nurse Checks on Polio Patient in Iron Lung- Photograph from 1940 VINTAGE EVERYDAY There are two types of poliovirus vaccines that have been in use for decades. One is inactivated, or killed, and given by injection, and the other is live but attenuated, or weakened, and given by mouth. They each have their own set of advantages and disadvantages. The inactivated vaccine was developed in the 1950s, and it has proven to be very safe and effective. Because the virus is killed, there is no chance of mutation causing a reversion to the original pathogenic form and causing disease. However, the protection it provides is only capable of preventing the individual from developing severe disease or paralysis, it does not prevent infection or transmission. The oral attenuated vaccine, on the other hand, is capable of preventing transmission as well as disease. Developed in the 1960s, the live virus vaccine was essential in curbing the polio pandemic by preventing further spread. Despite this, it is not the perfect solution. The principal complication has been the worldwide emergence of 555

cases of circulating vaccine-derived poliovirus (cVDPV) from the oral poliovirus vaccine. Although this vaccine has been modified to limit pathogenicity, it has the ability to mutate, reverting to a pathogenic form capable of causing severe disease and paralysis. The infection can then spread to and between people who have been vaccinated with the inactivated version of the vaccine. Although incidence of cVDPV is rare, the recent increase in cases as a result of mutation in the live attenuated virus is cause for concern. In 2021, there were 614 reported cases of cVDPV, largely in Africa and the Middle East. Furthermore, cVDPV can spread just as easily as the wild type, and communities with low vaccination rates and waning immunity are especially susceptible. This includes countries experiencing political conflict such as Yemen and now Ukraine, where weakened healthcare systems mean that children are missing routine vaccinations and national reporting networks are operating at limited capacity. Due to the clear need for a new and improved vaccine option, there is a novel live oral polio vaccine in development, termed nOPV2. It is very similar to the original live attenuated vaccine, but with some changes that greatly improve genetic stability, meaning mutations leading to reversion to a pathogenic form are much less likely. Under direction of the World Health Organization’s Emergency Use Listing procedure, the Global Polio Eradication Initiative has begun dispersal of the vaccine. While we are still in the early stages of mass administration, nOPV2 is a promising solution to the recent resurgence. It is estimated that there would be 16 million people paralyzed today by polio if not for vaccination, not to mention countless lives lost. If the goal of worldwide eradication of poliovirus is successful, it will be only the second human infectious disease ever fully eradicated, with smallpox being the first. The new attenuated oral vaccine with further safeguarding against mutation means this future is still in sight. Hopefully, the strategies behind vaccine development and dispersal against polio can aid in the fight against newly emerging enteroviruses from the same family. This article is featured on Forbes.org, and can be read online here: There May Be A New Polio Epidemic On Its Way- If So, What We Can Do: Part III

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Origin Of Virus Variation: Real-Time Evolution Of SARS-CoV-2 In An Immunocompromised Patient Forbes | June 30, 2022 | Article

Evolution optimizes all creatures, from viruses to elephants, to their environment. Where do new variants come from, especially Omicron which is so different from previous variants? There may be a clue in a long study of an immunocompromised patient. Most cases of Covid-19 last roughly two weeks from infection to symptoms subsiding. The timeline may be slightly longer or shorter from person to person, but the general timeframe of infectivity to the development of symptoms is typically congruent. However, in some rare cases, individuals may be infected for months. These patients are often immunocompromised, and their immune systems cannot clear the virus and end the infection. As the virus remains within the body, it sometimes adapts to the weakened immune system of the host, developing new mutations to equip itself against host defenses better. This is one of many avenues for new variants of SARS-CoV-2 to emerge. Here we analyze this event in a long-term Covid patient in Austria and the mutations developed by her infection with SARS-CoV-2. The woman in question was treating lymphoma with immunesuppressing drugs when she became infected in late 2020 before most variants of concern or interest were circulating. Rather than dissipating in two or three weeks, the infection remained for over seven months, despite mild symptoms. Throughout the infection, Dr. Sissy Sonnleitner would collect over twenty samples for genetic sequencing. They specifically analyzed the Spike protein, as this region of the virus has an immediate impact on infectivity and immune evasion, two characteristics crucial to the success of a viral variant. Sonnleitner found roughly twenty mutations throughout infection, though not all persisted. Some samples included transient 557

mutations that were not identified in later samples. Other mutations persisted in a few following samples, and some lasted through the course of infection. Sonnleitner was observing viral Darwinism in real-time. Most notably, Sonnleitner noted mutations that would later be found in Omicron strains and other variants of concern. Those later found in Omicron are highlighted by E484K, P681H, and deletions at positions 141-144.

Below is a detailed comparison between the Spike proteins of the Austrian patient and the first Omicron strain BA.1. We note that mutations outside of the Spike protein are as important to viral characteristics such as immune evasion, virulence, and pathogenesis, but those mutations in the Austrian woman’s samples were not included at this time.

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FIGURE 2: Venn diagram comparison of Spike protein mutations in Omicron BA.1 and the Austrian ... [+] ACCESS HEALTH INTERNATIONAL

FIGURE 3: Spike protein comparison between Omicron BA.1 and the Austrian patient. Mutations in blue ... [+] ACCESS HEALTH INTERNATIONAL

The SARS-CoV-2 virus adapts to survive. If it had remained the original Wuhan virus without mutation, it likely would have been extinguished by immune memory from previous infections, vaccines, and antibody therapies. However, it mutated to overcome these immune defenses, leading to waning vaccine immunity and mass reinfections. Within an immunocompromised person, the mutation process is accelerated because the virus has extended exposure to a weakened immune system, like a boxer practicing on a punching bag before their next fight. This environment allowed for mutations that rapidly enhanced infectivity and immune evasion, like the well-known mutations N501Y and E484K, which were later noted in various significant strains. This is but one example of potentially thousands of months-long cases of Covid-19. An effective surveillance method for emerging variants that we have championed for over a year is monitoring these 559

immunocompromised patients. Seven mutations found in the Austrian woman were later found exclusively in Omicron strains. There are tens, if not hundreds of millions of immunocompromised people worldwide who are in danger of longterm Covid infection. Local health officials should be notified when an infection lasts more than a month in one of these hosts, and samples should be extracted regularly for sequencing. We know of at least one study at Harvard University tracking long-term immunocompromised patients to be on the lookout for the next Omicron-like variants, which may trigger successive waves of the pandemic with increased infectivity and virulence. This article is featured on Forbes.org, and can be read online here: Origin Of Virus Variation: Real-Time Evolution Of SARS-CoV-2 In An Immunocompromised Patient

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How Common Is Long Covid? More Common Than You’d Think Forbes | June 30, 2022 | Article

This summer appears more relaxed and free than ever. All US mask mandates have lifted, restaurants and businesses have opened their doors, and flights have removed requirements for Covid testing. But behind the veil of normalcy is Long Covid, a post Covid condition with varying long-term symptoms. Using new data from the National Center for Health Statistics (NCHS), this article will highlight the prevalence of Long Covid and emphasize the importance of mitigating your risk. How Common is Long Covid? It’s hard to say exactly how common Long Covid is. Early statistics suggest 10 to 30% of COVID survivors develop Long Covid, but how does this translate to the wider US population? Recent data from the National Center for Health Statistics (NCHS) helps paint a clearer picture. The NCHS collaborated with the US Census Bureau, which has been collecting information on COVID-19 through 20 minute online surveys since April 2020. These surveys are sent to American households to understand the pandemic’s effects on physical and mental wellness, employment status, education disruptions and more. The most recent iteration included questions on post-covid symptoms which lasted three months or longer. 62,000 adults responded to this Household Pulse Survey. According to the results, 19% of Covid survivors are currently experiencing Long Covid symptoms. At almost one in five adults, the survey provides a much larger estimate than the 10% initially believed. The NCHS data also illustrates how expansive Long Covid’s effects are. An estimated 7.5% of all US adults, around 20 million people, currently have Long Covid symptoms. This is compared to the 14% of US adults who report having Long Covid symptoms at

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one point in the pandemic. So while symptoms resolve for some, millions of people continue to feel effects post Covid infection. There’s a chance these data are conservative. The survey reported less adults having Covid than seroprevalence estimates, suggesting there’s a pool of people who have Covid that have yet to be accounted for. Children were also excluded from the count. At present, this study provides the most accurate and detailed estimates of Long Covid’s burden in the US. The NCHS will release more Long Covid survey results on July 20 and August 17th. How to Stay Safe Relaxing restrictions fail to accommodate the risk of developing Long Covid. The Household Pulse Survey shows how millions of people have and had Long Covid symptoms. These numbers will only continue to grow if proper precautions are not taken. Considering that the condition can be caused by asymptomatic, mild and severe SARS-CoV-2 infection, and that vaccination only reduces the risk of getting Long Covid by 15%, the best way to prevent Long Covid is to avoid contracting Covid. I have often invoked the weather analogy as a guide to reducing your risk of contracting Covid. Just as one checks the weather to prepare for the week, you, too, should check your local COVID-19 cases to see what protective measures to take. A Covid “weather” app has yet to be finalized. Until then, the best tool to monitor local Covid risk is the CDC’s community level surveillance of COVID19. A green zip code equates to a light rain. You have relatively low risk in your county, and would likely be fine with minimal protections. Continue to stay up-to-date with your vaccines and boosters, and maintain improved ventilation indoors if possible. A yellow forecast spells medium risk. Well-equip yourself for Covid-19 as one would for steady rainfall. In addition to the previous measures, I recommend mask wearing in crowded areas. Immunocompromised individuals or those at high risk for disease should take precautions by having a plan for rapid testing; ask your primary care physician if you qualify for treatments like oral antivirals and monoclonal antibodies should you contract Covid. If your community is red, spare no measures, as this emulates dangerous rainfall. Limit non-essential indoor gatherings if possible. It’s best to wear a mask in public in these cases, even if others are 562

vaccinated; it is still possible to catch Covid if you are vaccinated. If you do suspect you have Covid at any point, test promptly, isolate appropriately, and wear a mask to protect others. Take-Aways A generally blasé attitude towards Covid-19 infection may stir trouble. Although many have resigned to contracting the virus months back into the pandemic, the public eye should not discount the real threat of Long Covid. Given its prevalence and its difficulty to treat, the best method to avoid Long Covid is to prevent getting Covid-19 to begin with. More information on Long Covid can be read in my book, A Family Guide to Long Covid. This article is featured on Forbes.org, and can be read online here: How Common Is Long Covid? More Common Than You’d Think

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July 2022

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The Many Faces Of Omicron Forbes | July 06, 2022 | Article

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FIGURE 1: Cryo-EM structure of the Omicron BA.1 spike protein. (A) A schematic diagram illustrating ... [+] MANNAR ET AL.

It is worth noting that Omicron has a number of mutations outside the Spike protein as well. These impact viral characteristics including replication efficiency, pathogenesis, and virulence. There are 19 mutations in proteins other than the Spike throughout the genome, including Orf1a, Orf1b, E, M, Orf8, and N.

FIGURE 2: BA.1 Non-Spike mutations.

ACCESS HEALTH INTERNATIONAL On top of its infectivity is Omicron’s immune evasion of natural immunity, vaccine-induced immunity, and monoclonal antibody immunity, notably for this discussion. Monoclonal antibodies target specific structures, often in the Spike protein. For the first year of 566

the pandemic, scientists made antibodies for the original Wuhan version of SARS-CoV-2. However, with every Spike mutation, the structure of Spike slightly changes. Think of a key in a lock. They made a key for the lock, but the lock changed shape in the meantime. Then the scientists created a new key for the new lock, but the lock was constantly shifting, and the cat-and-mouse game continued. An early study on the neutralization of Omicron BA.1 by various approved and in-progress monoclonal antibodies found that 26 of 29 lost some or all neutralizing potency against the new strain. Later Omicron subvariants likely have increased immune escape and may reduce the potency of these antibodies even further. Neutralizing efficiency only seems to be growing worse as Omicron evolves into more mutated strains such as the latest BA.4 and BA.5. These later variants are more heavily mutated in the receptor-binding domain than BA.1, meaning antibodies that target the receptor-binding domain will have a tougher time binding and neutralizing the new variants.

FIGURE 3: Omicron BA.1, BA.4, BA.5, and BA.2.75 Spike proteins compared.

ACCESS HEALTH INTERNATIONAL The continued mutation of Omicron is also leading to lower vaccine potency against later strains. For instance, three doses of Pfizer vaccination is almost 3x less effective against BA.4 or BA.5 than it is against BA.1, BA.2, or BA.3.

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FIGURE 4: IC50 values for the indicated viruses using serum obtained from vaccinees 4 weeks after ... [+] TUEKPRAKHON ET AL.

All is not lost. Among those that can neutralize Omicron was 35B5, a potent, broadly-neutralizing monoclonal antibody effective against all known variants. Rather than making a new key for the lock, 35B5 essentially blows the lock off the door. This antibody targets specific amino acids in the N-terminal domain of the Spike that are regularly unmutated. This indicates that the unmutated structure at those positions is critical for virus function, such as N165 and N234, which act together as a molecular switch for the Spike’s changing up and down conformations. By targeting unmutated positions in not only Omicron but all variants, 35B5 breaks the lock off the door, future-proofing against more heavily mutated variants that may come our way in the coming months or years. In our opinion, all monoclonal antibodies should pursue the broad-neutralization strategy. Three more recently unveiled antibodies have shown promise in early in vitro testing. The first two, Cv2.1169 and Cv2.3194, were isolated by researchers at the Pasteur Institute in recent months. Both antibodies are notable because they cross neutralize both BA.1 and BA.2,

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indicating a wide net of neutralization. Cv2.1169, specifically, demonstrated therapeutic efficacy in animal models as well. The third, SP1-77, was recently detailed by a team of researchers at the Harvard Medical School and Duke University Medical School. This antibody also cross-neutralizes BA.1 and BA.2, as well as all other variants of concern. The unique antibody was generated via humanized mouse model and blocks membrane fusion rather than RBD-binding. The more weapons in our arsenal, the better. We will also describe the latest in the Omicron family: BA.2.75. You may find descriptions for other notable Omicron family members, such as BA.4, BA.5, and BA.2.12.1 in previous articles. This article is featured on Forbes.org, and can be read online here: The Many Faces Of Omicron

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NYC To Offer Instant Access To Paxlovid At Mobile Testing Sites, But Messaging Around The Drug Needs To Improve Forbes | July 07, 2022 | Article

Mayor Eric Adams has announced that New York City will be the first to dispense the antiviral drug Paxlovid, free of charge, at mobile testing units to those who test positive as part of the Test-toTreat program. This is a sorely needed initiative that addresses some of the equity and access issues with Test-to-Treat as cases are rising once again in New York. The participating mobile testing units managed by NYC Test & Trace Corps will now include a clinician on to provide instant access to prescriptions for no-cost, antiviral medications for eligible New Yorkers who test positive at the sites. The mobile units will partner with local pharmacies to provide immediate distribution of medication from the prescription. Paxlovid reduces the risk of severe disease, hospitalization, and death but is most effective when taken at the earliest stage of infection, which is why instant access is so critical. Currently, there are only three mobile testing units offering this service, but Adams plans to expand this service to more than 30 units through July 2022. New York Residents who don’t have a primary care doctor, cannot afford the co-pays or fees associated with a doctor's appointment or are not able to access these mobile testing units can call 212-COVID19 (212-268-4319) to be connected with a clinician. This kind of program should be replicated across the country to ensure swift access to testing and treatment. The recent decision by the FDA to authorize pharmacists to prescribe Paxlovid will also increase access across the country, especially within healthcare deserts and rural areas. While federal emergency aid for testing, vaccines, and therapeutics is stalled, these free city-run Test-to-Treat sites that 570

dispense Paxlovid will be crucial for uninsured New Yorkers. A federal fund that made it possible for laboratories to perform tests free of charge for uninsured people across the country has not been accepting claims since March 2022. This means that certain providers are no longer able to provide testing to everyone free of charge, regardless of their insurance status. However, even as we begin to address issues of equity and access. The drug Paxlovid still has some serious messaging and awareness issues. A report by STAT states that many healthcare providers are conflicted on basic questions such as who they should be prescribing the drug, for what duration, and at what stage during the infection. The FDA authorization is broad and states that Paxlovid can be prescribed within five days of symptom onset to people 12 and older who have tested positive and are “at high risk for progression to severe Covid-19. High risk is a broad category and can include the immunocompromised to people who have asthma, obesity or diabetes to people who are over age 65. However, there are many reports of younger people under 65 with high-risk conditions such as asthma who are being denied a prescription to Paxlovid. Healthcare providers need clearer guidelines and prescription criteria with up-to-date data to make the best decisions for their patients. Unlike vaccines, public messaging around Paxlovid is limited and many patients infected with Covid-19 are not even aware that Paxlovid exists or is a treatment option. Many patients have only heard about Paxlovid rebound, making them hesitant about the drug. While I am not a medical doctor, and further research is needed, my interpretation of current data as a virologist suggests that Paxlovid should be prescribed for a 10-day course instead of the usual 5-day course to avoid the rebound effect. As pandemic fatigue prevents many from precautions such as mask-wearing and avoiding unventilated indoor spaces, we need to ensure that swift and equitable testing and treatment are implemented to control rising case counts. The test-to-treat model is of the most positive innovations to emerge in response to the Covid pandemic and it is my hope that it will eventually be used for other infectious diseases. This article is featured on Forbes.org, and can be read online here: NYC To Offer Instant Access To Paxlovid At Mobile Testing Sites, But Messaging Around The Drug Needs To Improve 571

Research On Flies Provides Hope For Brain Repair Forbes | July 11, 2022 | Article

This story on neuro-regeneration is part of an extended series on Regenerative Medicine. For other stories on this topic see williamhaseltine.com and search for Regenerative Medicine. My definition of Regenerative Medicine is any medical modality that returns us to normal health when we are damaged by disease, injured by trauma, disadvantaged by birth, or worn by time. Modalities include: chemicals, genes, proteins and cells used as drugs, gene editing, prosthetics, and mind-machine interfaces. The Challenge of Brain Repair One of the most significant challenges in medicine is the ability to repair brain damage. Damaged brain cells do not normally regenerate and can lead to permanent motor and cognitive impairments. The study of brain cell regeneration has been ongoing for many years. However, recent scientific advances using a distant model organism—the fruit fly—may offer a new window into how brain cells could be regenerated in humans. Fruit flies, while very physiologically different than humans, have proven to be a valuable model for a number of developmental processes in the past. Now, it seems that they may serve as a beneficial tool to study regenerative processes as well. Studies have shown that the brain contains a natural ability to partially produce new neurons in response to injury. This involves the activation of specialized cells called neural stem cells. Unfortunately, neural stem cells are rarely, if ever, fully activated. This means that while many neurons begin the process of regeneration, only a fraction of fully functional neurons is ever produced. But how, exactly, does the brain activate neural stem cells and how could we promote this process to increase the brain’s ability to repair damage? A recent paper by scientists at the Champalimaud Foundation in Portugal may bring us closer to the answer. To

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explore these questions, Simões et al. began by looking at brain injuries in adult fruit flies. Neural Stem Cells are Activated by Distant Signals Scientists created small lesions in the adult flies’ brains to determine how neural stem cells would respond. In line with previous studies, Simões et al. found that after inflicting brain injuries, neural stem cells commonly expressed markers of mitosis. This indicated that the neural stem cells had been activated and recruited to repair the injury. Surprisingly, neural activation occurred even though very few activated neurons were in direct contact with the injured brain tissue.

Figure 1: Neural stem cells in injured brains exhibit the green PH3 marker of mitosis. The injury site is marked by the white dotted line.SIMÕES ET AL., CELL PRESS (2022), DOI: HTTPS://DOI.ORG/10.1016/J.DEVCEL.2022.05.015

From this, Simões et al. speculated that stem cell activation occurs by distant signals that travel from the injured brain tissue to the neural stem cells. To test this, the researchers used genome-wide profiling tests to determine which genes were most active after 573

injury. They found that there were 74 upregulated and 8 downregulated genes following brain injury. Excitingly, most of the differentially expressed genes were associated with an injury-induced secretome—a set of proteins that is secreted into the extracellular space and can diffuse through the brain to interact with a variety of cells. A Crucial Role of Swim Transporter Proteins One protein detected in the secretome caught the researchers’ attention— Secreted Wingless-interacting molecule (swim). The swim protein specializes in moving proteins from one location to another. Researchers believed that swim may be the key to understanding how injury in one part of the brain can affect nearby cells as well as more distant cells. To test the role of swim in injury-induced neural stem cell activation, Simões et al. inhibited swim in some flies before inducing brain lesions. After analyzing the number of neural stem cells that had been activated after injury, they found that when swim was inhibited, far fewer neural stem cells exhibited signals of mitosis. These results showed that the swim protein was a critical aspect of neural regeneration after brain injury. But where was swim produced? Simões et al. noticed that when the brain was injured, a particular cell type jumped into action. These were a type of glial cell. Glial cells have a very diverse set of functions. Along with responsibilities related to immunity and inflammation, glial cells provide physical/metabolic support to neurons and they help with neuron insulation, communication, and the transport of nutrients. Now, it seems that glial cells may also play a role in neural regeneration by producing the swim protein. The last piece of the puzzle was to determine which signaling molecules swim was transporting to the neural stem cells. Long-range Signaling of Wg Proteins One of the primary mechanisms by which neural stem cells are activated is through the Wnt signaling pathway. Wnt is a family of proteins that exists in both humans and flies. The family of Wnt genes and proteins are important for organogenesis during the process of embryo formation. They specifically activate stem cells to initiate the process of creating new organs. Flies contain a particular type of Wnt protein called wingless (Wg). Wg proteins themselves 574

do not diffuse widely from their place of origin in the brain. How then, is it possible that they may exhibit long-range effects in the brain? In a second paper published in the Proceedings of the National Academy of Sciences, researchers hypothesized that swim might transport Wg proteins, allowing the protein to send long-range signals. The researchers first isolated Wg proteins and analyzed them for the presence of any residual molecules that may have been attached to the protein. To their surprise, they found peptides related to the swim transport molecule. Continuing their exploration of swim-Wg interactions, researchers then measured how well the two molecules bonded to each other. In line with their hypothesis, the two proteins bonded very favorably. These two experiments confirmed that swim binds to Wg. With the knowledge that swim binds to Wg, Simões et al. continued their investigation by exploring how the distribution of Wg protein in the brain was influenced by brain injury. In the absence of brain injuries, Wg is highly localized and is observed in only a few cells. When Simões et al. induced brain injuries, high levels of Wg protein were exhibited in the injured areas. When researchers analyzed the distribution of Wg in flies with or without the swim transporter protein, they found that swim played a crucial role in increasing the distribution and signaling of Wg in the brain. Strikingly, researchers also noticed that Wg-expressing neurons and glial cells were located in very close proximity, forming neuroglial clusters. After conducting further experimentation, it was clear: when brain tissue is injured, the unique neuro-glial clusters secrete Wg which is then transported by swim proteins to activate neural stem cells and promote brain tissue regeneration.

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Figure 2: Neurons (yellow) and glial cells (purple) form neuro-glial clusters and secrete Wg and swim protein. swim protein transports the Wg to neural stem cells to induce neurogenesis.SIMÕES ET AL., CELL PRESS (2022), DOI: HTTPS://DOI.ORG/10.1016/J.DEVCEL.2022.05.015

Injury-induced Swim Activity is Conserved in Mammals When these experiments were repeated in mice, researchers discovered that results were consistent. Simões et al. found that when traumatic brain injury was induced in mice, glial cells in the hippocampus were similarly activated and produced increased levels of swim protein. This suggests that the secretion of swim protein following injury is conserved in mammals. Conclusion This study represents significant progress in understanding how brain cells could potentially be regenerated. As we continue to grow our knowledge of neural stem cells and their activation mechanisms, we also come closer to developing therapeutics that could effectively treat brain damage. However, more research must be conducted to determine whether this mechanism of brain cell regeneration is 576

consistent in humans. While humans do not contain Wg protein, our bodies do produce multiple proteins from the same Wnt family. It will be interesting to determine whether any of the Wnt family proteins in humans may play a similar role to Wg in human neural cell regeneration. This article is featured on Forbes.org, and can be read online here: Research On Flies Provides Hope For Brain Repair

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BA.2.75: A Dark Horse In The Covid Pandemic Forbes | July 11, 2022 | Article

Far from concluding, the Covid pandemic seems to be picking up speed with new variants. The BA.5 variant is spreading rapidly in Europe and North America, potentially infecting as many or more people as the original Omicron virus from which it is derived. A second variant, BA.2.75, has been detected in India and is rising quickly. We have previously described BA.4 and BA.5; here, we focus on the novel BA.2.75. Omicron BA.1 variant emerged in late 2021 with substantial genetic and virological differences. BA.1 was quickly followed by the genetically distinct descendant BA.2. Figure one denotes the initial rise of BA.1, quickly followed by BA.2. These represented the bulk of cases during the winter months of early 2022 when confirmed cases in the United States peaked at around 1 million per day. BA.4 and BA.5, which are very similar, derive from BA.2, as does BA.2.75, although independently. There is no relation between Omicron BA.1 and the other major variants, Alpha, Beta, Gamma, Delta, etc. BA.4/BA.5 is the dominating strain of Covid-19 today, and we believe BA.2.75 may join them.

FIGURE 1: BA.2.75 was independently derived from BA.2, significantly differing from BA.4 and BA.5.

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FIGURE 2: Cases in the United States designated by variant type.

STEBBING Later Omicron variants, such as the currently dominating BA.4 and BA.5 strains, appear capable of reinfection of those previously infected with an earlier strain. Those infected with BA.1 were susceptible to BA.2, and so on. One report indicates that BA.5 was between 14.3 and 16.8-fold more resistant to Evusheld and Sotrovimab antibody treatments than previous variants. This resistance, in addition to new mutations, is likely related to sensitivity to a membrane protease, TMPRSS2, that is associated with cell membrane infectivity. BA.5 has a greater sensitivity to TMPRSS2 inhibitor Nafamostat, meaning that BA.5 is slightly less infectious relative to BA.1 and BA.2, but could be more virulent and immune evasive, akin to the Delta variant of 2021.

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FIGURE 3: (A) Shift in linear regression between virus infectivity and diagnostic PCR Ct values ... [+] AGGARWAL ET AL.

The latest in the line of Omicron variants is BA.2.75, which has a distinct set of Spike mutations in addition to those found in BA.2, BA.4, BA.5, and other mutations outside the Spike protein, indicating it was independently derived from BA.2.

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BA.4 and BA.5 are very similar, differing by only a few mutations in the structural proteins N and M, nonstructural protein NSP4, and accessory proteins Orf6 and Orf7b, as described in a previous discussion (Figure 3). These differences seem to grant BA.5 a replication advantage, outpacing BA.4 in North America and Europe (Figure 2). Both silent and amino acid altering mutations in the Orf1ab replication complex, structural proteins, and accessory genes can increase the viral fitness of SARS-CoV-2. It remains to be determined which mutation grants BA.5 the replication advantage it possesses.

FIGURE 4: Non-Spike protein mutations in BA.4 and BA.5 DR. TULIO DEL OLIVERA

BA.2.75 is still very new. It was recently discovered in India, followed by ten countries soon after. The World Health Organization has already warned about BA.2.75 and continues to monitor the variant as it spreads to more regions of the globe. While confirmed cases due to BA.2.75 are relatively low, numbers are expected to increase in the coming weeks. Not only is BA.2.75 a derivative of BA.2, but it is distinct from BA.4 and BA.5.

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FIGURE 5: Venn diagram comparison of BA.4/BA.5 mutations versus BA.2.75. Those on the left are ... [+] ACCESS HEALTH INTERNATIONAL

The Spike is the most heavily mutated protein in the Omicron family of variants. This pattern holds with BA.2.75. There are 36 mutated amino acids in the BA.2.75 Spike protein. Some early variants, such as Alpha, did not even carry 36 amino acid mutations in the entirety of their genome. BA.2.75 has this amount in the Spike protein, which accounts for roughly 10% of the SARS-CoV-2 genome.

FIGURE 6: BA.2.75 Spike protein mutations. Those in red are found in BA.2.75. Those in blue are ... [+] ACCESS HEALTH INTERNATIONAL

The unique mutations of the BA.2.75 Spike protein are isolated to the N-terminal and receptor-binding domains. These are regularly the most heavily mutated regions of the virus as they are typical targets for neutralizing antibodies derived from infection, vaccines, and monoclonal treatments, meaning mutations may allow the virus to overcome neutralization. These regions also play a significant role in the virus’s transmissibility, speaking to how later variants grow more infectious. The new additions fall into two categories: new mutations and reversions. We only find one reversion in BA.2.75, which is Q493. 582

In BA.2, this position was mutated from glutamine to an arginine (Q493R), a common mutation in circulating variants throughout the pandemic. Reversions are uncommon, as a mutation often confers some virological advantage over the original amino acid. However, this reversion may interact complimentarily with a new unique mutation to confer a more significant advantage than Q493R could have on its own. Aside from the reversion, there are eight mutations in the BA.2.75 Spike that are unique from BA.2. In addition, all but one of these eight mutations (K147E, W152R, F157L, I210V, G257S, G339H, and N460K) are uncommon in any previous variant of concern or interest. We note that F157L was previously detected in a minor African strain A.23.1. Mutations occur at position I210 in the African A.30 and French B.1.640 strains but to different mutations (I210N and I210T, respectively). Additionally, position W152 is mutated in the Epsilon strain of 2021, but to W152C. To have such unique mutations at this stage of the pandemic when the virus has mutated into hundreds, even thousands of competing strains is astounding. These mutations likely have health officials on high alert, as a wealth of new mutations could indicate increased transmissibility or immune evasion if the variant catches fire like its predecessor. The new lineage also has five new additions outside of the Spike protein. Four lie in the Orf1ab replication-transcription complex, and the last lies in the Envelope protein.

FIGURE 7: BA.2.75 mutations outside the Spike protein. Those in red are found in BA.2.75. Those in ... [+] ACCESS HEALTH INTERNATIONAL

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Two of the unique mutations, S403L and P822S, lie in NSP3. This protein is involved in the formation of the structure of the double-membrane vesicle replication compartment. It also inhibits interferon activity by direct cleavage of IRF3 and antagonizing MDA5 activation. These mutations could therefore be involved in virus replication and immune evasion activities. We additionally note N118S in NSP8, G671S in NSP12, and T11A in the Envelope protein. NSP12 is the RNA polymerase that drives viral replication and transcription, while NSP8 is a structural cofactor for that machinery. The Envelope is involved in viral assembly, budding, and viral porin activity. More research is needed on these unique mutations to determine their exact advantages, but they are likely involved in these processes. You can read more about the functions of each protein in our book Natural Immunity. There may be several mutations that do not change amino acids in the coding sequence, known as synonymous mutations, but we do not have access to those at this time. Even as we write this article, the Omicron family is everevolving. In addition to BA.2.75, recent reports from India suggest that there are accompanying lineages BA.2.74 and BA.2.76 that are circulating concurrently with BA.2.75. As of yet, the exact sequences are unavailable to view on the GISAID SARS-CoV-2 database, though the researchers suggest they share the same Spike protein, implying that differences lie outside the Spike just as BA.2.75 differs from earlier variants. To summarize, having just recovered from the first Omicron wave of BA.1 and BA.2 at the start of the year, as well as the BA.2.12.1 wave in the United States and elsewhere, the world is now facing two additional variants, which may individually or collectively surpass the first wave in magnitude. In the United Kingdom epidemic alone, infections jumped several hundred thousand in previous weeks due to the new strains. Were the US to face similar increases, daily rates could be in the millions, exceeding the peak of the Omicron wave in mid-January. The impact of BA.5 and BA.2.75 on health outcomes, hospitalization, and death remains to be seen. All countries but China have abandoned public mitigation measures, meaning Covid safety now falls on the individual, which is a sorry state of affairs in the face of the continued onslaught of SARS-CoV-2. 584

This article is featured on Forbes.org, and can be read online here: BA.2.75: A Dark Horse In The Covid Pandemic

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Long Covid And Its Unequal Burden Forbes | July 12, 2022 | Article

All countries have health disparities, or unfair and avoidable differences in health status between groups. As defined by WHO, these differences come “from the social conditions in which people are born, grow, live, work and age,” factors which extend beyond an individual’s control. The United States is no exception. The beginning of the Covid-19 pandemic provided a clear example of how health disparities exist for certain racial and ethnic groups in the US. Black, Latino and American Indian persons experienced higher rates of Covid-19 infection, hospitalization and death than white, non-hispanic persons. Genetic predisposition to health conditions like lung disease and hypertension contribute to these numbers, but greater still are the societal factors which influence these statistics. Marginalized groups are more likely to live and work in crowded settings and to have less income and wealth. As a result these groups were, and still are, more likely to catch Covid-19 than others, but are less equipped to face the economic strain which accompanies testing, treatment and recovery. The current state of the pandemic, now two and a half years in progress, has left at least 20 million people Covid survivors in the US facing post-Covid Conditions—otherwise known as Long Covid. And while it is uncertain whether people of diverse backgrounds have higher genetic risk of developing the condition, communities of color will likely face exacerbated effects of Long Covid as a consequence of persisting health inequities. In particular, the heightened probability of infection, reduced access to primary/specialized care, and diminished economic position faced by minority populations will translate to a more arduous road to Long Covid recovery. Heightened probability of infection Long Covid can develop from asymptomatic, mild and severe acute Covid-19. There currently is no cure-all for the condition; 586

treatment is offered based on the displayed symptoms and any traceable organ damage. Correspondingly, the best method to avoid Long Covid at present is to prevent getting Covid-19 to begin with. In this regard, racial and ethnic minorities are at greater risk of SARS-CoV-2 infection than others due to work and living conditions. As explained in the paper Long Covid and Health Inequities: The Role of Primary Care, people of color are overrepresented in high risk and essential occupations. Healthcare workers, transport workers, and members of the service industry come in contact with large quantities of people but have less ability to physically distance themselves. Adequate proper personal protective equipment (PPE) may not be given. These factors increase the chances of contracting Covid-19 and subsequently Long Covid. The environment at home also contributes to the likelihood of acquiring Covid-19. The high-density areas and crowded homes occupied disproportionately by vulnerable groups make it easier to spread Covid-19 and harder to isolate. Evidently, racial and ethnic groups face obstructions even at this first line of defense. Reduced access to primary/specialized care Once post Covid complications emerge, the critical next steps are to diagnose and treat symptoms and/or organ damage. A primary care physician (PCP) familiar with a patient’s health history would be more equipped to notice the unusual symptoms of Long Covid and refer the individual to specialized services, such as a multidisciplinary Long Covid clinic. Although this process proves difficult for most people with Long Covid and similar chronic conditions, people of color face additional barriers which aggravate access to treatment. A 2020 poll by the African American Research Collection found Black, Native and Latino Americans reported having less access to a primary care doctor than their white counterparts. A possible contributor is geographic constraints. A disproportionate number of Black and minority ethnic groups live in medically underserved areas which lack access to primary care. Even if primary care is available, inadequate public transportation can delay medical care.

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Access to care is further complicated by perceived levels of discrimination and medical mistrust—all which racial and ethnic minorities experience disproportionately. Location impacts specialist care, as well. Several US states only have one multidisciplinary Long Covid clinic, if at all. Essential workers will have a harder time requesting time off work and traveling to necessary appointments. Dr. Monica Lypson states it best in this STAT article: “People who were putting our groceries on the shelves, people in the transportation industry like Metro or bus drivers — the idea that you’d be on the phone trying to find a clinic that potentially was an hour to four hours away from you is a lot.” Coupled with geographic constraints is health insurance access. People of color are more likely to lack health insurance, as many jobs held by non-white groups do not provide sufficient healthcare benefits. This is especially true for Latinos. Recent results from the NCHS Household Pulse Survey estimate up to 17% of Hispanics or Latinos are currently uninsured. This is compared to 12% of people of multiple races, 10.6% of Black Americans, and 7% of white Americans. Considering that Long Covid symptoms often take months to resolve, the ability to pay for continued treatment will disadvantage minorities preferentially. Diminished economic position All stages of Long Covid diagnosis and treatment are intrinsically impacted by one’s economic means. People with Long Covid must cover the costs for doctor’s visits, medication, therapy, and transportation. The most common symptoms of Long Covid— fatigue, post-exertional malaise, brain fog and shortness of breath— often force people to leave work, suspending one’s income stream (at least temporarily). Money, therefore, is a major deciding factor in Long Covid recovery. Many racial and ethnic groups are less likely to possess the financial cushion needed to support those with Long Covid. As previously mentioned, minorities are overly represented in frontline, essential work. These positions earn less and are often inflexible to schedule changes. Temporary leave for recovery may not be an option. For some, it is better to suffer at home than risk losing their jobs or endangering household income by seeking treatment. Trevon Logan and Bradley Hardy explore how economic inequalities create disproportionate health burdens on Black 588

Americans in their essay, Racial Economic Inequality Amid the COVID-19 Crisis. Logan and Hardy emphasize how Black Americans face higher income volatility, higher unemployment, and greater levels of food and housing insecurity than many other Americans. These factors left Black Americans more vulnerable to acute Covid-19 crises; similarly, the lack of economic buffers for Black families will translate to worse Long Covid outcomes for this minority. Future Considerations Topics of health inequity and Long Covid still require investigation. Some studies such as UCSF Clinical trial on Long Covid are striving to increase minority representation in their Long Covid research, but for now, much of what is known is based on research with stronger representation from higher income, predominantly white cohorts. Despite this fact, one can expect disadvantaged groups to bear a heavier burden caused by Long Covid by inferring known associations between race/ethnicity, health disparities, and acute Covid-19. The pandemic is far from over—vaccines do not yet protect against infection, and statistics on new cases and hospitalizations are rising with the spread of Omicron variants BA.5 and BA.275. The population of Americans experiencing Long Covid symptoms will likely continue to grow. Now more than ever will the country need health policies which lessen cost barriers to care, improve insurance coverage, and bolster primary care systems. A stronger foundation in equitable health policy will not only help our disadvantaged communities weather the current Covid-19 storm, but also prepare the country for any possible future pandemics. This article is featured on Forbes.org, and can be read online here: Long Covid And Its Unequal Burden

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More Danger Ahead With BA.5: Covid-19 Reinfection Doubles The Risk For Death, Blood Clots, And Lung Damage Forbes | July 13, 2022 | Article

A recent preprint concludes that two or more Covid-19 reinfections double the risk for death, blood clots, and lung damage among other negative health outcomes, demonstrating the clear and present danger of reinfection. The most recent Omicron variants have an enhanced capacity to evade our innate immune defenses. BA.4 and BA.5 are continuing that trend with replication of the sub variants associated with reduced activation of epithelial innate immune responses compared to earlier BA.1 and BA.2 sub variants. With BA.5 responsible for over 54 percent of all COVID infections and BA.4 accounting for over 17 percent, the risk of reinfection is especially high even for those who were more recently infected with BA.1 or BA.2. We need to take action now to prevent reinfections and ensure that they do not cause long term health impacts for broad swathes of the population.

FIGURE 1: Cases in the United States designated by variant type from April 22nd to July 2nd, 2023

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JUSTIN STEBBING This is the first study to characterize the health risks of reinfection, an understudied area. The study uses the health records of more than 5.6 million people treated in the Veterans Affairs Health System. The study population was broken down into 257,427 participants with one documented SARS-CoV-2 infection and 38,926 participants who had two or more SARS-CoV-2 reinfection and 5,396,855 participants with no record of positive SARS-CoV-2 infection as a control group. Among those who experienced reinfection, 36,417 people had two infections, 2,263 people had three infections, and 246 people had four or more infections. The median time between the first and second infection was 79 days and between the second and third was 65 days. The authors found those with two or more documented infections had more than double the risk of dying and three times the risk of being hospitalized within six months of their last infection. They also had higher risks for lung and heart problems (cardiovascular disorders, coagulation, and hematologic disorders) fatigue, digestive and kidney disorders, diabetes, musculoskeletal disorders, mental health disorders, and neurologic problems (as represented in Figure 2 below). The risks were most pronounced in the acute phase, yet most were still evident at six months after reinfection.

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Cumulative risk and burden of sequelae in people with one, two, and three or more SARS-CoV-2 infections compared to a noninfected control. Risk and 6-month excess burden of all-cause mortality, hospitalization, at least one sequela, and sequelae by… ZIYAD AL-ALY, BENJAMIN BOWE, YAN XIE ET AL. OUTCOMES OF SARS-COV-2 REINFECTION, JUNE 2022 Many leaders within the US and globally have currently opted for more relaxed or even non-existent Covid-19 policies, relying on vaccines to reduce the risk of death and hospitalization. The New York City Department of Health recently removed its color-coded Covid-19 alert system from the website, stating that they were reevaluating the system. A move that seemed to contradict the rising case counts and current seven-day positivity rate of 14%. 592

In the face of rising case counts, and this disturbing new data on reinfection it would be foolish to rely purely on medical interventions such as vaccines and antiviral drugs alone. While there may be little appetite left for measures like lockdowns and social distancing, there is still plenty we can do to reduce transmission and the overall health burden of the virus. Reducing transmission is the only way to slow down viral evolution and prevent more deadly and immune-evading variants. Issuing N95 masks free to the entire population, reinstating mask mandates, and installing proper ventilation and filtration (HEPA filters) in schools and workplaces, along with CO2 monitors are just a few noninvasive measures we can take to reduce transmission. We also need consistent local and national data reporting and free PCR testing and rapid tests for all regardless of insurance status so individuals can make well-informed choices about their own levels of risk. This article is featured on Forbes.org, and can be read online here: More Danger Ahead With BA.5: Covid-19 Reinfection Doubles The Risk For Death, Blood Clots, And Lung Damage

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Pasteur Institute Scientists Discover SARSCoV-2 Broadly-Neutralizing Antibody Forbes | July 13, 2022 | Article

Cv2.1169 antibody binding to the SARS-CoV-2 Spike protein. PLANCHAIS ET AL. This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. From the early days of the Covid pandemic, monoclonal antibodies have been billed as an effective means to treat and possibly prevent SARS-CoV-2 infections. The originally sparkling promise has been substantially tarnished as one monoclonal drug after another has succumbed to the rapid variation of the SARS-CoV-2 virus, rendering them essentially useless for treatment. Now a global search for new antibodies that hone in on regions of the virus Spike protein that are required for effects and, therefore, resist change is in effect. We have already discussed one such antibody, 35B5, as described by a group of virologists from China. Here we discuss another such antibody as described by scientists at the Pasteur Institute in Paris, France. 594

Isolation of the Virus As described by Planchais et al. in the Journal of Experimental Medicine last month, the two antibodies Cv2.1169 and Cv2.3194 carry a notable headline: they potently neutralize both Omicron BA.1 and BA.2. Planchais et al. observed and cloned 102 human SARS-CoV-2 Spike monoclonal antibodies from the memory B cells of 10 convalescent Covid-19 patients. Most of the 102 mAbs are bound to the S2-region of the Spike protein, meaning nonbinding to the receptor-binding or N-terminal domain. None of these S2 mAbs were neutralizing against the Wuhan strain in vitro. Roughly one-third of the remaining receptor-binding and Nterminal domain mAbs neutralized SARS-CoV-2 in vitro. The most potent of these were Cv2.1169 and Cv2.3194. Further analyzing both antibodies, the researchers found that both were fully active against previous variants of concern Alpha, Beta, Gamma, and Delta, as well as the earliest strains of Omicron BA.1 and BA.2. Between the two, 1169 most potently neutralized all variants from the Wuhan strain up through BA.2.

FIGURE 1: Heatmaps comparing the binding (left) and RBD-ACE2 blocking capacity (right) of ... [+] PLANCHAIS ET AL.

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The researchers examined 1169 in greater detail as it neutralized recent variants more effectively than 3194, including Delta+, BA.1, and BA.2. Planchais et al. further enhanced 1169 with a purified Jchain containing IgA dimers as 1169 as a monomeric IgA antibody displayed muted effects against SARS-CoV-2. In other words, they greatly enhanced its neutralization against early and later variants by modifying the structure of the cloned antibody. Cv2.1169 Protection in Animal Models The Cv2.1169 antibody showed positive therapeutic effects during in vivo mouse and hamster SARS-CoV-2 infection models—small rodents infected with SARS-CoV-2 mirror the disease course of a mild-to-moderate case of Covid-19 in humans. A single injection of Cv2.1169 significantly reduced pulmonary viral infectivity and RNA levels. Intra-lung viral infectivity and RNA loads also reduced dramatically after using the 1169 antibody. These results were repeated with mice infected with Beta, among the most pathogenetically severe variants to circulate. The mice made a full recovery after treatment with 1169.

FIGURE 2: Schematic diagram shows the experimental design of Cv2.1169 antibody therapy in ... [+] PLANCHAIS ET AL.

Structure of Cv2.1169 Spike Binding The Cv2.1169 antibody binds in a set of three to the trimer of the SARS-CoV-2 Spike protein receptor-binding domain in the up configuration. 596

FIGURE 3: Cryo-EM map from the tri-S ectodomain in complex with Cv2.1169

PLANCHAIS ET AL. Via cryo-electron microscopy, we can see the exact contact points of 1169 to the SARS-CoV-2 Spike protein, illuminating targets for current and future Covid-19 therapies. These contact points include Y473, S477, T478, F486, N487, and Q493.

FIGURE 4: Close-up of the RBD-Cv2.1169 interface.

PLANCHAIS ET AL. Potential Escape from Cv2.1169 Inspection of the GISAID database shows that mutations involving the binding sites for Cv2.1169 occur but in relatively low frequency. However, there is more immediate cause for concern as some binding sites are altered in the most recent iterations of Omicron BA.4, BA.5, and BA.2.75.

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Y473 is unmutated in all Omicron strains, S477 is mutated to S477N in all Omicron strains, T478 is mutated to T478K in all Omicron strains, and N487 is unmutated in all Omicron strains. Because these are consistent, they should not have an altering effect in later Omicron variants. F486 is unmutated in BA.1 and BA.2, but is mutated to F486V in BA.4 and BA.5. Q493 is mutated to Q493R in all strains but BA.2.75, in which the position remains Q493.

FIGURE 5: Table of Mutational differences in 1169 contact points between different Omicron strains.

ACCESS HEALTH INTERNATIONAL Whether these changes are significant enough to cause later Omicron variants to escape Cv2.1169 is unknown, so we implore further research to be conducted on Cv2.1169 to analyze the neutralization of newer strains. This is but one of many potential broadly neutralizing antibodies that could work against currently circulating and future variants. We will continue to cover these antibodies as they are released because rather than playing catch up with SARS-CoV-2, we must start future-proofing our treatment arsenal as variants continue to arise. This article is featured on Forbes.org, and can be read online here: Pasteur Institute Scientists Discover SARS-CoV-2 BroadlyNeutralizing Antibody

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Robotic Arms Allow Partially Paralyzed Man To Feed Himself Forbes | July 14, 2022 | Article

This story on neuro-prosthetics is part of an extended series on Regenerative Medicine. For other stories on this topic see williamhaseltine.com and search for Regenerative Medicine. My definition of Regenerative Medicine is any medical modality that returns us to normal health when we are damaged by disease, injured by trauma, disadvantaged by birth, or worn by time. Modalities include: chemicals, genes, proteins and cells used as drugs, gene editing, prosthetics, and mind-machine interfaces. Thanks to the work of scientists at Johns Hopkins University, a partially paralyzed man was able to feed himself using just his mind and a pair of robotic arms. The 49-year-old man suffered from a spinal injury nearly thirty years ago, leaving him with limited upper body mobility and an inability to use any of his fingers. Now, with the help of an advanced brain-machine interface, the man can command robotic prosthetic arms to cut and feed him food simply by making subtle movements with his wrists and hands in response to audio prompts such as “select cut location” or “moving food to fork”. Brain-machine interfaces allow for direct communication between a person’s brain and a computer. When a microelectrode array is implanted into a person’s brain, their brain signals can be recorded and sent to a computer. The computer then decodes these signals and translates them in real-time into commands or external functions. In the past, brain-machine interfaces have restored some independence to those living with sensory or motor deficits by helping them speak, write, or perform other tasks they are normally unable to. However, most brain-machine interface systems that involve prosthetic limbs can only perform very simple movements and do not allow the user to customize the prosthetic’s actions in any way. In a recent paper published in the journal Frontiers in Neurorobotics, scientists at Johns Hopkins describe how they 599

developed an advanced brain-machine interface and robotic prosthetics system that not only moves in more flexible ways but allows the user to customize the robotic prosthetic’s actions to their liking. How many ways can you move your wrist? Most people will report 3 unique types of movement: side to side, up and down, and rotational movements. These movements are called degrees of freedom. One of the largest challenges of creating robotic prosthetics with human-like dexterity is to maximize the degrees of freedom that each joint in a prosthetic limb has while maintaining the user’s ability to control each type of movement. Previous work has successfully developed fully controlled robotic prosthetics with 7-10 degrees of freedom. However, creating a brain-machine interface that interacts with two robotic prosthetics at a time would require the Johns Hopkins team to develop a way for the user to control as many as 24 degrees of freedom. To address this challenge, the researchers sought to use a system called collaborative shared control. Shared control systems are a strategy in which the brain-machine interface user and a semiautonomous robot work together to accomplish tasks. Collaborative shared control occurs when only a subset of a prosthetic’s degrees of freedom are controllable by the user during task-specific times. For instance, if a prosthetic was tasked with picking up an object, the user may have control over the prosthetic’s side-to-side movements to determine the position that the prosthetic lifts the object from. By using a collaborative shared control system, researchers could allow the user to customize and control the robotic prosthetic’s movements while still minimizing the user’s workload. After determining their control strategy, the next step was to determine how the user could communicate specific actions to the robotic arms. Since the 49-year-old test subject could perform subtle movements with his wrists and hands, the team decided to use muscle movement signals from his hands and wrists to control the prosthetics. For instance, an open palm would cause the robot to move upwards. By pinching two fingers together, the user could command the robot to move downwards. Since a key feature of a collaborative shared control system is that the user only has control during task-specific times, the Johns Hopkins team programmed the computer to play audio prompts 600

when the robotic arms began to perform a task. These audio prompts would give the user an opportunity to respond with hand/wrist gestures, allowing the user to guide the prosthetic’s positioning during the task. After lots of practice using the dual brain-machine interface and robotic prosthetics system, the 49-year-old test subject was able to successfully communicate specific neural signals to the computer using his hand/wrist gestures 85% of the time and was ready to participate in a more complicated experiment. The culmination of this study was a self-feeding experiment. A dessert pastry was placed on a table between two robotic prosthetic arms equipped with a fork and a knife. The participant was then tasked to cut a piece of the pastry and bring it to his mouth using neural signals from his hand/wrist gestures. A trial would be considered fully successful if the participant was able to cut the pastry and fully complete the self-feeding portion of the task.

Figure 1: Self-feeding taskHANDELMAN ET AL., FRONTIERS IN NEUROROBOTICS (2022) DOI:HTTPS://DOI.ORG/10.3389/FNBOT.2022.918001

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After calibrating the robotic prosthetics and performing thirtyseven trials total, researchers found that the participant was able to successfully cut off a reasonably sized piece of food and bring it to his face without dropping it in seven of the trials. In seven partially successful trials, the participant was able to cut the food, but the food fell off the fork. In a total of twenty-six out of the thirty-seven trials, the participant was able to demonstrate control of both prosthetics. While these results are preliminary, this study marks a crucial step in advancing the fields of neurorobotics, smart prosthetics, and regenerative medicine. As brain-machine interfaces and robotic prosthetics continue to advance, our ability to return greater independence and function to those who are either disabled or suffer from sensorimotor deficits will only grow. This article is featured on Forbes.org, and can be read online here: Robotic Arms Allow Partially Paralyzed Man To Feed Himself

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Should You Get Vaccinated For Covid-19? Yes. Will It Protect You From Long Covid? Probably Not. Forbes | July 14, 2022 | Article

The United States is amidst a new Covid-19 wave. BA.2.12.1, the previous variant of concern, has been outcompeted by highly transmissible Omicron sub variants BA.4 and BA.5. BA.4 and 5 contribute 16 and 65 percent respectively to the 110,000+ Covid-19 cases seen daily—a conservative estimate considering at-home testing results are not included. And infection rates are forecasted to rise. These two variants are more likely than BA.2.12.1 to lead to vaccine breakthrough infections. As a result, even persons up-to-date on vaccines or recently recovered from Covid-19 are susceptible to reinfection. It is unknown if BA.4/5 cause less severity of disease than their predecessors. What is known is that, while vaccination does not protect against infection, it seems to successfully prevent hospitalizations and deaths. Latest CDC statistics show a 3.1% uptick in hospitalization rates, averaging over 5,700 hospitalizations a week. The current 7-day moving average of new deaths (273) actually decreased 20.9% from the previous week. Although this level of hospitalization and death is still unacceptable, it is comparatively modest to prior waves. With 78.4% of the US population protected with at least 1 dose of a Covid-19 vaccine, 67% fully vaccinated, and 47.9% boosted, the wide-spread adoption of vaccines has likely dampened the increase in observed rates. Vaccination + Long Covid The consequences for Covid-19 may appear mild, but each infection has the risk of developing serious post Covid complications, otherwise known as Long Covid. There are several circulating definitions for Long Covid. In my book A Family Guide to Long Covid: Questions and Answers, I define Long Covid broadly to highlight several long-term complications, such as organ damage 603

caused by acute Covid-19. More generally, it is characterized by a heterogeneous collection of symptoms which arise after SARSCoV-2 infection has cleared and linger for a long time. Fatigue, shortness of breath, and brain fog are commonly reported symptoms which, depending on severity, can intensely disrupt daily life for months (or more). Current covid-19 vaccines prevent hospitalization and death— do they also prevent Long Covid? A study published in The Journal of the American Medical Association confirms an existing observation: that vaccination provides little protection against developing Long Covid. This study observed 739 Italian health care workers who had Covid-19 but were not hospitalized for it. 229 individuals from this group had Long Covid. The authors defined Long Covid as any SARS-CoV-2-related symptom which lasted more than four weeks. Individuals with asymptomatic Covid-19 were included in 739 member cohort although they could not, by the study’s standards, develop Long Covid (note that asymptomatic cases have been observed to lead to Long Covid beyond the scope of this study). All health care workers completed surveys—which included a list of SARS-CoV-2 symptoms and their duration along with vaccination status—and were tested every one to two weeks for positive SARS-CoV-2 results. Some mentioned symptoms are fatigue, loss of concentration, shortness of breath, and problems sleeping.

FIGURE 1: The correlation between Long Covid prevalence and vaccination status, as referenced by "Association Between BNT162b2 Vaccination and Long COVID After

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Infections Not Requiring Hospitalization in Health Care Workers. JAMA" ACCESS HEALTH INTERNATIONAL...Insert Text Above

From the survey results, the authors found that the number of vaccine doses was associated with lower Long Covid prevalence. Unvaccinated health care workers reported a 41.8% incidence of Long Covid; those with one dose reported 30%. Patients with two doses reported a 17.4% incidence, and those with three reported 16%. According to these results, vaccines can only reduce one’s chances of getting Long Covid by about 24-5% at best, and only if the individual is fully vaccinated and/or boosted. All administered doses in this study were of Pfizer’s BNT162b2 vaccine. This data supplements findings by a study of US veterans which saw a 15% reduction of Long Covid after vaccination. The scale of the veteran’s study was much larger. However, the age skewed towards older persons and included a low number of women (only 8-10%). This is an important distinction, as women are more likely to develop Long Covid symptoms after infection compared to men. The two studies suggest that vaccination can reduce one’s chances of Long Covid, but the protection gained is not strong. Individuals with at least two vaccine doses may benefit slightly from this correlation. Is it worth getting vaccinated? Ultimately, should you still get vaccinated and boosted? Generally, yes. Vaccination is essential to reducing one’s chances of hospitalization and death. However, if you’re looking to get boosted to avoid Long Covid, it’s best to temper expectations. Recent studies suggest that current Covid-19 vaccines offer meager protection from Long Covid. The best means of avoiding Long Covid will be to use preventative measures as needed—such as mask wearing and social distancing—to prevent getting Covid-19 to begin with. This article is featured on Forbes.org, and can be read online here: Should You Get Vaccinated For Covid-19? Yes. Will It Protect You From Long Covid? Probably Not.

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Are Chronic Infections Responsible For SARSCoV-2 Variants? Forbes | July 15, 2022 | Article

This article is part of an ongoing series investigating the link between chronic infections and the emergence of new SARS-CoV-2 variants. Here, we give an overview of a novel preprint by the Grubaugh Lab at the Yale School of Public Health. The next article will provide a deep dive into the various mutations that emerged during the chronic infection and their implications for viral fitness. In the spring of 2022, a group of researchers at the Yale School of Public Health happened upon a mystery: their genomic surveillance dataset was picking up a SARS-CoV-2 lineage, B.1.517, thought to have gone extinct in the United States and globally as far back as April of 2021. How had this supposedly dead strain made its way into their database? The researchers traced the sequences to an immunocompromised individual in Connecticut, USA. They discovered that this patient, who was battling lymphoma and had previously undergone a stem cell transplantation, was suffering from a chronic SARS-CoV-2 infection — more than 470 days after initially contracting Covid-19, the virus continued to circulate throughout their body. As part of the Yale SARS-CoV-2 Genomic Surveillance Initiative —which was established with the emergence of the Alpha variant— 30 nasal swabs had been collected from the immunocompromised patient between February 2021 and March 2022. This allowed Chaguza et al. to derive whole-genome sequences of the virus, giving them a sense of how it evolved over the course of the infection. Their findings are published on the preprint server medRvix, and offer deep insights into the potential origins of SARS-CoV-2 variants. Although rare within the global population as a whole, chronic infections of this type are not uncommon amongst the immunocompromised community. Often, the immune systems of 606

such patients cannot clear the virus and end the infection, meaning the virus continues to replicate for weeks, months, or even years. The danger? As the virus remains within the body, it has the time to adapt to the weakened immune system of the host, developing new mutations to equip itself against host defenses better. This is precisely what Chaguza and colleagues saw unfold in the Connecticut patient. They tested a subset of 12 nasal swabs for viral load — the amount of virus present in the body— and found that the individual had high levels of infectious viral copies for nearly the entirety of their infection (Figure 1). Not only does this confirm long-term viral replication, it also suggests the patient may transmit the virus to others for the duration of their infection. Added to this risk is the fact that, barring a first week of mild respiratory symptoms, the patient remained asymptomatic. This means immunocompromised patients could very easily remain unaware of their chronic SARS-CoV-2 infection, continue interacting with others as usual, and transmit the virus all the while.

FIGURE 1. (A) Timeline showing clinical history of the patient from the earliest time they tested ... [+] FROM: “ACCELERATED SARS-COV-2 INTRAHOST EVOLUTION LEADING TO DISTINCT GENOTYPES DURING CHRONIC INFECTION” CHAGUZA ET AL. 2022

A second crucial finding of the study is that the evolutionary rate of the virus, which describes the speed at which it mutates, proved to be significantly higher in the immunocompromised patient than in the general population — roughly twice as fast as the average global SARS-CoV-2 evolutionary rate. 607

Predictably, this goes hand in hand with the emergence of new variants. The researchers witnessed the formation of three distinct viral genotypes over the duration of the chronic infection, all of which had upwards of ten amino acid mutations (Figure 2). These persisted for extended periods of time, implying they were advantageous to viral fitness, likely improving immune escape. Some of the mutations that kept popping up had already been seen in other variants of concern; a spike protein substitution, E484K, seen in the Beta, Gamma, Eta, Iota, and Mu variants, for example. This points towards a third crucial finding: a single chronic infection can give rise to multiple unique variants.

FIGURE 2. (C) Time-resolved phylogeny of the chronic infection samples with branch lengths scaled by ... [+] FROM: CHAGUZA ET AL. 2022

Curiously, the three genotypes remained present in the immunocompromised patient at the same time, rather than replacing one another (Figure 2). This suggests they may have been inhabiting different tissues or cells within the body, allowing them to coexist. Still, the variants would switch in and out, taking turns being the dominant genotype: genotype 1 remained dominant from day 79 to 247, followed by a volatile period during which the three genotypes frequently jumped back and forth in dominance. For example, in the span of only 100 days —day 281 to day 381— genotype 1 and genotype 2 alternated between being the dominant genotype a total of five times. During the later stages of infection genotype 3 briefly rose to dominance, only to again be replaced by genotype 2. Although unexpected, it is not entirely unprecedented. A similar scenario was observed in a study of an immunocompromised patient in London, England. For the first 57 days of infection, there was little change to the overall structure of the SARS-CoV-2 population. 608

Then, following treatment with a highly potent preparation of antiSARS-CoV-2 antibodies from three different patients, a dominant viral genotype suddenly emerged. This lasted until the patient’s antibody levels began dropping again, at which point the virus with the immune escape genotype began to fade. It returned full force during a final, unsuccessful course of antibody treatment. The simultaneous circulation of different genotypes presents the additional risk of recombination, a process whereby different viral strains exchange genetic information, creating new opportunities to overcome selective pressures. Although no recombination was seen in this particular case, we know that it is common amongst coronaviruses — especially when leaping from other animals into humans, the defining changes often happen via recombination, and SARS-CoV-2 is no exception. This is because recombination acts as a very quick way of sharing vast amounts of genetic information, helping viruses to diversify their genomes in large strides rather than small steps. Such large jumps make it extremely difficult for our immune system to keep up, with prior infection offering only little guidance. Another surprising finding: the Spike protein did not have the highest frequency of nonsynonymous changes. Synonymous changes are minor mutations to the viral genome that do not alter the amino acid sequence of a protein — loosely, they do not impact the shape and function of a protein. Nonsynonymous changes, on the other hand, are mutations that do impact the amino acid sequence. The higher the frequency, the higher the selective pressure for that protein. Often there is high selective pressure for the genes encoding the Spike protein, since any advantageous mutations will lead to higher infectivity. But instead of Spike, the accessory protein Orf10 saw the highest frequency of nonsynonymous changes, followed by the accessory protein Orf6 and the envelope protein. Orf6 is closely linked to immune evasion, and Orf10 is closely linked to immune suppression. This suggests that mutations to these genes may convey additional advantage; this may simply be a special property of immune selection in immunocompromised persons, but it may also reflect important contributions of these genes to overall viral fitness in a broader population. The full list of nonsynonymous changes in each of the three genotypes can be seen below, in figures 3, 4, and 5. 609

FIGURE 3. Mutations to the SARS-CoV-2 genome seen in genotype 1 (V1/blue), genotype 2 (V2/pink), and ... [+] SOURCE: ACCESS HEALTH INTERNATIONAL

FIGURE 4. Mutations to the SARS-CoV-2 Spike protein in: genotype 1 (V1/blue), genotype 2 (V2/pink), ... [+] SOURCE: ACCESS HEALTH INTERNATIONAL

FIGURE 5. A schematic of the overlap in mutations between the three novel genotypes found in the ... [+] SOURCE: ACCESS HEALTH INTERNATIONAL

Take-Aways The study by Chaguza et al. supports the hypothesis that chronic infection of immunocompromised individuals may be one of the 610

primary vectors for the emergence of novel, unpredictable variants. Theirs adds to a long list of studies documenting similar cases; 24 confirmed occurrences so far, but the actual number is likely to be much higher. I have analyzed a few of these, including the Boston, Pittsburgh, Italy, and Austria examples. But, how big of a problem is this really? So far there have been 550 million confirmed Covid-19 cases. Realistically, this is a vast underestimate. The actual number likely sits somewhere between 3 and 5 billion. Even if only 1% of those are immunocompromised, that leaves us with around 30 to 50 million individuals susceptible to chronic infection and, by extension, the incubation of new variants. There are around 37.7 million people living with HIV alone, not to mention other immunocompromised communities including cancer patients, organ transplant recipients, and those suffering from autoimmune disorders. We need to make sure that we prioritize the treatment of immunocompromised patients, helping them clear their infection as quickly as possible. We also need to double down on global SARSCoV-2 surveillance, particularly whole-genome sequencing. Without a solid surveillance infrastructure in place, we become blind to what may be lurking in wait for us around the corner. Both Alpha and Omicron are presumed to have come to us from chronic infections, let’s not make the same costly mistake again. This article is featured on Forbes.org, and can be read online here: Are Chronic Infections Responsible For SARS-CoV-2 Variants?

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New SARS-CoV-2 Variant BA.2.75 Evades All Approved Monoclonal Antibody Therapies Forbes | July 20, 2022 | Article

Viral variation has proved to be a critical weak point in our approach to medical solutions for controlling Covid-19. Over the last two and a half years, we've seen successive waves of reinfection by new variants of those who've been previously infected, those who have been vaccinated and boosted, and those who have been infected, vaccinated, and boosted as well. Behind this unfortunate dynamic is the dramatic variation in the structure of the virus exterior, specifically the Spike protein, which plays a critical role early in infection by binding to the cell surface and forcing entry. Antibodies that recognize this structure can block infection. However, changes in the exterior structure negate antibody collections in convalescent sera and monoclonal antibodies from binding and neutralizing the virus. A recent study by Yamasoba et al. summarizes the effectiveness of existing monoclonal antibodies against a successive set of virus variants, namely the BA.2 variant, which first emerged in late 2021 and quickly spread around the world, driving the most infectious wave of the virus to date, BA 4/5, which are the predominant strains circulating at the time of writing, and BA.2.75, a new sublineage of BA.2 which is likely more infectious and immune evasive than its predecessors, suggesting it may be the predominant variant in the coming weeks and months.

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FIGURE 1: Neutralization assay was performed using pseudoviruses harboring the SARS-CoV-2 Spike ... [+] YAMASOBA ET AL.The ability of the virus to evade natural immunity from the previously infected and vaccinated is also reflected in its ability to escape a host of specific monoclonal antibodies. As is clear from Figure one, the later Omicron viruses evade monoclonal antibodies much more effectively than early strains.

Immediately, we note that five antibodies: adintrevimab, bamlanivimab, casirivimab, etesevimab, and imdevimab failed to neutralize any of the three Omicron sublineages. Casirivimab and imdevimab, as well as etesevimab and bamlanivimab, are designed to be used in tandem in an antibody cocktail, yet their combination antibodies were just as ineffective. Adintrevimab is intended for individual use, meaning its neutralization potency for the strains circulating today is nonexistent. These were among the first monoclonal antibodies developed, rationalizing why they are so ineffective against recent strains. This leaves five individual monoclonal antibodies. Regdanvimab, sotrovimab, and tixagevimab did not neutralize the previously circulating BA.2 and the currently circulating BA.4/5. However, the three effectively neutralized the BA.2.75 pseudovirus. This suggests that if BA.2.75 became the dominant strain in the coming weeks and months, these three monoclonal antibodies could be effective treatments for those suffering from Covid due to this strain. Of the two remaining antibodies, cilgavimab poorly neutralized BA.2 and BA.4/5 but was 24.4-fold worse against BA.2.75. Although bebtelovimab effectively neutralized BA.2 and BA.4/5, it 613

again was much worse against BA.2.75, this time 21.2 to 25.6-fold. Despite poorly neutralizing BA.2.75 compared to BA.4/5, bebtelovimab still neutralized the strain better than any other antibody. Even newer generations of viruses recently detected in South Africa with more extensively mutated Spike proteins, against which bebtelovimab and others may perform even more poorly. New variants evading monoclonal antibodies should come as no surprise. After infection, the convalescent sera of a recovered patient contains many antibodies designed to inhibit the virus the host just overcame. For the virus to reinfect, it must mutate considerably to evade the convalescent antibodies. Monoclonal antibodies are effectively the same as convalescent antibodies on an individual scale. They are designed to overcome a virus by binding to specific amino acids on the Spike. If the virus mutates enough, the monoclonal antibody can no longer bind. This is how the cat and mouse game of developing antibodies and the virus mutating has continued for two and a half years. What then can be done? The search is on for monoclonal antibodies that recognize regions of the virus that are critical to the virus life cycle and therefore are resistant to most mutations. In other words, scientists worldwide are rushing to identify and develop antibodies with broadly neutralizing capabilities, i.e., antibodies that recognize highly conserved sequences of the Spike protein that may overcome all viral variants. The good news is that many such antibodies have already been identified. We recently described the Cv2.1169 antibody discovered by scientists at the Pasteur Institute and will continue to detail others as data is released. Whether these antibodies recognize and neutralize the latest variants such as BA.2.75 remains an open question. A second potential solution is to use extensive combinations of functional monoclonal antibodies. While many fail to neutralize, some retain neutralizing capability against the latest variants, and new monoclonal antibodies are constantly advancing. Combining two, three, or four antibodies into a single treatment may suppress infection. Our hope remains high for monoclonal antibodies as a short-term relief for those infected and, in the long run, as a prophylactic against infection in the first place.

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This article is featured on Forbes.org, and can be read online here: New SARS-CoV-2 Variant BA.2.75 Evades All Approved Monoclonal Antibody Therapies

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How to advocate for yourself as you struggle with long COVID New York Daily Post | July 21, 2022 | Article

It is estimated that between 10% to 30% of those infected with COVID-19 go on to experience long COVID symptoms. Yet the road to diagnosis for many such patients is often challenging. Long COVID hasn’t yet been clearly defined for health-care providers or patients and screening protocols are still evolving. Public health messaging about the risks of long COVID is also very limited. Many patients have reported having their symptoms dismissed by providers or are not aware that their symptoms may be linked to a prior COVID infection. A correct diagnosis is critical to unlocking treatment options, eligibility for clinical trials and financial coverage through insurance. A diagnosis also allows some to seek necessary disability benefits or work accommodations from the agencies that determine benefit eligibility. Thankfully, there are steps that all patients can take to advocate for a correct diagnosis. Patients should begin by tracking their symptoms. This can be done through a journal, worksheet or a smartphone app. The goal is to be consistent and descriptive when tracking your symptoms. Consider questions like: What are you doing when symptoms occur? Are you active or resting? Is it night or day? What if anything alleviates your symptoms? What makes them worse? How do your symptoms impact your daily living and general functioning? The type of care long COVID patients need depends on their symptoms and will often be multidisciplinary. Some patients see pulmonologists, who specialize in lung conditions, or cardiologists, who specialize in the heart. Other patients see physical therapists to work through limitations in mobility and speech therapists to regain the ability to speak during episodes of brain fog. It can be helpful to begin with a primary-care physician who can conduct initial exams and make referrals. You should bring your symptom tracker to your appointment and be prepared to share your 616

medical and surgical history, family history and social history (i.e. alcohol and drug use). Your medication list should also include any supplements or homeopathic remedies you are taking or have taken. It is important to establish with your physician how any new symptoms have disrupted your daily lifestyle and productivity prior to COVID-19 infection. As long COVID is currently a diagnosis of exclusion, meaning we assign it when we can’t find any other explanation for a set of symptoms, all of this rich information will help your doctor rule out other conditions. Experiencing long COVID symptoms (especially for those who have previously had a clean bill of health) can often feel overwhelming and isolating. You may want to consider joining patient advocacy and peer support groups such as Body Politic, Survivor Corps and the Patient-Led Research Collaborative to connect with others who are experiencing the same issues. If you feel your doctor is dismissing your symptoms and concerns, you may want to bring a family member or a friend to appointments who can help you advocate and take notes. You can also hire a professional patient advocate, or some nonprofits will match patients with patient advocates free of charge. Patient advocates are often former nurses or physicians or sometimes they are people whose own personal experiences have instilled in them a passion for advocacy. The Patient Advocate Foundation and the National Association of Healthcare Advocacy are great resources to learn more about patient advocates. And take heart that, while psychological symptoms can be involved, from everything we know, long COVID is not simply caused by stress or anxiety. If your doctor insists what you are experiencing is merely anxiety, you may need to change providers. Survivor Corps maintains a database of post-COVID clinics across America which is a good place to start your search. Long COVID is a complex illness and new data and insights are being uncovered every day. But by securing a diagnosis, patients can take their first steps toward recovery. This article is featured on Forbes.org, and can be read online here: How to advocate for yourself as you struggle with long COVID

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Can Inflammation Lead To Cognitive Issues? Past Research Says 'Yes', With Implications For Covid-19 Forbes | July 21, 2022 | Article

One of the most worrying consequences of Covid-19 is persistent cognitive impairment, including “brain fog”, difficulty concentrating, and poor short-term memory. These symptoms can be debilitating, often interfering with everyday tasks and forcing individuals to take extended periods of time off work. Understanding this damage, both in the short and long term, is a pressing challenge. Here, I discuss a few discoveries from the past two decades that may help us with this task. In a follow-up article, I will discuss how these findings have been used to study the effects of mild SARS-CoV-2 infection on the cognitive health of mice, with potential implications for Long Covid. “Chemo Brain” and the Hippocampus The first contribution comes from a body of work produced during the early 2000s by a group of researchers at Stanford University’s Department of Neurosurgery. Spearheaded by neuroscientist Michelle Monje, the group set out to pinpoint the mechanisms underlying the cognitive dysfunction associated with cranial radiation therapy, a kind of cancer treatment used to prevent the spread of cancer cells to the brain. This decrease in mental sharpness has since become well-established —now referred to as “cancer-therapy-related cognitive impairment” (CRCI), or “chemo brain”— and can happen in response to other cancer treatments as well, such as chemotherapy. The hippocampus is a complex brain structure closely involved in processes of both memory and learning, two areas heavily impacted by dysfunction following cranial radiation therapy. Dr. Monje and colleagues focused their attention on the hippocampus, discovering that cranial radiation therapy leads to significant neural cell dysfunction; in particular, rat models demonstrated that 618

irradiation was interfering with the formation of new neurons, known as neurogenesis. The continued production of new neurons is vital to healthy cognitive function. In some hippocampal regions, the number of newborn neurons was reduced by as much as 52% compared to non-irradiated levels. So, a decrease in the number of new nerve cells in the hippocampus led to the cognitive dysfunctions associated with “chemo brain''. But, what was causing the decrease? In a paper published in the winter of 2003, Dr. Monje returned with an answer: inflammation. Given that cranial radiation therapy kills dividing cancer cells in the brain, it brings with it a degree of damage to other cells as well. For example, the endothelial cells of the small blood vessels in the brain. This damage can activate immune cells called microglia, which in turn trigger small signaling molecules called cytokines that stimulate the inflammatory response. The neuroscientists hypothesized that the increase in cytokines, and by extension inflammation, that accompanies irradiation may be enough to throw the microenvironment of the brain out of balance, leading to the inhibition of neurogenesis in the hippocampus. To test this theory, the researchers injected rats with bacterial lipopolysaccharide (LPS) to stimulate systemic inflammation. Injection of LPS into the peritoneal cavity —which wraps around many of our abdominal organs, including the stomach and small intestine— causes an inflammatory cascade that eventually induces the release of pro-inflammatory cytokines in the brain, simulating what one might see after cranial radiation therapy. The rats were then treated with a chemical compound, bromo-deoxyuridine (BrdU), that is used to study the proliferation of cells. After 6 full days of exposure, the small team of experts analyzed the proliferative cells in the hippocampus. They noticed an immense reactivity in the microglial cells of the hippocampus; a 240% increase in the density of active microglial cells. In normal, healthy animals, very few activated microglial cells are found. This is because microglia are some of the major immune cells of the brain, in charge of destroying invading microbes, disposing of damaged or dying cells, and stimulating proinflammatory cytokines. As such, unless something has gone wrong

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and the central nervous system is damaged, microglia will be mainly dormant, performing only routine homeostatic functions. Crucially, microglial activation was accompanied by a 35% reduction in the formation of new, mature neurons in the brain of irradiated rats. Plotting the relationship between microglial inflammation and neurogenesis yielded a striking negative correlation — the higher the load of activated microglia, the weaker the formation of new neurons (Figure 1). This suggests that chronic inflammation can lead to a dysfunction in neural stem cells, suppressing neurogenesis and worsening cognitive function. One of the cytokines stimulated by microglial cells, called interleukin-6 (IL-6), was associated especially strongly with a decline in neurogenesis. Exposing progenitor neural cells to IL-6 in vitro led to a 50% reduction in new neurons, implicating IL-6 as a key player.

FIGURE 1. Inflammation negatively correlates with the accumulation of new neurons. The fraction of dividing cells adopting a neuronal phenotype is inversely proportional to total number of activated microglia per dentate gyrus. Each data point represents one irradiated animal. Control irradiated animals, black diamonds; indomethacin-treated irradiated animals, gray squares. FROM: “INFLAMMATORY BLOCKADE RESTORES ADULT HIPPOCAMPAL NEUROGENESIS” MONJE ET AL. 2003

To confirm that inflammation was responsible for the interference with neurogenesis, the researchers treated mice they had injected with LPS with a common non-steroidal anti-inflammatory drug, indomethacin. This lowered activation of microglial cells and 620

lowered systemic inflammation, successfully restoring hippocampal neurogenesis. All told, the work by Dr. Monje and her colleagues helped link systemic, general inflammation to cognitive dysfunction, laying part of the groundwork for current research on SARS-CoV-2-induced cognitive issues. Chemokines and Cognitive Decline with Age In 2011, a different group of researchers in the Department of Neurology and Neurological Sciences at Stanford University released a study investigating the mechanisms underpinning the cognitive decline that comes with aging. Chemokines—a group of cytokines principally responsible for stimulating cell migration— were found to be a driving force of dysfunction, with C-C motif chemokine 11 (CCL11) sitting at the steering wheel. To study the decline in neurogenesis observed during aging, Villeda et al. made use of laboratory technique called parabiosis. This is when two organisms are surgically connected so that they share the same bloodstream. Surgical union allows researchers to study the effects that cells circulating in the blood of one animal have on the other animal — it can be used to pinpoint the impacts of secreted factors, such as cytokines and chemokines. The scientists connected young mice that displayed healthy cognitive function and normal neurogenesis to old mice suffering from poor memory and impaired neurogenesis (Figure 2). As a control, they also connected young mice to young mice, and old mice to old mice. The scientists connected young mice that displayed healthy cognitive function and normal neurogenesis to old mice suffering from poor memory and impaired neurogenesis (Figure 2). As a control, they also connected young mice to young mice, and old mice to old mice.

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FIGURE 2. Schematic of the parabiotic pairings used by the researchers to study the impacts of aging on cognitive function. FROM: “THE AGEING SYSTEMIC MILIEU NEGATIVELY REGULATES NEUROGENESIS AND COGNITIVE FUNCTION” VILLEDA ET AL. 2011

After being paired to the old mice, the young mice displayed a decrease in the production of new neurons as well as a decrease in the proliferation of neural progenitor cells. The opposite occurred in the older mice, who saw a slight reversal in their cognitive decline due to improvements in hippocampal neurogenesis. Mice paired with other mice their age saw no such changes. The same results held true when blood from old mice was directly injected into younger mice. In both cases, reduced neurogenesis following exposure to the blood from old mice also led to functional impairments in cognition, as measured by fear conditioning and memory tests. This indicates that some systemic, age-dependent blood-borne factors were to blame for the sudden differences in neurogenesis. Vidella and colleagues analyzed the blood of the old mice, the young mice, and the young-old parabiotic pairs in order to find the exact molecules associated with worsening neurogenesis. They compared the levels of 66 different cytokines, chemokines, and other signaling molecules between the different groups. Six of these stood out, elevated in both unpaired old mice and in young mice who had been surgically paired with old mice: CCL2, CCL11, CCL12, CCL19, haptoglobin and β2-microglobulin (Figure 3).

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FIGURE 3. Venn diagram of results from aging and parabiosis proteomic screens. In gray are shown the seventeen age-related plasma factors that correlated most strongly with decreased neurogenesis, in red are shown the fifteen plasma factors that increased between young isochronic and young heterochronic parabionts, and in the brown intersection are the six factors elevated in both screens. Data from 5–6 mice per age group. FROM: VIDELLA ET AL. 2011

The presence of CCL11 was surprising, as it had not previously been linked to aging. Curious to see whether this was the case in humans as well, the researchers analyzed blood samples from people aged between 20 and 90. Indeed, CCL11 levels in humans also increased with age. Upon injecting live young mice with CLL11, the researchers noted a decrease in neurogenesis. Injection of CLL11 in combination with an anti-CLL11 antibody negated the effects. The CCL11-induced impairment of neurogenesis was also associated with a functional decline in cognition. Implications for Covid-19 Together, these various studies hint at an important point: systemic inflammation itself can slow the production of new neural cells in the hippocampus, which is in charge of memory and learning. That is, a viral infection could lead to cognitive dysfunction even in the absence of direct infection of the brain or central nervous system. Covid-19, as already mentioned, is characterized by high levels of pro-inflammatory cytokines and chemokines. Might these play a role in the cognitive impairments suffered by many Covid-19 and Long Covid patients? The circumstantial evidence is strong. 623

In a coming article, I explain how scientists at the Yale School of Medicine used these past findings as a springboard for insightful new research on Covid-19-induced cognitive issues. This article is featured on Forbes.org, and can be read online here: Can Inflammation Lead To Cognitive Issues? Past Research Says 'Yes', With Implications For Covid-19

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Learn from past pandemic mistakes and authorize second boosters for all Forbes | July 23, 2022 | Article

The U.S. is experiencing a surge in COVID-19 cases, driven by the highly transmissible BA.4 and BA.5 variants. We need to use all the tools available to protect ourselves from severe disease and death and to protect our health care systems from becoming overwhelmed as hospitalizations increase. That’s why I am calling for the Biden administration, the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA) to authorize and recommend second boosters for all Americans. A fourth dose of the mRNA vaccines is one tool that can be deployed swiftly to save lives and hospital beds. Reports from early July state that several top officials, such as Dr. Ashish Jha, the White House’s coronavirus response coordinator, and Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, support this initiative, citing that immunity from booster shots received in 2021 is waning. Yet critics say there is not enough data to support the decision, and by recommending second boosters now, the CDC may weaken its argument for reformulated booster shots in the fall. Unfortunately, this narrative reminds me of the bungled messaging on masks. In March 2020, the CDC unequivocally advised the public not to wear masks, fearing that many would hoard them, resulting in a shortage of masks for health care workers. When it became impossible to ignore the role of asymptomatic transmission, the CDC changed its guidance, but the damage was done, trust was lost and confusion was wrought in the earliest days of the pandemic. The same mistakes were made again in May 2021 when the CDC prematurely announced a sweeping relaxation of face mask guidelines for those who were fully vaccinated to encourage uptake of the vaccine, leaving many vulnerable to the delta surge. We then

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witnessed the same scenario with a delayed booster rollout and omicron in December 2021. Rather than trying to predict how the public will react to public health recommendations, we must give them equitable access to the tools to protect themselves and their communities. This is how we rebuild trust in public health recommendations after several years of confusion, in which public health agencies often gave conflicting recommendations. Empower the public with facts about the benefits of second boosters and let them make the decision for themselves. An ongoing randomized clinical trial in the United Kingdom found that the fourth dose of the mRNA vaccines can both boost cellular and antibody-mediated immunity as effectively as the third dose. Recent data from the CDC suggest that the risks of dying from COVID is reduced 29-fold for those over 50 years old who have had two booster shots. The difficult reality of life with COVID-19 means we are forced to make a myriad of risk assessments each day, especially as protective measures and mandates are rolled back around the globe. Fortunately, the calculus involved in getting a second booster is simple. With new data about the risk of death, blood clots and other negative health outcomes doubling with COVID-19 reinfection and the disabling symptoms associated with long COVID, we must take every opportunity to protect ourselves. This article is featured on Forbes.org, and can be read online here: Learn from past pandemic mistakes and authorize second boosters for all

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New-Wave Materials Help Create Mini Functional Kidneys Forbes | July 26, 2022 | Article

Figure 1: Kidney structures found in lab-grown organoids SPRANG, EINDHOVEN UNIVERSITY OF TECHNOLOGY (2022), LINK: HTTPS://RESEARCH.TUE.NL/EN/PUBLICATIONS/BIOMATERIALSFOR-KIDNEY-ORGANOID-BASED-REGENERATIVE-THERAPY

We may be one step closer to developing artificial kidneys as a replacement for kidney donor transplants. Recently, the Eindhoven University of Technology published a paper detailing an improved method of growing kidney organoids. This may serve as a large stepping stone towards developing complex kidney organoids that can be used as a substitute for kidney transplants. Chronic kidney disease affects over thirty-seven million people in the United States and occurs when the kidneys are damaged and slowly lose their ability to function. Kidney transplants are a viable treatment for chronic kidney disease. However, the number of individuals that require transplants remains substantially larger than the current supply of donor organs. A potential solution to this organ shortage is the use of kidney organoids. Much like Lego pieces can be combined to construct buildings, organoids are small components of organs that are grown within a petri dish. As different types of kidney organoids are 627

developed and used in tandem, scientists hope that they can eventually combine each critical organoid building block to form full, working kidneys. One of the most important structures found in the kidneys is the glomerulus. The glomerulus is responsible for filtering out waste products and extra water in the blood. While scientists have previously developed kidney organoids, many of them have lacked functional glomeruli. As part of his Ph.D. research, Johnick van Sprang aimed to grow kidney organoids that contain functional glomeruli.

Figure 2: Limitations of current kidney organoidsSPRANG, EINDHOVEN UNIVERSITY OF TECHNOLOGY (2022), LINK: HTTPS://RESEARCH.TUE.NL/EN/PUBLICATIONS/BIOMATERIALSFOR-KIDNEY-ORGANOID-BASED-REGENERATIVE-THERAPY

So, how are organoids made? Organoids begin at the stem cell. When stem cells are acquired from a patient’s blood or bone marrow, the cells can be reprogrammed into earlier, embryonic stages of cell development. With careful nurturing, the stem cells can then be transformed into nearly any cell type in the body. Once all the correct cell types have been assembled, scientists can use them to create the critical microstructures of an organoid.

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Figure 3: Kidney organoids are grown from stem cellsSPRANG, EINDHOVEN UNIVERSITY OF TECHNOLOGY (2022), LINK: HTTPS://RESEARCH.TUE.NL/EN/PUBLICATIONS/BIOMATERIALSFOR-KIDNEY-ORGANOID-BASED-REGENERATIVE-THERAPY

Scientists have already discovered how to nurture stem cells into the matrix of cells that are usually found in the kidneys. However, one of the key aspects of forming functional organ components is to provide the cells with the correct environment or structure. Sprang believed that if the cells were placed in an environment that was similar to human tissue, then they would develop as they would in the human body—forming glomeruli in the process. To test this, Sprang began by looking at a material called ureidopyrimidinone. The molecules of this material are able to recognize each other and self-assemble into fiber-like structures. When these fiber-like structures come together in a gel state or solid, they resemble the fibrous components of natural tissue. This new material is also held together by very weak, low-energy bonds. This means that the bonds between its molecules break and reform more easily, making the material very adaptable and dynamic. Depending on how the material is modified and processed, its fibers can exist as a solid, a flexible gel, or it can simply float in solution.

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Figure 4: Ureido-pyrimidinone can exist as a solid, a hydrogel, or it can simply float in solution.SPRANG, EINDHOVEN UNIVERSITY OF TECHNOLOGY (2022), LINK: HTTPS://RESEARCH.TUE.NL/EN/PUBLICATIONS/BIOMATERIALSFOR-KIDNEY-ORGANOID-BASED-REGENERATIVE-THERAPY

By engineering an environment using a material that resembles human tissue, Sprang hoped that he could simulate the body’s natural environment and structural cues to promote proper growth and the development of an advanced kidney organoid. In addition, by processing this new material to transition from a solution to a gel state after the organoid was introduced, the fibers of the material could travel into the kidney organoid while still in a solution state. If successful, this would mean that the material could deliver structural cues to the organoid’s surface as well as to the cells inside the organoid. After testing this method on the growth of several kidney organoids, the results were in. Surprisingly, the material and its structural cues allowed the kidney organoids to grow three times as many functional glomeruli as previous kidney organoids. These results represent a significant step forward in developing functional elements of organs from organoids. As the fields of biomaterials and organoid research continue to advance it will be interesting to see how organoids continue to evolve and how they may help to increase the accessibility of organ transplant treatments. This article is featured on Forbes.org, and can be read online here: NewWave Materials Help Create Mini Functional Kidneys

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies III Forbes | July 26, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. Alongside vaccines, monoclonal antibodies have been billed as the most effective means of treating and preventing Covid-19. This rang true for the first several months of the pandemic, as antibodies designed to neutralize the SARS-CoV-2 Spike protein were significantly effective. However, as the virus mutated, the potency of antibodies declined while virus immune evasion rose. To counter this trend, there is now an ongoing search for broadly neutralizing antibodies that can overcome not just a single strain but all strains of SARS-CoV-2, current and future. In this series, we have discussed several pan-variant monoclonal antibodies, all of which show promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Luo et al.: the SP1-77 antibody. SP1-77 Antibody Origin The typical search for monoclonal antibodies includes the collection of sera from Covid-19 patients and isolating the often dozens or hundreds of distinct antibodies found, testing them individually on pseudoviruses with the SARS-CoV-2 Spike for binding capacity, then sorting for the best neutralizers. A team of researchers from the Massachusetts Institute of Technology took a different approach. Rather than a blind treasure hunt in the convalescent sera of a Covid patient, the researchers developed a specialized mouse model modified with human gene segments VH1-2 and Vκ1-33. These genes are associated with complementarity-determining-region-3 (CDR3) sequences. In 631

essence, this modification results in a far more diverse B cell response when exposed to pathogens like SARS-CoV-2, leading to the identification of more unique and distinct monoclonal antibodies to test. Luo et al. immunized the mouse models by exposing them to the Wuhan Spike twice over four weeks. All mice involved developed strong resistance to the SARS-CoV-2 Spike, yielding 96 antibodies for the researchers to test. SP1-77 Antibody Neutralization Of the 96 candidates, the researchers narrowed the field down to nine that targeted the Spike protein specifically and then down to three that potently neutralized the Wuhan strain of the virus: VHH7-5-82, VHH7-7-53, and SP1-77. These three were tested for neutralization against current and previous variants of concern, both pseudotype and live viruses.

FIGURE 1: Immunizing the mouse model with SARS-CoV-2 spike or RBD elicited potent and broad ... [+] LUO ET AL.

Figure one demonstrates the neutralizing prowess of SP1-77. In the pseudotyped virus assays, SP1-77 neutralized all variants of concern tested to varying degrees, including Alpha, Beta, Gamma, Delta, and both previous and current Omicron strains. Alternatively, the other two antibodies tested failed to neutralize any Omicron strains and struggled against many other previous variants of concern. The live virus neutralization paints a similar picture. Omicron was omitted from the live virus tests for safety, but SP1-77 again neutralizes Alpha, Beta, Gamma, and Delta, whereas the others again struggle. 632

SP1-77 Antibody Target A closer examination of the SP1-77 antibody via cryo-electron microscopy reveals an alternate solution to neutralization aside from inhibiting ACE2 binding. SP1-77 targets sites on the opposite side of the receptor-binding domain from the ACE2 binding site. In particular, the antibody epitope targeted positions ranging from 339346, 440-450, and 499. In most variants of concern, these amino acids are unmutated, with the lone exception being G339 and G446 in some Omicron strains being changed.

FIGURE 2: Binding footprint of SP1-77 as compared to other neutralizing antibodies S309 and ... [+] LUO ET AL.

FIGURE 3: Cryo-EM structures of the SP1-77 Fabs in complex with the full-length S trimer in the ... [+] LUO ET AL.

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Further analysis found that the antibody binding results in either the blockage of S2’ cleavage and/or inhibition of S1 dissociation. What does this mean? After the virus binds a human ACE2 receptor, the S1 and S2 portions of the Spike must disengage for membrane fusion to occur. If the antibody is blocking either S2’ cleavage or S1 dissociation, it is inertly preventing the virus from fusing with the host cell, preventing that virus from replicating within the host cell and moving on to another. We have previously described two other antibodies that bind to different sites. The first, 35B5, targets the N-terminal domain positions N165 and N234, which act together as a molecular switch for the Spike’s changing up and down conformations. The second, Cv2.1169, targets a different region of the receptor-binding domain than SP1-77, namely contact points including Y473, S477, T478, F486, N487, and Q493. We will describe more in the coming days and weeks. Echoing Luo et al., SP1-77 represents an opportunity for highly potent monoclonal treatments. In addition to being an effective antibody on its own, due to its distinct residue targets, SP1-77 could be easily paired with one or two other antibodies to create a highly potent antibody cocktail for the treatment and prevention of Covid19. Such cocktails should be immediately investigated. Additionally, the novel humanized mouse model is a triumph of antibody discovery technology. The methods by Luo et al. should be further explored in all antibody discovery trials, potentially leading to dozens of antibodies to aid the millions continuing to get sick in this extended pandemic. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies III

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies IV Forbes | July 28, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. Monoclonal antibody treatments remain our greatest asset in the fight against Covid-19, though in recent months, the effectiveness of these treatments has waned. New variants with more mutations have evolved to overcome antibodies from monoclonal therapies, vaccines, and prior infections. The search for monoclonal antibodies that neutralize not one but all strains is underway to counter these new variants. In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Zhou et al.: the ZCB11 antibody. ZBC11 Antibody Origin Hong Kong researchers used an uncommon antibody identification method in their study. Antibodies described previously in this series were identified either from the sera of those naturally infected with Covid-19 or from a specially-engineered mouse model. Here researchers use the sera of mRNA vaccine inoculated patients as the basis for antibody collection. Collecting the sera of 34 Pfizer vaccinated subjects, Zhou et al. found that only two of the 34 samples had neutralizing activities against all variants of concern included in the study, notably Alpha, Beta, Gamma, Delta, and Omicron BA.1. Subject #26 displayed the highest neutralization titers against Beta and Delta. They showed above-average neutralization of Alpha, Gamma, and Omicron.

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Zhou et al., therefore, took a closer look at the sera of this vaccine for broadly neutralizing antibodies.

FIGURE 1: Serially diluted plasma samples subjected to neutralization assay against the pseudotyped ... [+] ZHOU ET AL.

The researchers collected another blood sample 130 days after the second vaccination. Fourteen antibodies were identified, while only seven (ZCB3, ZCB8, ZCB9, ZCB11, ZCC10, ZCD3, ZCD4) showed positive responses to WT spike. They narrowed these seven to four by limiting their search to receptor-binding domain-specific antibodies that displayed neutralization against the wildtype Wuhan strain, namely ZCB3, ZCB11, ZCC10, and ZCD3. Along with a control antibody, ZB8, these four moved onto VOC neutralization assays. ZCB11 Antibody Neutralization

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Zhou et al. conducted both pseudovirus and live virus assays for the four antibody candidates. Against the Alpha, Beta, Gamma, Delta, and Omicron BA.1 pseudotypes, ZCB11 was the only candidate to neutralize all variants and consistently outperformed other antibodies when they did neutralize. Against authentic viruses, this result was replicated to an even greater degree. ZCB11 consistently and effectively neutralized all variants in the lineup, including Omicron BA.1, BA.1.1, and BA.2. We note that the currently circulating BA.4 and BA.5 were not included in the neutralization assays. Still, the potent neutralization of BA.2 and all earlier strains strongly indicates ZCB11’s broad neutralization.

FIGURE 2: Neutralization IC50 values of NAbs against the wildtype, Alpha, Beta, Gamma, Delta, and ... [+]

ZHOU ET AL. ZCB11 Antibody Target Alongside many other monoclonal antibodies, The ZCB11 antibody targets the receptor-binding motif. Cyro-electron microscopy analysis of the antibody revealed the antibody binding to the Spike protein in the “up” conformation, wherein the Spike is preparing to bind the ACE2 receptor of the host cell.

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FIGURE 3: Cryo-EM density map of spike trimer in complex with ZCB11 Fab. Two of three different ... [+] ZHOU ET AL.

Why does this antibody neutralize much more effectively than previous monoclonal treatments when they all bind the receptorbinding domain? We can attribute this to two things. One reason for the broad activity found with ZCB11 is that most of the amino acid contacts are highly conserved among all known coronavirus sequences. The antibody footprint binds the Spike amino acids D420, L455, F456, N460, A475, S477, T478, F486, and N487. Three of these, S477, T478, and F486 are mutated in the latest Omicron strains to S477N, T478K, and F486V. In the GISAID SARS-CoV-2 sequence database, all positions aside from the three Omicron exceptions are mutated less than 5,000 times in over 12 million sequences, meaning the footprint of this antibody is highly conserved. Notably, Zhou et al. found that S477N and T478K increase the binding affinity between the antibody and Spike, rather than interfering with neutralizing capability. They postulate that this is at least a partial explanation for the antibody's substantial neutralization of the variant. 638

The second reason is that most of the amino acid positions in ZCB11 are relatively unmutated in natural strains. Previous antibodies have contact points at major residues of mutation like N501 and E484, but ZCB11 mostly lacks contact points at major mutational residues. As we recommend with all broadly neutralizing antibodies, there is no reason to limit treatment to just one. An antibody cocktail of two or three monoclonal antibodies covering a broad footprint of conserved residues could be a powerful weapon against current and future strains, which are sure to continue mutating to evade immunity. We must prioritize and expedite these antibodies' production as the pandemic continues to rage. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies IV

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A Cooling Implantable Device For Pain Relief Forbes | July 29, 2022 | Article

This story on pain relief is part of an extended series on Regenerative Medicine. For other stories on this topic see williamhaseltine.com and search for Regenerative Medicine. My definition of Regenerative Medicine is any medical modality that returns us to normal health when we are damaged by disease, injured by trauma, disadvantaged by birth, or worn by time. Modalities include: chemicals, genes, proteins and cells used as drugs, gene editing, prosthetics, and mind-machine interfaces. Scientists at Northwestern University have recently engineered a new pain-relieving device that uses temperature control to block pain signals to the brain. The device is a small, flexible implant that could serve as a replacement for opioids or other highly addictive medications. The misuse of opioids is an ongoing crisis in the United States that has only grown since the onset of the Covid-19 pandemic. Despite this, there are very few alternative treatments for pain relief that are as effective, leading to widespread usage of opioids and increased rates of opioid addiction and overdose. Previous studies have shown that pain signals can be blocked from the brain by using cooling implants that lower the temperature of specific nerves. By decreasing their temperature, researchers can decrease the activity of the nerves and inhibit the release of pain signals. However, cooling implants have historically been bulky, imprecise, and have required additional surgeries to remove once the patient no longer needs them. Now, in a paper published in the journal Nature, Reeder et al. describes how they developed a new pain-relieving implant that is not only highly precise but can also be absorbed by the body after use, by-passing the need for any extraction surgeries. To address the challenges of previous cooling implants, Reeder et al. sought to develop a device that would be flexible enough to wrap around specific pain signaling nerves. This would allow the device to be highly precise and target only one nerve at a time. To 640

do so, researchers began by looking at a category of materials called elastomers. Elastomers are very flexible and can be made from plastic, rubber, or resin. They can also be water-soluble, meaning that if a water-soluble elastomer were implanted in the body it would dissolve over time.

Figure 1: The implant wraps around specific nerves for precise, localized cooling.

REEDER ET AL., NATURE (2022), DOI: 10.1126/SCIENCE.ABL8532 But how could an elastomer-based device cool the nerve? Reeder et al. navigated this challenge by taking inspiration from microfluidic systems. A microfluidic system occurs when tiny channels are etched into a material, allowing small amounts of fluid to run through the channels. By incorporating a microfluidic system into the elastomer material, Reeder et al. was able to pass a coolant through the entirety of the implant, ultimately cooling the nerve that the device wrapped around. After incorporating an additional electronic system within the device so that researchers could track the temperature of the nerve and control how much coolant was pumped through the implant, the device was ready to begin testing in rats.

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Figure 2: The implant contains microfluidic channels and an electronic system for temperature control.

REEDER ET AL., NATURE (2022), DOI: 10.1126/SCIENCE.ABL8532 Researchers successfully implanted the cooling device in rats with leg-based nerve injuries, wrapping the device around their sciatic nerve which controls muscles in the lower body. After cooling down the sciatic nerve for eight minutes, researchers found that activity in the nerve had decreased by 92%. In addition, the amount of time it took for activity to travel through the sciatic nerve was significantly longer. After the device had been turned off and the nerve had returned to normal body temperature, Reeder et al. found that activity had also returned to normal levels.

Figure 3: The device was tested on rats with leg-based nerve injuries.

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REEDER ET AL., NATURE (2022), DOI: 10.1126/SCIENCE.ABL8532 While more research must be done to determine whether or not the results of this study are consistent in humans, these experiments are promising and suggest that we may be close to developing a new method for pain management. Hopefully, as innovative pain-relief devices continue to be developed, we will not only see the progression of interesting science but also how it may help curb the opioid crisis and limit access to highly addictive medications. This article is featured on Forbes.org, and can be read online here: A Cooling Implantable Device For Pain Relief Covid-19, Gender And Immune Response: What’s The Relationship? Forbes | July 29, 2022 | Article This is the first installment in a two part series which analyzes biological sex differences in immune responses to SARS-CoV-2 infection. This article focuses primarily on Covid-19 related viral entry as well as innate and adaptive immune responses Covid-19 and their correlation to epidemiological evidence. Part two will highlight the role of sex hormones in SARS-CoV-2 immune responses, examine sex differences in response to several vaccines, and consider their possible therapeutic implications.

Paper cut out illustration of a man and woman facing each other GETTY

Men and women experience Covid-19 differently. Epidemiological studies show that, while males and females have similar infection rates, males are more likely to experience severe 643

Covid-19 and die from SAR-CoV-2 infection. Women tend to have better prognoses; pregnant females are the exception, as they have an increased risk of severe illness, hospitalization, intensive care unit (ICU) admission, mortality and preterm delivery. After excluding societal and behavioral factors, a question remains: what are the biological mechanisms driving these observed differences? Ho et al. attempt to answer this question in their review, The Immune Response to Covid-19: Does sex matter? They consider several biological mechanisms in their work. This article will specifically examine differences in three stages of SARS-CoV-2 immune response: viral entry, activation of host innate immunity, and activation of adaptive immunity. Sex Differences in SARS-CoV-2 Immune Response Immune interaction to SARS-CoV-2 entails viral entry, then recognition of the virus and activation of host innate immunity, followed by activation of adaptive immunity. Ho et al. found several potential mechanisms in these three stages which may explain the stronger immune responses seen in females. Viral Entry In viral entry, SARS-CoV-2 attaches to and enters the host cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptors in upper respiratory tract cells. ACE2 receptors usually decrease inflammation, but the binding alters this function. As a result, ACE2 receptors critically influence SARS-CoV-2 entry to cells and can worsen SARS-CoV-2-caused tissue damage through inflammation. Ho et al. explain some known sex differences in ACE2 levels which could contribue to in the worse clinical outcomes seen in men. Some studies show that men express higher levels of ACE2, a factor which might increase vulnerability to infection. Males and females have similar soluble ACE2 (sACE2) levels up until 12 years old; after, male sACE2 levels exceed levels found in females. One paper demonstrated that females required a lower dose of ACE inhibitors to achieve optimal therapeutic effect. These data demonstrate potential in targeting ACE2 for Covid-19 treatments and for study on SARS-CoV-2 viral susceptibility, but Ho et al. note that more research is needed to further understand this relationship. Innate Immunity

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Sex differences were also found when comparing innate immune responses. The initial innate immune response involves viral detection, interferon (IFN) production and inflammasome activation. Innate immune responses are considered crucial to determining disease outcome. Ho et al. found that females exhibit stronger innate immune responses than their male counterparts. This is seen in sex-specific expression of toll-like receptor 7 (TLR7). Toll-like receptor 7 is important for detecting single RNA viruses such as SARS-CoV-2. It is believed that the more toll-like receptor 7 is expressed, the quicker Covid-19 can be recognized and cleared from the system. Toll-like receptor 7 expression can be upregulated by female sex steroid estrogen. It is also thought to escape X chromosome inactivation in some cells; by evading inactivation, toll-like receptor 7 is therefore expressed more highly in females, who have two copies of the X chromosome. Interferon (IFN) production involves the creation of proteins called cytokines which aid viral suppression after viral recognition. Chemokines, such as interferons, are a subclass of cytokines which encourage immune cells to move towards a target. Important to note are plasmacytoid dendritic cells (pDCs), immune cells which secrete interferons in response to viral infection. Women have higher plasma concentrations of IFNα and can produce more IFNα from plasmacytoid dendritic cells (pDCs) than men due to estrogen. They express greater INF regulatory 5 (IFN5), a significant transcription factor in IFN signaling, in their plasmacytoid dendritic cells than males as well. In contrast, one study found that autoantibodies inhibited type I IFN signaling in older males with severe Covid-19. The last consideration of innate immunity revolves around proinflammatory cytokines. Clinical studies associate increased inflammatory cytokine levels with severe Covid-19 pathology. In some cases, elevated cytokine levels lead to a cytokine storm: the flooding of cytokines in the bloodstream that damages tissues and organs. In this regard, males typically have higher levels of innate proinflammatory cytokines such as interleukin 8 (IL-8) and 18 (IL18) than their female counterparts. They also have higher serum levels of IL-8, IL-18 and chemokine ligand 5 (CCL5). A significant 645

correlation exists between high IL-8 levels and reduction in antiviral white blood cells. In comparison, research suggests the lower levels of cytokine levels—interleukin 6 (IL-6) especially—seen in women with Covid-19 are associated with better clinical outcomes; this is unusual, as women typically have stronger cytokine responses than men. Adaptive Immunity Adaptive immunity is a specialized mechanism which deploys immune cells (lymphocytes) and antibodies to target and destroy pathogens directly. Ho et al. observed sex differences in antibody production, T cell responses and the epigenetic status of immune cells. Generally, females show higher antibody-mediated immune responses to viral infection and vaccination. This positive effect could be tempered by the greater autoreactivity also noted in this sex. Elevated humoral responses in women could be influenced by several estrogen-mediated mechanisms, including but not limited to germinal center formation, selection against autoreactive B cells, and epigenetic accessibility of B-cell specific loci. Sex-different T cell interactions seem to impact Covid-19 recovery. Men with Covid-19 have weaker T cell activation during early disease than women; in comparison, elderly women with early Covid-19 have more robust T-cell activation. Weak T cell activation, in conjunction with lower lymphocyte count, higher neutrophil-to-lymphocyte ratio, and greater serum C-reactive protein (CRP) concentrations, could explain the poorer outcomes seen in males with Covid-19. Ho et al. state more research is necessary to understand sex differences in the role of T cells in acute infection, lung injury, and vaccine targets. Another divergence seen in adaptive immunity is in the epigenetic status of immune cells—in other words, physical changes in immune cell DNA structure which do not impact its genetic sequence. One example of this is aging. Between ages 62 to 64 years, males undergo changes in epigenetic landscape which majorly impact the immune system. There is increased expression of innate proinflammatory genes and decreased expression of adaptive immune system genes. In addition, B cell levels and naïve T cell levels decline faster in older men than in their female counterparts. Women exhibit similar epigenetic changes approximately five to six 646

years later than men. A possible biological mechanism is the overexpression of immune genes on the X chromosome of T cells. This overexpression seems to correlate with incomplete X inactivation—as similarly seen in toll-like receptor 7 expression— and epigenetic modifications. Conclusions Current knowledge of immunity suggests that strong innate immune responses likely contribute to the lower disease severity and mortality outcomes associated with females. Elevated levels of tolllike receptor 7 and IFNα, along with decreased levels of interleukin 6, may correlate to better prognoses in women. On the other hand, the worse clinical outcomes observed in men could be explained by their higher levels of ACE2 and epigenetic changes in their immune cells, These mechanisms leave important clues to understanding the relationship between biological sex and immune responses to SARSCoV-2 infection, but these associations are not linear. More research is needed to further our understanding. This article is featured on Forbes.org, and can be read online here: Covid-19, Gender And Immune Response: What’s The Relationship?

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August 2022

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Even Mild Covid-19 May Cause Lasting Brain Fog (Part 1) Forbes | August 01, 2022 | Article

This is part two of a series focusing on cognitive dysfunction and inflammation. The first article can be read here. For additional writings on inflammation and Covid-19, please see my website: www. williamhaseltine.com Although inflammation is a common feature of many infections, viral and otherwise, SARS-CoV-2 elicits a particularly intense inflammatory response. Exactly why remains unclear. What is becoming increasingly clear, on the other hand, is that much of the damage associated with Covid-19 can be traced back to this hyperinflammation. This holds true both in the short term, during acute infection, as well as the long term, during Long Covid, or Post-Acute Sequelae of SARS CoV-2 infection (PASC). Cognitive impairments such as “brain fog”, difficulty concentrating, and poor short-term memory make up some of the more worrying Long Covid symptoms. Often, these are severe enough to interfere with daily tasks, with significant impact on quality of life. In a collaborative study, researchers at Yale University and Stanford University have uncovered one plausible cause: inflammation. A heightened state of inflammation during Covid-19 can trigger an overabundant activation of microglial cells —the main immune cells of the central nervous system— leading to dysregulation of the cells required for healthy cognitive function, including oligodendrocytes and neural precursor cells. Strikingly, Fernández-Castañeda et al. found that even mild cases of Covid-19 set off enough inflammation to produce impairments to cognition and brain health. Here, I give an overview of the first part of their findings — microglial reactivity— and in a follow-up article, I discuss how this impacts brain health. Microglial cells are a type of macrophage found within the brain and spinal cord, where they make up 10-15% of all cells. As 649

macrophages, their role is to actively devour and destroy any invading microbes. Additionally, they produce a variety of signaling proteins, known as cytokines and chemokines, to help stimulate the inflammatory response and direct other immune cells to areas in need of protection. Despite existing to protect us, reactivity of microglial cells has also been associated with cognitive issues; inflammation is crucial to helping us clear infections, but too much of it can have devastating consequences, especially in an environment as sensitive as the brain. Cancer-therapy-related cognitive impairment is one such example, with microglial reactivity in the hippocampus driving neural dysfunction. The overlap of symptoms with Long Covid — including the presence of a continuous “chemo fog”— made Fernández-Castañeda and colleagues suspect that a similar mechanism may be at play. To test this theory, the experts turned to mouse models. They exposed a group of mice carrying human angiotensin converting enzyme 2 (ACE2) —the primary portal of entry for SARS-CoV2— to a mild SARS-CoV-2 infection that lasted no longer than a week. The mice exhibited no observable symptoms and no weight loss. Importantly, the mice were engineered to only carry ACE2 receptors in their respiratory tract, strictly limiting infection to the nose, throat, and lungs. This made it so that the virus had no direct access to the central nervous system of the mice, meaning any cognitive dysfunction had to instead be a result of knock-on effects. Despite the mild, essentially asymptomatic infection, all of the mice displayed elevated proinflammatory cytokine and chemokine levels, both in the blood and in cerebrospinal fluid. These included: IFN-γ, IL6, TNF-α, CXCL10, CCL7, CCL2, CCL11, GMCSF, and BAFF. Certain cytokines remained elevated up to seven weeks after the initial respiratory infection. As touched on in a previous article, IL-6 has been implicated in cognitive dysfunction following cranial radiation therapy. The chemokine CCL11, in turn, has been implicated in the cognitive decline that accompanies aging. Of note, even though CCL11 levels in the blood had normalized seven weeks after initial infection, in the cerebrospinal fluid they were higher seven weeks after infection than they were seven days after infection. 650

Next, the scientists set out to check whether the prolonged surge of cytokine levels in the cerebrospinal fluid correlated with any noticeable changes in the brain. Compared to control mice, which also carried human ACE2 in the respiratory tract but were infected with a mock virus, those who had been exposed to SARS-CoV-2 displayed increased microglial reactivity in subcortical white matter. White matter makes up the deeper tissues of the brain and serves to connect —and enable communication between— different parts of the central nervous system, particularly the spinal cord and the gray matter regions that comprise the surface of the brain. Think of it as a kind of super highway. White matter is crucial to healthy cognitive function, and changes to white matter can impact the efficiency and speed with which “messages” can be sent throughout the central nervous system — a highway full of potholes can only accommodate so much traffic. Although the total number of microglia remained the same across both Covid-19 and control mice, the number of activated microglia was markedly higher in the sick group. As with cytokine levels, reactive microglial cells persisted for up to seven weeks after infection. Fernández-Castañeda and colleagues also had the rare opportunity to study microglial cell reactivity in human samples. The researchers analyzed the subcortical white matter of nine individuals who were SARS-CoV-2 positive at the time of death, as confirmed by nasal PCR tests. Although not necessarily mild cases, since all nine individuals died of complications related to infection, none of them died while hospitalized and only two required ICU admission. Their lungs also displayed either no or only moderate signs of damage. Still, mirroring what was seen in the mouse models, all nine patients had elevated levels of microglial activation when compared to a control cohort (Figure 1).

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FIGURE 1. (M) A side-by-side comparison of microglial cell activation in the gray and white matter regions of the brain in a Covid-19 and a non-Covid patient. (N) A bar graph quantifying the differences in microglial cell activation. FROM: “MILD RESPIRATORY COVID CAN CAUSE MULTI-LINEAGE NEURAL CELL AND MYELIN DYSREGULATION” FERNÁNDEZ-CASTAÑEDA ET AL. 2022

To get a more precise understanding of the state of microglial cells following Covid-19, the scientists performed single-cell RNA sequencing. This is an extremely fine-grained technique that lets you see the full range of genes expressed within a single cell. Analyzing the data for around 6000 individual microglial cells, FernándezCastañeda et al. noticed an upregulation of genes associated with inflammation, including cytokine production and cytotoxicity. This was accompanied by a downregulation of genes associated with regular homeostatic functions — the maintenance of appropriate connectivity between neurons, the pruning of rarely used synapses, and the nurturing of vital synapses. Essentially, genes that help keep things in balance and support cognitive health. Following a run-in with Covid-19, the gene profile of the reactive microglial cells ends up closely resembling that of microglial cells linked to Alzheimer’s disease as well as those linked to aging, both of which go hand-in-hand with cognitive decline. So, mild Covid-19 can trigger an inflammatory response that makes its way into the brain and activates microglial cells. These cells then stimulate additional pro-inflammatory molecules and general cytotoxicity that can persist for up to seven weeks after infection. The next article in this series focuses on the consequences of such sustained microglial reactivity, particularly in the white matter regions of the brain. 652

This article is featured on Forbes.org, and can be read online here: Even Mild Covid-19 May Cause Lasting Brain Fog (Part 1)

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies V Forbes | August 01, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. As novel SARS-CoV-2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal therapies that worked against earlier variants struggle against current strains. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all strains to counter these new variants. In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Dacon et al.: the COV44-62 and COV44-79 antibodies. COV44-62 and COV44-79 Antibody Origin Researchers from the National Institute of Health and Scripps Research Institute began their search for monoclonal antibodies by a traditional method: examining the plasma samples of 142 previously infected donors. They scanned these samples not only for SARS-CoV-2 recognition but also for six other human coronaviruses, including SARS-1 and MERS. Of the 142 samples, 19 recognized SARS-CoV-2 and at least two other betacoronaviruses. By finding antibodies that recognize multiple betacoronaviruses, the likelihood increases of finding antibodies that recognize various strains of SARS-CoV-2 as well.

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From these 19 samples, they identified a staggering 673,671 IgG B cells. They tested for reactivity and binding specificity to a panel of coronavirus Spike proteins to narrow this pool. They eventually found a set of six monoclonal antibodies that bound all seven human coronaviruses tested.

FIGURE 1: Heat map representing the binding of broadly reactive mAbs to spike proteins from ... [+] DACON ET AL.

COV44-62 and COV44-79 Antibody Neutralization Introducing the set of six monoclonal antibodies to a neutralization assay of pseudotyped betacoronaviruses, Dacon et al. found that two antibodies, COV44-62 and COV44-79, showed the broadest functional neutralization, disabling SARS-CoV-2, SARSCoV-1, and HCoV-OC43, as well as the alphacoronavirus HCoVNL63 and HCoV-229E.

FIGURE 2: Antibody titer neutralization of pseudotyped coronaviruses. DACON ET AL.

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Perhaps more notably for our search, both COV44-62 and COV44-79 strongly neutralized a host of SARS-CoV-2 variants. In more pseudotype assay neutralization tests, COV44-62 and COV4479 effectively neutralized Alpha, Beta, Gamma, Delta, Mu, Omicron BA.1, BA.2, and BA.4/5. Neutralization of Omicron BA.4/5 is significant as they are still the majority sequence fueling infections worldwide as this is written in late July. In general, COV44-62 achieved neutralization at lower concentrations, indicating a more efficient antibody, but we note that COV44-79 neutralizes BA.4/5 more efficiently than COV44-62.

FIGURE 3: Neutralization of SARS-CoV-2 variants of concern (pseudovirus) by COV44-62 and COV44-79. DACON ET AL.

COV44-62 and COV44-79 Antibody Target While the rest of the antibodies in this series have also neutralized many variants of concern, what makes these two antibodies particularly special is their variant neutralization and Spike residue target. Most monoclonal antibodies target amino acids on either the receptor-binding domain, the N-terminal domain, or a combination of the two. These two domains facilitate ACE2 binding to the host 656

cell, and blocking these connections is often an effective way to prevent host cell infection. The COV44-62 and COV44-79, as well as the other four antibodies from the broadly recognizing set, all prefer to bind in the S2 portion of the Spike, specifically the fusion peptide. The two antibodies bind from amino acid positions 812-830 in the SARSCoV-2 Spike protein.

FIGURE 4: Fusion peptide conserved sequence in the SARS-CoV-2 Spike protein. DACON ET AL.

Dacon et al. note that critical binding residues include R815, E819, D820, L822, and F823. These five residues are amongst the most conserved in the coronavirus genera Spike protein, all of which are conserved in at least 34 of 35 coronavirus species. This also holds true for SARS-CoV-2 variants of concern; none carry mutations at these five amino acids. In fact, in the GISAID SARS-CoV-2 sequence database, all five mutations are identified less than 5,000 times among 12 million sequences, many of which are likely dead viruses. 657

Early in vivo trials on Syrian hamster models found that those treated with COV44-79 recovered from moderate to severe symptoms within 3-7 days, and those treated with COV44-62 recovered in 5-7 days. As we recommend with all broadly neutralizing antibodies, there is no reason to limit treatment to just one. An antibody cocktail of two or three monoclonal antibodies covering a broad footprint of conserved residues could be a powerful weapon against current and future strains, which are sure to continue mutating to evade immunity. We must prioritize and expedite these antibodies' production as the pandemic continues to rage. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies V

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Even Mild Covid-19 May Cause Lasting Brain Fog (Part 2) Forbes | August 03, 2022 | Article

This is part three in a series focusing on cognitive dysfunction and inflammation. The first article can be read here, and the second can be read here. For additional writings on inflammation and Covid-19, please see my website: www. williamhaseltine.comFor many people, an encounter with Covid-19 brings with it lingering cognitive symptoms, generally described as “brain fog”. And on occasion, these symptoms may last for months on end. A novel study describes why and how this might be happening. In a previous article, I gave an overview of one part of the researchers’ findings. To recapitulate in brief: SARS-CoV-2 infection can provoke serious inflammation which, through a series of “signaling cascades”, can make its way into the central nervous system, including the brain. Once there, inflammation triggers the activation of immune cells called microglia, which go haywire, causing additional inflammation that disturbs the sensitive microenvironment of the brain. Worryingly, even mild Covid-19 can trigger this domino effect. But in what ways, exactly, does microglial reactivity damage the brain, and how might this damage contribute to cognitive dysfunction? Fernández-Castañeda et al. suggest two main mechanisms of damage: first, a decrease in the production of new neurons in the hippocampus of the brain, known as neurogenesis; and second, loss of oligodendrocytes, which produce the myelin that keeps neuronal communication nice and snappy. In either scenario, symptoms akin to brain fog would be expected. In the context of impaired neurogenesis, a small signaling protein called C-C motif chemokine 11 (CCL11) —already associated with cognitive decline in elderly persons— was identified as a key culprit. Neurogenesis The hippocampus is a brain structure closely involved in both learning and memory-formation. Neurogenesis, in turn, is 659

speculated to be a process through which old or damaged neurons are replaced by fresh neurons, helping hippocampal functions run smoothly. The generation of new neurons is measured by quantifying Doublecortin-positive cells. With the exception of bacterial and archaeal cells, all cells contain a scaffold-like structure —known as the cytoskeleton— made up of interlinking protein filaments. This helps lend cells their shape and lets them maintain their internal organization. Microtubules are one kind of filament. In cells that are going to become neurons and in immature neurons, the protein doublecortin (DCX) helps regulate the formation of microtubules. It is most frequently seen during the embryonic stage of development, at which point the neurons of the brain’s cortical structures are just being formed. But it has also been observed in the hippocampus of certain adult mammals, including humans and various laboratory animals. Given its role in the formation of neurons in the embryo, its presence in the adult brain is thought to indicate continued formation of new neurons even in adulthood. As such, measuring the level of doublecortin in the brain has become a standin for measuring the quantity of cells scheduled to become neurons. Studying the hippocampus of mice that had been infected with SARS-CoV-2, Fernández-Castañeda and colleagues witnessed a significant drop in the amount of cells that expressed doublecortin — which is to say, a significant drop in the formation of new neurons. This was noticeable as early as seven days post infection, and remained the case up to seven weeks after infection. And there was a clear inverse correlation between neurogenesis and reactivity of microglia: the higher the number of reactive microglial cells, the lower the number of new neurons. Looking to pinpoint the driving force behind the atrophying of hippocampal neurogenesis, the group of experts turned to the chemokine CCL11. Although only one of many signaling molecules released as part of the inflammatory response, it is found in particularly high levels in the cerebrospinal fluid of mice recovering from Covid-19. It has also previously been linked to age-related cognitive decline. Indeed, healthy mice injected with CCL11 displayed an increased number of activated microglial cells in the brain. Further implicating the chemokine, the increase was localized specifically to 660

the white matter of the hippocampus, with no reactivity in cortical or other subcortical white matter. Activation of microglia was soon accompanied by a drop in neurogenesis, confirming the researchers’ hunch and identifying CCL11 as a prime suspect. Spurred on by this finding, the researchers also explored whether CCL11 might be associated with the occurrence of cognitive symptoms in Long Covid patients. To do this, they studied blood samples of Long Covid patients who reported cognitive impairments and compared them to blood samples of those who did not have any cognitive symptoms. As expected, patients lacking cognitive symptoms had lower levels of CCL11 circulating in their blood, again implicating the chemokine. An important caveat: the differentiation of stem cells into new, functional neurons in the adult human hippocampus continues to be a point of active debate. There is a growing body of research to support the possibility, but it is far from being consensus. This does not mean that microglial cell activation and inflammation are not key factors in cognitive dysfunction following Covid-19, rather it means we have to also be open to the fact they may be causing damage via a different mechanism. Oligodendrocytes and Myelin Loss One alternative explanation involves the loss of oligodendrocytes in response to activated microglial cells. The main function of oligodendrocytes is the generation of myelin, which wraps tightly around axons. Axons are the “wires” of the central nervous system, enabling the transfer of information in the form of electrical impulses. Myelin serves to insulate and protect these axons, helping to increase the rate at which information can be passed along. The researchers noticed that excess activation of microglial cells following Covid-19 correlated with a noticeable loss of mature oligodendrocytes in subcortical white matter — in infected mice, up to a third of oligodendrocytes had been lost within seven days of initial infection, and this persisted for weeks after the clearance of the virus. As expected, the decrease in oligodendrocytes was also accompanied by a loss of myelin. And again, this lasted for weeks following infection. Speaking at a conference on Long Covid organized by the Global Virus Network, one of the senior authors of the study, 661

Stanford neuroscientist Dr. Michelle Monje, mentioned that: “Disruption of myelin homeostasis and plasticity is profoundly disruptive to neural circuit function. It changes the timing, it changes the synchrony and coordination of neural impulse conduction. This slows cognitive processing time, it causes difficulty in multitasking, it impairs attention, it impairs learning and memory — many of the symptoms that people experience both after cancer therapy and after Covid-19.” This is reflected by data from demyelination diseases such as multiple sclerosis and from data on demyelination and declining cognitive function with age. Implications The work by Fernández-Castañeda et al. gives us new insights into one of the potential causes of cognitive dysfunction following Covid-19. Crucially, their findings suggest that SARS-CoV-2 doesn’t even need to enter the central nervous system or directly infect the brain, it can cause lasting damage simply by stimulating our inflammatory response. This can happen even after mild Covid19, where infection is restricted to the trachea and lungs. But there is good news: the resulting damage to myelin as well as the loss of neurogenesis are likely reversible. Based on evidence from cancer-therapy-related cognitive impairment —which shares many symptomatic and mechanistic parallels to Covid-19-induced cognitive impairment— the researchers suspect that blocking inflammation in the brain should restore myelin and neuron production. Although we don’t yet have a targeted, cytokinespecific anti-inflammatory strategy, it’s only a matter of time. This article is featured on Forbes.org, and can be read online here: Even Mild Covid-19 May Cause Lasting Brain Fog (Part 2)

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Covid-19, Gender And Immune Response: What’s The Relationship? (Part Two) Forbes | August 04, 2022 | Article

This is the second installment in a two part series which analyzes biological sex differences in immune responses to SARS-CoV-2 infection. Part one focuses primarily on Covid-19 related viral entry, innate and adaptive immune responses to Covid-19 and their correlation to epidemiological evidence. This article will highlight the role of sex hormones in SARS-CoV-2 immune responses, examine sex differences in response to vaccines, and consider their possible therapeutic implications. Covid-19 disease severity and mortality differ between men and women, but the reasons for such differences are not well understood. Part one of this series delves into sex differences in response to SARS-CoV-2 infection and notes how stronger immune responses seen in females likely contribute to the better outcomes observed. This second and final installment will analyze two more elucidating factors: the role of sex hormones on SARS-CoV-2 immune responses and sex differences in immune responses to vaccines. These components, in particular, pose potential therapeutic directions for treating and understanding Covid-19. Sex Hormones and SARS-CoV-2 Immune Responses

FIGURE 1: Men possess higher levels of androgens such as testosterone and dihydrotestosterone. Women, in contrast, have elevated levels of estrogen and progesterone.

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The first section of this article will concentrate on the role of androgens, estrogen and progesterone on Covid-19 disease progression and outcomes. CREDIT: HO ET AL.

Androgens In their review The Immune Response to Covid-19: Does sex matter?, Ho et al. analyze the complex relationship between sex hormones and SARS-CoV-2 immune response. They first consider androgens—such as testosterone and dihydrotestosterone—which males possess higher levels of than females. Ho et al. find that androgen receptor expression may impact two essential enzymes to SARS-CoV-2 viral entry: furin and transmembrane serine protease 2 (TMPRSS2). Furin is a calciumdependent enzyme which cleaves the spike protein into the configuration needed for priming and activation. Transmembrane serine protease 2 (TMPRSS2) primes the SARS-CoV-2 protein for entry into host cells. The theory is, since increased androgen receptor expression can upregulate furin and TMPRSS2, the higher androgen receptor expression seen in men increases their susceptibility to severe forms of Covid-19. Although the clinical association observed between androgenic alopecia and severe Covid-19 would suggest this mechanism to be true, studies on androgen deprivation therapy (ADT) in prostate cancer patients with Covid-19 do not necessarily support this claim. Androgen deprivation therapy reduces the number of androgen receptors available for activation through medicine or surgery. The therapy was expected to decrease androgen receptor expression in prostate cancer patients with Covid-19, thereby restricting androgen regulation of TMPRSS2 and reducing the risk of SARS-CoV-2 infection. In contrast to this notion, the treatment did not improve infection risk, ICU admission, hospitalization or mortality in comparison to controls. Randomized clinical trial results with antiandrogens, medicines which block androgen receptors and inhibit androgen synthesis, further complicate these associations. One randomized controlled trial revealed that Covid-19 patients given nitazoxanide/azithromycin therapy with antiandrogen dutasteride experienced decreased viral shedding, inflammatory markers and time-to-remission compared with placebo; another found that antiandrogen proxoludamine reduced the 30-day hospitalization rate 664

and risk ratio amongst men with Covid-19. On the other hand, a third trial with enzalutamide increased Covid-19 related hospitalization stay. Male sex steroids seem to perform varying roles with respect to Covid-19. The culminating conclusion from these studies suggests that both low and high androgen levels can correlate with poor Covid-19 prognoses. As Ho et al. state in their review, further investigation in this arena is needed. Estrogens Female sex hormone estrogen appears to mediate several beneficial immune responses. A study of hospitalized Covid patients correlated higher estradiol levels to decreased disease severity. And as mentioned in part one, estrogen promotes strong immune responses in women and likely contributes to the observed discrepancy in innate and adaptive immune responses between sexes. Inflammation in female innate immune responses reduces when estrogen activates anti-inflammatory cytokines, inhibits the nuclear factor kappa B (NF-B) pathway, and decreases the release of inflammatory cytokines. Women also have better priming of adaptive immune responses to viruses. This is thought to be influenced by estrogen; estrogen can help regulate immune cells called plasmacytoid dendritic cells (pDCs) which, in turn, promote the production of interferon alpha, an important antiviral cytokine in innate immunity. These mechanisms may translate to the better disease outcomes witnessed in women than men with Covid-19. Estrogen has also been found to regulate several proteins which are involved in SARS-CoV-2 viral entry: furin, TMPRSS2, angiotensin converting enzyme 2 (ACE2) and a disintegrin and metalloprotease 17 (ADAM17). It, too, suppresses immune enzyme dipeptidyl peptidase 4 (DPP4), thereby blocking another potential means of SARS-CoV-2 viral entry. Researchers are exploring possible therapeutic applications for estrogen in Covid-19 interventions. Two examples include a study on the effect of selective estrogen receptor modulators on Covid-19, and a randomized control trial analyzing the efficacy of an estradiol/progesterone therapy in reducing disease severity in hospitalized Covid patients. Progesterone

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Ho et al. complete their study of sex hormones in Covid-19 with progesterone. Progesterone levels tend to be higher in women than men and are associated with general anti-inflammatory effects. These anti-inflammatory effects include but are not limited to the ability to increase T regulatory cells, enhance antiviral immune pathways and disrupt endocytic pathways used by viruses to enter host cells. It is hypothesized, therefore, that progesterone may decrease the risk of hyperinflammation and SARS-CoV-2 related cytokine storm. There is therapeutic potential in administering progesterone to treat Covid-19. A study of hypoxemic men hospitalized with Covid19 observed that short term subcutaneous progesterone decreased hospitalization stay and supplemental oxygen needed. Additional research is needed to understand the specific mechanisms at work and its promising impacts on Covid-19 treatments. Sex Differences in Vaccine Immune Responses Vaccines are crucial to Covid-19 control and have been invaluable in reducing lives lost to severe forms of the disease. As a result, Ho et al. emphasize the importance of understanding sex differences in response to Covid-19 vaccines. They state, “sex differences should be taken into account as a biological variable for adjusting sex-personalized vaccine dosage and considering vaccine efficiency.” These considerations seem most pertinent to women. Two studies, one systematic review and one meta analysis, found that vaccination prevented Covid-19 disease less effectively in women than in men. Similarly, a 2021 CDC report observed that women received 61% of administered Covid-19 vaccines at the time yet accounted for 79% of adverse events. The discrepancies in vaccine response could be due to several factors—age, hormonal differences (as explored in this article) and sex differences intrinsic to SARSCoV-2 immune response (see part one of this series)—but more studies are needed to clarify these possible correlations. Conclusions Contemporary research reveals that sex hormones and biological sex do influence immune responses and vaccines, although specific mechanisms have yet to be fully understood. Ho et al. call for biological sex to be considered in basic, translational and clinical Covid-19 research. More extensive research on biological sex and Covid-19 could open potential therapeutic avenues and improve the 666

specificity of those strategies—be it through the use of sex hormone therapies or through the adjustment of vaccine dosage based on gender. This article is featured on Forbes.org, and can be read online here: Covid-19, Gender And Immune Response: What’s The Relationship? (Part Two)

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Even Mild Covid-19 May Cause Lasting Brain Fog (Part 3) Forbes | August 05, 2022 | Article

This is the fourth article in a series focusing on cognitive dysfunction and inflammation. The first part can be read here, the second can be read here, and the third, here. For additional writings on inflammation and Covid-19, please see my website: www. williamhaseltine.com In previous installments of this series, we looked at new research that suggests even mild SARS-CoV-2 infection can impair cognitive function. The first step in this chain of events happens when the virus sets off an intense inflammatory response that eventually reaches the brain. Once there, inflammation triggers reactivity of microglial cells —the resident immune cells of the central nervous system— which produce yet more inflammation and interfere with the normal functioning of important cells, including oligodendrocytes and neural precursor cells (Figure 1).

FIGURE 1. A schematic representation of the mechanism underlying cognitive dysfunction following mild Covid-19, as described by Fernández-Castañeda et al. (2022). SOURCE: ACCESS HEALTH INTERNATIONAL

But given that all of this happens off the back of inflammation, which we experience during any number of infections or injuries, wouldn’t we expect to see something similar unfold in other viral diseases? As part of their research, Fernández-Castañeda et al. asked 668

themselves this same question. In the search for an answer, they turned to influenza. Like Covid-19, the flu has also been associated with cognitive and neurological issues. And like SARS-CoV-2, influenza is rarely neuroinvasive — even though both viruses can infect the brain directly, as can many other viruses, it seems to happen only infrequently. It could be, then, that influenza causes cognitive issues through a similar mechanism as SARS-CoV-2. To find out, the researchers exposed a group of mice to the H1N1 strain of influenza. This strain is not neuroinvasive in mouse models, meaning infection is restricted to the trachea and lungs. To further keep things consistent with their Covid-19 experiments, the researchers made sure that the mice suffered only mild flu, as evidenced by minimal sickness behaviors and minimal weight loss. Despite largely mild illness, the mice showed signs of elevated inflammation, with a noticeable increase in cytokine and chemokine levels. The researchers found these both in the blood as well as in the cerebrospinal fluid, indicating that inflammation had made its way into the central nervous system. The influenza-induced cytokine profile closely mirrored that of mice with mild respiratory Covid-19 (Figure 2). In both cases, C-C motif chemokine ligand 11 (CCL11) was present in cerebrospinal fluid from seven days all the way to seven weeks after infection. In fact, CCL11 levels were higher at seven weeks than they were at seven days.

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FIGURE 2. Cytokine/chemokine profiles following mild Covid-19 and mild flu. Notice CCL11, present in both cases seven days post infection (7DPI) as well as seven weeks post infection (7WPI). FROM: “MILD RESPIRATORY COVID CAN CAUSE MULTI-LINEAGE NEURAL CELL AND MYELIN DYSREGULATION” FERNÁNDEZ-CASTAÑEDA ET AL. 2022

This means the first part of the mechanism driving cognitive impairment after Covid-19 —the peripheral inflammation that triggers neuroinflammation— is also present during infection with influenza. What about the remaining elements? In the context of Covid-19, elevated CCL11 levels were associated with an increase in reactivity of microglial cells. Studying the white matter of those mice exposed to the flu, the group of researchers discovered a similar increase in microglial reactivity; 670

although the total number of microglial cells remained the same in both the flu group and the control group, the number of activated microglial cells was noticeably higher in the mice recovering from illness. The increase in activated microglial cells was accompanied by both damage to oligodendrocytes and by a decrease in the production of new neurons in the hippocampus, called neurogenesis. The exact same pathology seen in the Covid-19 mice. Neurogenesis in the hippocampus was impaired as early as seven days following influenza infection, and this persisted until at least seven weeks after infection. Microglial cells in this region remained reactive throughout. Unlike the Covid-19 mice, however, those exposed to the flu showed signs of recovery to oligodendrocyte numbers. Although there was a marked drop at seven days postinfection, the oligodendrocyte population had normalized by seven weeks. This correlated with a steady reduction in microglial reactivity during the same period. So although they share the same underlying mechanism, the cognitive dysfunction at the hands of influenza differs to that caused by SARS-CoV-2; they both trigger a lasting drop in neurogenesis in the hippocampus, but only SARS-CoV-2 tiggers additional lasting damage to the production of oligodendrocytes. Implications The consequences of viral infections can extend for many weeks, months, and even years after infection. As far as cognitive impairment is concerned, these findings identify systemic inflammation —ultimately manifest in the brain— as a driving factor. Not all viruses are going to have the same impact since not all of them set off the same magnitude of inflammation. This holds true even for different strains of the same virus — although influenza as a whole is not particularly known for sparking neurological issues, the strain that caused the 1918 Spanish flu pandemic led to a sharp uptick in Parkinson’s disease. As the strain slowly decreased in virulence, the intensity of the inflammatory response followed suit and so too did the frequency of neurological and cognitive symptoms. But it may only be a matter of time before another virus with such capability arises. This is evident with SARS-CoV-2, which has by and large been characterized by the potency of the inflammatory response it triggers; 671

in a sense, it’s a hallmark of the virus. This manifests in cytokine storms, it manifests in hypercoagulation, and it manifests in worryingly high levels of cytokines and chemokines circulating in the blood and in the cerebrospinal fluid. Although we can expect slight differences across variants, the common theme is long-term cognitive and functional damage — some of which shows up immediately, and some of which may only show up much later. This article is featured on Forbes.org, and can be read online here: Even Mild Covid-19 May Cause Lasting Brain Fog (Part 3)

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Loss Of Smell Linked To Long Term Covid Cognitive Impairment Forbes | August 09, 2022 | Article

Very little is known about the long term effects of Covid-19, especially in relation to the brain. Who gets what symptoms may feel like a mystery. And with the current seven day average of new cases hovering around 1,000,000 cases worldwide, the issue of long term sequelae only grows more pertinent as infections increase. In this regard, a study presented at this year’s Alzheimer's Association International Conference (AAIC) may have uncovered a key to solving this riddle: loss of smell. Study Design The Argentinian research team investigated the long term Covid-19 cognitive impairment in older adults through a one-year prospective study design. All 766 participants were randomly invited from the health registry in Jujuy, Argentina, which holds all Covid19 testing information for its region. Investigators split the group by polymerase chain reaction (PCR) testing status: 88.4% who had Covid-19 and 11.6% without—in other words, the control group. The adults’ ages ranged between 55 to 95 years old, with the mean age landing on 66.9 years old. More than half of the group, 57% specifically, were female. The group averaged 10.4 years of education; the Argentine education system consists of 12 years of school before university. The researchers followed recommended measures from the Alzheimer's Association Consortium on Chronic Neuropsychiatric Sequelae of SARS-CoV-2 infection (CNS SC2) to evaluate the long term cognitive consequences of Covid-19. They tested four cognitive domains: memory, attention, language and executive function (ex: flexible thinking, self-control and working memory). The degree of anosmia (loss of smell) was confirmed through an olfactory test. An individual would attempt to identify three distinct odors; depending on the results, the team categorized the anosmia as either nonexistent, mild, moderate or severe. 673

Study Results The investigators stratified cognitive performance by number of impacted domains: normal cognition, memory-only impairment (single domain; 11.7%), impairment in attention and executive function without memory impairment (two domains; 8.3%), and multiple domain impairment (11.6%). Study investigator Gabriela Gonzalez-Alemán, PhD, told Medscape Medical News that the participants displayed “a predominance of memory impairment as would be seen in Alzheimer's disease,” with a large group presenting “a combination of memory and attention problems.” None of the controls had olfactory dysfunction, but 40% of the study sample notably did. Furthermore, all participants with severe cognitive impairment also had anosmia. In this study, the degree of anosmia—rather than the severity of Covid-19—significantly predicted cognitive impairment. This is of particular importance to this study group, as cognitive impairment and loss of smell can be persistent for those over 60 years of age. Researchers also collected participant vaccination status through a one year phone survey. The majority of the participants received vaccinations. Around 71.8% of the study cohort had three vaccine doses, while 24.9% had two. Of those groups, around 12.5% of individuals with three doses were infected, and 23.3% of participants with two doses were reinfected. Possible Implications Instead of disease severity, loss of smell seems a more promising avenue for predicting who develops persistent cognitive changes after SARS-CoV-2 infection. The presented study results provide an intriguing foundation for further investigation. As described by Dr. Gonzalez-Aleman in Neurology Today, anosmia could be a sign of SARS-CoV-2 infection entering the brain through the olfactory bulb, or a sign of a continuing disease process after infection. With additional research, the hope would be to more thoroughly understand this correlation and thus develop a means to prevent such brain damage. This article is featured on Forbes.org, and can be read online here: Loss Of Smell Linked To Long Term Covid Cognitive Impairment

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Advanced Technology (CRISPR) Shows That Mucus Is Your Body’s First Line Of Defense Against Viruses Forbes | August 11, 2022 | Article

This story on CRISPR is part of an extended series on Regenerative Medicine. For other stories on this topic see williamhaseltine.com and search for Regenerative Medicine. My definition of Regenerative Medicine is any medical modality that returns us to normal health when we are damaged by disease, injured by trauma, disadvantaged by birth, or worn by time. Modalities include: chemicals, genes, proteins and cells used as drugs, gene editing, prosthetics, and mind-machine interfaces. One of the key mysteries of SARS-CoV-2 is why it seems to infect some people more seriously than others. While vaccines have provided much-needed protection against the virus, there is still a need to develop better drugs to treat those who do become infected. A primary method to develop drugs that fight viral infection is to determine what a virus needs to replicate in the body. Up until now, most studies have focused solely on SARS-CoV-2 and its own methods of attack. However, infections require cooperation between the human body’s cells along with the virus. In fact, viruses rely on many cellular structures within the host cells to replicate. So how can we decode SARS-CoV-2 and its interactions with our own bodies? One way to do this is to conduct a systematic study of all the genes in cells that are known to interact with SARS-CoV2. By inhibiting the majority of genes while leaving some active, scientists can pinpoint exactly which genes and cellular structures affect SARS-CoV-2 infections. Fortunately, with a new gene-editing tool called CRISPR, researchers at U.C. Berkeley were able to do just that. After conducting a study of genes found in lung cells, Biering et al. discovered a naturally occurring protein in the body that may have the ability to inhibit SARS-CoV-2 infections. This study is the first

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to examine how SARS-CoV-2 interacts with human lung cells, marking a critical advancement in SARS-CoV-2 research. How does CRISPR work? CRISPR technology is a recent advancement in gene-editing. The technology consists of two components: Cas9, which is an enzyme that cuts DNA, along with a short RNA segment that guides Cas9. When the guide RNA locates its complementary sequence in DNA, it binds to the targeted gene and acts as a signal to the Cas9 enzyme. Once Cas9 locates and binds to the guide RNA, the enzyme can then cut the entire DNA sequence at that specific location. This allows scientists to knockout genes entirely or edit the genome by inserting new genes.

Figure 1: CRISPR consists of Cas9, an enzyme that cuts DNA, and guiding RNA which directs Cas9 to specific locations in the DNA strand.GENOME RESEARCH LIMITED

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Biering et al. used CRISPR technology to study genes that exist in human lung epithelial cells. Epithelial cells line the walls of the lungs and are responsible for producing mucus and for initiating several immune responses. Lung epithelial cells also express receptors such as ACE2 which are known to affect SARS-CoV-2 infections, making the cells a valuable model for SARS-CoV-2 infections. After studying the effects of each gene found in the lung epithelial cells, Biering et al. discovered several genes that seemed to impact SARS-CoV-2 infections. These included known genes such as those responsible for the ACE2 receptors, but also some new players. One of the most influential categories of genes was a group responsible for producing proteins called mucins. Mucins are the most abundant protein found in mucus and can either be secreted to form gels that span the surface of the lungs or can exist within epithelial cell membranes. Interestingly, Biering et al. found that only mucins within the cell membrane seemed to have a direct effect on SARS-CoV-2 severity.

Figure 2: Mucins can either be secreted into extracellular space or exist within the membrane KUFE, NATURE REVIEWS CANCER (2009), DOI: HTTPS://DOI.ORG/10.1038/NRC2761

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To determine if the membrane-bound mucins could have an anti-viral effect on SARS-CoV-2 infections, Biering et al. then overexpressed the mucins. To their surprise, the membrane-bound mucins were successful in decreasing the severity of SARS-CoV-2 infections. These results were consistent across prevalent SARSCoV-2 variants as well. These results prompted the researchers to test the relationship between membrane-bound mucins and SARS-CoV-2 in vivo. When Biering et al. compared mice with inactivated mucin genes to those with normally functioning genes, they found that mice with inactivated mucin genes had much greater levels of viral infection contained in their lung tissue.

Figure 3: Mice without mucin-producing genes (KO; right) contained higher amounts of SARS-CoV-2 infection (red) within their lung tissue. BIERING ET AL., NATURE GENETICS (2022), DOI: HTTPS://DOI.ORG/10.1038/S41588-022-01131X

But how did the mucins defend against SARS-CoV-2? Since only membrane-bound mucins affected SARS-CoV-2 infections, Biering et al. hypothesized that membrane-bound mucins somehow block the virus from entering the cell membrane. Surprisingly, after using microscopic imaging techniques to examine the interactions between SARS-CoV-2 and mucins, researchers found that the cells with overexpressed mucins did indeed inhibit viral entry. 678

More research must be done to determine whether the interactions between mucins and SARS-CoV-2 are consistent in humans. However, the results of this study are promising and could lead to new, effective treatments that have the potential to decrease the severity of SARS-CoV-2 infections. This article is featured on Forbes.org, and can be read online here: Advanced Technology (CRISPR) Shows That Mucus Is Your Body’s First Line Of Defense Against Viruses

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Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 1) Forbes | August 12, 2022 | Article

This is a short series about a recent success on the road to developing a much sought-after broadly neutralizing vaccine against all influenza A viruses. If successful, it may act as a precursor to a truly universal flu vaccine, one that protects against all types and subtypes of the virus. The breakthrough may also provide a blueprint for developing a Covid-19 vaccine that retains its efficacy in the face of new variants. Here, I give a brief overview of the history and nature of influenza viruses, including why it has been so difficult to develop successful vaccines. In a follow-up story, I will discuss strategies that have been used to try to overcome these challenges, and crucially, their shortcomings. And in the last installment, I offer a detailed analysis of one of the latest —and most promising— advances in the field. Understanding Influenza With Covid-19 on the scene, talk of other respiratory viruses has taken a backseat. But this doesn’t change the fact that they are as present as ever, lurking in the background. Influenza is one such virus, causing an estimated 1 billion cases globally every year — of these, upwards of 500,000 end up being fatal. Although lockdowns and social distancing efforts aimed at curbing the spread of Covid19 also helped put a dent in seasonal influenza epidemics, the flu is now returning with full force (Figure 1).

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FIGURE 1. Number of specimens positive for influenza by subtype globally, from August 2012 to August 2022. SOURCE: FLUNET (WWW.WHO.INT/TOOLKITS/FLUNET). GLOBAL INFLUENZA SURVEILLANCE AND RESPONSE SYSTEM (GISRS). DATA GENERATED ON 08/10/2022

There are seven types of influenza, four of which can infect mammals: A, B, C, and D. Influenza A and B are the most common, driving yearly seasonal flu epidemics. Influenza C infections in humans are rare and are thought to cause relatively mild disease. Influenza D mainly infects cattle with no recorded human infections to date. On the whole, influenza A viruses are considered the biggest threat. All of the major influenza pandemics of the last century have been caused by influenza A viruses, the most notable being the Spanish flu pandemic of 1918 which led to the death of roughly 50 million people, one tenth of the entire global population at the time. Influenza A viruses are also particularly adept at evading our immune system; they have a high rate of genetic mutation, resulting in a process known as antigenic variation. This is what allows the virus to reinfect us again every season, it is what makes the virus volatile and capable of causing the occasional pandemic, and it is what makes it so difficult for us to develop long-lasting vaccines. To help explain antigenic variation, it’s worth quickly looking at the structure of the virus (Figure 2). First, all influenza viruses have single stranded, negative-sense RNA. This is the genetic material used by the virus to replicate. In the case of influenza A, the genome is broken into eight separate segments. Surrounding and protecting these RNA strands is the viral envelope. The envelope contains two key surface proteins hemagglutinin (HA) and neuraminidase (NA) — the former helps the virus enter our cells, and the latter helps newly-formed viral particles leave the cell.

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FIGURE 2. Schematic of influenza A virion structure. SOURCE: TIMONINA/SHUTTERSTOCK.COM

There are 18 different subtypes of the hemagglutinin surface protein, aptly named H1 through H18. These are split into two groups, organized according to their evolutionary history. Crucially, immunity against one group does not confer immunity against the other group. Any universal influenza A vaccine has to account for this fact. Neuraminidase, in turn, has 11 different subtypes — N1 through N11. It is on the basis of these surface protein subtypes that influenza A viruses are classified. For example, the viral subtype that caused the 1918 Spanish flu had an H1 hemagglutinin protein and a N1 neuraminidase protein, making it an influenza A (H1N1) virus. The influenza virus that caused the 1968 pandemic, in contrast, had a different HA subtype, H3, and a different NA subtype as well, N2. As a result, it is classified as influenza A (H3N2). These are the two most common subtypes of influenza A, responsible for the majority of yearly infections. Influenza A subtypes can be broken down even further into strains. This is determined by the sum of their genetic material, known as the genome. Two viruses may have the same broad hemagglutinin and neuraminidase subtypes, but have subtle differences throughout their genome. For example, the H1N1 subtype was responsible for both the 1918 and 2009 influenza

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pandemics, but in each case it was a different strain of the virus (Figure 3).

FIGURE 3. A rough timeline of the most recent major influenza pandemics. Genetic mutations characterizing each pandemic strain are labeled in the center of the viral particles; each line represents an RNA segment, which encodes different proteins carried by the virus.

FROM: “THE EVOLUTION AND FUTURE OF INFLUENZA PANDEMIC PREPAREDNESS” HARRINGTON ET AL. 2021 So, how does all of this relate to antigenic variation and why does it make it difficult for us to develop a universal influenza A vaccine? The HA and NA surface proteins are recognized by our immune system as foreign substances —known as antigens. Detection of antigens in our body leads to the activation of various immune responses aimed at clearing the threat. One kind of immune response involves training B cells to produce antibodies that specifically target the antigens in question. In a sense, our B cells develop a type of “immune memory” towards the virus. Doing so allows our immune system to react more quickly and effectively in case of reinfection, blocking the virus before it has the chance to get us sick. As hinted at above, influenza A viruses are notorious for their ability to mutate. This happens when the virus makes small “mistakes” while copying its genetic information during replication. Over time, these small mutations to the genome can accumulate, changing the structure of various parts of the virus, including HA and NA. If it changes enough, the virus will be able to evade our immune system; our B cells will no longer be able to recognize the antigens, nullifying what they may have learned during a prior

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infection. This is a type of antigenic variation called “drift”, and it is the reason we can repeatedly become sick with the flu. Vaccination against influenza works by exposing our immune system to killed or weakened forms of the virus. In the same way a natural infection would, this helps our immune system build up a memory of the viral antigens and teaches it to keep an eye out for them in the future. But like the antibodies produced after natural infection, those produced in response to vaccination are also rendered useless over time by antigenic drift; as before, the antigens become too different to be recognized, forcing B cells to rebuild their immune memory all over again. This is why scientists have to develop a new flu vaccine every year, so that our immune system can build up its memory based on the most recent circulating influenza antigens. There’s also another, more drastic kind of antigenic variation called antigenic “shift”. Whereas drift happens over an extended period of time, via the accumulation of small changes, antigenic shift can lead to substantial changes very quickly. How? Usually when a cell is infected by an influenza virus, eight gene segments come in, these eight segments are copied and packaged into a newly formed viral particle, and the new particle then leaves the cell to go infect other cells. But sometimes a single cell —whether human or avian or porcine— can become infected by two different influenza A viruses at the same time. In such cases, the two viruses can end up “exchanging” RNA segments with one another. This is called reassortment, a process that leads to the creation of a third, novel virus that’s distinct from both of the parent strains. Often this happens when an influenza A virus jumps between different animal hosts. For example, the 1957 influenza pandemic was caused by a novel subtype, H2N2, composed partly of genes from the 1918 strain and partly from new genes gained while infecting birds (Figure 3). Such sudden jumps in genetic code can lead to influenza viruses that look very different to those usually circulating, rendering the surface proteins unrecognizable to our immune system. Once again, our immune system is confronted with what is essentially an entirely new threat and has to build its immune memory from the ground up. Whereas antigenic drift is responsible for yearly seasonal flu

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outbreaks, antigenic shift is often responsible for new influenza pandemics. The challenge: any universal influenza A vaccine has to successfully account for both antigenic drift and antigenic shift. So far, this has proven very difficult. In the next article, I go over some of the more popular vaccination strategies aimed at overcoming these obstacles. This article is featured on Forbes.org, and can be read online here: Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 1)

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Fixing Our Public-Health Data Problem Project Syndicate | August 16, 2022 | Article

Easily accessible, accurate health data is necessary to understand clinical information in real time, prevent medical errors, and make public-health decisions before, during, and after times of crisis. But as COVID-19 and monkeypox have shown, the US and others are falling far short of this standard. FAIRFIELD COUNTY, CT – With COVID-19 still an everpresent threat and monkeypox cases rising alongside existing chronic health epidemics, building strong, responsive public health systems has never been more important. But it will require developing effective data management, pursuing sustained investment and training, and building trust through clear communication and community engagement. Here, I want to focus on the data problem. Standardized, comprehensive health data is at the heart of any public-health system, whether the task is conducting infectiousdisease surveillance or understanding why some diseases affect certain populations more than others. Easily accessible, accurate health data is necessary to understand clinical information in real time, prevent medical errors, and make decisions about public-health measures before, during, and after times of crisis. Our response to the COVID-19 pandemic has clearly been hampered by the slow and inconsistent reporting of critical publichealth data. In the absence of universal standards, many public-health systems and practitioners rely on outmoded forms of communication such as paper and fax, which do not allow for rapid sharing. More than two years into the pandemic, we still cannot easily draw on the latest data to answer basic questions, such as what COVID-19’s mortality rate is according to patient variables. In the United States, a recent report from the Commonwealth Fund Commission on a National Public Health System finds that, “Only 3 percent of local health departments reported that information systems are all interoperable, a limitation that hampers both daily prevention work and coordinated responses.” Because 686

America has a federated model for public health, relying on the efforts of dozens of different state and territorial health departments, it lacks the capabilities of more integrated national systems like those found in England and Israel. Hence, US vaccine regulators have had to rely on Israeli and UK data, rather than data on the US population. The Commonwealth Fund commission calls for a “truly national public health system that functions day to day, with coordinated leadership at the federal level and with consistent state and local capacity.” But it is careful to acknowledge that, despite the need for better national coordination, local and state health departments should maintain their current level of control, since they ultimately know their own communities best. One way to achieve this balance is by setting comprehensive and binding national standards for state and local health data, rather than incentivizing voluntary, piecemeal forms of data exchange. Though hospitals and other health-care providers have shared data throughout the pandemic, the information they have reported tends not to be linkable across different systems, owing to the use of different definitions and different methods of collection. This makes it challenging and time-consuming for recipients at either the state or federal level to receive, aggregate, and analyze current data. Thus, in addition to setting standards for the data, we need to develop and implement national standardized data-collection and data-sharing systems. This would involve universal identifiers for individual patients and end-users, as well as a standardized infrastructure to integrate laboratory results from equivalent tests. We also need to ensure that local, state, and tribal health departments have consistent funding to participate fully in any such initiative. There is evidence that this approach can work. For example, owing to the Obama administration’s efforts, the number of hospitals using electronic records grew from 9% in 2008 to 84% in 2015. Unfortunately, his administration underestimated the critical need for standardization. The same data-collection issues that hampered the response to COVID-19 are also occurring with monkeypox, further highlighting the urgent need for reforms. While the Centers for Disease Control and Prevention recently shared a first public look at monkeypox case demographics, the agency has detailed information 687

on only about half of reported cases, because deciding whether to share the data is completely up to state-level authorities. But even for states that are motivated to share data with the CDC, the current infrastructure is flawed. While the CDC does have data on how monkeypox vaccines have been distributed among the states, it has no data on who has been vaccinated, and it does not yet have the authority to collect such data. This lack of local data makes it difficult to forecast the trajectory of the disease or to make public-health recommendations based on the efficacy of the vaccines. Moreover, while the CDC is focused on modernizing access to reportable disease data and laboratory test results, it is underutilizing other rich data sources. These include records on visits, hospitalizations, medications, and outcomes from payers (including the Centers for Medicare and Medicaid Services), and figures from electronic health-records systems and health information exchanges. While ensuring patient privacy is paramount, these data can be anonymized to highlight trends and provide valuable insights into public-health issues beyond infectious-disease surveillance. For example, Indiana’s health system exchange has made it possible to monitor not just statewide pediatric asthma trends but also surges in teen lung injuries from vaping. Finally, public-health departments can also exchange anonymized data with other government departments – such as those overseeing housing and education – to understand how social determinants of health are linked to certain populations and clinical trends. Implementing these changes and developing an accessible, standardized national public-health database could transform millions of health outcomes and save many lives. But it won’t happen overnight. The work must start immediately. This article is featured on Project Syndicate, and can be read online here: Fixing Our Public-Health Data Problem

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Controlling COVID-19: What Can We Learn From Previous Pandemics? Precision Medicine | August 17, 2022 | Article

Every virus has its preferred niche — a particular host or demographic it is more inclined to infect and a particular set of strategies and tactics it uses to achieve this. A virus develops a niche over millions of years, all the while adapting and mutating in response to host immune defenses. The longer a virus has to get to know a particular host species, the more attuned it becomes. It learns the nuances of the immune system in question, and critically, how to circumvent them; slowly, it focuses its blows on the chinks in the armor, slashes at the Achilles’ heel. But this doesn’t happen in isolation. As the virus adapts, so too does the host immune system it attacks. The two go hand-in-hand. Human history cannot be separated from this intricate dance with viruses. The COVID-19 pandemic — just like the Spanish flu pandemic of 1918, the polio epidemics of the 1940s and 1950s, and the ongoing HIV/AIDS crisis — makes this crystal clear. With advancements in modern medicine and public health, we have the possibility to shift the tide in our favor. Antiviral drugs and vaccines play an especially important role in this process. If before our immune system was fighting with a wooden club, we now have the means to supply it with a shield and sword. Even so, the effectiveness of vaccines and antivirals fluctuates according to the virus at hand. This means that, although there are general guidelines and preventative measures that can be applied to pandemic preparedness across the board, each virus will have its own specific dynamics that require us to make adjustments at a more granular level. Studying previous pandemics can help us understand how differences in viral niches and viral strategies may influence what kind of response is needed, particularly in regards to the role of vaccines and antiviral drugs. Nature knows best: vaccines

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When discussing successful vaccination campaigns, it is impossible not to make mention of poliomyelitis. Polio, as it’s more commonly known, is a disease caused by a small, single-stranded RNA virus. For the most part, people infected with the poliovirus are none the wiser: 70% of poliovirus infections are asymptomatic and a remaining 20-25% are only mildly symptomatic. But at the tail end of distribution, there are those who go on to develop fatal complications. Around 1% of those infected end up suffering from paralytic polio, the most severe form of the disease1. This is characterized by excruciating pain that radiates through the back and lower limbs. The hallmark of paralytic polio, and the reason it is so infamous, is eventual paralysis of limbs. This happens when the virus travels from the bloodstream into the spinal cord, infecting and damaging motor neurons. Paralysis can extend to the diaphragm, leaving certain patients unable to breath on their own. Without access to a respirator, this quickly leads to death. Some individuals may recover, but if motor function doesn’t return to normal within the span of a year, it is generally accepted that paralysis will be lifelong. Despite the fact that only a small subset of those with polio suffer serious disease, the ease and extent to which it spreads causes huge issues—a small subset of a very large number quickly becomes quite large itself. At the height of the global polio epidemics, more than half a million people were left paralyzed or dead by the virus each year2. The development of vaccines in the 1950s and the 1960s, however, has allowed us to eradicate poliovirus from endemicity in all but two countries, Afghanistan and Pakistan. The first vaccine for polio, developed in the 1950s by Jonas Salk, helped protect against severe disease and paralysis, but did not prevent transmission. It works by exposing our immune system to an inactivated, or killed, version of the poliovirus. Because the inactivated polio vaccine (IPV) uses killed virus, there is no chance of viral mutation causing a reversion to the original pathogenic form. Although not perfect, the vaccine quickly helped curb the worst of the polio endemics. A second polio vaccine was developed in the 1960s by Albert Sabin. Unlike its predecessor, it has the distinct advantage of also preventing transmission. The oral polio vaccine (OPV) uses an attenuated, or weakened, version of the poliovirus. 690

This elicits a stronger, more robust immune response. The fact it can prevent transmission truly kickstarted poliovirus eradication, lending solidity to what may have initially seemed a pipe dream. Like poliovirus, many SARS-CoV-2 infections are asymptomatic. Even more cause only mild disease. But for a few, infection will lead to serious, life-threatening disease. Unlike poliovirus, SARS-CoV-2 continues to circulate globally at nearly the same rate despite the development of vaccines. Why? Because poliovirus and SARS-CoV-2 inhabit very different niches and make use of different strategies. In a sense, the two viruses work on opposite sides of the street. Poliovirus, by and large, relies on what we call “the fountain of youth,” i.e., the fact that new human beings are born all the time. Infants and young children have a naive immune system, making them more susceptible to infection. Poliovirus exploits this fact, and it does so extremely well: the overwhelming majority of polio cases are seen in children under the age of five. Crucially, polio is a “oneand-done” kind of virus. Once someone has been infected, they essentially develop lifelong immunity. This is why complications are only seen in the immune-naive population — all others who were already infected and had no issues are no longer at risk. This leads us to a fundamental lesson in vaccinology. If in nature you become immune by infection, then we can generally make an effective vaccine; if in nature you do not become immune by infection, then it is generally very difficult to make an effective vaccine. This is why the vaccines we have for SARS-CoV-2 only protect against disease, not against infection and transmission. Unlike poliovirus, SARS-CoV-2 has developed extremely precise strategies to evade and suppress our immune system, allowing it to infect a large spectrum of people3. This includes adults with fully developed immune systems. As such, it thrives in a much more immuneexperienced niche than poliovirus. The complex immunoevasive abilities of SARS-CoV-2 mean it can infect, reinfect, and then reinfect again. This is complicated by a further tactic: antigenic variation. Essentially, mutations to the genetic material of the virus that cause its surface proteins, or antigens, to undergo structural changes. These changes render the virus unrecognizable to our immune system, 691

despite having been exposed to a previous version of the virus. Even within the same variant family the changes can be large enough to dodge our built up immunity. The result? Annual or bi-annual waves of infection spearheaded by new variants that our immune system cannot defend against. This is evidenced by Omicron BA.5 and BA.2.75, which are causing new spikes in cases just as the wave caused by the original Omicron variant begins to recede. The difficulty of containing SARS-CoV-2 via vaccination should come as no surprise. Influenza virus has been with us for a lot longer, and still it infects upwards of 1 billion people every year, causing roughly 300,000 to 650,000 deaths4. The reason is similar: influenza mutates at an extremely high rate, creating new sub-clades all the time. These sub-clades can then go on to reinfect us in a cyclical fashion, known as flu season. We have vaccines for the flu, but again they only stop disease, not transmission. In fact, the H1N1 subtype of the influenza A virus, which is to blame for most infections, is a descendant of the virus that caused the 1918 Spanish flu pandemic. So even a hundred years later, we continue to feel its effects. Vaccination campaigns were indispensable to controlling and resolving the polio epidemics of the 1940s and 1950s. They have continued to be central to eradication efforts ever since. A vaccine that helps prevent transmission is still far away in the case of SARSCoV-2, if it ever arrives. This difference between poliovirus and SARS-CoV-2 drives home a key point: we cannot rely solely on vaccines to contain or eradicate SARS-CoV-2. Future pandemics may be similar in this regard — within the influenza family, too, there are many strains that we know little about and that may not permit us the luxury of eradication simply through vaccination. Polio aside, there is another epidemic we can learn from, one where there are no vaccines to speak of. Lessons from HIV/AIDS: prophylactic drugs On June 5, 1981 the United States Centers for Disease Control and Prevention (CDC) published a report describing five cases of a rare lung infection, pneumocystis carinii pneumonia, in the Los Angeles area5,7. All five cases were in young gay men who were in good health before the onset of symptoms. Along with the pneumonia, all of the men also had other unusual infections. Their immune systems were clearly not working as they should be. At the 692

time of publication, two of the men had already passed away. The remaining three men passed away shortly after. A year later, in September of 1982, the CDC gave the disease an official name: acquired immunodeficiency syndrome (AIDS). It took until 1984 for the cause of AIDS to be discovered, a retrovirus initially labeled HTLV-III, now known as human immunodeficiency virus (HIV). More than 40 years have passed since that initial report and HIV/AIDS continues to be a public health issue in many parts of the world. There is no cure for the disease and, as mentioned, we do not have a vaccine. I suspect we may never develop a vaccine like the ones we have for polio. This has to do with the particularly crafty nature of the virus. Like influenza and SARS-CoV-2, and unlike poliovirus, HIV infection does not lead to lasting immunity. On the contrary, HIV infection means persistent, life-long infection; our immune system just can’t get rid of the virus, allowing it to replicate and circulate indefinitely. Once HIV has entered the body, it quickly begins infecting immune cells — the very cells that we usually rely on to fight infection. Its primary targets are T helper cells. These have a number of important immune functions, including: the activation of small signaling proteins, called cytokines, that help initiate the inflammatory response and call other immune cells into action; the production of killer T cells which destroy cancer cells, cells infected by pathogens, and cells damaged through other means; and finally, they also maximize the antibacterial activity of phagocytes, which ingest foreign invaders. HIV infection of T helper cells destroys them, leaving them functionally useless. With time, the number of T helper cells drop below a critical level, causing a complete attrition of cell-mediated immunity. This leaves HIV-positive individuals at an extremely high risk of opportunistic infections and cancers. As with SARS-CoV-2, HIV is a master of evading our immune system. The immune cells it infects act as a disguise, providing the virus with a reservoir to replicate undisturbed and unseen. As long as there is a single latent reservoir, the infection can bounce back to full force without much trouble. Added to this is the fact it replicates at break-neck speed, brute forcing its way past our immune system, which simply can’t keep up. And not only is the replication cycle fast, it also churns out massive amounts of mutations. This leaves us with many different strains, all circulating at the same time. For now, 693

this combination of factors has bested all attempts at vaccine development. Regardless, we have made tremendous progress. The biggest contribution, aside from the fervent activism that set things in motion, has come from investment in and development of pre-exposure prophylaxis (or PrEP) medicine. PrEP medication helps prevent HIV infection and can be taken by HIV-negative individuals at high-risk of exposure. We also have effective treatment options for HIV-positive patients: antiretroviral therapy. These suppress viral load to undetectable levels, eliminating the risk of the virus being transmitted to an HIV-negative partner. Together, the two drug therapies make for a powerful combination — one helps prevent those who don’t have the virus from contracting it, the other helps prevent those who do have the virus from passing it on. At the personal level, these drugs mean that HIV infection can now be managed, allowing HIV-positive individuals to live long, healthy lives. At the population level, these same drugs act as a containment mechanism, serving to curb the spread of the virus. The blueprint is clear: learn as much about a given pathogen as we possibly can and then use this newfound knowledge to develop prophylactic drugs that help render it de facto irrelevant. This is what has been working for HIV/AIDS, it is what will work for SARSCoV-2, and it is also what will work for future pandemics that cannot be solved solely through vaccination. Of course, this won’t happen overnight; it requires persistent effort, resources, and funding. It also requires international collaboration and, perhaps most importantly, a certain amount of political will. References 1. Mehndiratta, M. M., Mehndiratta, P., & Pande, R. (2014, October). Poliomy elitis: Historical facts, epidemiology, and current challenges in eradication. The Neurohospitalist. Retrieved July 2022. 2. Canadian International Immunization Initiative (CIII) & Canadian Public Health 3. Association (CPHA). (2004, December). The end of polio is within our grasp… but 4. major challenges remain to reach goal by 2005. Retrieved July 2022.

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5. Haseltine, W. A. (2021, August 12). How Sars-CoV-2 Evades And Suppresses The Immune System (Part 1). Forbes. Retrieved July 2022. 6. World Health Organization. (2019, March 11). WHO launches new global influenza strategy. Retrieved July 2022. 7. A Timeline of HIV and AIDS. HIV.gov. (2022, April 29). Retrieved July 2022. 8. Ståhl, P.L., et al., Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science, 2016. 353(6294): p. 78-82. 9. Centers for Disease Control and Prevention. (n.d.). Pneumocystis pneumonia — Los Angeles 1981. Centers for Disease Control and Prevention. Retrieved July 2022. William R. Haseltine, PhD, is chair and president of the think tank ACCESS Health International, a former Harvard Medical School and School of Public Health professor and founder of the university’s cancer and HIV/AIDS research departments. He is also the founder of more than a dozen biotechnology companies, including Human Genome Sciences. This article is featured on Precision Medicine, and can be read online here: Controlling COVID-19: What Can We Learn From Previous Pandemics?

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An End To HIV In Botswana, Why Can’t We Accomplish The Same In The US? Forbes | August 17, 2022 | Article

Botswana has surpassed the UN's 95-95-95 goals on HIV. Researchers say that the country is on track to end the public health threat of AIDS by 2030. A truly remarkable feat, considering that Botswana is ranked among the top four countries in the world most affected by HIV and AIDS. Botswana has exceeded the UNAIDS targets, with 95 percent of HIV-positive people being aware of their status, 98 percent of those diagnosed on medication, and 98 percent of those under treatment showing signs that the virus is being suppressed in their blood and therefore cannot transmit the virus (U=U). Botswana Harvard AIDS Institute virologist, Madisa Mine presented the 5th Impact Survey at The 24th International AIDS Conference in Montreal, Canada. The survey has not yet been published. The survey was a nationally representative sample of 14 763 adults with ages ranging from 15–64 years who filled in questionnaires at home and were tested for HIV, viral load, and the presence of antiretrovirals. The accomplishment is the result of a national response strategy spanning two decades. The success of the strategy is attributed to the sustained political focus from three different Presidents (initiated under President Mogae in 1996), strong domestic and foreign investment, a collaboration between political and scientific leaders, and an evidence-based, inclusive, guideline setting with an openness to learn from international experts. The evolving partnership between the Botswana government and Harvard AIDS Insitute has also played a significant role in delivering high-quality health research, training, practical interventions, and infrastructure. The partnership was first established when Former President of Botswana Sir Ketumile Masire met with Harvard Professor Dr. Max Essex to sign a renewable memorandum of understanding in 1996, resulting in a collaboration 696

between Botswana’s Ministry of Health and the Havard AIDS Institute to implement HIV/AIDS research and training initiatives. This program then become the Botswana Harvard AIDS Institute Partnership and has been renewed every five years since. In 1997, the Botswana Harvard AIDS Institute commenced its first study at Harvard to characterize the molecular and biologic traits of HIV in Botswana. Another critical element of the response was investing in a strong health system. In 2000, the Botswana Harvard AIDS Institute Partnership commenced work on state-of-the-art BotswanaHarvard HIV Reference Laboratory, that was was the largest such laboratory dedicated to HIV/AIDS research and diagnosis in Africa at the time. The laboratory has allowed Botswana to carry out critical epidemiologic work in the country and launched ongoing projects aimed at curbing mother-to-child transmission of HIV, understanding resistance and adherence to antiretroviral drugs, and conducting genomic analysis of HIV-1C, the viral subtype predominant in southern Africa. Today, the lab employs 350 full-time employees, with local researchers taking the lead. When the Covid-19 pandemic struck, the lab was also well equipped to pivot to genomic surveillance and was credited with identifying the Omicron variant. On my visits to Bostwana, I was impressed with the remarkable reach of the lab. Even those living in isolated communities were able to participate in clinical trials. In 2002, President Mogae announced an HIV/AIDS program called Masa, which translates as New Dawn. The same year, Botswana became the first African country to implement a test-totreat program and provide free anti-retroviral drugs and introduce routine HIV testing in medical appointments, unless the patient explicitly refused. Decriminalizing same-sex relationships also helped reduce the stigma associated with HIV and reduced barriers to care. The Botswana Harvard AIDS Institute worked closely on this program with the Botswana government, and President Mogae commended Dr. Max Essex for his commitment to building local capacity by training health care workers and scientists from Botswana and surrounding countries. Funding for the program also came from a private-public collaboration called The African Comprehensive HIV/AIDS 697

Partnerships between the Government and the Bill & Melinda Gates Foundation, and Merck, which donated the drugs. Additional support came from PEPFAR and the Global Fund. The affordability of HIV drugs in the US and in many other parts of the world is a huge barrier to prevention and treatment. The commercial price for a pre-exposure prophylaxis HIV drug cabotegravir has been set at $3,700 per dose in the US, which is equivalent to an annual price of $22,200 per person. Calculations by the Clinton Health Access Initiative found that generics companies could make cabotegravir for $23 per person. The results of a randomized clinical trial showed that the drug is more effective at preventing infection than the standard means of pre-exposure prophylaxis, a daily oral dose of tenofovir disoproxil fumarate and emtricitabine currently in use around the world. In 2003, the Government launched a National Strategic Framework that provided clear guidance for ministries, districts, non-governmental organizations, and the private sector, allowing them to work collaboratively. Working with the Botswana Harvard AIDS Institute researchers, Botswana also focused on eliminating mother-to-child transmission of HIV, by treating all pregnant and breastfeeding women living with HIV with a highly effective lifelong triple antiretroviral treatment regimen at the time of diagnosis. In December, 2021, Botswana received an award from WHO for cutting child transmission rates to less than 5% after achieving transmission rates of 2·2%, down from 9% a decade earlier. While significant foreign aid investments were made to develop the initial infrastructure for the program, the willingness of the Bostwana government to invest heavily in the program is impressive and important for the longevity and sustainability of the program. UNAIDS estimates that around 60% of investment into HIV came from the national government, 30% from PEPFAR, and 10% from the Global Fund. Egypt’s remarkable initiative 100 Million Healthy Lives is another example of how chronic diseases such as Hepatitis C can be virtually eliminated in a large population. Egypt previously had the highest rate of Hepatitis C in the world. In 2018, Egypt launched the 100 Million Healthy Lives program. The goal was to screen all Egyptians over the age of 12 for active hepatitis C virus replication 698

along with other chronic conditions such as hypertension, diabetes, and obesity. Treatment was offered for free in government clinics for those who tested positive for hepatitis C, hypertension, and diabetes; free counseling was available for those considered obese. Approximately 4 million people with active hepatitis C were identified and treated with the antiviral medication Sovaldi (sofosbuvir), a nucleotide analogue that inhibits the polymerase enzyme of hepatitis C and blocks its replication, effectively eliminating hepatitis C from Egypt. Once initiated, the program was completed in 18 months. The cost to the Egyptian government was about $45 per 3-month treatment. Patients electing to receive treatment at a private clinic paid $75. The program was primarily funded by a World Bank loan of $530 million. About half of the loan was to expand screening and treatment and the remainder was for health system strengthening. If both Botswana (a middle-income country) and Egypt (a lowincome country with a large population of over 100 million) can accomplish such a significant reduction of chronic diseases like Hepatitis C and HIV, why can’t Europe and the United States? Their success provides proof of principle and a model that could be adapted and replicated globally. Both the strategy employed in Botswana to reduce transmission of HIV and Egypt’s 100 Million Healthy Lives initiative serves as proof of principal for how Hepatitis C, HIV and other chronic diseases could be eliminated in the United States and around the world. This article is featured on Forbes.org, and can be read online here: An End To HIV In Botswana, Why Can’t We Accomplish The Same In The US?

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Will Covid-19 Vaccines Continue To Protect Us From Hospitalization And Death?? Forbes | August 17, 2022 | Article

Vaccines are integral to our control of Covid-19—if not for preventing infection, at least for preventing severe illness and death. But what if vaccine-induced immunity lost its efficacy against new variants on all accounts? Along with earlier work published by MIT, a recent study from Cardiff University places this thought into the realm of possibility. In their paper, Dolton et al. investigate the origins of a SARS-CoV-2 spike protein mutation and its impacts on T cell immune responses in recovering Covid-19 patients and vaccine es. Their analysis revealed that the SARS-CoV-2 spike protein mutation appears invisible to CD8+ T cells, thus dodging immune responses from previous infection and vaccines. About the Study Immune mechanism: CD8 T cells The efficacy of vaccines depends on adaptive immunity, the ability of the body to recognize organisms and structures not previously encountered. Adaptive immunity can be split into two classes: humoral immunity driven by antibodies, and cell-mediated immunity driven by “helper” CD4 T cells and “killer” CD8 T cells. While both contribute to the body’s adaptive immune mechanisms to counter foreign invaders, in the context of the Covid-19, cellmediated immunity seems the more necessary component to SARSCoV-2 recovery. Current understanding dictates that much of the body’s ability to prevent infection comes from the humoral response; it’s now been very well documented that successive variants of SARS-CoV-2 evade by mutations primarily in the Spike protein. Each one of these successive waves of new infections allow the virus to infect people who were previously infected as well as evade the waning immunity such as provided by vaccines. However, these vaccines have proven effective in dramatically reducing, but not entirely eliminating, serious illness requiring hospitalization and death. The ability of 700

vaccines to do so is thought by most researchers to be the result of recognition and clearance of the virus through cell-mediated immunity, primarily through CD8+ killer T cells.

Computer illustration of T-cell receptors (purple) binding to MHC-antigen complexes (orange and green). The antigen (light green) is a peptide broken down from a tumour cell, bacteria or virus. A number of cells present antigens to T cells via membrane bound major histocompatibility complex (MHC) molecules (orange). After binding to the MHC-antigen complex, the T-cell receptor sends a signal cascade into the T-lymphocyte cell (top, in green) that activates an immune response. GETTY For this study, Dolton et al. focused solely on CD8+ T cell responses. Cell-mediated mechanisms differ from that of humoral immunity. Unlike antibodies, cytotoxic T cells cannot bind and recognize antigens directly. They require antigen presenting cells (APC) to break down foreign proteins into smaller protein fragments and to present the resultant peptides on its cell surface for T cells. Major histocompatibility complexes (MHC) present the resulting peptides to the cytotoxic CD8+ T cells. MHC class I molecules present to cytotoxic CD8+ T cells in particular. Here, the investigators examined killer T cell responses through the most prevalent MHC class I allele in humans—HLA A*02—which occurs in 40% of individuals.

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Figure 2: Mapping of mutants onto Spike prefusion structure (PDB 6VXX). Mutation P272L is indicated in red. DOLTON ET AL.

Spike Epitope Mutation P272L After selecting their scope—CD8+ T cell responses observed through the HLA A*02 allele—the researchers identified which SARS-CoV-2 protein mutation to investigate. Considerations began in people recovering from Covid-19. One study revealed that the most frequent CD8 T cell responses in HLA A*02+ convalescent patients recognized regions of the virus within Spike residues 261-280, as well as ORF1ab residues 3,881-3,900. From this pool, Dolton et al. selected Spike residues 269-277 (YLQPRTFLL) for analysis. The most prevalent mutation in this T cell epitope is an amino acid substitution of proline for leucine, denoted as YLQLRTFLL or P272L mutation. Study Results Tracking the P272L Spike protein mutation The next step entailed contextualizing mutation P272L by noting its first appearance and frequency in several SARS-CoV-2 variants. The team compared global genome sequencing datasets of 702

Spike residues 269-277 starting from January 1st, 2022 through the course of the pandemic.

Figure 3: SARS-CoV-2 viral dynamics of P272L Spike mutation in Western Europe during a period of 16 epidemiological weeks. The figure includes the first recorded appearance of P272L on March 8th, 2020, and continues up until November 28th, 2021. The increased frequency of Spike mutation P272L, coupled with its tendency to emerge independently in other variants of concern beyond B.1.117, suggests it could confer some selective advantage. DOLTON ET AL.

They found that the two most frequently occurring mutations in this region occurred at position 272, with P272L widely distributed internationally. The mutation was first detected in lineages B1 and B.1.1.263 of March 2020; by June 2020, it appeared in B.1.177 and spread extensively throughout Europe (as shown in Figure 3). Additional analysis revealed the P272L mutation recurred frequently and independently in other variants. Dolton et al. report that P272L ranked in the top 15 Spike mutations observed globally. In 2022, P272L spontaneously arose in variants of concern including B.1.1.7/Alpha, B.1.351/Beta, B.1.617.2/Delta, P.1/Gamma, and BA.1/Omicron. The mutation also transmitted locally. Examples include Campania, Italy and Nebraska, USA. Variants carrying the 703

mutation comprised 32% and 29% (respectively) of reported sequences between February 2021 to June 2021. T cell immune response in convalescent patients and vaccinees With the epidemiological context established, the question remained: how important is Spike epitope YLQPRTFLL? The investigators sought the answer in two ways: by comparing T cell responses to the founder and variant epitopes in convalescent patients; and by comparing T cell responses to the founder and variant epitopes in vaccinees with no prior history of Covid-19. To accomplish the first objective, the researchers recruited healthcare workers who had Covid-19 more than 28 days before sample collection in 2020. A polymerase chain reaction (PCR) test confirmed the status. The donors were screened for HLA A*02+ through antibody staining. The team confirmed the wide response to the epitope, as twelve out of the thirteen patients who tested positive for HLA A*02+ also had CD8 T cells stained with HLA A∗02:01-YLQPRTFLL tetramer. In contrast, the P272L mutant failed to elicit a response. The YLQLRTFL tetramer did not stain T cells in four convalescent patients. Moreover, a T cell line created from tetramer+ patients did not respond to the mutant despite more than 128 different T cell receptors. Surrogate infected cells with full-length spike and the P272L substitution also failed to recognize T cell clones, supporting the hypothesis that mutant P272L escapes all T cell receptors raised against the Wuhan sequence—a troubling find, as the Wuhan spike is the antigen in all current vaccines. Dolton et al. also tested T cell responses in vaccinated individuals with no history of Covid-19 and screened positive for HLA A*02+. All seven vaccinees received either the AstraZeneca (ChAdOx1 nCoV-19) vaccine or the Pfizer (BNT162b2 2) vaccine. All participants had T cells which recognize the founder epitope—but do not recognize the P272L mutation. While it is possible that previously infected or vaccinated individuals could raise an entirely new response to the P272L Spike mutant, and that the YLQLRTFLL sequence could fall into a naïve TCR repertoire “blind spot” that most individuals cannot recognize, Dolton et al. ultimately conclude that T cells specific to spike epitope 269–277 cannot recognize the P272L spike. This, in turn, suggests 704

that such a mutant could escape immunity induced from current interactions of AstraZeneca and Pfizer vaccines. Possible Implications The emergence of a T cell escape variant of concern may be impending. The studies of Naranghai et al. and Dolton et al. reaffirm this possible peril. Mutation P272L increased in frequency in B.1.177, and emerged independently in other SARS-CoV-2 lineages. Eventually, a T cell escaping mutation could confer a strong selective advantage. As Dolton et al. mention in their work, “As the pandemic progresses, and SARS-CoV-2 reaches an optimal position with regards to ACE2 binding and cell entry, it is likely that other selective advantages (such as T cell escape) will become more important in the mix of potential advantages that the virus could develop to enable it to persist as a human pathogen.” What of our best protections against Covid-19, vaccines and immunity from prior infection? These defensive measures may already be weakening. Evidence from Naranbhai et al. suggests that spike mutations in Omicron variant B.1.1.529 can reduce protection in about 21% of people previously infected and/or vaccinated. And should P272L or a similar variant emerge, we can expect even more T cell evasion than already exists. Given this data, it would be advisable to monitor Spike epitope 269–277 and other viral proteins (ex: ORF1ab residues 3,881-3,900) for possible T cell escape. And Covid-19 vaccines, despite their ability to drastically reduce hospitalization and death, are not impervious to the threat of antigenic variation of SARS-CoV-2 spike. In the future, boosters may need to be altered to anticipate variants primed for T cell escape. This article is featured on Forbes.org, and can be read online here: Will Covid-19 Vaccines Continue To Protect Us From Hospitalization And Death??

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Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 2) Forbes | August 18, 2022 | Article

This is a short series about a recent breakthrough on the road to developing a much sought-after broadly neutralizing vaccine against all influenza A viruses. If successful, it may act as a precursor to a truly universal flu vaccine, one that protects against all types, subtypes, and lineages of the virus. The breakthrough may also provide a blueprint for developing a Covid19 vaccine that retains its efficacy in the face of new variants. In the first part of this series, I gave a brief overview of the history and nature of influenza viruses, including why it has been so difficult to develop successful vaccines. The next few articles discuss some of the attempts that have been made to overcome these challenges, including their shortcomings. And in the last installments, I will offer a detailed analysis of the latest —and most promising— advances in the field. The Seasonal Approach Picking up where we left off in the previous article, any successful influenza vaccine has to account for the ability of influenza viruses to mutate. Genetic mutations to vital proteins can lead to antigenic variation — changes to parts of the virus that our immune system relies on to stimulate its “memory”. Although various different parts of the virus serve as antigens, the surface proteins that help it enter and exit host cells are some of the most important. Changes to these proteins can prevent our antibodies from recognizing the virus, rendering them unable to block its spread. Antigenic variation is responsible for influenza reinfections, leading to seasonal flu outbreaks. In an attempt to circumvent the issue of antigenic variation, vaccine manufacturers update the flu shot each year based on the latest circulating influenza strains. The idea is to expose our immune system to the antigens it is most likely to encounter during flu season, helping it to build up its antigen-specific defenses in advance — once

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our immune system has built up its memory, it can jump into action straight away should we become infected. Which influenza strains ultimately get used to make the yearly flu shot is decided on the basis of data collected throughout the year by the World Health Organization’s (WHO) Global Influenza Surveillance and Response System (GISRS). This surveillance and response system is made up of roughly 150 different laboratories spread across the globe, each of which gathers thousands of influenza samples from sick patients. The most prevalent viral strains are then shared with five WHO Collaborating Centers for Influenza, which perform further analysis. Two times a year —once in preparation for flu season in the Northern Hemisphere, and another in preparation for flu season in the Southern Hemisphere— Directors of the WHO Collaborating Centers, Essential Regulatory Laboratories, and representatives of a few of the smaller national laboratories come together to: “review the results of surveillance, laboratory, and clinical studies, and the availability of flu vaccine viruses and make recommendations on the composition of flu vaccines.” Once the WHO vaccine composition committee has made its recommendations, each country makes a final decision on which viruses they will choose to use in their flu vaccines. In the United States, all influenza vaccines are “quadrivalent”, meaning they contain four different influenza viruses. This is done to broaden protection against the various influenza subtypes and lineages known to drive seasonal outbreaks: influenza A (H1N1), influenza A (H3N2), influenza B/Victoria, and influenza B/Yamagata. Quadrivalent vaccines will also protect against any other influenza viruses that are antigenically similar. Although this may seem like a relatively reliable process, there is one glaring drawback to the seasonal vaccination approach: vaccines produced in this way are nowhere near as effective as we might hope. At best, they protect 60% of people from illness, but this number can, and often does, drop much lower. For the influenza A (H3N2) subtype, vaccine effectiveness hovers around 33%. Of course, any protection is better than no protection, but it is still suboptimal— remember, these numbers represent best case scenarios, years where the viruses selected for use in vaccines are well matched to those that actually end up circulating during the flu season. So, where are things going wrong? 707

Missing the Target: Egg-based Vaccines Selection of candidate vaccine viruses (CVVs) is only one part of the equation, growing them is another. This is no simple feat considering they need to be available in bulk, enough to make millions of vaccines. For the past 70 years, the majority of manufacturers have turned to chicken eggs in order to achieve the necessary growth (Figure 1). The candidate vaccine viruses are injected into fertilized hen’s eggs and left to incubate for a few days. During this period, the viruses are able to replicate. The fluid in the eggs is then extracted and the viruses are “killed” (inactivated). Finally, the antigen of choice —usually the hemagglutinin surface protein— is isolated from the killed viruses and purified, making it ready for use in vaccines. Even now, most flu vaccines continue to be egg-based.

FIGURE 1. An overview of the steps involved in producing egg-based vaccines. SOURCE: ESCO VACCIXCELL (HTTPS://ESCOVACCIXCELL.COM/APPLICATIONS/TECHNOLOGYADAPTION-SCALE-UP/EGG-BASED-INFLUENZA-VACCINES)

But there are two issues with this approach. First, growing the viruses in eggs is a fairly slow process. This means the selection of candidate vaccine viruses has to happen far in advance of flu season, to make sure manufacturers have enough time to produce the amounts needed. In the six to nine months it takes to grow and purify enough virus, the wild type influenza strains continue to mutate and change. If these changes impact the antigen, the wild type viruses may escape the immunity that the vaccines provide us, 708

reducing their effectiveness. When this happens, the viruses are referred to as “escape mutants”. A growing body of research suggests that a second factor may be even more important: egg-adapted changes. Because the candidate vaccine viruses are human influenza viruses, growing them in chicken eggs carries the risk that they adapt to the new immune niche while replicating. The immune niche of chickens is different to that of humans, so adaptations that improve viral fitness in chickens may result in genetic and antigenic changes to the viruses. As before, these changes can lead to a drop in vaccine effectiveness, since the vaccine strains no longer resemble the circulating wild type strains; the egg-adapted vaccines end up training our immune system to recognize the wrong viruses, thus hampering its ability to respond efficiently come flu season. “Egg Substitutes”: New Ways of Growing Candidate Viruses In response to these issues, manufacturers have tried to develop new production methods that avoid using chicken eggs to culture candidate viruses. This search has led to a cell-based approach and a recombinant approach (Figure 2).

FIGURE 2. Timeline of current influenza vaccine production methods. Schematic overview of egg-based, cell-based and protein-based (recombinant) influenza vaccine production. SOURCE: “BETTER INFLUENZA VACCINES: AN INDUSTRY PERSPECTIVE” CHEN ET AL. 2020

Cell-based vaccines are produced using candidate viruses grown in mammalian cells rather than chicken eggs. Aside from this, the 709

manufacturing process between the two is virtually identical: candidate vaccine viruses are grown in mammalian cell cultures by the CDC, these are then handed over to private manufacturers who inoculate the viruses into mammalian cells, the viruses are left to replicate for a few days before being harvested, and finally, purified. Although approved in 2012, it wasn’t until this past 2021-2022 flu season that fully egg-free, cell-based vaccines were produced — previously, the initial production of candidate vaccine viruses by the CDC was still done using fertilized hen’s eggs, and only after being handed over to the private sector were the viruses mass-produced in mammalian cells. Using the cell-based approach eliminates egg-adapted changes in candidate viruses, keeping the viruses as close as possible to the wild type influenza strains predicted to circulate during flu season. An added benefit of cell-based vaccines is that the production process can be scaled up more quickly; mammalian cells can be frozen in advance to ensure steady supply, which could prove especially useful during pandemic outbreaks. In theory, the lack of egg-adapted changes should improve vaccine effectiveness. But what about in practice? Although there still hasn’t been enough research for a clear consensus to develop, initial findings suggest the difference in effectiveness is modest at best, and statistically insignificant at worst. This hints that eggadapted changes might not play as important of a role as initially suspected; low vaccine efficacy can occur even when eggs are not used in the manufacturing process. That said, the 2021-2022 flu season marks the first time truly egg-free cell-based vaccines —in which all four viruses are derived entirely through cell-based methods— were used, so perhaps future research will yield different results. For now, things don’t look too promising. Recombinant vaccines provide a third option, and manage to overcome a crucial issue faced by the other two options: the lengthy, tedious virus production process. Whereas egg- and cell-based vaccines depend on candidate virus samples, recombinant manufacturing skips this step. Instead, recombinant vaccines are made by isolating the gene that makes the hemagglutinin surface protein from a wild type influenza virus. Once isolated, this gene is combined with a different kind of virus, called baculovirus. The new virus is known as a “recombinant” baculovirus and it is used to ferry 710

the gene that makes the hemagglutinin antigen into a host cell line. As soon as the gene enters the cells, they begin to mass produce the hemagglutinin antigen. The antigen can then be extracted and purified before being assembled into a vaccine. Given that they are entirely egg-free and don’t require candidate virus samples, recombinant vaccines bypass the issue of eggdependent changes. Due to the speed of production, there is also a decreased risk of escape mutants developing. As before, there is a paucity of comparative research, making it difficult to draw any firm conclusions, but early findings suggest recombinant vaccines may be more effective than traditional egg-based and cell-based vaccines, including improved antibody production. Takeaway Developing consistently protective influenza vaccines has proven difficult, with effectiveness frequently hovering somewhere between 40 and 60%. Too low, considering the threat posed by influenza. A big part of the challenge is the mutability of the virus; it is constantly changing, making it hard for our immune system to keep up and retain useful “memories” of previous encounters. In response, public health agencies and scientists around the world develop new vaccines every year that prime our immune systems for the latest circulating strains. Sometimes scientists miss the mark with their predictions, in which case the circulating influenza strains do not match up with those in the vaccine, undermining vaccine effectiveness. At other times predictions are right on the money, but the vaccine production process impairs effectiveness either by being too slow and giving the wild type viruses time to mutate again, or because of mutations to the candidate vaccine strains during massproduction in chicken eggs. Cell-based and recombinant vaccines aim to resolve the issues on the production side of things. The former by skipping the need for eggs, and by extension, the threat of egg-adapted changes. The latter by skipping the need for eggs as well as cutting down the time it takes to produce the vaccines, reducing the risk of escape mutants. Despite these advances, vaccine effectiveness has not yet seen the boost it needs. The below table gives a summary of the advantages and disadvantages associated with these three production processes.

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FIGURE 3. Advantages and disadvantages of strategies for influenza virus vaccine production. SOURCE: “THE EVOLUTION OF SEASONAL INFLUENZA VIRUSES” PETROVA ET AL. 2017

The next article in this series will look at two additional technologies: intranasal vaccines and mRNA vaccines. Might they succeed where the more traditional strategies have wavered? This article is featured on Forbes.org, and can be read online here: Getting A Grip On Influenza: The Pursuit Of A Universal Vaccine (Part 2)

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How To Mend A Broken Heart Forbes | August 20, 2022 | Article

This story on artificial hearts is part of an extended series on Regenerative Medicine. For other stories on this topic see williamhaseltine.com and search for Regenerative Medicine. My definition of Regenerative Medicine is any medical modality that returns us to normal health when we are damaged by disease, injured by trauma, disadvantaged by birth, or worn by time. Modalities include: chemicals, genes, proteins and cells used as drugs, gene editing, prosthetics, and mind-machine interfaces. Heart disease affects approximately 82.6 million people in the United States and is the leading cause of death among both men and women. One solution for those suffering from advanced heart failure is heart transplantation. Unfortunately, there is currently a nationwide shortage of human donor hearts. Scientists have attempted to create artificial hearts or use pig organs in lieu of human hearts for transplantation surgeries. However, current methods to produce artificial hearts are generally unsuccessful and the use of pig organs for transplants can lead to serious infections. Now, a group at Harvard University is tackling this issue through a new, innovative method of growing artificial hearts. By building an artificial structure and implanting cardiac cells, researchers were able to grow the cardiac cells in a pattern that mimicked the natural organization of muscles in the heart. This study serves as a significant stepping stone toward developing artificial hearts that are fully functional. The heart is largely made from muscles arranged in a helical fashion. When the heart contracts, its helically-patterned muscles engage in a twisting motion to push blood out of the heart. In fact, this helical patterning is predicted to be a crucial characteristic of healthy, functioning hearts. Many individuals who suffer from cardiac dysfunction also exhibit abnormal muscular patterning.

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Figure 1: Human hearts engage in a twisting motion to pump blood through the body.BUCKBERG, THE JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY (2002), DOI: 10.1067/MTC.2002.122439

In the past, several studies have attempted to grow artificial hearts with helical patterning by using 3D printers. These studies have largely been unsuccessful because 3D printers are unable to achieve the tiny details of the heart’s structure within a reasonable amount of time. For instance, a 3D printer could take hundreds of years to print even a small component of the heart’s structures with enough detail for cells to grow in the correct patterns. So, how did scientists at Harvard University achieve this feat? Knowing that a simple 3D printer has significant limitations, Chang et al. turned towards a different technique: fiber-spinning. Fiber-spinning is a method that uses similar materials to 3D printers but can produce much finer, high-resolution structures. Traditionally, materials are heated and extruded from a tiny hole to create singular fibers at a microscopic scale. The fibers can then be collected or processed to form 3D structures.

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Figure 2: Fiber-spinning involves extruding polymers from a tiny hole to create microscopic fibers.MARTINS D, ET AL. (2019) BIODEGRADABLE POLYMER NANOFIBERS APPLIED IN SLOW RELEASE SYSTEMS FOR AGRIFOOD APPLICATIONS. POLYMERS FOR AGRI-FOOD APPLICATIONS. SPRINGER INTERNATIONAL PUBLISHING

Fiber-spinning can create structures with very high resolutions. However, traditional methods of fiber-spinning are often imprecise and would not be able to form the consistent helical patterns of the heart. This prompted Chang et al. to engineer a new method of fiber-spinning that would not only allow them to create the heart’s 3D structure at a microscopic scale but would also be precise enough to form the heart’s helical patterning. Chang et al. created a new fiber-spinning device with two major design features. First, instead of simply extruding the material haphazardly in one direction, the fiber-spinning device contains a “spinneret” that spins at high speeds. When the heated material is pushed into the device, the fibers are then extruded through a small hole in the side of the spinneret. This causes the fibers to collect in a cloud around the device. Chang et al.’s second innovation was to include a strong stream of air at the top of the spinneret that could align the fibers to resemble the striations of muscles. From this, Chang et al. could

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collect the fibers at an angle, ultimately creating the helical patterns of cardiac muscle.

Figure 3: Chang et al. used a spinneret and focused airstream to create 3D structures of the heart.CHANG ET AL., SCIENCE (2022), DOI: 10.1126/SCIENCE.ABL6395

Using this method, Chang et al. was able to create 3D frames that resembled human heart ventricles. When the frames were seeded with human cardiac cells, the resulting tissues maintained the helical patterning of the frame. Surprisingly, after 3 to 5 days of growing cardiac cells on the 3D frames, Chang et al. observed spontaneous contractions that resembled the natural activity of the human heart. This indicated that Chang et al.’s model ventricles could be used to study how muscle patterning affects heart function. To investigate this question, Chang et al. created model ventricles with helically aligned cells as well as ventricles with abnormal, circumferentially aligned cells.

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Figure 4: Chang et al. created model ventricles with helically aligned cells and circumferentially aligned cells.CHANG ET AL., SCIENCE (2022), DOI: 10.1126/SCIENCE.ABL6395

The researchers then suspended both model ventricles in a liquid containing fluorescent beads. By tracking the displacement of the beads, Chang et al. could determine how many were pumped through the ventricles at a time. This strategy allowed the researchers to calculate the overall volume of liquid the model ventricles could pump. After testing both the helically patterned ventricle and the abnormally patterned ventricle, Chang et al. found that the helically patterned ventricle was able to pump significantly higher volumes of liquid. This demonstrated that abnormal alignment of cardiac cells does, in fact, decrease the heart’s ability to function. Finally, not only was Chang et al. able to create model heart ventricles that could contract, but by using their innovative fiberspinning method, the researchers were able to recreate all four chambers of the heart. These individual chambers were then assembled to ultimately create a full-sized model of the human heart.

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Figure 5: Chang et al. successfully created all four chambers of the heart to assemble a fullsized model. CHANG ET AL., SCIENCE (2022), DOI: 10.1126/SCIENCE.ABL6395

Overall, this study represents significant progress in our ability to create a fully functional artificial heart. While more work must be done to expand functional model ventricles into full-scale heart models, this study demonstrates real promise for the use of innovative fiber-spinning techniques for complex whole-organ formation. This article is featured on Forbes.org, and can be read online here: How To Mend A Broken Heart

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Health Messaging in the Disinformation Age Project Syndicate | August , 2022 | Op-ed

Unclear, opaque public-health messaging has been a major problem throughout the COVID-19 pandemic, sowing confusion and making the crisis worse than it needed to be. To regain the public's trust, health officials must start learning from their mistakes and stop confusing credibility with infallibility. FAIRFIELD COUNTY, CT – As the director of the Centers for Disease Control and Prevention, Rochelle Walensky, recently acknowledged, poor public-health communication and messaging throughout the COVID-19 pandemic has damaged the public’s trust in health agencies and institutions. This, in turn, contributed to wellknown problems such as vaccine hesitancy, noncompliance with mask recommendations and other protective measures, and general misinformation about the virus and how it is transmitted. According to a 2021 poll from the Robert Wood Johnson Foundation and the Harvard T.H. Chan School of Public Health, only 52% of Americans now place a great deal of trust in the CDC, and only 37% have much confidence in the National Institutes of Health or the Food and Drug Administration. State health departments fare little better. They are trusted by just 41% of Americans, with local health departments trusted by 44%, and the same poll shows that positive ratings of the public health system declined from 43% to 34% between 2009 and 2021. Clearly, public-health agencies need to win back the public’s trust, not just to combat crises like COVID-19 and monkeypox, but also to address a wider range of ongoing health issues. This process must start with a commitment to community engagement, partnerships across other sectors such as housing and education, effective communication at every level, and transparency and integrity in decision-making. Public-health officials often must base their recommendations on incomplete data; as the data evolve, so will the recommendations. However, in a misguided effort to appear authoritative, public719

health officials are rarely transparent about the nuances and fluid nature of what they are communicating. A perfect example is the early advice on how SARS-CoV-2 is transmitted. The CDC was adamant that the coronavirus was spreading on surfaces and not through the air, rather than acknowledging that airborne transmission was still a strong possibility. This approach bred confusion and distrust, because the CDC eventually had to change its advice (as it should have foreseen). After acknowledging that SARS-CoV-2 was spreading through droplets, it finally also conceded that it was spreading through aerosol particles. As this example shows, credibility often gets confused with infallibility, resulting in public-health officials who may be slow to admit mistakes – further undermining their credibility. Transparency is key, especially at a time when peddlers of online misinformation will seize every opportunity to discredit public-health officials. Successful public-health communication establishes credibility by being effective, not by being unchanging. Another cornerstone of sound communication is clarity. Publichealth officials must explain how data and recommendations relate to people’s everyday lives. Whether the information is correct or incorrect is a moot question if the public doesn’t understand what is being communicated. Here, US officials failed the messaging test again when they did not make clear that COVID-19 vaccines’ effectiveness was measured by hospitalizations, not infections. The public believed that vaccines would block transmission and infection; but when the Delta and Omicron variants emerged and caused breakthrough infections to surge, distrust and “booster-shot fatigue” duly followed. As of August 3, only 32% of Americans had received their first booster shot. In this case, public-health officials could have used the example of the Salk polio vaccine to assure the public that a vaccine doesn’t need to prevent infection or transmission outright to eradicate a disease. Or, they could have emphasized how much the vaccines reduce the burden on our hospitals. Unfortunately, other historical lessons seem not to have sunk in. Many public-health officials have been committing a grave error by stigmatizing monkeypox as a disease that only threatens gay, 720

bisexual, and other men who have sex with men. Yet while it is true that this population has been disproportionately affected by the current outbreak, monkeypox can be transmitted in any situation where there is close skin-to-skin contact with lesions. By depicting monkeypox as a sexually transmitted infection, public-health officials could give people the false impression that they are not at risk, preventing them from seeking a diagnosis or isolating if they do contract the virus. The situation is not dissimilar to the (incorrect) early messaging about HIV/AIDS spreading only among homosexual populations. More broadly, public-health messages are best understood and most likely to be believed when they come from trusted individuals within the communities that need to be reached. The messenger is often as important as the message, especially in communities where structural racism and historical traumas have left people disinclined to trust medical authorities. Rather than issuing authoritative statements and assuming that they will be heeded, local public-health officials should think of their messaging as being part of an inclusive conversation. They should seek out community voices and trusted advocates such as faith leaders, shelter managers, and food-bank directors to collaborate on messaging and reaching populations that may be vulnerable to health disparities. Another good approach is the one pioneered by the Ryan White HIV/AIDS Health Services Planning Councils. These are community groups appointed by local officials whose members represent the general public, people living with HIV, funded service providers, and other health and social service organizations. Planning Council members work together to identify the care needs of people living with HIV. They then determine which services are highest priority, and how much funding should be committed to each. This model of inclusive decision-making could be applied more broadly to public-health planning and resource allocation. The current climate of “alternative facts” and rampant disinformation presents many challenges to effective public-health communication. But by learning from past mistakes and developing messages that are clear, inclusive, and conveyed by the right sources, we can start the difficult but necessary process of rebuilding trust in public-health agencies before the next big crisis strikes. 721

This article is featured on Forbes.org, and can be read online here: Health Messaging in the Disinformation Age

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Solving The Question Of Covid Variant Increased Fitness Is Like Deciphering A Rubik’s Cube Forbes | August 25, 2022 | Article

The ability of one virus to replace another is determined by its ability to evade the immune system of those previously infected or vaccinated, as well as intrinsic replication properties. It is essential to distinguish between these two properties as they work cooperatively to determine how fast a variant spreads through a population. Most studies throughout the pandemic have focused on the ability of the virus to evade the immune system because of the importance of prior immunity. However, a few studies have investigated the intrinsic replication properties of the virus. Two recent studies have revealed previously unknown epidemiological properties. The first by Wu et al. revealed that the incubation periods of successive variants are growing shorter. Their meta-analysis of 141 studies throughout the pandemic showed that the average incubation period of all cases was 6.57 days. For the Alpha variant specifically, the period was 5.00 days. For Beta, it was 4.50 days. For Delta, it was 4.41 days. For Omicron, it was only 3.42 days. This means later variants get the host sick sooner after the initial infection, and the virus moves much quicker from host to host. A second paper by Reuschl et al. demonstrated that the ability of the virus to suppress interferon, the first line of defense for innate immunity, is increased amongst the latest variants. These two properties are most likely associated with proteins other than the Spike. Specifically, the non-Spike structural proteins, namely E, M, and N, and the accessory proteins Orf3a through Orf10. In a series of elegant experiments described by Syed et al., the Gladstone Institutes have examined the intrinsic replicative properties of the virus in the absence of the Orf proteins using a 723

simplified virus-like particle. Virus-like particles are constructed only to contain specific proteins, allowing isolated testing of viral characteristics such as replication using luciferase enzymes to measure results. In a previous study, the same group of researchers found that the N protein was critical to the virus replication of the Alpha variant. They found that the four mutations in most natural variants increase mRNA delivery and expression by about 10-fold, whereas other mutations such as R203M increase virus production between 50 to 150-fold, displaying the significant role the structural proteins can play in transmission pathogenesis.

SYED ET AL.

FIGURE 2: N Protein Enhanced Replication. (A) Map of SARS-CoV-2 N domains showing the locations of ... [+] SYED ET AL.

In their recent study, they expand to analyze more recent variants and look at the effects of other proteins on replication outcomes, specifically, the structural proteins E, M, N, and S. Using virus-like particles, they could individually examine the impact of mutations in each structural protein. First investigating the 724

S protein, they found that the infectivity of the Delta Spike drops significantly compared to the ancestral B.1, whereas the Omicron Spike is roughly equivalent. Next, they examined the N protein of the three viruses and B.1.1, which includes mutations R203K and G204R. The B.1.1 N is roughly six-fold more infectious than B.1, Delta N is 23.5-fold more infectious, and Omicron N is 26-fold more infectious, indicating the heavily mutated Delta and Omicron N proteins contribute significantly to virus infectivity. Syed et al. then tested the M and E proteins, which is conjunction drop infectivity of both Delta and Omicron four-fold as compared to B.1. However, the Omicron M protein on its own remained as infectious as the B.1 isolate, indicating that the Omicron E protein contributes dropping infectivity. Finally, they analyzed the different viruses with a complete set of S, N, E, and M proteins. B.1.1 was 5.3-fold more infectious than the ancestral B.1 strain, whereas Delta was only 3.7-fold more infectious, and Omicron was 4.6-fold more infectious (Figure 2).

FIGURE 3: Omicron structural gene variants alter the infectivity of SC2-VLPs. SC2VLPs assembled in ... [+] SYED ET AL.

They also found that the cleavage and processing of Spike varied both between different S variants and when coexpressed with differing M and N variants. For example, Spike was less efficiently cleaved when all S, N, M, and E, were Omicron variants rather than an isolated Omicron Spike (Figure 3). FIGURE 4: Western blot of cell lysates from cells transfected to generate VLPs stained for N, S, and ... [+]

SYED ET AL. These results amplify the notion that N protein mutations play an essential role in the increased fitness of the virus. They also reveal 725

complex interactions between the structural proteins, suggesting epistatic effects of mutated viral proteins working together to create a more infectious virus. For instance, introducing E, M, and N to an S protein may increase overall viral expression, or in some cases, it may even reduce the replication level. These experiments add substantially to our understanding of the fitness of variants and how one virus can displace another. It involves not only immune evasion but also the ability of the virus to replicate and compensatory mutations throughout the genome that contribute to the virus’s ability to spread throughout a population. To solve a Rubik's Cube, you must have the nine pieces of each of the six sides correctly aligned. This is a good analogy for viral fitness. In addition to having optimal mutations in the Spike protein, there must also be optimal mutations in the E, M, and N proteins as well. In addition, there are many more dimensions to consider, as SARS-CoV-2 has roughly 30 proteins throughout the genome, akin to an immensely complicated 30-sided Rubik’s Cube. We await further studies that include the effects of accessory genes that are well known to play a role in the ability of the virus to counteract the immune system and possibly increase the rate of virus spread. This information would be critical to understanding current variants and what to expect from future iterations that may be more fit and possibly more dangerous or virulent. This article is featured on Forbes.org, and can be read online here: Solving The Question Of Covid Variant Increased Fitness Is Like Deciphering A Rubik’s Cube

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T Cells Play An Important Role In Immunity, But Without B Cells Cannot Provide Protection From Covid-19 Forbes | August , 2022 | Article

When invaded by a virus such as SARS-CoV-2, the body engages multiple layers of defense to ward off the intruder, just as a moat and various defensive walls serve to protect a castle. The first line of defense is innate immunity, followed by adaptive immunity. Adaptive immunity concerts two separate arms—humoral immunity driven by antibody-producing B cells, and cell-mediated immunity driven by “helper” T cells and “killer” T cells. In healthy people, the collaboration of both branches leads to the recognition and suppression of pathogens and infected cells. Typically some of these protections crumble with age, leaving the body more susceptible to disease. But for certain immunocompromised people, parts of the adaptive immune system may be missing altogether. A study published in the journal Viruses seeks to understand what happens to T cell responses if the B cell defense of adaptive immunity is severely compromised. In these immunocompromised patients, could T cells alone fend off SARS-CoV-2 infection? The results suggest that T cells make a valiant effort to ward off the serious effects of SARS-CoV-2 infection. Yet, in the absence of B cells, T cells alone may not be enough to surmount the virus. All about T cells The scientists at the University Medical Center HamburgEppendorf narrow their analysis of adaptive immune responses on cell-mediated immunity. The cell-mediated immunity operates mainly through two types of T cells: CD8+ “killer” T cells and CD4+ “helper” T cells. While killer T cells eliminate host cells infected by viruses and other pathogens, helper T cells indirectly aid antiviral processes by stimulating B cells or killer T cells. Helper T cells themselves must be activated to gain function. To activate a CD4+ T cell, a naive T cell must encounter an antigen 727

presenting cell (APC) with its corresponding antigen. The antigen is presented by a class II major histocompatibility complex (MHC-II) molecule. The T cell receptor (TCR) on the naive T cell binds to the antigenic peptide and causes differentiation. The binding process is illustrated in Figure 1.

Figure 1: The T cell differentiation process. An antigen presenting cell (APC) breaks down foreign proteins into smaller fragments within the cell, depicted as yellow rectangular antigens. The smaller peptide, shown as a small yellow circle, is presented by a class II major histocompatibility complex (MHC-II), highlighted in red. The T cell receptor (TCR), illustrated in purple, on the naive T helper cell can then bind to the antigen and stimulate differentiation.

WIKIPEDIA

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The differentiation of CD4+ T naive cells into several different kinds of T cells, including effector T cells, effector memory cells and central memory T cells. GOLUBOVSKAYA & WU It is believed that differentiation converts the naive T cell into either an effector T cell or a memory T cell, and thus gains a corresponding function. CD4+ effector T cells accomplish the supportive work done by T cells. Examples of an effector T cell include follicular helper T cells (TFH) which help B cells produce antibodies. CD4+ Memory T cells are more long-lasting and can trigger a recall response that accelerates B cell defense mechanisms to a familiar antigen. About the study In this study, authors Cords et al. analyzed the frequency and phenotype of CD4+ T cell responses specific to SARS-CoV-2. For this, the team collected and tested peripheral blood mononuclear cells from two Covid-19 patients. First of the two patients was a 53 year old female with follicular lymphoma, a type of slow growing B cell cancer. This immunocompromised woman received a chemotherapy regimen and an anti CD20 (B-cell depleting) monoclonal antibody treatment (obinutuzumab) every two months for her cancer. In March 2020, 729

she suffered from severe Covid-19 and was treated with antiviral drug remdesivr and a transfusion of convalescent plasma. SARSCoV-2 virus was detected in her sputum samples for almost three months. Notably, throughout the entire study this patient had undetectable B cell levels and a generally reduced level of T cells. The second Covid-19 patient was a 50 year old immunocompetent female. She caught Covid in October 2020, but only had mild symptoms which did not require hospitalization. A polymerase chain reaction (PCR) test confirmed positive Covid-19 status for both women. The researchers monitored CD4+ T cell responses in the first patient over the course of 391 days; in the second, for 159 days. Study Results Through major histocompatibility complex (MHC) staining, the investigators analyzed the frequency of CD4+ T cells which target a specific binding site on the SARS-CoV-2 membrane protein. The team found that the immunocompromised participant had a CD4+ T cell frequency almost 7.5 times greater than that of the immunocompetent patient, as visualized on the right in Figure 3. These elevated levels generally remained stable throughout the entire study. This outcome suggests that T cell immune responses can successfully respond to SARS-CoV-2 specific antigens in the absence of B cells and B cell—T cell interaction.

Figure 3: The frequencies of Tetramer+ CD4+ T cells in the peripheral blood of the index patient (red) and the reference patient (blue), depicted longitudinally (left) and pooled for each patient (right). The graphic clearly illustrates the higher and more sustained CD4+ T levels observed in the immunocompromised patient. This patient had a mean of 0.163% and a range between 00.447-0.453%.

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CORDS ET AL. The frequency of specific T cells differed between the two patients. For example, the investigators observed a reduced frequency of circulating T follicular helper cells (cTFH) in the immunocompromised patient (4.72% versus 21.5%). Follicular T helper cells typically collaborate with B cells to produce antibodies. Given this, the anti CD20 immunotherapy may have impaired the differentiation pathway of circulating T follicular helper cells as a result of the lack of B cells to interact with. So, although T cell responses were plentiful in the B cell depleted participant, the absence of B cells seems to have impacted what kind of T cells circulate the peripheral blood.

Figure 4: Development of IL7R↵ (CD127) negative SARS-CoV-2-specific effector CD4+ T-cells of the index (red) and the reference patient (blue). Dotted lines indicate the last positive PCR result.

CORDS ET AL. The team noticed a difference in the expression of IL-7 receptors (CD127+), which are often used as markers for long-lived memory T cells. Both patients had reduced proportions of memory T cells while they displayed symptoms. This frequency increased to bulk CD4+ T cell levels when the immunocompetent patient spontaneously cleared the infection; in comparison, the immunocompromised patient saw a similar increase only after receiving remdesivir and a plasma transfusion. The inverse of this relationship is illustrated in Figure 4, with the percentage of CD127731

T cells—instead of CD127+ T cells—displayed for the reference patient (in blue) and for the index patient (in red). Figure 4 demonstrates how the increase of CD127+, thus the decrease in CD127- T cells, occurs faster in the immunocompetent participant. This delayed contraction of effector cells seemed largely influenced by therapeutics. As the authors posit, there could be a specific T cell memory at play which can only maintain homeostasis in immunocompromised patients; here, T cell constructed homeostasis appears to keep SARS-CoV-2 replication at bay, but cannot do more without additional help. This could explain why the SARS-CoV-2 infection was so prolonged in the immunocompromised patient until the implementation of antivirals. Take-aways Cords et al. provide a valuable addition to current understandings of cell-mediated immune responses to Covid-19 in the absence of B cells, which helper T cells usually support. The study has clear implications for people with cancer, chronic inflammatory diseases or an organ transplant—people who may have a compromised antibody system. Beyond this scope, the results remain relevant due to decreasing humoral response seen in typical Covid-19 protections: natural infection and vaccines. Previous studies have shown that antibody protection attained through natural infection and vaccines wanes within the span of a few months. Additionally, the SARS-CoV-2 virus appears adept at changing the conformation of its spike protein—the binding site which current vaccines target—and thereby dodging antibody recognition. Despite antibody responses dwindling rapidly, vaccines and natural infection remain highly efficacious in preventing severe disease and death. This remarkable ability to protect against hospitalization appears to rely upon cell-mediated immune responses rather than humoral responses. In this study, the authors found that B cell depletion caused by anti CD20 immunotherapy did not encourage viral replication, and that cell-mediated responses can be activated without B cells present. The lack of a humoral response, however, may have altered the kind of T cell response seen. Most importantly, the delayed contraction of effector T cells suggest that helper T cells must be integral to suppressing SARS-CoV-2, but alone cannot overcome the illness without therapeutic intervention. Although more investigation with 732

a wider cohort is needed, it would appear that cell-mediated immunity can indeed provide protection against viruses without B cells—just not enough to surpass the illness. This article is featured on Forbes.org, and can be read online here: T Cells Play An Important Role In Immunity, But Without B Cells Cannot Provide Protection From Covid-19

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Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 3) Forbes | August 29, 2022 | Article

This is a short series about a recent breakthrough on the road to developing a much sought-after broadly neutralizing vaccine against all influenza A viruses. If successful, it may act as a precursor to a truly universal flu vaccine, one that protects against all types, subtypes, and lineages of the virus. The breakthrough may also provide a blueprint for developing a Covid-19 vaccine that retains its efficacy in the face of new variants. In the first part of this series, I gave a brief overview of the history and nature of influenza viruses, including why it has been so difficult to develop successful vaccines. Here, and in the previous article, I discuss some of the attempts that have been made to overcome these challenges, including their shortcomings. And in the last few installments I will offer a detailed analysis of the latest —and most promising— advances in the field. Understanding mRNA Technology Traditional influenza vaccines are based on “killed” influenza viruses. Inactivated viruses cannot cause infection or disease, but when injected they rally our immune system into action all the same. While our immune system clears the viruses, it learns about them. Next time we’re exposed, it is already primed to defend against infection. But there are a couple issues with influenza vaccines that rely on inactivated viruses. For one, they take a long time to produce. Candidate vaccine viruses need to be grown, either in fertilized hen’s eggs or in mammalian cells, before being inactivated and purified for use in vaccines — this is a biological production process, and can take up to six months to complete. This means the wild type viruses circulating across the globe can continue to mutate while the vaccines are being made. If the viruses change enough, they may be able to evade the immunological “memory” the vaccines would

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provide us. Slow production times also render inactivated vaccines poorly suited to pandemic response, where speed is of the essence. Enter messenger RNA (mRNA) technology. Unlike traditional flu vaccines, mRNA vaccines do not depend on the cultivation of viruses. Instead, they are based on the synthesis of carefully designed mRNA. All of the cells in the human body contain proteins. These are the building blocks of life, involved in almost all of our body’s vital processes. Messenger RNA, in turn, is crucial to the synthesis of proteins. Our cells need instructions to build proteins, and mRNA provides these instructions in the form of genetic information. In a sense, mRNA acts as a kind of blueprint. Vaccines can take advantage of this, using mRNA to instruct our cells how to produce a protein —or even just a small section of a protein— that is unique to whatever virus the vaccines are meant to protect us from. In the case of Covid-19, for example, vaccines include mRNA that teaches our cells to make the SARS-CoV-2 spike protein, which the virus uses to bind to our cells. Our immune system then recognizes these proteins as foreign entities and quickly gets to work, as it would against inactivated viruses or the wild-type virus itself. Lightning Fast Manufacturing Messenger RNA vaccines can be manufactured extremely quickly because they bypass the need for inactivated viruses or virus antigens. Unlike traditional vaccines, mRNA vaccines are synthesized chemically. The process is more akin to how a pharmaceutical drug like aspirin is produced than it is to how current flu shots are produced. As soon as researchers have a complete sequence of a virus’ genome, they can isolate the section they want to use for vaccination —usually, the gene corresponding to the surface protein the virus uses to bind and enter our cells— and artificially synthesize it in the lab. The gene can then be inserted into a small, circular piece of DNA known as a plasmid. Plasmids are easy to replicate, allowing researchers to produce large amounts of the target viral gene at once. Finally, the plasmids are put into a reactor with a molecule that transcribes the viral RNA into mRNA. Any remaining plasmid material is broken down and the mRNA put through a series of purifiers before being packaged into the protective lipid shell. The 735

whole process takes a matter of weeks rather than months. Or maybe even less: in 2013, a team of scientists at Novartis generated an mRNA vaccine candidate in the span of eight days. Messenger RNA technology skips a large chunk of the usual vaccine manufacturing process by recruiting our very own cells as factories to make the antigen “onsite”, instead of having to produce it externally and administer it after the fact. All that’s needed for the mRNA vaccines is the relevant sliver of genetic material and a protective lipid shell. The rest of the work is done by us, inside our cells. The protective lipid shell is crucial. Messenger RNA on its own is very fragile and any free floating or “misplaced” mRNA molecules in our body are quickly chopped up and broken down by enzymes. Added to this is the fact that mRNA cannot enter cells on its own, meaning it would never even be able to deliver the necessary genetic instructions. Lipid nanoparticles (LNPs) form a bubble around the delicate mRNA, helping to shield it and shuttle it into our cells (Figure 1). A big part of the reason mRNA technology wasn’t introduced into the mainstream until recently is because finding the right combination of lipid nanoparticles had given researchers difficulties. In 2004, while working on small-interfering RNA (siRNA), which can selectively silence genes in mammalian cells, researchers at Alnylam Pharmaceuticals made a breakthrough: attaching a cholesterol molecule to the siRNA allowed it to pass through cell membranes. A later breakthrough improved on this discovery by adding a transient positive charge to the lipid nanoparticle, creating a bubble around the siRNA that protected it and helped ferry it into cells more effectively. Although designed for siRNA, the same delivery technology opened the door for improved mRNA therapeutics. In fact, Pfizer and Moderna used very similar lipid nanoparticles to ferry the mRNA in their respective Covid-19 vaccines. And according to Alnylam, the lipid nanoparticle formulation is too similar to its own, leading them to file a lawsuit against the two pharmaceutical companies.

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FIGURE 1. A schematic diagram of lipid nanoparticles used to protect and shuttle mRNA (red) during vaccination. FROM: “NANOMATERIAL DELIVERY SYSTEMS FOR MRNA VACCINES” BUSCHMANN ET AL. 2021

Messenger RNA vaccines may soon become even quicker and cheaper to produce by implementing self-amplifying mRNA (samRNA) technology. With conventional mRNA vaccines, the amount of mRNA in the vaccine is the amount of mRNA that will end up in the cell, and by extension, the amount of antigen produced. Self-amplifying technology combines the mRNA with enzymes from alphaviruses, which help the mRNA make copies of itself once inside the cell (Figure 2). This means fewer mRNA molecules need to be used per vaccine. The samRNA vaccines may also induce a more robust immune response, as the mRNA stays in the body for a longer period of time.

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FIGURE 2. samRNA delivery system (left) compared to conventional mRNA delivery system (right). FROM: “NANOPARTICLES AS ADJUVANTS AND NANODELIVERY SYSTEMS FOR MRNA-BASED VACCINES” ALFAGIH ET AL. 2020

Faster production should allow manufacturers to keep up with new viral mutations; as soon as a variant of concern pops up, researchers could begin production of a bespoke vaccine to neutralize it. In the case of influenza, mRNA technology would improve the odds of vaccines being well matched to the circulating wild type viruses — something we continue to have trouble with, even as recently as this past flu season. Drawbacks? Unlike Covid-19 outbreaks, which are usually driven by one dominant circulating variant, seasonal flu outbreaks are characterized by multiple cocirculating strains. These strains are derived from the four main influenza viruses: influenza A(H3N2), influenza A(H1N1), and two influenza B viruses. To account for differences in the hemagglutinin surface protein between strains, traditional flu shots are “quadrivalent”, meaning they contain an inactivated version of each of the four viruses. But a synthesized mRNA sequence can only make one particular protein; an effective mRNA influenza vaccine would need four different sequences to make four different HA proteins. A 2020 study led by researchers at the Icahn School of Medicine at Mount Sinai suggests this shouldn’t be an issue. The team of scientists manufactured an mRNA vaccine against 738

an influenza subtype that encoded four different proteins at once. They have since repeated the experiment for two additional influenza subtypes, and they plan to test a combined vaccine that encodes for a total of 10 to 12 different antigens across subtypes. This is part of a larger movement towards mRNA flu shots. A more serious concern is that mRNA technology comes at the cost of thermostability. The delicate nature of mRNA renders it thoroughly unstable at room temperature, leading to degradation and loss of quality. In general, mRNA vaccines need to be stored and transported well below freezing — Pfizer’s Covid-19 vaccine needs to be stored at -94 Fahrenheit, colder than Antarctic winter. This makes global distribution and storage a serious challenge, especially in areas without ready access to electricity. But, this too, is already becoming a problem of the past. A process known as lyophilization, or “freeze-drying”, allows researchers to dehydrate the mRNA solution and turn it into a powder. This powder is stable at room temperature and can be mixed with diluents just before administration. In 2017, researchers used this method to produce a rabies mRNA vaccine that retained its efficacy after being stored at 113 degrees Fahrenheit for 12 months. Even when stored at 158 degrees Fahrenheit for three months, the vaccine managed to protect mice from disease. In India, Gennova Biopharmaceuticals has just received emergency use authorization for their lyophilized Covid-19 mRNA vaccine, which can be stored between 35 to 45 degrees Fahrenheit without degradation. Instead of sub zero temperatures, standard refrigeration will do just fine. What remains unclear is whether mRNA technology by itself could address a major issue plaguing current influenza vaccines: waning immunity. A 2018 meta-analysis of influenza vaccine durability found that protection can vanish as early as 90 days post vaccination. Recombinant and inactivated vaccines both suffer from this issue, even though they trigger immune responses in different ways. If the Covid-19 mRNA vaccines are anything to go by, waning immunity will likely continue to be a factor even if influenza vaccines made the switch to mRNA technology. The next article in this series will look at the nasal spray flu vaccine —live attenuated influenza vaccine [LAIV]— which has

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faced a bumpy road ever since it was first introduced. What did it promise, and why has it fallen short? This article is featured on Forbes.org, and can be read online here: Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 3)

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Increased Disease Potential Of Covid Variant BA.5 Currently Circulating In The United States Forbes | August 29, 2022 | Article

The following is a continuation of a discussion on variant pathogenicity begun in part one. Throughout the Covid-19 pandemic, many iterations of the SARS-CoV-2 virus have dominated over others. The B.1 variant of 2020 was overtaken by the Alpha variant in early 2021. Alpha was overcome by Delta later that Summer. Next, Delta was pushed aside for Omicron BA.1 in late 2021, followed by BA.2 shortly thereafter. Now, BA.5 dominates the global virus landscape. There are two driving forces behind the success of a SARSCoV-2 variant over another. The first is a variant’s ability to infect those previously infected or vaccinated and the second are the virus’s intrinsic pathogenic properties. Here we focus on the latter, specifically in reference to the latest Omicron variants. A recent study by Tamura et al. focuses on the latter, specifically on how Omicron subvariants, including BA.5, compare pathogenetically to earlier virus variants. Understanding the pathogenic dynamics of emerging variants may inform drug and vaccine development, treatment regimens for those with moderate to severe disease, and surveillance for variants yet to come. As there have been nearly 600 million confirmed cases of Covid19 (meaning the accurate count is almost certainly in the billions), hundreds, if not thousands, of SARS-CoV-2 variants are circulating at any given time. However, the pandemic's number of variants of concern or interest lies under 20. These viruses have some discernable features that make them more prevalent than others. Tamura et al. focus this line of reasoning on BA.5: the most widespread SARS-CoV-2 variant at the time of writing. They analyzed BA.5 alongside BA.2, BA.1, and B.1.1 as a control. Lung Cell Disruption Pathogenicity is a combination of traits that result in a wide variety of virological outcomes. One of the most critical outcomes 741

is infection impact on the lung. Tamura and colleagues evaluated the effect of different variants on respiratory epithelial and endothelial barriers. Using in vitro airway simulations, they found that B.1.1 and BA.5 exhibited significantly higher disruption than BA.1 or BA.2. They note that a significant amount of virus gathers in the blood vessel channels of respiratory cells in BA.5 and B.1.1, more so than BA.1 and BA.2. This indicates that the later Omicron variant, BA.5, has a more significant respiratory impact than BA.1 or BA.2.

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FIGURE 1: Airway-on-a-chip analysis. A medium containing SARS-CoV-2 was injected into the airway ... [+] TAMURA ET AL.

Animal Model Observations Tamura and colleagues then monitored each virus's pulmonary in vivo dynamics in infected hamster models with the same virus variants. Notably, they found that subcutaneous oxygen saturation 743

in BA.5 was lower than BA.1 and BA.2, but not quite to the level of B.1.1. Again, this indicates the more severe pathogenicity of BA.5 as compared to earlier Omicron variants.

FIGURE 2: Syrian hamsters were intranasally inoculated with saline (n = 6, uninfected control), ... [+] TAMURA ET AL.

The researchers also noted a higher concentration of N protein in some variants over others. The N protein oversees a significant portion of virus pathogenicity, meaning a higher concentration would imply more severe symptoms. BA.2 and BA.5-infected subjects had a higher concentration of N protein in bronchial cells as compared to BA.1, but still lower concentrations than B.1.1. After five days, BA.1 and BA.2 N protein was hardly detectable in the lungs as compared to BA.5 and B.1.1. Echoing previous observations, BA.5 appears more pathogenically adept than BA.1 or BA.2, particularly in the lungs.

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FIGURE 3: Syrian hamsters were intranasally inoculated with B.1.1 (n = 4), BA.1 (n = 4), BA.2 (n = ... [+] TAMURA ET AL.

The researchers examined lung tissue inflammation over time based on their previous observations. As expected, B.1.1 displayed the most significant lung inflammation, followed by BA.5, BA.2, and BA.1. Lung inflammation leads to many of Covid’s most notable symptoms, including fatigue, difficulty breathing, dry cough, chest tightness, and chest pain. Syncytia Formation and Cleavage Efficiency Two indicators for the severity of disease outcomes are syncytia formation and cleavage efficiency. They observed the reduced size of syncytia formation in the Omicron sublineages compared to the B.1.1 control. Syncytia are cellular structures formed by the fusion of uninuclear cells. Upon further examination, BA.2 and BA.5 were far more syncytia efficient than BA.1. FIGURE 4: Syncytia formation in vitro for infected VeroE6/TMPRSS2 cells.

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TAMURA ET AL. Cleavage efficiency in B.1.1 was highest among the four subjects, but BA.2 and BA.5 outranked BA.1. What does all this mean? A unique trait of the Omicron family is the lack of efficient cleavage present in earlier variants. Cleavage allows for more efficient fusogenicity in host cells, leading to more severe disease. This is why Omicron was considered slightly less intense in terms of illness than Delta and other variants. However, the increased syncytia efficiency of BA.2 and BA.5 over BA.1 indicates that fusogenicity is increasing in later Omicron strains as opposed to BA.1. This means that despite the lacking cleavage in the variants, they are evolving to be more severe in terms of disease, meaning more intense complications for patients. Using the data from this study, we can infer that despite its parental lineage’s reduced pathogenicity, BA.5 evolved to become more pathogenic. That suggests that the cases of BA.5 circulating today exhibit more severe symptoms on average than cases from the BA.1/BA.2 wave in early 2022. While further study would be required, we hypothesize that emerging strains of SARS-CoV-2 such as BA.2.75 and BA.4.6 would follow a similar trendline of increased pathogenicity as the virus continues to mutate and evolve. While earlier Omicron-induced Covid-19 symptoms may have been quelled by the standard remedies of rest, Tylenol, Advil, and so on, BA.5 cases are much more likely to require more advanced interventions like monoclonal antibody treatments and antiviral drugs. As such, the production and price of such products must be improved to satisfy the increased need in the coming weeks and months. This article is featured on Forbes.org, and can be read online here: Increased Disease Potential Of Covid Variant BA.5 Currently Circulating In The United States

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September 2022

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Five Days Is Too Short! Study Says To End Covid-19 Isolation Based On Rapid Tests Results Instead Forbes | September 01, 2022 | Article

The party on Friday was a blast except for one thing: you think you caught Covid. Saturday you felt fine, but you realize that the runny nose and cough you developed Sunday afternoon may not be so innocent. One of your roommates returns from work Monday morning with a rapid antigen test for you to use, and turns out the suspicion is true. You suppose you should isolate, but for how long? Until you feel better? Should you just wait for five days and assume all is well? And what day would you even count from? From Friday, the start of the party; Sunday, the day the coughing began; or maybe Monday, when the antigen test came back positive? Most of us are concerned about how long we should isolate for when Covid positive. Current guidelines set by the UK’s National Health Service (NHS) and the US’s Centers for Disease Control and Prevention (CDC) suggest a baseline of five days. These policies developed in response to the urgent need for healthcare and essential workers to return to their positions as soon as possible, regardless if some fraction would remain contagious. A new study published in The Lancet Respiratory Medicine applies real-world data to answer the questions of how and when to safely leave Covid-19 isolation. The team discovers, in particular, that the fixed-date recommendation releases a significant number of people from selfisolation who are likely still infectious, and that the best practice is to test out of isolation with rapid antigen tests. Natural Covid-19 infection study In this study, Hakki et al. analyzed the infectious period of SARS-CoV-2 in a real-world community setting over several months. Enrollment occurred twice, once between September 2020 and March 2021, and once between May 2021 and October 2021. The involved household and non-household contacts had mild cases 748

of Covid-19, all confirmed through a polymerase chain reaction (PCR) test. The participants filled out a daily diary of symptoms and were considered symptomatic if they experienced one of three hallmark Covid-19 symptoms prior to Omicron—fever, cough or loss/change in smell or taste—or at least two of the following symptoms: muscle aches, headache, appetite loss, sore throat. The team tracked the viral RNA load, or the amount of virus detectable in an infected individual, for the duration of the illness through daily reverse transcription PCR tests (RT-PCR). Plaque assays measured the level of infectious SARS-CoV-2 in samples. The participants also performed self-administered Covid-19 lateral flow tests—otherwise known as rapid antigen tests—throughout. This information, when paired with the RT-PCR results, helped determine how effective Covid-19 antigen tests are at detecting SARS-CoV-2 viral proteins.

A photo of a lateral flow test, otherwise known as a rapid antigen test. Using a nasal swab sample, the test detects whether or not antiSARS-CoV-2 viral proteins are present in the body. Unlike with polymerase chain reaction (PCR) tests, the results appear quickly and directly on the testing apparatus. (Photo by JENS SCHLUETER / AFP) (Photo by JENS SCHLUETER/AFP via Getty Images) AFP VIA GETTY IMAGES Study Results 749

The investigators narrowed the cohort size to those with positive PCR results and a detectable viral growth phase (an observed low initial viral load followed by an increase). A total of 57 cases were analyzed. This number was further reduced to a cohort of 38 who had a definitive symptom-onset date and shed culturable virus within the sampling period. The group had mixed vaccination status, but there were no significant differences in demographics between the vaccinated and the unvaccinated. Most participants were white, middle aged (between 29-49 years old), and of a healthy BMI.

Figure 1: The survival probability of infectious virus presence, as determined by plaque assays. When analyzed from first positive PCR result and from symptom onset, it is clear that infectious virus survives well within the first five days (in blue); after five days, the

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probability of survival drops off, but is still significant, especially six and seven days (in red) after positive PCR result/symptom onset.

HAKKI ET AL. Very few people—only 20% of cases (7 out of 35 participants)— shed infectious virus before developing symptoms. On average, infectiousness peaked three days after symptom onset. These levels decreased over the course of the illness; figure 1 illustrates this decline well by graphing the chances for infectious SARS-CoV-2 virus to survive in the body based on symptom onset and first positive PCR result. By Day 5, however, about 62% of the cohort (22 of 34 cases) still continued to shed infectious virus. By Day 7, 23% of the cohort (8 of 34 cases) still shed infectious virus. If someone with Covid-19 leaves isolation after five days, as mentioned in NHS guidelines, likely this person would continue to be infectious. This could be especially ill advised if there are people at home who are highly at risk for Covid-19. Hakki et al. also investigated the efficacy of lateral flow tests. The sensitivity of rapid antigen tests was found to be poor at the start of illness but high after peak infectious levels (67% versus 92%). Given this finding, the researchers suggest that rapid antigen tests are unreliable for early diagnosis of Covid-19 but useful for testing out of isolation.

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Figure 2: A graphical summary of the study’s results by the Imperial College London. Based on study results, it is ill advised to assume that infectiousness ends after five days of Covid-19 sickness. To best decide when to leave isolation, a daily rapid test should be taken after Day 5. If the results come back negative two days in a row, it is safe to leave isolation.

IMPERIAL COLLEGE LONDON Link Added So, how long should you isolate for?

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Although the CDC recommends that isolation end for people with symptomatic Covid-19 at Day 5 if fever free for 24 hours or if symptoms improve, recent data suggests that this may be unwise. According to this study by the Imperial College London, selfisolation should begin once symptoms begin. Day 0 represents the first day symptoms appeared—rather than when the positive test came back—and Day 1 would be a full day afterwards. In the best case scenario, suspicions of Covid-19 would be confirmed with a PCR test rather than a rapid antigen test, which appears to be less effective around symptom onset. Most people will still be infectious at Day 5, albeit much less so than originally. To leave isolation after the fifth day, the researchers suggest using a rapid antigen test once a day; if the test returns negative two days in a row, it should be fine to resume social activities (see summary in Figure 2). This contrasts CDC recommendations which use testing to determine when to stop wearing masks after isolation ends on Day 5. This guidance is based on cohorts sick with Pre-alpha, Alpha and Delta variants of SARS-CoV-2. The omicron variants now in circulation—predominantly variants BA.5, followed by BA.4.5 and BA.4—seem to have lower viral loads and shed for less time than the variants in the study. Although this may suggest that Omicron variants are less infectious, these protocols still apply to those wishing to safely exit isolation. Testing out of isolation diminishes the chances of spreading Covid-19 to loved ones. Using a fixed date, in contrast, can be particularly disadvantageous for those in households with people at risk of Covid-19 and for people taking Paxlovid, a Covid-19 antiviral. The most recent example of this risk is President Biden and First Lady Jill Biden, who experienced a relapse in symptoms due to Paxlovid rebound and had to extend their isolation. At the end of the day, the best course of action would be to prepare for the worse. Prevent infection to begin with by wearing a mask in crowded locations. If possible, get vaccinated and boosted to reduce the odds of developing severe Covid-19. And lastly, store a couple of unexpired rapid antigen tests at home so that, should symptoms arise, it can be easy to know when to safely leave isolation. This article is featured on Forbes.org, and can be read online here: Five Days Is Too Short! Study Says To End Covid-19 Isolation Based On Rapid Tests Results Instead 753

Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VI Forbes | September 01, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV-2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants. In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we analyze two new antibodies described in a study by Low et al. that target a conserved site not only in SARS-CoV-2 variants, but in many similar coronaviruses as well. Antibody Discovery Low et al. began their search in a familiar fashion: isolating memory B cells from convalescent and vaccinated SARS-CoV-2 patients. This yielded many thousand samples from 43 individuals. Next, the researchers introduced the samples to an ELISA panel of many viruses, testing for cross-reactivity to a broad range of coronaviruses. The tested viruses included SARS-CoV, MERSCoV, human coronaviruses OC43, HKU1, NL63, and 229E. A group of seven antibodies emerged cross-reacted against all Spike proteins tested, including alphacoronaviruses NL63 and 229E. The 754

antibodies were sorted into three groups depending on their broad binding capacity, with Group 3 designated for those that bound all tested coronaviruses.

FIGURE 1: Monoclonal antibody cross-reactivity of a panel of coronaviruses. The colored dots ... [+] LOW ET AL.

Antibody Neutralization The next step was to examine those that broadly bound for neutralizing capability. Testing all seven Group 3 antibodies for neutralization capabilities, Low and colleagues narrowed their search to two: C77G12 and VN01H1. The VN01H1 antibody neutralized all viruses examined, specifically SARS-CoV-2, SARS-CoV, MERS-CoV, NL63, and 229E from the binding assay and bat virus WIV-1 and merbecovirus PDF-2180. C77G12 neutralized the binding assay betacoronaviruses but to the highest degree of all Group 3 antibodies. Hamster Study Extending their studies to in vivo examination, they found that the Gamma variant SARS-CoV-2 virus in infected hamsters was potently neutralized by both antibodies, as were authentic Omicron BA.1 and BA.2 viruses. The researchers found that prophylactic administration of either C77G12 or VN01H1 reduced viral RNA copies and lung titers and ameliorated lung pathology at statistically significant levels. Notably, earlier SARS-CoV-2 variants such as Alpha, Beta, and Delta were not examined in the study, nor were later Omicron variants, including the currently dominant BA.5 or BA.2.75 lineages. Antibody Binding Site Low et al. examined the exact binding of the antibodies by cryoelectron microscopy, a method of freezing small proteins and enzymes to enable a high-definition image of very small biological 755

masses. Such broad cross-reactivity with a wide variety of SARSCoV-2 relative viruses would require a highly conserved region of the Spike to bind. C77G12 and VN01H1 bind amino acids 811 through 825, which are highly conserved among all genera of the Orthocoronavirinae subfamily.

FIGURE 2: C77G12 and VN01H1 fusion peptide binding map. LOW ET AL.

This conservation holds with all circulating SARS-CoV-2 variants to date. In the GISAID SARS-CoV-2 database containing 12.8 million sequences, the antibody binding epitope amino acids are only mutated a few thousand times.

TABLE 1: Antibody epitope residue mutations in the GISAID database as of 8/31/2022.ACCESS HEALTH INTERNATIONAL

The Low antibodies share a very similar epitope to the Dacon antibodies, described in a previous entry in this series. Both target 756

the highly conserved fusion peptide and many of the amino acid contact points overlap between the two, and as such, both are viable candidates for monoclonal treatment in the months to come.

FIGURE 3: Venn diagram comparing the amino acid footprint of the Low antibodies and the Dacon ... [+] ACCESS HEALTH INTERNATIONAL

Notably, the epitope target is buried toward the core of the Spike trimer and is highly inaccessible. How, then, do these antibodies bind a hidden target? Low and colleagues identified a crack in the fusion peptide’s defenses using transfected human embryonic kidney cells. During ACE2 binding, conformational shifting of the Spike trimer exposes the cryptic epitope. The coronavirus Spike can be in one of two conformations based on its infecting host stage. The “down” conformation is before ACE2 binding occurs, and the “up” conformation is as ACE2 binding occurs. The receptor-binding domain attaches to the ACE2 receptor, revealing the fusion peptide, at which point C77G12 and VN01H1 can bind. Antibody-Mediated Phagocytosis

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Based on personal communications with the authors, we also note preliminary evidence that the antibodies may induce antibodydependent cellular phagocytosis. ADCP is a mechanism of elimination by which the monoclonal antibodies target cells to promote clearance from the body by phagocytic immune cells. The phagocyte engages with the antibody and engulfs the infected cell, leading to quicker, more favorable outcomes for the patient. Summary The Low study adds another useful pair of broadly neutralizing monoclonal antibodies to our growing armamentarium of SARSCoV-2 antibody treatments and prophylactics. In tandem with the Low antibody, additional antibodies could be used in combination to target other binding epitopes or trigger conformational changes to maximize antibody potency. Combination antibody therapies present the best weapon to overcome the virus and clear infected cells in Covid-19 patients. We recommend immediate high scale production of such combinations as the pandemic continues to rage in its third year. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VI

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Hopeful New Entry In The Race For A Universal Covid Vaccine Forbes | September 02, 2022 | Article

A recent paper published by a team of scientists in Sweden boasts the ambitious title: “A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells”. This claim of a universal SARS-CoV vaccine would be good news if the paper measured up to its title. The study offers some interesting insights and may be of use for the creation of more broadly active SARSCoV vaccines. However, the approach of this group of scientists seems hopeful if nothing else. To understand the issue with their approach, let’s take a step back. Current variations of the SARS-CoV vaccine rely on a singular feature of the virus: the spike protein. There are two widely accepted forms of these SARS-CoV vaccines. These are the adenovirus vector vaccine and the mRNA vaccine. Both the adenovirus vector vaccine and the mRNA vaccine rely on introducing a slightly modified version of the spike protein to the body, causing the body to respond by creating antibodies against the spike protein. These vaccines have proven to be highly effective at reducing serious illness and death across a wide variety of SARS-CoV variants. Unfortunately, the immunity they provide against infection and transmission is short-lived and seemingly depends on very high levels of antibodies. When the level of antibodies begins to fall or when a new spike protein variant appears and is unrecognizable to our antibodies, protection against infection quickly fades. This means that there exists a need to create a vaccine that can protect the body from all SARS-CoV variants and that maintains high levels of antibodies. The research group from Sweden proposes to address this question in two different ways. First, they include DNA from the most reactive portion of the spike protein in their new vaccine. This section of the spike protein is called the receptor binding domain (RBD). The receptor binding 759

domain directly binds to the ACE2 protein on the cell surface and allows for viral entry and infection. By including three variations of these spike protein receptor binding domains, the researchers hoped to account for three common variants of the virus. These variants were the original Wuhan strain along with the Alpha and Beta variants. Second, researchers also included DNA from two additional proteins that are highly conserved across the SARS-CoV variants. These are the membrane protein (M) and the nucleocapsid protein (N) from the original Wuhan strain of the virus. They coupled the M and N DNA with the spike protein receptor binding domain DNA into a single antigen. Both the M and N proteins have some broadly cross-reactive epitopes across strains of betacoronaviruses.

Figure 1: The “universal” vaccine contains the DNA of three spike protein receptor binding domains (RBD) to account for three variants of SARS-CoV. The vaccine also contains additional membrane and nucleocapsid viral protein DNA.APPELBERG ET AL., EMBO MOLECULAR MEDICINE (2022), DOI: 10.15252/EMMM.202215821

Researchers believed this strategy would work because the receptor binding domain has previously been shown to naturally fold into its proper, functional configuration. Presumably, the M and N proteins would do the same. To administer the vaccines, the researchers constructed the genetic material into a DNA plasmid with a promoter signal that would allow the DNA to be transcribed and translated into protein. They also optimized the codon usage of the receptor binding domain and the M and N proteins so that the proteins could be more easily expressed in human cells. In their experiments, immunization took place via electroporation. Electroporation is a DNA vaccine delivery system that uses an electrical pulse to create temporary pores in cell membranes. This allows the DNA plasmid to pass through the cell 760

membrane and into the cell. The cells could then use the DNA to form antibodies and induce an immune response. After testing their vaccine on mice and rabbits, the researchers found that they were able to produce antibodies for the three spike protein variants and the N protein, but not the M protein. In mice, these antibodies were able to effectively neutralize the original Wuhan strain of the virus as well as the Beta variant of SARS-CoV2. In rabbits, the antibodies effectively neutralized the Delta and Omicron variants of SARS-CoV-2. When mice were tested with a lethal dosage of the SARS-CoV2 Beta variant, the DNA vaccine fully protected the mice against lethal infection. After staining the mice’s organs to determine if viral infection spread throughout the body, researchers also found that the vaccine minimized viral replication in the upper airways and spleen. While their results seem to be promising, there are still a number of questions as to whether this vaccine will translate to a universal vaccine against all SARS-CoV-2 variants. First, it is unclear whether or not the spike protein receptor binding domains along with the M and N proteins are sufficient to prevent reinfection. The unmentioned challenge of creating a universal vaccine against SARS-CoV-2 is that reinfection occurs frequently. In other words, prior infection with all viral proteins including M and N does not provide long-lasting protection. Why do the authors assume that the antibodies that they include will perform any better than natural infection? Based on this observation, it is not evident that the addition of M and N antibodies would offer the protection that this team of researchers hopes for. Whether or not a variety of spike protein receptor binding domains will offer broader protection against SARS-CoV-2 variants is also unclear. However, there is some hope of success for this theory, and it is currently being tested by several researchers in the field. Another question is whether DNA-based vaccines are effective at all. Although there are a number of DNA vaccines in clinical trials, none have been approved by the United States FDA. In fact, only one has been given emergency-use authorization by India for Covid-19. It would also be interesting to know how broadly neutralizing this vaccine is against the newest variants that have been sweeping the world: BA.5 and BA.2.75 761

Overall, this study may contribute some insights into how we can create a universal vaccine, however, the title of the paper is highly misleading. Using a variety of spike protein receptor binding domains may offer broader protection against SARS-CoV-2 variants, but additional experiments on primates are necessary to determine whether this theory could hold in humans. While including more viral proteins in our vaccine strategy may seem like a solution, there is still much work to be done to understand how we can prevent reinfection before a universal vaccine is possible. We will wait with interest and hope for the results of this DNA vaccine. This article is featured on Forbes.org, and can be read online here: Hopeful New Entry In The Race For A Universal Covid Vaccine

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Long Covid Is Keeping Millions Of People Out Of Work Forbes | September 02, 2022 | Article

The Covid-19 pandemic has triggered a number of labor shortages over the past few years with reasons ranging from a lack of childcare, safety concerns about Covid-19 exposure, less willingness to accept poor labor conditions and pay, burnout, and increased mental health strain in some industries. A new Brookings report finds that Long Covid is keeping as many as 4 million people out of work. Lost wages are estimated at between $170 billion and $230 million a year, a significant economic burden at a time when the cost of living is rising steeply. The Brookings report uses updated data from Census Bureau’s Household Pulse Survey in June, which included four new questions about Long Covid to better understand the condition’s prevalence and impact. The survey found that 16.3 million people (around 8%) of working-age Americans currently have Long Covid. Brookings corroborated those findings with a recent Federal Reserve Bank of Minneapolis study, which found that 24.1% of people who have contracted Covid-19 experienced symptoms for three months or more. According to the CDC, about 70% of Americans have contracted Covid-19. This percentage translates to 34 million working-age Americans experiencing Long Covid symptoms. The same study found that 50% of respondents had recovered from Long Covid, leaving approximately, around 17 million people who may currently have Long Covid. However, the experience of each person who is afflicted with Long Covid is often unique. Some may have more severe and disabling symptoms that prevent them from working entirely, some may have milder symptoms or employer accommodations that allow them to maintain their regular employment, some may work reduced hours or some may be forced by dire financial circumstances to keep working despite severe illness. A July 2021 study from the Patient-Led Research Collaborative found only about 27% of long 763

Covid patients worked as many hours as they did before falling ill, and approximately 23% weren’t working at all, as a direct result of long Covid. In order to accommodate these diverse experiences in their estimates, Brookings drew data from the Minneapolis Federal Reserve Bank, United Kingdom’s Trades Union Congress, and a Lancet study about the extent of work reductions. This data resulted in estimates of 2 million, 3 million, and 4 million full-time equivalent workers out of the labor force due to Long Covid. The midpoint of this range (3 million full-time equivalent workers) is 1.8% of the entire U.S. civilian labor force. While this number may seem high, it is very similar to an estimate by Harvard University economist David Cutler who used a different methodology. His study cited research that 12% to 17% of Covid-19 patients are still experiencing three or more symptoms 12 weeks after onset and the labor force reduction among those with significant impairment is 70%. He then used Covid-19 case counts and labor force participation rates to estimate that 3.5 million people are out of work due to long Covid. Culter estimated lost wages at approximately $200 billion per year. It is also important to remember that, staggering as they are, these estimates do not represent the full economic burden of Long Covid. They focus only on lost wages and not the reduced capacity of caretakers and the significant healthcare costs for Long Covid patients. In August, the Biden Administration released a report on Services and Supports for Longer-Term Impacts of Covid-19, but up until this point, policymakers and public health have been primarily focused on the loss of life from Covid-19 and not on the longer-term loss of health and quality of life. As more people are infected and reinfected with Covid-19, rates of Long Covid and the accompanying economic burden will continue to rise. If the long Covid population increases by just 10% each year, in 10 years, the annual cost of lost wages will be half a trillion dollars. The Brookings report makes several suggestions on how we can urgently address this crisis. The first is focused on better, long-term measures to prevent people from being infected with Covid-19. Prevention in public health is always better and more cost-effective than a cure. While many are weary of protective public health 764

measures at this stage in the pandemic, there are still many noninvasive tools we can use. These include wearing masks in poorly ventilated public spaces and increasing ventilation and air quality in spaces like schools, daycares, and offices. We need a paradigm shift to prioritize clean, pathogen-free air in the same way we ensure food safety and water quality. The goal should be the explicit inclusion of protection against indoor air hazards (including airborne infection control) in the statements of purpose and definitions of all relevant building design and engineering standards, regulations, and codes. Comprehensive ventilation standards must be developed by professional engineering bodies. New approaches must be developed to encourage the implementation of standards, one option is implementing “ventilation certificates” similar to food hygiene certifications for restaurants. Over time, all new buildings would ideally be designed to ensure good indoor air quality, while existing buildings will be retrofitted. Many cite significant costs as a barrier to accomplishing this. But such a system will not only reduce Covid infections but rates of other airborne diseases. The economic losses of even an average flu season cost the United States $11.2 billion due to reductions in productivity and absenteeism. However, even with the best prevention efforts, some will still continue to be afflicted with Long Covid. We need thousands of well-trained, knowledgeable, affordable physicians to treat and support the growing Long Covid patient community. We also need wider access to disability insurance, expanded sick leave, and employers who are willing to make necessary accommodations. Only with such systematic and institutional changes can we reduce the tremendous financial burden on Long Covid patients and the broader economy. This article is featured on Forbes.org, and can be read online here: Long Covid Is Keeping Millions Of People Out Of Work

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Covid-19 Infection Increases Risk And Excess Burden Of Cardiovascular Disease Forbes | September 02, 2022 | Article

With new research on Long Covid emerging every day, it is becoming increasingly clear that Covid-19 infection impacts our health beyond the acute stage of the illness. A study demonstrates that infection with Covid-19 impacts the risk of cardiovascular events up to 12 months post-infection, regardless of age, race, sex, and other cardiovascular risk factors. This study emphasizes the recognition of and the need for more effective strategies to address the long-term effects of Covid-19. A group of researchers from the Clinical Epidemiology Center in Saint Louis, Missouri investigated the risk and excess burden of cardiovascular disease following the acute phase of Covid-19. In the study, over 150,000 veterans who had recovered from infection were compared with non-infected peers, in addition to a prepandemic control group. Xie and colleagues followed these three groups for twelve months and conducted a thorough analysis to estimate the risk and associated burden of cardiovascular outcomes. Who and What Factors were Considered? Xie and colleagues obtained information from databases managed by the United States Department of Veteran Affairs to construct their three cohorts. Of the 6,241,346 veterans who encountered the department of Veteran Affairs in 2019, 162,690 had a positive Covid-19 test between March 1st 2020 and January 15th 2021. 153,760 of such individuals were alive 30 days after their positive test date and selected into the Covid-19 test group. Researchers based the average date of the positive test for the cohort, T0, based on the distribution of the positive Covid-19 test dates. 5,960,737 veterans who encountered the department of Veteran Affairs in 2019 and were alive by March 1st, 2020; 5,806,977 of such individuals were not part of the Covid-19 group and were selected into the contemporary control group. Xie et al randomly chose the average enrollment date for the cohort, T0, which would allow for 766

the participant distribution to be identical to the distribution of the Covid-19 group. In doing so, the contemporary control group and the Covid-19 cohort had similar follow-up times. Xie et al selected individuals who encountered the department of Veteran Affairs in 2017 to be part of the pre-pandemic cohort. Of the 6,461,205 veterans, 6,150,594 of them were alive by March 1st 2018. 6,008,499 were not included in the Covid-19 cohort and were further chosen to be included in the pre-pandemic group. Researchers randomly selected the average enrollment date for the group, T0, which would allow for the participant distribution to be the same as the distribution of the Covid-19 group. This action similarly ensured that the historical control group and Covid-19 group had identical follow-up times. The cardiovascular outcomes assessed were based on Xie and colleagues previous work regarding Long Covid. These outcomes include cerebrovascular disorders, dysrhythmia, inflammatory heart disease, ischemic heart disease, thrombotic disorders, and other cardiovascular diseases. Researchers conducted a follow-up for each cardiovascular outcome that the participant had no prior history with one year before their enrollment date. The follow up period began 30 days after the average enrollment date, T0, and ended by October 31st 2021. Xie et al considered pre-defined and algorithmically selected variables to account for any baseline differences between the cohorts. Previous studies have shown that race, sex, body mass index, Area Deprivation index, smoking status, frequency of hospitalization, and use of long term care can influence risk and associated burden of cardiovascular outcomes. Xie and colleagues identified cancer, chronic kidney disease, diabetes, and several other comorbidities as variables to also consider. In addition, they adopted an algorithm that determined the top 100 variables with the highest risk relative to the cardiovascular outcome and cohort. The program factored the diagnoses, medications, and lab abnormalities common in at least 100 members of the cohort. Researchers tested each cardiovascular outcome within each cohort independently so the algorithm could be applied. Recovered from Infection Vs. Never Infected Vs. PrePandemic: What Happened?

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In the study, Xie et al estimated the risk, burden, and excess burden up to 12 months post infection of cardiovascular outcomes through inverse probability weighting, a common method used to estimate the probability of exposure observed for a particular person and using the value as a weighting factor in further analyses. In this case, researchers calculated a propensity score, which describes the chance of being selected to the target population, for individuals who had no prior history (up to one year before enrollment) for each specific cardiovascular outcome. These scores estimated the probability of being a veteran who encountered the department of Veteran Affairs in 2019 based on the pre-defined and algorithmically selected variables; they were then used to calculate the inverse probability weight of being part of the sample who interacted with the department in 2019. Additionally, researchers applied the weight scores in hazard ratio models for each cardiovascular outcome. These models estimated the risk of each cardiovascular complication caused by Covid-19, using death as the other, competing risk. Among the pre-specified cardiovascular outcomes, those who recovered from the acute phase of infection had higher risk of all pre-specified cardiovascular outcomes in comparison to those who were not infected. The composite scores for each group of cardiovascular outcome were above one, indicating increased risk: cerebrovascular disorders scored 1.53±0.16 , dysrhythmias scored 1.69± 0.11, inflammatory heart disease scored 2.02 ± 0.53, ischemic heart disease scored 1.66± 0.28, thromboembolic disorder scored 2.39± 0.24, and other cardiovascular disorders scored 1.72± 0.14 . In particular, veterans who survived the first 30 days following a positive test result exhibited higher risk of myocarditis (5.38 ± 3.79), cardiac arrest (2.45 ±0.81), cardiogenic shock (2.43 ± 1.30), and pulmonary embolism (2.93 ± 0.42). Xie et al estimated the associated burden of cardiovascular outcomes caused by Covid-19 per 1,000 people at twelve months based on the differences between the estimated rate of Covid-caused cardiovascular outcomes caused in the Covid-19 cohort and the contemporary control cohort. They found similarly high and excess burdens with all cardiovascular outcomes. The associated excess burden for heart failure (11.61 ± 2.78) and atrial fibrillation (10.74 ± 2.3) were especially high. 768

Taking care setting into consideration, risks and associated burdens persisted among those who were not hospitalized for infection, which gradually increased with severity of infection. Participants who were not hospitalized, representing the majority of the US general population, had higher risk and excess burden for cardiovascular disease than those who were not infected by Covid19. Those who were hospitalized for infection had higher risk and associated burdens than those who did not, and those who were admitted to intensive care had the highest risk of cardiovascular disease and excess burden. Relative to the pre-pandemic control group, participants who recovered from Covid-19 infection had higher risk and excess burden of any pre-specified cardiovascular disease per 1,000 individuals in one year. The results of each assessment were consistent to what was found when comparing the covid-19 cohort with the contemporary control group. A few studies suggested a potential association of some Covid vaccines and a very rare risk of heart or pericardium inflammation; Xie et al conducted two analyses to diminish any potential impact vaccine exposure may have had on cardiovascular outcomes. In their first test, researchers excluded participants who received their first dose of a Covid-19 vaccine. In the second assessment, they considered vaccination status as a time-sensitive covariate. In both analyses, Covid-19 was associated with higher risk of both heart and pericardium inflammation. This set of findings reinforces the importance of getting vaccinated. Xie and colleagues validated their analytical approach by testing variables with expected outcomes. They tested the association between Covid-19 and the signature risk of fatigue; Covid-19 increased the risk of experiencing fatigue, as expected. Researchers tested the association between receiving a flu shot on evennumbered versus odd-numbered calendar days and the pre-specified cardiovascular outcomes. Following the same analytical approach and resources as the study, they found no significant association between the even versus odd-numbered calendar day of the influenza shot and pre-specified cardiovascular outcomes. The cardiovascular disease risk associated with Covid-19 infection further highlights how we need a coordinated global response strategy to urgently address the challenges of dealing with 769

the long-term health effects of Covid-19. Physicians should also be adjusting their screening questions to include past infection with Covid-19 and assess for all Long Covid symptoms including cardiovascular. Early identification, diagnosis, and treatment of heart disease are essential to lessen the risk of adverse health impacts. This article is featured on Forbes.org, and can be read online here: Covid-19 Infection Increases Risk And Excess Burden Of Cardiovascular Disease

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII Forbes | September 6, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV-2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants. In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we analyze two new antibodies described in a study by Fenwick et al. that target a conserved site in SARS-CoV-2 variants. Antibody Discovery Collecting sera samples from over 100 donors of varying vaccine and infection status, Fenwick and colleagues focused their search on the sera of a post-infected donor who had received a full dose of Moderna mRNA vaccine, yielding six monoclonal antibodies for cloning and testing. The six clones were introduced to a binding assay against a panel of SARS-CoV-2 variants to whittle further the field, including the original Wuhan Spike, Alpha, Beta, Gamma, and Delta. Two antibodies, P2G3 and P5C3, outperformed the five others and

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became the focal point of examination for Fenwick and colleagues moving forward. Neutralization Comparison to Existing mAb Therapies Currently available monoclonal antibody treatments, such as the AstraZeneca cocktail (cilgavimab + tixagevimab), the Regeneron cocktail (casirivimab + imdevimab), and Sotrovimab, are not effective against the Omicron family of variants, including BA.1, BA.2, BA.5, and BA.2.75, with the possible exception of bebtelovimab and sotrovimab against BA.2.75. Not only does the P2G3 antibody bind and neutralize the Wuhan, Alpha, Beta, and Delta variants to a greater degree than any of the above-listed monoclonal treatments. It also wholly neutralizes live Omicron BA.1 and BA.2 virus. Both alone and in combination with P5C3, the P2G3 antibody’s IC50 values consistently remain under .025 µg/ml.

FIGURE 1: Heat map showing IC50 and IC80 neutralization potencies for the indicated mAbs in the live virus.

FENWICK ET AL. Notably, Fenwick and colleagues did not include neutralization data for the currently circulating BA.5 or BA.2.75 variants. However, they suggest that the novel mutations in those viruses would likely not impact neutralizing activity. Regardless, a further examination should be taken to confirm that hypothesis. Antibody-Dependent Cellular Cytotoxicity and AntibodyDependent Cellular Phagocytosis The researchers next examined the antibodies for antibodydependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Both function to clear infected cells from the host. ADCC is the 772

enabling of host immune cells to kill virus-infected cells, whereas ADCP is the enabling of host immune macrophages to absorb and kill infected cells. They found that P2G3-mediated ADCC was more robust than any other monoclonal antibody tested, including the other cloned antibodies and the AstraZeneca and Regeneron cocktails. For ADCP, Fenwick and colleagues found greater activity in the P2G3 + P5C3 combination rather than in isolation, adding a significant data point in favor of the tandem. Live Animal Studies To examine the use of P2G3 as both a prophylactic and therapeutic in live hosts, Fenwick and colleagues administered the antibody to both hamster and macaque models. In hamsters, the antibody was administered two days before infection with the Wuhan virus. After four days of infection, only one of six infected hamsters had detectable infectious virus in the lungs, which was a massive four-log reduction from the control group. In the macaques, the P2G3 antibody was administered 72 hours before infection with the BA.1 virus. The researchers found that peak viral load in the treated macaques was roughly half that of the control group, suggesting P2G3 maintains therapeutic efficacy even against later Omicron viruses.

FIGURE 2: (A) Overview of study design for the SARS-CoV-2 NHP challenge model. (B) Tracheal swabs (left), nasopharyngeal swabs (middle), and bronchoalveolar lavages (BAL, right) performed during the course of the study were evaluated for viral copies

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FENWICK ET AL. Binding Epitope P2G3 and P5C3 fall into the antibody group that binds the receptor-binding domain, many of which we have described in this series.

FIGURE 3: Cryo-EM composite density map of the full-length Omicron spike bound to one P5C3 and three P2G3 Fab fragments. Spike protomers are colored in green, orange, and blue, P5C3 Fabs in dark and light orange, and P2G3 in black and grey. FENWICK ET AL.

Using the widely adopted method of cryo-electron microscopy, Fenwick and co. found the exact binding epitope of the antibodies. The two antibodies in conjunction interact with residues 344-347 and 440-451 for a total of 16 receptor-binding domain contact points. According to the GISAID sequence database, these residues are highly conserved, aside from three notable exceptions, all commonly found in the major versions of the Omicron family. However, as the neutralization assays demonstrate, these mutations clearly do not impede Omicron neutralization.

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TABLE 1: Antibody epitope residue mutations in the GISAID database as of 9/2/2022.

ACCESS HEALTH INTERNATIONAL The Fenwick antibodies share many overlapping residues with antibodies we have previously described in this series. Most notably, the Luo and Wang antibodies. All three are viable receptor-binding domain targeting antibodies that would prove vital to combination therapy in tandem with fusion domain and S2 targeting antibodies.

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FIGURE 4: Venn diagram comparing the amino acid footprint of the Fenwick antibodies, the Wang antibody, and the Luo antibody. ACCESS HEALTH INTERNATIONAL

The researchers also make a note of P2G3’s angle of attack. Many monoclonal antibodies are structured such that they can only bind at certain angles to the receptor-binding domain when it is in a specific conformation, whether up or down. This includes the Regeneron cocktail REGN10987, which can only bind to the up-RBD. In contrast, P2G3 can bind in either conformation from a different angle, giving the antibody more flexibility and potentially one catalyst behind its broad neutralization.

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FIGURE 5: Fab binding angle of attack to the RBD is defined as the line connecting the centroid of the Fab to the centroid of the surface area of the RBD that the Fabs bury. The angle of attack of P2G3 is compared to that of other class 3 antibodies FENWICK ET AL.

Conclusion While this antibody lacks data on the latest versions of Omicron sweeping the world, its binding and neutralization speak for themselves. It shares many binding residues with antibodies that we know are effective against the latest variants and outcompetes many commercially available monoclonal therapies. Add this antibody to the growing list of weapons we can use to combat the everpresent SARS-CoV-2 virus as Omicron continues to infect, mutate, and spread. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII

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Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 4) Forbes | September 08, 2022 | Article

This is a short series focusing on the challenges of developing effective influenza vaccines. In the first part of this series, I gave a brief overview of the history and nature of influenza viruses, including why they represent a tricky target for vaccine manufacturers. In the previous two articles —which can be read here and here— I discussed some of the attempts that have been made to overcome these challenges. This article focuses on another such attempt: intranasal vaccination. Finally, the last few installments will offer a detailed analysis of the latest, and most promising, advances in the development of universal vaccines. Nasal Vaccination: Straight to the Source Current influenza vaccines are reasonably effective at reducing the risk of severe illness, hospitalization, and death, but they need to be updated on a yearly basis to remain protective. The influenza surface proteins that vaccines use to teach our immune system how to defend itself can mutate rapidly, leading to a mismatch between the vaccine antigens and those actually in circulation. A good season will see around 60% of people protected by the flu vaccines. A poorly matched season can see this number drop as low as 20%. Even when the vaccines are well matched to the circulating strains, they still need to elicit a high concentration of neutralizing antibodies to be effective. Antibodies bind to the influenza surface protein and prevent viral entry into cells, creating a barrier between virus and host. Unfortunately, these wane very quickly — a recent meta-study, performed by scientists at the University of Michigan, Ann Arbor and Northwestern Memorial Hospital, found that the protection offered by the flu shot can be lost completely within a span of 90 days. Most flu vaccines are administered by intramuscular injection, delivering the antigens deep into the muscle tissue. This raises circulating antibody levels throughout the body; a generalized, systemic response. Over the years there have been a number of 778

suggestions that a vaccine administered intranasally may offer better protection. The flu, being a respiratory virus, is most at home in the nose and the throat. It spreads by aerosol droplets when people talk, sneeze, or cough. Vaccinating through the nose —in the form of a spay, rather than injection— would more closely mimic influenza’s natural route of infection. Ideally, the corresponding antibody response would be localized, offering highly targeted protective immunity at the very source of infection. Understanding Mucosal Immunity Why exactly is it thought that nasal administration could improve vaccine efficacy? To understand this, we need to take a closer look at something called mucosal immunity. Our skin is one of the first lines of defense against injury and infection, acting as a physical barrier that prevents unwanted entry. But some areas of our body need to allow for a degree of exchange between inside and outside. Broadly, these include: the gastrointestinal tract, the urogenital tract, and the respiratory tract. Each of these inhabits a strange in-between space— on the one hand they are inside our body, but on the other they are constantly exposed to the outside world, rendering them particularly vulnerable. To protect against foreign threats, these areas are covered by a lining known as a mucous membrane, or mucosa. As with our outer skin, part of the mucosa’s protective function lies in acting as a physical barrier. To this effect, the membrane is covered in mucus, which helps trap and slow the advance of microbes — a runny nose is our body’s attempt at expelling microbes once they’ve been trapped, so too is phlegm. But aside from this passive form of protection, the mucous membrane is also packed full of pockets of mucosa-associated lymphoid tissue (MALT), which contain all kinds of immune cells (Figure 1). This layer is known as the lamina propria. It includes B and T lymphocytes, roughly three quarters of which reside in our various mucosal regions. B cells produce antibodies, which can bind to viral particles and prevent them from entering our cells, blocking infection. T cells help kill off cells that have already been infected, curbing viral spread. They also recruit additional immune cells to areas of infection, speeding up viral clearance. Along with B and T cells, mucosa-associated lymphoid tissue is also home to natural killer 779

cells and macrophages, which directly engulf and neutralize any pathogens trying to pass through the epithelium. Finally, dendritic cells act as a kind of surveillance system that modulates the specifics of our immune response on a case-by-case basis. Dendritic cells also present naive B and T cells with foreign antigens, prompting them to differentiate and provide the most specific possible immune response against the microbes at hand. The close proximity of mucosal immune cells to the membrane surface carries with it a distinct advantage: they can jump into action more quickly than they would be able to in non-mucosal regions of the body. They don’t need to waste important time traveling to far off sites of infection, since they are already at the main portal of entry.

FIGURE 1. An overview of the various cells and processes involved in mucosal immunity. The mucus membrane stretches from the mucus layer to the bottom of the lamina propria. SOURCE: WIKIPEDIA, MADE BY OLGAMATVEEVA

Crucially, there exists a class of antibodies —immunoglobulin A (IgA)— that is only produced by B cells in the mucosal membrane. Immunoglobulin A can take on one of two forms: serum IgA, which circulates through the blood as one might expect, and secretory IgA (sIgA), which is made on the underside of the mucosal membrane and is transported across the membrane to the mucosal surface. A key component of this process is the polymeric Ig receptor (pIgR) that binds to the IgA, enabling the antibody to be absorbed by the epithelial cell barrier and ferried through to the surface. As the IgA 780

exits the epithelial cell, the polymeric Ig receptor is cleaved off, preparing the antibody for action (Figure 2). An additional strength of sIgA is the fact that it is a dimer, meaning it is composed of two identical molecular IgA parts, held together by a small joining-chain (JC). Whereas monomeric antibodies have two binding sites, dimeric sIgA has four. This is suspected to improve its ability to bind to antigens, allowing for quicker viral clearance.

FIGURE 2. Generation and Transportation of Secretory IgA “In this example of a mucosal epithelial cell lining a body tract, dimeric sIgA binds via its J chain to a polymeric Ig receptor (pIgR) expressed on the cell surface. The polymeric sIgA molecule is endocytosed by pIgR, transported across the cell (transcytosis), and released into the lumen of the tract. During this release, the pIgR is enzymatically cleaved so that the polymeric IgA and a pIgR fragment (secretory component) remain attached and are released together as secretory IgA.” FROM: “CHAPTER 4, PRIMER TO THE IMMUNE RESPONSE (SECOND EDITION)”

So, unlike most other antibodies, sIgA is stationed on the outside of the body. Here, IgA can bind to microbes —including viruses— before they even have a chance to enter the body. This prevents the microbes from binding to our cells, and by extension, protects us from infection. Akiko Iwasaki, Sterling Professor of Immunobiology and Molecular, Cellular, and Developmental Biology at Yale University, describes it as “putting the guard outside of the door instead of inside the door where antibodies normally work, inside the body.”

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Problems? The first nasal flu vaccine, FluMist, was approved by the U.S. Food and Drug Administration (FDA) in 2012. By 2016 it had been pulled off the market, not to reappear until 2018. The reason? Decreased vaccine effectiveness. Combined data from 2013 to 2016 indicates that nasal spray vaccine effectiveness was roughly 26% in children between the ages of 12 and 17. This is compared to 51% effectiveness for inactivated vaccines, administered intramuscularly. Although these low numbers were brushed off as being a result of poorly matched vaccine virus strains, there may also be other reasons the nasal spray influenza vaccine hasn’t quite lived up to expectations. For one, IgA is typically quite short-lived; it is produced only for a brief period after exposure. Any barrier immunity that the nasal spray vaccine does offer us will likely fade as soon as IgA numbers drop. And durability is already a point of concern with traditional, inactivated flu vaccines, which depend on the longer-lived circulating IgG class of antibodies for protection from infection. So, we cannot expect it to be all that durable. Another worry is that the nasal spray vaccine may be cleared by our immune system before it has the chance to complete its job. Adults who have previously been exposed to the flu —and that means more or less every adult— will still have at least partial mucosal immunity against the influenza viruses. The attenuated, or “weakened”, viruses used in the nasal spray vaccine might simply be getting neutralized before they can infect nasal cells, preventing our B cells from updating sIgA to match the viruses circulating that season. On a more theoretical note, one might also question the belief that vaccine-induced mucosal immunity can provide lasting protection if natural infection, through the same path of entry, fails to do so. Many people are infected by influenza on a seasonal basis, with waning immunity and viral mutation leading to renewed vulnerability year after year. This happens even though they would have built up some degree of mucosal immunity during prior infection. If prior infection through the nose doesn’t protect us from reinfection, why would a nasal vaccine? This hints at a larger issue: we still know very little about the dynamics of mucosal immunity and about the nuances of nasal 782

vaccination. Speaking at the White House Summit for the Future of Covid-19 Vaccines, Dr. Anthony Fauci stressed that we still lack validated animal models to help us sample and quantify mucosal immune responses. Similarly, we lack clear correlates of protection for intranasal vaccines. Although a simplification, IgG levels following intramuscular injection are associated with improved vaccine effectiveness; we don’t yet have enough data to make such inferences in the case of intranasal vaccines. Knowledge about clinical trial designs and endpoints is also lacking, further complicating the development and adoption of nasal vaccines. There has been tremendous excitement around mucosal vaccines, with many in the field pinning their hopes on long-term protection from infection as well as severe disease. The experience with FluMist serves as a caution that the high hopes for nasal vaccination may not be realized. We will soon have data from two new efforts. In China, CanSino Biologics has just had their inhaled Covid-19 vaccine authorized for emergency use. And in India, Bharat Biotech’s Covid-19 nasal vaccine was authorized for restricted use. Hundreds more are in development. Time will tell if they live up to their promise to prevent infection and reduce transmission. The next article in this series will look at a shift in strategy: moving away from yearly vaccines that try to closely match predicted wild type viruses, and instead, attempting to create vaccines that neutralize a broad array of influenza viruses even in the face of continued viral mutation. So called “universal” influenza vaccines. This article is featured on Forbes.org, and can be read online here: Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 4)

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Public-Health Funding Today Keeps the Doctor Away Forbes | September 09, 2022 | Article

Public-health funding often competes with many other demands on government budgets. But by investing in robust public-health staffing and infrastructure, the United States and other countries can significantly reduce the amount of money spent on treating preventable diseases, ultimately freeing up funds for other purposes. FAIRFIELD COUNTY, CONNECTICUT – With COVID19 still an ever-present threat and monkeypox cases rising alongside existing chronic epidemics, building strong, responsive public-health systems has never been more important. In addition to improving data management (which I addressed in a previous commentary), we also need sustained investment and training in the public-health workforce. Public health has always suffered from chronic underfunding, partly because the social and economic benefits of investing in preventive care are difficult to quantify or invisible to the untrained eye. Successes in containing disease outbreaks or reducing mortality rates often go unnoticed. Unfortunately, it often takes a massive failure of prevention to get policymakers and the public to recognize the need for greater preparedness. Americans spend significantly more on medical costs than do people in other similar wealthy countries, yet still have lower life expectancy, higher rates of chronic disease and maternal mortality, and fewer doctors per capita. America’s underinvestment in public health is a major reason. Researchers developing cancer treatments have far greater access to funding than those working on cancer prevention. You know you have a flawed system when you can’t mobilize investments that will pay for themselves – which is precisely what most outlays for public health will do. According to a systematic review of 52 interventions published in 2017 in the Journal of Epidemiology and Community Health, health-protection programs 784

(including vaccinations) saved an average of $34 for every $1 spent on them. Despite this massive return on investment, a 2020 investigation by Kaiser Health News and the Associated Press found that per capita funding for state public-health departments in the United States dropped by 16% between 2010 and 2019, while spending for local health departments fell by 18%. Many health departments thus were already in dire shape when COVID-19 arrived. While Congress invested heavily in public health during the pandemic, federal funding is still following a familiar boom-and-bust cycle. The federal government will spend money to address a specific crisis, but it will not sustain anywhere close to the same level of funding after the emergency ends. Local and state health departments are left to bootstrap their operations until the next crisis, for which they will not have had the resources to prepare. We are now entering a new bust phase, with many local health departments approaching a COVID-19 funding cliff. Emergency funding has been spent or is expiring, even though core publichealth services such as testing, reporting, and vaccinating are still needed to address a high caseload. Public-health departments need to provide these services in addition to all the other functions they are charged with, including but not limited to managing water safety, issuing death certificates, tracking sexually transmitted diseases, and preparing for other infectious-disease outbreaks. A lack of consistent and predictable funding also means that health departments are unable to recruit, retain, and invest in a skilled workforce. Emergency funds for personnel sometimes remain unspent, because local public-health departments are wary of hiring people who will then become permanent fixtures on their payrolls after the additional funding runs out. Owing to these staffing limitations, public-health agencies will instead simply shift personnel from one agenda to another. For example, some public-health departments are now being forced to divert staff working on syphilis and chlamydia in order to conduct outreach, contact tracing, and vaccine campaigns for monkeypox. These staffing issues, together with an environment of political pressure, harassment, and personal threats from the public, have led to an exodus of public-health officials in recent years, following a

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similar exodus of physicians and other frontline workers during the pandemic. A bad problem is fueling a worse one. In a major new report, the Commonwealth Fund Commission on a National Public Health System recommends that Congress provide an additional $4.5 billion annually for guaranteed – not discretionary – federal funding for public health. That would cover the difference between what the US currently spends on publichealth personnel ($19 per capita) and what it needs to spend ($32 per capita). But this figure is primarily based on personnel; it almost certainly needs to be higher to cover core infrastructure such as material, equipment, and training. Moreover, the Commonwealth Fund also recommends that the US Department of Health and Human Services establish a national continuing-education and training system, in coordination with schools and public-health programs, and together with state, local, tribal, and territorial health authorities. The report also advocates public-health training in other departments (such as those focused on education, housing, and criminal justice) to encourage collaboration and address issues like the social determinants of health. Public-health funding often competes with many other demands on government budgets. But by investing in robust staffing and infrastructure, we can significantly reduce the amount of money we spend on treating preventable diseases, ultimately freeing up funds for other purposes. More importantly, we can develop systems that are well prepared to control outbreaks of emerging diseases and address ongoing chronic illnesses, giving all citizens a chance to benefit from longer, healthier lives. This article is featured on Forbes.org, and can be read online here: Public-Health Funding Today Keeps the Doctor Away

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VIII Forbes | September 12, 2022 | Article

This is part of a continuing series describing antiviral antibodies to prevent and treat SARS-CoV-2 infections. In this series, we will discuss the fundamental nature of virus evolution, how SARS-CoV-2 has mutated to evade neutralizing antibodies, and our latest attempts to fight against these mutations with more recent and improved antibody candidates. SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV-2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants. In this series, we have discussed several pan-variant monoclonal antibodies, all of which show promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we describe one promising new antibody described in a study by Ishimaru et al. that targets a conserved site in SARS-CoV2 variants. Antibody Discovery Ishimaru and colleagues began searching for antibodies in previously infected, two-dose-vaccinated patients with the Pfizer mRNA vaccine, examining the sera of three patients led to ten antibodies cloned for further testing. Three of the ten stood out in ELISA binding assays: MO1, MO2, and MO3. All three efficiently bound the D614G variant of SARS-CoV-2 and Omicron BA.1 and BA.2. MO1 and MO2 also bound to the Delta variant. Most notably, MO1 effectively bound

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the pervasive BA.5 variant driving the majority of infections worldwide at the time of writing.

FIGURE 1: The binding of three mAbs to the SARS-CoV-2 spike ectodomains of the D614G, Delta, BA.1, ... [+] ISHIMARU ET AL.

Antibody Broad Neutralization Ishimaru and colleagues next performed neutralization examinations on the three antibody candidates to determine if they killed virus particles to the same efficiency as they bound. MO3 was the worst of the three, failing to neutralize even D614G strongly. In second place was MO2, which eliminated Omicron BA.1 and BA.1.1 with excellent efficiency, and D614G, Delta, and BA.2 to a lesser degree. The true standout of the group was the MO1 antibody. It strongly neutralized the D614G, Delta, BA.1, BA.1.1, BA.2 variants, and BA.5 easily in the 10-100 ng/mL range. Most of the monoclonal antibodies described in this series strongly neutralize BA.1 and BA.2. However, they drop off significantly against BA.5. Furthermore, Ishimaru and colleagues note that MO1 also binds and neutralizes BA.2.75 with significant efficiency, another feat often lost among other monoclonal antibody candidates.

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FIGURE 2: The neutralizing activity of MO1 against D614G, Delta, BA.1, BA.1.1, BA.2, or BA.5 as ... [+ ]ISHIMARU ET AL.

Binding Epitope Unfortunately, the exact binding map of MO1 is unavailable at the time of writing. Cryo-electron microscopy analyses are underway as MO1 is further investigated as a vital anti-Covid monoclonal antibody treatment or prophylactic. The researchers note that the antibody binds the receptor-binding domain and likely the receptor-binding motif, the contact region between the virus and the human host cell. As the exact amino acids are unavailable, we cannot compare MO1 to other similar monoclonal antibodies described previously. However, given the antibody's substantial neutralization of BA.5 and BA.2.75, MO1 likely avoids major mutation sites in the latest Omicron variants, such as L452R and F486V. Conclusion When the binding map becomes available, it would be worth investigating a combination antibody that includes MO1. While the receptor-binding domain is one of the most highly active regions of the virus in a mutational context, MO1’s neutralization of the latest 789

Omicron variants suggests an evasion of these mutations. Pairing MO1 with one or two others could yield a powerful treatment for Covid-19 as of yet unseen in the ongoing pandemic. This article is featured on Forbes.org, and can be read online here: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII

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Molecular Jujitsu: SARS-CoV-2 Co-Opts Host Defense Forbes | September 14, 2022 | Article

Host defenses and the viruses countermeasures are critical to understanding infectious disease. To survive a host must be able to ward off the pathogen. In turn the pathogen must be able to counter the host defenses. In the midst of the Covid pandemic, we are living with the consequences of this eternal host/pathogen struggle. We adapt to the virus through our innate, acquired, medical and social defenses. The virus simultaneously adapts to our evolving response. Science brings an important weapon to this battle by understanding the means by which the SARS-CoV-2 virus counters our host defenses. In an earlier book, Natural Immunity and Covid19, we reviewed numerous means by which the SARS-CoV-2 virus counters our innate immune defenses and how drugs may play an important role in tipping the balance in our favor. Here we describe a recent study published in the journal Nature which investigates one of the virus’s central means to counter innate immunity: the caspase-6 defense back-flip. The Caspase-6 Back-Flip Innate immunity is our first line of defense against new and recurring infections. Innate immunity relies upon an elaborate network of sensors to detect the presence of foreign molecules and to trigger nonspecific defenses against any invader. Rapid suicide of the infected cell is one of many effective innate immune strategies. An infected host cell triggers its own destruction before the virus has a chance to multiply and spread throughout the body. The process is mediated by proteins called caspases. Caspases modulate three specific forms of cell death: apoptosis, programmed cell death without inflammation; necroptosis, cell death accompanied by fever; and pyroptosis, cell death accompanied by inflammation. These enzymes degrade multiple structures within cells to accelerate cell death. Caspase-6 is one such immune defense to infection by a variety of pathogens. For example, caspase-6 is 791

central to our immune defense against influenza. Cells which lack caspase-6 are hypersensitive to infection and death by influenza viruses. Absent caspase-6 cells and animals normally resistant to influenza quickly succumb when challenged. Coronaviruses are an anomaly. Chu et al. report that caspase-6, rather than resisting coronavirus infection, is in fact required for successful replication of SARS-CoV-2, SARS-CoV-1, MERS, and several human cold-causing coronaviruses. Contrasting its role in influenza viruses, caspase-6 appears to be a necessary component in the replication. The investigators provide a detailed description of this miraculous jujitsu using a key component of the host’s own immune defense to defeat that very defense. Nucleocapsid (N) protein

Figure 1: Illustration of different SARS-CoV-2 proteins, including spike (S) protein, matrix (M) protein, envelope (E) protein and nucleocapsid (N) protein. The nucleocapsid protein, depicted in purple, binds to the RNA genome sequence to package and protect the genome in a “beads-on-a-string” conformation. INVIVOGEN

The nucleocapsid protein (N) plays a key role in this remarkable saga. The N protein is the most abundant protein found in infected cells. This protein serves at least two essential roles in the virus life cycle. First and foremost, the N protein binds to viral RNA and packages it into the mature virus particles. But that is not all it does. 792

A series of experimental studies on SARS-CoV-2 and other coronaviruses illuminates another role of the N protein: suppression of the innate immune response.

FIGURE 2: Nucleocapsid (N) protein, denoted in purple, can disrupt the interferon pathway by meddling with Tripartite Motif Containing 25 (TRIM25), interferon regulatory transcription factor 3 (IRF3), as well as transcription factors STAT1 and STAT2. ACCESS HEALTH INTERNATIONAL

The N protein dampens innate immunity in several ways, as summarized in Figure 2. It does so by suppressing the production of interferons (IFN), an essential protein key in triggering both innate and adaptive immune responses, and by inhibiting the induction of interferon stimulated genes. For example, N protein can block phosphorylation of RIG-1, a receptor critical to the interferon signaling cascade. N protein also blocks phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), a key regulator of interferon messenger RNA production. Lastly, the N protein can suppress the production of interferon responsive genes by inhibiting activation by phosphorylation of transcription regulators STAT1 and STAT2. N Protein Cleavage by Caspase-6 Earlier studies found that caspase-6 can cleave N proteins of the MERS and SARS-CoV-1. The study described here extends those results to SARS-CoV-2. 793

Chu et al. reveal that caspase-6 cleaves the SARS-CoV-2 N protein—both as an isolated protein as well as in cell culture. Inhibiting caspase-6 similarly inhibited N protein cleavage. They further outlined that N protein cleavage is specific to caspase-6 and to no other cellular caspase. How Does N Protein Cleavage Stimulate SARS-CoV-2 Replication? A mystery remained: how does cleaving N proteins potentiate virus replication? Chu et al. answered this question by generating N protein mutants to potentially alter known caspase-6 cleavage sites. The team identified the cleavage site in SARS-CoV-2 and noticed its location in the linker region between the two distinct ends of the N protein (see Figure 3B). They found only a small fraction of the total N protein produced by the infected cell is cleaved; the vast majority remain intact and participate in the assembly of infectious virus particles.

FIGURE 3A: An simplified graphic of coronavirus N protein anatomy. The two distinct ends—the amino (N) terminal and the carboxyl (C) terminal—are joined by this flexible linker region. Caspase-6 can cleave N protein at this site and produce two N fragments. FIGURE 3B: Several N protein phosphorylation sites are located only a few base pairs away from the caspase-6 cleavage site in SARS-CoV-2.

ACCESS HEALTH INTERNATIONAL When asked which of the two protein fragments were responsible for immune suppression, Chu et al. made a somewhat puzzling discovery. Both halves—the amino terminal fragment (amino acids 1-215) and the carboxyl terminal fragment (amino acids 794

216-419) were equally immune suppressive. This is surprising as the two proteins are different in sequence and structure (Figure 3A, 3C). N Protein Fragment Binding to IRF3 The team demonstrated that both cleaved N products equally suppress innate immunity by acting as interferon antagonists for IRF3. Both fragments selectively bind IRF3 as judged by coimmunoprecipitation. Both also inhibit IFR3 phosphorylation and translocation into the nucleus where it’s required for initiation of interferon messenger RNA. Caspase-6 is required for virus replication and pathogenesis Is N protein cleavage required for immune suppression? The authors find that, yes, this is the case. Mutations in N that prevent cleavage abrogate N immune suppression. Similarly drugs that inhibit caspase-6 almost limit viral replication in cells and in infected animals. Caspase-6 knockouts in mice SARS-CoV-2 replication and pathogenesis. This result emphasizes the difference between the influenza and coronaviruses, as the same knockout stimulates influenza replication and pathogenesis. Genetics of the N Protein

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FIGURE 4: The proportion of total number of Covid-19 variant sequences (not cases) over time, graphed for both the United States and the United Kingdom. The wavelike replacement of one variant over the other is apparent. COVARIANTS.ORG

A salient feature of the Covid pandemic is the replacement of one viral variant for another (Figure 4). Successive variants evolve along several quasi-independent dimensions. Most famously, variants alter recognition of neutralizing antibodies that bind to the exterior spike protein (reference Figure 1). Recently we have come to recognize additional properties of each new variant. Each new variant replicates more rapidly in the infected host, shortening the incubation period from infection to transmission. Moreover, each new variant suppresses interferon induction more vigorously than its predecessor.

TABLE 1: SARS-CoV-2 variants of concern and the associated mutations in the N protein.

ACCESS HEALTH INTERNATIONAL Jennifer Doudna’s laboratory made a remarkable observation. All successful SARS-CoV-2 variants have mutations in the linker region 796

of the N protein (Table 1). Syed et al. report these mutations singly and collectively accelerate the rate of virus replication both in the context of virus-like particles and in isogenic virus strains in cell culture. We note that these mutations are located in the linker region, proximal to the N protein caspase-6 cleavage site. Specifically, they reside between amino acids 198-205, close to the amino (N) terminal to the amino acid 215 (see Figure 3B). We speculate that these mutations accelerate caspase-6 cleavage of the N protein, thereby accounting for the observed increase in suppression of interferon induction and increase in replication/fitness of the variants. Such is an easily tested hypothesis. Viral Fitness We postulate that fitness is a complex property of each variant conveyed by multidimensional adaptation. Examples include mutations that change protein sequences as well as those that alter RNA cis-acting sequences which affect virus replication and transcription. We suggest that, as this elegant work on the N protein reveals, viral fitness be considered in the context of all the virus genes and cis-acting regulatory sequences in addition to the alterations in Spike protein that have received so much attention to date. Implications for Therapeutics and Beyond This study emphasizes the importance of the N protein in the life cycle of the virus. Without caspase-6 or the proper cleavage site, the SARS-CoV-2 virus is not capable of replicating well in cells. This raises the possibility that drugs that inhibit caspase-6 should dampen SARS-CoV-2 replication. Other targets—such as N protein interaction with IRF3—should be explored as well. This article is featured on Forbes.org, and can be read online here: Molecular Jujitsu: SARS-CoV-2 Co-Opts Host Defense

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Nasal Vaccines May Not Be The Game Changer We Think They Are Forbes | September 14, 2022 | Article

With four new vaccines provisionally approved for nasal delivery in China (inhaled), India, Iran, and Russia, and over 100 mucosal vaccines in development globally, many are pinning their hopes on nasal vaccines becoming the tool that finally halts transmission of the Covid-19 virus. I do not share the same unbridled enthusiasm that nasal vaccines will provide long-lasting protection from infection and protection from all variants for several reasons. Nasal vaccines work by targeting the thin mucous membranes that line the nose, mouth, and lungs. The working theory is by prompting immune responses where SARS-CoV-2 first enters the body, mucosal vaccines could block infection and transmission to other people, which the current intramuscular vaccines do not. The localized mucosal immune cells, known as tissue-resident memory T and B cells, function differently from the circulating T cells and B cells. Tissue-resident memory B cells produce antibodies called secretory immunoglobulin A (IgA). When they are intertwined with the layers of the respiratory tract, they might be able to stop pathogens quickly, but it is not clear how well secretory IgA will protect against SARS-CoV-2. The majority of research on mucosal immunity has been done on cells that line our intestines, not the nasal mucosa, which remains largely unexplored. Almost all Covid infections begin with nasal exposure. We now know that such exposure does not offer long-lasting protection from re-infection, much less protection from new variants as they arise. If natural intranasal exposure does not give the protection we hope for, why should a vaccine administered via the same route do so? By changing the route of administration, we still do not solve the issue of the ever-evolving viral variants of SARS-CoV-2 that are skilled at evading and suppressing our immune systems. Previous attempts to develop a nasal vaccine for influenza have also not succeeded. FluMist is one notable example. During the 798

2013-2014 flu season, the nasal spray showed no measurable effectiveness against the pandemic H1N1 virus in kids ages 2 to 8. That was the predominant type of influenza virus circulating that year. In the 2016-2017 season, the CDC found FluMist's efficacy among children 2-17 was only 3 percent, providing no protective benefit. While the exact reasons for FluMist’s poor performance are unknown, matching the strains used in the vaccine to the predominant strain during the flu season still remains a challenge for all influenza vaccines, regardless of the route of administration. Both China and India have provided limited data from trials of their mucosal vaccines. Data from a phase II trial of CanSino’s inhaled vaccine found that when given as a booster, the vaccine raised blood-serum antibody levels significantly more than a CanSino intramuscular booster injection. Indian vaccine developer Bharat compared its intranasal vaccine to Covaxin, a Covid-19 intramuscular injection available in India, by measuring antibody levels in the blood, deeming it successful but did not publicly release the results of the trial. Neither has been compared to mRNA intramuscular injection, currently the gold standard for Covid-19 vaccines. Even less data is available on the efficacy of the other mucosal Covid-19 vaccines. Iran approved a Covid-19 vaccine administered as a nasal spray and made by Razi Vaccine and Serum Research Institute in Karaj in October 2021. More than 5,000 doses have been delivered to the public. Russia’s health ministry is reported to have approved an intranasal-spray version of Sputnik V, but neither country has published data on efficacy in humans. We can always hope for the best with the development of nasal vaccines, but we must also prepare alternatives. In the past few months, there has been remarkable progress in the creation of broadly neutralizing monoclonal antibodies. These monoclonal antibodies have the potential to neutralize all known SARS-CoV-2 variants and other related coronaviruses, including SARS-1 and MERS. A strategy for Covid control is possible using combinations of these antibodies in tandem with long-awaited highly effective small molecule antiviral drugs for the treatment and pre-and postexposure prophylaxis. With the ever-increasing burden of Long Covid and fatigue around public health measures such as mask-

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wearing, there is an acute need for drugs to treat and prevent SARSCoV-2 infection. This article is featured on Forbes.org, and can be read online here: Nasal Vaccines May Not Be The Game Changer We Think They Are

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Will The BA.4.6 Variant Drive The Next Wave Of The Covid-19 Pandemic? Forbes | September 16, 2022 | Article

Here, we draw attention to a variant, BA.4.6, which is the latest variant spreading in the United States. We note in this summary that BA.4.6 contains five amino acid changes distinct from the currently dominant BA.5 variant and seventeen nucleotide changes, which all may act to improve viral fitness and trigger a new wave of the pandemic. The World Health Organization sees the light at the end of the tunnel. The expression that comes to mind for Covid-19, stemming from the days of the Vietnam War protests, recalls a different perspective: I see a tunnel at the end of the tunnel. In particular, given our current state of understanding of viral variants, there is reason to believe that the next few years will closely resemble our recent experience of successive waves of new variants, each well adapted to spread in a previously vaccinated and/or infected population. Coronaviruses are very well adapted to reinfection of adult immunocompetent mammals that have been previously and recently infected by their predecessor, such as the Influenza virus. In this respect, we should constantly look for new variants that may trigger yet another wave. Figure 1 summarizes the contemporary history of SARS-CoV-2 variants.

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FIGURE 1: Successive waves of SARS-CoV-2 variants in the United States and the United Kingdom.

NEXTSTRAIN.ORG

FIGURE 2: BA.4.6 gaining ground on BA.5 in the United States

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CDC BA.4.6 and BA.5 Between the currently rampant BA.5 and the emerging BA.4.6, there are only seven amino acid differences throughout the entire genome. Although the focus is typically on the spike protein, five of seven lie outside the spike. These include a deletion in NSP1, an amino acid in the membrane protein that is unmutated in BA.4.6 but is changed in BA.5, and three amino acid substitutions in Orf6, Orf7b, and the nucleocapsid (Figure 3).

FIGURE 3: BA.4.6 Genomic mutations. Additional mutations in BA.4.6 as compared to BA.5 are ... [+] ACCESS HEALTH INTERNATIONAL

In NSP1, BA.4.6 contains a deletion from positions 141-143. This mutation is observed in its parent, BA.4, but not in other Omicron variants. All Omicron variants contain the nearby mutation S135R. The deletion may affect NSP1’s immune suppressive functions, including mRNA translation efficiency, interferon inhibition, and phosphorylation inhibition of STAT1 and STAT2. In the membrane protein, we note a significant difference between BA.5 and BA.4.6. In The prevalent BA.5, we note the D3G mutation, which is not found in BA.4.6. This difference may impact RIG-I/MDA-5 signaling, MAVS binding, and inhibition of downstream TBK1 signaling. In Orf6, BA.4.6 contains D61L, which is common to all Omicron variants barring BA.5. While BA.5 is the most pervasive Omicron variant currently, that does not necessarily mean D61L in Orf6 detracts from the virus’s virological capabilities. This mutation may impact interferon inhibition, inhibition of nuclear translocation of STAT1, inhibition of TBK1 and IRF3, inhibition of nuclear 803

translocation of IRF3, inhibition of karyopherin, and blockage of nuclear import and export. In Orf7b, BA.4.6 contains L11F, which again comes from the parental BA.4. The mutation is absent in other Omicron variants. Orf7b is theorized to inhibit certain immune responses, but these are structural assumptions rather than experimental deductions, so further research is needed on this protein and mutation. In the nucleocapsid protein, we observe P151S, which comes from BA.4 as well and is absent in other Omicron variants. The N protein is a significant player in inhibiting the host's innate immune response. P151S may enhance interferon inhibition and RIG-I suppression of phosphorylation and nuclear translocation of STAT1 and STAT2. Another mutation of note lies in the NSP5 protease. NSP5, or M Pro, has been shown in SARS-CoV-2 to be much more active in cellular protein cleavage than in previous coronaviruses. The mutation, P132H, is native to Omicron variants and may affect a critical region of the virus’s M Pro, potentially increasing the efficiency of cleavage of the viral proteins and regulatory proteins NEMO, optineurin, and RIPLET. These proteins are central to the activation of interferon-stimulated genes. The amino acid change imposes structural constraints on this critical region. The impacts of mutations in NSP5 are worthy of a more detailed study. The BA.4.6 genome differs from BA.5 in the spike protein by only two amino acid mutations: R346T and N658S (Figure 4).

FIGURE 4: BA.4.6 spike protein mutations. Additional mutations in BA.4.6 as compared to BA.5 are ... [+] ACCESS HEALTH INTERNATIONAL

Position 346 lies in the receptor-binding domain, the contact region between the virus and the host ACE2 receptor. Mutations in 804

this region can impact transmissibility by increasing ACE2 affinity and may also boost immune evasion by blocking certain antibody binding sites on the spike. Position 658 lies close to the S1/S2 cleavage point and may impact cleavage efficiency, though little is known about its exact impact. Below is a Venn diagram demonstrating the overlap between BA.4.6 and earlier Omicron variants, namely BA.5 and BA.2.12.1 (Figure 5). Note the high degree of overlap between BA.4.6 and BA.2.12.1, as BA.4 is derived from the BA.2 lineage.

FIGURE 5: Venn diagram comparing the amino acid substitutions in the BA.4.6, BA.1, and BA.2.12.1 ... [+] ACCESS HEALTH INTERNATIONAL

We also note several synonymous mutations throughout the genome that do not impact amino acids but may still play an essential role in virulence and immune regulation via transcription sequences and other avenues (Figure 6). There are four synonymous mutations in NSP3, two in NSP12, and one in each of NSP2, NSP4, NSP5, NSP8, NSP10, NSP15, M, NSP6, NSP7b, and N. Research by Thorne et al. indicates that these mutations can initiate significant regulatory effects, particularly in structural and accessory proteins. These mutations are in addition to one mutation, C241T, present in all variants in the 5’ untranslated region. This section of the virus is difficult to analyze as it is not typically recorded. FIGURE 6: BA.4.6 Synonymous mutations.

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ACCESS HEALTH INTERNATIONAL We note that the ability of the virus to strengthen immune suppression may be directly related to the fitness of the virus in the population. Viral fitness is akin to a Rubik’s Cube. As the Cube requires each square to be in the correct formation to complete the puzzle, variants may have different mutations working epistatically to yield more efficient viral fitness. BA.4.6 Immune Resistance While BA.4.6 is still relatively uncommon in terms of global infections, some researchers have noted it as a variant worth examining, specifically in reference to its evasion of commercially available antibody treatments. Jian et al. of Peking University in Beijing tested BA.4.6 against a panel of therapeutic monoclonal antibodies alongside its parent BA.4 (Figure 7). Of the 15 singular and combination treatments, only seven treatments displayed any degree of neutralization. Only three displayed substantial neutralization: Bebtelovimab, Cilgavimab, and the Tixagevimab + Cilgavimab combination. Against BA.4.6, the results are even further diminished. Both Cilgavimab and the Tixagevimab + Cilgavimab combination lose all neutralizing capacity against BA.4.6. Six antibodies display at least minor neutralizing capacity, but only one, Bebtelovimab, strongly neutralizes the variant.

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FIGURE 7: Neutralizing activities against SARS-CoV-2 D614G and BA.4/5 subvariants pseudovirus of ... [+] JIAN ET AL.

While we are lucky that we still have an available antibody that neutralizes the variant, continued use of Bebtelovimab may result in the mutation of variants to overcome the treatment, similar to weeds evolving to overcome specific weed killers. We must continue developing new treatments that maintain a wide variety of targets if we control the spread of emerging variants. Broadly Neutralizing Monoclonal Antibodies The next step for Covid-19 treatment will be the mass production of monoclonal antibodies, both singularly and in 807

combination. Although the data on their ability to neutralize BA.4.6 is not yet available, it is noteworthy that the binding sites for many broadly neutralizing antibodies are conserved in BA.4.6, except for Luo et al., Fenwick et al., and Wang et al. We have previously pointed out the importance of using combinations. Specifically, we may use a variety of broadly neutralizing antibodies that minimizes mutations in amino acid binding sites. For example, the combination of Li et al., Dacon et al., and Bebtelovimab, which we know, neutralizes BA.4.6 to a strong degree.

FIGURE 8: A proposed combination of antibodies for use against BA.4.6 infection.ACCESS HEALTH INTERNATIONAL

Implications Ultimately, we will not know the potential impact of BA.4.6 for some time. It may fade into relative inconsequence along with many variants that have come before it, but it may also emerge as the newest variant of concern. As such, we must prepare for the latter, continuing to identify and develop treatments that can neutralize the variant. This article is featured on Forbes.org, and can be read online here: Will The BA.4.6 Variant Drive The Next Wave Of The Covid-19 Pandemic?

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Reimagining Alzheimer’s (Part 1) Forbes | September 16, 2022 | Article

Alzheimer’s disease affects over 5 million individuals in the United States and has proven to be an enigma in the scientific world. Historically, scientists have believed that Alzheimer’s disease was solely caused by two dysfunctional proteins in the brain called ßamyloid and tau. Now, recent discoveries have thrown into question this fundamental assumption about Alzheimer’s disease and its origins. For decades, scientists have approached Alzheimer’s research by searching for ways to prevent the unusual activity of amyloid-beta and tau. While several medications and antibody treatments have been developed to intercept the activity of these proteins, many of them are unsuccessful and do not prevent the major cognitive decline associated with Alzheimer’s disease. Due to this, there are serious suspicions in the scientific world that there may be more to the story of Alzheimer’s than ß-amyloid and tau. In this short series, we will introduce some of the latest interesting work surrounding Alzheimer’s disease, its origins, pathology, and potential avenues for treatment. In the first installment of the series, we offer a brief overview of Alzheimer’s disease in general. Clinical View Alzheimer’s is categorized as a specific form of dementia. As such, the most marked clinical symptom of Alzheimer’s disease is a decline in cognitive abilities. This includes loss of memory, difficulty thinking, along with changes in behavioral and social skills that can prevent an individual from living independently. Alzheimer’s disease typically affects the elderly, but there exist rare cases of early-onset Alzheimer’s that impact those in their 30s or 40s. While Alzheimer’s is often confused with other forms of dementia, it uniquely targets regions of the brain responsible for learning, especially at the onset of the disease. This includes the hippocampus and the entorhinal cortex. The hippocampus is the 809

primary brain region responsible for short/long-term memory and spatial memory. Some of the first symptoms of Alzheimer’s are shortterm memory loss and disorientation. The entorhinal cortex specializes in pattern recognition and the perception of time.

Figure 1: Alzheimer's predominantly affects the Hippocampus and Entorhinal cortex.RASLAU ET AL., AMERICAN JOURNAL OF NEURORADIOLOGY (2014), DOI: 10.3174/AJNR.A4169

Alzheimer’s is also characterized by its very gradual onset and cognitive decline. Most other forms of dementia either involve abrupt changes in cognitive abilities or they simply affect different regions of the brain. The most recent diagnostic criteria for Alzheimer’s disease suggest that the disease progresses through three fundamental stages: a preclinical period with no obvious symptoms, a middle stage of mild cognitive impairment, and a final stage marked by severe symptoms of cognitive decline. During severe stages of Alzheimer’s, patients often lose their ability to respond to their environment whatsoever and become unable to control their movements. As cognitive abilities worsen, communicating also becomes painful. Ultimately, Alzheimer’s disease is lethal and one of the leading causes of death in the United States. Biological View But what do all these clinical symptoms mean, biologically? The human brain typically shrinks during healthy aging. However, those 810

with Alzheimer’s disease experience a much higher degree of brain atrophy. This is because the primary biological feature of Alzheimer’s disease is widespread injury and loss of neurons in the brain. When individual neurons stop functioning, this impacts the connections between neurons that allow our brain cells to communicate, metabolize, and repair themselves, leading to severe brain atrophy.

Figure 2: Alzheimer's causes severe brain atrophy.

HTTPS://WWW.NIA.NIH.GOV/HEALTH/ALZHEIMER S-DISEASE-FACT-SHEET One of the primary challenges of Alzheimer’s disease is that biological symptoms often begin to develop during the preclinical period before patients exhibit any obvious cognitive symptoms. By the time patients notice symptoms of cognitive decline and seek clinical help, damage to the neurons has already occurred and Alzheimer’s has taken hold of the brain. This has not only prevented researchers from pinpointing exactly what causes Alzheimer’sinduced brain atrophy but also makes it very difficult to develop preventative treatments. Despite these limitations, scientists have found that two primary indicators for Alzheimer’s disease progression are amyloid plaques

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and tau tangles. This discovery is what led to the belief that ßamyloid and tau proteins are the root causes of Alzheimer’s. Both amyloid plaques and tau tangles are caused by abnormal clusters of protein fragments. Amyloid plaques are built out of a protein called ß-amyloid. ß-amyloid is produced when a much larger protein called amyloid precursor protein (APP) is broken down. APP is expressed in many tissues but is especially found within the synapses of neurons. In healthy brains, APP is cut into fragments of protein that specifically contain 40 amino acids. These fragments are called ßamyloid-40. ß-amyloid-40 is regularly cleared from neural synapses and does not cause damage to the brain. In those with Alzheimer’s, when APP is cut into ß-amyloid, the ß-amyloid fragments can sometimes be slightly longer than average. These longer chains of protein consist of 42 amino acids and are referred to as ß-amyloid-42. ß-amyloid-42 is chemically stickier and tends to aggregate. When ß-amyloid-42 aggregates, the brain is unable to clear the protein properly, leading to the growth of amyloid plaques that exist between neurons.

Figure 3: Alzheimer's is linked to the formation of amyloid plaques in the brain.

HTTPS://WWW.STATNEWS.COM/2019/04/30/AMYLO ID-FAILURES-NEW-TACK-TREATING-ALZHEIMERS/ These plaques disrupt cellular function, though it is unclear exactly how. Amyloid plaques typically begin to develop during earlier stages of Alzheimer’s but stop developing when severe stages of Alzheimer’s set in. 812

Tau tangles, or neurofibrillary tangles, are accumulations of a protein called tau that collect inside of neurons. Neurons contain structures called microtubules that help transport nutrients throughout the cell. In a healthy brain, tau proteins can help microtubules maintain their structure. In patients with Alzheimer’s, however, tau proteins undergo unusual chemical changes. These changes lead to the tau proteins being unable to hold their own structure or support the microtubules. Instead, the tau proteins become messy tangles that accumulate inside our neurons. Some researchers have speculated that the unusual chemical changes that cause tau tangles are due to specific mutated genes. However, much like amyloid plaques, the exact cause of these changes is unclear.

Figure 4: Alzheimer's disease has been linked to the formation of tau tangles/neurofibrillary tangles.

HTTPS://WWW.BIO-RADANTIBODIES.COM/BLOG/HAUNTED-BRAIN-GHOSTTANGLES.HTML Abnormal tau accumulation has been shown to increase brain cell damage. Unlike amyloid plaques, tau tangle accumulation continues throughout the course of Alzheimer’s. While these two features of Alzheimer’s disease are the most widely known and accepted markers for Alzheimer’s progression, they do not paint a whole picture of the disease. 813

Both amyloid plaques and tau tangles were previously suggested to be the root cause of Alzheimer’s and cognitive decline. Now, more recent research suggests that this may not be the case. Studies conducted by scientists at NYU Langone and the Nathan Kline Institute in New York suggest that cell damage and disruption occur long before amyloid plaques or tau tangles begin to appear. Other studies have found that some patients exhibit amyloid plaques but no symptoms of cognitive decline. Additional studies have discovered other irregularities in the brain associated with cognitive decline and Alzheimer’s. Alzheimer’s disease is a pervasive mystery in the scientific community. However, with decades of research and recent innovations, scientists have uncovered several clues as to how and why Alzheimer’s might develop. In the next installment of this series, we will be analyzing multiple theories of Alzheimer’s pathogenesis and the biology behind them. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 1)

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Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 5) Forbes | September 19, 2022 | Article

This is a short series focusing on the challenges of developing effective influenza vaccines. In the first part of this series, I gave a brief overview of the history and nature of influenza viruses, including why they represent a tricky target for vaccine manufacturers. In the following three installments—which can be read here, here, and here— I discussed some of the attempts that have been made to overcome these challenges. The following articles will focus on one of the more promising approaches to date: universal vaccines. Vaccine Platform vs. Vaccine Antigens Vaccines work by exposing our immune system to viral proteins. Our immune system then recognizes these proteins as foreign molecules —antigens— and springs into action, neutralizing the threat. As part of this process, immune cells known as B and T lymphocytes build up an “immunological memory” of the antigen in question, helping them to react more quickly and more efficiently should we become reinfected. Many of the recent advances in vaccine technology have focused on changing the way the antigens are delivered to our immune system. Recombinant vaccines, mRNA vaccines, and liveattenuated mucosal vaccines, to name just a few. With recombinant and mRNA vaccines in particular, the benefits are most acutely seen on the production side of things: both production times and production costs are cut back significantly by these vaccine platforms. They also provide scalability, allowing for improved pandemic preparedness. But in and of themselves, changes to the delivery method have not yielded substantial changes to vaccine effectiveness. At least not in the case of influenza. True, faster production times will help scientists keep the antigens used in the vaccines well matched to those of the viruses actually circulating during flu season. 815

Unfortunately, even when well matched, influenza vaccines are currently 60% effective at best. How the antigen is delivered is one side of the coin, what antigen gets chosen in the first place is the other side. Delivery platforms continue to evolve, yet progress in antigen selection has been lagging. To optimize our influenza vaccines, we need both. Targeting Conserved Regions: HA Stalk and M2e Whether egg-based, cell-based, recombinant or otherwise, most of the currently available flu shots use the hemagglutinin (HA) surface protein as their antigen of choice. This makes sense; influenza viruses depend on the hemagglutinin protein to gain entry into our cells. Block the protein, and you have a chance at blocking infection. The issue with using hemagglutinin as the primary antigen is that it is extremely mutable. The structure of the protein can change to avoid previously established immunity, including B cell-derived antibodies. Thus, the seasonal flu cycle and yearly flu shots. The universal vaccine approach aims to change things. Instead of using the head of the hemagglutinin protein as the antigen, researchers working towards an universal flu vaccine look for viral proteins, or specific protein regions, that are highly conserved across strains and across time — essentially, antigens that only very rarely mutate. If successful, universal vaccines could provide a much broader immunity, eliminating the need for new vaccines every year. So far, there have been two promising contenders: the stalk portion of the hemagglutinin protein and, for influenza A viruses, the extracellular region of the Matrix-2 protein (M2e). The hemagglutinin surface protein can be separated into two regions, one being the highly mutable head (HA1) and the other being the stable stem (HA2) (Figure 1). The head region contains the receptor binding domain (RBD) which the influenza viruses use to bind to our cells. Using this area as the reference antigen creates a highly specific immune response, with antibodies that can block the RBD and, if present in sufficient numbers, may prevent infection. But the specificity comes at the cost of breadth. Even a single mutation can throw off our immune system, rendering the antibodies practically ineffective.

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FIGURE 1. “The influenza virus with surface proteins hemagglutinin (HA) and neuraminidase (NA) is shown in the left panel. The middle panel shows the X-ray structure of the HA trimeric glycoprotein. The right panel shows the phylogenetic tree of HA, group 1 (colored blue) and group 2 (colored red). The subtypes that have been confirmed in humans are boxed.” FROM: “ANTIBODY CROSS-REACTIVITY TO HEMAGGLUTININ PROTEIN ANTIGENS DEMONSTRATES FEASIBILITY FOR DEVELOPMENT OF A “UNIVERSAL” INFLUENZA A SYNTHETIC PEPTIDE VACCINE” JIANG ET AL. 2015

Where the HA head domain is used to bind to our cells, the stem region is used for fusion — the process by which the virus “injects” its genetic material into the host cell cytoplasm, kicking off viral replication. Briefly, once a viral particle has bound to a host cell it is “ingested” by a section of the cell membrane that then buds off to form a membrane “bubble”. The resulting bubble is called a vesicle, and foreign substances internalized in this way are usually promptly digested and disposed of by the cell. But influenza viruses are crafty and have developed a way to use the acid that normally helps our cells digest such vesicle contents to instead free up the RNA inside the viral particle. This is where the stem portion of the hemagglutinin protein comes into play. Once the RNA has been freed up, the HA stem is used as a lever of sorts to pull the viral membrane into the cellular membrane, finally creating a “tear” that lets the genetic material escape into the cytoplasm. Once there, the virus can begin replicating before spreading to other cells. A vaccine that targets the HA stem and blocks it from initiating fusion would interrupt the replication process, preventing further infection and stopping the virus in its tracks. Indeed, researchers have 817

already developed vaccines based on “headless” HA proteins as antigens, but results so far have been slightly underwhelming; although the protection the vaccines have provided has been fairly broad, it has not been very strong. Mice inoculated with vaccines based on HA stalk as an antigen still lose noticeable amounts of body weight, a common symptom of illness. This has been particularly true for influenza A viruses with group 2 HA proteins (see figure 1), which also happen to be those causing the majority of seasonal infections. In response, attention has also been directed towards the matrix2 (M2) protein (Figure 2). This protein spans the membrane of the influenza viral particle and functions to internalize the acid I mentioned above. Without the M2 protein, the viral RNA cannot be untethered from its membrane and replication is once again impaired. Vaccines that target the M2 ectodomain (M2e) have fared better than those that target the HA head alone.

FIGURE 2. Viral particle of influenza with its membrane proteins: hemagglutinin (HA), neuraminidase (NA), matrix protein 2 (M2); and associated proteins: matrix protein 1 (M1), ribonucleoprotein (RNP). A) A model of the viral particle. B) Viral budding. FROM: “INFLUENZA M2 PROTON CHANNELS” PIELAK ET AL. 2011

More recently, efforts have been made to merge HA stem and M2e into one single molecule, to boost immunogenicity. Linking them chemically has yielded promising results, with higher protective potency than HA stem or M2e vaccines alone. This suggests a way forward. The next article in this series will provide a detailed analysis of a recent success in the search for an effective universal flu vaccine: genetically linking M2e and HA stem. 818

This article is featured on Forbes.org, and can be read online here: Getting a Grip on Influenza: The Pursuit of a Universal Vaccine (Part 5)

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SARS-CoV-2 Main Protease Suppresses Innate Immunity By Cleaving Proteins Required For Interferon Induction And Inflammation Forbes | September 22, 2022 | Article

Many viruses rely on proteases to process polypeptides into smaller proteins required for replication and virus production. SARS-CoV-2 is no exception. It contains two proteases in the long open reading frame of ORF1A1B that encode for 15 proteins which need proteolytic processing. Main protease, or Mpro, is the cysteine protease responsible for most of these cleavages. A series of previous studies have shown that this target site for Paxlovid plays an additional role in the virus life cycle: suppression of the innate immune system. In a previous publication we described how SARSCoV-2 is a master at suppressing the innate immune system—both through poly-functional proteins, proteins serve both more than one function in the virus life cycle, as well as a series of accessory genes which primarily alter innate immunity. Here we discuss studies on main protease (Mpro), and its activities.

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FIGURE 1: Mpro, denoted as NSP5, disrupts the interferon pathway of innate immunity at three previously known points. The protease interferes with typical functions of RIG-I, TBK1 and IRF3, which can lead to consequences in downstream signaling and interferon production.

ACCESS HEALTH INTERNATIONAL Also known as 3C-like protease or non-structural protein 5 (nsp5), Mpro blunts the interferon pathway of innate immunity at three points. It inhibits ubiquitination of RIG-I, a pattern 821

recognition receptor needed to identify pathogens, and inhibits phosphorylation (activation) of transcription factors TBK1 and IRF3 (see Figure 1). In addition to cleaving virus proteins, Mpro released into the host cell has the potential to cleave cellular proteins. In this study published in the journal Proteomics, Koudelka et al. address what cellular proteins can be cleaved by Mpro and if these cleavages may partly account for the ability of SARS-CoV-2 and other coronaviruses to suppress innate immunity. To do this, the researchers employ unbiased techniques to analyze the structure and binding of SARS-CoV-2 Mpro in lung cells. The technique, coined N-terminomics, identifies the substrate (protein target for Mpro) and corresponding cleavage site. With this, the team could better understand how SARS-CoV-2 Mpro suppresses host immune responses. According to their research, Mpro is able to cut several cellular proteins involved in innate immunity. As a result, this tampers with innate immune responses which stimulate interferons, proteins which signal the presence of foreign invaders; induce interferonstimulated genes to inhibit viral replication; and stoke inflammation to counter present viral intruders. NEMO—NF-κB Essential Modulator

FIGURE 2: Nonstructural protein 5, or Mpro, can blunt the branch of innate immunity which stimulates inflammation. NEMO, short for NF-κB Essential Modulator, is a crucial component to this defense.

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ACCESS HEALTH INTERNATIONAL NEMO, short for NF-κB Essential Modulator, is a protein which is essential for inflammation. As seen in Figure 2, NEMO must be activated in order for several other proteins in the inflammation cascade to function. Koudelka et al. discover that Mpro cleaves NEMO between Q231/V232, likely blunting this innate immunity pathway. This could correlate to observed symptoms in COVID-19 patients. A disrupted NF-κB pathway can lead to overactive inflammation, as often observed in chronic inflammatory diseases. Following this line of thought, Mpro could possibly contribute to the heightened inflammatory response seen in those with COVID-19. OPTN—Optineurin NEMO is not the only host protein that Mpro can cut. SARSCoV-2 Mpro is able to cleave optineurin (OPTN), a protein coding gene. OPTN is needed to transfer inactivated TANK-binding kinase I (TBK1) to the golgi apparatus. There TBK1 is activated, triggering interferon regulatory factor 3 (IRF3) to action and thus the production of interferon. Mpro can cleave optineurin at two different sites, likely dampening the activity of TBK1 and thus all other signals downstream. TRIM25 & RIPLET As previously mentioned, Mpro can prevent ubiquitination of RIG-I. This study further outlines this mechanism by clarifying how Mpro cleavage accomplishes this task. Akin to tagging a product for processing, ubiquitination coordinates host proteins for localization, activation and/or deactivation. The team found that Mpro can cleave several ubiquitin ligases which mediate this process. They point out TRIM25 and RIPLET in particular, which mediate the ubiquitination of RIG-I and therefore likely impact interferon production. EIF4G1—eukaryotic translation initiation factor 4 The team found that Mpro also cleaves eukaryotic translation initiation factor 4 (EIF4G1), a modulator for human cell growth. The authors posit that cleaving this protein may result in host cell shut-off, as similarly demonstrated for picornavirus. Possible cleavage of STAT2 Last in the list, the authors note that transcription factor STAT2 has been cleaved by Mpro in pig coronaviruses. STAT2 induces 823

interferon-stimulated gene expression. While it is possible that SARS-CoV-2 Mpro cleaves this protein, too, the team could not observe this effect using mass spectroscopy. The cleaved, resultant peptide is too small to be detected with this method. Little Changes, Big Impacts Alongside identifying cellular substrates for Mpro, the team also conducted a comparative analysis of cleavage sites in SARS-CoV-2 and SARS-CoV-1 in vitro. They uncovered that SARS-CoV-2 Mpro cleaved in far greater amounts than its predecessor. SARSCoV-2 Mpro had 391 cleavage events as compared to 130 events for its SARS-CoV equivalent. SARS-CoV-2 Mpro could also cleave a wider spread of amino acids at the P2 region, as visualized in the frequency diagram in Figure 4.

FIGURE 4: A frequency diagram of found cleavage specificity sites between (A) SARSCoV-2 (n=391) and (B) SARS-CoV (n=130). The comparison of cleavage site specificities demonstrates how slight changes in protein structure can significantly impact whether or not a protein can be cleaved.

CREDIT: KOUDELKA ET AL.Link Added This finding is surprising because Mpro in both coronaviruses share highly similar amino acid sequences (96% similarity) and catalytic efficiency. One would expect the amount of cleavage events to be comparable. The authors suggest that the three824

dimensional shape of the binding sites may be a contributing factor. For example, at residues 45 to 51 SARS-CoV Mpro forms a right handed 310 helical structure. This finding underscores the importance of protein structure and sequences, and how little changes in these areas can cascade to vastly different resultant products. Mpro and SARS-CoV-2 Viral Fitness

TABLE 1: SARS-CoV-2 variants of concern and mutations in NSP5, otherwise known as Mpro or main protease.

ACCESS HEALTH INTERNATIONAL

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FIGURE 5: Mpro bonds with other Mpro structures to create dimers, here composed of Mpro chain A and Mpro chain B. The C terminal tail of the dimer is important for Mpro to bind with other dimers and afterwards bind with NEMO. The C terminal tail shown here is located between 𝜷𝜷 strands Gly109-Tyr118 and Ser121-Arg131 in the neighboring dimer.

HAMEEDI ET AL.Link Added We note that all omicron variants have mutation P132H. This mutation substitutes an uncharged proline, which would constrict the protein structure, with a positive histidine. The different charge likely results in a major structural change near what appears to be a critical region for SARS-CoV-2 Mpro (see Figure 5). A recent paper found that SARS-CoV-2 Mpro binds with itself, forming a dimer with a C-terminal tail when interacting with NEMO; one of the C terminal tails nestles between 𝜷𝜷 strands Gly109-Tyr118 and Ser121Arg131 in the Mpro neighboring dimer. A mutation near this site could impact Mpro’s ability to cleave NEMO and possibly other host proteins. 826

We speculate that this change may increase the efficiency of proteolytic cleavage of proteins such as NEMO, thereby improving the ability of the omicron series of viruses to suppress the immune system and accelerate their fitness. We pointed out a similar phenomena in an earlier article in which N protein cleavage facilitates virus replication by innate immune suppression. Conclusions Koudelka et al. bring additional specificity and understanding to how Mpro contributes to viral replication. The typical role of Mpro as a cysteine protease is to mature other SARS-CoV-2 proteins and aid processing of translated viral proteins. However, similarly to N protein, it seems Mpro does double duty by also cleaving cellular proteins essential to innate immune signaling. Alongside studies on SARS-CoV-2 spike and nucleocapsid, additional investigation could further elucidate the full scope of Mpro’s immune suppressive qualities, as well as identify whether mutations in Mpro significantly contribute to SARS-CoV-2 viral fitness. This article is featured on Forbes.org, and can be read online here: SARS-CoV-2 Main Protease Suppresses Innate Immunity By Cleaving Proteins Required For Interferon Induction And Inflammation

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Waiting In The Wings: A Potential New Variant Of SARS-CoV-2 Forbes | September 22, 2022 | Article

Just as a chameleon changes its color to avoid predators, SARSCoV-2 continues mutating to enhance its infectivity and immune evasion. As President Biden declares the pandemic over, the virus continues to evolve and infect steadily. 2022 is the year of the Omicron family of variants, and another version has emerged, adding itself to the evergrowing list. Here, we describe the new Omicron variant, BA.2.75.2, and why we caution it at this early stage of its detection.

FIGURE 1: Continued evolution of SARS-CoV-2 spike protein.

JUSTIN STEBBING As its name implies, BA.2.75.2 is derived from its parent: BA.2.75. At the time of writing, BA.2.75 accounts for less than 1% of global SARS-CoV-2 infections, though the ancestral BA.2 variant drove the major wave of infections in early 2022. Why are we 828

interested in BA.2.75.2 if its direct parent is not widely prevalent? The answer lies in its immune evasion. BA.2.75 is among the most immune evasive SARS-CoV-2 variants, evading both the host immune response and antibody responses from vaccines and monoclonal treatments. The BA.2.75.2 variant takes the blueprint of its parental lineage and adds three new mutations to the landscape, notably located in the spike protein, one of the major players in immune evasion.

FIGURE 2: BA.2.75.2 spike protein mutations. Mutations in red are those added in the BA.2.75.2 spike ... [+] ACCESS HEALTH INTERNATIONAL

Two of the three additional mutations in BA.2.75.2 are found in the receptor-binding domain. The first, R346T, lies in the receptorbinding core, stabilizing the binding domain and connecting it to the rest of the spike. The second, F486S, lies in the receptor-binding motif, the region that makes contact with the host ACE2 receptor. Position 486 is one of the residues directly touching the ACE2 receptor, meaning mutations here are highly impactful for ACE2 affinity. Together, these two mutations may significantly increase the virus's immune evasiveness and infectivity. The third mutation lies far from the receptor binding domain in the S2 subunit of the spike. S2 is the area closer to the membrane of the spike, impacting fusion post-contact with the host cell. Mutations in this region are much less common than in the receptorbinding or N-terminal domains. The mutation, D1199N, may have several effects on fusion and immune evasion, but more research would be needed to say definitively.

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FIGURE 3: BA.2.75.2 genomic mutations.

ACCESS HEALTH INTERNATIONAL No new mutations are found outside the spike protein. We discussed the ancestral mutations in BA.2.75 that were passed down to BA.2.75.2 in a previous article. Among Omicron variants, BA.2.75 evades commercially available monoclonal antibody treatments with excellent efficiency.

FIGURE 4: BA.2.75 monoclonal antibody evasion. YAMASOBA ET AL.

BA.2.75.2 continues the trend.

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FIGURE 5: BA.2.75.2 monoclonal antibody evasion.

SHEWARD ET AL. Among commercial antibodies, Bebtelovimab is the only treatment that neutralizes BA.2.75.2. GSK’s S2K146 antibody mildly neutralizes the variant, and all others do not neutralize to a significant extent. There are many broadly neutralizing antibodies in production around the world that target conserved residues in the SARS-CoV2. The correct combination of such antibodies could neutralize BA.2.75.2 with solid efficiency. For example, the combination of Li et al., Dacon et al., and Bebtelovimab, which we know, neutralizes BA.2.75.2 substantially. The only overlap between BA.2.75.2 mutations and the residues targeted by this combination lies in the receptor-binding domain, which Bebtelovimab overcomes with strong efficiency.

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FIGURE 6: A proposed combination of antibodies for use against BA.2.75.2 infection.

ACCESS HEALTH INTERNATIONAL We also note emerging cryptic virus lineages from wastewater that share many similarities to BA.2.75.2. There are at least 11 consensus mutations between the cryptic viruses and this version of Omicron solely in the spike protein from positions 413 to 505. There are certainly many more mutations throughout the genome that align with BA.2.75.2 that are not yet available. The similarities suggest that Omicron variants are widely pervasive throughout the United States and novel versions of Omicron will continue to arise as infections continue.

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FIGURE 7: Mutational comparison of BA.1, BA.2.75.2, and sewer viruses from positions 413 to 505 in ... [+] ACCESS HEALTH INTERNATIONAL

Ultimately, time will tell if BA.2.75.2 becomes the next to trigger a significant wave of SARS-CoV-2 infections. We must remain vigilant, as a new wave is more likely than not with the continued evolution of the virus and the return to regular operation so much of the world is undergoing. This article is featured on Forbes.org, and can be read online here: Waiting In The Wings: A Potential New Variant Of SARS-CoV2

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We need resilient health systems to address the dual crisis of infectious and chronic diseases Forbes | September 26, 2022 | Article

With our health systems strained by the concurrent outbreaks of monkeypox, polio and COVID-19, chronic diseases are not receiving the attention they deserve. But as we continue to face ongoing infectious disease threats, we need to build resilient health systems that are equipped to face both public health emergencies and ongoing population health challenges. Pre-pandemic, chronic disease was already a serious problem in the U.S. According to the Centers for Disease Control and Prevention (CDC), one in six adults has a chronic disease, and four in 10 adults have two or more chronic diseases. These include diabetes, heart disease, cancer, chronic lung disease, stroke and chronic kidney disease. Chronic diseases represent seven of the top 10 causes of death in the United States. The COVID-19 pandemic has starkly affected chronic disease directly and indirectly through disruption to preventive care and disease management and by contributing to high morbidity and mortality rates. Heart disease, diabetes, cancer, chronic obstructive pulmonary disease, chronic kidney disease and obesity are all conditions that increase the risk for severe illness from COVID. We have also witnessed the birth of new chronic disease in “long COVID,” which affects nearly one in five Americans. A growing number of studies has shown that COVID can increase a person’s risk of diabetes, even months after infection. A Lancet study found that people who were infected with COVID were about 40 percent more likely to develop diabetes up to a year later than others in the control groups. For every 1,000 people studied in each group, roughly 13 more individuals in the COVID group were diagnosed with diabetes. Even people who had mild

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infections and no previous risk factors for diabetes had increased odds of developing the chronic condition. Several studies have also highlighted how the pandemic has created a barrier to preventive cancer care. A 2021 study published on the pandemic’s impact on cancer services in Louisiana and Georgia found there were nearly 30,000 fewer cancer pathology reports than in 2019, representing a 10 percent decline. Many reported delaying or missing preventive care appointments due to fear of exposure to the virus in 2020. Without responding to the dual crisis of infectious disease threats like COVID and chronic disease, each will continue to amplify the negative effect of the other. This will only put further strain on our health systems, ultimately creating barriers or reduced care capacity for other health care issues. Our health care system needs to align incentives to encourage payers, providers, employers and individuals to better prevent, detect, treat and manage chronic diseases before they become acute, costly problems. This begins with increasing access and removing barriers to primary care doctors and complete integrated preventive care. Primary care doctors are critical to helping patients prevent and navigate chronic disease and providing referrals to other specialists who can assist with their conditions. According to a Kaiser Family Foundation poll, one-fourth of adults and nearly half of adults under 30 don’t have a primary care doctor. This care disparity is worse for minorities. A 2020 poll by the African American Research Collection found that Black, Native and Latino Americans reported having less access to a primary care doctor than their white counterparts. One positive impact of the pandemic has been the uptake of telemedicine, particularly for those in rural areas or health care “deserts.” New technological advances can also expand the role that telemedicine plays in at-home care delivery. Remote patientmonitoring devices allow providers to monitor patient progress remotely and receive alerts if there is an issue. To continue to reap the benefits of telemedicine, we need to make the emergency authorizations permanent and ensure payment parity for providers. Equitable access to the internet for all Americans is also necessary to reduce care disparities. 835

Standardized, interoperable health care data systems will also help providers reduce inefficiencies and improve the health system’s ability to proactively identify risk and coordinate care. By investing in emerging technology tools such as big data analytics and genomic testing, providers can conduct early outreach and consistently follow-up, monitor and manage patients more effectively in their homes, while cultivating a deeper understanding of how, why and where chronic diseases develop. By investing in resilient health systems to address public health emergencies and chronic disease, we can encourage healthy longevity for all. Dr. William Haseltine is president of ACCESS Health International and will be moderating two expert panels at the Metabesity 2022 conference on how lessons from the COVID-19 pandemic can prepare us to address the ongoing epidemic of chronic diseases. This article is featured on Forbes.org, and can be read online here: We need resilient health systems to address the dual crisis of infectious and chronic diseases

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Is A Universal Influenza Vaccine —One Shot For All Strains— On The Horizon? Forbes | September 26, 2022 | Article

This is the second of two articles on universal flu vaccines, both of which are part of a larger series on influenza vaccines and influenza treatments. Previous articles can be read here: Part 1, Part 2, Part 3, Part 4, Part 5. For many years there has been an obvious need for an influenza vaccine that neutralizes not just one strain, but many different strains across both major influenza types. This includes providing protection against as-yet undiscovered strains. Current vaccines have not achieved this, requiring yearly administration to keep pace with viral mutations. But over the last decade there has been great effort to develop such a universal flu vaccine, with much of it now beginning to pay off. Two Targets Are Better Than One The key problem so far has been that flu vaccines provide a potent but highly specific immune response; they work decently well, but only against those strains that have been included in the vaccine. Should a new strain of influenza appear, or one that scientists hadn’t predicted, then vaccine efficacy is left seriously jeopardized. To combat this issue, scientists have begun looking towards highly conserved regions of the viral genome. These are regions that are shared across many strains and that only very rarely mutate. Two areas of the influenza virus particle are particularly promising targets: the stem portion of the influenza surface protein hemagglutinin (HA), and the extracellular region of the matrix-2 protein (M2e). Both of these proteins play a crucial role in viral replication — M2 helps untether the viral RNA from the surrounding viral membrane, and HA stem (HA2) creates the opening through which the RNA can then enter the host cell cytoplasm. Antibodies that bind to and block either of these proteins impair viral replication. Here, we describe a new vaccine candidate

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developed by Subbiah et al. that combines these two attractive targets into a single, highly engineered protein. Design of the Antigen: Perfecting the “Headless” HA Protein Interest in the stem portion of HA has its roots in the discovery of human broadly neutralizing antibodies (bnAbs) that target the region. This discovery revitalized hopes of developing universal flu vaccines by giving researchers an entry point for the production of a universal antigen. Since the full HA protein only rarely elicits broadly neutralizing antibodies, various approaches have emerged to expose and properly present the stem domain to our immune system. One of the more successful strategies involves knocking off the HA head domain while ensuring structural stability of the leftover HA stem. But this is easier said than done. The head of the HA protein stabilizes the prefusion protein resulting in a tightly coiled triplex of three proteins. Broadly neutralizing monoclonal antibodies only recognize this tightly coiled triplex and are lost once the fusion process commences or the head of the HA protein is removed. To perfect the hemagglutinin portion of their antigen, Subbiah and colleagues drew upon previous research by Impagliazzo et al. and Yassine et al. Both groups managed to engineer stable, headless HA stem antigens —called “mini-HAs”— that exhibited structural and antibody binding properties similar to full-length HA. Antibodies produced in response to these mini-HAs managed to protect against a wide range of HAs in mice and nonhuman primates. Figure 1 outlines the various stages of Impagliazzo et al.’s design, culminating in the creation of a stable, headless "mini-HA" stem that retains all the relevant neutralizing binding sites. Figure 2 outlines the design stages of Yassine et al, who achieved great minimization of the HA protein, ultimately succeeding in having the stem domain contribute 94% of the total surface area of their antigen.

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Forming a Stable Trimer and Preventing Protein Aggregation Influenza hemagglutinin is naturally a trimeric molecule, meaning it is composed of three distinct hemagglutinin proteins bound together. In order to accurately mimic this three-part 839

structure, Subbiah et al. excluded the C-terminus of the HA stem portion and instead replaced it with the foldon sequence of a virus that infects bacteria. When added to the end of a monomer that has a propensity to dissociate, the foldon sequence stabilizes it and allows it to form a trimer (Figure 3). Failure to do so may lead the headless HA stem antigen to lose its structure, and by extension its epitope sites. If our antibodies cannot bind to the stem, they cannot learn about its structure and thus cannot take on the specific shape needed to protect us from future reinfection.

FIGURE 3. (A) A trimeric fiber loses its structure and breaks down into unfolded monomers, leading to irreversible protein aggregation. (B) The addition of the foldon sequence allows monomers to return to their trimeric structure, preventing loss of structure and subsequent protein aggregation. FROM: "FOLDON-GUIDED SELFASSEMBLY OF ULTRA-STABLE PROTEIN FIBERS" BHARDWAJ ET AL. 2009

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The researchers also added specific point mutations along HA1 head and HA2 stalk to prevent protein aggregation issues. All of the mutations were in regions outside of those recognized by broadly neutralizing antibodies. Each one of the mutations was in a long helical region with a tendency to self-aggregate. By changing the amino acids in these stretches, they substantially reduced nonspecific aggregation and instead favored the stable form of the protein. In addition to this, they mutated a cysteine residue and replaced it with a serine residue. Again strengthening the stability of the protein and minimizing the chance of any unwanted disulfide bonds from forming. Addition of M2e To broaden the neutralization of their antigen, and increase its potency, the researchers added in two identical segments of the M2 channel protein. The segments they chose correspond to a 23 amino acid-long sequence that encodes the epitopes for the extracellular region of the protein. This area protrudes from the viral membrane, allowing it to be bound by neutralizing antibodies. Crucially, M2e has previously been shown to confer cross-group immunity. Influenza A viruses are split up into two groups according to phylogenetic differences in their HA protein. Usually immunity against group 1 HAs (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18) does not confer immunity against group 2 HAs (H3, H4, H7, H10, H14, H15). This means that infection by an influenza A/(H3N2) virus, for example, generally does not confer protection against a strain of influenza A/(H1N1). Finally, Subbiah et al. connected the various domains of the M2e-H3 construct using flexible linkers, which are small amino acids that function like bungee cords; they keep the proteins tethered together but are flexible enough not to interfere with the folding process. The full genetic sequence of the M2e-H3 antigen can be seen in Figure 4, and the structure of the antigen —as compared to wild type hemagglutinin— can be seen in Figure 5.

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Production and Purification of the Antigen For ease of purification of the antigen, the researchers added a histidine tag (6xHis) to the N-terminal of the M2e-H3 stem domain. A histidine tag is made up of a DNA sequence that encodes six to nine histidine residues. These histidine residues bind to several different types of immobilized metal ions. So, once a protein has been “His-tagged” it can be easily identified and separated from 842

other proteins by exposure to such metal ions. The histidine tag can then be removed from the protein or it can be retained, either way it does not affect the antigenicity of the protein. Next, Subbiah et al. synthesized a codon-optimized gene encoding the M2e-H3 stem protein. This gene is then inserted into escherichia coli (E.coli) for quick and easy mass production. Does it Retain Antigenicity? After extraction and purification, Subbiah et al. began testing the M2e-H3 stalk molecule for its ability to be recognized by antibodies, known as antigenicity. They exposed the chimeric molecule to antibodies known to target the stem portion of the hemagglutinin protein as well as antibodies known to target M2e; in both cases they witnessed high reactivity. The scientists recorded similar results from antisera — blood samples from mice that had previously been infected with an influenza virus and had built up a corresponding antibody response. Importantly, the construct retained its antigenicity when exposed to influenza A viruses belonging to different HA groups. And, even after storage at 122 degrees Fahrenheit for 11 days, the antigen continued to elicit strong antibody reactivity, suggesting solid thermostability. Animal Models Having confirmed that the M2E-H3 stem protein can be recognized and bound by antibodies, Subbiah and colleagues moved on to vaccination trials. Often, vaccines based on viral proteins instead of inactivated or attenuated viruses require an adjuvant to help stimulate a stronger immune response, and by extension, better protective efficacy. For their experiments, the researchers used a particularly strong adjuvant similar to the AS01 liposome adjuvant licensed for use in herpes Zoster vaccination. This is a double-edged sword: the adjuvant ensures a quick immune response, but because of its strength, it also comes at the cost of side effects. Although the side effects are not much cause for concern —headache, fever, chills, and so on— they are uncomfortable all the same. Using a different delivery platform, like mRNA technology, may help circumvent these issues in the future. The adjuvanted M2e-H3 stalk vaccine was administered to mice as two doses, a primary vaccination followed by a boost two weeks later. Following the first dose, there was a noticeable increase in antibodies specific to M2e and HA stem regions. Addition of the 843

boost further increased antibody titers 10 fold. Antisera built up in response to M2e-H3 exposure managed to protect against both group 1 and group 2 influenza A viruses. Subbiah et al. suspect this might be explained by antibody-dependent cellular cytotoxicity (ADCC), a mechanism of immune defense whereby effector cells recognize antibodies stuck to antigens and actively break down the foreign threat. Their suspicions were proven right by western blotting, which showed that M2e antibodies, in particular, raised ADCC activity 20 fold. To compare protection against an M2e only vaccine, the group of researchers designed another chimera protein combining M2e fused with the receptor binding domain (RBD) of SARS-CoV-2 spike protein. When exposed to a lethal dose of influenza A/HK/H3N2 virus, all of the mice inoculated with M2e-H3 stem survived, suffering only moderate body weight loss of around 10%. On the other hand, none of the mice inoculated with the M2e-only vaccine survived the infection (Figure 6).

Along with a robust antibody response, M2e-H3 stalk vaccination also triggered a strong T cell response. Where antibodies can bind to pathogens to prevent infection, T cells are in charge of getting rid of already-infected cells, curbing the spread of the virus. Indeed, the vaccine significantly reduced the amount of virus in the lungs of infected mice within a span of six days. Depleting T cell levels in vaccinated mice before exposing them to a lethal challenge with influenza A H7N9 produced twice as much weight loss (12%) than in mice that had not been depleted of their T cells. Implications The work by Subbiah et al. acts as a clear proof of concept: using M2e and HA stalk regions as antigens can stimulate broad 844

neutralizing immunity. Crucially, this immunity should hold up to new strains, as it targets those viral regions that only very rarely mutate. Although their work focused specifically on influenza A viruses, there is no reason to think that, given enough time and research, we won’t be able to produce a truly universal influenza vaccine. The progress that has been made is in very large part due to the development of monoclonal antibodies that are broadly neutralizing against a wide array of influenzxa strains. These antibodies have provided a key guide to the design of vaccine antigens to help elicit immune responses with a similar breadth of neutralizing capabilities. This elegant work that has developed over the past ten years may prove to be a reliable framework for the development of similarly universal vaccines to some of our most vexing problems, including HIV and SARS-CoV-2. Such attempts are already underway for Covid-19, and proved to be the missing part of the puzzle for the design of effective vaccines against respiratory syncytial virus (RSV). This article is featured on Forbes.org, and can be read online here: Is A Universal Influenza Vaccine —One Shot For All Strains— On The Horizon?

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CDC Study Says Isolating In A Separate Room Is Most Effective Against Household Transmission Of SARS-CoV-2 Forbes | September 29, 2022 | Article

A new CDC study shows that isolating in separate rooms significantly reduced the odds of SARS-CoV-2 transmission between household members. Isolating in separate rooms proved to be the most effective mitigation measure against onward SARSCoV-2 virus transmission regardless of age, gender, vaccination status, and other risk factors. While vaccination was found to reduce the risk of infection, it did not appear to reduce transmission within the household. Both vaccinated (primary vaccination series) and unvaccinated adults and children with varying demographic factors were found to transmit the virus at similar rates. The study serves as a reminder that we should not ignore public health and nonmedical interventions such as masking and isolating, even as mandates are dropping and much of the world is experiencing fatigue. The study used data from 513 households with children under the age of 18 and 2,053 people. Data was collected from August 2020 to August 2021 in Utah, September 2020 to August 2021 in New York City, and November 2020 to October 2021 in Maryland. This means the study was conducted before the emergence of the Omicron variant and the availability of vaccines for most children in the United States. To assess how SARS-CoV-2 virus was transmitted in households, study participants self-collected nasal swabs weekly and with the onset of acute illness, which were sent to a laboratory for PCR testing. The study followed participants through the Alpha and Delta waves. Consistent PCR testing allowed the researchers to capture both asymptomatic and symptomatic infections. Participants also completed weekly questionnaires about symptoms experienced

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in the preceding week and, if symptomatic, what preventive measures they took, such as isolating or wearing a mask. Primary cases were defined as participants with the first symptom onset or positive SARS-CoV-2 test, whichever occurred earlier. However, as several household members were often infected at the same time, co-primary infections were excluded from the transmission analysis. The researchers found that 1 in 4 households experienced onward spread of SARS-CoV-2 among household contacts. In addition to isolating in separate rooms, household members should also remain in isolation until they test negative on a rapid antigen test, despite the current recommendation of 5 days. A recent study found that 62% of patients were still shedding infectious viruses by day 5, and 23% were still shedding infectious virus by day 7. Those taking Paxlovid should also be aware of the possibility of a rebound infection and return to isolation if necessary. By employing these mitigation strategies, we can ensure that we are minimizing the chances of spreading Covid-19 to our loved ones and co-habitants. This article is featured on Forbes.org, and can be read online here: CDC Study Says Isolating In A Separate Room Is Most Effective Against Household Transmission Of SARS-CoV-2

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Covid Virus Accelerates With Each New Variant Forbes | September 30, 2022 | Article

Far from subsiding, SARS-CoV-2 is rapidly mutating worldwide, in multiple directions, with the commonality that each new variant is more fit and spreads through a previously infected and vaccinated population more rapidly. Since late 2021, the Omicron variant has dominated SARSCoV-2 infections, fueling the massive wave of cases in January and onward. From the parental Omicron BA.2 lineage came BA.5, which currently accounts for most infections. Though while BA.5 was headlining SARS-CoV-2 infections, a cacophony of sibling variants was mutating and emerging behind the scenes, leading to a massive pool of variants being identified around the globe.

FIGURE 1: Emergence of new Omicron sublineages over time as new mutations emerge in the Spike ... [+]JUSTIN STEBBING

One of the most disturbing attributes of these later variants is increased growth rates as compared to earlier lineages. For instance, four Omicron sublineages, BA.2.75, BA.2.75.2, BQ.1, and BA.2.3.20, have a growth rate advantage of between three and 15 848

percent per day as compared to the predominant BA.5 lineage BA.5.2. For context, BA.5 is regarded as having a significant growth rate advantage over BA.2, which is the variant largely responsible for the most infectious period during the pandemic.

FIGURE 2: Growth rate advantage of four emerging Omicron variants of SARS-CoV2. JUSTIN STEBBING

These variants, as compared to BA.5, contain a few variable mutations in the spike protein that may account for increased viral fitness. The table below compares the additions and reversions in each strain as compared to their shared parent BA.2.

TABLE 1: Emerging mutations of new Omicron variants. Those in parentheses indicate reversions to the ... [+] ACCESS HEALTH INTERNATIONAL

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Increased viral fitness via advantaged growth rate can yield a slew of Covid-19 implications. For instance, one of the reasons the Omicron variant created so many new infections, over 800,000 confirmed cases per day in the United States at its peak and likely millions more, is because the variant was capable of infecting those with previous infections and those who had been previously vaccinated. The mRNA vaccines widely available in the United States never effectively prevented infections, to begin with, as they were more tailored to prevent moderate and severe disease outcomes if an infection occurs. This trend continued with the Omicron variants, but infections were far more common. Those fully vaccinated and boosted were much more likely to be shielded from severe disease and death, but not so much infection.

FIGURE 3: Deaths averted by widespread vaccine use.

JUSTIN STEBBING Emerging variants, such as the four described as more fit, can reinfect those previously infected with an earlier Omicron variant. Confirmed Covid cases have been on the decline in the United States for the past couple of months, though this may be attributed to fewer testing centers and reduced data reporting. The winter months throughout the pandemic have been a breeding ground for the virus as populations move indoors more 850

often as temperatures drop and indoor winter holidays are celebrated. While infections are unlikely to be averted this winter as Covid regulations are at their most relaxed since before the pandemic began, individuals can take measures to protect themselves to the greatest extent possible. Most notably, one can now receive an updated booster vaccination targeted toward the Omicron variant. While extensive testing and data on the viability of these vaccines to prevent infection are unavailable, it is very likely that the additional dose of the vaccine will strongly increase protection against severe disease and death. While many, including the President, suggest that the pandemic is concluding, those who regularly check the numbers would disagree. Be sure you and your loved ones are vaccinated as possible this upcoming winter as these new variants begin to rear their ugly heads. This article is featured on Forbes.org, and can be read online here: Covid Virus Accelerates With Each New Variant

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CDC Study Says Isolating In A Separate Room Is Most Effective Against Household Transmission Of SARS-CoV-2 Forbes | September 30, 2022 | Article

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Two Are Better Than One: Expanding Our Covid-19 Vaccine Antigens Forbes | October 03, 2022 | Article

New research published in Science Translational Medicine suggests an mRNA vaccine that targets both the SARS-CoV-2 spike protein (S) as well as the nucleocapsid protein (N) may offer stronger and broader protection than current, spike-only vaccines. This research opens the possibility that one vaccine may protect against current and future variants. Rationale for Inclusion of Nucleocapsid Protein One of the most difficult challenges of the Covid-19 pandemic is accounting for the rise of new variants. Successive waves of infection and hospitalization are driven by the emergence and spread of fitter strains. These quickly push out prior strains as they rise to dominance. The Omicron family of viruses has asserted itself as king of the hill in 2022. In large part, this is because the lineage has proven especially adept at evading vaccine induced immunity. And, because Omicron sublineages can be as different from one another as the Alpha variant was from Delta, prior infection with one Omicron virus does not necessarily mean you will be protected against any of its relatives. Current mRNA vaccines work by exposing our bodies to the SARS-CoV-2 spike protein, which the virus depends on to bind and eventually enter our cells. Antibodies that block the spike protein can block infection. The issue is that the spike protein is prone to mutation — its structure can change a lot without sacrificing functionality. By extension, vaccines based only on the spike protein risk losing efficacy when confronted with new variants. The nucleocapsid protein is a structural protein with an integral role in viral assembly and the packaging of genetic material (Figure 1). It is 90% conserved between SARS-CoV-1 and SARS-CoV-2, compared to 76% for the spike protein. These two features combined make it a very promising target for vaccine design.

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Added to this is the fact the nucleocapsid protein has been shown to elicit a powerful T cell response. Where B cells produce antibodies that can bind to viral particles before they enter cells, T cells are in charge of destroying host cells already infected with the virus; the stronger the T cell response, the better the body can contain the spread of the virus and clear the infection. Analyses of people infected with SARS-CoV-1 have indicated that N-specific T cell immunity can be very long lasting, with some individuals retaining memory T cells up to 17 years after initial infection. These same T cells managed to recognize the SARS-CoV-2 nucleocapsid protein, mounting a quick and specific immune response.

FIGURE 1. A schematic diagram of the SARS-CoV-2 viral particle, with its various proteins clearly marked. FROM: “SARS-COV-2 NUCLEOCAPSID PROTEIN IS DECORATED WITH MULTIPLE N- AND O-GLYCANS” SUPEKAR ET AL. 2020

Animal Models: mRNA-N Vaccine First up, Hajnik et al. tested their vaccine for its ability to produce an immune response, known as immunogenicity. For this, they separated mice into a control group, which was vaccinated with a saline solution, and a test group, which received the mRNA-N vaccine. The mRNA-N vaccine was administered intramuscularly in two doses, an initial prime and a boost three weeks later (Figure 2). Two weeks after the booster shot, the scientists euthanized the mice and analyzed their blood, with a focus on T and B cell responses. 856

FIGURE 2. Experimental design and timeline. Two groups of BALB/c mice (n = 7) were intramuscularly vaccinated with PBS (Mock) or mRNA-N vaccine (1 μg) at weeks 0 and 3. At week 3 before booster vaccination, blood and serum samples were collected for analysis of antibody response. Two weeks after booster vaccination (week 5), mice were euthanized and subjected to immune analysis.FROM: “DUAL SPIKE AND NUCLEOCAPSID MRNA VACCINATION CONFER PROTECTION AGAINST SARS-COV-2 OMICRON AND DELTA VARIANTS IN PRECLINICAL MODELS” HAJNIK ET AL. 2022

Compared to the mock group, mice vaccinated with the mRNA-N vaccine displayed strong CD4+ and CD8+ T cell responses. CD4+ T cells, also known as helper T cells, activate a number of other immune cells and help orchestrate the immune response. CD8+ T cells, or killer T cells, actively recognize and destroy infected host cells, slowing the spread of the infection. The N-specific T cells expressed three important signaling molecules: interferon-γ (IFN-γ), tumor necrosis factor–α (TNF-α), and interleukin-2 (IL-2). Interferon-γ is the primary activator of immune cells called macrophages, which engulf and destroy invading microbes. Tumor necrosis factor–α is a vital component of our inflammatory response, and can induce regulated cell death in infected or damaged tissues. And interleukin-2 helps stimulate the growth and proliferation of T and B cells. Hajnik et al. also witnessed a clear induction of nucleocapsidspecific immunoglobulin G (IgG) antibodies following vaccination. However, although these antibodies could bind the nucleocapsid protein, they did not have any neutralizing potency, meaning they cannot help prevent infection of our cells. Next, they tested their vaccine against live infection in mice and hamsters. They used the same timeline as they did for their immunological analysis: one prime dose followed by a booster three weeks later. They infected the mice with a mouse-adapted SARSCoV-2 strain two weeks after administration of the booster shot. They did the same for the hamsters, albeit with the Delta variant, since hamsters are susceptible to wild-type SARS-CoV-2 infection. 857

Two days post infection, the researchers looked at viral loads in the lungs of the mice and hamsters. Compared to the control group, mice and hamsters vaccinated with mRNA-N showed a reduction in viral RNA and in infectious viral titers. This reduction was statistically significant, but only modest. Curiously, no such reduction was seen when the vaccine was administered intranasally instead of intramuscularly. Intranasal administration also failed to induce any antibody response. To pinpoint the protective effect of the vaccine, Hajnik and colleagues depleted CD8+ T cells in one group of hamsters. They did this by administering antibodies that bind to CD8+ T cells, effectively blocking them and impairing their function. Depletion of CD8+ T cells almost entirely eliminated the protective effects of mRNA-N vaccination, with no noticeable reduction of viral loads in the lungs. This implicates N-specific CD8+ T cells as the key component of viral control. One important drawback is that there was no noticeable reduction of viral loads in the upper respiratory tract following mRNA-N vaccination. The scientists suggest this may be due to the vaccine’s inability to stimulate neutralizing antibodies. Animal Models: mRNA-N+S Vaccine Given their initial success with the nucleocapsid-based vaccine, Hajnik et al. created a bivalent mRNA vaccine that targets both the nucleocapsid protein as well as the spike protein. To test the efficacy of the combination mRNA-N+S vaccine, they compared it to an mRNA vaccine containing only the spike protein (mRNA-S) and to a mock vaccine based on saline solution. Again they used a mouse model as well as a hamster model. Both the mRNA-N+S vaccine and the mRNA-S vaccine managed to successfully control the infection, with almost no detectable infectious virus in the lungs. A more fine-grained analysis using reverse transcription polymerase chain reaction (RT-PCR) technology revealed that the combination vaccine outperformed the spike-only vaccine; the spike-only vaccine managed to reduce viral RNA copies in the lungs to barely detectable levels, but the combination vaccine was able to completely eradicate viral loads in the lungs (Figure 3).

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FIGURE 3. A comparison of viral RNA copies in the mouse lungs (log10 viral copies per milligram) between different groups at 2 DPI. FROM: HAJNIK ET AL. 2022

The vaccines fared just as well against the Delta virus in hamster models. But again, the mRNA-N+S vaccine outperformed the spike-only vaccine, and this time by a larger margin. Where the mRNA-S vaccine managed to reduce lung viral RNA copies 57fold compared to mock, the mRNA-S+N vaccine managed to do so by 770-fold. Still, both vaccines protected against lung damage, including bronchiolitis and interstitial pneumonia. Addition of the spike protein to the vaccine also helped improve its efficacy in the upper respiratory tract. As with the nucleocapsidonly vaccine, the spike-only vaccine was not that effective in clearing the virus from the nose and throat, with only a five-fold decrease in viral mRNA copies four days after initial infection. Compare this to the mRNA-N+S vaccine, which already provided an eleven-fold decrease two days after initial infection. This rose to a 98-fold decrease by four days post infection. So, the bivalent nucleocapsid/spike vaccine provides stronger and faster control of SARS-CoV-2 Delta in the lungs and upper respiratory tract compared to the spike-only vaccine. What about Omicron? 859

To test the breadth of the immune response elicited by the mRNA-N+S vaccine, Hajnik et al. also exposed hamsters to the Omicron variant (BA.1). They split the hamsters into four groups: one that received a mock vaccine, one that received a two microgram dose of spike-only vaccine, one that received a four microgram dose of spike-only vaccine, and finally one that received the mRNA-N+S vaccine, which included two micrograms of each protein. At two micrograms, the spike-only vaccine induced modest viral clearance from the lungs; a 12-fold reduction in viral RNA copies two days after infection. Little changed with the four microgram dose, which produced only modest viral control very similar to that of the smaller dose. The mRNA-N+S vaccine, in contrast, managed to completely clear viral RNA copies from the lungs by day two of the infection. The same held true for viral titers, with four out of five hamsters having no detectable levels of infectious virus. Results in the upper respiratory tract mirrored those seen with Delta: the spike-only vaccine only weakly reduced viral RNA copies compared to the mock vaccine. The combination vaccine again outperformed the others, managing to produce a 3-fold decrease in viral copies two days after initial infection. This suggests a combination vaccine may be better suited to reducing viral shedding and possibly onwards transmission of the virus. Implications This work by Hajnik et al. has laid the groundwork for the development of more broadly neutralizing Covid-19 vaccines, ones that can remain effective even in the face of continued viral variation. Research like this sets us on the path for the generation of a truly universal SARS-CoV-2 vaccine. Successes like these reemphasize the importance of continued funding and research on coronaviruses; the more we know about a virus, the easier it is to produce targeted and lasting interventions. This is true for vaccines and prophylactic drugs alike. This article is featured on Forbes.org, and can be read online here: Two Are Better Than One: Expanding Our Covid-19 Vaccine Antigens

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Saline Nasal Irrigation After Covid-19 Diagnosis Reduces Hospitalization Forbes | October 03, 2022 | Article

A new study from the Medical College of Georgia at Augusta University has found that irrigating your nose twice a day with a saline solution after testing positive for Covid-19 can decrease your chances of hospitalization and death in higher-risk patients. Participants in the study who performed nasal irrigation were more than eight times less likely to be hospitalized than the national rate. Only 1.3% of study participants who used nasal irrigation were hospitalized after testing positive for Covid-19. While 9.47% of Covid patients from a national CDC dataset who did not use nasal irrigation were hospitalized. Irrigating your sinuses is a simple practice anyone can implement at home using a neti pot, sinus rinse bottle, or syringe bulb available at a pharmacy or retailer like Target or Walmart. You can buy premade mixes or simply mix a half-teaspoon of salt and a half-teaspoon of baking soda in a cup of boiled and then cooled or distilled water. The water needs to be boiled or distilled to ensure it is sanitary. Many people often use these devices to clear up allergies, colds, or sinus infections. The study population was aged 55 and older and were enrolled within 24 hours of a postive PCR test between September 24 and December 21, 2020. Among 826 screened, 79 high-risk participants with a medium age of 64 and BMI of 30.1 were enrolled and randomly assigned to add 2.5 mL povidone-iodine 10% or 2.5 mL sodium bicarbonate to 240 mL of isotonic nasal irrigation twice daily for 14 days. To verify irrigation, patients uploaded pictures of used irrigation materials. A researcher called the patient or their designated contact on days 2, 7, 14, and 28 to verify irrigation and hospitalization. Nasal irrigation under pressure has been demonstrated to safely reduce the duration and severity of both Coronaviridae and illnesses like flu with shorter incubation periods. Sodium bicarbonate and 861

povidone-iodine were chosen due to prior research supporting the virucidal activity of povidone-iodine against MERS and SARSCoV-2 and the possible impact of alkalinization via sodium bicarbonate to reduce SARS-CoV-1 viral cell fusion and entry. By day 28, there was one emergency department visit and no hospitalizations in the group irrigating with sodium bicarbonate and one hospitalization in the povidone-iodine group. There were no deaths in either group. Eleven particpants reported irrigation-related complaints and four discontinued use. Symptom resolution was more likely for those reporting twice-daily irrigation. While prior research has found that almost half of those 50 and older had continued symptoms 14–21 days after diagnoses, only 13% of participants in the study had symptoms at day 28. The study has limitations in terms of generalizability and risk of bias in the comparison dataset. The CDC national data did not serve as a matched control group. However, the participants in this study were actually at greater risk for severe disease than the general population in the CDC dataset due to age and obesity. Prior studies have also have found that saline can help combat COVID-19. A 2020 study shows that gargling with a saline-based solution can reduce viral load. A 2021 study suggests saline can be used as a “first-line intervention for COVID-19. The results of these studies suggests twice daily nasal irrigation is a safe, over-the-counter measure that can be used reduce disease severity. This article is featured on Forbes.org, and can be read online here: Saline Nasal Irrigation After Covid-19 Diagnosis Reduces Hospitalization

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Reimagining Alzheimer’s (Part 2): Breaking The Barrier Forbes | October 03, 2022 | Article

In the first installment of this series about Alzheimer’s disease, I discussed a general overview of Alzheimer’s along with its most wellknown biological indicators. Here, I take a deeper dive into the disease by focusing on a recent paper that explores the primary genetic risk factor of Alzheimer’s. This risk factor is APOE4. Now, researchers believe that APOE4 may induce Alzheimer’s disease by damaging the brain’s main defense system—the blood-brain barrier. Mutations in the apolipoprotein E gene (APOE) are one of the strongest risk factors for late-onset Alzheimer’s. For many years, how these mutations in the APOE gene contributed to Alzheimer’s was not understood because the function of the gene wasn’t originally thought to involve the brain. Now, one particular mutation of the gene called APOE4 is known to be the biggest risk factor for Alzheimer’s and amyloid plaques, but it is still unclear how. In a recent paper, researchers at the University of Southern California describe how APOE4 may cause molecular changes to a structure in the brain called the blood-brain barrier, leading to cognitive decline in Alzheimer’s patients. This discovery is an exciting clue into the root cause of cognitive decline in patients with Alzheimer’s. The APOE gene is responsible for producing proteins that help carry cholesterol and other types of fat in the bloodstream. This means that APOE plays a large role in regulating the fats/lipids in our brain. Interestingly, fats and lipids help to maintain the brain’s primary defense system: the blood-brain barrier. The blood-brain barrier is composed of several types of cells that regulate which substances can reach the brain from the bloodstream.

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Figure 1: The blood-brain-barrier is composed of several types of cells that regulate which substances reach the brain from the bloodstreamOBERMEIER ET AL., NATURE MEDICINE (2013), DOI: 10.1038/NM.3407...

Mutations in the APOE gene have previously been linked to damage in the blood-brain barrier as well as the development of beta-amyloid plaques. However, up until now, there has been no large-scale molecular analysis to study exactly how APOE4 affects this crucial barrier and how it may cause cognitive decline. So how did Barisano et al. approach this question? To explore the effects of APOE4 in the blood-brain barrier, Barisano et al. induced the APOE4 gene in mice and compared them to control mice with the typical APOE3 version of the gene. First, the researchers were interested in determining how APOE4 might affect the expression of other genes in the brain. After running a full genetic analysis of the mouse brains, Barisono et al. found that of over 200 genes that were different between the mice, the majority had an increase in expression. As the mice aged, those same genes continued to be expressed in overabundance. An additional large set of genes were also overexpressed as the animals aged. What are these genes? When the overexpressed genes were analyzed, they were surprised to find that a number of these genes had previously been identified as being critical for maintaining the function of the bloodbrain barrier. This included several different categories of genes that 864

encoded proteins for ion channels, proteins that support the structure of the blood-brain barrier, and molecules that move nutrients and other compounds across the blood-brain barrier. This study focuses on two types of cells in particular: endothelial cells which line the blood vessels and pericytes which play an important support function for the vasculature system throughout the body. Barisono et al. found that proteins responsible for the structure of endothelial cells were especially impacted by APOE4. The proteins affected by APOE4 in pericyte cells were those that regulate gene expression. Since both endothelial cells and pericytes are crucial to the function of the blood-brain barrier, these results further confirmed that APOE4 likely leads to blood-brain barrier dysfunction and breakdown. The next question that Barisono et al. sought to tackle was: how could APOE4 decrease cognitive ability in Alzheimer’s patients? Researchers suspected that changes in the blood-brain barrier may actually affect the network of connections between brain cells/neurons. These connections between brain cells are called synapses. One of the ways to measure cognitive ability is to look at the synapses of the brain and analyze whether or not they are functioning properly. In particular, the researchers focused on one protein called postsynaptic density protein 95 (PSD95). PSD95 plays a large role in the synapse by supporting the structure and positioning of many receptors, membrane channels, and signaling proteins that are involved in communication between neurons.

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Figure 2: Researchers were interested in exploring the effect of APOE4 on neural synapses—the connections between neurons in the brain.UNIVERSITY OF MARYLAND, LINK: HTTPS://LIFESCIENCES.UMARYLAND.EDU/NEUROSCIENCE/RESE ARCH-FOCUS-GROUPS/SYNAPSES—CIRCUITS/...

One of the most remarkable observations of the study was how the APOE4 gene affected mice as they aged. In younger mice, there was very little difference in cognitive abilities between the APOE4 and control mice. As the mice aged, the cognitive abilities of the APOE4 mutated mice became significantly worse. Middle-aged control mice did not display the same cognitive decline as the mutated mice. In line with this observation, Barisono et al. also found that the PSD95 protein also displayed abnormal function in middle-aged APOE4 mice, but not in control mice. These results suggest that APOE4 mice develop decreased brain connectivity after blood-brain barrier breakdown, leading to significant cognitive deficits. For the past several decades, people have focused on tau and beta-amyloid as the major points of intervention for Alzheimer’s. This study offers the possibility that alterations in the blood-brain barrier may precede observed changes in amyloid plaques and tau. If this is the case, this study warrants a much deeper investigation into how the blood-brain barrier is disrupted and how it may lead into perceived changes in the brain and cognition.

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Hopefully, as our molecular understanding of Alzheimer’s disease continues to grow, we will be able to develop treatments that can prevent the onset of Alzheimer’s or decrease its detrimental cognitive effects. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 2): Breaking The Barrier

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Broadly Neutralizing Monoclonal Antibodies For Covid-19 Treatment, Prevention, And Vaccine Design Forbes | October 05, 2022 | Article

Recent progress on monoclonal antibodies raises the possibility that the means to prevent and treat SARS-CoV-2 infections may soon be at hand. The hope arises from discoveries of antibodies that have the potential to neutralize all known SARS-CoV-2 variants and other related coronaviruses, including SARS-1 and MERS. A strategy for Covid control is possible using combinations of these antibodies for the treatment and pre-and post-exposure prophylaxis. Such treatments may eventually be combined with highly active antiviral drugs to end the pandemic. There is an acute need for drugs to treat and prevent SARSCoV-2 infection regardless of variants. Current vaccines dramatically reduce hospitalization and death from multiple variants. However, protection from infection and transmission diminishes with time and as the virus mutates. Recurrent viral variants are common despite multiple vaccine boosts and prior infection. The infection of hundreds of millions of people dramatically increases the risk that even more transmissible and virulent variants may arise, not to mention the ever-increasing burden of Long Covid. The possibility of increased virulence is real. Recall that SARS-1 and MERS kill 10% and 30% of those infected, respectively. The recently discovered broadly neutralizing monoclonal antibodies provide near-term hope effective for variant-independent prevention and treatment as we await the discovery of highly active antiviral drugs. Monoclonal Antibodies that Neutralize SARS-CoV-2 Monoclonal antibodies are one of the most powerful tools for treating viruses and other infectious diseases. They target specific surface structures and either eliminate the virus from the host bloodstream or destroy the infected cell in which it resides. An advantage and disadvantage of monoclonal antibodies reside in their 868

specificity. Monoclonal antibodies target specific structures on the surfaces structures leading to their destruction and clearance. Mutations that alter the structure of the binding site render monoclonal antibodies useless. Pharmaceutical and biotechnology companies have developed monoclonal antibodies that neutralize SARS-CoV-2. These antibodies have been directed at the Spike protein on the virus's exterior. This is a favored target, as studies show that greater than ninety percent of naturally-occurring antibodies that neutralize SARS-CoV-2 are directed against the Spike protein. The targets in the Spike protein are the primary receptor-binding domain (RBD) and the secondary N-terminal domain (NTD). The Spike is an intertwined trimer of three S proteins. Each of the three is composed of two subunits: S1, the membrane distal region, including the receptor-binding and N-terminal domains, and S2, the membrane-proximal protein. The receptor-binding domain atop S1 can assume two configurations: up, capable of binding the ACE 2 receptor, or down, non-binding (Figure 1).

FIGURE 1: Schematic of 2019-nCoV S primary structure colored by domain. Domains that were excluded ... [+] WRAPP ET AL.

The original monoclonal antibodies approved for clinical use were met with great success, potentially neutralizing the virus both singularly or in combination. Unfortunately, the utility of these antibodies waned quickly. The virus has mutated significantly over 869

the last two and a half years, leading to evasion of the immune response to infection. These mutations also abrogate neutralization by many of the FDA-approved monoclonal antibodies. This seems to be a never-ending game of catch-up. First, the virus changes, then new antibodies are created to recognize the new variants. Next, variants mutate to evade natural and monoclonal antibody immunity. This endless cycle resulted in the rapid deterioration of antibody potency against themes' recent variants (Table 1). Note that even the most broadly neutralizing antibody available today, bebtelovimab, has reduced activity against the BA.2.75 variant currently circulating in Asia and Europe.

TABLE 1: Neutralization by available antibody treatments against the latest Omicron variants as ... [+] ACCESS HEALTH INTERNATIONAL

Discovery of a Set of Broadly Neutralizing Antibodies To circumvent this cycle, researchers worldwide sought and found monoclonal antibodies that recognize highly conserved regions of the virus to neutralize most, if not all, variants. Some also neutralize SARS-1, MERS, and related Bat betacoronaviruses, and some even neutralize human betacoronaviruses that use the ACE2 protein as the receptor (Table 2). These antibodies have multiple origins, from convalescent and vaccinated volunteers to mice, alpacas, and macaques.

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TABLE 2: Neutralization of early SARS-CoV-2 variants, Omicron family variants, other ... [+]

ACCESS HEALTH INTERNATIONAL Despite differing binding sites on the Spike protein, the broadly neutralizing antibodies share one common property. Most amino acid contacts between the antibody and the spike are highly conserved, not only amongst all SARS-CoV-2 variants but also with the closely-related human and bat coronaviruses. Specifically, mutations in the amino acid binding sites are exceedingly rare in the over 12 million sequences deposited in international databanks. These amino acids likely perform a critical function. For example, the monoclonal antibody COV44-62/79 targets highly conserved the fusion peptide. The 35B5 antibody binds a crucial region that serves as a hinge for the up-down configuration father receptor binding domain. Wang et al. report that binding to the region dissociates the trimer. The Camelid antibodies lock the receptor binding domain in the down, inactive configuration. The Li antibody binds the very highly conserved linear epitope in the S2 region near the virus membrane Table 3 details seven antibodies' amino acid binding sites.

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TABLE 3: Broadly neutralizing antibodies and their amino acid epitope footprints.

ACCESS HEALTH INTERNATIONAL At the time of writing, there is another preliminary antibody candidate by Cao et al., SA55+SA58, which shows strong Omicron neutralization. This set binds the receptor-binding domain in a similar configuration to the Fenwick antibodies P2G3/P5C3. We will do additional in-depth analysis of this new set at a later time. We have reached out to the researchers behind each antibody to discern their current status. While none of those described here are clinically approved or are currently undergoing human trials, many of the authors indicated that after further preliminary data collection, they anticipate clinical trials in the coming months. New Monoclonal Triads for Treatment and Prevention The protean nature of SARS-CoV-2 to sustain viable mutations in the Spike protein merits caution. A strategy of combining three of the broadly neutralizing antibodies rewires an escape variant that alters three highly conserved bindings sites simultaneously, an 872

unlikely possibility. Examples of sets of such antibodies are shown in figure 3.

ACCESS HEALTH INTERNATIONAL

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FIGURE 3: (A) Binding epitopes of the bebtelovimab, Li, and Dacon/Low antibodies. Bebtelovimab in [+] ACCESS HEALTH INTERNATIONAL

We note there are a number of mutations common to binding sites in bebtelovimab. Specifically, key residues such as N440, Q498, and N501 are commonly mutated in the latest Omicron family variants. In the future, more mutations in these residues may occur in emerging variants, potentially rendering bebtelovimab less effective. Furthermore, cryptic sewershed variants described by Marc Johnson and colleagues display similar mutations throughout the receptor-binding domain. Were these variants to gain traction, bebtelovimab’s neutralization capacity would likely be severely hampered due to mutations in key binding sites. Treatment and Prevention Figure 4 illustrates these three uses for Covid-19. For treatment, monoclonal antibodies must be used in the first five or six days of infection, as the virus concentration decreases sharply in most people after five days. Post-exposure prophylaxis is the prevention of infection and disease in people known to be exposed to the virus. The duration of 874

such treatment with antiviral drugs and monoclonal antibodies should be no longer than ten days, the maximum incubation period for signs and symptoms of Covid-19. The third use, pre-exposure prophylaxis, is the use of long-acting monoclonal antibodies and highly-active antiviral drugs for those under conditions of high risk of infection. This may include people living in close quarters, for example, military bases, ships, sea cruisers, schools and hospitals, and nursing homes. High-risk situations may also pertain to entire communities where the infection rate exceeds five percent of the local population.

FIGURE 4: Covid-19 infection/symptom duration after monoclonal antibody treatment.

ACCESS HEALTH INTERNATIONAL Delivery At present, most monoclonal antibodies require intravenous infusion. However, some monoclonal antibodies, such as Evusheld, are administered via an intramuscular injection. Engineering the proposed combination antibody to be administered by intramuscular or subcutaneous injection would significantly increase widespread public access and acceptance of the drug. Caveats One caveat is that each of the broadly neutralizing antibodies described (and there will be more) was developed by different independent labs and may be licensed to diverse biotechnology and pharmaceutical companies. Development of the ideal antibody cocktails may require that the pharmaceutical industry and the NIH 875

work in tandem. Solutions to such issues have been successfully addressed to develop effective combination therapies for cancer and HIV using the global National Institutes of Health office network. A second caveat is potency. Some of the antibodies are active in the IC50 in the low nanogram range. Others are significantly less potent. All the antibodies described here may require additional engineering for potency, extended half-life, and antibody-dependent cellular cytotoxicity and phagocytosis—all possible with extant technology. The cost to manufacture is another caveat. At present, the price of monoclonal antibody treatments is loosely tied to manufacturing costs. Antibodies can be produced at a cost of about $250 a gram. At this price, the manufacturing cost of a potent triad dose might well be under $100. Summary Combinations of broadly neutralizing monoclonal antibodies administered in tandem with long-awaited highly effective small molecule antiviral drugs are our best chance to deliver the final knockout blow to Covid-19 by preventing infection, transmission, and disease. This article is featured on Forbes.org, and can be read online here: Broadly Neutralizing Monoclonal Antibodies For Covid-19 Treatment, Prevention, And Vaccine Design

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Supercharging mRNA Vaccines With SelfAmplifying RNA Technology Forbes | October 06, 2022 | Article

This article is part 2 of a series on next-generation mRNA vaccines. The first installment can be read here. The unprecedented speed with which we developed vaccines to help us control and curb the Covid-19 pandemic is in large part thanks to years of research on messenger RNA (mRNA) technology. As impressive as this effort has been, and as effective as the vaccines are, it by no means marks the pinnacle of mRNA technology; on the contrary, this initial success has emboldened further innovation, and the next-generation of mRNA vaccines is beginning to take shape. Self-amplifying mRNA (saRNA) represents one such advancement. What are self-amplifying mRNA vaccines and how do they compare to conventional mRNA vaccines? What are the benefits of self-amplifying mRNA? Messenger RNA: A Blueprint for Protein Synthesis All of the cells in the human body contain proteins. These are the building blocks of life, involved in almost all of our body’s vital processes. Messenger RNA (mRNA), in turn, is crucial to the synthesis of proteins. Our cells need instructions to build proteins, and mRNA provides the cell the information it needs to make proteins — one mRNA for each protein. The primary function of a viral vaccine is to expose our immune system to a virus, and its various proteins, in a controlled way, allowing us to build up immunity without causing actual infection. Should we re-encounter the virus down the line, our body is already primed to fight off the infection. But there are many different ways of exposing our immune system to viral proteins. Live-attenuated vaccines, for example, do so by introducing a live but weakened form of the virus into our body. Inactivated vaccines, like the yearly flu shot, rely on a killed version of the virus — it doesn’t cause infection, but all of the structural elements remain intact, giving our immune system a 877

chance to learn what it needs to effectively combat the virus. Subunit vaccines, in turn, introduce only fragments of the viral particle, usually some part of the surface protein. Nucleic acid vaccines, which is the umbrella under which mRNA vaccines sit, rely on a different strategy: delivery of genetic material that encodes the target viral protein (Figure 1). Essentially, mRNA is used to instruct our cells how to produce the desired viral protein — or even just a small section of the protein. In the case of Covid-19, vaccines include mRNA that teaches our cells to make the SARS-CoV-2 spike protein, which the virus uses to bind to our cells. Our immune system then recognizes these proteins as foreign entities and quickly gets to work, as it would against inactivated viruses or the wild-type virus itself. The use of messenger RNA as a vaccine allows for quick manufacturing and rapid upscaling; scientists do not need to spend long periods of time growing full viruses, nor do they even need to spend time producing specific proteins. Instead, they can just chemically synthesize the mRNA that encodes the desired protein, and once injected, our own cells do all of the manufacturing for us by translating the mRNA and producing the corresponding antigen.

FIGURE 1. A COMPARISON OF VACCINE PLATFORMS INCLUDING VACCINES DERIVED FROM THE VIRUS ITSELF AND ARE FORMULATED AS A PART OR WHOLE MODIFIED VERSION OF THE VIRUS (LEFT) AND NUCLEIC ACID VACCINES, SUCH AS MRNA VACCINES (RIGHT). FROM: “AN UPDATE ON SELF-AMPLIFYING MRNA VACCINE DEVELOPMENT” BLAKNEY ET AL. 2021

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For a long time, mRNA vaccine efficacy was hindered by serious limitations: mRNA is naturally fragile and prone to instabnility, and any misplaced mRNA—including extracellular mRNA from vaccines—is easily recognized by the body and subsequently broken down by enzymes. This is where Katalin Karió and Dre Weismann’s groundbreaking research in the early 2000s comes into play. The two University of Pennsylvania scientists discovered that by substituting uridine with pseudouridine, a nucleoside naturally found in RNA, lowers mRNA’s tendency to trigger our immune system while simultaneously improving its stability and translational capacity. But even when vaccine-derived mRNA manages to avoid detection, it faces another issue: on its own, the mRNA struggles to enter cells. To overcome this, most current mRNA vaccines make use of a protective “bubble” called a lipid nanoparticle. This bubble covers the mRNA, preventing unwanted detection, and helps ferry it into host cells. The combination of these two breakthroughs forms the basis for today’s successful mRNA vaccines. But there continues to be clear room for improvement. Synthesized mRNA still only stays in our cells for one or two days, at most, before being broken down. The antigen proteins it produces during this period stay around only slightly longer. This means our immune system has a short window of opportunity to educate itself on the nuances of the viral protein at hand — the corresponding immune memory we build up is not as strong as it could be, given more time. What is Self-Amplifying mRNA Technology? For the most part, self-amplifying mRNA is very similar in structure to conventional, synthesized mRNA. Both are made up of a genetic sequence that encodes the desired viral antigen. This includes: the two ends of the mRNA called the five- and threeprime untranslated regions (5’ UTR, 3’ UTR) and, a poly(A) “tail” at the end of the 3’ UTR, which stabilizes the structure, and a fiveprime “cap”, which lends the sequence additional structural stability. Self-amplifying RNA vaccines also encode four extra nonstructural proteins derived from an alphavirus that drive self-replication.(Figure 2).

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FIGURE 2. COMPARISON OF CONVENTIONAL (A) AND SELFAMPLIFYING (B) MRNA SEQUENCES USED FOR VACCINE PRODUCTION. NOTICE THE ADDITION OF FOUR ALPHAVIRUS NONSTRUCTURAL PROTEINS IN SELF-AMPLIFYING MRNA, WHICH COME TOGETHER TO FORM AN RNA REPLICASE. FROM: BANKLEY ET AL. 2021…

When an alphavirus infects a host cell, the first section of its mRNA encodes and synthesizes these four nonstructural proteins, which come together to form what is called an RNA-dependent RNA polymerase, or RNA replicase. Think of the RNA replicase like a transportable photocopier, except instead of copying paper it copies viral RNA. As soon as it is finished forming, the replicase begins printing out copies of the viral mRNA, including the downstream, structural proteins — those proteins that actually make up the viral particle. As a result, what was originally one single strand of viral mRNA is continuously multiplied into many copies. These can then all go on to be translated and synthesized into proteins by the host ribosome. Self-amplifying mRNA technology takes advantage of this transportable mRNA photocopier by swapping out the alphavirus structural proteins with the desired vaccine antigen. The same massproduction of mRNA occurs, but this time with mRNA that encodes the vaccine antigen (Figure 3). The result? More copies of mRNA and therefore more copies of the target antigen, expressed for a longer time.

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FIGURE 3. COMPARISON OF INTRACELLULAR DYNAMICS OF SELF-AMPLIFYING MRNA VACCINES (LEFT) AND CONVENTIONAL MRNA VACCINES (RIGHT). FROM: “THE NEXT GENERATION OF RNA VACCINES: SELF-AMPLIFYING RNA” ANA BLAKNEY 2021

Advantages of Self-Amplifying mRNA Vaccines Like mRNA vaccines, the manufacture of self-amplifying mRNA is entirely synthetic. Manufacturing does not require growth in any living cell. It is entirely a chemical process. Production is rapid and flexible and can be adjusted rapidly to respond to new demands. The manufacturing infrastructure and quality control are greatly simplified as compared to traditional vaccines and are affordable for low-income countries. The self-amplifying mRNA vaccines do not require chemically modified nucleotides —the substitution of uridine with pseudouridine— as once the RNA enters a cell it begins to continually produce new copies of messenger RNA to replace those degraded. Hence, the raw materials are unrestricted and widely available. This also means that much less mRNA can be used per vaccine dose, allowing for even quicker and cheaper production. Compared to the Pfizer/BioNTech and the Moderna mRNA vaccines, which use 30 and 100 micrograms, respectively, a recently tested selfamplifying RNA vaccine used only 0.1-10 micrograms — this means the same batch of RNA could produce between 10 and a 881

thousand times as many doses of saRNA vaccines as conventional mRNA vaccines. The requirement for lower doses offers the opportunity to create a variety of different antigens in one vaccine. A single dose may include several variants of the same virus or of different viruses and even bacterial antigens. Self-amplifying mRNA vaccines may not require liquid nanoparticle formulations. In subsequent installments of this series, we will describe two new vaccines, one composed of naked, unmodified RNA and another in which the RNA is located outside, not within, the lipid nanoparticle. The last and possibly most important difference is that selfamplifying mRNA vaccines produce the antigen for 20 to 26 days compared to the relatively short period of 2 to 3 days, as is typical of conventional mRNA vaccines. The length of exposure to an antigen can make a qualitative difference. Longer exposure to an antigen allows the antibody response to mature, to make higher affinity more potently neutralizing antibodies, as well as antibodies that may elicit cellular cytotoxicity as well. Moreover, longer antigen exposure is important for generating memory T cells required for long-lasting memory. As a consequence, self-amplifying mRNA vaccines offer the promise of enduring protective immunity from infection and disease. The installments of this series will describe the use of selfamplifying RNA for the design of Covid-19 vaccines. This article is featured on Forbes.org, and can be read online here: Supercharging mRNA Vaccines With Self-Amplifying RNA Technology

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Self-Amplifying mRNA Vaccine Receives EUA Nod From Indian Regulators Forbes | October 14, 2022 | Article

This is the third article in a series on next-generation mRNA vaccines. The first two installments can be read here and here, respectively. Messenger RNA (mRNA) technology has been central to the global response to Covid-19, enabling rapid manufacture of effective vaccines. But the virus continues to evolve, learning to outmaneuver prior immunity, whether from infection or from vaccination. To keep up, researchers and manufacturers alike have been developing the next generation of mRNA vaccines. Here, we take a look at one such example: India’s first home-grown mRNA vaccine, Gemcovac-19, manufactured by Gennova Biopharmaceuticals. The Office of the Drugs Controller General of India (DCGI) has granted the vaccine emergency use authorization (EUA), and an Omicron-specific booster shot is already in the works. Self-amplifying RNA Technology The first next-generation feature of Gennova’s vaccine comes in the form of self-amplifying RNA technology. Where traditional Covid-19 mRNA vaccines are based only on the genetic sequence that encodes the spike protein of SARS-CoV-2, Gemcovac-19 includes a sequence of alphavirus RNA that encodes four nonstructural proteins. Once inside a host cell, these non-structural proteins come together to form a molecule called an RNA replicase — a transportable “photocopier” that prints out multiple copies of the mRNA sequence (Figure 1). This means each mRNA sequence included in the vaccine is able to make copies of itself, increasing the amount of antigen produced. It also extends the duration of mRNA translation over a longer period of time; usually the vaccine-derived mRNA is degraded after a day or two, limiting protein expression to two to three days at the most. However, self-amplifying RNA technology can extend this timeframe —both mRNA and protein production— to up to almost a month. 883

FIGURE 1. A) Conventional mRNAs encode the vaccine immunogen and flanking 5′ and 3′ UTRs. An antigen or immunotherapy is translated from the nonreplicating transcript. B) Self-amplifying RNA encodes 5′ and 3′ CSE sequences, the nsP1-4 genes, a subgenFROM: “SELF-AMPLIFYING RNA VACCINES FOR INFECTIOUS DISEASES” BLOOM ET AL. 2020…

Self-amplifying RNA technology comes with a number of benefits. For one, because each mRNA sequence will produce multiple copies of itself, manufacturers can use a much smaller amount per vaccine dose. The smaller dose enables multiple different strands of mRNA —each encoding its own antigen— to be included in one vaccine. The dose-sparing approach also significantly reduces the cost of production; the price of the vaccine drops accordingly, making it more accessible to low- and middle-income nations that may not be able to afford the conventional Pfizer/BioNTech or Moderna counterparts. The second major advantage is on the immunogenicity front: since self-amplifying mRNA allows more of the target antigen to be produced, over a longer period of time, it gives our immune system a better chance to learn what it needs to develop a highly targeted immune response. Longer exposure to an antigen allows the antibody response to mature, to make higher-affinity, more-potently neutralizing antibodies, as well as antibodies that may elicit cellular cytotoxicity. Moreover, longer antigen exposure is important for generating memory T cells required for long-lasting memory. Finally, the inclusion of alphavirus RNA, which mimics viral replication, may act as a natural adjuvant by stimulating pattern recognition receptors. Lyophilization: Making Storage and Transportation Easier A drawback of first generation mRNA vaccines is that they need to be stored at extremely low temperatures. Pfizer/BioNTech’s Covid-19 vaccine, for example, has to be kept at minus 103 degrees Fahrenheit (around minus 75 degrees Celsius); in a conventional 884

refrigerator, the vaccine degrades within five days, and at room temperature, this drops down to a mere six hours. This poses logistical challenges when it comes to transportation and storage of the vaccine, especially in areas where the necessary “ultra coldchain” infrastructure —the network of refrigerators, freezers, cold boxes, and cold rooms used to keep the vaccines at the right temperature— is lacking. Gennova’s vaccine mitigates this issue through lyophilization, or freeze-drying. This is a process by which solvent is gradually removed from the vaccine solution (Figure 2). Once the solution has been frozen, which is the first part of the process, it is then put in a low-pressure chamber to get it to “jump” from a solid state directly into a gaseous state. This jump removes roughly 95% of the solvent, leaving behind a chalky powder. The remaining liquid is removed in a second drying cycle.

FIGURE 2. A schematic diagram of the three-part lyophilization process.

SOURCE: CYTIVA LIFE SCIENCES… The vaccine powder that remains after freeze-drying is significantly more temperature stable than the liquid solution. Compared to the sub-zero temperatures required for first-generation mRNA vaccines, the Gemcovac-19 vaccine can be stored between 35 and 46 degrees Fahrenheit (two and eight degrees Celsius) 885

without any issue. Instead of special freezers, it can just be kept in a normal refrigerator. Even without any refrigeration, the lyophilized vaccine can maintain its efficacy for an extended period. This provides countries that may not have the infrastructure in place to accommodate the ultra cold storage requirements of conventional mRNA vaccines with a more stable, equally effective counterpart. Lyophilization also reduces weight and volume, cutting down on shipping costs and again making it more accessible to lowand middle-income countries. Intradermal Delivery Boosts Immunogenicity and Improves Uptake One additional feature that sets Gennova’s vaccine apart from the older, first-generation Covid-19 mRNA vaccines is the fact that it is administered intradermally (Figure 3). Our skin is made up of three layers: the outermost protective layer called the epidermis, a middle layer called the dermis, and a fatty layer at the bottom called the hypodermis or subcutaneous tissue. Most Covid-19 vaccines are injected into muscle tissue, which lies well beneath the skin. This has been the standard approach for a long time. But intradermal delivery holds a few distinct advantages.

FIGURE 3. Comparison of different injection angles.

SOURCE: TECHNOFLEX… The dermis layer of skin is tightly packed with immune cells, including antigen-presenting cells (APCs). These play a vital role in the initiation and modulation of our immune system, including our adaptive immune response. In particular, various different types of dendritic cell latch onto antigens and present these to naïve T cells, 886

helping to shape and mature a targeted cellular immune response. Since intradermal injection lets the antigen be produced in close proximity to these antigen presenting cells, it may lead to a quicker and more fine-tuned adaptive immune response. A recent study comparing intradermal and intramuscular administration of the SARS-CoV-2 receptor binding domain (RBD) confirmed as much, describing improved T cell responses after injection into the dermis. Intradermal injection also promises to cut down on costs and boost production, since a smaller dose triggers the same immunogenicity as a larger dose administered via traditional, intramuscular injection. Finally, intradermal administration comes with the added benefit of needle-free delivery options, such as jet injectors (Figure 4). These use springs or compressed gas to produce a narrow, high-pressure stream of fluid that penetrates the skin and delivers the vaccine. The lack of a needle helps improve uptake, especially among younger and needle-averse populations. The World Health Organization (WHO) has sponsored the introduction of needle-free delivery systems for use with inactivated polio vaccines in Somalia and Pakistan. The needle-free vaccination campaign in Pakistan saw uptake increase by 18.4% compared to the previous year. Both caregivers and vaccinators overwhelmingly preferred needle-free delivery compared to needle and syringe, with 100% of surveyed caregivers admitting they would be more likely to bring their children back for a future vaccination campaign that used needlefree injectors. Needle-free delivery also nullifies the possibility of needle reuse and subsequent cross contamination, which continues to be a commonplace issue in many parts of the globe, with up to 1.3 million deaths a year attributed to such practices. Needle-free delivery also precludes accidental needlestick injuries, which again can lead to cross contamination and infection. Needlestick injuries are also prevented by bypassing the need for sharps disposal, which is often a complex and expensive process prone to errors and mishandling.

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FIGURE 4. A schematic diagram of a PharmaJet Tropis needle-free injector.

SOURCE: PHARMAJET... Implications Gennova Biopharmaceuticals’ new Covid-19 vaccine, Gemcovac-19, represents a step towards next-generation mRNA vaccines. The use of self-amplifying RNA technology allows for cost-effective vaccine dosing and gives our immune system more time to develop high-affinity antibodies and mature T cells. The fact it is freeze-dried makes it easy to transport and store, including in areas that may not have ready access to cold-chain infrastructure. And needle-free intradermal delivery enables quick and safe administration, which may help to improve uptake, improve cellular immunity, and reduce the risk of cross contamination. This article is featured on Forbes.org, and can be read online here: SelfAmplifying mRNA Vaccine Receives EUA Nod From Indian Regulators

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Progress In The Search To Reverse AgeRelated Vision Loss Forbes | October 15, 2022 | Article

Thanks to new techniques in regenerative medicine we are now closer to a future where your own cells can be used to restore the sense of vision. A recent paper from Ripolles-Garcia et al. at the University of Pennsylvania School of Veterinary Sciences describes a novel approach to both the creation and surgical implantation of such cells directly into affected areas of the retina. Macular degeneration is a leading cause of blindness. Over 15 million Americans over the age of 50 experience some form of agerelated macular degeneration, a genetically linked disease characterized by the loss of vision. The most common form, dry macular degeneration, develops as a consequence of widespread damage to photoreceptor cells within the central region of the retina, called the macula. As the site with the greatest concentration of these light sensing cells, degeneration of photoreceptors in the macula gradually alters central vision, and in rare cases, can lead to complete blindness. Remarkably, even as photoreceptor cells degenerate with age, studies show that the retina’s internal structure remains intact. More than 270 genes have been linked to macular degeneration and other inherited retinal diseases. Additional environmental factors, such as diet and smoking, can also increase your risk of developing macular degeneration with age. Despite the prevalence of age-related macular degeneration, however, there are no readily available treatments to restore vision. The team from the UPenn School of Veterinary Science and corresponding institutions has made considerable progress in the search to cure macular degeneration and other causes of vision loss. Ripolles-Garcia et al. demonstrates that retinal precursor cells derived from tiny samples of human pluripotent stem cells can effectively replace damaged photoreceptor cells. Further optimizing this technique to ensure integration with the rest of the retina could restore the sense of vision in millions of people. 889

Stem Cells for Regenerative Retinal Therapy New techniques in regenerative medicine have made it possible to take small samples of your own cells and revert them back into stem cells capable of regenerating into almost any type of cell in the body. For retinal therapies, this means first creating pluripotent stem cells (iPS cells) then applying cell specific treatments that gently nudge them into becoming retinal precursor cells. Ripolles-Garcia et al., in their experiment, injected these precursor cells directly into the retina with the goal of replacing damaged photoreceptors. There are, however, major challenges to using stem cells for regenerative retinal therapy. First, to make up for the widespread prevalence of damaged receptors, large amounts of retinal precursor cells need to be delivered directly into the subretinal space without harming other structures in the eye. Second, ensuring the survival of these cells is dependent on avoiding activation of the innate immune system. Finally, the cells must be able to form connections with the rest of the retina to recover the sense of vision. This study uncovered an innovative approach for not only transplanting stem cell-derived photoreceptor cells into the retina but also avoiding immune rejection. Ripolles-Garcia et al. recruited dogs with retinal mutations similar to those associated with human retinal diseases and treated them with retinal precursor cells derived from human pluripotent cells. This was then followed by a daily regimen of immunosuppressive drugs. These cells were remarkably able to make meaningful connections with the rest of the eye, leaving one question yet to be answered, “can retinal transplants actually restore the sense of sight?” Delivery The first challenge this study had to overcome was how to safely deliver a sizable amount of photoreceptor precursor cells into the subretinal space. To allow a wide-gauge cannula to reach the back of the retina, some of the vitreous humor, the gel-like substance between the lens and the retina, usually has to be removed. Displacing the vitreous gel, however, can cause newly injected retinal cells to backflow into the rest of the eye, which further obstructs vision. Instead, Ripolles-Garcia et al. designed a smaller cannula that injected cells directly to the back of the retina without removing any of the vitreous layer.

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FIGURE: CARTOON IMAGE OF SUBRETINAL INJECTION INTO THE RETINA.

FROM:” "GENE THERAPY AND GENOME SURGERY IN THE RETINA” DECARLO ET AL. 2018 The stem cell-derived retinal precursor cells used in this procedure were tagged with fluorescent dye before injection into the retina, enabling researchers to track their location and distribution over time with non-invasive imaging. In one of their first observations, imaging revealed that gravitational effects contributed to an unequal distribution of cells across the retina. Researchers argued that these effects could be mitigated in clinical settings by allowing individuals to lay horizontally in the days following the procedure. Avoiding Immune Rejection A total of seven normal dogs and three dogs with retinal mutations were divided into two groups: those that continuously received a triple cocktail of anti-inflammatory, immunosuppressive drugs post-transplant and those not under any medication. Blood and urine samples collected throughout the evaluation period 891

confirmed that this high immunosuppressive regimen was well tolerated in this experimental group. In both groups, Ripolles-Garcia et al. observed an initial loss of transplanted photoreceptor cells in the early days following injection. The animals that did not receive the immunosuppressive regimen, however, experienced continued loss of these cells, until the fluorescently tagged retinal precursor cells were no longer detectable. Furthermore, researchers found that both normal and mutant dogs in this group showed signs of a robust innate immune response, given by the enhanced presence of macrophages, and activated microglia in the capillaries surrounding the retina. This robust immune response, concurrent with the complete loss of stem cellderived photoreceptors, in dogs that did not receive antiinflammatory drugs suggests that immune suppression is critical for avoiding immune rejection and prolonging the survival of transplanted cells. Integration For the final challenge in this study, researchers had to ensure that the photoreceptor cells that survived transplantation could integrate into the retina to restore vision. Within the group that was on the immunosuppressive regimen, the normal dogs had smaller fluorescently tagged clusters over time, which was not accompanied by an inflammatory response. The clusters of transplanted cells in the dogs with retinal mutations, on the other hand, remained unchanged. It is likely that the donated cells were filling the gaps left from degenerated photoreceptors requiring less remodeling of the retinal architecture. As William Beltran, one of the senior authors of this study and professor of ophthalmology at Penn Vet, says, “What we showed was that, if you inject the cells into a normal retina that has its own photoreceptor cells, the retina is pretty much intact and serves as a physical barrier, so the introduced cells don’t connect with the second-order neurons in the retina, the bipolar cells. But in three dogs that were at an advanced stage of retinal degeneration, the retinal barrier was more permeable. In that environment, cells had a better ability to start moving into the correct layer of the retina.” The donated cells that extend connections to internal structures of the retina are then in a perfect position to send visual information to the brain. 892

Conclusion This approach is still far from being used for treating retinal degeneration in humans. For one, the extent of immunosuppression needed to support these cells may not be practical, not to mention safe, in humans. The dogs recruited for this study were on immunosuppressive drugs for the duration of the study, until they were euthanized at its conclusion. It is unclear how long an individual would have to be on an immunosuppressive regimen to avoid immune rejection. Also, given the nature of using animal subjects, we still do not know whether donating new cells will restore vision. This may be a question that can only be answered through clinical trials. A regenerative therapy that replaces damaged photoreceptors with retinal precursor stem cells could someday restore sight to millions of people. This study has set the stage for further optimization of not only the delivery of these cells but also their long-term survival in future clinical trials. This article is featured on Forbes.org, and can be read online here: Progress In The Search To Reverse Age-Related Vision Loss

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Children Experiencing Respiratory Trouble Post-Covid-19 Improve With The Use Of An Inhaler And Exercise Regimen Forbes | October 15, 2022 | Article

While children infected with Covid-19 generally report milder symptoms compared to adults, a subset goes on to experience persistent symptoms after infection. The most common of these symptoms is pediatric dyspnea, otherwise known as shortness of breath or difficulty breathing. However, there is limited research describing post-covid pediatric dyspnea, and a lack of objective criteria to diagnose the condition may cause some providers to assume a psychological trigger, especially when spirometry values are normal. A new case study published in the Journal of Allergy and Immunology highlights the importance of investigating beyond spirometry value and not assuming a psychological component for the best possible patient outcome. I’ve outlined a summary of the case study using the pseudonym “John” for the patient. In November 2020, 17-year-old John found himself with a sore throat. The next day, he experienced coughing, abdominal pain, myalgia, and chest tightness. He took a rapid antigen test and confirmed that he was positive for Covid-19. He prepared himself to ride the grueling wave of infection. Over the next three weeks post-infection, John became easily fatigued. He developed a severe case of shortness of breath and a lower tolerance for exercise, which persisted over the next 5 months. He set an appointment with his primary care provider and was later prescribed a fluticasone metered-dose inhaler 110 and an albuterol metered-dose inhaler, both of which are typically used to treat lung diseases like asthma. He later switched over to a fluticasone/salmeterol 115 metered-dose inhaler. On medication, John noticed an improvement in exercise tolerance; he was proud to walk on the treadmill for 5 minutes before feeling winded. 894

John had no medical history of wheezing episodes or recurrent bronchitis. He has never been diagnosed with asthma and was not allergic to 23 aeroallergens. His normal forced expiratory volume (REV) in 1 second was 97%, which was above the normal of 79%, and his FEV to forced vital capacity ratio was 0.81, which was also above the normal of 0.79. John did not have any significant anxiety or depression. So, what happened? John developed moderately increased hyperinflation, with a normal residual volume to total lung capacity ratio of 24, which is below the normal of 30. Through impulse oscillometry testing, he showed an increased percent of peripheral airway resistance at 44% total airway resistance, when the average is less than 30%. Additionally, there was significant reversibility of small airway reactance at 56% below baseline, which is higher than normal (less than 40% below baseline). John’s results suggested small airway dysfunction, which is commonly observed in asthma. He left the hospital with a different prescription and recommended a gradual increase in exercise. In 3 months, John saw a notable improvement in exercise and shortness of breath. He attributed his positive results to physical conditioning, diet changes, and better adherence to his medication regime. Oscillometry testing showed a lower peripheral airway to total airway resistance ratio (down to 12.5% total airway resistance). John had a slight, but still high reversibility of small airway reactance when given albuterol. Overall, this showed that small airway obstruction and airway hyperinflation greatly influenced labored breathing and exercise intolerance. John’s experience is among the many children with post-covid dyspnea and low exercise tolerance whose health improved following physician intervention. At National Jewish Health, the leading respiratory hospital in the US, children with post covid dyspnea were evaluated, and a majority were found with increased residual volume and/or residual volume to total lung capacity ratio, a high proportion of peripheral to total airway resistance, or large AX reversibility -all signs indicating airway obstruction. When prescribed an inhaler and exercise regimen, most children saw their health gradually improve with treatment duration. Although some symptoms resolved due to the disease prognosis, providers believed that the treatment advanced the recovery rate. 895

Covid-19 may persist beyond infection, lasting months, and contribute to respiratory complications. For the best possible health outcome, you should visit a specialist if your child or teen has trouble breathing during and following Covid-19 infection. They can recommend an appropriate course of treatment to help your child recover. If that provider suggests a psychological trigger, encourage them to investigate further or find a new provider. This article is featured on Forbes.org, and can be read online here: Children Experiencing Respiratory Trouble Post-Covid-19 Improve With The Use Of An Inhaler And Exercise Regimen

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Reimagining Alzheimer’s (Part 3): The APOE Story In Alzheimer’s And Other Diseases Forbes | October 17, 2022 | Article

This article is the third installment in my series on Alzheimer’s disease. Read more about Alzheimer’s disease in part 1 and part 2 of the series. Alzheimer’s is a serious disease that affects nearly 6 million people in the United States. Interestingly, some people have a much higher chance of developing Alzheimer’s than others, especially as they age. A principal risk factor of Alzheimer’s disease depends on a single gene called Apolipoprotein E (APOE). By having two copies of a particular APOE variant called APOE4, you become twenty times more likely to develop the disease. Although APOE4 is not the only inherited cause of Alzheimer’s, it is by far the most predominant. For this reason, APOE demands some special attention as to what it is and how it works. Housekeeping functions of APOE Lipids APOE proteins are an essential component of our body’s ability to process fats/lipids. APOE proteins collect the circulating lipids in the body and help distribute them across cells. In the brain, APOE is produced by cells called astrocytes. Astrocytes are one of the most abundant cells in the brain and play a supportive role for neurons and their signals. When APOE proteins are formed in the brain, they collect several lipids in the body to form lipid complexes. Then, the APOE proteins bind to receptors on the surface of cells. This allows the cells to internalize the free-floating lipids and use them to form structural components of the cells or to store them as free energy. Cholesterol A secondary responsibility of APOE is that it helps the body process and distribute cholesterol. Much like how the protein distributes fats to cells, APOE is also responsible for collecting freefloating cholesterol and distributing it among cells in the body. Cholesterol is an essential component for cell membranes and can

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help cells produce hormones in the body. It is also a major determinant of heart disease. Inflammation While lipid and cholesterol processing are very important functions of APOE, studies have suggested that APOE may play a significant role in regulating inflammation as well. Recent research suggests that APOE regulates one of the primary inflammation pathways in the brain—the classical complement cascade— by binding to a protein complex called C1q. When the classical complement cascade is activated, a series of proteins including C1q begin to bind/react with each other. This activity sends a signal to distant, inflammatory immune cells and communicates where an infection is located. While sending a signal to the distant immune cells, proteins in the classical complement cascade will simultaneously tag or label infected cells to be eliminated by the immune cells. Once inflammatory immune cells have been recruited to the site of infection, the immune cells take over and eliminate any of the infected cells tagged by the complement cascade proteins. By binding to the complement cascade C1q, APOE can effectively stop inflammatory reactions in the brain. Variations in the APOE gene APOE may play an essential role in the body, but what is its association with Alzheimer’s disease? There are three known variants of the APOE gene: APOE2, APOE3, and APOE4. Each of these variants has its own important physiological consequences for those who carry them. This is especially relevant to those who contain two copies of the same variant. APOE4 Currently, the APOE4 variant is the greatest known risk factor for Alzheimer’s disease. Those who only have one copy of the APOE4 variant are three times more likely to develop Alzheimer’s disease during old age. This risk increases dramatically for those who contain two copies of the APOE4 variant. As mentioned, those with two copies of the APOE4 variant are twenty times more likely to develop Alzheimer’s disease. Having two copies of the APOE4 variant also increases the risk of developing early-onset Alzheimer’s. Early-onset Alzheimer’s affects people between the ages of 30-60 years old. APOE3 898

The APOE3 variant is the most common version of the gene and is considered neutral. The APOE3 variant has not been associated with any risk for disease. APOE2 APOE2 is the rarest variant and its physiological consequences are somewhat unknown. Interestingly, studies have suggested that APOE2 may play a protective role against Alzheimer’s disease. There is evidence to suggest that containing even one copy of the APOE2 gene can reduce risk of Alzheimer’s disease. Other studies have found that APOE2 is associated with a greater risk for diseases such as type 2 diabetes and psoriasis.

Worldwide frequency and disease relevance of APOE alleles. INFORMATION SOURCE: WIKIPEDIA... Evolutionary origins of APOE variants Where did these variants come from? Approximately, 7.5 million years ago, primates and humans split into two unique species. The APOE4 variant was one of the first APOE variants that were found in the hominid line but not in primates. Over the course of the next few 100,000 years, the APOE gene continued to mutate and create the two other major APOE variants that exist in modern day. These mutations began approximately 220,000 years ago. The APOE4 variant underwent a single amino acid substitution mutation. The building blocks of proteins in the body are amino acids. When a single amino acid is changed, this can affect the overall function and structure of the protein. For APOE4, an uncharged 899

cysteine amino acid was substituted with a positively charged arginine amino acid. This resulted in significant physical changes and subsequently formed the APOE3 variant. 140,000 years later, the APOE3 variant underwent an additional amino acid substitution mutation. This mutation involved substituting a different positively charged arginine amino acid for a neutral, uncharged cysteine amino acid. This additional mutation resulted in the APOE2 variant of the gene. Physiological consequences of APOE variants across populations Although scientists have demonstrated that different APOE variants can increase or decrease the risk for certain diseases, it is important to note that none of the APOE variants are associated with the onset of Alzheimer’s disease 100% of the time. This means that while the APOE4 variant significantly increases the risk of developing Alzheimer’s disease, not everyone who contains one or two copies of the APOE4 variant will develop Alzheimer’s. In fact, of those who have two copies of the APOE4 variant, 40% do not develop any symptoms of Alzheimer’s disease whatsoever. This observation is especially true across different populations. For instance, while the Nigerian population has the highest percentage of individuals with the APOE4 variant, it has one of the lowest occurrences of Alzheimer’s disease. This suggests that there are several other genetic or biological factors that play a role in determining whether an individual develops Alzheimer’s disease. Final Comments While none of the APOE variants are associated with Alzheimer’s disease 100% of the time, if your family members have Alzheimer’s, it is advisable to undergo genetic testing to understand your own APOE profile. Genetic tests are relatively straightforward and can be done through a simple blood or saliva test. DNA from these samples is then isolated and specific genes within the DNA can be detected. There is more research to be done about the variants of APOE and how they affect the onset of Alzheimer’s disease. Fortunately, the foundational knowledge and science that exists today allow us to understand the risks of developing Alzheimer’s disease and hopefully, prepare for the future. 900

This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 3): The APOE Story In Alzheimer’s And Other Diseases

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CAR T Therapy [Part III]: A New Direction for Multiple Myeloma Treatment Forbes | October 21, 2022 | Article

This is the third installment in a series on the advances CAR T, a remarkable immunotherapy treatment dubbed a “living drug.” The first in the series lays the foundation for understanding how CAR T works, while the second outlines its uses for B cell cancers. This third installment highlights recent advances in treating multiple myeloma. Multiple myeloma is a relatively uncommon yet serious disease estimated to impact more than 30,000 US citizens this year. Although several treatment options exist, the illness is considered incurable as most treatments do not resolve the condition permanently—including the most recent advancements with CAR T cells. Here, we describe an approach using a different variant of CAR T cells for multiple myeloma that holds promise for those with treatment-resistant forms of the disease. What is Multiple Myeloma? Multiple myeloma (MM) or myeloma is a cancer of the plasma B cells found in the bone marrow. Although these white blood cells typically produce antibodies, for people with multiple myeloma, the plasma cells multiply faster than the body can handle, produce abnormal antibodies, and set the body out of balance. The illness can spread to other organs through the bloodstream, and masses of plasma cells may form in the bone marrow or soft tissues, as well. The illness usually occurs to people 60 years and older, and is unlikely to develop in individuals under 40 years of age. The symptoms can be widely varying—some even report having no symptoms at all—but most with this disease experience bone pain and fatigue. Other common complications include anemia, kidney problems, or thickened blood. Without treatment, the prognosis is poor. However, with the advent of chemotherapy and more advanced medicines, survival is usually four to five years. If diagnosed early, the five-year survival rate exceeds 77%. 902

Patients with active myeloma first receive a combination of drugs to target the abnormal cells. Another alternative is chemotherapy. For example, I contributed to the creation of Velcade, a chemotherapy medicine which slows or stops the growth of myeloma cells. Stem cell transplants, steroids and even CAR T therapy—a newer medical technology which alters patient cells in the lab and infuses them back into the body to fight the cancer— may be tried as other potential options. Unfortunately, once a therapy fails, the body typically becomes resistant to its reintroduction and thus loses efficacy. The Current Reality of CAR T Therapy CAR T therapy has recently been approved to treat multiple myeloma, but it is only considered after four or more refractory lines of treatment—in other words, when other four or more options fail to achieve lasting remission. The two existing CAR T therapies on the market target B cell maturation antigen (BCMA), an antigen expressed on the surface of malignant plasma cells; in this piece, the antigen will be referred to as Target 1. A Chimeric Antigen Receptor T cell derives its name from the synthetic combination of T cell and antibody properties. Patient T cells are taken from the body and modified to detect Target 1 through an antibody-based fusion receptor (scFv). The lysing process relies on signaling from the T cell. As seen in Figure 1, when the CAR T antigen receptor binds with Target 1 on the cancer cell, the CAR T cell releases chemicals to trigger the cancer cell’s death.

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FIGURE 1: The FDA has approved two CAR T therapies for multiple myeloma. While the signaling domains differ between the two, both therapies target BCMA, an antigen found on the surface of myeloma tumor cells. Abbreviations: MM, multiple myeloma; scFv, single chain variable fragment (fusion protein which detects BCMA) BO YU, TIANBO JIANG & DELONG LIU

Clinical trials have confirmed these CAR T therapies as safe to use and capable of producing results. A study of Ide-cel found 73% of participants had a decrease in their cancer. Even more successfully, a study of Cilta-cel saw a 98% response rate, with 78% of patients showing no signs of cancer in their bone marrow. The outstanding issue with both treatments, however, is that relapse does eventually occur—around 8.8 months later for Ide-cel, and 22 months later with Cilta-cel. With relapse and treatment resistance a prevailing concern for multiple myeloma treatments—not just CAR T—researchers are seeking new ways to sustain longer remission and increase survivability when other alternatives are exhausted. One possible method is to enhance current CAR T protocols with a new therapeutic target. Methods In their study, Mailankody et al. consider the safety of an alternative antigen target. The target is known as G protein-coupled receptor, class C, group 5, member D (GPRC5D), but shall be referred to as Target 2 for simplicity. Despite its unknown function 904

in tissues, it poses as a promising CAR T antigen target due to its presence in several myeloma cell lines and in bone marrow plasma cells. The team chose a second generation CAR T design for their product. Second generation CAR T cells contain a single costimulatory domain (shown in blue in Figure 2) inside the T cell to extend the life of the cell once in the body. The chimeric antigen receptor in this study is tailored to find cancer cells that express Target 2 (denoted in green in Figure 2). As depicted in Figure 3, the researchers first collected patient T cells through leukapheresis. They modified the T cells, expanded them to large numbers, and then infused the CAR T cells back into the body after completing preparatory chemotherapy. The patients received an escalating dose of the trial CAR T infusion, totalling to four doses.

FIGURE 2: Later generations of CAR T therapy include co-stimulatory signaling domains to improve T cell expansion after infusion and T cell survival once in circulation. BRENTJENS R, ET AL. “DRIVING CAR T CELLS FORWARD.” NAT REV CLIN ONCOL. 2016 13, 370–383.

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FIGURE 3: For this study, T cells were extracted with leukapheresis before being modified and expanded to detect GPRC5D. NOVARTIS

Results A total of 17 participants received CAR T therapy, all who have previously tried five different kinds of multiple myeloma treatment. The majority of participants developed cancer resistance to their last line of treatment; this includes a group of individuals who previously received Target 1 CAR T therapy. On the whole, this study successfully confirms Target 2 CAR T therapy as safe and effective, particularly for individuals who have already received Target 1 CAR T cell therapy or have run through several other therapeutic options. Around 78% of patients had a partial response or better, and 59% had a very good partial response or better. The therapy was effective even ten months after infusion for some individuals. Common CAR T therapy side effects include cytokine release syndrome and immune effector cell-associated neurotoxicity (ICANS). While both conditions can be reversed with prompt treatment, the severity of both side effects can range from mild to life-threatening. Most participants experienced milder cytokine 906

release, with the exception of one patient who experienced lifethreatening side effects. All with side effects were treated. Future Directions Mailankody et al. demonstrate that Target 2 CAR T therapy can effectively treat multiple myeloma. If Target 1 CAR T therapy fails, Target 2 appears to be a viable alternative. The results also suggest that using Target 1 and Target 2 CAR T therapies in succession could lead to positive outcomes. A third alternative is to enhance the T cell design further to allow for tandem targeting; an ideal synergy could be attained if T cells were fitted with both Target 1 and Target 2 receptors, hopefully resulting in longer remission periods and increased survival. This article is featured on Forbes.org, and can be read online here: CAR T Therapy [Part III]: A New Direction for Multiple Myeloma Treatment

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Introducing A “Naked” Self-Amplifying RNA Vaccine Candidate Forbes | October 21, 2022 | Article

The following two articles on a controllable self-replicating RNA vaccine form part of a broader series on next-generation mRNA vaccines. The first three installments can be read here, here, and here, respectively. A second generation of Covid-19 mRNA vaccines is making its way onto the scene. Common to many of them is the use of selfamplifying RNA technology, which maximizes antigen expression. Now, researchers have developed a self-amplifying RNA vaccine with a number of exciting new characteristics. First, it doesn’t require any of the nucleobase modifications that conventional mRNA vaccines depend on for improved stability. Second, it simply uses “naked” RNA injected directly without a protective lipid nanoparticle. Third, the vaccine is designed to be administered into the outer layer of the skin —the dermis— for improved cellular immunity. And fourth, it has been optimized to suit the temperature range of the surface of the body, rather than the core of the body. Here, we take a look at how the vaccine designed by Amano et al. differs from conventional mRNA vaccines and even other selfamplifying RNA vaccines. In a follow-up article we will take a look at what impact these changes have, focusing on results in animal models. What is Self-Amplifying RNA Technology? Messenger RNA (mRNA) vaccines work by exposing our cells to the genetic material needed to build a target antigen. In the case of SARS-CoV-2, this is usually the spike protein that the virus uses to bind and enter our cells. Essentially, mRNA vaccines take advantage of our cells’ own protein-making machinery to produce SARS-CoV-2 proteins “in-house”. Self-amplifying RNA technology further optimizes this process. Where conventional mRNA vaccines only include the genetic information for the antigen protein, self-amplifying RNA vaccines include a sequence of alphavirus RNA that encodes four non908

structural proteins. These nonstructural proteins come together once in the cell to form an enzyme called RNA replicase — think of this as a genetic photocopier. The RNA replicase makes many copies of the target mRNA, each of which can then go on to be translated into the antigen (Figure 1).

FIGURE 1. (Left) Conventional, synthetic mRNA technology (Right) Self-amplifying RNA technology. FROM: “Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses” VOGEL ET AL. 2017

No Need for Lipid Nanoparticles or Nucleobase Modifications Self-amplifying RNA technology, as epitomized by this vaccine, undercuts two of the current shibboleths typical of conventional mRNA vaccines, including Pfizer’s and Moderna’s. For one, selfamplifying RNA vaccines work perfectly well without any nucleobase modifications — no need to substitute uridine with pseudouridine, for example. Additionally, it appears that in some circumstances self-amplifying RNA technology allows us to dispense 909

with lipid nanoparticle formulations entirely, and that is without great sacrifices to uptake or gene expression. This vaccine is developed using “naked” RNA alone. A recent report, published in the journal Current Opinion in Virology, outlines the advantages of self-amplifying RNA as well as the pros and cons of different delivery methods, such as administration into the skin rather than the muscle tissue. The report also compares and contrasts the results of using naked RNA with the results of using RNA complexed to lipid nanoparticles. Engineering functional lipid nanoparticles is no easy task, with many promising candidates ultimately falling short during in vitro testing. Added to this is the fact that manufacturing reliably highquality lipid nanoparticles, and the raw materials from which they are made, is a difficult process and can act as a limiting factor in vaccine production. By using RNA derived from an alphavirus, Amano et al. avoid the need for lipid nanoparticles or nucleobase modifications, and in doing so, bypass the above mentioned manufacturing bottlenecks. The alphavirus RNA takes on a functional role similar to that of the lipid nanoparticles: shielding the target mRNA from degradation and helping it to get into our cells. Even if initial uptake is lower because of the lack of a lipid nanoparticle, this is made up for by the self-amplifying mechanism of the alphavirus RNA. Intradermal Administration … With a Twist Unlike most vaccines, which are administered into muscle tissue deep underneath the skin, Amano et al. designed their vaccine to be delivered into the dermis layer of skin. This has a number of distinct advantages, not least because 40% of our antigen-presenting cells (APCs) are found in the dermis. Antigen-presenting cells are a class of immune cells that help expose our adaptive immune system — our B and T cells— to foreign proteins. Another benefit is that intradermal injection can make use of alternative, pain-free injectors. The Gennova Gemcovac-19 vaccine, for example, is administered intradermally using a needle-free injector called the PharmaJet Tropis. Amano et al. instead opt for a microneedle approach — a tiny needle, only one fiftieth of an inch in length (0.6mm), is used to inject the vaccine solution into the skin. This enables easy and consistent administration.

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But the dermis has a lower temperature than does the rest of the body, on average hovering around 91 degrees (33 degrees Celsius) instead of 98 degrees Fahrenheit (37 degrees Celsius). In order to tailor their vaccine to the environment of the dermis, the researchers systematically mutated the non-structural proteins (nsP1-4) of the alphavirus vector. After each mutation, they checked to see the expression levels of the target antigen gene at 91 and 98 degrees Fahrenheit. Amano et al. finally found a mutant version of the RNA —a five amino acid insertion in the non-structural protein 2 of the alphavirus— that functions between 86 and 95 degrees Fahrenheit (30 and 35 degrees Celsius) but is inactivated at 98 degrees and above. They called this new design controllable self-amplifying RNA (c-saRNA). The rationale for temperature sensitivity given by Amano et al. is threefold: first, more efficient RNA replication in the skin; second, reduction of the risk of aberrant RNA replication throughout the body — if the RNA were to accidentally make its way beyond the dermis, into organs or other tissues, it would simply not be activated, preventing any replication; and third, it may reduce injection-site adverse reactions since excessive inflammation would raise the temperature of the skin and inactivate the vaccine. To test whether controllable self-amplifying RNA works as intended in practice, the group of researchers exposed mice to an intradermal injection encoding a bioluminescent luciferase enzyme (c-saRNA-LUC). The luciferase enzyme produces light when exposed to lucifern, allowing researchers to track its presence in the body over time. Importantly, the RNA was kept in its “naked” form, without the addition of any lipid nanoparticles or other transfection agents that are usually used to improve uptake. They compared the results to conventional, nucleoside-modified mRNA encoding the same luciferase enzyme. The expression of luciferase driven by the mRNA lasted around a week. But the expression of luciferase driven by the controllable self-amplifying RNA carried on for another three weeks, reaching almost a month of continuous expression (Figure 2). On top of the extended duration, the controllable self-amplifying RNA injection yielded 10- to 100- fold higher expression levels than the mRNA injection. All of the luciferase expression was restricted to the injection site, with no unintended expression in other areas of the 911

skin nor in any other part of the body, including internal organs. The temperature-sensitive safety-switch had clearly done its job. Even direct intramuscular injection with the controllable selfamplifying RNA did not yield any luciferase activity (Figure 3).

FIGURE 2. mRNAs, either mRNA-LUC or c-srRNA1-LUC, were formulated as naked RNAs, without LNP or other transfection reagents, in Lactated Ringer’s solution. 5 μg of mRNAs were injected intradermally into a single site on the right hind limb of CD-1 outbred mice (Day 0). Luciferase activity was visualized and quantitated by using a bioluminescent imaging system, AMI HTX (Spectral Instruments Imaging, Tucson, AZ) from Day 1 through Day 26. (A) Pictures of mice that received intradermal injection of mRNA-LUC. (B) Pictures of mice that received intradermal injection of c-srRNA1LUC. FROM: “Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity” AMANO ET AL. 2022

FIGURE 3. A five microgram dose of controllable self-amplifying RNA (c-saRNA) encoding a bioluminescent luciferase enzyme was injected into mice either intradermally (left) or intramuscularly (right). As intended, intramuscular injection does not show any luciferase expression, indicating the temperature-based “safety-switch” the researchers designed had kicked in. FROM: AMANO ET AL. 2022

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Given that controllable self-amplifying RNA (c-saRNA) technology is a ‘plug-and-play’ system, it can be used to express any number of viral proteins as antigens. In a follow-up article, we will look at the results of using controllable self-amplifying RNA (csaRNA) technology in combination with: mRNA encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein, mRNA encoding the nucleocapsid protein (N) of SARSCoV-2, mRNA encoding the N protein of SARS-CoV-2 plus the N protein of MERS-CoV, and finally, mRNA encoding the RBD and N proteins of both SARS-CoV-2 and MERS-CoV. This article is featured on Forbes.org, and can be read online here: Introducing A “Naked” Self-Amplifying RNA Vaccine Candidate

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How Enhanced Ventilation And Air Filtration Can Fight Covid-19 Forbes | October 21, 2022 | Article

At a time when Covid fatigue levels are high, improving ventilation is a highly effective, non-invasive strategy to stop the transmission of Covid-19. A body of scientific literature reveals that particles of SARS-CoV-2 can remain in the air for hours at a time. In a 2021 literature review, researchers summarize the effectiveness of air management tools within the perspective of varying scales and settings of exposure. Covid-19 Particles in the Area: How BIG or small are they? How covid-19 particles originate affects their size and momentum in the air. Particles can be released by the force of speaking, coughing, or even breathing. In a 2022 study investigating how airborne SARS-CoV-2 particles travel, researchers studied the viral amounts within fine and coarse respiratory droplets produced in breathing, talking, and singing. They found that 59% of their participants released detectable levels of SARS-CoV-2 RNA in respiratory aerosols, with greater levels recorded from talking or singing samples. Fine-sized particles (≤5 µm) contributed to 85% of the viral loads found. Further, this suggests that fine covid-19 particles are catapulted in the air by force necessary to vocalize and are able to travel more deeply into the lungs due to their size. Air management to control Covid-19 transmission can be accomplished in several ways: Ventilation: Ventilation can increase air exchange and directional airflow, lowering the risk of covid-19 exposure. In conditions where “natural” ventilation, or access to the outdoors, is not possible, proper ventilation can dilute (if not effectively redirect) contaminated air in indoor settings. A number of studies have demonstrated a statistically significant increased risk of infection in poorly ventilated areas compared to well-ventilated conditions. 914

MERV Rating Effectiveness PLATINUM AIR CARE In a separate literature review, researchers also highlighted the association between improved ventilation and student performance. A body of literature has shown a reduction in health effects and student absences associated with increased ventilation rates. Although improved ventilation causes an increase in energy consumption, the net annual costs are estimated to be less than .1% of the typical public spending on primary and secondary education in the United States (Fisk et al, 2017). The total expense of improving ventilation, further, is a minor consequence compared to the benefits provided. Filtration Filtration has shown to be very effective in removing respiratory aerosols, minimizing exposure and infection risk. Products as affordable as MERV 13 filters are reported to capture at least 50% of .3 to 1um, 85% of 1-3 um and 90% of 3-10 um particles (Mesquita et al, 2021). In cases where covid-19 particles are on a super micron scale, MERV 11 provides comparable filtration efficiency with lower airflow resistance, requiring less energy. MERV filters ratings 13 and above are certified to capture submicron particles, which can be beneficial even in spacious settings. A growing number of publications have shown quality filtration systems in schools and hospitals have effectively decreased airborne particles. One study found a 95% reduction in particles sized 0.0110 um after 37 minutes of four portable HEPA cleaners in an active, relatively large classroom setting.. Teachers and students that were 915

involved reported little disturbance by the noise of the commercial air cleaners. In clinical settings, where it is very important to circulate clean air, studies have shown that air filtration systems are effective in removing covid-19 particles as well other harmful airborne particles, like influenza or dust. Far UVC light Ultraviolet C-radiation light has been used for decades among scientists to quickly destroy microorganisms, including bacteria and viruses, to sterilize their benchwork. It involves using high energy, short wavelength radiation to effectively damage the genetic material found in the organisms neutralizing them. Studies have shown that far ultraviolet C-radiation lamps within exposure can have strong efficacy rates and work well against variants of covid-19, influence, and other drug-resistant bacteria without harming humans. Installing some lamps on walls or ceilings can irradiate the upper half of an indoor space, thereby disinfecting the air within the proper limitations that can later be recirculated through fans or ventilation systems. One study has actually shown that installing UVC light in three distinct climates reduced air concentration of pathogens within a range of 50%-70%. As air temperature and air velocity play a role in UVC light effectiveness, there was a median percentage of 65% in the reduction of airborne particles when focusing on the different environments of Houston, Los Angeles, and New York. This suggests that UVC light systems are comparable to filtration systems and effective in a wide spectrum of environments. A Call to Action Governments worldwide have heavily invested efforts in food safety, sanitation, and drinking water for the sake of public health. It is time we give the same priority to achieving clean, pathogen-free air in buildings and indoor public spaces. Many critics of such a policy will cite significant costs as a barrier, However, the economic losses of even an average flu season cost the United States $11.2 billion, primarily due to reductions in productivity and absenteeism. The total cost of the Covid-19 pandemic in 2020 was estimated to be more than $16 trillion, or approximately 90% of the annual gross domestic product of the US in October 2020. The costs will only continue to grow. It is in the best interest of our children, adults, and future generation to invest in quality air management tools now. 916

The goal should be the explicit inclusion of protection against indoor air hazards (including airborne infection control) in the statements of purpose and definitions of all relevant building design and engineering standards, regulations, and codes. Comprehensive ventilation standards must be developed by professional engineering bodies. New approaches must be developed to encourage the implementation of standards, one option is implementing “ventilation certificates” similar to food hygiene certifications for restaurants. Like many other issues of public health, the Covid-19 pandemic has highlighted the desperate need to prioritize air quality in indoor spaces. This article is featured on Forbes.org, and can be read online here: How Enhanced Ventilation And Air Filtration Can Fight Covid-19

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Broadly Neutralizing SARS-CoV-2 Antibodies From Immunized Macaque Monkeys Forbes | October 21, 2022 | Article

Monoclonal antibodies have been effective in both preventing and treating SARS-CoV-2 infections. However, just as SARSCoV-2 evolves to evade immune responses by vaccines and natural infection, so too do variants arise that evade neutralization by specific monoclonal antibodies, hence the search for antibodies that broadly neutralize regardless of the source. We have previously described eight such antibodies derived from infected humans, mice, and camelids. Here we describe a different approach in which antibodies are isolated from monkey subjects. In the search for such antibodies, He et al. discovered a series of antibody candidates induced in macaque monkeys. The researchers immunized macaques over a ten-week period and drew the antibodies from sera post-inoculation (Figure 1A). They used a recombinant prefusion-stabilized soluble S-protein plus a saponin (SMNP) adjuvant as their vaccine, yielding strong antibody responses in the animals. Neutralization of SARS-CoV-1 and SARS-CoV-2 Not only did the enhanced adjuvant immune serum grant the macaques with significant virus neutralization of SARS-CoV-2 but the treatment also neutralized macaques infected with SARS-CoV1, indicating broad neutralization capability (Figure 1B). This response is in stark contrast to human natural infection, which rarely induces cross-neutralizing activity against SARS-CoV-1. Inoculation with mRNA SARS-CoV-2 did not yield SARS-CoV1 neutralization in monkeys or humans, and inoculation with the enhanced adjuvant S protein only yielded a significant SARS-CoV1 response in monkeys. The sera of adjuvant vaccinated monkeys bound and neutralized both viruses with great consistency (Figure 1C).

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FIGURE 1: (A) The SARS-CoV-2 S-protein prime-boost immunization in rhesus macaques and sampling ... [+] HE ET AL.

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Isolation of Monoclonal Antibodies The intention behind using enhanced adjuvant S protein as a vaccine in monkeys is to enable enhanced memory B cell responses, from which strong antibody candidates can be isolated. These differ from most monoclonal antibody candidates, which derive from plasma cells. The researchers isolated many monoclonal antibody candidates from the macaque sera, but two stood out for binding and neutralization: K288.2 and K398.22. Both bind and neutralize a wide variety of viruses, including both SARS viruses and bat viruses WIV1 and RaTG13. Additionally, both neutralize variants of SARS-CoV-2 from the wildtype through Delta effectively. However, K288.2 loses neutralization capability against BA.1, whereas K398.22 maintains it. Figure 2 below illustrates both antibodies' similar but slightly adjusted binding actions, which is likely the culprit for K288.2 losing neutralization against Omicron, binding to the wrong residue. Cyro-Electron Microscopy: The Angle May Make All The Difference The researchers note that differences in binding capacity between different viruses may be attributed to inherited differences between macaque and human sera from the immune system. Each species uses a different immunodominant germline gene segment in its antibody development, leading to different approach angles and binding modes when introduced to the receptor-binding domain of SARS-CoV-2 and others.

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FIGURE 2: S-protein binding angles of approach were compared between macaque IGHV3-73–encoded RBD ... [+] HE ET AL.

High-Resolution X-Ray Crystallography X-ray crystallography allows researchers to observe specific amino acid binding residues as the antibodies come in contact with the virus. Not only do K288.2 and K398.22 bind conserved regions in the receptor-binding domain that are less often targeted in other monoclonal candidates, such as 373-375, 404-408, and 502-508, but there is additional evidence suggesting that the macaque germline introduces a specifically unique binding motif not found in humanderived antibodies, E33 in CDRH1, which perhaps is another cause for the bolstered SARS-CoV-1 neutralization in the macaque antibodies.

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FIGURE 3: Structures of SARS-CoV-2 RBD in complex with K288.2 and K398.22 antibodies. HE ET AL.

Caveats The first caveats come from the isolation of the antibodies themselves. The sera was developed from an irregular and powerful adjuvant that was administered over a much longer 10-week period, meaning comparison to mRNA-derived sera in a more consistent four-week period is not directly apples to apples. These antibodies would be of much greater significance if they demonstrated binding and neutralization of later Omicron variants such as BA.5, BA.2.75, or BA.4.6, though there have been no published results discussing these variants. Through personal communications with the authors, we have confirmed that K398.22 neutralization drops significantly against BA.2, which does not bode well for later Omicron variants. Speculation The macaque isolation process may be a favorable system for isolating new monoclonal antibodies to be used as drugs. The Omicron viruses circulating today have been selected against the human immune responses, not macaque. This study shows that the inherited germline sequences in macaque antibodies may give them a leg up against emerging variants tailored to evade human 922

antibodies. That is not to say that K288.2 and K398.22 evade currently circulating strains. Rather, the method used to discover these two may yield others yet to come and the macaque isolation process is one that should be thoroughly investigated. This article is featured on Forbes.org, and can be read online here: Broadly Neutralizing SARS-CoV-2 Antibodies From Immunized Macaque Monkeys

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Reimagining Alzheimer’s (Part 4): Cautious Optimism For A New Alzheimer’s Disease Treatment Forbes | October 22, 2022 | Article

This article is the fourth installment in my series on Alzheimer’s disease. Read more about Alzheimer’s disease in part 1, part 2, and part 3 of the series. The pharmaceutical and biotech companies, Eisai and Biogen recently revealed promising clinical trial results for a drug that could potentially treat Alzheimer’s disease. In their clinical study, patients treated with the drug lecanemab experienced a 27% slower cognitive decline from Alzheimer’s disease than control groups. This treatment has been heralded as a breakthrough in Alzheimer’s disease research. Some scientists have cited the study as proof of one of the predominating theories of Alzheimer’s disease and its origins—the amyloid theory. This leads us to two questions: what makes lecanemab unique and are these clinical trial results truly a historical breakthrough in Alzheimer’s research? The biological origins of Alzheimer's disease have puzzled scientists for decades. One of the leading theories of Alzheimer’s disease is the amyloid theory. Amyloid plaques are one of the biological indicators of Alzheimer’s disease progression and are caused by abnormal amyloid protein fragments. Amyloid fragments are typically soluble and can easily be cleared from the brain. However, sometimes these protein fragments become sticky and aggregate, forming dense plaques that the brain cannot remove. These plaques are hypothesized to induce cognitive decline in Alzheimer’s patients. In recent years, several treatments have been developed to minimize the occurrence of amyloid plaques in the brain. Unfortunately, these treatments have had little to no effect on the cognitive decline seen in Alzheimer’s patients. Studies have also 924

shown that Alzheimer’s patients can have a significant number of amyloid plaques without exhibiting any cognitive symptoms. This has caused a substantial amount of doubt within the scientific community that amyloid plaques are truly the cause of cognitive symptoms in Alzheimer’s patients. Now, clinical results indicate that lecanemab has a different effect than other medications. Although lecanemab also targets amyloid in the brain, it seems to be effective at slowing the cognitive symptoms of Alzheimer’s disease. So, what makes lecanemab different? One of the unique aspects of lecanemab is that it targets amyloid fibrils rather than amyloid plaques. Amyloid fibrils occur at an earlier stage of amyloid plaque formation and consist of much smaller clumps of amyloid protein. Researchers have suggested that lecanemab may be a more effective treatment for Alzheimer’s disease simply because it targets amyloid fibrils before they develop into amyloid plaques. Others have speculated that the reason why lecanemab is effective at treating cognitive symptoms is not that it targets amyloid fibrils at all, but because one of the effects of lecanemab is that it increases levels of soluble amyloid in the brain. Previous studies published by the University of Connecticut’s College of Medicine have shown that regardless of the buildup of amyloid plaques in the brain, people with higher levels of soluble amyloid were cognitively normal, while those with low levels of soluble amyloid experienced cognitive impairment. The team at the University of Connecticut has reasoned that the increase of soluble amyloid caused by lecanemab is what ultimately makes it an effective treatment for Alzheimer’s. So, is lecanemab truly a historical breakthrough in Alzheimer’s disease research? While lecanemab seems to support the notion that amyloid plays a role in the origins of Alzheimer’s disease, it is still unclear whether these clinical trials alone support the amyloid plaque theory. Similarly, there still exist several limitations to the results of this clinical study. A primary limitation is that this is an early-stage study with a limited number of participants. The difference in cognitive decline between Alzheimer’s patients taking lecanemab and the control group was statistically significant. However, by clinical standards this difference was relatively minimal. That is to say, some patients 925

experienced a slower decline in cognitive abilities, but many did not. Additionally, the study was conducted for only eighteen months. Whether lecanemab will continue to be an effective treatment for a longer time and for a greater number of people is unknown. A second limitation is that while lecanemab targets amyloid, there are other biomarkers and contributors to Alzheimer’s disease progression that the clinical study does not consider. For example, another significant biomarker for Alzheimer’s disease is tangles of tau protein in brain cells. These tau tangles are arguably more associated with cognitive symptoms than amyloid plaques, yet the clinical study of lecanemab does not take tau tangles into account. A contributor to Alzheimer’s disease progression is neuroinflammation which has been theorized as the main trigger for cognitive decline in Alzheimer’s patients. Without taking these aspects of Alzheimer’s disease into account, the results of the trial do not paint a full picture of the disease and its potential origins. The final limitation of these clinical trial results is that the study was only conducted on Alzheimer’s patients with early or mild cognitive impairment. This means that the results of the study are not generalizable to all Alzheimer’s patients and may only help those who are in the early stages of the disease. While lecanemab offers a beacon of hope for the development of effective Alzheimer’s disease treatments, there is still much more work to be done to understand precisely why lecanemab might be effective and what the origins of Alzheimer’s disease are. This clinical trial is a significant step in the right direction, but more information is needed before calling lecanemab a breakthrough in Alzheimer’s research. As lecanemab is one of the few drugs made available to clinicians and to the public, it is likely to stir a significant market. However, whether lecanemab will be worth the risks and costs to individuals and to society remains an open question. Only time will tell. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 4): Cautious Optimism For A New Alzheimer’s Disease Treatment

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As Protection From Current Covid-19 Monoclonal Antibody Treatments Fades, The Discovery Of A New Class Of Antibodies Brings Hope Forbes | October 26, 2022 | Article

The latest Omicron subvariants are evolving to become more immune evasive, rendering monoclonal antibody treatments such as Evusheld and the forthcoming bebtelovimab ineffective against new strains. With surges predicted in the winter and few mitigation measures left in place, over 17,000 immunocompromised Americans and older populations in nursing homes who rely on these treatments could be left vulnerable to severe disease and high mortality rates. As reported in STAT news, Biden administration officials are “racing to game out other antibody options [to protect these populations].” However, there is hope in the discovery of at least two new classes of antibodies, which appear to be impervious to new virus mutations. They could provide a long-term solution to the issue of SARS-CoV-2 drug resistance fueled by evolving variants. Unlike previous waves driven by a single ominous variant, current and future waves will be driven by multiple subvariants of Omicron. Each new subvariant accumulates mutations in similar parts of the receptor binding domain, a critical spot in the spike protein where virus-blocking antibodies dock, continuing to enhance the virus’s immune dodging capabilities.

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FIGURE 1: Visualization of the continued evolution of SARS-CoV-2 variants.

NEXTSTRAIN.ORG The most concerning of the emerging immune-evading variants are BQ.1.1 and XBB. The two variants currently represent under 5% of cases in the United States and worldwide, but that figure is likely to rise. In addition to the various mutations established in their ancestral strains, BQ.1.1 and XBB carry several new mutations in the spike protein that enable their further evasion of approved monoclonal antibodies, including, but not limited to V213G/E, G339D/H, and F486V/S.

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FIGURE 2: Comparative spike mutations of BQ.1.1, XBB, and Delta variants of SARS-CoV-2. Those.[+] ACCESS HEALTH INTERNATIONAL

All previously approved monoclonal antibodies work by binding the receptor-binding domain at positions mutated in these new strains, suggesting that they will lose most, if not all, neutralization against new subvariants. However, two new groups of monoclonal antibodies in development may circumvent these and future Omicron variants by binding at alternative conserved sites to the receptor-binding domain that rarely mutate. The first set is antibodies that bind the fusion peptide and surrounding region. Two examples are COV44-62/79 by Dacon et al., and VN01H1/C77G12 by Low et al. These two antibodies have binding epitopes between positions 812 and 824. As with most variants throughout the pandemic, BQ.1.1 and XBB lack mutations in this range, meaning the antibodies likely maintain neutralization capabilities despite the highly mutated spike protein. The second type of antibody binds in the lower S2 region proximal to the membrane. An example in this set CV3-25 by Li et al. CV3-25 binds from positions 1149 to 1165, which is unmutated in both BQ.1.1 and XBB. 929

FIGURE 3: Schematic representation of the fusion and membrane-proximal S2 binding sites.

WRAPP ET AL. Not only could these antibodies neutralize all SARS-CoV-2 variants, but other related coronaviruses such as MERS and SARSCoV-1. Figure 4 below shows the conservation of amino acids between different coronaviruses in these regions.

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FIGURE 4: Conservation of amino acid sequences among different coronaviruses in (A) the fusion ... [+] LI ET AL.

These antibodies are not approved, nor have they been approved for human trial, but ongoing experiments in mice and hamsters show great promise in their Omicron neutralization. Most significantly, COV44-62/79 demonstrated substantial neutralization even against BA.5. Using combinations of antibodies could also further protect against SARS-CoV-2 drug resistance. We can create an insurance policy against future mutated variants by pairing conserved targets. Another issue is that the strength of binding of non-receptorbinding domain antibodies is lower than those that bind the receptor-binding domain. The binding strength can be boosted through modification of the variable end of the antibody. They can be further optimized by increasing half-life by up to three months or improving cell toxicity by modifying the stem portion of the antibody known as the Fc receptor. All such techniques are wellestablished and reasonable methods to be explored. Here we have discussed the use of monoclonal antibody drugs for the prevention and pre and post-exposure and treatment of Covid-19. The alternative approach, which has been successful for HIV, is the use of small-molecule drugs to prevent and treat AIDS and other HIV-related complications. Currently, more than 30 drugs are approved for the prevention and treatment of HIV infection, some of which provide durable protection between two and six months with a single injection. It is clear that while we continue to pursue the development of broadly active monoclonal antibodies, we should accelerate our programs to discover and deploy highly effective, long-acting small molecule drugs targeted to Covid-19. 931

This article is featured on Forbes.org, and can be read online here: As Protection From Current Covid-19 Monoclonal Antibody Treatments Fades, The Discovery Of A New Class Of Antibodies Brings Hope

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The Dramatic Cost Of The Covid-19 Pandemic: A Historical Drop In Life Expectancy Forbes | October 27, 2022 | Article

While many would like to forget this dark chapter of Covid-19 in our history and move on, we will likely be experiencing Covid19 itself and the aftershocks for generations to come. Not just because of the impact of Long Covid but also because of a dramatic drop in life expectancy. New data from the CDC shows that U.S. life expectancy dropped by a total of 2.7 years between 2019 and 2021 to 76.1 years, the lowest number since 1996. Not only is this very distressing, but certain races and ethnic groups continue to be disproportionally affected by a decrease in life expectancy.

Life expectancy by birth, by sex: United States, 2000-2021 NATIONAL CENTER FOR HEALTH STATISTICS, NATIONAL VITAL STATISTICS SYSTEM, MORTALITY Despite being the richest country in the world, the U.S. has one of the lowest life expectancies of any developed country. The US has also seen one of the largest declines in life expectancy among such countries during the pandemic, according to World Bank data. 933

By contrast, Japan has 36.73 Covid deaths per 100,000 people compared to the US’s 324.25 deaths per 100,000 people.

Life expectancy changes in 2019–2020 and 2020–2021 across countries. The countries are ordered by increasing cumulative LE losses since 2019. The two line segments indicate the annual changes in LE in 2020 and 2021. Red segments to the left indicate SCHÖLEY, J., ABURTO, J.M., KASHNITSKY, I. ET AL. LIFE EXPECTANCY CHANGES SINCE COVID-19. NAT HUM BEHAV (2022). HTTPS://DOI.ORG/10.1038/S41562-022-01450-3 However, the US is not the only country to experience a drop in life expectancy. Globally, research from Nature shows that only four countries, Belgium, France, Sweden, and Switzerland, have returned to pre-pandemic levels of life expectancy. The researchers calculated life expectancy using a technique called a period life table. The technique requires researchers to imagine a group of 100,000 hypothetical infants and apply the death 934

rates observed for the real population in 2021 for each year of those infants’ lives. The data produced is not the life expectancy for actual babies born in 2021 but rather how life expectancy rates would apply to various age groups at a specific point in time Life expectancy in the U.S. was on a gradual upward trajectory in the past century, with a few notable exceptions, including the 1918 influenza pandemic, World War II, and the HIV crisis. The Covid-19 pandemic has eroded much of that progress, with over 50 percent of the total causes of death contributing to the change in life expectancy in the United States in 2020 and 2021 directly related to Covid-19 infection. Unintentional injuries driven largely by drug overdoses also contributed to 15 percent of causes of death contributing to the change in life expectancy. Increases in deaths from heart disease, chronic liver disease, and suicide also contributed. All of these conditions are part of what I refer to as inter-related Covid syndemics. The impact of Covid-related Post Traumatic Stress can not be underestimated. There were 91,799 drug overdose deaths in the U.S. in 2020, a 30% increase from 2019, which began accelerating in March 2020, and rates of mental health disorders have also surged during the pandemic. Americans have reported rates of depression and anxiety six times higher than in 2019. Creating integrated healthcare systems that treat the physical, mental and social health of patients will be critical in fighting the syndemics such as PTSD and addiction that accompany the Covid-19 pandemic. The drop in life expectancy would have been greater if it had not been partially offset by declines in influenza and pneumonia deaths, which were likely reduced by pandemic-related precautions such as masking and social distancing. However, with few protective measures left in place against respiratory illnesses and a severe influenza season expected based on data from Australia’s season, these numbers are likely to rise. What is particularly disturbing is how we continue to see certain races and ethnic groups continue to be disproportionally affected by Covid-19. Non-Hispanic Native American and Alaska Native peoples saw the biggest decline in life expectancy of 6.6 years. Life expectancy decreased by 4.2 years for the Hispanic population, by four years in the non-Hispanic Black population, by 2.4 years in the non-Hispanic white population, and by 2.1 years in the nonHispanic Asian population. In 2021, the Native American and Alaska 935

Native populations had the lowest life expectancy of any race or ethnicity: 65.2 years. This is equivalent to the life expectancy of the total U.S. population in 1944.

US Life Expectancy by Race or Ethnicity, 2006-2021 NATIONAL CENTER FOR HEALTH STATISTICS, NATIONAL VITAL STATISTICS SYSTEM, MORTALITY Many Native American and Alaska Native individuals, along with Hispanic and Black people, suffered disproportionately high death rates during the pandemic’s first year because many worked in essential jobs with a high COVID exposure risk. The researchers from the CDC study suggested that Indigenous peoples were disproportionately affected because of high rates of chronic disease and poor healthcare access before the pandemic. We need far greater public awareness, community relations, and accountability in our health systems to ensure that these disparities do not continue to widen. This article is featured on Forbes.org, and can be read online here: The Dramatic Cost Of The Covid-19 Pandemic: A Historical Drop In Life Expectancy

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How Strength Training Can Help Post-Covid Recovery Forbes | October 28, 2022 | Article

Covid-19 infection affects each person differently, and the recovery journey is no different. Although you may want to hit the ground running following infection, it is important to listen and adapt to your body’s needs. Consult with your primary care physician about what steps are appropriate or may enhance your recovery. Resistance training has shown to be beneficial and a potentially interesting activity to consider. When done correctly, strength exercises improve mental and functional capacities across all age groups. Below, I highlight existing literature describing the benefits of strength training when done alone, or in a combination with aerobic exercise. What We Already Know A cannon of literature has demonstrated the health benefits of strength training, especially resistance exercise. In a meta-analysis studying participants who were healthy, “non athletic,” and at least 18 years of age, researchers found resistance training helpful in reducing resting blood pressure, improving flow-mediated dilation (reducing the risk of poor cardiovascular outcomes), enhancing cardiorespiratory fitness, and favorable blood biomarkers (especially among older adults). In a separate literature review focusing on mental health benefits associated with strength training, researchers highlight how such exercises have consistently been associated with reduced anxiety symptoms, small to moderate improvements in cognition, positive changes in overall self-esteem and aspects of quality of life and mental health among healthy adults. When done alone or in some combination with aerobic exercise, resistance exercise has also benefited muscle performance and overall quality of life. In a study focusing on the effectiveness of implementing a telerehabilitation exercise program in primary care among patients previously infected with Covid-19, researchers 937

found significant improvements in fatigue and dyspnea, as well as increases in muscle growth and handgrip strength when participants followed a mixed low-or-high intensity aerobic exercise with resistance training. No Need for Fancy Equipment or a Gym Membership Resistance training can be done in the comfort of your home or on the grass at a nearby park. A separate literature review highlighted how such exercises can be well adapted in many different environments! While single-joint exercises like quad extensions or biceps curl are found to make no significant impact on health, multijoint exercises like squats and push-ups are said to produce sufficient muscle gain. In addition, stationary bike training, use of elastic bands, and plyometric training work well. Listen To Your Body; No Size fits all Exercise Regimen Exists Unlike aerobic exercise, resistance training requires less cardiovascular demand, which is a great benefit when resuming (or beginning) to exercise following infection or having to be done in a mask. When done properly, strength training is safe and feasible for all. Short intervals of training periods can induce significant improvements in physical fitness among post-Covid-19 patients. A growing number of studies have found exercise intensity ranging from 30 to 80% of 1RM for resistance exercises and from 3 to 5 on the modified Borg scale for aerobic exercise serve well to post-Covid patients. Specifically, exercise programs based on a combination of low volume resistance exercises (e.g., 1–2 sets of 8–10 repetitions at 30–80% of 1RM) and aerobic exercise (e.g., 5 to 30 min at moderate intensity) may help on the road recovery for post-Covid-19 patients. The combination increases muscle strength, reduces activityinduced shortness of breath and fatigue, and perceptions of quality of life. No individual is the same, however, especially when it comes to exercise. It is important to listen to your body’s needs and work at a sustainable pace. It may be wise to avoid high-intensity aerobic exercise and resistance training, or long sessions of exercise; such activities may induce immunosuppression and increase vulnerability to re-infection. Consult with your primary care provider about next steps to take on your recovery journey from Covid-19. 938

This article is featured on Forbes.org, and can be read online here: How Strength Training Can Help Post-Covid Recovery

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Salt, Fat And Sugar: How Americans Became Addicted To Eating Forbes | October 30, 2022 | Article

“Why can’t I stop myself from overeating?” This is a question that for many cannot be answered simply by a lack of motivation, or genetics. It is the feeling of wanting to eat that last bite of dessert, even though you know that you were full halfway through dinner. Logically, you know that your body does not need any more food, but there seems to be an internal force that fixates on how good that next bite will taste. David Kessler, in his New York Times Bestseller The End of Overeating, describes how the American food industry constructed and continues to profit from this dissociation. The former FDA commissioner lays out the science behind high-caloric food and the brain’s reward systems that has led to an epidemic of overeating. Although more than a decade has passed since this book was published, millions of people continue to struggle with chronic overeating that for some has led to uncontrollable weight. The compulsion to overeat often does not neatly fit into any defined eating disorder. As a result, individuals are blamed for their own lack of motivation to control their weight. Restraining yourself from eating junk food is difficult because that is how it was engineered. This is something that Dr. Kessler writes about firsthand. Regardless of the numerous studies that reveal how sugar and fat hijack your brain’s reward system to crave more, even the former FDA commissioner and physician admits that its pull can sometimes be too much to resist. In this first installment of a multi-part series on the science behind why we overeat, we highlight the lessons from The End of Overeating and Dr. Kessler’s own love/hate affair with food. What is “overeating”? Simply put, overeating is defined as eating more food that your body needs to the point where you feel uncomfortably full. Many of us can recall a time when you were on vacation or at 940

Thanksgiving dinner where you loosened your belt buckle or undid the first button on your pants to make room for another plate of food. Overeating happens unconsciously and often people do not realize that they have eaten beyond their body’s needs until it is too late. Overeating can be a symptom but not, in of itself, be diagnosed as an eating disorder. In extreme cases of binge eating disorder, for example, a person experiences episodes where they compulsively eat large amounts of food in a short period of time, to the point where it causes significant emotional distress. In an effort to offset the consequences of weight gain, an individual may be compelled to purge or take other drastic steps to avoid weight gain. Most people that overeat do not qualify for binge eating disorder. Unless you feel like you cannot physically control yourself from eating and feel a sense of shame or guilt during and after binge eating episodes, then you likely do not have this serious medical condition. Even though overeating is not a defined eating disorder, people that routinely overeat may still experience a lot of anxiety surrounding food. Individuals that appear to be overweight or obese are not the only ones affected. Even people with a relatively normal weight can struggle with a preoccupation with food. Individuals that are prone to overeating often describe feeling like they cannot stop thinking about food. Immediately after finishing a meal, they cannot help but think about the next meal or thing to snack on. Their entire day seems to revolve around eating. What makes some people prone to overeat and not others? Although genetics plays a role in weight maintenance, there is no “fat gene.” In fact, for most of human history, weight across the population remained relatively stable. Obesity was a rare occurrence, and the majority of people ate only until they felt satiated. In the past hundred years, however, the commercialization of the food industry and the outsourcing of home cooked meals to restaurants dramatically increased the weight of the average American. Food not only became more accessible, but it also became a lot cheaper to produce. To cut costs even further, the food industry increasingly incorporated cheap sugar and fat into the American diet. As Dr. Kessler argues, “the problem is not weight, the problem is what we're eating.” It’s Not You, It’s The Food Industry 941

The American diet is designed to appeal to all your senses. Think of your favorite fast-food chain. The first step into the restaurant you get a whiff of that all too familiar smell of grease that automatically makes your mouth water. As you study the menu, plump images of artistically curated dishes draw your attention. Should I also get a side of fries? Those milkshakes look good, too. Just as you place your order, you hear the sound of burgers sizzling on the grill and potatoes entering the fryer. Oh, this is going to be good. In no time, your order is ready. You search for the perfect table, fantasizing about how good that first bite will be. And it is. The first bite melts into your mouth, taking almost no effort to chew. The perfect combination of salt, fat and sugar dances across your taste buds. It goes down almost too easily and before you know it you have eaten hundreds of calories in just one meal. The food industry knows what we like and how to keep us coming back. In his book, Dr. Kessler talks directly to leaders of some of the most successful chains across the country. “What creates irresistibility is caring, attention, visual appeal, and the appeal of aroma, texture, and consistency,” says Jerilyn Brusseau, the woman behind Cinnabon. “Those fat-on-sugar-on-fat-on-salt-on-fat combinations generate multiple sensory effects. Which is just what the industry wants,” Dr. Kessler adds. It is not just fast food. Let’s take a Snickers bar. A nougat cookie bar baked with loads of sugar and fat is then covered with peanuts that are fried in more oil and coated in salt. If that was not enough, a heavy layer of caramel is poured onto the nougat before the entire bar is dipped into a thick milk chocolate mixture. How can anyone resist? In his book, Dr. Kessler extensively interviews Gail Vance Civille, a food consultant that serves as the founding president of Sensory Spectrum Inc.. “The genius of Snickers, explained Civille, is that as we chew, the sugar dissolves, the fat melts, and the caramel picks up the peanut pieces so that the entire candy is carried out of the mouth at the same time,” Dr. Kessler writes. High-calorie foods look, feel and taste good because they tap into our carnal desires to seek pleasure. Like sex or drugs, it targets areas in our brain that keep us wanting more, even after our stomachs are full. Conclusion 942

We cannot eliminate the reward pathway that drives our desire to eat, nor would we want to. Unlike other high-salience stimuli like drugs and alcohol, we need to eat every day to survive. Being aware of these reward pathways, may help us understand why some foods are irresistible and how we may be able to override the impulse to overeat. Admittingly, that is easier said than done. Next in this series, we will take a closer look at how sugar, fat and salt hijack our brains to keep us wanting more. This article is featured on Forbes.org, and can be read online here: Salt, Fat And Sugar: How Americans Became Addicted To Eating

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Covid-19 Infection Linked With Poor Cardiovascular Outcomes And Death Forbes | October 31, 2022 | Article

A new UK study shows that being infected with Covid-19 is linked to an increased risk of poor cardiovascular health and death. The risk is greatest within the first 30 days of infection and for those whose infection required hospitalization, but the risk also remains heightened for those who did not experience a severe infection. When compared with a matched control group of peers who didn’t catch the virus, those infected with Covid-19 were three times as likely to have a blood clot in a vein and more than 10 times as likely to die of any cause. This study is the latest in a rapidly growing body of research demonstrating that Covid-19 infection impacts our health well beyond the acute stage of the illness. A different preprint also concludes that two or more Covid-19 reinfections double the risk for death, blood clots, and lung damage, among other negative health outcomes, reinforcing the fact that Covid-19 is by no means the mild virus that many are treating it as. With surges predicted in winter, we must remain vigilant and take steps to protect ourselves, including wearing high-quality N95 masks, avoiding crowded spaces, and remaining aware of the seropositive rate in your community so you can modify your behavior accordingly and ensuring you receive boosters every 4 months. The researchers used data from 53,613 UK Biobank participants. The UK Biobank tracks the health and survival of its participants through medical records and death registration data. 17,871 participants were diagnosed with a Covid-19 infection between March 2020 and March 2021, and 35,742 were not. Of the 17,871 Covid-19 cases, 2,701 required hospital admission for their infection; 866 were admitted to hospital for another condition; and 14,304 didn't need hospital treatment. All participants were tracked until a cardiovascular problem arose, they died, or until the end of March 2021, providing an average of 141 days of 944

monitoring but ranging from 32 to 395. The cardiovascular outcomes considered included heart attack; stroke; heart failure; atrial fibrillation (irregular heartbeat); blood clot in a vein); pericarditis (inflammation of the heart lining); death from any cause; death from cardiovascular or ischemic heart disease. Those hospitalized because of Covid-19 had higher risks of all the outcomes considered. They were more than 27 times as likely to develop a blood clot in a vein, more than 21.5 times as likely to be diagnosed with heart failure, and 17.5 times as likely to have a stroke. The risk of newly diagnosed atrial fibrillation was nearly 15 times higher, the risk of pericarditis nearly 14 times higher, and that of a heart attack nearly 10 times as high. Those admitted with Covid-19 as a primary reason were 118 times as likely to die as those who didn't need hospital treatment, while those for whom Covid-19 was a secondary diagnosis were 64 times as likely to die. The majority of cardiovascular disease diagnoses, especially atrial fibrillation, blood clots in a vein, pericarditis, and death from any cause, occurred within the first 30 days of infection and among those admitted to the hospital for Covid-19 as the primary reason. However, the heightened risk remained beyond 30 days, particularly for heart failure, atrial fibrillation, blood clot in a vein, pericarditis, and all-cause deaths compared to the population who were not infected with Covid-19. Based on the results of this study, I recommend that everyone who has been infected with Covid-19, mild or otherwise, get a cardiovascular workup within 12 months of infection. I urge anyone who has unexplained cardiovascular symptoms after a Covid-19 infection to take them seriously and seek medical care immediately. Physicians should also be adjusting their screening questions to include past infection with Covid-19 and assess for all Long Covid symptoms, including cardiovascular. Early identification, diagnosis, and treatment of heart disease are essential to lessen the risk of adverse health impacts. Further research is also required to understand if specific interventions are needed to mitigate the risk of a blood clot in a vein associated with Covid-19. This article is featured on Forbes.org, and can be read online here: Covid-19 Infection Linked With Poor Cardiovascular Outcomes And Death 945

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CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example Forbes | November 1, 2022 | Article

This is the fourth installment in a series on the advances CAR T, a remarkable immunotherapy treatment dubbed a “living drug.” Here we draw attention to promising findings for CAR T lupus treatment. The foundations of CAR T as well as applications for B cell cancers and multiple myeloma can be found in earlier works in the series. The previous installments in this series have focused on CAR T therapy as a cancer treatment. A recent study published in the journal Nature Medicine highlights the potential of CAR T therapy beyond this realm—specifically for lupus and other autoimmune diseases. What is Lupus? Lupus (systemic lupus erythematosus) is an autoimmune disease that affects women approximately ten more than men, and is characterized by the overproduction of antibodies that attack the body's own tissues. Lupus symptoms—ranging from mild to lifethreatening—often come and go, making the condition hard to diagnose. Characteristic signs such as fatigue, muscle pains, joint pains and fever also coincide with symptoms of other diseases.

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FIGURE 1: LUPUS SYMPTOMS AND COMPLICATIONS

VECTORMINE Current Lupus Treatments Although lupus has no cure, modern day symptomatic treatments ensure a normal life expectancy for 80-90% of people with lupus. One of our successes at Human Genome Sciences, a company I founded and served as Chair and CEO, was the use of genomics to discover and bring to market the first drug to treat lupus: Benlysta. Although the medicine proved to be effective, for some with lupus even the strongest drugs offer no relief. CAR T Therapy for Lupus In their study, Mackensen et al. test the effectiveness of CAR T therapy for treatment-resistant forms of lupus. The theory derives itself from CAR T cells’ ability to kill cells. In lupus, B cells produce antibodies that attack the body and trigger inflammation (Figure 2). Using CAR T therapy, the researchers aimed to purge the B cell lineage, allowing the body to restore B cells de novo. 948

To do this, the researchers first collected patients’ white blood cells. The patients then underwent lymphodepletion, the use of chemotherapy drugs (i.e. fludarabine and cyclophosphamide) to preferentially kill B cells. As seen in Figure 3, this drug regimen leaves room for the later infusion of engineered T cells, but can be very dangerous if the immune system is too thoroughly depleted.

FIGURE 2: (A) ANTI-CD19 CAR T CELLS TARGET CD19 OFTEN FOUND ON NAIVE B CELLS AND PLASMABLASTS. (B) LYSING THESE B CELLS PREVENTS THE PRODUCTION OF AUTOANTIBODIES—SPECIFICALLY ANTI-DNA ANTIBODIES AND ANTI-RNA BINDING PROTEIN ANTIBODIES—THAT ARE MAJOR DRIVERS IN LUPUS INFLAMMATION. NOTE THAT LONG-LIVED PLASMA CELLS WITHOUT CD19 WOULD NOT BE DETECTED AND CAN CONTINUE AUTOANTIBODY PRODUCTION.

MACKENSEN ET AL

FIGURE 3: THE CAR T CELL PROCESS INVOLVES EXTRACTING T CELLS FROM THE BODY, SYNTHETICALLY ADDING A RECEPTOR THAT DETECTS AND BINDS TO ANTIGEN CD19, AND THEN

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INFUSING THE MANUFACTURED CELLS BACK INTO THE BODY AFTER PREPARATORY CHEMOTHERAPY.

SEC The team altered patient T cells with new genetic information. The new, chimeric T cell products contained a new receptor—a single-chain variable (scFv) fragment poised to detect CD19expressing cells—a 4-1BB costimulatory domain and a CD3 intracellular domain. Figure 4 illustrates these cellular components. The antibody-derived receptor and additional costimulatory structure do not naturally occur on T cells, lending the chimeric nature the therapy is coined after (Chimeric Antigen Receptor T cells).

FIGURE 4: A SECOND GENERATION CHIMERIC ANTIGEN RECEPTOR T CELL COMBINES THE SIGNALING MACHINERY OF A T CELL WITH AN ANTIBODY-DERIVED RECEPTOR.

S. E. LINDNER ET AL.... Results Five patients with severe, treatment-resistant lupus (four women and one man) participated in the study. Lupus impacted several of their organs, including the kidney, heart, lungs, and joints. In addition, these patients did not respond to steroids, antimalarial drugs and other immunosuppressive medicines.

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Each of the patients received a transfusion of modified T cells after chemoablation treatment. The chemoablation successfully depleted patient B cells while T cell numbers remained within normal range. Moreover, the team could no longer detect malignant autoantibodies (ie. anti-double stranded DNA antibodies). The participants’ responses to vaccines also remained largely unchanged, suggesting that the CAR T therapy correctly targeted detreminal B cells without damaging the entirety of the immune system. Three months later, prior symptoms including kidney inflammation, arthritis, fatigue, and heart fibrosis disappeared, and all other immunosuppressive drugs could be discontinued. The symptoms did not return even when B cells began to reconstitute months later. Remission was defined by DORIS, a standardized criteria used to measure lupus symptom severity. Future Possibilities for CAR T This study demonstrates how CAR T can send treatmentrefractory lupus to remission. This is a first hopeful step. The search is now on for ways to improve CAR T induced remission for prior B cell ablation using a cocktail of cytotoxic drugs. The study also opens the door to the possibility of applying CAR T to other difficult to treat autoimmune diseases. This article is featured on Forbes.org, and can be read online here: CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example

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"Naked" Self-Amplifying RNA Vaccine Shows Promise As A Booster, Inducing Strong Cytotoxic T-Cell Responses Forbes | November 02, 2022 | Article

This is the second of two articles on a temperature-sensitive selfreplicating RNA vaccine; click here for part one. They form part of a broader series on next-generation mRNA vaccines. The first three installments can be read here, here, and here, respectively. RNA vaccines have revolutionized traditional vaccine technologies. The current generation of Covid-19 mRNA vaccines rely on messenger RNA that encodes the viral spike protein encased in a lipid particle injected into the muscle. The results have been spectacular in producing powerful, but unfortunately, short-lived protection from infection. The search for even more effective, second-generation, mRNA vaccines is underway. Self-amplifying RNA vaccines, described in this series, offer one promising new approach. A recent preprint by Amano et al. describes one such approach using self-amplifying RNA with some surprising properties. The vaccine candidate uses natural messenger RNA with no modified bases. The mRNA is naked, unprotected by a lipid nanoparticle coat. Moreover, the RNA is injected directly into the skin instead of the muscle. Additionally, the self-amplifying vector is temperature sensitive, active only at the lower temperature of the skin, not at the higher temperature of internal organs and muscle. In a previous article, I covered the how and why of the new vaccine — how it was designed, and the rationale behind it. Here, I take a look at some of the preliminary results, all derived from mouse models. Induction of Robust CD8+ T Cell Immunity The design of the initial vaccine candidate is illustrated in figure 1B. The vaccine is designed to express only a small part of the SARS-CoV-2 spike protein, the roughly 176 amino acid long receptor binding domain (RBD). By contrast, approved mRNA 952

vaccines encode the entire 1273 amino acid long spike protein (Figure 1A). The receptor binding domain is the part of the spike protein that enables it to dock to our cells, initiating viral entry.

FIGURE 1. (A) Diagram of c-saRNA1-RBD vaccine design, including the mutation to NSP2 that confers temperature sensitivity and the antigen construct (CD5 signaling peptide fused to the N terminus of the receptor binding domain of the SARS-CoV-2 proteiSOURCE: ACCESS HEALTH INTERNATIONAL (FIGURE 1A ADAPTED FROM “STRUCTURAL AND FUNCTIONAL INSIGHTS INTO THE SPIKE PROTEIN MUTATIONS OF EMERGING SARS-COV2 VARIANTS” GUPTA ET AL. 2021)

The researchers administered the vaccine to mice intradermally, just underneath the surface layer of the skin, using a microneedle injector commonly used for cosmetic purposes. Fourteen days later, they drew blood samples and analyzed them for immune markers. They then administered a second dose two weeks later and again waited fourteen days before drawing blood samples for analysis. Amano et al. discovered indicators that a strong, selective T-cell response had taken place (Figure 2). In particular, they noted a sharp 953

increase in RBD-specific CD8+ cytotoxic T cells, also known as killer T cells, which bind to infected cells and break apart their cell membrane. This destroys the infected cells, stopping viral replication and curbing the spread of infection. The researchers also witnessed a robust type 1 CD4+ helper T-cell response. Type 1 helper T cells regulate immunity by activating macrophages, which devour foreign pathogens, and by activating killer T cells. This is in contrast to type 2 helper T cells, which instead regulate the immune response by prompting B cells to differentiate into antibody-producing plasma B cells. Markers for type 2 helper T cells suggested only weak activation.

FIGURE 2. (A) Experiment design. (B) Cellular immune responses two weeks after the first dose of the temperature-sensitive self-amplifying RNA vaccine or placebo vaccine (PBO). IFN-y acts as a marker for CD8+ and type 1 CD4+ T cells; IL-4 acts as a mFROM: “CONTROLLABLE SELF-REPLICATING RNA VACCINE DELIVERED INTRADERMALLY ELICITS PREDOMINANTLY CELLULAR IMMUNITY” AMANO ET AL. 2022

As expected, the weak type 2 helper T-cell response coincided with a lack of humoral immunity; the researchers did not find any RBD-specific antibodies following a two-dose regimen of their temperature-sensitive self-amplifying RNA vaccine. Naked RNA for Prime/Boost Vaccinations 954

Many viral vaccines are spread across two or more doses. The first of these is referred to as a prime, and the follow-up dose is called a booster. Some vaccines may be better suited as a prime than a booster, or vice versa. Amano et al. tested their temperature-sensitive self-amplifying RNA vaccine in both capacities. To see how it did as a prime, they first administered two doses of the vaccine fourteen days apart, drawing blood for analysis after each injection. They then waited three weeks before administering a booster dose of recombinant RBD protein. This was compared to a control group, which was given a placebo vaccine as a prime but the same RBD booster. The mice receiving the temperature-sensitive vaccine displayed a rapid spike in RBD-specific antibodies following the booster dose (Figure 3). In the placebo group, the change in antibody titers was minimal. This suggests that, although the temperature-sensitive vaccine on its own fails to stimulate antibody production, it does manage to prime a secondary antibody response. Interestingly, this held true even in mice exposed to a booster shot of the receptor binding domain of the Delta variant instead of the receptor binding domain of wild type SARS-CoV-2. Despite the structural differences between the two spike proteins, the vaccine still managed to prime the humoral response, with a noticeable increase in Delta-specific antibodies post-booster.

FIGURE 3. Antibody titers following a placebo vaccine/RBD boost regimen compared to antibody titers following a temperature-sensitive self-amplifying RNA vaccine (EXG-

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5003)/RBD boost regimen. Black arrows indicate the first two doses of vaccine (Day FROM: AMANO ET AL. 2022...

Having shown their vaccine can act as a prime, Amano et al. next tested to see if it could be used as a booster shot to raise T-cell responses and stimulate a secondary antibody response. They injected mice intradermally with wild type SARS-CoV-2 RBD protein plus adjuvant as the prime vaccine. After two weeks, the mice were given a booster shot of either a placebo dose, a second dose of the RBD subunit vaccine plus adjuvant, or a dose of the temperature-sensitive self-amplifying RNA vaccine encoding the RBD. They also developed and tested an omicron-specific booster (EXG-5003o) by switching out the wildtype RBD of their selfamplifying RNA platform and replacing it with omicron RBD instead. Mice that received the placebo booster did not manage to mount a cellular immune response. Those that received a second dose of the RBD subunit vaccine as a booster did mount a cellular immune response, but only weakly. As expected, the temperature-sensitive vaccine managed to induce robust T-cell responses. The omicronspecific booster induced the strongest T-cell responses. The researchers also measured and compared antibody titers two weeks after the booster had been administered (Figure 4). Here, the temperature-sensitive booster managed to induce a humoral response on par with that of the adjuvanted RBD protein booster. The omicron-specific booster did not fare so well, likely because of the structural differences between the wild type and omicron spike proteins.

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FIGURE 4. The levels of serum antibodies against the RBD protein of the SARSCoV-2 (original strain), measured by an ELISA assay. The levels of antibodies are represented by the OD450 measurement. The average and standard deviation (error bars) of…FROM: AMANO ET AL. 2022

Although the vaccine may not be best suited as a stand-alone vaccine it holds promise as a booster, successfully inducing both humoral and cellular immunity. An Improved Vaccine Amano et al. created a second version of their temperaturesensitive self-amplifying RNA platform in which they switched to a different viral vector. The original alphavirus vector they used contained a common mutation (A551D) as well as a truncated three prime untranslated region. For their new vector, they used an unmutated wild type strain of the virus, including a full-length threeprime untranslated region. The strain they settled on is especially adept at avoiding our innate immune response, giving the virus more time to spread the target antigen before being discovered. The new vector design significantly boosted T-cell responses. Compared to the first version of their vaccine, the updated vector yielded a three-fold increase CD8+ killer T cells (Figure 5). And crucially, compared to a conventional, non-temperature-sensitive self-amplifying RNA vaccine, the researchers witnessed a six-fold increase in T-cell responses. 957

FIGURE 5. (A) Schematic of the experiment design (B) Graphical representation of Tcell responses post vaccination. From left to right: PBO, placebo; saRNA0, conventional self-amplifying RNA vaccine; c-saRNA1, initial iteration of the temperature…FROM: AMANO ET AL. 2022...

Expanding Breadth: Testing a Nucleocapsid-based Vaccine Despite the many uncertainties of the ongoing Covid-19 pandemic, one thing is clear: SARS-CoV-2 has the capacity to evolve and adapt. Wave after wave of infections driven by new variants confirms this. A key goal has been to develop vaccines that confer broad immunity that holds up to these continuous mutations. So called “universal” SARS-CoV-2 vaccines. Amano et al. adapted their temperature-sensitive self-amplifying RNA vaccine platform to fit this need. Instead of targeting the receptor binding domain, which is part of the mutation-prone spike protein, they simply switched out the mRNA to one that encodes the full-length nucleocapsid (N) protein. This is the most highly conserved SARS-CoV-2 protein, only rarely changing across variants. The nucleocapsid protein is also the most abundant of the 958

viral proteins, with the membrane and spike proteins coming in a close second. Both of which make it a viable target. The researchers designed two versions of this vaccine, one that encodes just the full-length nucleocapsid, and another that encodes the full-length nucleocapsid plus the human signaling peptide, CD5 (Figure 2).

FIGURE 2. Schematic representation of the genomes of the two nucleocapsid-based controllable self-amplifying RNA vaccines. Notice that EXG-5005 has the additional CD5 signaling peptide attached to it. FROM: AMANO ET AL. 2022...

The vaccine without the signaling peptide induced only a weak cellular immune response in mice, with lackluster recruitment of both killer T cells and helper T cells. Increasing the dose had no effect. On the other hand, the vaccine that included the signaling peptide induced a robust T-cell response, suggesting that a signaling peptide is required for the effective stimulation of cellular immunity. Given their initial success, Amano et al. also designed a vaccine intended to protect against SARS-CoV-2 variants and other betacoronaviruses — SARS-CoV-1, MERS-CoV. The nucleocapsid proteins of SARS-CoV-1 and SARS-CoV-2 are quite similar, but that of MERS-CoV is only 48% identical to the SARS-CoV-2 nucleocapsid. In order to account for this difference, the researchers fused together the nucleocapsid proteins of SARS-CoV-2 and MERS-CoV, producing a chimera nucleocapsid protein (Figure 3). Again, they also included the CD5 signaling peptide.

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Intradermal administration of the vaccine managed to induce a strong T-cell response against both the SARS-CoV-2 and the MERS-CoV nucleocapsid proteins.

FIGURE 3. A schematic drawing of EXG-5006, encoding a fusion protein of CD5 signal peptide, nucleoprotein of SARS-CoV-2, and a nucleoprotein of MERS-CoV. FROM: AMANO ET AL. 2022

Amano et al. designed one additional vaccine candidate that combined all of the previous elements (Figure 4): SARS-CoV-2 RBD, SARS-CoV-2 nucleocapsid, MERS-CoV RBD, MERSCoV nucleocapsid, and the CD5 signaling peptide. As expected, the vaccine managed to stimulate cellular immunity against all of the viral proteins encoded in the construct, indicating its promise as a truly universal pan-betacoronavirus vaccine.

FIGURE 4. A schematic diagram of EXG-5008, encoding a fusion protein of the signal peptide of CD5, the RBD and nucleoprotein of SARS-CoV-2, and the RBD and nucleoprotein of MERS-CoV. FROM: AMANO ET AL. 2022

In Vivo Challenge: Tumor Cells Next, Amano et al. set out to determine whether the activated CD8+ T cells were successfully destroying infected cells, and by extension, suppressing the spread of the infection. To do so, they engineered tumor cells that expressed the nucleocapsid protein of SARS-CoV-2 and of MERS-CoV. Upon injection, these tumor cells begin to grow and proliferate rapidly, mimicking the dynamics of a viral infection. The researchers administered an intradermal dose of the temperature-sensitive self-amplifying RNA vaccine encoding the MERS-CoV and SARS-CoV-2 nucleocapsid protein. Three weeks later, they injected the engineered tumor cells.

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The tumor cells quickly took hold and proliferated in mice that received only a placebo vaccine. After a period of twenty days, all of them had died from the tumor growth. On the other hand, the growth of tumor cells was largely suppressed in those inoculated with the temperature-sensitive self-amplifying RNA vaccine, with a final survival rate of 40%. The mice that survived were injected with a second round of tumor cells 140 days after initial vaccination; again, tumor growth was quickly suppressed and all of the mice survived. Takeaways The most startling result is the fact that unmodified “naked” RNA —without the support of lipid nanoparticles— was able to induce a powerful CD8+ T-cell response. That alone suggests that this approach may prove valuable in adding additional protection to current vaccine strategies. The experiments also reveal a few weaknesses. Most notably, the absence of a strong humoral immune response. One might argue that the absence of a humoral immune response could be blamed on the use of the receptor binding domain alone, instead of the fulllength spike protein. The scientific literature suggests otherwise, with an abundance of studies confirming high neutralizing antibody titers following injection with an recombinant RBD subunit vaccine. The fact that the nucleocapsid protein-based vaccine also failed to induce a neutralizing antibody response illustrates a shortcoming of this approach as a stand-alone vaccine. That may in part be compensated by use of this vaccine as either a primer or a boost to strengthen the CD8+ T-cell responses of conventional mRNA vaccines, or more traditional vaccines that use attenuated or killed viruses. An additional caveat concerns the temperature-sensitivity of the vaccine; Amano et al. optimized it for skin temperature, which is lower than our core body temperature. This may account, at least in part, for the absence of humoral immunity, since the lymph nodes and germinal centers are located well within the body and function at core body temperature. Germinal centers act as a kind of training camp for B cells. It is where they proliferate and mutate to develop high affinity antibodies against an invading pathogen. If the vaccine antigen is inactive in the germinal centers, then this crucial step of the humoral immune response is omitted.

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Future research should aim to test the temperature-sensitive selfamplifying RNA vaccine platform designed by Amano et al. against in vivo viral challenge with SARS-CoV-2, which is notably lacking from this series of experiments. Additionally, it would be very interesting to compare these results with those achieved by conventional, non-temperature-sensitive self-amplifying RNA vaccines and even with those achieved by traditional mRNA vaccines. In some respects, it is surprising that this data was not included in this study. This article is featured on Forbes.org, and can be read online here: "Naked" Self-Amplifying RNA Vaccine Shows Promise As A Booster, Inducing Strong Cytotoxic T-Cell Responses

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New Monoclonal Antibody Cocktail Neutralizes Lassa Virus Forbes | November 03, 2022 | Article

Mean predicted Lassa risk map for West Africa from the Model 2 series with two absence and one presence clusters, with positive localities indicated by stars. ROGERS ET AL. RISK MAPS OF LASSA FEVER IN WEST AFRICA What is Lassa Virus? Lassa Virus is a pathogen responsible for Lassa hemorrhagic fever, which afflicts between 300,000 and 500,000 people per year, 963

commonly in West African countries, including Nigeria, Liberia, Sierra Leone, Guinea, and Ghana. Lassa fever has a mortality rate of roughly one percent. Pregnant women are at the greatest risk of death, with up to a 90% fatality rate. The symptomatic cases present issues such as fever, headaches, vomiting, and muscle pain. Spread typically occurs via contact with infected mice excrement or urine, though direct contact spread from person to person is also common. Unfortunately, a vaccine is not yet available for the virus, and antivirals are weak at best. Lassa Virus, like SARS-CoV-2, is an RNA virus that constantly mutates. A therapy or prophylactic to fill the void of Lassa Virus drugs is needed. Lassa Virus Monoclonal Antibody Therapy Fortunately, a new candidate may be just around the corner. A group of researchers from the La Jolla Insitute for Immunology in California led by Dr. Erica Ollmann Saphire have identified a cocktail of three antibodies that bind and neutralize the Lassa Virus. This same group discovered a broadly accepted cocktail of monoclonal antibodies for Ebolavirus, which we have previously described. This cocktail arrived just in time as the current to treat the current epidemic caused by the Sudan strain of Ebolavirus in Nigeria. The new Lassa Virus therapy, denoted Arevirumab-3, is comprised of three distinct antibodies, each one binding distinct regions of the Lassa Virus glycoprotein. The Lassa Virus spike protein comes in a set of three identical subunits embedded into the membrane. The virus is synthesized as a single long polypeptide and is cleaved into three parts, a leader sequence, GP1 which encodes the receptor-binding function, and GP2 embedded into the membrane (Figure 1).

FIGURE 1: (A) Schematic of LASV GPC primary structure showing positions of subunits and N-linked glycans. SSP, stable signal peptide; TM, transmembrane domain; C-tail, cytoplasmic tail; SKI-1/S1P, subtilisin kexin isozyme-1/site-1 protease; Asn, aspa PNAS

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(B) LASV spike glycoprotein model. PNAS 8.9F inhibits virion-cell attachment by blocking the GPC/α-DG interaction The first antibody, 8.9F, binds the top of the spike trimer, stretching across all three portions of the glycoprotein. 8.9F is an unusual antibody as the single antibody binds all three faces of the trimer. The antibody also binds the three cleavage sites of the GP1 subunits. It binds the portion of the glycoprotein that attaches to the host cell, directly inhibiting virion-cell attachment by mimicking the cell alpha-dystroglycan receptor. The 8.9F antibody also specifically binds a glycan, N119, which is required for neutralization activity. This glycan is required for alpha-dystroglycan receptor-binding, so 8.9F’s recognition is surprising. A native N89 glycan plays a central role in 12.1F/LASV GPC recognition The second antibody, 12.1F, binds to a separate site on GP1, directly interacting with N-glycans for neutralization activity. The antibody binds more membrane-proximal and has direct interaction with six crucial amino acid residues and critical glycans N89 and N109. To infect a cell, the Lassa Virus must bind the cell membrane and be engulfed by a cellular endosome. Then the protein undergoes 965

a structural change to bind the LAMP-1 protein in the endosome interior. Binding to LAMP-1 is essential for fusion of viral and cell membranes and entry into the cytoplasm where replication occurs. 37.2D neutralizes LASV by locking the GPC trimer in an inactive configuration. The third antibody, 37.2D, binds two adjacent subunits of GP2. The antibody stretches across subunits, locking the trimer in place by binding conserved peptides and conserved glycans N390 and N395. 37.2D is special in that it attacks GP2 at a unique angle, stabilizing the pre-fusion trimer, preventing fusion activation and, therefore, infection. These three mechanisms work together to yield a highly effective antibody cocktail.

FIGURE 2: Cryo-EM density maps of native LASV GPC in complex with 8.9FscFv/37.2D-scFv or 12.1F-scFv/37.2D-scFv. GP1 subunits: cyan; GP2 subunits: pink; N-linked glycans: red; TM: gray. PNAS

FIGURE 3: (A) Structural model (PDB ID: 7UOV) showing the GPC-binding orientation of one 12.1F-scFv molecule. (B) Epitope of 12.1F on the GP1 protomer. (C) Focused GP1-binding region by 12.1F. Residues on interfaces are indicated. GP1: blue; 12.1F HC PNAS

One critical question for monoclonal antibody treatment, is can the virus escape via mutation in the binding sites? Dr. Ollmann 966

Saphire's team shows that such is the case for treatment with a single 8.9 F antibody. However, no resistance mutations arose when using the cocktail of all three antibodies, nor was the virus capable of escaping. The Arevirumab-3 antibody may provide a long-awaited answer to the lack of treatment and prevention of Lassa infections. This is particularly important for cases involving pregnant women and their fetuses. It will be important to reduce the costs of this antibody cocktail as much as possible so that it is available when needed in West Africa. Current technologies allow monoclonal antibodies to be produced at $200 and $250 per gram. The next step in the development of the control of Lassa Fever is the development of a vaccine effective against all Lassa Virus variants. This work may serve as a guide for the creation of such a vaccine. This article is featured on Forbes.org, and can be read online here: New Monoclonal Antibody Cocktail Neutralizes Lassa Virus

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Study Finds That Regular Physical Activity Enhances Vaccine Effectiveness Against Covid19 Forbes | November 04, 2022 | Article

A recent case-controlled study in South Africa finds that regular physical activity enhances vaccine effectiveness against Covid-19. While the positive impact of regular physical activity against severe Covid-19 outcomes is well understood, the association between regular physical activity and vaccine effectiveness is underexplored. The study predominantly used physical activity data from 196,444 participants and concluded that increased levels of regular physical activity improved the protective effects of vaccination against Covid-19 hospitalization and exhibited a dose response. Researchers conducted a negative case-control study and a retrospective analysis from data collected by Discovery Health and Vitality. A negative case-control is commonly used to assess the effectiveness of the annual influenza vaccine; studies would calculate the odds ratio of testing positive when vaccinated against specific pathogens and testing negative when vaccinated against a specific pathogen. In this case, researchers were interested in estimating the hospitalization admission for Covid-19 among those who are vaccinated and unvaccinated based on their “regular” physical activity levels. Collie et al. used anonymized data from Discovery Health and Vitality and derived information from members who had a partnership from mid-February to late October 2021. Participants were at least eighteen years old and categorized into one of the three physical activity levels based on their average monthly physical point allocation two years prior to the study: low activity (less than 60 minutes of at least moderate intensity activity per week), moderate activity (those engaging in 60 and 149 minutes of at least moderate intensity activity per week), and high activity (engaged in at minimum of 150 minutes of at least moderate activity per week). To 968

qualify for the study, participants had to submit their proof of Covid19 infection at the beginning of the study, given that they did test positive up to 3 months prior. Vaccinated participants were healthcare professionals who were vaccinated as the result of a separate study. Unvaccinated participants were a mix of healthcare and nonhealthcare professionals that are members of Discovery Health and Vitality. Through stratified random sampling, roughly proportional ratios of unvaccinated to fully vaccinated participants within each physical activity group. There was a slight difference in the biological sex between the three groups: 64.1% of the low-activity group were female, compared to the 56.4% and 50.9% of the moderate and high-intensity physical activity groups. Participants in the moderate and high activity groups also had slightly higher Covid-19 risk factors on average compared to the low acting group. Collie et al conducted three sensitivity analyses to investigate the vaccine effectiveness among and within the three groups in relation to their physical activity levels. In the first sensitivity analysis done, the vaccine effectiveness among the fully vaccinated participants in each group compared to the full population. No statistical differences were found, suggesting that the sample was representative of the population. Collie et al conducted a single multinomial Poisson regression model, which estimates the relative risk of hospitalization given the physical activity level. Compared to unvaccinated individuals with low physical activity, there was a 91.5% vaccine effectiveness for vaccinated individuals with high physical activity. Additionally, using unvaccinated individuals with low physical activity as baseline, there was a difference of vaccine effectiveness between vaccinated individuals in the high intensity physical activity and vaccinated individuals in the moderate intensity exercise of about 28.1%. This sensitivity measure also found the difference between hospitalization vaccine effectiveness between those who were vaccinated at least 28 days prior to the study with low physical activity and those who vaccinated at least 28 days prior to the study with high physical activity to be 25.8%, which aligns with the main study’s findings. In a third sensitivity analysis, Collie et al used Bayesian modeling, allowing to test the hypothesized causal relationships between vaccine effectiveness and physical activity levels. This analysis 969

determined a 25% difference in vaccine effectiveness between vaccinated participants performing high-intense physical activity and vaccinated participants performing moderate-intensity physical activity, relative to those unvaccinated with low activity. It also demonstrated the main difference to be 25.8%, which supports findings from the multinomial position regression analysis and main study’s findings. Collie et al found that vaccine effectiveness and physical activity seem to have a dose-response relationship, where increased regular physical activity enhances vaccine effectiveness against Covid-19 hospitalization. Among the fully vaccinated, vaccine was most effective among those with high activity levels (85.8%), which was notable higher than those with low physical activity (60%). The difference of 25.8% was supported by the two sensitivity analyses, and these findings further validate the World Health Organization recommendation of regular physical activity (150-300 minutes of moderate to intense activity per week), as it may have meaningful health benefits like preventing severe disease. Collie et al’s findings align with existing literature focusing on increased vaccine effectiveness with regular physical activity. One study measured antibody levels among 898 patients with autoimmune rheumatic disease roughly two months after becoming fully vaccinated with the CoronaVac vaccine. After adjusting for covariates, active patients exhibited greater antibodies and geometric mean titers(GMT) compared to their counterparts with sedimentary or very low physical activity. Researchers also noticed a doseresponse relationship between physical activity and presence of Covid-19 antibodies and GMT: patients performing at least 350 minutes of physical activity a week had more Covid-19 antibodies and higher GMT compared to the least active group exercising at most 30 minutes a week. This study suggested that a lifestyle incorporating greater physical activity may help protect immunocompromised communities by enhancing Covid vaccine immunogenicity. In a separate study, physical activity was associated with enhanced antibody presence up to 6 months post-vaccination among immunocompromised patients. Through logistic regression analysis, higher rates of seroconversion and neutralizing antibodies were found among patients with higher levels of regular physical activity. 970

Although Collie et al encourages increased regular activity to maximize the protection of the Covid-19 vaccine, it is important to recognize that the study is not without limitations. This study was done prior to the emergence of Omicron. Additionally, it is likely that participants in the sample were of higher socioeconomic status, which is an inadequate representation of the entire population of South Africa. A healthier diet and better sleeping patterns may also contribute to higher vaccine effectiveness among the highly active participants compared to their low physical activity counterparts. Lastly, the biological mechanism behind vaccine effectiveness and physical activity remains unclear, in large part because of the lack of immunogenicity and cell-mediated response analyses within the study. Additional studies are needed to validate the study’s main findings. This article is featured on Forbes.org, and can be read online here: Study Finds That Regular Physical Activity Enhances Vaccine Effectiveness Against Covid-19

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Female Healthcare Workers Have Faced Heightened Moral Distress Psychology Today | November 07, 2022 | Article

KEY POINTS • Moral distress occurs when someone is aware of the ethical action to take but is constrained by internal or external factors from taking it. • Many women healthcare providers felt unable to provide an ethical standard of care while maintaining COVID-19 prevention protocols. • Some underlying constraints pre-dated COVID-19 and are notably gendered, requiring structural change to aid COVID-19 recovery efforts. New research shows that female healthcare providers were disproportionally affected by moral distress during the COVID-19 pandemic compared to their male counterparts. The authors concluded that female healthcare providers were more likely to experience a “double dose” of moral distress both at home and in the workplace. Moral distress is a complex and challenging problem affecting healthcare providers more frequently during a crisis or disaster but also during circumstances such as end-of-life care or understaffing. It should be distinguished from the term "burnout," which is often used to describe the effects of ongoing stress in the workplace, but the two conditions can overlap. Moral distress occurs when someone is aware of the ethically correct action to take but is constrained by internal or external factors from taking it. It can include physical, emotional, and psychological symptoms, such as headaches, palpitations, gastric upset, anger, guilt, frustration, withdrawal, and depression. In addition to these symptoms, when left unaddressed, moral distress can affect the quality of care patients receive and result in healthcare providers leaving the profession. I discuss the impact of 972

moral distress, moral trauma, and moral injury on healthcare providers and the broader population during the COVID-19 pandemic in my book, COVID-Related Post Traumatic Stress Disorder (CV-PTSD): What It Is and What to Do About It. The research was based in British Columbia, which had some of the highest COVID-19 case numbers (84,569 confirmed cases) in Canada during the first year of the pandemic. The researchers conducted 16 focus groups with 66 participants, 12 semi-structured interviews, and 10 key informant interviews with those in management positions in the health system and representatives of unions and professional organizations between December 2020 and March 202. Healthcare providers interviewed included workers from community health, long-term care, nurses, and midwives. Pre-COVID-19, many of the women interviewed already worked in environments where external constraints related to the increasing privatization of healthcare restricted their ability to provide quality care. In 2019, the province had the lowest number of registered and licensed practical nurses working in direct care in Canada, leading to severe staffing shortages. There was also a significant gender wage gap of 15.8 percent in 2020, with a disproportionate number of women fulfilling lower-paid positions, such as care aids, and men dominating leadership positions. Women in Canada also remain the primary care providers within households and families, doing 2 to 3 times more unpaid care work than men. The closures of schools and childcare centers and mandatory isolation periods have continued to impose heightened care burdens on women. A survey conducted in April 2021 found 71 percent of mothers were “at the breaking point” due to stress and anxiety. Researchers investigated how participants responded to challenges related to moral events, which were categorized as constraints, conflicts, dilemmas, or uncertainties. Moral constraints at work and home Moral constraint is understood as the inability to carry out a preferred personal moral requirement due to external or internal constraints. Many participants spoke about how inadequate staffing and a lack of PPE led to moral constraints related to the quality of care. During the early stages of the pandemic, midwives couldn't

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access PPE from the government supply, forcing them to source and reuse their own PPE. All participants noted that, as women, they were primarily responsible for unpaid care in their families and that unpaid care work had increased dramatically due to COVID-19-related childcare, schooling, and service interruptions, as well as due to the needs of vulnerable family members. Many reported feeling guilty that they could not adequately support their children's well-being and education during the pandemic. Moral conflict at work and home Frontlines female healthcare workers often felt decision-makers, including supervisors or managers, were too distanced from the realities of care work to understand the consequences of COVID19 protocols. Many female healthcare workers reported that attempts to adapt their schedules and accommodate childcare duties faced resistance at work. A moral dilemma at work and home Women healthcare providers felt unable to provide an ethical standard of care while maintaining COVID-19 prevention protocols. Nurses were instructed to spend as little time as possible with patients to reduce transmission risk when patients needed increased emotional support because they were isolated from their families. With insufficient PPE in the workplace, they also felt they were putting their families at heightened risk. Moral uncertainty at work Constantly changing information about COVID-19, particularly early on, made it difficult for healthcare providers to know how to best protect their patients/residents. They linked uncertainty and lack of communication to distress. While some sources of moral distress were entirely out of the control of healthcare providers, many also reported actions that they took to fight moral distress. Midwives collaborated with hospital managers and other HCPs to successfully advocate for access to government-supplied PPE, and workers in long-term care joined unions to advocate for improved staffing policies. Many also increased counseling sessions or began therapy during the pandemic. Unfortunately, midwives were the only healthcare providers who reported not having access to employer-provided mental health support or extended benefits. They were forced to pay out of pocket 974

for mental healthcare, often racking up credit card debts and causing an additional source of stress. However, our responses to this crisis must go beyond short-term mental health interventions to address the underlying constraints, many of which pre-date COVID-19 and are notably gendered, around working conditions and investments in the care economy. Such structural change will not only strengthen COVID-19 recovery efforts but will also better prepare health systems for future pandemics. This article is featured on Psychology Today, and can be read online here: Female Healthcare Workers Have Faced Heightened Moral Distress

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CAR T Therapy For Cardiac Fibrosis: A New Method Forbes | November 09, 2022 | Article

This story in the CAR T series delves into recent adaptations to treat the heart. Earlier installments cover the fundamentals of CAR T, as well as its applications for B cell cancers, multiple myeloma and lupus. CAR T therapy, a “living drug,” traditionally involves isolation and purification of T cells outside the body. The cells are then modified with a synthetic receptor and then re-infused into the body for treatment of cancers. Researchers have now successfully demonstrated that T cells can be modified in vivo by mRNA technology, bypassing the need for extraction, chemotherapy and reinfusion. Although this method proves effective in treating mice with scarred hearts, considering fibrosis contributes to over 800,000 deaths worldwide, the study contains great potential for human treatment. A Damaged Heart The heart, flexible yet strong, circulates blood through the body by pumping blood through its chambers. Aging and injury tamper with this function, creating scarred and thickened tissue called fibrosis. Although fibrosis occurs normally when healing, a highly fibrotic heart loses its elasticity; the stiffened tissues and interrupted electrical signaling prevent proper contractions of the heart (see Figure 1). Cardiac fibrosis is highly associated with heart disease and heart failure. Cardiac fibrosis has no “cure-all” treatment. Early detection improves prognosis, but options dwindle as damage progresses irreversibly. People with advanced cardiac fibrosis may take drugs which antagonize overstimulation of the heart or might even require heart valve replacement.

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FIGURE 1: Comparison of the cells in a healthy heart and the cells seen in cases of cardiac fibrosis. Note the thickened extracellular matrix, which impedes on normal heart function in several ways.

CREDIT: Liu et al. https://www.sciencedirect.com/science/article/pii/S0169409 X21001009 How CAR T Cells Work In their study, Rurik et al. explore a new method to directly counter cardiac fibrosis. This method builds upon the basics of CAR T: the use of T cells with a synthetically engineered receptor to target and kill specific cells. CAR T is approved to treat people with certain lymphomas, leukemias, and multiple myeloma. Figure 2 illustrates this process. In these cases, the desired T cells are extracted from the patient’s body. Synthetic mRNA is inserted into the cell with a retrovirus, a virus commonly used in gene therapy to permanently change other cells’ genomes. The altered and expanded cells are then infused back into the body after preparatory chemotherapy. These T cells target either CD19 or BCMA, two antigens found on malignant B cells.

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FIGURE 2: The CAR T process has several steps. T cells must be extracted from the blood, then genetically modified with a new receptor and expanded to great numbers. The patient prepares with chemotherapy before the CAR T cells are introduced into the bloodstream.

CREDIT: Cleveland Clinic https://my.clevelandclinic.org/health/treatments/17726-car-tcell-therapy The benefit of inserting genetic information with a retrovirus lies in its permenance. The CAR T cells can expand and persist in the body for a long time after infusion, continually fighting the cancerous cells they encounter. However, this is of no benefit to researchers hoping to fight cardiac fibrosis. If T cells continuously target fibrotic cells, they would impair normal healing processes and potentially induce autoimmunity. Rurik et al. employ an elegant solution which shortens the CAR T cells’ active duty, thereby circumventing the extraction process altogether. New CAR T Cell Design The team adapted mRNA delivery technology seen in current COVID-19 vaccines and applied it to basic Chimeric Antigen 978

Receptor design. The mRNA does not integrate into the T cell genome, allowing for temporary transcription of the mRNA and transient expression of the new receptor. CD5 Lipid Nanoparticles (LNP) The authors adopted a strategy to introduce the chimeric receptor to T cells in the body rather than extracting and purifying them outside the body. To accomplish this aim, they first synthesized mRNA that encodes a receptor against fibroblast activation protein (FAP), a protein expressed on activated fibroblasts responsible for fibrosis. They purified the mRNA and packaged the engineered mRNA into standard lipid nanoparticles (LNP). The team then decorated the lipid nanoparticle surface with CD5 targeting antibodies to direct lipid uptake. The integration of CD5 antibodies allowed the lipid nanoparticles to target antigen CD5 naturally expressed by T cells once injected into the body; the CAR T cells are made after a single shot. Chimeric Antigen Receptor The chimeric antigen receptor contains a single chain variable fragment (scFv) derived from fibroblast activation protein monoclonal antibodies; this recognition domain enables the CAR T cell to target cells which express fibroblast activation protein. The CAR design also includes CD28 and CD3z signaling domains in the cytoplasm. All three components are mouse-specific. Not illustrated in Figure 3 is an added small peptide which prevents immune suppression. (“prevents adenosine- and prostaglandin E2-mediated suppression”)

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FIGURE 3: The mRNA encoded for a chimeric antigen receptor composed of a fibroblast activation protein (FAP) antigen recognition domain and a CD28 and CD3z signaling domain in the cytoplasm. The team also included an additional peptide to prevent immune suppression.

CREDIT: CreativeBiomart https://www.creativebiomart.net/Targets-of-CAR-T-CellTherapy.htm Genetic Integration In Vivo The team found that lipid nanoparticles could successfully deliver the mRNA package to T cells, as seen in Figure 4. The killer T cell absorbs the lipid nanoparticle by endocytosis. The lipid particle then degrades and the synthetic mRNA releases into the cell. Finally, the cellular machinery reads the genetic instruction and briefly produces the receptor against fibroblast activation protein. This is possible with both animal and human T cell cultures.

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FIGURE 4: To create a CAR T cell with transient CAR expression, a lipid nanoparticle (LNP) with the desired genomic information is absorbed by the T cells through endocytosis. Once inside the T cell, the lipid nanoparticle degrades and releases the mRNA which encodes for the desired receptor. The expressed receptor allows the T cell to detect fibroblast activation protein located on the surface of many activated fibroblasts.

CREDIT: Rurik et al. https://www.science.org/doi/10.1126/science.abm0594 Transitory CAR Expression Unlike traditional CAR T cells that carry a chimeric receptor encoded by DNA inserted into the genome, these CD5+ T cells carry mRNA only transiently. The mRNA is not integrated into the cell’s genome and remains stuck in the T cell cytoplasm before degrading. This is ideal; fibroblast activation protein receptors must be expressed briefly as longer expression may harm other tissues. Results The research team assessed the efficacy of the CAR T cells in different conditions. When they treated the cells in tissue culture, more than 80% of T cells expressed the chimeric antigen receptor and could effectively kill target cells with fibroblast activation protein. The team then tested this model on mice with cardiac fibrosis. The mice received medication to injure the heart and induce scarring. After one week, the team administered the lipid-mRNA injection. Consistent CAR expression was noted 48 hours after injection, and disappeared after one week. The results were impressive. The function of the heart’s largest chamber improved, in some cases returning to uninjured levels. Similarly, the amount of blood filling the heart normalized to safe volumes. The therapy notably reduced the thickness of the heart. 981

Finally, although the mass of the largest chamber did not normalize, it trended towards improvement. One caveat in lipid-CAR T cell delivery is that some cells, perivascular fibroblasts, do not express fibroblast activation protein. In consequence, these cells were not impacted by CAR T cells and some fibrosis persisted. No overly toxic side effects were noted. Trogocytosis A key observation of effective CAR T therapy is the ability of the modified T cells to take small bites of the target cell—a phenomenon known as trogocytosis. Deriving “trogo” from the Greek word “to bite,” trogocytosis entails one cell nibbling another and, in the process, transferring the surface molecules from one to the other. The researchers found evidence of CAR T cells “nibbling” the activated fibroblasts and retaining the stolen antigens (illustrated in Figure 5), suggesting that the T cells successfully adopted the chimeric antigen receptors in vivo.

FIGURE 5: Trogocytosis occurs when a cell ingests small “bites” of another cell, thus taking the surface molecules from one cell and expressing them on its own cell surface. Here, the T cell detects the activated fibroblast, ingests parts of its surface, and then expresses the ingested surface molecules.

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CREDIT: Rurik et al. https://www.science.org/doi/10.1126/science.abm0594 Future Implications CAR T therapy revolutionized cancer treatment with its efficacy and innovation. Combining mRNA technology to this therapy creates a temporary version of this “living drug” that does not sacrifice on quality. The therapy is well tailored to heal mice with damaged and scarred hearts, and widens the possibilities to treat other non-cancerous human ailments. If translated to clinical settings, transient CAR T therapy may be less expensive and more readily available than its traditional counterpart. This article is featured on Forbes.org, and can be read online here: CAR T Therapy For Cardiac Fibrosis: A New Method

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The Best Reason To Keep Up To Date With Boosters: Covid-19 Protection From Infection, Hospitalization And Death All Wane Over Time Forbes | November 10, 2022 | Article

A report by the United States Centers for Disease Control and Prevention (CDC) makes clear that vaccines can only protect us if we let them: just as protection from infection wanes over time, so too does protection against serious disease and hospitalization. The message is clear, if we want to keep our guard up against the virus, we need to keep up with our booster shots. What follows is an overview of the latest data on vaccines, immunity, and risk mitigation. Protection Against Infection The gold standard for any vaccine is protection against the acquisition of infection — if you can’t get infected, you can’t get sick. But for most respiratory viruses, this is a high bar to set. Diseases like polio and measles grant us lifelong, “sterilizing” immunity after infection. By extension, so do the vaccines that protect against them. But unlike polio or measles, infection with a respiratory virus does not provide us with lasting immunity. This is why, for example, we have yearly flu seasons, with reinfections a common occurrence. Vaccine-induced immunity wanes accordingly. Think of this as a general rule, and a thorn in the side of vaccinology: if natural infection doesn’t grant lasting immunity, it is going to be difficult to design vaccines that do. Covid-19 is no exception. Recent data from Qatar suggests that pre-omicron infections are only 35.5% effective at protecting against symptomatic reinfection with the BA.4 or BA.5 subvariants. This plummets to 27.7% when asymptomatic infections are taken into consideration as well. And making it clearer still, reinfection with the same variant has also been documented.

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The main driver of reinfections is viral mutation — changes to the structure of SARS-CoV-2, in particular the spike protein it uses to bind to our cells, allow the virus to bypass previously built-up immune responses. In brief, our immune system doesn’t properly recognize the virus because of the mutations. Even without the presence of new variants, reinfections can still occur. Although we do not yet have an exhaustive understanding of the biomarkers associated with immune protection, known as “correlates of protection”, there is enough evidence to suggest that neutralizing antibody titers play a key role. These antibodies home in on the spike protein and bind to it, preventing the virus from entering our cells. The higher the number of neutralizing antibodies, the higher the protection against infection. But neutralizing antibody titers begin to drop off significantly —up to a five-fold decrease— within three to four months of vaccination. In both cases, the best way to mitigate the risk of another bout of Covid-19 is staying up to date with your booster shots. Since antibody titers begin to wane as quickly as three months following the last dose, I would recommend getting a follow-up booster every four to six months. Variant-Specific Booster Shots High levels of neutralizing antibodies are crucial, but they are only as useful as they are “accurate.” All the antibodies in the world won’t do any good if they can’t bind to the relevant antigen. Mutations, by changing the structure of the antigen, make things difficult for our antibodies. Best case scenario, the antibodies still bind the antigen, but only more weakly than before. Worst case scenario, the antibodies no longer fit the antigen at all. It is for this same reason that we need to update our flu vaccines every year, to make sure the antigens in the vaccine match those of the latest strains in circulation. When a mismatch does occur, we end up with a more severe flu season. Again, Covid-19 is no exception. For most of the pandemic, we have relied on first-generation Covid-19 vaccines to also act as boosters. Generally, these vaccines are formulated on the spike protein of the wild type SARS-CoV-2 strain. But this strain has long since been outcompeted by wave after wave of variants, all of which, owing to mutations, have slightly 985

different spike proteins. The original vaccines have become poorly matched to the circulating strains. Fortunately, the mRNA vaccine platform is extremely adaptive and can quickly be updated to accommodate new variants; the mRNA sequence encoding the original, wild-type spike protein simply needs to be swapped out for a sequence that encodes the spike protein of the dominant variant. Or if not swapped out, then added in along with the sequence encoding the original spike protein. Earlier this fall, the FDA authorized two such “bivalent” booster shots. One from Pfizer-BioNTech and one from Moderna. In both cases, the booster vaccine has been formulated to contain the genetic sequence for the wild-type spike protein as well as the sequence for the Omicron BA.4/BA.5 spike protein. We are now beginning to see the data trickle in. Despite a rocky start, with preliminary results suggesting no difference in protection between the original, monovalent vaccines and the updated, bivalent booster shots, the tides are turning. A recent press release from Pfizer reports that people aged 55 and up enjoyed a four-fold increase in neutralizing antibody titers against Omicron BA.4/BA.5 compared to those who received a booster shot of the original Covid-19 mRNA vaccine. Compared to those who received no booster shot, neutralizing antibody titers a month after vaccination were 13 times higher in adults older than 55. Younger adults also saw the benefits, with a nine-fold increase in antibody titers. In a study posted on the preprint server bioRxiv, researchers discovered that the bivalent booster shot also remains effective against the newest members of the Omicron family, BA.2.75.2 and BQ.1.1. Good news, considering these two sublineages are quickly rising to dominance across the globe. Although these are still the early days, and we need to wait for real-world data to start flowing in before making any firm conclusions, these initial results are promising. They suggest that updating boosters to match circulating variants is a viable strategy: variant-specific boosters ensure not only high levels of neutralizing antibodies, but high levels of the right kind of neutralizing antibodies. Vaccine Protection Against Serious Disease, Hospitalization, and Death also Wane with Time

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The above doesn’t come as anything new — it has been clear that neutralizing antibody titers fade over time, and along with them, protection against infection. We also knew that novel variants can render old vaccines ineffective. But the latest CDC report challenges one of our standing hopes: that even as protection against infection fades over time, infection against severe disease and hospitalization remains strong. Their work suggests otherwise. The CDC researchers collected data through the Influenza and Other Viruses in the Acutely Ill (IVY) network, consisting of 21 large hospitals spread across 20 different cities in 18 states. Founded in 2019, the initial purpose of IVY was to track influenza vaccine effectiveness among patients admitted to the intensive care unit (ICU). Following the outbreak of Covid-19, the initiative expanded to also enroll hospitalized Covid-19 patients. Their latest analysis is based on a group of 4,730 adult, immunocompetent patients enrolled between December 26, 2021 and August 31, 2022. Enrolled patients were then split into four different groups, depending on their vaccination status: 1) those who had not been vaccinated before contracting the virus, 2) those who had received two doses of the original mRNA vaccines at least fourteen days before contracting the virus, 3) those who received two doses plus a booster dose of the original mRNA vaccines at least seven days before disease onset, and 4) those who received two doses plus two booster shots of the original mRNA vaccines, again at least seven days before disease onset. The team of scientists found that, in those who received two doses, vaccine effectiveness against hospitalization during the period of BA.1/BA.2 predominance hovered around 63%. After 150 days, vaccine effectiveness dropped down to 34%. In the group that received two doses plus a booster dose, vaccine effectiveness against hospitalization started at 79% before dropping to 41% after 120 days (Table 1).

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TABLE 1. Vaccine effectiveness against hospitalization during Omicron BA.1/BA.2 wave, 7 to 150 days since last dose and 150+ days since last dose. SOURCE: ACCESS HEALTH INTERNATIONAL

A similar waning of protection against hospitalization was seen during the BA.4/BA.5 wave of infections. Here, vaccine effectiveness in the two-dose group began at 83% but plummeted to a mere 37% after 150 days. After two doses and a booster shot, vaccine effectiveness stood at 60% before dropping down to 29%. And vaccine effectiveness after two doses and two booster shots stayed at roughly 60% for the duration of 120 days (Table 2).

TABLE 2. Vaccine effectiveness against hospitalization during Omicron BA.4/BA.5 wave, 7 to 150 days since last dose and 150+ days since last dose. SOURCE: ACCESS HEALTH INTERNATIONAL

Clearly we can’t rely on prior vaccination to provide us with failsafe protection against severe disease and hospitalization if our last booster shot was more than 120 days ago. The booster shots work, but we can’t expect miracles; you wouldn’t expect a flu shot from last year to protect you this year, and it’s no different with Covid19. Until the development of long-lasting, “universal” coronavirus vaccines, our best strategy for protection against infection and hospitalization is staying up to date with our booster shots. Even better if they are variant-specific. This comes on top of continued mask-wearing, social distancing, and a general avoidance of crowded indoor spaces. This article is featured on Forbes.org, and can be read online here: The Best Reason To Keep Up To Date With Boosters: Covid-19 Protection From Infection, Hospitalization And Death All Wane Over Time

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Reimagining Alzheimer’s (Part 5): Setback For Potential Alzheimer’s Treatment Forbes | November 12, 2022 | Article

This article is the fifth installment in my series on Alzheimer’s disease. Read more about Alzheimer’s disease in part 1, part 2, part 3, and part 4 of the series. In the last installment of my series on Alzheimer’s disease, I discussed a new experimental treatment for Alzheimer’s called lecanemab. This September, clinical trial results suggested that lecanemab could effectively decrease cognitive deficits in Alzheimer’s patients. Unfortunately, it now seems that lecanemab may have played a role in the death of one of its clinical trial participants, introducing new skepticism as to whether the medication is safe for use. While the death is still under investigation, this unfortunate event emphasizes that all medications come with their own risks that can be hard to predict. The death was mainly attributed to significant swelling and bleeding in the patient’s brain. The patient was regularly taking a blood thinning medication that may have exacerbated their symptoms. Eisai, one of the drug manufacturers, was quick to point out other factors unrelated to lecanemab that may have played a role in the patient’s death. In particular, the patient was reported to have experienced several falls, a heart attack, a respiratory infection, and multiple stroke-like events within the past few months. But how could lecanemab itself have contributed to the death of the patient? Lecanemab works by alleviating the burden of amyloid protein in the brain and is an antibody-based drug. This means that it directly identifies and binds to harmful amyloid proteins in the brain to eliminate them. A significant drawback of antibody-based Alzheimer’s drugs is that they are associated with the risk of brain bleeding. This is not the first time something like this has happened. When antibody-based Alzheimer’s drugs were first created, one of the 989

major side effects was that a subset of patients developed lesions in their brains. The lesions were generally small but would cause swelling in the brain. This phenomenon was labeled amyloid-related imaging abnormalities (ARIA). As of now, there are several procedures used to monitor ARIA in patients taking antibody-based Alzheimer’s drugs, but because ARIA is typically asymptomatic and rare, it hasn’t stopped the progress of most clinical trials. Since less than 10% of patients who received lecanemab during early trials developed any sign of ARIA, Eisei and Biogen plan to continue their clinical trials of lecanemab and will now be including patients on blood thinners. This will hopefully allow the biotech and pharmaceutical companies to fully establish what the risks are for patients taking lecanemab, especially patients that represent a more diverse trial population with different pre-existing conditions. The question that remains is: what exactly does this mean for the development of Alzheimer’s drugs? This recent update in lecanemab’s clinical trials is a setback for the drug, but the search for an effective Alzheimer’s disease treatment is still promising. In 2021, an experimental drug called Aduhelm was granted accelerated approval by the FDA—making Aduhelm the first Alzheimer’s medication in nearly two decades to be approved by the government agency. Like lecanemab, Aduhelm is an antibody-based treatment. The drug works by stimulating the immune system and using the body’s defensive immune cells to target and break down the plaques. In addition to Aduhelm, Gantenerumab is a promising antibodybased treatment that is in the midst of a long-term clinical study. The overall goal of the study is to follow patients taking the drug over the course of four years to determine how the drug improves memory, daily function, and overall cognition. Researchers are also developing new ways to approach Alzheimer’s disease treatments. Beyond antibody-based treatments, there are several ongoing studies whose primary goal is to determine whether approved diabetes medications may alleviate symptoms of Alzheimer’s disease. Why diabetes? In the past decade, Alzheimer’s has been associated with a decreased number of insulin receptors in the brain. Insulin is the primary hormone that controls the amount of sugar in your bloodstream and is a key player in type 2 diabetes. Interestingly, evidence suggests that resistance to insulin may increase the 990

formation of amyloid plaques. Now, because Alzheimer’s disease seems to decrease the number of insulin receptors in the brain, scientists are suggesting that Alzheimer’s may lead to insulin resistance and that this may induce the formation of amyloid plaques and cognitive decline. In spite of last month’s unfortunate news, there is still reason to be hopeful for the development of effective Alzheimer’s disease treatments. As we continue to probe the mysterious symptoms of Alzheimer’s disease, we come closer and closer to finding treatments that could alleviate symptoms, reduce the cost of care, and help millions of people suffering from this disease. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 5): Setback For Potential Alzheimer’s Treatment

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New Monoclonal Antibody For Treatment Of Malaria Forbes | November 14, 2022 | Article

Monoclonal antibodies are among our greatest assets in treating and preventing virus-induced disease. While the spotlight has focused squarely on Covid-19 monoclonal antibodies throughout the pandemic, antibody candidates for other severe pathogens have also made strides forward. Here we describe a new antibody candidate that neutralizes a parasitic foe that has circulated much longer than SARS-CoV-2 and is responsible for hundreds of thousands of deaths annually: Malaria. While the incidence of Malaria is much lower than 20 years ago, poverty-stricken countries, particularly in Africa, continue to struggle with the disease. Progress has halted in recent years due to the lack of effective treatments for the disease. Existing monoclonal antibody treatments are few and far between, and vaccines are relatively ineffective. While newly emerging vaccines backed by the WHO show more promise, Malaria expert Dr. Umberto D’Alessandro notes we should use all the tools we can gather, specifically in reference to monoclonal treatments. A new study by Kayentao et al. from the Mali Malaria mAb Trial Team discusses the phase two clinical trials of the CIS43LS antibody, which is already demonstrating protection against controlled malarial infection in phase one. The new antibody was discovered back in 2018 by Kisalu et al. of the National Institutes of Health. It was isolated with other antibody candidates from humans vaccinated with the malarial vaccine RTS,S/ASO1. Protection from this vaccine wanes over time, but the researchers discovered that the isolated antibody CIS43 demonstrated extended protection against malaria in mice. They found that the antibody preferentially binds a junction between two critical domains of the malaria parasite, resulting in remarkably high binding affinity and the inhibition of cleavage, rendering the pathogen inert. The antibody targets the sporozoite 992

form of malaria, which is the infective stage. Figure one demonstrates how the antibody holds the sporozoite in the uncleaved form at the junctional region. The parasite cannot bind and spread within the human liver due to the lack of cleavage. This antibody target is notable as it's the same target used in the new GSK malarial vaccine: Mosquirix.

FIGURE 1: The three developmental stages of the P. falciparum parasite that could be targeted by ... [+]

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WELLS ET AL. The antibody was also modified with an LS mutation to increase its half-life. First discovered by Zalevsky et al. in 2010, LS mutations refer to a set of two mutations in an antibody, M428L and N434S. These two mutations decrease the antibody dissociation rate and increase Fc binding affinity to human receptors 11-fold. In various experiments, LS mutations to the Fc receptor yield a 2-to-3-fold increase in antibody half-life, which has led to LS mutations becoming common practice among monoclonal antibody developers to extend the efficacy of their treatments. CIS43LS then underwent phase one trials, described in an August 2021 paper by Gaudinski et al. While the trial was relatively limited with 25 participants, Gaudinski and colleagues found that none of the participants who received CIS43LS displayed malarial parasitemia 21 days after controlled human malarial infection. These results were encouraging, as the RTS,S/ASO1 malarial vaccine’s efficacy ranges from only 30-60%. The Mali Malaria mAb Trial Team expands Gaudinski’s controlled phase one trial to a more uncontrolled setting in phase two. 330 adults across Mali received either 10 mg of CIS43LS per kilogram of body weight, 40 mg, or a placebo over a six-month period. Of the 110 participants randomly assigned to the 40 mg group, 20 (18.2%) were infected within the six-month period. In the 10 mg group, 39 (35.5%) were infected. In the placebo group, 86 (78.2%) were infected. The final efficacy of the 40 mg treatment, as compared to the placebo, was 88.2% within a 95% confidence interval, while the 10 mg treatment was slightly lower at 75%. The only notable side effect was the risk of moderate headache in the 40 mg group, which was reported 3.3 times as often as the placebo group. Aside from headaches, no significant side effects were noted, and no safety concerns were observed. Both 75% and 88.2% efficacies for CIS43LS are far higher than the 30-60% efficacy of existing malarial vaccines. Upon approval, the greatest challenge for the antibody will be keeping the production cost low, as those needing the treatment most reside in lower-income nations. Current technologies allow monoclonal antibodies to be produced at $200 and $250 per gram. 994

Ensuring the treatment reaches those that need it most without a high financial barrier to entry is crucial if the drug is to be successful. This article is featured on Forbes.org, and can be read online here: New Monoclonal Antibody For Treatment Of Malaria

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CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment Forbes | November 23, 2022 | Article

Here we describe the integration of CRISPR gene-editing technology to improve CAR T therapy design. Other alternative CAR T designs include mRNA vectors to create temporary CAR T cells and the use of antibody switches to control CAR T cell activation. Previous installments discuss the fundamentals of CAR T and its applications for B cell cancers, multiple myeloma, lupus and the heart. Researchers find that combining novel gene-editing CRISPR technology with CAR T therapy could simplify and improve CAR T therapy in one fell swoop. Traditional CAR T Therapy A remarkable feat in cancer care, today people with difficult-totreat blood cancers can receive CAR T therapy, a personalized “drug” made from their own immune cells. Chimeric Antigen Receptor T cell (CAR T) therapy relies on extracting a patient's immune cells and modifying them in the lab with a new, synthetic receptor. The new receptor allows the white blood cell to target and destroy cancer cells once re-infused back in the bloodstream. Evoking the patched image of a mythical chimera, these receptors merge signaling machinery typical of a T cell with an antibodyderived detection region to create a powerful “living drug” which continually expands inside the body. Figure 1 highlights the basic design of a CAR T cell, while Figure 2 illustrates the step-by-step process in more depth.

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FIGURE 1: A T cell and a chimeric antigen receptor combine to create a CAR T cell. Most approved CAR T therapies employ a similar CAR design. The receptor usually consists of an antigen-recognition domain (scFv), two co-stimulatory molecules to help the cell expand and persist inside the body, and a CD3 T cell signaling subunit to activate the cell upon binding.

MESOTHELIOMA.COM Link Added

FIGURE 2: In CAR T therapy, 1) T cells are first isolated from the patient’s collected blood. 2) In the lab, these T cells are genetically modified to equip the desired chimeric

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antigen receptor. 3) Scientists multiply or expand the number of CAR T cells to millions before 4) the lympho-depleted patient receives the CAR T infusion. 5) The modified T cells circulate the blood, targeting and eliminating encountered cancer cells.

CANCER.GOV Link Added Gene Editing with Viral Vectors To craft CAR T cells, the very genes of the T cells must be altered to express the chimeric antigen receptor. Gene editing, therefore, provides the foundation for the therapy. Integrating CAR genes normally requires the use of a viral vector. Retroviruses in particular have the unique ability to insert and meld their own foreign genetic material into human cells permanently. This allows viruses to use host machinery to produce viral proteins. Scientists have repurposed this strength to deliver CAR genes into T cells. An inactivated form of the virus is filled with genetic material which encodes for CAR. The desired genes are then transferred from the virus into the T cells through a process called transduction (see Figure 3). As if reading biological instructions, the T cell uses the genetic information to construct the receptor before expressing it onto the cell surface.

FIGURE 3: How to modify T cells to express chimeric antigen receptors. A) Essential viral genes are removed and replaced with a vector carrying CAR genes. B) The produced viral particles transduce the genetic information encoding CAR, ultimately enabling the T cell to create and place the new receptors on its cell surface.

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MORGAN AND BOYERINAS

Figure 4: Retroviral transduction of T Cells. Translation of the synthetic genes results in the addition of chimeric antigen receptors on the T cell surface.

MOLECULAR THERAPY, 2016 Link Added The industry standard may depend on viral vectors, but the procedure lacks in some aspects. This stage of the CAR T process is the most time-consuming and expensive; it can take a year or longer to produce a batch of viral vectors, and can cost up to $50,000 per dose. For these reasons researchers now hope to turn to CRISPR technology, a recent scientific breakthrough in gene editing, to resolve these issues. Enter CRISPR/Cas9 Gene Editing CRISPR originates from organisms such as bacteria and plays a major role in their defense. The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats—in 999

essence, they are short, repeating DNA sequences which read the same forwards or backwards, similarly to words such as “MADAM” or “DEED.” Sandwiched between these repeats are protospacers, a genetic history of viruses the bacteria encounters (see Figure 5). When a virus tries to insert its genetic information into the bacteria, the bacteria can recognize the sequence from its protospacer catalog. The bacteria transcribes the protospacer DNA into RNA; this RNA guides enzymes such as Cas9 to the viral DNA to cut and deactivate it. The same CRISPR/Cas9 interface can also snip human DNA. As seen in Figure 6, an RNA guide can be made to cut DNA at a specific site. The broken DNA, eager to repair itself, can easily adopt a new DNA sequence in that location. Translating this concept to CAR T therapy, researchers could modify T cell DNA directly to express a new receptor. Synthesizing an RNA guide is cheaper and more efficient than cultivating retroviral vectors. If successful, CRISPR could simply solve two major drawbacks associated with CAR T therapy: price and timeto-delivery.

FIGURE 5: CRISPR consists of spacers—unique, virus-derived DNA sequences— sandwiched between short, repeating sequences of DNA.

SCIENCE BUDDIES Link Added

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FIGURE 6: Researchers leverage CRISPR/Cas9 to cut and insert genes at a desired site on the genome. The guide RNA directs the Cas9 enzyme to snip the DNA at a specific location.

LABIOTECH Conclusion CAR T therapy, although a triumph of human engineering in its own regard, still has room for improvement. There is potential to propel CAR T design forward by integrating contemporary innovations such as CRISPR/Cas9 technology. Although this method still requires T cell manipulation outside the body, this change could streamline the process while becoming more accessible. The most critical step now is to test the feasibility of this concept. The next installment in the series will explore the latest clinical results from PACT Pharma and the University of California, Los Angeles on their CRISPR/CAR T dual interface. This article is featured on Forbes.org, and can be read online here: CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment

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Do You Have The “Thin” Gene? Forbes | November 23, 2022 | Article

Happy Thanksgiving! As you dig into your Thanksgiving dinner, a time when many of us overeat, you may well ask: Will I regret it tomorrow when I check my weight? Will it be ok? New research suggests that it may depend on whether you have a single “C” or a “T” at a critical position in one of three billion inherited units in your DNA! A new study has identified a gene that predisposes some individuals to being thin. Those that carry this gene have an easier time staying fit and a lower risk of significant weight gain. In the previous installment of this series on overeating, we reviewed the lessons from Dr. David Kessler Bestseller, The End of Overeating, which reveals why some of us cannot help but overeat. Most people logically can differentiate between foods that are “good” vs. “bad” for your body. The food industry, however, spends millions to keep consumers addicted to salt, fat and sugar. While other factors including lifestyle choices can overall affect weight, this study suggests that genetics may play a larger role in weight management than previously thought. Recruiting over 790 healthy volunteers for this study, the Spanish team found that approximately 60% of Europeans have a variant of the so-called “thin gene” that lowers their risk for obesity. Having a C vs. the T variant of the FNIP2 gene was shown to correlate with a lower body mass index (BMI) and higher metabolism. After adjusting for age and sex, the results proved to be even more significant. Those that were homozygous for C gene variant, meaning that they inherited the same copy of this gene variant from both parents, had the more optimal fat to muscle mass ratios, compared to those that were heterozygous, or only had one copy of the “thin gene.” Table: Predicted distribution of the C vs. T allele among humans, compared to the study cohort. ACCESS HEALTH INTERNATIONAL 1002

does this gene do? The FNIP2 “thin gene” is involved in regulating a key metabolic pathway called mechanistic target of rapamycin complex 1 (mTORC1). Through a cascade of enzymes and proteins, this cell signaling pathway senses incoming nutrients, energy levels, and growth factors to either promote or limit cell growth. In other words, it tells your body to grow or produce more fat cells in response to the type of foods that you eat. This gene is, therefore, involved in various processes on the cellular levels that influence appetite, energy storage, and metabolism among other functions. Fernandez et. al found that having the T variant of this gene correlated with lower expression of FNIP2 proteins. In fact, individuals with two copies of the T allele had significantly less of these proteins in their blood compared to those that had at least one C allele copy. Together these findings suggest that increased expression of this protein may reduce the risk of obesity. How can one gene variation have such a large effect on weight? Fernandez et. al argue that the difference between a T vs. C allele in this region of the FNIP2 gene impacts how post-transcriptional factors regulate the expression of FNIP2 proteins. When the T allele is present, miR-181b-59, a microRNA gene regulator, strongly binds to the FNIP2 gene and prevents expression of the protein as demonstrated by the figure below. miR-181b-59, however, does not bind to the C allele, which enables FNIP2 to be transcribed and expressed. Figure: Illustration summarizing the methods and results from this investigation. When the T allele is present, miR-181b-59, a microRNA gene regulator, strongly binds to the FNIP2 gene and prevents expression of the protein as demonstrated by the figFROM: “FOLLICULIN-INTERACTING PROTEIN FNIP2 IMPACTS ON OVERWEIGHT AND OBESITY THROUGH A POLYMORPHISM IN A CONSERVED 3′ UNTRANSLATED REGION” FERNANDEZ ET. AL 2022... How big of a role does this one gene play in weight maintenance and metabolism? To confirm their findings in humans, Fernandez et. 1003

al developed a mouse model in which they “knocked-in,” or inserted, the C gene variant into the animal’s FNIP2 DNA sequence. Just as they observed in the healthy volunteers, mice that had two copies of this gene had lower fat content, especially the female mice. Mice with two T alleles, on the other hand, developed and sustained more fat cells. This suggests that a T vs. C allele in the FNIP2 protein gene may be a key predictor for being overweight or obese. Obesity remains one of the most imminent threats to public health in America. People with poor fat to muscle mass ratios not only have a higher risk of metabolic complications, including insulin resistance, cardiovascular disease, and cancer, but also risk more severe complications associated with Covid-19 and other viral infections. Although lifestyle choices, such as high calorie diets and limited physical activity, can increase the risk of being overweight or obese, emerging research shows that genetics may be a key predictor of weight maintenance. As the group from Spain’s IMDEA Food Institute argues, “Obesity is preventable, but causes and consequences must be deeply understood to design efficient preventative measures and tools.” Although genetics are not destiny, the findings from this study suggest that the amount of FNIP2 proteins in the blood may serve as a useful measure for key metabolic markers. This sets the stage for a future in which obesity can be treated through gene therapeutic interventions that can give you the “thin gene.” Next in this series, we will take a deeper look at how genetics drives cellular and metabolic processes that regulate our appetites. This article is featured on Forbes.org, and can be read online here: Do You Have The “Thin” Gene?

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Hope For A New Treatment On The Horizon For Zika Virus Forbes | November 23, 2022 | Article

Researchers from Duke University, UC Berkeley, Purdue University, and elsewhere have made what will likely be a significant breakthrough in drug development to treat Zika Virus infections. They discovered an unusual monoclonal antibody, not of the common immunoglobulin G family, but rather immunoglobulin M. This antibody is extraordinarily potent in neutralizing the Zika Virus in tissue cultures, as well as in live animal experiments. Monoclonal antibodies are among our greatest assets in treating and preventing virus-induced disease. While the spotlight has focused squarely on Covid-19 monoclonal antibodies throughout the pandemic, antibody candidates for other severe pathogens have also progressed. Here we describe a new antibody candidate that neutralizes the Zika Virus, which is responsible for thousands of infections annually. While not nearly as prevalent as its peak in 2016, documented Zika Virus infections still occur in over 80 countries, with roughly 18,000 cases per year. Infections most often occur in areas closer to the equator, as mosquitoes are the primary mode of transmission for the virus.

FIGURE 1: Tropical regions where Zika infection is common due to regional moquitoes.

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The incubation period for Zika Virus disease is between three and 14 days, at which point symptomatic patients yield fever, rash, conjunctivitis, joint pain, and headache for up to a week. One of the more severe Zika Virus complications occurs in pregnant hosts. Roughly 14% of host fetuses develop severe brain and eye defects. Many cases result in stillbirth, premature birth, or miscarriage. Researchers Singh et al. aimed to discover a monoclonal antibody treatment to placate the virus that still rages in tropical regions. The scientists were surprised to find that a specific type of antibody, immunoglobulin M (IgM), was particularly active concerning Zika Virus immunity for the fetus during pregnancy. The vast majority of antibodies are immunoglobulin G (IgG). While most antibodies are a single ‘Y’ shaped monomer, the IgM antibody presents in a set of five, or a pentamer. IgM antibodies are the largest produced antibody and are the first to respond to initial exposure to an antigen. The monomers are bound to their adjacent monomer by a disulfide bond, and a joining chain keeps the large antibody intact.

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FIGURE 2: Immunoglobulin M antibody type (top) and immunoglobulin G antibody type (bottom). ... [+] ARTUR JAN FIJALKOWSKI

Singh et al. extracted plasma IgM to test for binding and neutralization from a cohort of 10 pregnant Brazilian women during the 2015-2016 Zika Virus outbreak. They eventually isolated one Zika Virus monoclonal antibody candidate, DH1017.IgM, which demonstrated the most robust Zika-neutralizing capacity in early testing. In addition to neutralizing the parental Zika Virus, DH1017.IgM also neutralized ZIKV PRVABC59 and ZIKV H/PF/2013, two prominent variants of the parental virus. DH1017.IgM also had a lower mutation rate than other antibody candidates in early testing, meaning neutralization capacity is unlikely to change due to chance mutations in the antibody. Notably, Singh et al. found that the pentameric form of the IgM is crucial to DH1017 binding. As a Fab fragment, meaning the arms of the ‘Y’ shape solely, the antibody bound poorly. DH1017 bound roughly 20-fold stronger as an IgG monomer, but as an IgM pentamer, the antibody bound more than five-fold stronger than the monomer. In parallel, neutralization for IgM was 40-fold stronger than the IgG monomer. The researchers conducted further tests on mice models to determine in vitro neutralization for DH1017. They found that the 1007

IgM antibody protects against severe and lethal cases of Zika Virus infection at lower 50 or 100 microgram doses but protects against viremia much more efficiently at higher doses. Again, the researchers found that the IgG monomer version of DH1017 fails to achieve the marks set by the pentameric IgM.

FIGURE 3: Neutralizing potency of (A) the DH1017 antibody fragment, (B) the DH1017 IgG antibody, and ... [+] SINGH ET AL.

Singh et al. note that the five-armed IgM antibody “may contact up to five epitope pairs compared to a single epitope pair for the bivalent DH1017.IgG.” This would explain the vastly increased binding and neutralization. Picture a chest with a single lock and a chest with five locks. Which is more secure? The researchers also found that the IgM may bind epitope pairs across different virus particles, creating a cross-linked virion epitope. Now picture multiple chests chained and locked together. The IgM antibody form presents fascinating advantages over its IgG counterpart. The DH1017 antibody, like many antibodies treating viruses primarily impacting lower-income nations, would need to be produced at a low cost to ensure those who need the treatment most could afford it. Current technologies allow monoclonal antibodies to be produced at $200 and $250 per gram. DH1017 could be a godsend for those still impacted by Zika Virus, particularly pregnant women in low-income countries. In a broader sense, this study opens a new avenue for monoclonal antibody development. It is clear that pentameric IgM antibodies bind and neutralize much more effectively than monomeric IgG antibodies. It would be relatively straightforward to convert potent IgG antibodies to IgM by replacing the Fc portion of the antibody. 1008

If researchers could harness that advantage and engineer antibodies to other global antigens, such as SARS-CoV-2, the reward would be well worth the effort. This article is featured on Forbes.org, and can be read online here: Hope For A New Treatment On The Horizon For Zika Virus

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The N Protein, A New Target For Anti-Covid Drugs Forbes | November 29, 2022 | Article

A cross-institutional effort between researchers at Weill Cornell Medicine and Duke University has yielded exciting progress in the search for new therapeutics against Covid-19. Where much of our current anti-SARS-CoV-2 arsenal is built around the virus’ spike (S) protein, many other proteins contribute to the viral life cycle as well. This includes the nucleocapsid (N) protein, which packages viral genetic material and helps suppress the host immune system, allowing the virus to replicate undisturbed. Yaron et al. show that inhibiting phosphorylation of the nucleocapsid protein successfully interferes with replication, leading to a significant reduction in viral RNA and infectious viral particles. Their work provides a clear path for future drug development. In fact, an FDA-approved kinase inhibitor used for the treatment of non-small cell lung cancer (NSCLC) proved effective against SARS-CoV-2. These advances have been made possible by decades of research on a process called protein phosphorylation: when a phosphate group gets added to a protein and subsequently modifies its structure and function. A single protein may have multiple phosphorylation sites, which act as on/off switches for specific functions. The “switches'' are activated by a family of proteins known as kinases. Blocking kinases can inhibit protein phosphorylation, and by extension, certain protein functions. This has proven an effective strategy at developing drugs against a number of diseases, including various cancers, autoimmune diseases, cardiovascular diseases, and the list goes on. To our good fortune, Yaron et al. have now extended this approach to the realm of Covid-19, with promising results. Understanding the Nucleocapsid Protein Despite heavy focus on the SARS-CoV-2 spike protein, the nucleocapsid protein is actually the most abundant protein in infected cells. And along with the spike protein, it is the other major 1010

immunogen. Its primary function is to bind to viral RNA and package it into fully-assembled viral particles. Aside from this, the nucleocapsid protein also plays a crucial role in the suppression of our initial immune response by blocking the stimulation of interferons (IFN) — antiviral proteins released as warning signals by infected cells. This also has downstream effects, since interferons often stimulate additional genes involved in antiviral immune defense. Recent findings by the laboratory of Jennifer Doudna further emphasize the importance of the nucleocapsid protein: all successful SARS-CoV-2 variants have mutations in the linker region of the protein, and these mutations directly affect the speed of viral replication. Phosphorylation of the Nucleocapsid Protein The first step in determining which host kinases to use for drug development is determining the phosphorylation sites of the intended target. To do so, Yaron et al. infected human lung cells with SARS-CoV-2 and then extracted and analyzed them. Although phosphorylation sites were detected across many different SARS-CoV-2 proteins, the nucleocapsid was the most heavily phosphorylated of them all. In particular, an area of the nucleocapsid protein called the serine/arginine (SR)-rich domain, located within the linker region of the protein. The same linker region that Jennifer Dounda’s lab identified as being critical to viral replication. Even compared to other regions of the nucleocapsid protein, the SR-rich domain was the most densely phosphorylated (Figure 1).

FIGURE 1. Phosphorylation sites in SARS-CoV-2 nucleocapsid protein. The majority are localized within the SR-rich domain of the linker region. S, serine; T, threonine; Y, tyrosine.

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FROM: “Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication” YARON ET AL. 2022 Identifying Target Kinases Determining the phosphorylation sites of the SARS-CoV-2 nucleocapsid protein is only one part of the equation, identifying the host kinases involved in the process is another. In order to successfully block phosphorylation and interfere with viral replication, you need to first know which kinase is triggering the phosphorylation. Human kinases are picky. Generally they have distinct preferences for or against amino acids at their phosphorylation sites. These preferences are referred to as a kinase’s substrate motif. Think of this as a lock and a key: the substrate motif is the lock, and the kinase the key that interacts with it. Researchers have developed techniques that allow them to match kinases with their corresponding substrate motif — matching lock and key. Over time, this has led to a large library of kinase substrate motifs. For example, kinases known to phosphorylate serine-arginine rich proteins. Along with being picky, kinases also like to band together. As such, proteins are often phosphorylated at various different sites in an ordered manner. In a phenomenon called phospho-priming, phosphorylation of a protein by one kinase “primes” the substrate by creating a knock-on motif that can be recognized by a second kinase: the first phosphate added facilitates the addition of others. The result? A domino-like cascade of phosphorylation. Targeting an upstream kinase, one of the first primers, can effectively interrupt a cascade before it begins. Drawing on previous research, Yaron and colleagues identified two promising kinase families for drug development: glycogen synthase kinase-3 (GSK-3) and serine-arginine protein kinases (SRPKs). Both kinase families had previously been implicated in phosphorylation of the SARS-CoV-1 nucleocapsid, and recent studies suggest they may play a similar role in phosphorylation of SARS-CoV-2 nucleocapsid. They discovered that SRPK1 acts as the primer kinase, kicking off the chain. Once the substrate has been primed, GSK-3 comes in to further phosphorylate the SR-domain. Finally, one additional kinase —casein kinase I (CK1)— rounds it off. The latter two both 1012

depend on the upstream SRPK1 kinase in order to be able to phosphorylate the nucleocapsid protein. Knock out SRPK1 and you take out the rest of them as well. To test the phosphorylation cascade model they had developed, the scientists analyzed purified SARS-CoV-2 nucleocapsid protein that had been incubated with three kinases, one from each of the kinase families: SRPK1, GSK-3α, and CK1ε. Nucleocapsid protein incubated with SRPK1 showed clear indicators of phosphorylation. In contrast, incubating the nucleocapsid protein with either of the other two kinases —in the absence of SRPK1— resulted in little to no phosphorylation. Interventions: SRPK1 Inhibitors Yaron et al. next tested two well-known synthetic SRPK1 inhibitors, SPHINX31 and SRPIN340 , for their ability to interfere with phosphorylation and interrupt replication. Indeed, cells treated with the inhibitors indicated a significant reduction in viral replication. In both cases, the levels of viral RNA and viral infectious particles were markedly lower than in the control group. The researchers then combed through the FDA databases to find an approved drug that may provide similar benefits. They found an inhibitor of a different kinase, anaplastic lymphoma kinase (ALK), used to treat non-small cell lung cancer that, despite not directly targeting SRPKs, inhibits these as well. As before, treatment with the kinase inhibitor reduced viral RNA and infectious titer. Even when infected with alphacoronavirus HCoV-229E, a very distant cousin of SARS-CoV-2, inhibition of SRPK1/2 reduced replication by 1000-fold. Takeaways Building off years of prior research, this work by Yaron et al. opens the door to a new approach for the treatment of Covid-19: inhibition of SRPK1/2 host kinases. With resistance against monoclonal antibodies on the rise, and resistance against Paxlovid sure to follow, new therapeutic interventions are sorely needed. While we wait for more Covid-19-specific kinase inhibitors to be developed, we might consider repurposing existing inhibitors to treat those most at risk, including upwards of 17,000 immunocompromised patients. The more drugs we have available to us, the more prepared we are to confront viral variation. Kinase inhibitors are a welcome addition to the quiver. 1013

This article is featured on Forbes.org, and can be read online here: The N Protein, A New Target For Anti-Covid Drugs

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Covid-19 And Children: What We Know Forbes | November 30, 2022 | Article

Less is known about the impact of Covid-19 infection among children compared to adults. Why do children experience milder symptoms on average compared to adults? Do children experience Long Covid, and what makes them different? What is MIS-C and what is its prevalence? A recent perspective published by Science explores current research and provides answers to these questions. Why can Covid-19 be milder in children? There are several theories as to why Covid-19 can be milder in children. One explanation is that a child's innate immune system mounts a more vigorous attack at the onset of infection. A child’s acquired immunity changes throughout life and is found to be initially less extensive compared to adults. In particular, some studies have found that children have weaker memory T-cells and lower neutralizing and Fcγ-receptor activating responses compared to adults. Fcγ-receptors play a critical role in triggering the adapted immune system, and a lower activation provides a less robust cell immunity response. Additionally, cytokine interferon levels, which are heavily involved with inflammation, have been found to be agedependent.

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DRANOFF 2004... A study on mucosal responses at the time of Covid-19 diagnosis found that RNA sequencing data and measurement of cytokines had a much more brisk response in the nasal mucosa of children compared to adults. This finding can possibly be attributed to the “trained” immunity resulting from more frequent respiratory infections in children, which increases the baseline or standard innate activity in children. The developing nature of a child’s acquired immune system is also hypothesized to contribute to mild cases. Studies have found that children have an increased frequency of naive T-cells, less natural killer cells, and lower frequency of cytotoxic T cells compared to adults. Additionally, the T-cell receptor declined clonal expansion among children who were previously infected by Covid19 compared to adults. Why do some children develop MIS-C? Multisystem Inflammatory Syndrome in children (MIS-C), is a post-Covid infection condition where different systems of the body become inflamed. It can be seen in children 6 to 12 years old and occur within 3 to 6 weeks following Covid-19 infection. MIS-C symptoms include fever, atypical fatigue, red eyes, strong abdominal pain, severe or worsening vomiting or diarrhea, and rashes. The underlying causes have yet to be identified, but current research finds that increased immune cells and cytokines are involved. Some studies have found increased activation of neutrophils, monocytes, T-cells, B-cells, natural killer cells, and dendritic cells, all of which regulate immune system function, in patients with MIS-C. Additionally, there is higher frequency of cytokines, large proteins involved in pro- or anti-inflammatory factors, and chemokines, which are involved in inducing cell migration; meaning that the higher concentrations of cytokines and chemokines direct activated immune cells to inflamed organs, exacerbating the current state. Some patients with MIS-C have autoantibodies, or antibodies that attack self-antigens made by the body; there is insufficient evidence as of right now, however, to conclude whether autoantibodies occur as a result of the inflamed organ state or drive autoimmunity. If a child is suspected of having MIS-C, they should immediately seek medical care. In the United States, roughly 3 in 10,000 people 1016

under the age of 21 were reported to have been diagnosed with a case of MIS-C infection, and there is an approximately 0.8% chance of mortality. MIS-C is a treatable condition with a high recovery rate. Healthcare providers may administer anti-inflammatory drugs to reduce inflammation in the body and protect vital organs from permanent damage. Studies have also found that Covid-19 vaccination as an effective means to reduce the risk of MIS-C, in part by reducing the risk of Covid-19 infection. Becoming vaccinated also reduces the incidence rate of MIS-C. Long Covid in Children and Young People Most children and adults fully recover from Covid-19. However, some individuals may experience symptoms beyond the average recovery period (3 to 4 weeks). This condition is known as Long Covid. At least 200 different symptoms of Long Covid have been reported, and it has affected every organ system within the body. Depending on the methodology, cohort, and definition, global prevalence estimates of Long Covid among children are between 1:4 and 1:100. In the UK alone, 120,000 children experienced long Covid in 2021, with 26,000 having symptoms persisting for more than 1 year. Children with Long Covid can be grouped based on their ability to return to "normal" life and the presence of additional postinfectious complications. Children may experience debilitating symptoms, such as unexplained fever, fatigue, pain, cognitive difficulties, and post-exertional symptom exacerbation, that interfere with daily life activities. Some post-infectious complications include MIS-C, acute neurological disease, and myocarditis, and can last a few weeks to many months past Covid-19 infection. There are currently no established biomarkers that can identify Long Covid, making it challenging to diagnose. The first research definition of Long Covid was published this past July; in alignment with the clinical case definition released by the World Health Organization, it defined Long Covid as a “post-Covid infection condition with at least one physical symptom persisting 12 weeks past infection and cannot be explained by an alternative diagnosis.” A global agreement for a pediatric Long Covid definition is expected in 2023.

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On a global front, we must work together and invest resources to understand the full implications of Covid-19 among children. In doing so, we can optimize treatments and create more effective therapies, enhancing recovery and reducing the prevalence of Long Covid. This article is featured on Forbes.org, and can be read online here: Covid-19 And Children: What We Know

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December 2022

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Crimean-Congo Hemorrhagic Fever Virus Monoclonal Antibodies: A Work In Progress Forbes | December 01, 2022 | Article

Crimean-Congo Hemorrhagic Fever is among the deadliest diseases in the world; a tick-born disease touting a mortality rate of up to 40%. First discovered almost eight decades ago, there is no effective treatment at present. Here we describe a work in progress of attempts to develop monoclonal antibodies to prevent and treat this deadly disease. CCHFV Background The virus was first identified in Crimea in 1944 and later in the then-Belgian Congo in 1967. More recent reports date the virus as far back as ancient Celtic settlements between 1500 and 1100 BCE. Since its discovery, outbreaks have plagued dozens of countries in Eastern Europe, the Middle East, Africa, and South Asia. The most substantial was an outbreak in Turkey from 2002 to 2008, in which 3,128 cases were reported.

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FIGURE 1: CCHFV distribution map.

CDC CCHFV is most commonly transmitted by ticks. In southeast Iran, 31 different species of tick carry the pathogen. The pathogen has also been found in hares, hedgehogs, rats, birds, and domesticated animals such as sheep, goats, and cattle. The virus is a member of the genus Orthonairovirus, family Nairoviridae of RNA viruses. Other viruses in this genus include the Dugbe virus, the Nairobi sheep disease virus, and the Kasokero virus. The figure below illustrates the basic structure of CCHFV.

FIGURE 2: CCHFV virus structure.

BENTE ET AL. Throughout the decades, attempts have been made at vaccines and treatments for the virus and its subsequent disease, but all were tabled due to low efficacy or associated toxicity. A new antibody treatment could be helpful for hundreds impacted by the disease annually and thousands that could be spared from infection altogether. CC5 Human Monoclonal Antibodies 1021

The first objective for researchers Durie et al. was to find a worthwhile target for monoclonal antibody treatment. All previous treatments had failed, so a novel site would be a quality starting point. Their strategy was to use a previously discovered antibody that prevented severe disease in mice against CCHFV, but failed to do so in humans. They analyzed the antibody, 13G8, and its primary binding site: CCHFV glycoprotein GP38.

FIGURE 3: Mouse monoclonal antibody 13G8 binding GP38.

DURIE ET AL. Using this site as a template, the researchers searched for human monoclonal antibodies targeting this same site. They pulled sera from six verified survivors of CCHFV infection and isolated a panel of antibodies for further testing from one patient: CC5. Of the seven antibodies isolated from CC5, all matched or exceeded the binding affinity of the mouse antibody 13G8. The three most potent binders overall were CC5-6, CC5-16, and CC517. Three of the seven human monoclonal antibodies directly competed with the binding epitope of 13G8: CC5-6, CC5-12, and CC5-17. This overlap would suggest that GP38 is a prime target for antibody binding moving forward for CCHFV treatments.

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Upon further cryo-electron microscopy experiments, Durie et al. found that CC5-17, one of the stronger binding human monoclonals, attacks GP38 at a differing angle of 22 degrees, which may account for the more thorough binding affinity.

FIGURE 4: Differing binding angles of the mouse antibody 13G8 and the human antibody CC5-17.

DURIE ET AL. The researchers also note the binding capability of CC5-17 to another closely related virus, the Aigai Virus. The GP38 binding site is shared between the two viruses, and minor mutational differences do not affect binding affinity. Unfortunately and unsurprisingly, the human antibodies were again non-neutralizing in human in vitro models. The antibody may still protect against severe disease and death in human CCHFV patients, but it seems the GP38 antibody binding site is exclusively nonbinding. This suggests another receptor could yield a more substantial neutralizing capacity when bound by an as-of-yetundiscovered antibody. The CC5 antibodies may have some diagnostic value but fall short as a tool for protection. Concluding Thoughts While it is disappointing that CC5-17 and its CC5 alternatives were not innately neutralizing, their shortcoming could enable another treatment's success in the future. The GP38 binding site is a 1023

prime target for broad binding affinity. GP38 likely has a significant role in virus maturation and localization of viral particles to the host cell's surface. A viable antibody candidate could disrupt this process and effectively neutralize the virus. GP38 could also serve as a starting platform for vaccine treatment soon. Otherwise, different binding sites should be investigated throughout the virus genome in the search for a treatment. This study confirms the possibility of highly effective antiCCHFV treatments; we need only find them. This article is featured on Forbes.org, and can be read online here: Crimean-Congo Hemorrhagic Fever Virus Monoclonal Antibodies: A Work In Progress

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Reimagining Alzheimer’s (Part 6): The Many Effects Of The APOE4 Variant Forbes | December 05, 2022 | Article

A recent paper published in the journal Nature has revealed a new mechanism by which the genetic risk factor, APOE4, may contribute to Alzheimer’s disease pathology. The E4 variant of the APOE gene is the predominant genetic risk factor for Alzheimer’s disease. Those who contain one copy of the E4 variant are three times more likely to develop Alzheimer’s, while those who contain two copies of the E4 variant are nearly ten times more likely to develop the disease. The E4 variant has been associated with Alzheimer’s disease for years. However, it is still not understood exactly why or how this genetic variant contributes to the debilitating biological and cognitive symptoms of Alzheimer’s. Now, studies conducted by a team at the Massachusetts Institute of Technology suggest that the E4 variant of the APOE gene disrupts how fat molecules are processed in the brain. It appears that the disruption of these fat molecules could be the fundamental reason why those that contain the E4 variant are more likely to develop Alzheimer’s symptoms including brain cell death, memory issues, and cognitive decline. To investigate the role of the E4 variant in Alzheimer’s patient's brains, the team at MIT began by examining the genetic makeup of thirty-two patients who had donated their brain tissue samples to Alzheimer’s research before passing away. The first subgroup of donors contained twelve individuals with two copies of the low-risk, E3 variant (E3/E3). The second subgroup contained twelve donors, each with one copy of the E3 variant as well as one copy of the E4 variant (E3/E4). The final subgroup consisted of eight donors with two copies of the high-risk, E4 variant (E4/E4). Only half of the donors in the E3/E3 and E3/E4 subgroups had been diagnosed with Alzheimer’s disease in their lifetime, while all the E4/E4 donors had been afflicted with Alzheimer’s. 1025

Figure 1: Brain tissue samples were taken from patients with different combinations of the E3 and E4 variants. Each subgroup of E3 patients contained those who had been diagnosed with Alzheimer's as well as those who hadn't.

EMILY FORDEN, POWERPOINT One of the primary questions the researchers were interested in investigating was whether the E3 and E4 variants were associated with abnormal cell activity. To determine this, the team at MIT took brain tissue samples from each subgroup and analyzed the tissue with an RNA-sequencing tool. This would allow the researchers to isolate specific types of cells within the brain and figure out which genes seemed to be expressed more or less across different brain cells. After running their analysis, the researchers found that the E4 variant significantly affected the expression of genes related to intracellular activities. Interestingly, this included increased expression of genes associated with inflammation and other immune-related pathways. Many studies have recently pointed to inflammation as a core feature of Alzheimer’s disease. These studies suggest that increased inflammation in Alzheimer’s patients may be linked to the development of both amyloid plaques and tau tangles and may exacerbate the cognitive symptoms of Alzheimer’s. Researchers also found that genes related to the transmission of information in brain cells were expressed less in patients with the E4 variant. Curiously, among all the cell RNA tested, differential gene expression was not only found in neurons—the primary brain cells that are responsible for information exchange—but was also found in other cells called oligodendrocytes. Oligodendrocytes are supportive cells in the brain whose main function is to insulate neurons. Neurons send information to each other through electrical signals in the brain. Much like a wire is insulated to allow for electricity to travel efficiently, neurons are 1026

often wrapped with fats called myelin for insulation and to allow information in the brain to travel more quickly. Oligodendrocytes have the essential function of wrapping myelin around neurons.

Figure 2: Oligodendrocytes have the essential function of insulating neurons with a fatty substance called myelin.

WIKIPEDIA When researchers investigated the effects of the E4 variant on myelin, they found that neurons seemed to be under-myelinated in patients carrying the high-risk variant. To delve deeper into how oligodendrocytes themselves were being affected, researchers examined the genomic data of isolated oligodendrocytes. They soon 1027

found that the E4 variant was associated with increased expression of cholesterol-related genes in oligodendrocytes. Interestingly, there seemed to be an even greater association between cholesterol gene expression, the E4 variant, and other biomarkers of Alzheimer’s disease like amyloid plaques and tau tangles. Those who had both the E4 variant and some Alzheimer’s disease pathologies displayed the highest levels of cholesterol-related gene expression. This suggested that both the E4 variant and typical Alzheimer’s pathology like amyloid plaques or tau tangles had some effect on the regulation of cholesterol and lipid myelination. After examining the expression of cholesterol-related genes in the oligodendrocytes of E4 samples, researchers additionally found that only genes related to the formation of cholesterol were expressed more. In contrast, genes associated with the transport of cholesterol through oligodendrocytes were expressed significantly less. But how did these genetic profiles affect the function of oligodendrocytes in those with the E4 variant? To investigate how a decrease in cholesterol transport gene expression could affect the function of oligodendrocytes, the team at MIT began by examining where cholesterol was located within the oligodendrocyte cell bodies. By staining the cholesterol molecules within the brain samples of patients, the researchers were able to determine exactly where the cholesterol molecules were localized within the cell.

Figure 3: In APOE4 carriers, cholesterol (green) accumulates around the oligodendrocyte nucleus (blue). In contrast, cholesterol in APOE3/3 individuals is highly dispersed throughout the cell body.

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TSAI ET AL., NATURE (2022), DOI: 10.1038/S41586-02205439-W To their surprise, they found that patients with the E4 variant displayed an accumulation of cholesterol around the cell nucleus, where DNA is stored. This was unlike samples taken from E3 patients. In E3 patients, cholesterol was dispersed throughout the cell. These results suggested that those who contain the E4 variant seem to have abnormally functioning oligodendrocytes that produce more cholesterol and are unable to transport the cholesterol throughout the cell body, leading to a significant accumulation of cholesterol molecules. These unusual results prompted the research team at MIT to explore the relationship between cholesterol, oligodendrocytes, and Alzheimer’s disease more thoroughly. To delve deeper into the biological mechanisms that cause cholesterol to accumulate in the oligodendrocytes of E4 patient cells, the MIT lab set out to use stem cell technology to grow their own oligodendrocytes with either the E3 or E4 APOE variants. In the next installment of this series, we will discuss their fascinating discoveries using this stem cell model and a new route for potential Alzheimer’s treatments. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 6): The Many Effects Of The APOE4 Variant

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Symptomatic Covid-19 Infection Is Associated With An Increased Risk Of Overactive Bladder Symptoms Forbes | December 05, 2022 | Article

A new study finds that symptomatic Covid-19 infection is associated with the risk of developing or exacerbating overactive bladder symptoms. As an overlooked area of study, only a handful of investigations have focused on the impact of Covid-19 infection on the urinary system. Previous studies have found increased urinary tract symptoms, including the frequency, urgency, and waking up to urinate, post Covid-19 infection; they attributed the symptoms to inflammation and described the condition as Covid-Associated Cystitis (CAC). Overactive bladder is characterized by a frequent and sudden urge to urinate that can be difficult to control. A person with an overactive bladder may experience urgent incontinence or unintentional loss of urine immediately after feeling an urgent need to urinate, as well as the need to urinate more than typical throughout the day and night. Roberts and colleagues at the Oakland University William Beaumont School of Medicine investigated the incidence of developing or worsening CAC/Covid-related lower urinary tract symptoms between symptomatic and asymptomatic individuals with Covid-19. They additionally measured the change in the International Consultation on Incontinence Questionnaireoveractive bladder (ICIQ-OB) score for individuals with other health conditions and infected with Covid-19. Here, I provide an overview of their study’s findings and discuss some primary implications. Roberts et al retrospectively asked participants to score their overactive bladder symptoms at three different points in time using the International Consultation on Incontinence Questionnaire: pre1030

pandemic, two months following their Covid-19 infection (if they had a documented positive Covid-19 test result), and at the time of the study. The questionnaire helped evaluate the severity and “bothersome” characteristics of symptoms. Symptomatic individuals with Covid-19 have notably higher rates of worsening or developing overactive bladder symptoms compared to asymptomatic participants and those who tested negative for Covid-19. Following systematic analysis, Roberts and colleagues found that participants previously symptomatic with Covid-19 infection had a 36.6% incidence of developing covidassociated cystitis, with 22% of the group developing overactive bladder. Symptomatic Covid-infection was associated with a twofold increased risk of worsening overactive bladder symptoms in comparison to asymptomatic Covid infection and a three-fold increased risk to those who tested Covid-negative. From pre-pandemic to the time of study, Roberts et al found a 6.5 average point increase on the International Consultation on Incontinence Questionnaire-Overactive bladder among individuals who reported a positive Covid-19 test. The greatest changes regarding individual overactive bladder symptoms were the average increases in urgency (0.8 +/- 0.8) and frequency (0.8 +/- 0.9), followed by the average score increases for urge urinary incontinence (0.7 +/- 0.8) and nocturia (0.6 +/- 0.8) right after. Among participants with reported a positive Covid-19 test and other comorbidities, those with diabetes mellitus, chronic steroid, or immunosuppressed were more likely to experience to have increased average score differences compared to participants who tested negative for Covid-19 and without co-morbidities. As Roberts et al noted in their publication, some possible physiological explanations behind the association include inflammation and direct infection to urogenital organs. When SARS-CoV-2 invades the body, various immune cells activate and cause systemic inflammation. Moreover, individuals who were Covid-positive may develop bladder inflammation and thereby felt greater “bothersome” urinary symptoms. Alternatively, the release of proinflammatory cells, including cytokines, can trigger afferent nerves and detrusor muscle contractions that cause urinary discomfort symptoms. One additional biological explanation is the possibility that SARS-CoV-2 directly infects urogenital organs. 1031

Similar to the heart and lungs, bladder urothelial cells are rich with ACE2 receptors, which is one of the primary entryways for SARSCoV-2. Further, urothelial cells can be directly infected with Covid19, leading to inflammation and increasing the likelihood of bothersome urinary symptoms. Albeit interesting, we must heed Robert et al’s findings with caution. 31.9% of participants were Covid-positive, which is much higher than the rates reported in the literature. The difference in percentage suggests that selective bias is a confounder, as participants were invited to join the study because of past participation with the BLAST study and indicated a willingness to later join future research. Moreover, those who had symptomatic Covid infection or had tested positive for Covid were more likely to accept the study’s invitation. External stress from the pandemic was also likely experienced among participants, which can also be a factor in overactive bladder symptoms. Recall bias may have also confounded the study’s findings. Roberts et al. did not conduct a urinalysis, so symptoms may also be influenced by other infectious or inflammatory causes that may have been present. According to the most recent report by the Centers for Disease Control and Prevention, 1 in 13 adults in the United States have Long Covid symptoms. Such symptoms, like an overactive bladder, can severely disrupt a person’s daily life and well-being; additional research must be done to determine the pathophysiology between such symptoms and SARS-CoV-2 and help construct effective treatments. This article is featured on Forbes.org, and can be read online here: Symptomatic Covid-19 Infection Is Associated With An Increased Risk Of Overactive Bladder Symptoms

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Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T Forbes | December 06, 2022 | Article

Here we describe early clinical trial results on combination CRISPR and CAR T therapy, a sequel to an earlier, introductory piece. Other alternative CAR T designs include mRNA vectors to create temporary CAR T cells and the use of antibody switches to control CAR T cell activation. Previous installments also discuss the fundamentals of CAR T and its applications for B cell cancers, multiple myeloma, lupus and the heart. CAR T therapy can treat blood cancers by inserting new genes into a patient’s own immune cells using viruses. Early clinical trial results present an alternative that forgoes viral gene transfer: CRISPR technology. Such integration of CRISPR gene editing could improve the precision, speed and cost-effectiveness of CAR T cell production. In addition, researchers hope CRISPR will broaden CAR T therapy applications from blood cancers to solid tumors, which the engineered T cells notoriously have failed to target. Inserting Genes into CAR T Cells Chimeric Antigen Receptor T cell (CAR T) therapy genetically alters a patient’s T cells to recognize cancer cells and subsequently kill them. This engineered recognition relies on hybrid T cell receptors with antibody components to detect antigens, or biological tags, found on the surface of cancer cells (see Figure 1).

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FIGURE 1: ILLUSTRATION OF A CHIMERIC ANTIGEN RECEPTOR. THE STRUCTURE UTILIZES AN ANTIBODY-DERIVED DOMAIN TO DETECT SPECIFIC ANTIGENS, ALL WHILE LEVERAGING A T CELL CD3Ζ COMPLEX FOR ITS SIGNAL MACHINERY.

HUGHES-PARRY ET AL Researchers typically incorporate hybrid receptor genes into a CAR T cell via viral gene insertion. Despite its regard as a staple in cell therapy, retroviral gene transfer comes with several drawbacks. Viral vector manufacturing is expensive and time-consuming. The method lacks precision and could potentially allow an unwanted gene entry. Perhaps most limiting, it cannot be personalized to detect uncommon antigens. For this reason, all approved CAR T therapies in circulation target blood cancers that share a common antigen (usually CD19 or BCMA) rather than solid tumors, which greatly vary in antigen presentation. Standardizing a new means to insert genes would improve the accessibility, efficiency and usage of CAR T therapy. Innovating with CRISPR Gene Editing In their Phase I clinical trial, the researchers at PACT Pharma and the University of California, Los Angeles explore the possibility of a different type of CAR T therapy—one that creates a hybrid receptor with CRISPR gene editing. With CRISPR, the team selectively removed native T cell receptor genes and replaced them with new, cancer-fighting alternatives.

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The researchers began by searching and isolating a novel T cell receptor from the patient’s own immune system. First, they screened the patients by sequencing DNA from healthy blood samples and tumor biopsies; this step identified mutations which the tumor cells share but cannot be found in normal tissue. Algorithms then predicted which antigens would be present on the tumor. Next, the team copied the antigens and mixed them with different versions of HLA, a type of molecule needed to present antigens to T cells. This process revealed specific T cells which could react to this particular combination of antigen-HLA. Researchers copied up to three of the highly personalized receptor genes to be integrated into the T cells using CRISPR/Cas9. Figure 2 illustrates the subsequent process. The CRISPR/Cas9 interface knocked out two T cell receptor genes, TRCα and TRCβ (see Figure 3), and replaced them with three new receptor genes in a single step—decidedly more efficient than sourcing and cultivating retroviruses for gene transfer, as is currently standard in CAR T therapy. The researchers multiplied the T cells to great numbers. Finally, the patients underwent lymphodepletion chemotherapy before receiving up to three doses of their personalized CRISPR/CAR T cell infusion.

FIGURE 2: AN OVERVIEW OF THE CAR T THERAPY USING CRISPR TECHNOLOGY. GENES FOR TWO NATIVE T CELL RECEPTORS,

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TRAC AND TRBC, ARE REMOVED AND REPLACED WITH GENES FOR NEW AND PERSONALIZED T CELL RECEPTORS.

NATIONAL CANCER INSTITUTE

THE STRUCTURE OF A T CELL RECEPTOR (TCR), COMPOSED MAINLY OF DIMER TRCΑ AND TRCΒ AND ACCOMPANIED BY A CD3 PROTEIN COMPLEX. NOTE THE HUMAN LEUKOCYTE ANTIGEN (HLA) MOLECULE PRESENT ON THE ANTIGEN PRESENTING CELL, DENOTED IN PURPLE; THIS STRUCTURE IS NECESSARY FOR THE T CELL RECEPTOR TO RECEPTOR TO RECOGNIZE THE ANTIGEN PEPTIDE.

NESUN ET AL Results The researchers assessed the safety and dosage of combination CRISPR/CAR T therapy to treat 16 people with various kinds of solid tumors, including breast and lung tumors. All of the patients experienced drug resistance, and had already received five or more prior lines of therapy. The CRISPR/CAR T infusion did not prove overly dangerous. All the patients experienced typical side effects of lymphodepleting chemotherapy. One patient in particular experienced mild cytokine release—an expected side effect of CAR T therapy. Another experienced severe encephalitis. 1036

The combination CRISPR/CAR T therapy did not cure any patients. Through biopsies, the team found that the CAR T cells successfully multiplied and traveled into the tumors of eight participants. Four weeks after infusion, five participants had stable disease, meaning their condition did not change. The other eleven patients’ cancer worsened. Future Implications Gene integration via viral vectors establish the current standard for CAR T therapy, but could soon be replaced with cheaper and more efficient CRISPR gene editing. The clinical results demonstrate that tumor-specific CAR T cells can be made and used safely; that these CRISPR-edited immune cells can recognize solid tumor masses; and that this method holds potential to be effective against drug-resistant solid tumors. While this realm of research still warrants room for improvement, especially with more uniform tumors (ex: lung tumors only), this foundation sets an excellent springboard for advancements to come. This article is featured on Forbes.org, and can be read online here: Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T

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Covid-19 Treatments Like Paxlovid Are Being Underused Forbes | December 06, 2022 | Article

The antiviral drug Paxlovid has the potential to significantly reduce the mortality rate associated with Covid-19, but is underused by those who would benefit most. A recent CDC report notes that people over the age of 80 are less likely to receive Paxlovid than those under 65. From April to July this year less than one-third of America’s 80-plus-year-olds infected with Covid-19 ended up taking Paxlovid. A significant issue when those over 65 and especially those over 80 are making up a significant proportion of Covid-19 related deaths. An independent study published in the New England Journal of Medicine found that Paxlovid reduced hospitalizations and deaths in patients over 65 by 79 percent during the Omicron surge in Israel earlier this year. Pfizer’s own studies found that Paxlovid reduced the risk of hospitalizations and deaths by 88 percent. There may even be reason to also consider prescribing Paxlovid to younger populations or those without conditions that make them more vulnerable to severe disease. A recent large study found that people who took Paxlovid within a few days after being infected with the coronavirus were less likely to be experiencing Long Covid several months later. With no shortage of Paxlovid available in the US, this is worth exploring. There are several reasons why patients may hesitate to take Paxlovid, and doctors may be hesitant to prescribe it. We need to address each of these and create targeted outreach and communication to vulnerable groups who would benefit from Paxlovid. First, there is the issue of access, which is presently being addressed by the Test and Treat initiative, which provides free Covid testing and free immediate access to treatment for those eligible. There are also local hotlines such as New York City’s 212-COVID19 hotline, which provides free treatment to New Yorkers who 1038

don’t have a regular healthcare provider. However, greater efforts need to be made to address the lack of these resources in health care deserts and rural areas. Second, there is the more complicated issue of public perception. Paxlovid rebound is a common concern for hesitant patients and doctors. However, the high-profile rebound cases of President Joe Biden, First lady Jill Biden and Dr. Anthony Fauci and anecdotal evidence spread among communities and on social media may have contributed to a sense that Paxvlovid rebound is more common than it actually is. Regardless of the prevalence of rebound, a recent CDC report found that Paxlovid rebound is very unlikely to progress to severe illness. Paxlovid rebound is most likely due to Paxlovid working as it should but not for long enough. Paxlovid works by stopping viral replication. It is currently given for five days, but some people still have the virus in their bodies after five days. When Paxlovid is stopped, the virus starts replicating again. Paxlovid may need to be given for a longer period of time, such as 10 days. With no monoclonal antibody treatments currently authorized in the US for Covid-19 due to resistant Omicron sub-variants. Paxlovid remains a powerful tool against death and hospitalization. This article is featured on Forbes.org, and can be read online here: Covid-19 Treatments Like Paxlovid Are Being Underused

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Why The Covid Vaccines Work And How To Make Them Better Forbes | December 07, 2022 | Article

It's no wonder that by now, people are looking both to understand how the Covid vaccines work and to determine how best to improve them. Current mRNA vaccines are excellent at raising high neutralizing-antibody titers against the strain to which they were designed, namely the early Wuhan strain. However, antibody levels fade quickly after three to four months, yielding poor effectivity even against the original strain. Immunity quickly fades after vaccinations, boosters, and even after vaccination with the recent bivalent boosters. Furthermore, the original vaccines and the updated booster generate weak neutralizing activity against the latest variants. Bowen et al. have undertaken a systematic study to understand why the vaccines seem to work as well or poorly as they do and with the thought that they may be able to improve their activity. Background The mRNA vaccines that most Americans received over the past year and a half are prefusion spike protein from the SARS-CoV-2. When the virus infects a host, the S1 portion of the spike locks onto a host cell, and the S2 portion engages fusion mechanisms after undergoing a conformation shift. The vaccines teach the natural immune system to identify the spike and neutralize it before cell infection occurs.

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FIGURE 1: Structure of 2019-nCoV S in the prefusion conformation. (A) Schematic of 2019-nCoV S ... [+ ]WRAPP ET AL.

However, as the virus mutated over time in the form of variants such as Alpha, Delta, and now Omicron and its sublineages, vaccines became less and less effective at creating antibodies that recognize the modified spike. Researchers Bowen et al. from the Veesler lab at the University of Washington aimed to evaluate the influence of spike conformation on plasma-neutralizing activity through a series of spike protein experiments. In other words, where is the best target for vaccine-elicited antibodies, and during what conformation should those antibodies strike? Prefusion SARS-CoV-2 S stabilization reduces the fraction of antibodies recognizing an off-target conformational state To determine the importance of proline stabilization, the researchers generated in vitro models of the entire spike, the S1 portion containing the N-terminal domain and receptor-binding domain, the S2 portion pre-fusion, and the S2 portion post-fusion. Proline stabilization refers to inserting two prolines into a pivotal joint of the vaccine’s spike protein to stabilize the structure’s perfusion shape. The three that include the ‘2P’ mutation are Moderna’s mRNA-1273, Pfizer’s BNT162b2, and Novavax’s NVX-CoV2373. 1041

FIGURE 2: Prefusion and postfusion structures of the SARS-CoV-2 spike protein. The spike sheds a ... [+] BING CHEN

They gathered sera from a wide panel of subjects vaccinated with different vaccines, all of which were not infected before vaccination. The researchers introduced the differing sera to the different in vitro spike targets. Six of the seven vaccines examined yielded much stronger neutralizing activity against the whole spike and the S1 subunit. The lone exception was BIBP-CorV, an inactivated SARS-CoV-2 vaccine by Sinopharm.

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FIGURE 3: IgG binding titers elicited by SARS-CoV-2 infection or vaccination against prefusion S ... [+] BOWEN ET AL.

Bowen et al. note that proline-stabilized vaccines are among the most robust in S1 targeting and neutralization compared to S2 preand post-fusion. SARS-CoV-2 neutralization is determined by S1 subunit targeting antibodies Next, Bowen et al. affirm the necessity of S1 subunit targeting antibodies for virus neutralization. The researchers sorted the antibodies found in the sera of subjects who received Moderna or Pfizer’s mRNA vaccines by the binding site. After sorting, they could then deplete specific antibodies by spike target, whether perfusion spike, S1, prefusion S2, or postfusion S2. The results demonstrate that the prefusion spike and S1 are by far the most crucial to antibody neutralization of the virus. When those antibodies were depleted, neutralization fell dramatically, whereas when S2 pre- or post-fusion was depleted, neutralization remained roughly the same.

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FIGURE 4: Neutralization titers resulting from depletion of polyclonal plasma antibodies targeting ... [+] BOWEN ET AL.

What does this tell us about vaccines? They are most strongly neutralizing when introduced to the S1 subunit, namely the receptor-binding and N-terminal domains. Not surprisingly, these are the regions most heavily mutated in Omicron and its sublineages. SARS-CoV-2 variant cross-neutralization is determined by RBDspecific antibodies Finally, Bowen et al. show that the N-terminal domain is nearly inconsequential in terms of antibody neutralization. They conducted the same depletion experiment, this time comparing the N-terminal and receptor-binding domain targeting antibodies of the S1 subunit group. Upon depletion of the receptor-binding domain targeting antibodies against major variants, neutralization drops to near undetectable levels. The opposite holds true for N-terminal domain antibodies.

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FIGURE 5: Plasma neutralizing activity against Beta S VSV, Delta S VSV, and Omicron BA.1 S VSV after ... [+] BOWEN ET AL.

This tells us that N-terminal domain targeting antibodies elicited from vaccines are inconsequential in the neutralization of SARSCoV-2 and its variants. Paired with our newfound knowledge that S2-targeting antibodies are also far less involved in neutralization, we can conclude that receptor-binding domain targeting antibodies are our best bet at neutralizing the virus. This confirms our ongoing suspicion that the numerous receptor-binding domain mutations in Omicron and its sublineages are those responsible for the evasion of vaccine protection. Concluding Thoughts How can this study inform us of the current status of vaccines and how to move forward? The vaccines released in 2021 by Moderna, Pfizer, and others neutralize Omicron and its sublineages very little to not at all. The updated bivalent vaccines released in 2022 may be slightly better but still fall well short of the necessary mark. Bowen et al. provide more detail about why the vaccines work as well as they do but offer very little about how you might improve their activity. We need considerably more work to understand what we can do to either prolong immunity or amplify memory. Some are beginning to despair that vaccines alone will not be the answer for the limitation of Covid-19 symptoms and infections. Perhaps shifting focus to a combination of vaccines plus highly active and long-acting antiviral drugs may provide the answer in the long run. This article is featured on Forbes.org, and can be read online here: Why The Covid Vaccines Work And How To Make Them Better 1045

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The Fat Truth: You Feel It In Your Gut Forbes | December 07, 2022 | Article

With thanksgiving in the rear-view window, the last thing most of us want to do is think about food, not to mention what those calories did to our waistlines. From deep-fried turkey to loaded mashed potatoes to freshly baked apple pie, there is no doubt that fat had a starring role at dinner tables across America. Now, in the sobering light of post-thanksgiving food comas, we continue our series on the science behind overeating. In the first installment, we delved into Dr. David Kessler’s Bestseller, The End of Overeating, in which he frames fat, sugar, and salt as the main driver behind overeating. The food industry, as he argues, profits from the dissonance between logical restraint and a carnal drive to continue eating beyond the point of satiation. The second and most recent installment explored the genetic influences that predispose some people to being thin, regardless of exercise, diet, and other lifestyle factors. Genetics, however, are not destiny. For that reason, this installment will consider what actually happens when you consume high calorie foods. Fat, sugar, and salt contribute significantly to our daily calorie intake. There is something about fat, in particular, that makes us continue to crave it even after the point of feeling full. If you have ever had deep-fried onion rings or a plate full of French fries, the craving for more is not merely driven by taste. It is how these foods smell and feel in our mouths. It is how after just one taste you cannot help but devour the entire plate. In a recent paper published in Nature, investigators at Columbia University argue that restricting fat cravings may be outside our conscious control. The results of their experiments found that fat activates a post-ingestion gut-brain circuit that keeps us wanting more. Li et al. began their investigation by presenting their mice subjects with a choice between two drinking solutions, one sweetened with artificial sweeteners and the other saturated with fat. Presented with both options at the same time, the animals had an 1047

immediate preference for the artificially sweetened drink. Within 24 hours, however, their preferences considerably shifted to the extent that, by the 48 hours, the mice almost exclusively drank from the fat mixture. The animals appeared to have developed a strong appetite for fat over the sweet drink with an equal number of calories. Investigators concluded that there may be something unique about how fat interacts with the gut that allows it to influence our behavior. Activation of sweet taste receptors generated the initial preference for the artificial sweetener, but as the animals consumed more of the fat infused drink, it became clear to researchers that there may be a post-ingestion circuit sending signals to the brain. To be sure that this behavioral shift had nothing to do with taste, Li et al. genetically engineered mice without fat-activated taste receptors. Despite being blinded to its taste, the animals continued to exhibit a higher preference for fat, compared to the artificial sweeteners. While the mice ate, investigators observed an interesting pattern of neural activity in the brain. By placing electrodes on the animal’s heads, they were able to record what was happening in the brain while they ate different types of fat, compared to a texture matched non-fat drink. Only the fat-saturated drink mixtures were able to activate a particular part of the brain called the caudate nucleus, located deep within the brainstem. Caudate nucleus and other parts of the brainstem serve as keep connectors between the brain and the rest of the body.

Figure: Illustration of the caudate nucleus in red.

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WIKIPEDIA COMMONS/ LEEVANJACKSON In addition to its well understood role in movement, emerging studies suggest that the caudate nucleus plays a critical role in associative learning and reward-driven behaviors. This is also the part of the brain that has been shown to influence appetite and digestion via the gut-brain axis. Eating your favorite food tastes good because neurons in this brain region release dopamine neurotransmitters that teach your brain to associate that particular food with a good feeling. What connects the gut to the brain? As the longest cranial nerve, the vagus nerve stretches from the caudate nucleus and branches down the neck and torso. This is the superhighway that connects the brain to all your vital organs, including the gut’s digestive system. For example, while your body is at rest, the brain signals down the vagus nerve to lower your heart rate and slow your breathing in order to expend more energy digesting and extracting nutrients. If the brain can send down signals to increase food intake through this nerve, Li et al. hypothesized that fat-activated receptors in the gut can also influence how the brain responds to certain foods. For their next set of experiments, rather than allowing the animals to drink the solution, Li et. al inserted a feeding tube directly into the gut. What they observed not only reinforced the existence of a gut-brain circuit, but also called attention to the role of the caudate nucleus within this pathway. In fact, when investigators exposed these neurons to pharmacological blockers the prevented them from receiving signals from the vagus nerve, the mice no longer developed a strong preference for fat, regardless of how many times they were exposed to the fat infusions. The animals, however, still exhibited an initial preference for the artificial sweetener. To confirm these findings, Li et al. hoped to find that severing the vagus nerve disrupted the gut-brain axis. Just as they thought, cutting the vagus nerve effectively prevented the mice from developing fat preferences, as well as eliminated any gut-linked neural activity in the caudal nucleus. If the gut and the brain talk, the vagus nerve, as the main model of communication, may be a likely pharmacological target for treating obesity. Conclusion America has a fat problem. The USDA recommends that fats should only make up 30% of your calories for a day. Having too much fat in your diet, especially high-processed saturated fats, can 1049

lead to significant health problems, including high cholesterol and obesity. For someone who eats 2,000 calories, up to 66 g of fat should be consumed per day. Thanks to the food industry’s efforts to incorporate fat into as many foods and drinks as possible, many Americans eat much more than that. In a world where fat is added to nearly every meal, the challenge many Americans face is how to cap their daily fat intake. Many of the foods and drinks found on supermarket shelves and restaurant menus are infused with considerable amounts of fats that taste good, albeit terrible for our bodies. In Dr. Kessler’s book, The End of Overeating, he describes how the food industry attracts and hijacks all our senses through food science and skillful marketing. Li et al. now argues that we are biologically wired to crave fat. Eating food rich in fat triggers a deep seeded signal that takes tremendous willpower to override. Although we cannot completely get rid of the vagus nerve, this study offers the insight that one day the insatiable desire for high fat foods could be medically treated. This article is featured on Forbes.org, and can be read online here: The Fat Truth: You Feel It In Your Gut

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Six Potential Causes of Brain Fog in Covid-19 Patients Psychology Today | December 08, 2022 | Article

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Brain fog is a debilitating condition that affects mental abilities related to focus and attention. Potential causes of brain fog include inflammation, direct infection, and an autoimmune response, among others. The possible causes of brain fog and other neurological symptoms related to Covid-19 are not mutually exclusive.

A new research review has analyzed six potential causes for one of the most common yet elusive long Covid symptoms. Brain fog is an often debilitating condition affecting executive function, the set of mental abilities that includes focusing attention, holding information in the mind, and blocking out distractions. Despite affecting many with certain chronic illnesses and postviral syndrome for years, the condition has been poorly understood from a neurobiological and neuropsychological perspective with few tools for diagnosis or treatment. Doctors often diagnose it using the Montreal Cognitive Assessment, which was originally designed to assess elderly dementia patients. Patients are also assessed against the average population, not against their previous baselines. Neither are effective diagnostic tools for the diverse population affected. However, brain fog is receiving renewed attention and research focus because of its prominence in long Covid patients, bringing hope for better diagnostic tools and treatment. Researchers at Yale and Stanford University reviewed the literature relevant to cognitive dysfunction after Covid-19 infection and analyzed six potential causes for covid-related cognitive dysfunction. 1. Respiratory inflammation causing neuroinflammation and neural dysregulation The first potential cause identified is that the immune response by the respiratory system to SARS-CoV-2 may cause neuroinflammation, thereby increasing cytokines, chemokines, and 1051

immune cell trafficking in the brain and inducing reactive states of resident microglia and other immune cells in the brain and brain borders. The white-matter-selective microglial reactivity which appears after SARS-CoV-2 infection is also observed in other diseases such as cancer-related cognitive impairment. Deciphering the precise mechanisms by which respiratory inflammation triggers each aspect of neuroinflammation will be critical for developing therapeutic interventions to mitigate the effects of Covid-19 and other respiratory infections on the brain. 2. Direct infection of the brain Direct infection of the central nervous system by SARS-CoV-2 is possible, neuroinvasive infection can contribute to acute and chronic brain pathology through neuroinflammatory and cytotoxic mechanisms. Cases of SARS-CoV-2 neuroinvasion have been reported, with SARS-CoV-2 detected in brains, but durable infection was found to be uncommon amongst the research reviewed. This suggests that direct central nervous system infection is rare and does not account for the majority of neurological symptoms associated with Covid-19. 3. Autoimmune response Another possibility is that SARS-CoV-2 may evoke an autoimmune response against the nervous system. A review of the research points out that while autoimmune encephalitis can and does occur, it remains to be determined whether anti-neural antibodies occur commonly in mild to moderate Covid-19 and whether subclinical autoimmunity contributes to chronic neuroinflammation and lasting cognitive impairment. 4. Reactivation of latent herpesviruses The reactivation of latent herpesviruses, like the Epstein-Barr virus, may also trigger neuropathology. There are many viruses that remain latent in the brain. Epstein-Barr virus is a highly prevalent γherpes virus that persistently infects over 90% of the human population. Epstein-Barr virus may contribute to neuroinflammation in long COVID patients due to viral pathogenesis (viral proteins and viral transcription factors) and/or host immune response to EBV infection (cytokines, Th2 responses, and autoantibody production). A study that followed 309 Covid-19 patients found that Epstein-Barr virus at the time of Covid-19

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diagnosis was one of the four predictive factors for long Covid development. The herpes simplex virus type 1 and 2 can also result in herpes encephalitis, a life-threatening brain infection producing behavioral changes, seizures, and altered levels of consciousness. There are rare cases of herpes encephalitis occurring within the weeks following Covid-19 diagnosis. These studies suggest that certain herpesvirus reactivation is somehow more frequent in those who develop long Covid and could provide clues for investigation. 5. Cerebrovascular and thrombotic disease Another theory explored by Iwasaki and Monje is that cerebrovascular and thrombotic disease may disrupt blood flow, disrupt blood-brain-barrier function, and contribute to further neuroinflammation and ischemia of neural cells. Covid-19 increases the risk of ischemic stroke and other thrombotic complications, highlighting the potential thrombotic and vascular effects of Covid19 on the brain. 6. Hypoxia Finally, pulmonary and multi-organ dysfunction occurring in severe Covid-19 can cause hypoxemia, hypotension, and metabolic disturbances that can negatively affect neural cells causing cognitive impairment. Critical illness in general is associated with a high rate of persistent cognitive impairment. This hypothesis requires further testing but it explains the increased rate and severity of chronic neurological complications in people who survived more severe acute disease. All of these possible causes of brain fog and other neurological symptoms related to Covid-19 are not mutually exclusive, and many long Covid patients may be experiencing a combination, with varying frequency and timing. Based on the literature review, brain inflammation triggered by the immune response to the respiratory system infection and consequent dysregulation of neural homeostasis and plasticity is most likely a common cause in those who have experienced mild disease. Brain fog and other neurological symptoms associated with Covid-19 have a profound effect on patients' lives, often preventing them from working, pursuing passions or caregiving. Therapies that are effective in the treatment of cancer-therapy-related cognitive

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impairment and strategies to reset microglia to homeostatic, nonreactive states are avenues that should be explored for treatment. This article is featured on Forbes.org, and can be read online here: Risk for newly diagnosed diabetes after COVID-19: a systematic review and meta-analysis

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Extended Antigen Availability Improves Vaccine Protection Forbes | December 08, 2022 | Article

The mRNA vaccines we are currently using are not sufficient to stop the ongoing Covid-19 pandemic. Although they have been critical in the fight against SARS-CoV-2, they suffer from a glaring limitation: poor longevity. Wait long enough between booster shots, and you’re back to square one, once again susceptible to serious disease, hospitalization, and death. Even against the same strain of the virus, immunity wanes. Factor in viral mutation and the constant rise of new variants, and this trend is supercharged. Just within the Omicron family of variants, for example, many of the newer offshoots manage to evade immunity built up against their parent lineages (Figure 1). Generally, this happens through structural changes to the spike (S) protein that the virus uses to bind and enter host cells. If these changes are significant enough, our immune system —and in particular antibodies— simply can’t recognize the virus.

FIGURE 1. Neutralization of pseudotyped D614G and Omicron sub-variants by sera from five different clinical cohorts. Notice the marked drop in immune protection against newer Omicron sub-variants.

FROM: “Alarming antibody evasion properties of rising SARSCoV-2 BQ and XBB subvariants” WANG ET AL. 2022 Can we do better? Yes. Slow and Steady Wins the Race Adenovirus-vector vaccines, despite lower initial antibody titers, manage to retain their efficacy for a longer period of time than do mRNA vaccines (Figure 2). They may also elicit stronger CD8+ T 1055

cell responses, which are a crucial part of the adaptive immune response and a key player in protecting us during reinfection.

FIGURE 2. A comparison of vaccine effectiveness over time. The green line represents the Johnson & Johnson/Janssen adenovirus vaccine, the red line represents the Moderna mRNA vaccine, and the blue line represents the Pfizer/BioNTech mRNA vaccine. Adapted from: “Association of Primary and Booster Vaccination and Prior Infection With SARS-CoV-2 Infection and Severe COVID-19 Outcomes”

LIN ET AL. 2022. Courtesy: Dan H. Barouch, M.D., Ph.D. Are the adenovirus vector vaccines better than the mRNA vaccines? Epidemiological evidence suggests no. But they do hint at a potential way of strengthening immune memory: prolonging antigen availability. This is an area in which mRNA vaccines fall short. Despite seeding messenger RNA with synthetic nucleotides that stave off degradation, the mRNA lasts only one or two days following injection. By extension, the antigen proteins that the mRNA encodes are also of short duration, persisting no longer than three days. By contrast, adenovirus vector vaccines can continue to express the target antigen for a much longer time. This may be one of the reasons they induce longer-lasting immunity. A similar phenomenon can be observed with influenza, where data indicate the antigen must be present for at least seven to eight days for a robust memory response to develop. This is evidenced by the fact that germinal center activity doesn’t begin until around this time (Figure 3). Germinal centers act as a kind of boot camp for B cells, training them to recognize diverse regions of an antigen and to bind the antigen more tightly. B cells, in turn, secrete antibodies that bind to the virus, making it harder for the virus to bind host cells

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and, if present in large enough quantities, inhibiting viral entry wholesale.

FIGURE 3. Germinal center responses to influenza do not kick in until roughly seven days after initial infection. Germinal centers produce long-lasting, antibody-producing B cells that contribute to immune memory.

FROM: “The Multifaceted B Cell Response to Influenza Virus” BAUMGARTH ET AL. 2019 Another series of studies showed that primates immunized according to a slow-delivery strategy —one in which vaccine administration is spread out across multiple days— developed enhanced neutralizing antibody responses against human immunodeficiency virus (HIV) compared to primates following a traditional, single-dose immunization protocol —where the entire vaccine dose is administered in one sitting. Simply extending antigen availability boosted antibody production 10-fold. The same group of scientists has now published a new study investigating this process in more detail. Their work shows that a slow-delivery approach, with dose-escalation over the span of 12 days, can lead to broader, higher quality, and longer-lived immune memory. In particular, Lee et al. discovered that germinal centers stay active for far longer and produce a more diverse set of memory B cells when vaccine administration is extended over the span of a week or more. Here, we take a closer look at their findings and discuss the implications for future vaccine design. Slow-Delivery Vaccination Extends Germinal Center Activation To compare the impact of different vaccination strategies on germinal center activation and durability, Lee et al. separated rhesus monkeys into three groups. The first group received two averagesized doses of recombinant HIV envelope trimer MD39 protein, one in each arm. This was combined with a common adjuvant, called alum, to mimic conventional vaccination. The other two groups 1057

(groups 2 and 3) received the same recombinant protein dose, but formulated with an immune-stimulating adjuvant saponin/MPLA nanoparticle (SMNP). Instead of injecting the entire dose in one sitting, they split it up over a span of seven smaller doses, injected every other day for a 12-day period. As before, injections were administered in both arms. Monkeys immunized according to the traditional, single-dose approach showed a spike in total germinal center B cells three weeks post-inoculation. Only a small portion of these B cells effectively bound the envelope protein, but from week three onwards, their numbers began to fall quickly. Monkeys vaccinated according to the dose-escalation protocol —groups 2 and 3— displayed a steeper spike in total germinal center B cells than their counterparts. Of those, the frequency of envelopespecific B cells was also significantly greater, with a 7.8 fold increase over group 1. And most strikingly, the number of germinal center B cells continued to rise for many weeks. By week 10, the gap had increased to a 186-fold difference. Groups 1 and 2 received an additional booster dose 10 weeks after initial vaccination. Group 3 monkeys, on the other hand, did not receive an additional booster until 29 weeks after initial vaccination, giving the researchers a chance to study the impact of slow-delivery over a longer interval. Indeed, germinal center activity in group 3 monkeys was recorded for a total of 191 days, a full 27 weeks after the end of the initial priming period. Even at 191 days, the frequency of envelopespecific germinal center B cells was 27-fold higher than the peak frequency following conventional, bolus vaccination. Continued Germinal Center Activity Produces Better B Cells Extended germinal center activity overlapped with continued B cell evolution throughout. To retain efficacy in the face of viral mutation, B cells need to be able to recognize a broad variety of binding sites. They also need to bind to these sites tightly. Both diversity and affinity are improved through a process called somatic hypermutation (SHM). In a nutshell, B cells undergo rapid mutation and the resultant lineages are put into competition with one another. Those that best bind the antigen survive, the others are got rid of.

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In group-3 monkeys, germinal center B cell lineages that were initially unable to bind the HIV envelope trimer protein managed to do so several weeks later, indicating continued evolution. In some cell lines, affinity improved 1000-fold over a six month period. Similar somatic hypermutation was witnessed in memory B cells, improving both affinity as well as diversity. What About Antibodies? Germinal center activity is an important indicator of B cell maturation, but antibodies are the ultimate metric of vaccine efficacy. Having established that slow-delivery supercharges germinal centers, the researchers next analyzed how this prolonged activation shapes the antibody response. Only one monkey in the conventional vaccine group developed neutralizing antibodies towards the HIV envelope trimer protein, and this only after receiving the booster dose. Even then, titers were low. All of the monkeys in the slow-delivery vaccine groups developed neutralizing antibodies. Crucially, all of the monkeys in group 3 developed these antibodies after only primary immunization, before they even received a booster dose. The number of antibodies remained stable from week three all the way to week 29. Following administration of a booster dose, peak titers were 50 times as high as those in group 1. Clearly the slow-prime approach boosts antibody production, but what about antibody quality? Antibodies are only useful if they can properly bind to the circulating antigen, afterall. A key feature of antibody efficacy is antibody diversity; a single antigen can have multiple binding sites, or “epitopes”, and the more diverse the antibodies the better the chances of neutralizing the antigen. This is especially true given a phenomenon called “immunodominance”, which is the tendency for antibodies to target only certain binding sites, those deemed most important. This lets the virus escape the antibody response by mutating in just one area, something it cannot do as easily if it is targeted at multiple sites. Again, the slow-delivery approach proved superior: monkeys in group 2 and group 3 developed a more diverse pool of antibodies compared to those in group 1, targeting a larger array of antigen binding sites (Figure 4). Monkeys in group 3 displayed the greatest breadth of antibodies. 1059

FIGURE 4. Number of antigen binding sites recognized by monkeys in group 1, 2, and 3, respectively.

FROM: “Long-primed germinal centres with enduring affinity maturation and clonal migration” LEE ET AL. 2O22 Implications Vaccine development is a complex field with lots of moving parts; the seemingly smallest of differences —are you injecting into the muscle or into the skin?— can potentially change how well the final product protects people from disease. There are some clear factors that impact immune response: the size of the dose, what part of the virus a vaccine targets, what platform is used to present the viral antigen —inactivated or live-attenuated or mRNA or any of the other myriad options— and whether anything is added to stimulate the immune system, called an adjuvant. The work by Lee et al. adds to a growing list of research suggesting that speed of

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delivery and duration of antigen availability are another fundamental part of the equation. Their work also has important implications for our current vaccines against Covid-19. Practically speaking, few people are going to want to sit through seven “micro” vaccinations spread across 12 days. Given the clear benefits, this is an unfortunate limitation. However, there are several technologies that may be able to mimic this extended delivery schedule by prolonging antigen expression. Adenovirus vector-based vaccines, as mentioned above, are one such technology. But they come with a few drawbacks. Since adenoviruses are common, cold-causing viruses, many people have already been exposed to them during their lifetime. This means they have built up immune memory towards the viruses, making it difficult for the viral vector to ferry the target gene into our cells — the vector is quickly recognized, targeted, and cleared as soon as it enters the body. Second, there exists the serious, albeit rare, risk of blood-clotting, called vaccine-induced thrombocytopenia (VIT). This can happen when the vaccine is accidentally injected into the bloodstream, rather than muscle tissue. Self-amplifying RNA vaccines represent an alternative option. Where conventional mRNA vaccines only include the genetic information for the antigen protein, self-amplifying RNA vaccines include an additional sequence of RNA that encodes four nonstructural proteins. These nonstructural proteins come together once in the cell to form an enzyme called RNA replicase — think of this as a genetic photocopier. The RNA replicase makes many copies of the target mRNA, each of which can then go on to be translated into the antigen. This massively extends the time of antigen expression, potentially up to 26 days. Some manufacturers have already begun incorporating self-amplifying RNA into their Covid19 vaccine design. Although not a perfect stand-in for the dose-escalation strategy explored by Lee and colleagues, these technologies harness the benefits of extended antigen expression while maintaining realworld practicality. This article is featured on Forbes.org, and can be read online here: Extended Antigen Availability Improves Vaccine Protection Reimagining Alzheimer’s (Part 7): 1061

Cholesterol Abnormalities May Contribute To Alzheimer’s Disease Forbes | December 17, 2022 | Article

This article is the seventh installment in my series on Alzheimer’s disease. Read more about Alzheimer’s disease in part 1, part 2, part 3, part 4 , part 5, and part 6 of the series. An Introduction to APOE4 A recent study published in Nature provides new insights into genetic predispositions of Alzheimer’s disease. The E4 variant of the APOE gene is accepted as the predominant genetic risk factor for Alzheimer’s. Those who contain one copy of the E4 variant are three times more likely to develop Alzheimer’s, while those who contain two copies of the E4 variant are nearly ten times more likely to develop the disease. Unfortunately, while the E4 variant has been associated with Alzheimer’s for decades, it is still unclear exactly how it contributes to the debilitating biological and cognitive symptoms of Alzheimer’s. Now, a study conducted by Tsai et al. at the Massachusetts Institute of Technology shows that those who contain the E4 variant display abnormalities in cholesterol metabolism. The MIT team suggests that the disruption of cholesterol metabolism could be a fundamental reason why those with the E4 variant are more likely to develop Alzheimer’s disease symptoms. In my last article, I discussed Tsai et al.’s initial findings using brain tissue samples from Alzheimer's patients. After characterizing the genetic makeup of tissue samples from thirty-two Alzheimer’s patients, Tsai et al. discovered that the E4 variant of the APOE gene was associated with abnormal cellular activity. These abnormalities particularly affected a subset of brain cells called oligodendrocytes.

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Figure 1: Oligodendrocytes are supportive cells in the brain and are responsible for myelinating neurons.

ELECTROSPINNING COMPANY Oligodendrocytes are supportive cells in the brain whose main function is to insulate neurons. Neurons send information to each other through electrical signals in the brain. Much like a wire is insulated to allow electricity to travel efficiently, neurons are wrapped with fats called myelin. These fats provide insulation and allow information in the brain to travel more quickly. Oligodendrocytes have the essential function of processing fat molecules to produce myelin for neurons. Here, we will describe the additional experiments the MIT team conducted to study the relationship between the E4 variant, cholesterol, and oligodendrocytes. Studying oligodendrocytes with stem cells Tsai et al. began with a stem cell model. Stem cells are a unique type of cell that can be turned into nearly any other cell type in the body. When stem cells are converted to their embryonic state, they can then be exposed to chemical cues that nudge them into developing into nearly any other cell in the body, including oligodendrocytes. By using a stem cell model, the team could compare the effects of the E4 variant of the APOE gene in cells from a single patient. 1063

This would allow them to confirm that any abnormal activity displayed by oligodendrocytes was due to the E4 variant of APOE. Using gene editing tools like CRISPR, the team could also alter the oligodendrocytes so that they expressed two copies of either the E3 or E4 APOE variants. To examine the effect of the E4 variant in the stem cell model, the researchers first examined the fats present in the oligodendrocytes. Compared to the E3/E3 cells, the E3/E4 cells exhibited an increase in 88 different fat species. Only one fat species decreased within the E4/E4 cells. Upon further analysis of the two oligodendrocyte types, researchers found that genes responsible for transporting cholesterol in the E4/E4 oligodendrocytes were less active. This suggested that the E4 variant decreases the ability of oligodendrocytes to distribute cholesterol throughout the cell. To determine where cholesterol was accumulating, researchers stained the oligodendrocytes to visualize where the cholesterol was localized within the cell. The team found that cholesterol in E4/E4 oligodendrocytes seemed to accumulate more than E3/E3 cells. Surprisingly, researchers found that the cholesterol accumulated within a structure called the endoplasmic reticulum. Typically, cholesterol is stored within the oligodendrocyte’s cell membrane. These results demonstrate that E4/E4 oligodendrocytes display abnormal activity in cholesterol transport and storage. The E4 variant, oligodendrocytes, and myelination Since oligodendrocytes are responsible for myelinating neurons, researchers sought to explore how oligodendrocyte dysfunction may impair its ability to myelinate neurons. Tsai et al. speculated that E4 variant carriers may exhibit decreased myelin production. To explore this, the researchers stained myelin in postmortem tissues from patients that contained a combination of the E3/E4 variants and those who contained two copies of the E3 variant. The MIT team soon found that samples taken from patients with the E4 variant displayed a significant reduction in myelination. To confirm these results, the researchers then conducted these experiments on mice and found that E4/E4 mice exhibited much fewer myelin proteins than E3/E3 mice. Surprisingly, when inspecting the structure of myelin in E4 mice versus E3 mice, researchers discovered that myelin in E3 mice was much more 1064

densely packed than E4 mice, suggesting that there may be structural differences in myelin due to the E4 variant as well as a decrease in myelin production. This led to a key question: Was the decrease in myelin found in Alzheimer’s patients due to the abnormal activity of oligodendrocytes only? Or were other cells also contributing to the decrease in myelination? Studying myelination with a 3D stem cell model Tsai et al. approached this question by using stem cells to form both oligodendrocytes and neurons. The two cell types were cocultured and grown into 3D tissue that resembled the cellular organization of human brain tissue.

Figure 2: To explore the effects of the E4 variant on myelination, Tsai et al. used stem cells (iPSCs) to create a 3D co-culture of oligodendrocytes and neurons.

TSAI ET AL. NATURE (2022), DOI: 10.1038/S41586-02205439-W The team created two 3D cultures—one with two copies of the E4 variant and one with two copies of the E3 variant. After observing the model for 6 weeks, Tsai et al. found that in line with their suspicions, the E4/E4 culture produced significantly less myelin 1065

than the E3/E3 culture. To determine whether this difference in myelin was due to the oligodendrocytes or due to cellular processes within the neurons themselves, the team then conducted experiments where they co-cultured E4 oligodendrocytes with E3 neurons or E3 oligodendrocytes with E4 neurons.

Figure 3: E3/E3 oligodendrocytes produced structured myelin in the presence of E4/E4 neurons (top row). E4/E4 oligodendrocytes did not produce regular, structured myelin even in the presence of E3/E3 neurons (bottom row)

TSAI ET AL. NATURE (2022), DOI: 10.1038/S41586-02205439-W... Surprisingly, samples containing E3 oligodendrocytes engaged in regular myelination even with E4 neurons. In contrast, samples containing E4 oligodendrocytes consistently displayed abnormally low myelination levels. These results confirmed that the decrease in myelination displayed by those with the E4 variant of the APOE gene was, in fact, due to the abnormal activity of oligodendrocyte cells. So, what do these results mean for potential Alzheimer’s disease treatments? Having confirmed that the E4 variant of the APOE gene 1066

promotes abnormal cholesterol accumulation in oligodendrocytes and decreases myelination, Tsai et al. sought to determine how these findings may lead to new treatments for Alzheimer’s. Cyclodextrin: A potential treatment Using their stem cell model of oligodendrocytes, the researchers tested specific molecules known to inhibit the production of cholesterol and promote the transport of cholesterol. After two weeks of treatment, researchers found that one molecule called cyclodextrin was particularly effective. In cyclodextrin-treated E4/E4 oligodendrocytes, Tsai et al. found that oligodendrocytes exhibited a decrease in cholesterol production and accumulation. To examine the effects of cyclodextrin in live organisms, Tsai et al. applied the treatment to mice with the E4 variant. After treating the E4/E4 mice with cyclodextrin for eight weeks, researchers found that the treatment significantly reduced cholesterol production. The treatment also increased the transport of cholesterol and increased healthy myelination. Interestingly, cyclodextrin not only improved biological symptoms but seemed to support learning and memory. In an additional experiment, researchers conducted several learning and memory tests to see if cyclodextrin could improve cognitive function in E4 mice. To their surprise, E4 mice treated with cyclodextrin exhibited improvements in their learning and memory test results. These results contrasted with a control group of E4 mice who were not treated with cyclodextrin and exhibited decreased cognitive abilities. Overall, this innovative and exciting study points to a new theory for how the E4 variant of the APOE gene may induce or exacerbate Alzheimer’s disease symptoms by causing abnormal cholesterol metabolism and myelination patterns. Given the success of cyclodextrin in improving the biological and cognitive impacts of the E4 variant in both cells and mice, it will be interesting to see whether cholesterol metabolism and myelin-based treatments can improve the symptoms of Alzheimer’s disease in humans. This article is featured on Forbes.org, and can be read online here: Reimagining Alzheimer’s (Part 7): Cholesterol Abnormalities May Contribute To Alzheimer’s Disease

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CAR T Therapy To Treat And Cure Rheumatoid Arthritis Forbes | December 19, 2022 | Article

Here we describe the use of CAR T therapy to treat mice with experimental rheumatoid arthritis. Previous installments discuss the fundamentals of CAR T and its applications for B cell cancers, multiple myeloma, lupus and the heart, as well as the use of antibody switches to control CAR T cell activation and new research on combination CRISPR/CAR T therapy. A study published in the Journal of Immunology advances work towards a cure for rheumatoid arthritis via cell mediated therapies. Around 1.3 million people in the US have this autoimmune disease. The condition can severely impair quality and can reduce life expectancy by 3 to 10 years. The study builds upon a recent cancer innovation to effectively target the cells associated with disease progression. Although the results have not yet been verified in humans, the findings demonstrate the burgeoning potential of this treatment to address autoimmunity. Autoimmunity and Rheumatoid Arthritis Rheumatoid arthritis (RA) differs from the “wear and tear” arthritis most recognize. Rheumatoid arthritis occurs when the immune system wrongly attacks the body’s own joint tissues and leaves widespread inflammation in its wake. Figure 1 clearly illustrates how the injured joint tissue swells and erodes the bone. The affected joints often become painful, stiff and red as a result. Further side effects include fever, fatigue and weight loss. Existing treatment options usually involve medicines to suppress the immune system and slow down disease progression. However, these treatments do not address the underlying cause: the errant and self-reactive immune cells.

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FIGURE 1: With rheumatoid arthritis, the immune system attacks the synovial membrane, the thin tissue encapsulating the joint; the membrane becomes inflamed and causes bone erosion.

FLORIDA ORTHOPEDIC INSTITUTE Haywire Immune Mechanism Current understandings of autoimmune arthritis suggest that the disease relates to a set of genes called human leukocyte antigen (HLA) class II. Many versions of this gene exist. In general, reading these genes produces a variety of molecules which can present biological tags to nearby helper T cells, also known as CD4+ T cells. The system works similarly to a flag and pole. The biological tag—formally known as an antigen (Ag)—acts as a flag; the flag cannot be read unless a major histocompatibility (MHC) class II molecule lifts it up for helper T cells to see. Corresponding helper T cells can then recognize their “flag,” bind to it, and trigger an immune response. Considering that particular helper T cells react and progress rheumatoid arthritis, targeting these haywire CD4+ T cells could cease the undesired inflammatory response. 1069

Innovative CAR T Cell Targeting Researchers at the Memphis Veterans Affair Medical Center sought to target the pathogenic helper T cells responsible for rheumatoid arthritis. To accomplish this, the team designed a CAR T cell that autoreactive helper T cells can bind to. A typical Chimeric Antigen Receptor (CAR) T cell artificially combines an antigen-detecting region derived from antibodies with a signaling domain derived from killer T cells (see Figure 3). Once infused into the blood, the CAR T cells detect antigens on the surface of cancer cells and subsequently eliminate the cancer. As this method does not work against pathogenic CD4+ cells, the researchers developed their own chimeric antigen receptor in response. Figure 2 illustrates the team’s innovative design. The receptor maintains the same activation and costimulation domains seen in typical CAR T cells. The antibody component, however, is replaced with something new: a synthetic, rheumatoid arthritis-specific protein complex. The complex mimics a specific, rheumatoid associated “flag and pole” (MHCII and antigen) pair that autoreactive helper T cells in mice can recognize. Once bound, the CAR T cell releases chemicals to kill the bound helper T cell.

FIGURE 2: This study uses a chimeric antigen receptor which contains CD3ζ T cell machinery alongside CD28 costimulatory molecules. The most notable departure from cancer-based CAR T cell design is the major histocompatibility class II/antigen domain,

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which enables the cell to interact with pathogenic CD4+ T cells in mice. Abbreviations: CTL, cytotoxic T cell; Th, helper T cell; TCR, T cell receptor.

WHITTINGTON ET AL. Study Results The researchers found that the new CAR T cells could indeed be recognized by rheumatoid-associated helper T cells when tested in the lab; the helper T cells could only be stimulated if the corresponding antigen on the CAR T cell was present. The synthetic T cells also successfully eliminated the pathogenic helper T cells. The researchers then used the CAR T cells on mice immunized with collagen type II (CII). This induced autoimmunity similar to that of human rheumatoid arthritis. The team removed killer T cells from the mice, genetically modified them to express the DR1 chimeric antigen receptor, and reinfused them into the body. Overall, this new CAR T cell design seemed to effectively eliminate the target CD4+ T cells. The mice treated with the CAR T therapy had significantly reduced helper T cell responses—in some cases completely eliminating the them. As seen in Figure 3, the administration of CAR T therapy also reduced autoantibody responses linked to rheumatoid tissue damage. In addition, the treatment delayed the onset and severity of rheumatoid arthritis in mice when given early on in disease development. The eventual arrival of arthritis signaled that the CAR T cells, effective as they may be, circulated in the body for a shorter time than expected. The CAR T cell design may need further adjustments to support CAR T cell expansion inside the body.

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Mice treated with dr1-car t cells (in blue) produced fewer autoantibodies. WHITTINGTON ET AL. Future Directions This study illustrates that CAR T therapy can indeed translate from cancer to autoimmune treatment. The CAR T cells successfully targeted and eliminated self reactive CD4+ cells, as well as reduced autoantibody production. While the concept proves feasible in mice, the hope is to translate this work for human use. The results broaden the scope of possibility for treating and potentially curing rheumatoid arthritis and other autoimmune diseases. This article is featured on Forbes.org, and can be read online here: CAR T Therapy To Treat And Cure Rheumatoid Arthritis

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Successes Like The Covid-19 Vaccines Come From Long-Term Investments In Public Health Forbes | December 19, 2022 | Article

Despite the devastating impact of the Covid-19 pandemic, we must also celebrate our successes. The Covid-19 vaccines are nothing short of a medical miracle. A new report by the Commonwealth Fund has found that the Covid-19 vaccines have prevented more than 18 million additional hospitalizations and more than 3 million additional deaths in the United States alone. However, the success of these vaccines is largely due to longterm investment in fundamental biomedical science research and vaccine technology through the National Institute of Health and foundations like the Bill and Melinda Gates Foundation. The Covid-19 vaccines would not have been sequenced, developed, and authorized in record time if it were not for over 40 years of dedicated investment by the US in HIV research. Over decades, HIV researchers have developed a better understanding of the complex workings of the immune system, allowing them to map out in detail how HIV invades cells and replicates and identifying weak spots in the virus that can be targeted by drugs. Both HIV and SARS-CoV-2 are RNA viruses that can quickly mutate, making it challenging to develop interventions. Not only the mRNA technology used in the Pfizer and Moderna vaccines but also the adenovirus vector technology utilized in the AstraZeneca vaccine have evolved from past HIV research. In 2004, Merck worked with investigators funded by the NIAID to create a vaccine using a weakened common-cold virus called Adenovirus 5. This vector was used to deliver three genes found in HIV into the body, hypothesizing that the immune system would attack these foreign genes and learn to fend them off, preparing it for future encounters with actual HIV. However, the trial was abruptly ended when early results showed that the vaccine provided little protection and was likely making men more susceptible to HIV. But

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many others built on this research, developing vaccines that use different cold viruses, including AstraZeneca. This is why it is so critical to take a long-term, proactive approach to invest in public health rather than a reactive approach as emergencies and crises unfold. According to a systematic review of 52 interventions published in 2017 in the Journal of Epidemiology and Community Health, health-protection programs such as vaccinations saved an average of $34 for every $1 spent on them. This has proven to be true in the case of the Covid-19 vaccines, which has saved the U.S. $1.15 trillion in medical costs that would otherwise have been incurred. While significant investments were made early in the pandemic, federal funding is still following a familiar boom-and-bust cycle. Additional Covid-19 funding has been stalled in Congress since March this year. This is despite two new reports from Democrats in Congress, which recommended boosting federal funding for public health for multiple years alongside clarifying the role of the Strategic National Stockpile and modernizing the country’s outdated data and surveillance systems. We also have an opportunity to invest heavily in the development of new combinations of broadly neutralizing monoclonal antibodies, which have the potential to neutralize all known SARS-CoV-2 variants and other related coronaviruses, including SARS-1 and MERS. Like the vaccines, these antibodies could save millions of lives and could eventually be combined with highly active antiviral drugs to end the pandemic. But without the dedicated investment that Project Warp Speed and HIV research have received, these treatments will remain just a possibility. Consistent public health funding allows us to save lives and be prepared for the myriad of virological threats we will face in the future. This article is featured on Forbes.org, and can be read online here: Successes Like The Covid-19 Vaccines Come From Long-Term Investments In Public Health

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New Insights Into The Postfusion Structure Of SARS-CoV-2 Spike Protein Forbes | December 21, 2022 | Article

Covid-19 begins when the SARS-CoV-2 virus first encounters cells in the upper airway that express a suitable attachment site, the angiotensin converting enzyme 2 (ACE 2). Blocking binding and entry short circuits infection before it begins. That is why vaccines can be so effective in protecting us from disease. However, as we are learning, the duration of vaccine protection is so short and specific to each virus variant that we cannot quell the pandemic with vaccines alone. We need new highly potent, long-acting, safe antiviral drugs to protect us and end the pandemic. In my opinion the only way to find these drugs is to understand the virus in all its complexity. Fortunately, we have the tools of modern biomedicine to do so if we wish. A recent preprint from the Department of Molecular and Cellular Biology at Harvard University provides atomic-level information revealing new and unexpected aspects of the early critical steps of viral entry. Opening the Gates: Membrane Fusion Entry into the target cells requires much more than surface attachment. Both the virus and host cell are surrounded by a protective membrane. Viral entry begins when the virus binds to the surface of the target cell via the ACE 2 protein on the target cell surface. Binding is followed by fusion of the virus cell membrane to that of the host cell. Both binding and fusion are mediated by the tripartite spike protein embed in the virus membrane. The spike protein is originally made of a single long polypeptide. As the virus exits the cell the spike protein is usually cut at what is called the furin cleavage site, producing the amino-terminal S1 protein and the carboxyl-terminal S2 protein. The actual spike is a timer consisting of three S1 and three S2 proteins. In the pre-fusion state, the S1 protein wraps around, covering almost the entirety of the S2 protein. In Figure 1, regions of the spike protein gene are 1075

highlighted in different colors and identified by abbreviations that denote their function. Figure 2 depicts the structure of the prebinding S1/S2 trimer.

FIGURE 1. Schematic of the SARS-CoV-2 spike protein genome with its different domains and segments. S1/S2 represents the furin cleavage site, and S2’ represents the transmembrane serine protease 2 (TMPRSS2) cleavage site. Glycans are indicated by sma…

SOURCE: ACCESS HEALTH INTERNATIONAL (ADAPTED FROM: “CRYO-EM STRUCTURE OF SARSCOV-2 POSTFUSION SPIKE IN MEMBRANE” SHI ET AL. 2022)

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FIGURE 2. Ribbon diagram of SARS-CoV-2 spike protein. (Left) Complete prefusion spike protein, including both subunits. (Right) X-ray view of prefusion S2, buried within S1. Note, the receptor binding domains are at the top. The bottom extends throug…

SOURCE: ACCESS HEALTH INTERNATIONAL (ADAPTED FROM PROTEOPEDIA, BASED ON CRYO-EM WORK FROM “DISTINCT CONFORMATIONAL STATES OF SARS-COV-2 SPIKE PROTEIN” CAI ET AL. 2020) Upon binding to the ACE2 receptor on the target cell surface, all three S1 proteins separate from the complex, releasing the S2 trimer. The S2 protein is then cut by a cellular enzyme into a short amino-terminal fragment, which here we will call the SAT fragment, and the longer S2 protein. It is the S2 protein-SAT fragment complex that drives the viral-cell membrane fusion. Figure 3 illustrates the structure of one of the three S2-SAT fragments of the pre-fusion protein free of the surrounding S2. Note that the SAT-fragment remains tightly associated with the S2 protein post the second cleavage.

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FIGURE 3. Prefusion structure of one of three subunit 2 protomers of the SARS-CoV-2 spike protein, after subunit 1 has been removed by ACE2. Labels: 3H, three-helix segment; FPPR, fusion peptide proximal region; i-FP, the internal fusion peptide; b-F…

FROM: “CRYO-EM STRUCTURE OF SARS-COV-2 POSTFUSION SPIKE IN MEMBRANE” SHI ET AL. 2022 Fusion of two membranes, once begun, is an energetically favorable reaction. However the charged surfaces of the membranes repel one another and require additional energy to initiate the fusion process. That energy takes several forms. The initial step is provided by the S2 protein. Once free of S1, it springs into a fully open position, like a jackknife , embedding its amino-terminus into the juxtaposed host cell membrane, effectively harpooning the cell and tethering the virus to the cell. Some additional energy for the eventual fusion is derived from the association of the aminoterminus of the S2 protein as it buries itself in the cell membrane. The actual structure of this open structure remains to be resolved. We know much more about the next step. The S2-SAT fragment complex then folds back on itself, pulling the two membranes together (Figure 4). The energy for this reaction comes from the formation of a very stable final structure whereby each S2 complex creates a long alpha helix folded back upon itself. The final structure is a six-helix bundle embedded in both the viral and cell membranes. Multiple spike proteins working side-by-side in parallel 1078

create an open pore that then widens to permit entry of the virus genetic material into the interior of the cell.

FIGURE 4. Model for how viral fusion proteins function — For most class I fusion proteins, prior to triggering (i and ii), the receptor-binding subunit (deep purple, rb) clamps the fusion subunit (dark blue, f). Upon triggering, the receptor-binding…

FROM: “FUSION OF ENVELOPED VIRUSES IN ENDOSOMES” WHITE & WHITTAKER 2016 Postfusion Structure of the SARS-CoV-2 Spike Protein Although we have a solid sense of what the prefusion spike protein looks like, until now the same could not be said about the postfusion spike protein. Given the importance of membrane fusion to the viral life cycle, this represented a serious lacuna. Enter cryogenic electron microscopy (Cryo-EM), a technique that helps researchers determine the structure of biological molecules. In a nutshell, a sample of the target molecule is flashfrozen in a solution. The frozen solution is then blasted with an electron beam, which hits the molecules and passes through a downstream lens that creates a magnified image of the structure on a detector plate (Figure 2). A camera takes thousands of twodimensional images which, with the help of algorithms, can be layered into a three-dimensional model. The benefit of cryo-EM is that researchers can easily flash freeze the same protein at various different conformational stages, giving them a sense of how the structure changes over time. Shi et al. prepared the SARS-CoV-2 spike proteins for cryo-EM by wrapping them up in a lipid bilayer using scaffolding proteins, 1079

creating a so-called “nanodisc” — the scaffolding protein keeps the lipid bilayer, which mimics a cell’s membrane, tightly pressed up against the spike protein (Figure 3). Next, they exposed the nanodisc spike constructs to ACE2, triggering conformational changes and membrane fusion. After the first cleavage event, subunit 1 falls away while still attached to ACE2, leaving S2 exposed.

FIGURE 3. Schematic diagram of a membrane scaffold protein nanodisc. The membrane scaffold protein is highlighted in green, the lipid bilayer in gray, and the target membrane protein in orange.

SOURCE: CUBE BIOTECH The researchers placed the dissociated, nanodisc-wrapped S2 under the electron microscope. The resulting structure can be seen in figure 4, below.

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FIGURE 4. (Left) A ribbon diagram of the SARS-CoV-2 S2 spike protein trimer, postfusion. (Right) Same S2 spike protein postfusion, but one single protomer in isolation. Labels: 3H, three-helix segment; FPPR, fusion peptide proximal region; i-FP, the…

SOURCE: ACCESS HEALTH INTERNATIONAL (ADAPTED FROM: “CRYO-EM STRUCTURE OF SARSCOV-2 POSTFUSION SPIKE IN MEMBRANE” SHI ET AL. 2022). Homing In on the Fusion Peptide In an exciting “first”, Shi et al. successfully managed to resolve the membrane-interacting sections of the S2 fragment. Their work settles a long-standing point of debate regarding the final post fusion structure. Three different regions had previously been suggested as potential candidates for the fusion peptide, but none had been acceptably confirmed. The first is located upstream of the S2’ cleavage site, called the n-terminal fusion peptide (n-FP). The second candidate, just downstream of the S2’ cleavage site, has been called the “bona fide” fusion peptide (b-FP) — this area is highly conserved across coronaviruses. And finally, a region just upstream of heptad repeat 1 (HR1), called the internal fusion peptide (i-FP). Although the so-called “bona fide” region had previously been considered the likeliest candidate, Shi et al. show that it is actually only the internal fusion peptide (i-FP) that fully enters the target lipid membrane; neither of the other two candidates insert into the cell membrane in the postfusion conformation, making it unlikely that either acts as the true fusion peptide.

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The i-FP region of the postfusion structure extends from the central S2’ coil, creating one long, rigid alpha helix. Once inside the lipid bilayer, the internal fusion peptide makes a sharp U-turn back towards the outside of the cell membrane, creating a hook or hairpin shape. When the three protomers are put together in their natural trimeric conformation, the fusion peptides of the S2 come together to form a cone-like structure — the researchers speculate that this cone shape helps the spike protein penetrate the target cell membrane, like the tip of an arrow. Fusion Peptide as Docking Site Along with settling the location of the fusion peptide, Shi et al. also managed to solve another enduring question: does the fusion peptide interact with the transmembrane segment during fusion? It had been speculated that the two may link during the final stages of the fusion process, but again, direct structural evidence had been missing. Indeed, their work shows that the cone formed by the internal fusion peptides is bound by three transmembrane segments, forming a larger, nine-helix-bundle cone. This larger, transmembrane cone is itself capped by three copies of the cytoplasmic tail segment, which lie flat across the top (Figure 5). Both the transmembrane region and the cytoplasmic segment further stabilize the postfusion construct.

FIGURE 5. Ribbon diagram of the transmembrane region of the postfusion S2, with the cytoplasmic tail clearly visible, capping the structure. Heptad repeat 1 (HR1) in yellow, fusion peptide proximal region (FPPR) in green, internal fusion peptide (i-FROM: SHI ET AL. 2022

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An area previously considered separate to the transmembrane segment, called the pre-transmembrane segment, is shown in this model to lie fully within the membrane, extending the anchor by 13 amino acids and forming one single transmembrane structure. Implications In what can only be described as a tour de force, Shi et al. have given us a clearer picture of the SARS-CoV-2 spike protein in its postfusion conformation. Not only is this a first in the study of coronaviruses, but also in the study of class I fusion proteins as a whole. Using cryogenic electron microscopy, the researchers solved two enduring mysteries: the exact location of the fusion peptide, and whether or not the fusion peptide and the transmembrane segment interact. An unanswered question and a caveat remain. The first step of the fusion process is harpooning of the target cell by the S2 protein. The structure of the initial fusion intermediate is not resolved. We are free to speculate what it might be. A caveat is that the structural model developed by Shi et al. is solved for the S2 protein absent the S2’ cleavage (see figure 1, first and second cleavage). That leaves 147 additional amino acids covalently attached to the S2 protein. These are located immediately amino-terminal to what was previously known as the "bona fide” fusion peptide. Might the result of either the first intermediate or the final postfusion structure differ were amino acids 834-856 terminus of the S2 in the Shi et al. experiments to more closely resemble that of what is almost certainly the actual S2 protein of the infectious virus? This article is featured on Forbes.org, and can be read online here: New Insights Into The Postfusion Structure Of SARS-CoV-2 Spike Protein

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CAR T Therapy, A Promising New Therapy For Multiple Sclerosis? Forbes | December 21, 2022 | Article

Here we describe the use of CAR T therapy to treat multiple sclerosis in mice. Previous installments discuss the fundamentals of CAR T and its applications for B cell cancers, multiple myeloma, lupus, rheumatoid arthritis and the heart, as well as use of antibody switches to control CAR T cell activation and new research on combination CRISPR/CAR T therapy. A study of mice by the researchers at the Washington University School of Medicine suggests that CAR T therapy offers a new opportunity to treat and possibly cure multiple sclerosis. Around 1 million people in the United States have this autoimmune disease. The symptoms that arise—numbness, double vision, and tremor among others—impact the brain and nerves, and can potentially disable. Available treatment options only patch the problem and do not cure it. CAR T therapy may help piece together some missing clues. While the therapeutic is best known for its uses against cancer, it has shown recent promise to treat other autoimmune diseases including lupus and rheumatoid arthritis. Here, the researchers extend this potential to multiple sclerosis in their animal model of the disease—a hopeful step towards eventual success in humans. What is Multiple Sclerosis? Multiple sclerosis (MS) occurs when the immune system wrongfully perceives the body’s own cells as a threat. White blood cells called helper T cells or CD4+ cells go rogue and target the outer covering of the nerves. This covering, similar to insulation on wires, protects the nerve fiber inside (see Figure 1). When damaged, the nerves cannot properly send electrical signals to other parts of the body and thus impedes communication between the brain, spinal cord and other parts of the body. The degradation of the nerve sheath, otherwise known as demyelination, manifests signs variably from person to person. Multiple sclerosis-related nerve damage commonly causes numbness 1084

and tingling, fatigue or impaired vision among other symptoms. Although treatment options such as steroids or physical therapy can ease MS flare-ups and relapses, a cure has yet to surface.

FIGURE 1: With multiple sclerosis, the immune system targets the outer sheath covering the nerve and exposes the nerve fiber. This can cause a range of symptoms including numbness, tingling and pain.

MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH CAR T Cells Target Autoimmunity A recent study published in Science Immunology builds towards a possible cure for multiple sclerosis by using CAR T Therapy. The therapy relies on Chimeric Antigen Receptor T cells to detect and destroy malignant cells inside the patient’s body. To create CAR T cells, scientists attach new receptors onto a patient’s extracted killer T cells—usually this meshes the detection power of an antibody onto killer T cell machinery. The CAR T cell, once reinfused into the body, possesses a heightened ability to

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recognize and destroy cells with a specific biological tag, otherwise known as an antigen. This CAR T design has proven effective against cancer, but cannot be applied to autoimmunity without first changing the chimeric antigen receptor. Instead of targeting antigens on the surface of cancer cells, the CAR T cell must now target the selfreactive helper T cells which perpetuate the autoimmune disease. CAR T Cell Design for Multiple Sclerosis For this study, the researchers created a chimeric antigen receptor which can bind to MS-related helper T cell receptors in mice. The signaling molecules remained the same as usual. However, a protein complex replaced the oft-seen antibody-derived antigen detection region. Figure 2 illustrates this design. It utilizes a major histocompatibility class II (MHC II) molecule, a structure not found on killer T cells, alongside a peptide attached by a flexible linker. The team found that they could easily change the presentation—and thus the targeting—of this protein complex, pointing to the adaptable nature of the design. They programmed the CAR T cells to initially target MOG, a protein found on the surface of myelin that pathogenic helper T cells react to in mice models of multiple sclerosis.

FIGURE 2: Schematic of a peptide/major histocompatibility class II chimeric antigen receptor (pMHC II CAR). The signaling domain consists of a CD3ζ molecule and a CD28 costimulatory molecule. The pMHC II domain can be changed and personalized depending on the desired allele.

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YI ET AL. Success for CAR T Therapy The researchers tested the efficiency of this design in several stages. They first confirmed that the basic CAR T cell design seen in Figure 2 could target helper T cells with specific receptors in mice. Even without standard practices such as lymphodepletion, the CAR T cells eliminated almost all of the T cells they were programmed to target. The therapy only found partial success in mice induced with an autoimmune disease to mimic multiple sclerosis in humans. The CAR T cells did not efficiently eliminate their first target: 2D2 T cells known for inducing multiple sclerosis in animals. The authors surmised that, due to low-affinity between these harmful helper T cells and the MOG CAR T cells, the cells fail to bind strongly to each other and thus influences the response. Fascinatingly, the therapy significantly limited the severity of disease when injected at the first sign of illness. So while the CAR T cells failed to efficiently deplete low affinity pathogenic helper T cells, they reacted more readily to alternate T cells which can cause disease. The team then altered the CAR T cell design to further support the cells’ survival. In doing this, the CAR T cells gained higher sensitivity to both low and high affinity MOG-specific helper T cells. Through this process, the researchers discovered a two-part mechanism that drives multiple sclerosis in animals. Based on the responses from the CAR T therapy, the team realized that higher affinity pathogenic T cells seem to initiate the disease, while their lower affinity counterparts seem to perpetuate the condition. As a result, the most comprehensive use of CAR T cells takes into consideration different stages of disease. Implications This CAR T therapy mouse model leaves room to hope for a long-sought cure for multiple sclerosis. The researchers successfully crafted CAR T cells which can target either low and high affinity helper T cells responsible for multiple sclerosis in mice. From these observations, CAR T therapy could be strategically used to either prevent disease or mitigate existing symptoms. If translated to

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humans, CAR T therapy could be administered to either prevent MS flare-ups or to thoroughly address already-existing symptoms. This article is featured on Forbes.org, and can be read online here: CAR T Therapy, A Promising New Therapy For Multiple Sclerosis?

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New Monoclonal Antibody Fully Approved For The Treatment Of Covid-19 Forbes | December 22, 2022 | Article

The Food and Drug Administration has approved an early gift for those suffering from Covid-19 this holiday season. Tocilizumab, branded Actemra by pharmaceutical company Genentech, is a monoclonal antibody now approved for treating Covid-19 in hospitalized adult patients in moderate to severe conditions. All treatments to this point of the pandemic were only temporarily approved via emergency use authorization, speaking to the value of Tocilizumab. Here we discuss how the antibody was discovered and how it works. Unlike many monoclonal antibody treatments we discuss, Tocilizumab has a long history dating back 25 years. Clinical development of the antibody began in 1997 by Chugai Pharmaceuticals in Japan to treat rheumatoid arthritis. Chugai Pharmaceuticals and Genentech are now under the Roche Group umbrella, a Swiss multinational healthcare company. The researchers at Chugai Pharmaceuticals found that specific mouse antibodies contained anti-human IL-6 receptors. Interleukin6, or IL-6, is a secreted cytokine protein expressed by white blood cells. In terms of disease, IL-6 stimulates inflammatory processes when the body is exposed to various pathogens and clinical conditions such as diabetes, multiple sclerosis, and rheumatoid arthritis. Upon identifying the anti-human IL-6 receptors in mice, the researchers grafted the receptor onto a human IgG constant Fc region using recombinant DNA technology, effectively humanizing the receptor. Later research by Kishimoto et al. confirmed that the engineered mouse antibody receptor grafted to the human IgG constant Fc exclusively blocked IL-6 trans-signaling without affecting other significant pathways.

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FIGURE 1: Tocilizumab calms the inflammatory storm by blocking IL-6 receptors

FU ET AL. IL-6 inhibition sufficiently blocks inflammation without inhibiting other immune defenses against infection. Contrary to popular belief, many of the classic symptoms we experience when we are sick are not directly caused by an invading pathogen but rather consequences of our immune system fighting off the pathogen. Inflammation is one of the most prominent causes of body pain, fatigue, fever, rash, and other significant symptoms most endure during sickness. In severe cases, inflammation may cause severe pain, lack of appetite, and severe headaches and chills. To produce the drug en masse, Chugai Pharmaceutical used genetic manipulation of host cells. They encoded the genes that code for tocilizumab production onto a human host T-cell. The encoded T-cell is then transfected into Chinese hamster ovary cells. These cells are then manipulated to express countless tocilizumab genes, creating a master that can be copied several times. In 2005, Tocilizumab was approved in Japan to treat Castleman’s disease, a rare disorder involving hyperactive immune systems and chronic hyper-inflammation. By 2010, the drug was approved in the 1090

United States and the European Union for the treatment of rheumatoid arthritis. More recently, the drug was approved via emergency use authorization by the FDA in June 2021 for the treatment of Covid19 in hospitalized patients receiving systemic corticosteroids and require supplemental oxygen, non-invasive or invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). Its full approval by the FDA is a testament to the drug's effectiveness in hospitalized patients. While much of the world is content to declare the pandemic over, case numbers and hospitalizations beg to differ. We must continue to search for every available treatment and pursue their distribution with the same urgency we had in the early months of the pandemic. In the next article, will discuss tocilizumab’s efficacy for the treatment of Covid19, leading to its approval. This article is featured on Forbes.org, and can be read online here: New Monoclonal Antibody Fully Approved For The Treatment Of Covid19

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African Morgue Data Reveals A More Significant Covid-19 Death Toll Than Official Reporting Forbes | December 28, 2022 | Article

The low rate of reported Covid-19 infections, hospitalizations, and deaths in Sub-Saharan Africa throughout the pandemic, has raised many questions, especially when Omicron infections swept the globe. Africa has reported over 12 million Covid-19 cases and over 255,000 deaths since the beginning of the pandemic. While these numbers are significant, they pale in comparison to reported cases and deaths among countries in North America, Europe, and Asia. It is likely a lack of access to testing is distorting reported data. New data from a study led by Boston University School of Public Health researchers suggests that the reported Covid-19 death toll in Africa is substantially higher than official records indicate. PCR testing confirmed active Covid-19 infection in nearly 90 percent of deceased individuals at a morgue in Lusaka, Zambia. By contrast, only 10 percent of these individuals tested positive for Covid-19 while alive. The researchers further confirmed Covid-19 as the cause of death through lung biopsies on a select subset. The study was conducted during peak transmission periods between July 2020 and June 2021. The results of this study build upon the author’s previous postmortem surveillance of Covid-19 at the same morgue in Lusaka published in February 2021. The study found that 15-19 percent of 70 deceased individuals tested positive for the virus between JuneOctober 2020. The majority of deaths occurred outside of hospitals and in communities with very limited or even non-existent Covid19 testing. The results of these studies suggest that there is gross underreporting of cases and deaths in Africa due to limited resources and lack of access to testing. This represents a serious unacknowledged threat not only to the people of Africa and visitors 1092

to the country but also for the generation and evolution of new viral variants. Accurate Covid-19 mortality reporting is essential to informing the choices of the public health officials and individuals. By understanding the mortality risk associated with Covid-19, the public can assess their own risk and make use of life-saving tools like masks, vaccines, and antiviral drugs. Unfortunately, as we enter our fourth year of a pandemic that shows no sign of slowing down. Under-reporting of Covid data seems to be the new pandemic spreading across the world. China, once a leader in pandemic control, is now using a narrow definition for reporting Covid deaths, including only people whose death is caused by pneumonia and respiratory failure. At this stage of the pandemic, we now understand that issues like blood clots, heart problems, and sepsis can be directly caused by Covid-19 infection and have resulted in countless deaths around the world. China has also reduced their requirements for mass testing, quarantine, and isolation. According to the GISAID database, China is also not providing up-to-date information about current variants. China has submitted a total of just 667 Omicron sequences, compared to nearly two million from the U.S. As long as access to testing remains an issue, surveillance of Covid-19 mortality needs to include cases where infection is presumed but not confirmed and use a broader definition of what constitutes a Covid death. Only then can we help protect our communities from the risk of death from Covid-19 infection. In the US, the CDC has been updating cases and deaths on a weekly basis instead of a daily basis since late October. This time lag makes it far more difficult to predict risk and surges. State health departments also face an uncertain future regarding the funding they need to continue reporting. Other countries like Australia have also shifted to weekly reporting despite experiencing a higher mortality than any other stage in the pandemic. Reducing reporting capacity or underreporting globally at a time when Covid variants are evolving to become immune evasive is akin to walking into a storm without checking the forecast. This article is featured on Forbes.org, and can be read online here: African Morgue Data Reveals A More Significant Covid-19 Death Toll Than Official Reporting 1093

How Recently Approved Tocilizumab Treats Covid-19 Forbes | December 28, 2022 | Article

The treatment of severe Covid-19 has proven difficult over the past three years, illustrated most clearly by the lack of approved drugs to treat the disease. Recently, the Food and Drug Administration has just its third fully approved treatment, joining Baricitinib and Remdesivir. Baricitinib is approved for the treatment of COVID-19 in hospitalized adults requiring supplemental oxygen, non-invasive or invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). In contrast, Remdesivir is approved for all patients with mild-to-moderate COVID-19 who are at high risk for progression to severe COVID-19, including hospitalization or death. Tocilizumab, branded Actemra by pharmaceutical company Genentech, is a monoclonal antibody now approved for treating Covid-19 in hospitalized adult patients who receive systemic corticosteroids and require supplemental oxygen, non-invasive or invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO), akin to Barcitinib. All treatments to this point of the pandemic were only temporarily approved via emergency use authorization. In a previous article, we discussed the antibody’s discovery and mechanism of action. Here we discuss how Tocilizumab treats those suffering from moderate to severe Covid19. Chief among Covid-19 symptoms are inflammatory conditions such as body pains, headaches, fatigue, and others. Recent studies even link long-term Covid-19 inflammation to permanently impacting the lungs, kidneys, and brain. Treating inflammation in moderate to severe Covid-19 patients could quell both short and long-term consequences of infection. As a highly active anti-inflammatory monoclonal antibody treatment used for over ten years to treat Castleman’s disease, rheumatoid arthritis, and other forms of chronic inflammation, 1094

Tocilizumab was a prime candidate for treating Covid-19 inflammation. Tocilizumab is an IL-6 inhibitor, a secreted cytokine protein expressed by white blood cells. In terms of disease, IL-6 stimulates inflammatory processes when the body is exposed to various pathogens and clinical conditions such as diabetes, multiple sclerosis, and rheumatoid arthritis. Both the emergency use authorization issued for Tocilizumab in June 2021 and the recent full approval by the Food and Drug Administration were based on the University of Oxford-led trial RECOVERY and supported by Genentech-sponsored trial EMPACTA. In the RECOVERY trial, 4,116 adults with progressive Covid19 (defined as oxygen saturation <92% on room air or receiving oxygen therapy, and CRP ≥75 mg/L) received either the current standard of care or Tocilizumab treatment in addition to the current standard of care. Most patients also received systemic corticosteroids at the start of their disease progression. The cohort who received Tocilizumab had a mortality rate of 30.7%, whereas those who only received the standard of care had a mortality rate of 34.9%. Therefore the reduced risk of death from Covid-19 for those who received the drug was -4.1%. Notably, those who received systemic corticosteroids in addition to Tocilizumab saw even more robust results. Their reduced risk of death was -5.9%, suggesting the drug should be administered alongside corticosteroids in Covid-19 patients. We note that these are somewhat marginally positive reductions in the risk of death for a drug to receive full approval. The EMPACTA study delivered similarly modest results in a smaller 389-patient study. 12% of those receiving the drug required ventilation or died, whereas 19.3% of those receiving the placebo required ventilation or died. On Wednesday, December 21st, Genentech announced that Acterma, the clinical name for Tocilizumab, was fully approved for use in hospitalized adults. It joins Baricitinib as an approved treatment solely for hospitalized adults and Remdesivir, which remains the only approved drug for all ages. The emergency use authorization for Tocilizumab remains, allowing hospitalized patients as young as two years old to receive the drug. 1095

Currently, there are three fully approved drugs and 13 treatments approved via emergency use authorization. The marginal effect of Tocilizumab and the recent withdrawal of some antibody treatments from the Emergency Use Authorization list outlines the research difficulty for Covid-19 antivirals. This underlines the necessity to continue expanding our search for highly effective, safe, long-acting antiviral drugs that can be used singularly or combined to prevent and treat Covid-19. This article is featured on Forbes.org, and can be read online here: How Recently Approved Tocilizumab Treats Covid-19

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Synthetic Gene Circuit To Hone CAR T Therapy Forbes | December 29, 2022 | Article

Here we describe a new way to control CAR T therapy through synthetic gene circuits. For more on controlling CAR T therapy, read our article on the use of antibody switches to control CAR T cell activation. Previous installments also discuss the fundamentals of CAR T and its applications for B cell cancers, multiple myeloma, lupus, multiple sclerosis and the heart, as well as new research on combination CRISPR/CAR T therapy. While CAR T therapy represents one of the most impressive innovations in cancer care, the treatment can cause autoimmunelike side effects. Many have contended with the dilemma of maximizing the therapy’s benefits while minimizing its side effects. A recent advancement published in Science shows great promise in improving control of CAR T therapy with the hope to eventually benefit patients. Challenges of CAR T therapy Chimeric Antigen Receptor T cell therapy relies on engineering synthetic receptors onto a patient’s immune cells to recognize and eliminate a programmed target. The treatment has proven effective in targeting antigens, or biological tags, found on the surface of some blood cancer cells. Although successful in this regard, the modified T cells cannot be controlled once injected back into the body. This factor, coupled with other obstacles such its inability to target more than one antigen at once, leaves space to improve the treatment. Possible Solutions There are several budding research initiatives seeking possible solutions. One previously discussed method of controlling CAR T therapy involves creating CAR T cells which bind to antibody switches. Researchers found that a single infusion of antibody switches could help mitigate the worst of CAR T therapy’s toxic side effects. Although this method could potentially fight solid tumors by altering the antibody target, it has yet to be tested in animal or human models.

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A recent study from the scientists at Boston University offers an alternative and versatile platform which could transform CAR T therapy implementation. The team turns to synthetic gene circuits for answers and receives encouraging results. A New Alternative: Synthetic Gene Circuits In a gene circuit, the process of turning an input into a desired output can be controlled through the use of a synthetic regulator (see Figure 1). The synthetic regular can, in theory, tailor a cell’s gene expression to produce a desired result. The researchers in this study designed such a circuit using synthetic proteins and small molecule switches. To establish the circuit, the team relied on synthetic versions of proteins called transcription factors. Transcription factors can recognize certain DNA motifs and help convert that DNA into RNA, genetic instructions for protein synthesis. They leverage synthetic zinc fingers (SynZiFTR) in particular due to its compact size and human origin; Figure 2 illustrates the structure. Both factors together allow the protein to move efficiently in human cells while minimizing unwanted side effects. Additionally, several zinc fingers can be joined together to create a structure capable of recognizing potentially unique human DNA sequences in the genome, as seen in Figure 3. This circuit must be controlled using a gene switch. The team experimented with three clinically approved small molecule inducers to accomplish this task. The unique combination of zinc fingers and small molecule inducers allowed genes to be turned “on” with the introduction of the inducer, and turned “off” with its removal.

FIGURE 1: A synthetic gene circuit can alter a network of input signals to produce a custom response. Using this underlying base mechanism, CAR T therapy could be controlled through the use of synthetic zinc fingers to produce a more precise result.

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LI ET AL.

FIGURE 2: Representation of the Cys2His2 zinc finger motif. The zinc ion is represented in green.

WIKIPEDIA

FIGURE 3: Zinc fingers can be modularly arranged with unique DNA binding motifs to modulate transcription in a manner that does not impede other native functions.

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LI ET AL. Combining Synthetic Gene Circuits with CAR T Therapy How do these synthetic gene circuits fare when used on CAR T cells in vitro and in vivo? The team investigated this question in several stages using cell and animal models. The researchers first tested the efficacy of a gene circuit which controlled the expression of chimeric antigen receptors. They found that in a xenograft liquid tumor model, the engineered receptor’s expression could indeed be controlled in a drug-dependent manner. The treatment could be reinforced using the same stimuli and secondary infusion of chimeric antigen receptors with a different antigen target, demonstrating the adaptable premise of the platform. The platform produced similar results when tested on blood tumor models in mice, as well. The mice treated with drug inducer or a drug-increasing cocktail could clear the tumor, while those without had high tumor burden. This phase illustrated how gene circuits can be influenced in a drug-dependent manner in living creatures. One of the most fascinating findings of this study is the creation of a dual-switch gene circuit. Researchers crafted a gene circuit which impacted cytokines known to influence cellular proliferation. This cytokine circuit could successfully prime dual-switch CAR T cells in mice with leukemia; a secondary signal to encourage CAR expression sparked their anti-tumor activity. The sequential and synergistic effect of the dual-switch circuit can be seen in Figure 4. This duo reduced tumor burden in mice more successfully than untreated CAR T cells or CAR T cells induced with anti-tumor stimuli only.

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FIGURE 4: Dual-switch gene circuit creates a sequential and synergistic effect by triggering and encouraging cellular proliferation before activating T cell anti-tumor activity.

LI ET AL. Looking Forward The researchers here show how synthetic gene circuits can feasibly improve CAR T cell proliferation and anti-tumor activity in animal models. This promising platform could prove clinically viable once translated to humans. However, the implications of this study stretch beyond the rapidly growing field of CAR T therapy. The underlying mechanism could be customized to improve other gene and cell therapies, or combined with other powerful technologies such as CRISPR-Cas9 to achieve more bespoke results. This article is featured on Forbes.org, and can be read online here:Synthetic Gene Circuit To Hone CAR T Therapy

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Media Inerviews

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Lifting Mask Mandates

The Lead | February 10, 2022 | CNN Broadcast

Jake Tapper, CNN Correspondent 0:03 Dr. Haseltine is a former Harvard Medical School Professor known for his work in researching deadly viruses. Mr. Haseltine. Good to see you. Only today did the number of people in the hospital with COVID drop below 100,000. The CDC director said this trend's encouraging but she repeatedly said hospitalizations remain too high for states to lift the mask mandates. Do you agree? Dr. William Haseltine 0:26 I agree. If you take the United States as a whole, we have more cases than we added the peak last December, we have about 340,000 cases - that's reaching down to the peak. So we have a lot of COVID still in the country. An awful lot. Secondly, the number of people who died of COVID yesterday was about 3,300. That's a lot of people. And again, it's on par with the worst we've ever had. Now, I look at this as coming out of a tornado and going into a hurricane on average, but an average isn't your local area. So what people are going to decide to do is when people say it goes down 40%, 40% down from a tornado is not good. So that's what the CDC is looking at. But other people: look at what's happening in their local area. 1103

And I urge everybody to go online, we just go online and look up COVID by your zip code. It's right there on Google, you can find it. So that's what I would do. If you really want to know how dangerous it is. I have another comment to make about masks if I have a minute, and that is that masks are a good thing in general. This is flu season. A mask helps stop flu. This is RSV season, respiratory syncytial virus- that hurts very young children and older people, masks help that. I think that there's all this heat abouts masks, but we should begin to think about masks as just a regular part of life during the flu and cold season. And when COVID is there, it's even more important to think about it. So I think that's something that people should keep in their mind. Other countries do that around the world - in the winter, mask up - and it's not a big deal for kids. They just do. It's not a big deal for adults either. Jake Tapper, CNN Correspondent 2:15 Dr. Wollensky said the CDC is not there yet, when it comes to calls to change the guidance. Do you think the CDC is acting too slowly when it comes to changing guidance? Dr. William Haseltine 2:28 I don't think so. We see that the CDC only makes recommendations. In any event, we are a country where states and local communities have a great deal of power. It's not that it's not a federal mandate, that everybody wears masks. It's a strong recommendation. And I think that's the prudent thing to do when we have as much COVID right now, today, as we had at the peak in December, and much more than we had when people were worried about it before. So it's all relative. Yes, it's coming down. But it hasn't gone away. Jake Tapper, CNN Correspondent 3:00 Democrat Stacey Abrams is running for governor in Georgia, she came under fire for taking off her mask at a school event in which every child in the picture had a mask on. I want to note something she said in her apology about why she says she took her mask off, take a listen. Stacey Abrams 3:17 I approach the podium with my mask on, I follow the protocols. I told the kids I'm taking my mask off because I'm reading to kids who are listening remotely as well. And we were socially distanced, the kids were socially distanced for me. I told them, that's what I was 1104

doing. Protocols matter. And protecting our kids is the most important thing, and anything that can be perceived as undermining that is a mistake. And I apologize. Jake Tapper, CNN Correspondent 3:40 But the point that I think a lot of folks wonder about is - and I should note that she was talking to my colleague, Erin Burnett - and Stacey Abrams is making the point about trying to communicate with kids, taking off the masks, which I understand. But it should apply for more than just a concern for when a candidate is speaking to a classroom of kids. I think there are a lot of people very concerned about whether kids need to be masked up, whether kidstheir speech development and other socialization skills are being heard here. Do you have those concerns? Dr. William Haseltine 4:18 Well, first thing I'll say about the Stacey Abrams situation is a picture's worth 1000 words. And in this case is now a good picture. Simple as that. The second thing I would say about our kids, I have grandkids and they have no problem with a masks. There may be kids that do have problems with masks, I understand that. But the kids I see and all their kids class are very happy, seem to be happy wearing their masks. You know, kids are really adaptable. And I'm not sure that there's really good science behind the claims that the masks are impeding education. But does impede education is not being able to go to school, that you stay at home because your COVID, or somebody in your family has COVID. So I don't think the masks impeding education is really a hard, proven fact. And kids are very adaptable and the ones I've seen seem to adapt pretty well. Other people may have seen other situations. Jake Tapper, CNN Correspondent 5:12 Alright, William Haseltine. Thank you so much for your time today, sir. I really appreciate it.

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Discovery of New HIV Variant Sends Warning for COVID Pandemic Scientific American | February 17, 2022 | Article

As SARS-CoV-2, the coronavirus that causes COVID-19, has spread throughout the world, many observers have failed to take note of the millions of illnesses and deaths caused by HIV—another virus that has approached pandemic status during its history. Now an HIV variant that is more virulent and transmissible has been discovered in the Netherlands, where it apparently has been circulating for decades, according to new research. Luckily, none of the variant’s new mutations make it resistant to widely used therapies. But the finding may offer a warning for how the COVID pandemic could proceed in the coming months: viruses do not necessarily evolve to become milder. Without treatment, people infected with the newly identified HIV variant have more than three to more than five times higher levels of the virus in their blood, making them more infectious. Plus, their immune system deteriorates twice as fast, setting them on a course to potentially develop AIDS years earlier than people with other versions of HIV. These findings, published this month in Science, indicate the newfound variant carries more than 500 mutations scattered across its genome—though it is unclear how they enable the virus to cause more-severe disease. William A. Haseltine, an infectious disease researcher who founded Harvard University’s cancer and HIV/AIDS research departments and now chairs the think tank ACCESS Health International, has written extensively about the potential of SARSCoV-2 assume a more dangerous form. Haseltine spoke with Scientific American about why a more virulent variant of HIV—a virus that has been known for nearly half a century—is just coming into 1106

focus now, whereas the new coronavirus has spawned several “variants of concern” in a matter of months. [An edited transcript of the interview follows.] What does the discovery of a more virulent HIV variant tell us about how viruses, including SARS-CoV-2, evolve? We’ve known that all viruses adapt. The way they adapt is much like how we use artificial intelligence to solve complex problems: the machine throws a lot of random combinations at something, and the one that works best is the one that survives. With HIV, [its evolution has] been a long, drawn-out process because the virus is poorly transmissible. It takes, on average, 100 sexual contacts for a man to give it to a woman and 200 contacts for a woman to give it to a man. The process is a slower-evolving one— not because the virus doesn’t change but because the replication cycles can be quite long. For Omicron, those cycles can take hours or days at the most. The virus can be transmitted by a person simply breathing the air somebody else breathed a half an hour ago. In the past year or so, SARS-CoV-2 has produced several different variants of concern. How worried should we be that more virulent versions of the virus are yet to emerge? First of all, the virulence of SARS-CoV-2 has been very stable— with the exception of Delta, which is twice as likely to land you in the hospital. Delta was a warning shot across our bow, showing the virus can become both more transmissible and more virulent. There is nothing that we know of that restrains this virus from becoming as lethal as its cousin SARS-CoV-1. We still have no clue whether one genetic change or many make SARS-CoV-1 so much more virulent than SARS-CoV-2. As long as we are in the dark about what it is that determines the virulence of the virus, we have no idea which direction the next variant will come from. So I’ve been telling policy makers to be optimistic about the upside but prepare for the downside. Are some viruses poised to produce more variants of concern than others? Yes, RNA viruses [such as HIV and SARS-CoV-2], in general, make more mistakes [and thus enable more opportunities for evolution] than DNA viruses. Your cells have elaborate machinery to fix mistakes in DNA, which must last a long time to be inherited, whereas RNA is effervescent and plays a more transient role in 1107

cellular life. It turns out that SARS-CoV-2 has a proofreading enzyme that can correct mistakes, so people thought that would protect against variation. They were incorrect. One of the major errors people made in underestimating this virus was the extent to which it can change. HIV and SARS-CoV-2 are both RNA viruses. Do the factors driving their evolution differ, and if so, how? The selective pressure on a virus is to survive, just like it is for any other organism. What the virus wants to do is get from one person to another—get in and get out. Because HIV is so poorly transmitted, its best strategy is to get in and stay there for a very long time and rely on a predictable behavior—sex—to get out. For SARS-CoV-2, the virus depends for its existence on reinfecting people who have been infected the year before. We are fighting millions of years of evolution of an organism that knows how to fool our immune system and get into us again and again. One thing we have seen the viral variants doing is getting faster and faster [at transmission]. Delta is faster than Alpha. Omicron is faster than Delta. And BA.2 Omicron is faster than BA.1 Omicron. There are many ways for this virus to mutate to increase its transmissibility. Whether any of those will affect virulence is unclear. This interview originally appeared on The Scientific American, and can be read online here: Discovery of New HIV Variant Sends Warning for COVID Pandemic

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Mask Mandates

The Heat | February 20, 2022 | CGNTV Broadcast

Mike Walter 00:00 Many countries are ditching COVID-19 mask mandates, but medical experts are concerned because the number of cases are slowly going up. Hello, I'm Mike Walter sitting from in an eyeview, and this is The Heat. Mike Walter 00:12 This week a US federal judge struck down the mask mandate for planes and other forms of public transportation. Airlines quickly complied, allowing passengers to travel without masks. Spain ended its indoor mask rule after two years, and Israel will do the same this weekend. Brazil has also relaxed protocols and is holding its first and famous carnival for the first time in two years. But medical experts are concerned with the omicron sub variant, or the United States cases arising after a two month decline, causing many to ask, is it too soon to lower our guard? To help us answer that question? We have Dr. William Haseltine. He's the Chair and President of Access Health International and author of "Omicron: From pandemic to Endemic." Bill, what was your reaction when you heard this federal judge ruling a 59 page ruling striking down the mask mandate from Florida? Dr. William Haseltine 01:22 I was very concerned. I follow variants, as you probably know, pretty closely. And I'm watching the new variants of omicron - the sons of omicron, the daughters of omicron - begin to rise in many parts of the world. Not all parts yet, and not quickly, but rise seemingly inexorably. Ditching the mask mandate, I think, can only speed that rise. Mike Walter 01:51 Yeah. So what states here in the United States, will you be watching as these infections start to spread? Dr. William Haseltine 01:55 1109

Well, the typical pattern in the US is it starts first in the northeast, then it jumps over to the West Coast, sometimes to the northwest as well... then to the Midwest and South. And that's been the pattern for the last two years, through five successive waves of the pandemic. This one doesn't look nearly as abrupt as the onset of omicron in December and January, but it is certainly something to watch. Underlying this is the short period of time that either prior infection or vaccines are effective in preventing infection; they don't last very long. It would seem three to four months at the outset. And at the end of that period, the virus has an opportunity to reinfect without changing and if it does change - which it is changing - to reinfect all that all the all the better. Mike Walter 02:55 And anecdotally, I just heard from somebody today who said, they know some what to add to the shots, didn't get the booster, really had a tough go of it. Should we start looking at what's "fully vaxxed" differently? I mean, now, you could have three shots, you could have four shots, but people keep saying, "Oh, I got the two shots, I'm vaxxed." But is that the case? Dr. William Haseltine 03:15 Well, I think we now know that the Moderna and the Pfizer vaccines are three-dose vaccines. The difference is about tenfold in terms of short term protection. But the difference is much greater than that in terms of protection from serious disease. If you have three shots, you are about 90% protected from serious disease for probably at least a year and possibly longer. That means it's a very, very good idea. They get the three shots, and then the fourth shot and the fourth shot drops out even lower. That 10% drops to another 75% protection with four shots. And so against serious disease, not just against infection, against serious disease, which is what most of us are really concerned about. Sure, we can stand a cold, but we don't want to get serious disease. Mike Walter 04:11 Protection. That brings up the mask question. If you were to have a conversation with somebody, they were going to travel, get on a plane or a train, would you say, "Hey, I'd still wear the mask”? I mean, what's your suggestion to people watching? Mike Walter 04:23

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Let me ask you about this. Bloomberg was out with a piece headline "Biden team waivers on fight over masks after court setback" and the concern was, you know, do we want this to go all the way to the Supreme Court and then it kind of handcuffs the CDC in the future? And yet late today, it looks like the CDC is going to ask the Justice Department to pursue this in terms of an appeal. What are your thoughts on that? Because it seems as though the Supreme Court is tilted very much to the right, there is the possibility that they could handcuff the CDC moving forward. Dr. William Haseltine 04:23 Well, if you're going to take a train or a plane, I dare say double mask. I say that because these viruses, to give you a rough rule of thumb, whereas Wuhan might infect 1.2 people, not two or three, this current one will infect up to 12 people. It's much, much more infectious. And so if people aren't generally wearing masks, the only thing you can do is try to protect yourself. And that means wearing at least one, and I would say two masks at this point. If you are planning to be with a large group of people whom you don't really know - that means a crowd that means a bar. That means a plane, a train, a bus, etc. Dr. William Haseltine 05:35 Well, I think that's a very serious issue. In fact, it's a broader issue. Going back to very ancient times, Roman law, which is the foundation of much law around the world, was that public health is the greatest good; all other rights must give way to public health. That has been an operating assumption, from the creation of quarantines to the isolation of people who are ill and depriving them of other rights that they would normally have. And in this pandemic, we've seen the legal ability to enforce that ancient principle of protection of societies itself, erode. The latest example is this mask, but also there have been many other cases. So I think that we are emerging from COVID-19 in a much weaker position to protect ourselves from future pandemics, which will surely come. Mike Walter 06:37 Yeah, speaking of which, your book of course, "Omicron, from Pandemic to Endemic..." Is that where we are now? [Have] we entered the endemic phase? And do people really understand what endemic is? Dr. William Haseltine 06:47 1111

Well, we've entered the endemic phase because COVID is here to stay. Pandemic means a sweep through and it's gone. That hasn't happened with the flu. And now it's not going to happen, I think, with COVID-19. We know enough about it to know that it's here, it keeps changing. And we've infected not just our human population, we've infected broad swaths of the animals we live with: mice, deer, dogs, cats. I could make a very long list of the other animals that we've now infected. It's not just from bats anymore, we get back and forth. We know now that we give it to the minks, the minks give it back to us. We are now living in a new world, in which COVID-19, this particular coronavirus, is here to stay. Now endemic doesn't mean mild. Tuberculosis is endemic and still kills. Malaria is endemic and still kills. They don't get more mild. So there's no guarantee. All endemic means is it's here to stay. The severity will depend on: are we vaccinated and this virus relates to that vaccination. Are we vulnerable? Are we older? Are we overweight? Do we have diabetes? Do we have other immune deficiencies? Are we being treated for cancer? There are many other factors that go into vulnerability. And the virus is a combination between what this virus can and will do and how it changes, as it's always going to be with us, and who we are and how we behave. Mike Walter 08:32 Bill, I'm going to ask you about measuring this latest outbreak. I mean, you're talking about how infectious it is. But we've seen in Shanghai, a large number of people who are asymptomatic. In other words, they're walking around, they have no idea they have it. It's spreading like wildfire. As you said, it's very easy to spread. How much of a concern is that here in the United States as well? Dr. William Haseltine 08:53 It's a pretty big concern, because in addition to the short term consequences of COVID, there are the long term consequences. It's called Long COVID or post acute sequela of COVID (PASC). And we're not realizing that there are many, many long term consequences. It can affect the way you think and the way your brain is, it can age your brain about 10 years as people have found; if you look at somebody who's mentally confused after COVID three or four months later, it looks as if they have Alzheimer's -they don't, but it looks as in their blood as if they might. It can affect your heart, your kidneys, your pancreas. And of course, it's characterized by 1112

fatigue. There's some debate about whether 10 to 30% of people might suffer from Long COVID. And so when 100 million Americans are likely to have been infected at this point, at least, that's a lot of people. And we have to take long COVID very seriously. It's very hard for people to understand what chronic fatigue syndrome is. It's hard for doctors to treat it; they don't have a simple measure that says "yes, you have chronic fatigue syndrome." But those people who have it are really disabled. And it's something to contend with because you don't have to get sick to get Long COVID. You don't even have to know you've had COVID to get Long COVID. So we are now dealing with a new unknown that's going to be with us for a long time. So not only is the virus endemic, but the long term effects of what already happened are going to be serious. Mike Walter 10:38 Bill, it's always a pleasure talking to you. Thanks so much for your time. Dr. William Haseltine 10:40 Well, I wish I could be more cheerful. Thank you. Mike Walter 10:43 I wish you could too, but at least you're being honest. [END OF TRANSCRIPT]

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Impact of Long Covid on the Climate Crisis The Heat | September 22, 2022 | CGTN Broadcast

Oliver Diamond 00:39 Talk of the pandemic winding down, new data shows long COVID may linger on. Plus, later in the show, we go to the United Nations for a conversation with one of the leaders in the fight against climate change. Hello, I'm Oliver Diamond, this is The Heat. Oliver Diamond 01:12 Climate change is high on the agenda as global leaders gather for the annual meeting of the United Nations General Assembly. We'll have more on that later in the program. But we begin with the COVID 19 pandemic, the average daily death toll in the United States is more than 350. This, as the government estimates, seven to 23 million Americans have long COVID. William Haseltine is an expert on the subject. He is the chair and president of Access Health International, and author of the new book, A Family Guide to long COVID questions and answers. Bill, great to have you with us. Dr. William Haseltine 01:48 My pleasure. Thank you. Oliver Diamond 01:50 So let's start with the basics. What is long COVID? What are the symptoms and how long does it last? Dr. William Haseltine 01:56 Long COVID is the consequence of being infected by the SARS-CoV-2 virus. It takes multiple forms. First of all, shortly after you're infected you experience—or many people experience— something called acute COVID. Without a vaccine, it's about 20%—much slower if you've been vaccinated. That lasts up to a month, and after about a month to six weeks the most seriously affected will die. Dr. William Haseltine 02:21 But the long COVID symptoms begin later, I would say the next phase of it lasts for up to six months to a year. And that's a series of symptoms that are debilitating. You just feel different than you did 1114

before you were infected. That includes most prominently the symptoms of fatigue. And fatigue isn't just being tired after a long night. It's so tired, you can't get up, you can't work. And brain fog, it's not just feeling a little fuzzy, you have something called executive dysfunction, you really can't plan your day, you can't necessarily read; if you start to write an email, you may forget what you're doing halfway through. It can be very serious. Most people get over that, and about 20 to 30% of people have some of these symptoms. There are many other more minor symptoms, but those are the two major ones. That is of course, unless you suffered serious damage to your organs, either a heart attack or serious lung damage or diabetes or kidney disease. But about 2% to 4% of people are seriously impaired for up to two years, two and a half years now that we've had COVID around for that long. And that means they can't work. It's estimated that between three and 4 million Americans now are out of the job market. And we don't know for how long. Dr. William Haseltine 03:47 And then there is very Long COVID. That's something that we know with many viruses when you're infected, there can be repercussions from that infection, many years to decades in the future. For example, the 1918 flu epidemic caused a high incidence—10 times incidence—of Parkinson's disease many decades later. There's a whole family of serious consequences. And it's a big problem. Probably five to 6 million Europeans are out of work for that. And as the pandemic continues, the infections, as you pointed out, are infecting more people every day. It's pretty serious. I might also add that COVID affects children. 75% of American children have been infected, and up to a quarter of those have some symptoms at least six months later. Oliver Diamond 04:49 You know, that's right, those figures that the government is giving us...their estimate is that from seven to 23 million people have Long COVID. Is the government doing enough to combat this particular condition? I mean, could we see a specific vaccine that'll be created for this or perhaps some other kind of medication as well? Dr. William Haseltine 05:09 Well, the vaccines have some effect. The vaccines that have been introduced, particularly the mRNA vaccines, can reduce the incidence of long COVID by about 15%. That's not a lot. The thing 1115

that people have to remember is getting COVID is not a joke. It's not like a mild cold, it's much worse than that because of Long COVID. The startling fact is that you can have no obvious symptoms of the disease or a very mild disease, and you can get long COVID. You can be young, and get long COVID. In fact, the average age is in the 20s to 50 year olds; they're the ones that get long COVID. So as far as we know now, there's no medication specific to long COVID. A number of the symptoms people have— heart disease, diabetes, kidney disease—can be treated with very few, if no treatments really for the brain problem, except for more standard kinds of treatments associated with a management of chronic fatigue syndrome, for example. Oliver Diamond 06:20 So as you say, many young people are getting long COVID. This is a survey that's been done by the CDC and the United States Census Bureau, which tells us that— Well, firstly, it tells us that one in five Americans who had COVID still have long COVID. And then it also tells us that older adults are less likely than young adults to get long COVID. You just told us about that. And women are more likely than men to get long COVID. So what can we deduce from that? Why younger people and why women? Dr. William Haseltine 06:49 It's really a mystery. As we know, the serious part of COVID hospitalization and death, most serious parts result most heavily felt by the older population 65. And up and above 80, it's a pretty serious thing to happen. One in 10 will die if they're not vaccinated if you're over 80. But that's not true for long COVID. It follows what we call a "W" pattern, that is younger people, very young people are somewhat spared. However, then when you get to people who are 20 to 50, to 55, there's a peak, and then it fades away again, and we have no good explanation for that. Oliver Diamond 07:36 Now, in terms of people getting help, Scientific American had a report in which it says that medical discrimination and a lack of data also make it difficult for people of color to get help for post COVID problems. And the reason, one of the reasons, they say, is "systemic poor access to health care." How much of this are we seeing? Dr. William Haseltine 07:59

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We're seeing a lot of it. If you look at all the statistics, they show the COVID falls most heavily on the minority populations: the black population, the Hispanic population, the white population that is below a certain income level, are the most heavily affected populations... indigenous American populations. The statistics are really rough, but 20 times the rate in the indigenous populations, eight to 10 times the rate of serious infections in those and other populations. And it's reflective of a healthcare system which is inequitable. The United States healthcare system and health all together isn't the best—it is maybe the best of the world or as good as the best at the high end. But that's not uniform, the equity kind of services we know aren't uniformly available to many people. And it shows up in the overall health statistics of how long people live, infant mortality, maternal mortality—we're down around 34/35th in the world, so we're not doing well with respect to health outcomes. And that is even more reflective when a pandemic like COVID comes along. Those at the bottom of the pyramid who don't have access to high quality health suffer the most. It's something that we need to address as a country, as a people, urgently. Oliver Diamond 09:36 Bill, you were talking a moment ago about how long COVID has impacted the workforce. And I mentioned those figures that the government has given us between seven and 23 million people having long COVID. A million people are actually out of work right now, and I'm wondering what's the long term impact of long COVID on the economy, which is already having to deal with things like a potential recession with high inflation and the broader impact of the pandemic? Dr. William Haseltine 10:02 Well, there are multiple effects. First of all is the healthcare system. The health care system is, as we said, already faulty in some ways, and it's really not geared to helping very large numbers of people who are seriously incapacitated with no obvious remedy. That takes really intensive care to take care of these people who can't go to work because they have executive dysfunction—their brains, you can actually see damage to the brain. It's not imaginary. You can see for people are really fatigued, and we're familiar with chronic fatigue syndrome, for keeping people out of work. That's a big dent. We actually, although we have some people out of work, 1117

unemployment is really low. And it's very frequent. That means something everybody knows: it's very hard to hire people now, their jobs going begging. And this is really making that worse. Oliver Diamond 11:01 Bill there's a minute left. You know, last weekend, President Biden, in an interview here in the United States said that the pandemic is over. But the World Health Organization tells us that the end of the pandemic is in sight, but we're not there yet. What is your sense of where we are now? Dr. William Haseltine 11:19 Well, seeing the end of the pandemic reminds me of my days years ago as a Vietnam War protester, where they said there was a light at the end of the tunnel. There can be a tunnel at the end of the tunnel was there was then and that's how I see it. We do not know when this is going to end. We don't know when the next variant is going to appear only that is very likely that will. We desperately need new types of medication—not just the vaccines, we need antiviral drugs. And I think we're not doing enough to get those drugs that we need to put this thing to bed for good. Oliver Diamond 11:54 Oh, Haseltine. Thanks so much for joining us. Dr. William Haseltine 11:56 My pleasure. Thank you. Oliver Diamond 11:59 We need to take a break right now. When we come back: How is the UN responding? [END OF TRANSCRIPT]

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President Biden's "Cancer Moonshot" Initiative Alhurra | September 24, 2022 | Public broadcast

Alhurra Interviewer 01:27 Welcome back. American President Joe Biden launched Cancer Moonshot Initiative, which aims at creating an agency for advanced medical research in order to deal with the prevention, detection and cure and to reduce the number of fatalities of cancer, reducing it by 50%. In 25 years, this program was launched by Mr. Biden for the first time in 2016 when he was vice president, and aimed at pushing forward efforts to fight cancer. This time around, the program is relaunched, but the objective is to reduce the number of cancer fatalities by 50% over the next 25 years. Alhurra Interviewer 02:21 Among the types of cancer, lung cancer is considered the most virulent according to the statistics. Indeed, the survival chances do not exceed 19% and could dwindled down to 5% when it's detected at an advanced stage. The cancer research has to deal with the genes and proteins responsible for cancers especially breast cancer and skin cancer. However, the gene called Krass, which is responsible for the growth and the division of cells continue to be difficult to deal with. Alhurra Interviewer 03:06 KRAS gene is responsible for 32% of lung cancer and 90% of pancreatic cancer. However scientists believe that it's very difficult to deal with KRAS. In May of last year, the FDA approved the LUMAKRAS, which was produced by Amgen after the test showed that it can slow the growth of cancer. The medicine also showed the efficacy and dealing with colon cancer. The company said this is not a therapy, but it can slow the growth. And it is a good alternative for chemotherapy. President Biden, who lost his son to a brain cancer in 2015, announced this initiative on the 16th anniversary of the speech of John Kennedy in which he launched the initiative of moon landing. There is hope now that we can deliver on the promise of Mr. Biden. Alhurra Interviewer 04:31 1119

To shed light on this issue, we're joined through Skype from Connecticut by a professor and doctor William Haseltine, former Harvard Medical School professor. And from Washington, vice president of the Global Policy Institute, Mr. Paulo van Chirac. Welcome, gentlemen, we appreciate your time. Let me start with Dr. Haseltine. Dr. Haseltine, what is the technique exactly that we're talking about here for cancer? Dr. William Haseltine 05:09 There's been a number of programs like this starting with Nixon in 1971 with the war on cancer. You mentioned Joe Biden's moonshot in 2016. And now the renewed effort, and the creation of a new agency—ARPA H—designed to accelerate all types of applied knowledge to cure many types of diseases, including cancer. Dr. William Haseltine 05:35 Let's talk first about the goal: is it realistic to reduce the deaths from cancer by 50% in 25 years? Well, I think it is because of the last 20 years, in terms of deaths per 100,000 in the United States, we've achieved a 27% decrease. That was just recently announced. That's pretty impressive. And it's based on a combination of various things. Really good funding for fundamental cancer research, new breakthroughs—I would cite two in particular, one is the use of antibodies to activate the immune system, which has been silenced by the [] cells, and the use of monoclonal antibodies. it's a very big part of that. And now we have a new type of cell therapy called CAR T, which is using the person's own cells, activated by manipulation outside the body and reintroduced, which is having some pretty good results. Also, the kinds of cancers that you mentioned that are refractory to other cancer. So I think the goal is possible. It will require a steadfast investment—both in fundamental and applied research—as well as accelerating clinical trials to make sure that new drugs get the best chance for making a difference to patients. Alhurra Interviewer 07:12 Indeed. Thank you, Doctor. Let me go to Mr. Paulo, American President launched this initiative, Cancer Moonshot. What what do you expect the outcome to be if everything goes well? Paolo von Schirach 07:33 Well, look, Americans are proverbially problem solvers, right? We want a big challenge. And we want to say, "Okay, let's go do 1120

it." Now, I am not a scientist. And I defer to my esteemed colleague here, who knows everything. I know nothing about the science. But I know a little bit about public policy. And therefore, I would say that much of the success will depend not just on the quality of the scientists—that I'm sure are out there, many people extremely gifted with great ideas and new concepts—but how this is harnessed, how the funding will be distributed; whether it will be done in a timely fashion. Paolo von Schirach 08:18 I applaud the creation of this new agency. I know it's "sister" DARPA, Defense Advanced Research Project Agency, which is been the think tank at the laboratory of the Pentagon. Now, of course, defense and medicine are two entirely different fields. But if this ARPA H, where H stands for health, really is modeled after DARPA—that is, to try to get the best and the brightest to think out of the box, to be innovative, to dare to do the things that nobody would dare and try and have adequate funding distributed in an appropriate fashion according to the appropriate time sequence— well, then many, many things are possible. And I think that if this effort worked, in other words, if President Biden manages to harness this incredible talent that we have in this country...the private sector, in the universities, in the research institutions and the government, NIH, others—there's a long, long list. The problem is when everybody's doing something in isolation, if this could be somehow coordinated in some fashion, and people have the opportunity for cross pollination, for sharing in a timely fashion, their accomplishments, their lines of research, that in many, many new things are possible. Alhurra Interviewer 09:47 Mr. Paulo, what is the significance of the United States leading this effort given the financial costs? And do you think this will remain an American effort, or do you see down the road, a cooperation between America and other Western countries? Paolo von Schirach 10:11 Well look. America leads, America has resources—that's not the only country that has resources, clearly— but has more resources and more talent probably than other countries do. Eventually, any scientific discovery becomes the patrimony of science. You know, whatever, whatever will come out of all this, and I hope it will be 1121

fantastic results; and soon, even, they will be spread all over the world. This is not going to be "Okay, we invented it. It's only for Americans, nobody else gets it." It would be absurd. Everything is shared eventually. And there will be an opportunity, assuming really dramatic breakthrough —and I really hope they will be sooner rather than later—this will become the patrimony of the world. If America can lead, that's fantastic. If down the line, there will be cooperative arrangements with other countries with the European Union—I don't know, in Japan, with Australia, we've got friends in other countries. Well, that would be fantastic. But it's important to get started on this. And this is an immensely vast and challenging task. Thank you. Alhurra Interviewer 11:18 Let me go back to Dr. William Haseltine. Dr. Haseltine, regarding this initiative. We asked you earlier about the technique. But now we'd like to ask you about the steps and the focus. In other words, do you think this is going to concentrate on the prevention, on the detection or on the cure? Where do you see the focus? Dr. William Haseltine 11:46 It's clear that the focus for this part is on finding new cures and getting those cures tested. Not just in small groups, but there's a very important focus of this administration in particular on equity, making sure that all members of society, from all classes rich and poor, black and white, different colors are included in trials and will benefit from this. Dr. William Haseltine 12:13 Let me also comment on this concept that was referred to of DARPA is this is really the fourth such agency which looks ahead using the eyes and ears of our government and all of our best scientists to see what could be done next. First of all, there was ARPA, which led to the internet; there was DARPA, Defense Advanced Research Projects Agency; there's IARPA for our intelligence community new technologies, both for understanding what's happening in the world. And now I'm very pleased to see that there's an ARPA H. This has been a very good model for standing ahead, looking at the future and bringing that future into reality. Dr. William Haseltine 12:58 I have benefited in my career from the war on cancer, the special virus cancer program. And I can say we have all benefited recently 1122

by the almost miraculous speed at which COVID vaccines were developed. They are direct descendants of the war on cancer, the special virus Cancer Program, the work on HIV which benefited from the cancer work, and now the ability to develop a vaccine in record time. They all build on each other. And I'm very happy to see this new development, and I do hope we can reach the stated goal. Paolo von Schirach 13:36 Thank you very much to both my guests. Dr. William Haseltine, former Harvard Medical School professor and Mr. Paolo von Schirach, the president of the Global Policy Institute, the gentlemen We appreciate your time and insights. Thanks a lot. [END OF TRANSCRIPT]

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How the Pandemic Shortened Life Expectancy and New Drugs on the Horizon: COVID, Quickly, Episode 40 Scientific American | October 14, 2022 | Podcast

In this episode of the COVID, Quickly podcast, we talk about why we’ve had years shaved off our average collective life since 2020. Also, we talk about “mabs” and why you might want to know what they are. This article originally appeared on Politico, and can be read online here: How the Pandemic Shortened Life Expectancy and New Drugs on the Horizon: COVID, Quickly, Episode 40

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The Quest For New COVID-19 Solutions Science Friday | November 4, 2022 | Article

Ira Flatow 00:03 This is Science Friday, I'm Ira Flatow. As we head towards our third winter with COVID. This may be a good time to reflect on the paths taken to deal with the pandemic, the development of the vaccines distribution, and what may lie ahead. Case in point. Pfizer announced last week that your vaccines may no longer be free next here, retail price about 130 bucks per shot. So what does this all mean? Here to help explore the viral landscape as a scientist who has spent a lifetime researching and battling viruses, and who frequently writes about where he sees viral research headed? Dr. William Haseltine, Chair and President of Access Health International, former professor of Harvard Med School and Harvard School of Public Health, founder of several biotechnology companies. Including Human Genome Sciences, Dr. Haseltine's writings appear regularly on Forbes online. Welcome back to Science Friday. Dr. William Haseltine 01:00 Thank you, I'm happy to be back. Ira Flatow 01:05 Certainly lots of things have happened since we last spoke. Let's talk about some of this stuff. What is there about this disease that the public needs to know that you spend so much of your time writing about? Dr. William Haseltine 01:14 The first thing to know is about what it is?And I think most people don't really have a good grasp of that. It's a virus, like the flu virus is very well adapted to infecting an adult, a healthy animal and then reinfected them shortly thereafter. And we know that because it's natural habitat, our bats that's something most people don't know about bats. They live a long time, about the size of a mouse, or a rat but they live about 20 to 30 years. And these viruses infect them every year, year in and year out, kind of like our colds. But what does that mean? It means the ecological nature this virus is an adult 1125

healthy person that infects again, and again and again, even if it infected them before. And that means it's got lots of tools to do that. That's its niche. We happen to be the new target the new ecosystem. Ira Flatow 02:19 So we shouldn't be surprised and about how many times we can get infected? Dr. William Haseltine 02:22 No, just like the flu. It comes back and it changes. This is even more subtle than the flu is bigger. And science magazine had it right on the front of its cover a beautiful issue a couple of weeks ago, calling it an immune saboteur. Not only does it change its code, but it's got we're still counting I'm finally up to 35 to 40 different ways it can jimmy our immune system. So once it gets in, it shuts down your ability to see it until it gets out and then doesn't care what happens to you. Some of its cousins killed 10% of us some of his cousins him 30% of us were very lucky this one kills about 1% of us. Ira Flatow 03:06 Earlier in the pandemic, there was an idea circulating that as the virus mutates, it tends to get less severe has the has that been the case here? Dr. William Haseltine 03:16 You know, there's arguments about that. But let me just put an end to that myth. Think about tuberculosis. Think about malaria. Think about what smallpox did what it was rolling on. Did you get after 1000s of years, any less horrible? No. That is not the way pathogens go. They don't generally get weaker. So that's a myth. And it's, I think, a dangerous myth. What's the case for this? It's very hard to tell because the population has changed. Because tremendous experience of the population our this and there is some partial protection. There's one other thing that I think people should know. Right now we're in an era of complacency. I live amongst other places in New York City and the city as there's no virus around. There is virus around but the important thing to know is a vaccine will protect you for about two to three months from infection. It will protect you pretty well for five to seven or eight months from ending up in the hospital or dead. But then all bets are off the idea that there's perpetual protection from the worst thing this virus can do to you is not right. The latest data is coming out says that protection from hospital physician and even worse and are actually 1126

wanes, it just wanes more slowly. And that's what you would expect for a virus is the sad side all these tricks to come back and get you again and again. So the last thing I like to say that people should know is this thing is around to stay unless we learn how to put it to bed like we're learning for HIV to use drugs, as well as our immune system to fight it. This thing knows everything about our immune systems. It knows how to fight it. We've had to find new drugs that it's never seen before. And combinations of those that's what's work for HIV. That's what we have to do now. And we're doing a very poor job. What do you mean we're doing a very poor job of that. Please tell us what you're talking about? Well, you can count on your one hand, the number of drugs we have, and those aren't really great drugs as somebody who's worked on developing HIV drugs, both the theory and the practice. We have super dark. We now have a drug that you can get injected once every two months and you won't get infected. And you got the virus HIV it won't make you sick. We're on the brink of having a shot. That will do that every six months. We are so far away from that. Or this disease for example, Paxvolid. Paxvolid, it doesn't stop you from getting infected. Excellent. It does have an impact on keep you out of the hospital, but only about 50% We'd like you to wipe out the virus completely. And I haven't COVID last May. And I took Paxvolid for and I mentioned it because it's our best drug. I took Paxvolid, for 10 days, and it was virus positive for 15. Yes, it's an important drug to have and I urge everybody who gets infected take it because it does reduce your chance of ending up in the hospital. But it's not the powerful drug that we want. Why don't we have that powerful drug like we have with HIV? Well, it's taken us 40 years to get where we are. I think we can do it in five or six years if we really put our mind to it. But you know, developing drugs is a complicated business. I've been at it for a long time. And the way I like it too is like say we got to fix my Ferrari by throwing a wrench into the engine. Okay, it's a complicated machine. There is a strong sense that we're getting better. There's a lot bigger chance somebody up that I don't know about yet. So Gordon development is tough. And you've really got to have a lot of knowledge now. There's one or two little pieces of the SARSCoV-2 virus that we don't well enough to begin to do really rational argument design. One of us how it puts a cap on it completely 1127

differently from most other viruses, cells, but it gives us a great juicy target and we know every avenue involved in that process, and so we can start to make drugs but we know very little and say has a giant replication machine. It's got many moving parts, you can probably mess it up every single a lot of different ways. We only have one way to mess it up, which is very preliminary, some of it not working as well as we'd like. And so we just do a lot more research on this. We have the tools now. Thank goodness, we have so much better tools than we had at the outset of HIV. That is comparable, how much we could do and the time we can do it but we've got to we've got to do it now. COSTA we were talking about here is it drug companies the NIA community is there. Is there money on it is really, really not the drug companies yet no drug companies are able to take something that academics have shown will work and then turn it into a drug we need to companies and actually we need the companies to work more closely together. I don't really work for HIV. First of all money. The United States was willing to spend two to $3 billion a year on HIV research. Second, the collaboration a very tight collaboration between academia and business. In fact, I remember working with Dr. Fauci to create a special series of grants in which the federal government would give an academic laboratory money provided they have an industrial party partner, which would develop that drug. That's the kind of programs that really work we have a couple of those but we need to expand that. We don't even give money now for preparing our society for the next attack of a virus right is a series of politics and other questions, but it is the case and I've been working on health policy for a long time now to that there are great reports so Commonwealth Fund just did a really important report on the need for a powerful National Public Health Service. We don't have it. It's all balkanized. And we and that is a Commonwealth Fund report. My friend Peggy Hamburg shared it. She was former head of the FDA. It's a great report and shows you what we should do. Well, we do it. Same thing and as Academy of Medicine did a wonderful report on how we have to integrate our health care services across the nation to help people who are underserved and talking about the way we deliver health care now. Beautiful report tells us a clearer idea of where to go. It's now up to us as American citizens to make sure that our politicians do follow the directions. We need them to 1128

follow. You know, as well as I do that this country's in a difficult spot right now. Ira Flatow 10:27 Yeah, I you've written just living on a bit that new research published to the Science Translational Medicine, suggest an mRNA vaccine that targets both the SARS cov to spike protein, as well as something called the nucleo capsid protein may offer stronger and broader protection than current spike only vaccines. This research opens the possibility that one vaccine may protect against current and future variants. I'm asking is it possible to create a universal COVID vaccine? Dr. William Haseltine 10:59 Well, that's a thank you for asking. That's a tough question. I'm really very close. To my heart. I don't think it's possible. Let me just tell you what I was working on just before we started talking. It's a great paper a wonderful group, Dr. Sapphire in La Jolla has done some very beautiful work on developing antibodies, cocktails of antibodies that neutralize two really important viruses. When everybody knows about Ebola, and she's developed drugs that will probably work against this new Sudan strain. Very exciting. She's just recently published run on another nasty creditor out there called Lhasa. And she's got her Shark Tale of Three antibodies that you know, exactly, add them to add them which ones it touches. And the last thing and and probably stop that too. So I think we can do this. There are cocktails now, of monoclonal antibodies that are very broadly neutralizing, they neutralize not only SARS-CoV-2 but SARS one and MERS in some of these crazy math viruses. You can do this. Now. They're also beginning to use that knowledge to design vaccines. Is it possible to design vaccines but let me talk a little bit about some of the mRNA vaccine. We're really excited that RNA can now be used directly as a vaccine because it's really simple to make totally chemical process. And it's really fast and flexible. But the way it's currently being used with these modified nucleotides, you stick it in the RNA lasts a couple of couple of days, the protein is there for no more than two three days. And then your immune system can't sit anymore. There's now some real self amplifying messenger RNA. Really exciting, because little bit urinate and it makes a lot of urine if you don't put it in the muscle. You put it into the layer of the skin where 1129

most of your immune system can see it. And that is giving very, very good results. The antibodies lasts for up to a month, your body gets to see it and you can add lots of different proteins very, very easily, including what you mentioned the nucleo capsid and when you do that, you get some broad reactions that can react all the way across most of the size variants for all of them that I know about the MERS as well as SARS one so the I think there's some good things coming both of the vaccines and with monoclonal antibody cocktails, and hopefully eventually, the small martial arts. It's not impossible to put this thing behind us. It's just hard. Ira Flatow 13:38 This is Science Friday from WNYC studios. In case you just joined us we're talking with Dr. William Haseltine. about the future of the COVID pandemic and lessons learned. You've worked with many different viruses as we're talking about and many people, as you say know of your work with HIV. And I'm wondering what you can tell us what we can learn from those other viruses about living with COVID Well, we live with flu. Dr. William Haseltine 14:07 And we've lived with flu all our lives. I've been in bed for 5 to 10 days, five times. I've lived through a flu. I haven't liked it, but I've lived with it. I get my flu shots and most years they work and some years they don't. But the difference between this and so is this is the worst virus is much more lethal. It's already killing on average every year a lot more people than the flu does. It's not seasonal. It doesn't come along only in the winter. It can was a long, fall, spring summer and winter. So it's not seasonal life though, which makes it nastier and it affects many more organs. So yeah, we can live with it. But right now, we're living in sort of a low and the average is about five 600 people a day are dying. But when we do have an airplane fell out of the sky fell five 600 people every day. We were like it right but that's what's actually happening right now. It's not season. It's been pretty constant from me. And so yeah, we can live with it. Hopefully it won't get worse. You know the real nightmare for somebody like me who thinks about what these viruses can do is I know that with some very subtle change, this could go from filling 1% of us it's 20% to 30% or more. It's we don't know that's not going to happen. So the reason you hear people like me say we've really got to control this as much as possible, is to reduce that possibility. 1130

The more viruses that are out there and humans, the more they get into our animal environment, the more likely it is as something worse will happen. Not better. Worse. And I can give you have enough examples to curl your hair about how viruses get worse. One of the questions you asked me at the very beginning, we know how it happens. We know it can happen. And we're just trying it won't happen now. I would say something else to think about why we really have to pay attention to this. This is happening to us not because we're moving into new ecosystems. But because we are a new justice. I was born there might have been 2 billion people in the world. They're 8 billion today. That's a lot more people there are 5 billion single airplane flies you people taking flights a year. We move around a lot and give us his bats congregated together in a cave. When you're on one of those cramped airplanes it's like being in a Batcave and we're these viruses are taking advantage of a brand new ecosystem are a great food for viruses. And we've got to be able to protect ourselves to Haseltine you've given us a lot to think about both scary and hopeful. And I have more hope and I'm and the reason I do what I do is I think that there's a lot of hope that a lot of great people out there that I work with and we have really great scientists and great people working on this. Ira Flatow 17:15 So would it be fair to say you're more hopeful than fearful? Yes, cautiously optimistic that we can do better. Thank you very much for taking the time to be with us today. You're welcome. Thank you, Dr. William Haseltine. Chair and President of Access Health International This article originally appeared on Essence, and can be read online here: The Quest For New COVID-19 Solutions

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Simone Fishburn Interviews William A. Haseltine at BioFuture 2022 BioFuture 2022| August 17, 2022 | Youtube Video

Simone Fishburn 0:06 I'm Simone Fishburne, editor in chief at Biocentury. I'm here with Bill Haseltine—of course, a longtime, huge name and mover/driver in our industry. So I want to start with this: I'm gonna ask you something in a minute rather forward looking. But, you know, [having] founded Human Genome Sciences more than a couple of decades ago, when you were starting out thinking about where we are now—fewer, at that point to have projected—how are we doing compared with what you thought at the time? Dr. William Haseltine 0:50 Well, our technologies are even more powerful than I imagine. Let's go even further back. I had a decision to make as a young man, which fields go into: chemistry, physics, or biology. And I picked biology as a field that would move most quickly, I'm happy to say that was a very good choice. Now we go back to to 1992, I was creating Human Genome Sciences. And the idea was to provide new tools, so we could begin to work on any medical problem we wanted. And that worked very well. And now when I look at the tools that we have and at the production—the scientific and medical production is phenomenal. I would say it's beyond what I imagined. Better. So bioscience is moving in a more rapid way. Is that true of the pharma industry? Well, the hard part of Pharma is, once you have an idea, to make it work in a person. And the way I describe it over dinner to people who aren't scientists is like having a Ferrari 1820s, and throwing a wrench into the engine and hoping it gets better. We just don't know a whole lot. And you put something into our body, and it's likely to do more harm than good. And most of the time that happens. So that problem has not gone away. It's gotten a little better. There's still, as every CEO knows, it takes a lot of effort to get a drug to work in a human being and the human population, because we're pretty diverse. 1132

Simone Fishburn 2:28 So what I'm hearing is the science and in particular, the technology has really gone leaps and bounds. But we haven't necessarily made the progress we should have done in the business of making drugs and getting more efficient at making drugs, is that what you're saying? Dr. William Haseltine 2:44 That's a good summary. It's not so much more efficient, it still takes a long time. Now, there is one great exception, which we all know, which is the COVID vaccines, which shows things can go much, much faster when they have to. Simone Fishburn 3:00 All right, you've obviously been a huge pioneer in infectious diseases, HIV and AIDS. And so maybe you can tell us a little bit about your thoughts on next generation vaccines and what we should be thinking about. Dr. William Haseltine 3:14 We now know a lot about the vaccines we have. The good news is, for a few months, they really protect you from infection. And for a few months more—or five or six months more maybe—they protect you from serious disease and hospitalization. And no matter how many times you've been boosted, they poop out. We need to do better; we need to understand what it is that protects us from disease, even if we're infected. And we don't know that. Some people say it’s T cells, some say stick up your nose, breathe it through your mouth, swallow it—there's all sorts of ideas, but nobody really knows. We need better vaccines. There are some really new technologies for mRNA vaccines that are coming along. They're called self amplifying. So you don't need all these lipids, you don't need funny nucleotides, you just stick it into the skin—you just blow it into the skin, you don't even need to inject it. And it works pretty well; protein will be produced for a month or more. And I think those technologies are going to be the next generation. Whether they're going to solve the problem for vaccination, I don't know. From the very beginning, what I've said for about this disease is that it reminded me of HIV/AIDS. We don't have a vaccine for AIDS. We have vaccines for COVID. But they wear out. We need something better. We need really good drugs.

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We know how to make them from 40 years of HIV work, we need to start making them faster now. Simone Fishburn 4:39 So my last question is this. We know that historically, infectious diseases have been one of the biggest scourges—and credit to our industry that we managed to tamp down many of those. It's still a huge burden and yet, the last decade or more the pharmas/the industry has really gone away from infectious diseases. COVID changed that. Is that a blip? Or do you think that going forward, we're really going to see new energy put into infectious diseases— which still, on a global scale, are one of the most pressing health needs. Dr. William Haseltine 5:18 I hope it's not a blip. We've seen these blips before, we've seen the company that money go in. We've seen the money go and I can't tell you what it was like to be a Coronavirus researcher in 2008. Hey, all the money was gone. And it was a huge mistake. Simone Fishburn 5:43 Sorry to interrupt—because actually, it wasn't just then. Even with the SARS outbreak, with MERS, we went down this road and then they walked back. Dr. William Haseltine 5:52 All the money disappeared. That's 2015+. You just couldn't get money if you're a Coronavirus researcher. I made many friends in that community after COVID. And they all were busy ramping up their labs. But I can tell you, we know so little about that virus. Even today, I'm writing a textbook on molecular biology of COVID and another one on COVID writ large. And what we don't know will fill up two textbooks. But let me say something people should understand. We are the new favorite ecosystem for nasty bugs. There are 8 billion of us, 5 billion individual airplane trips a year. That is fat city for infectious diseases and it's not by chance that we're getting these. AIDs came because we connected travel with Sub Saharan Africa. This is spread around the world. I mean, I haven't seen a variant pop up in South Africa, and three months later we'd be devastated in New York, and then the west coast and then the rest of the world. We are connected. We have to take these seriously. People like me and a lot of people have been predicting something like this. But we're still 1134

predicting it. Those predictions haven't gone away just because we got one correct. There will be many more. And we need the industry to take this seriously. So next time we're hit with this, it's not terra incognita, like it was for Covid. We're filling in the blanks, but there are a whole lot of blanks to fill in still. Simone Fishburn 7:30 Well, I think you're one of the very notable chorus of people who's calling for this. And let's hope that this time, it's not a blip. Dr. William Haseltine 7:38 This is Biofuture, and we hope our future is better than a recent past. This interview can be found online here: Simone Fishburn Interviews William A. Haseltine at BioFuture 2022

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Ask the Doctor: Will We See A “Triple-demic” of COVID, Flu, and RSV? Plus, the Latest on the Zero-COVID Policy Protests in China Houston Public Media| November 30, 2022 | Podcast

Protests have erupted in China with many calling for an end to lockdowns following a surge of COVID cases despite the country's strict zero-COVID policy. Due to the rare nature of public protests in China, we talk with an expert over what has led to these protests and determine what we can expect from them. Plus, he shares his thoughts on what the future of COVID is here in the U.S. With the rising number of cases and hospitalizations from COVID-19, the flu, and RSV (Respiratory Syncytial Virus) in the U.S. Many have labeled this collision of illnesses the "Triple-demic." Due to the amount of sickness experienced during the winter months, we talk with an infectious disease expert who explains the levels of illness we're experiencing now and shares with us how we can stay safe during the holiday season. This podcast originally appeared on Houston Public Media, and can be found here:Ask the Doctor: Will We See A “Triple-demic” of COVID, Flu, and RSV? Plus, the Latest on the Zero-COVID Policy Protests in China

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Media Mentions

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Covid Cases Are Still Rising. Here’s When They Could Peak. Barron’s | January 7, 2022 | Article

The surge in cases related to the Omicron variant of Covid-19 could peak within a month in the U.S., with cases in the most impacted states starting to decline by the end of January, according to some experts. Earlier this week, the U.S. surpassed 1 million new daily Covid19 cases, with the seven-day average increasing by almost 100% to around 500,000 cases per day, said Dr. Rochelle Walensky, director of the Centers for Disease Prevention and Control. *** Viruses rely on their ability to evade immune responses, and the coronavirus and its variants are no exception, said infectious disease specialist and former Harvard Medical School professor William Haseltine. He expects to see even more variants as the virus looks for ways to bypass existing immunity, whether as a consequence of infection or vaccination. “We are basing our plan on hopes the virus will behave like we want it to, i.e.: become less and less deadly, but there’s no guarantee that that will happen,” he said. Experts likely will have a better idea of the future trajectory of the variant—and its impact—in the U.S. after January, Cleghorn said. Deaths and hospitalizations tend to lag case numbers, and many of the current figures still reflect Delta variant infections, he added. “Worst case scenario is that the economic and social impact continues to overwhelm our ability to manage Omicron,” he said. “Best case scenario is that everyone gets Omicron and we all are immune, but that is — I have no ability to say that.” This article can be read in full here: Covid Cases Are Still Rising. Here’s When They Could Peak.

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New HIV Variant Discovered: May be More Infectious and Severe Healthline | February 4, 2022 | Article

Testing an important tool against HIV Asked what the variant means regarding testing recommendations for at-risk populations, William A. Haseltine, PhD, chair and president of the global health think tank ACCESS Health International, said it depends on people’s behavior. “I learned early in the course of AIDS [that] human behavior is a lot more variable than I expected,” said Haseltine, who has authored numerous books, including Variants: The Shape-Shifting Challenge of COVID-19. “It depends on your activity, and how many different partners you have,” he said, adding that people who have multiple partners should undergo HIV testing more often. This quotation originally appeared on Healthline; the article can be read in full here: Background Briefing: January 13, 2022

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4th shots and COVID-19 boosters: Why and when best to get it Medical News Today | March 2, 2022 | Article

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Studies indicate that immunity conferred by a past Omicron infection is not enough to prevent reinfection or protect against other variants. Real-world data show that a third dose is needed for better protection against severe disease or hospitalization due to Omicron. A fourth dose may not be needed for everyone, but doctors strongly recommend it for at-risk groups. There is no global consensus about the timing of booster doses, but the minimum recommended interval is around 3–4 months.

The emergence of the Omicron SARS-CoV-2 variant has confirmed the need for vaccine boosters — to combat waning immunity and provide more robust protection against severe disease and death. Several studiesTrusted Source have shown that two doses of a COVID-19 vaccine do not provide enough protection against infection or severe disease from Omicron. More and more evidence

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supports a three-step approach, which might involve infection-plusvaccination hybrid immunity or triple immunization. Meanwhile, a growing number of countries are now exploring second boosters — or fourth doses — as a way of effectively neutralizing Omicron. Following in the footsteps of Israel, countries including Chile, Spain, Denmark, and Sweden are offering fourth doses to certain atrisk groups. In the United Kingdom, groups deemed most at risk are advised to have a second booster shot this spring. Depending on the person, this could be a fourth or fifth dose. Variations in guidance have raised questions about who needs a fourth dose and what the most effective approach to immunity might be. What data say about the 4th dose Data about the need for a fourth dose, so far, is lacking, but recently published research indicates that it can restore waning immunity. A trial with the Pfizer and Moderna mRNA vaccines found that a fourth dose provided a modest boost in protection against Omicron and restored antibodies to levels seen right after the third dose. However, this did not translate into significantly increased effectiveness and did not prevent infection with Omicron. Four doses of the Pfizer vaccine were 30% more protective against infection than three doses. This extra protection was 11% for Moderna’s vaccine. Dr. William Haseltine, a scientist, public health expert, and president of ACCESS Health International, said initial studies showed that the fourth dose provided an additional boost in antibody levels. While not huge, this boost was “measurable.” The additional dose could also reignite antibody production in the body when this begins to wane. According to a report by the U.K. Health Security Agency, the booster dose’s capacity to protect against symptomatic infection drops to 45–50% from 10 weeks onward. Another U.K. analysis estimated that protection would drop to around 40% by the fourth month after the third shot. 4th dose could benefit those at higher risk

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There is no hard evidence that a fourth dose is crucial, which means that it will likely be reserved for certain groups. Data suggest that it could benefit people with a high risk of severe illness and restore long-term protection. An observational study from Israel showed that a fourth dose conferred slightly higher levels of protection against both infection and severe disease for those aged 60 or over and considered at risk. The additional dose was administered at least 4 months after the third dose. Although the increase in protection may seem minimal, experts believe that it could make a large difference for high-risk groups. Dr. Haseltine said that traditional risk groups, such as people with asthma, obesity, or immune suppression, could benefit from a fourth dose. Do I need a booster if I had COVID-19? The biggest argument against boosters is that COVID-19 vaccines offer less protection against Omicron than they did against variants such as Delta or Alpha. While the effectiveness against infection is reduced, recent evidence shows that three doses are 99%Trusted Source effective at preventing hospitalization and severe disease across all populations. “With Omicron, it’s clear that two doses aren’t enough, and three doses are needed if you are going to have any protection at all,” Dr. Haseltine told MNT. There is also growing evidence that unvaccinated individuals with past SARS-CoV-2 infections are vulnerable to reinfection by both current and future variants. This is especially true for the Delta and Omicron variants. Research shows that a past Omicron infection does not protect against infections with other SARS-CoV-2 variants in people who have not been vaccinated. In the study, people who had been vaccinated then developed breakthrough infections not only had better humoral immune responses but also had cross-protective immunity to the Delta and Beta variants. Furthermore, having COVID-19, then receiving 3 doses of the vaccine increased protection to 71% from 44% for the unvaccinated with prior infections, according to the SIREN study. 0 seconds of 15 secondsVolume 0% Timing of boosters 1142

Having a booster too soon or too late after the primary series is another point of contention. A study conducted in Japan found that having a breakthrough infection shortly after vaccination did not boost antibodies by much. However, having an infection months after the last dose significantly increased protection, particularly against Omicron. “We do see that antibodies begin to wane after 3 months,” said Dr. Alexander Rodgers, an associate program director at Charles R. Drew University of Medicine and Science. Most countries offering the additional booster have adopted this time frame. “If you’re a person that is at higher risk, mostly because of your age or other factors, I’d recommend the fourth after 3 or 4 months. The reason for that is we know that the protective antibodies tend to wane pretty rapidly after 3–4 months,” said Dr. Haseltine. He said that for those at higher risk who may not have produced sufficient antibodies, he would recommend “a shorter rather than longer interval: 3–4 months rather than 6 months or longer.” Dr. Rodgers pointed out that optimizing the interval for antibody efficiency may be a more viable strategy for most countries. “If you’re in a country that has not yet approved the fourth dose, then it would be reasonable to try and push that third dose to the point of maximum efficiency of that second dose,” he told MNT. “That’s why I think there’s that variability — because countries are just trying to pick an arbitrary line in the sand with the best research and evidence that they have and be mindful of the resources that they have,” he added. How many boosters is too many? “Right now, three versus four versus five or even six [doses] more speaks to a country’s vaccine allocation and resources than it does to any hard evidence,” said Dr. Rodgers. He pointed out that more studies were needed to determine the optimal interval and number of doses. With the prospect of multiple boosters, peak immunity or an immunity threshold may also be possible. Research from China, which has not yet been peer-reviewed, suggested that immune response to COVID-19 could not be endlessly boosted, and there would be a “turning point.” There is also the issue of resource utilization and vaccine inequity. 1143

“When we’re thinking about the vaccine program, we’re really trying to reach maximum efficiency. I think that there are two types of efficiency, for the individual and from a global public health standpoint. From a global standpoint, the most important risk factors are preventing hospitalization and death,” said Dr. Rodgers. He pointed to two strategies: having some countries begin to administer fourth doses or acknowledging that this is a pandemic and focusing on getting first and second doses to large swaths of populations that have not yet had them. “Although the third and fourth doses do decrease the risk of hospitalization and death, the most dramatic improvement is after completing the initial two doses. The current vaccines are still effective in protecting against hospitalization and death — the two major points of concern.” The need for annual boosters will depend on how future variants differ from the initial strain, said Dr. Rodgers. “In the United States, the coronavirus is demonstrating this type of seasonality where our biggest waves are in the winter, similar to other cold and flu seasons. I wouldn’t be surprised if annual boosting or annual vaccine programs like for the flu become a common practice,” he said. This article originally appeared on Medical News Today, and can be read online here: 4th shots and COVID-19 boosters: Why and when best to get it

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I'm a Doctor and These COVID Symptoms Alarm Me Eat This, Not That! Health | March 8, 2022 | Article

Although COVID cases are declining and the surge is pretty much over, the virus is still very present and causing serious longterm effects for millions of people. Even for people who have experienced a mild case of COVID, they are at risk for severe health issues like heart damage, according to Dr. William Haseltine, a scientist who has worked on the forefront of medical research with cancer and AIDS, an internationally recognized expert on COVID, Harvard professor and author who spoke with Eat This, Not That! Health. Read below to find out the symptoms that are causing lifelong problems and what Dr. Haseltine believes people should know about the virus–and to ensure your health and the health of others, don't miss these Sure Signs You've Already Had COVID. 1. Why Do Some People Experience COVID More Severely Than Others? 2. Dr. Haseltine explains, "The answer has to do with either their age or underlying health conditions. The older you are the more likely you are to suffer serious consequences. The reason for that is because the older you are the weaker your immune system, both your innate and adaptive immune system. Then, there are also predisposing conditions – any immunological disorder, cancer treatments, morbid obesity, diabetes, and obstructive pulmonary disorders and a few others but those are the main ones." 3. How Long Do Lingering Symptoms Last? 4. Dr. Haseltine says, "They have lasted as long as we can measure them now from the very first people that contracted COVID. There are some people who have lingering symptoms for more than three years, and there is some permanent damage that can be done to several critical systems, particularly the heart." 5. Brain Fog and Fatigue 1145

6. According to Dr. Haseltine, "Some other people experience some very long-term brain fog, or mental dysfunction, that is sometimes so serious that they can't work. The most common long-term symptom is fatigue, and in some cases, the fatigue is so serious that they can't work. The overall phenomenon we call Long COVID or post-acute PASC." 7. Serious Heart Disease 8. "SARS-CoV-2 infections can damage heart muscle and affect heart function," says Dr. Haseltine. "There are several reasons for this. The cells in the heart have angiotensinconverting enzyme-2 (ACE-2) receptors where the coronavirus attaches before entering cells. Heart damage can also be due to high levels of inflammation circulating in the body. As the body's immune system fights off the virus, the inflammatory process can damage some healthy tissues, including the heart. SARS-CoV-2 infection can also affect the inner surfaces of veins and arteries, which can cause blood vessel inflammation, damage to very small vessels, and blood clots, all of which can compromise blood flow to the heart or other parts of the body." 9. Millions of Americans Are Expected to Have Serious Heart Issues Due to COVID 10. Dr. Haseltine reveals that the, "Most recent data from the US Department of Veterans Affairs on a very large number of people suggests that the serious cardiac symptoms affect 45 people out of 1,000 for a year or more, regardless of age, vaccination status and severity of disease when they caught it. Because there are estimates now of 140 million Americans, that means there are something like 6.3 million Americans are likely to have right now serious heart conditions for over a year. It is going to be a major problem for our health system and for people. And when I talk about major heart conditions, I mean heart attacks and other serious issues." 11. Diabetes 12. "SARS-CoV-2 infections may raise the risk of developing diabetes, because the virus can damage insulin-producing cells in the pancreas," states Dr. Haseltine.

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13. Harvard Health reports, "Experts have found that the virus that causes COVID-19 can directly attack insulin-producing structures in the pancreas. According to the NIH director's blog, researchers found that the virus, called SARS-CoV-2, affects the pancreas in three different ways. First, it may directly damage pancreatic beta cells, the ones that produce insulin, reducing their ability to make enough insulin to keep blood sugars controlled. Second, as the virus replicates in the pancreas, it also can damage the cells that directly surround the beta cells, which are needed for proper insulin release. Third, the virus also seems to reprogram surviving cells, making them malfunction, which can wreak havoc with blood sugar regulation." 14. What People Should Know 15. Dr. Haseltine explains, "For advice, I think health care systems should do a serious cardiac work up on all patients that have been infected by COVID. Patients should be aware that having COVID, even if it was the mildest of cases, puts them at equal risk for serious heart disease to someone that was in the ICU. It is a risk of about 5% of people, who have had any form of COVID, regardless of vaccination status. It's really confounding. People can't understand how all of this damage can occur to the heart if you've been vaccinated or had even a mild case. Long COVID is a very serious longterm consequence of this disease." 16. How to Stay Safe Out There 17. Follow the public health fundamentals and help end this pandemic, no matter where you live—get vaccinated or boosted ASAP; if you live in an area with low vaccination rates, wear an N95 face mask, don't travel, social distance, avoid large crowds, don't go indoors with people you're not sheltering with (especially in bars), practice good hand hygiene, and to protect your life and the lives of others, don't visit any of these 35 Places You're Most Likely to Catch COVID. This article originally appeared on the website Eat This, Not That! Health, and can be read online here and here.

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Worried About Long COVID? You May Be at Less Risk if You’ve Had Omicron Variant Healthline | June 17, 2022 | Article

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A new study found that the likelihood of developing long COVID was significantly less after having the Omicron variant of COVID-19 compared to having the Delta variant of the disease. • The researchers looked at data from over 56,000 adults in the U.K. • Doctors are still learning about how long symptoms may last, but there are treatment options available. What we know about long COVID William A. Haseltine, PhD, former professor at Harvard Medical School and Harvard School of Public Health and author of Omicron: From Pandemic to Endemic : The Future of Covid-19, said at one point it was suspected that long COVID was actually a psychological condition. “But those have been dispelled by the serious nature of some long-term symptoms, particularly neurological symptoms,” he said. “The second thing we understand,” he continued. “Is that acute COVID-19 can cause serious permanent damage to organs.” Dr. Haseltine said this includes damage to the brain and the heart, the lungs, liver, the pancreas, and the kidney. “Another way to define long COVID is a series of symptoms that occur following COVID-19, following resolution of the virus two to three months after the virus is gone,” he explained. According to Haseltine, when defined this way “you get somewhere between 30 and 50 percent of people have at least some long symptoms within three to six months.” He emphasized that a much smaller fraction of infected people, from two to five percent, have very serious, life-altering symptoms extending for a year or more. “Those include, in my mind, specific organ damage,” he noted. “Everyone is at risk of long COVID, as far as we know.” 1148

Haseltine explained that an exception to this is a type of long COVID in which someone experiences organ damage. “Organ damage is more likely associated with severe COVID19 requiring hospitalization and in some cases intensive care unit admission,” he said. But he emphasized that the “traditional symptoms” of brain fog, difficulty breathing, and extreme fatigue, aren’t associated with disease severity and almost anybody can develop those symptoms. “Almost anybody can suffer those long consequences regardless of the severity or any other pre-existing condition that we know,” said Haseltine. Could vaccination or boosters help people with long COVID? Haseltine confirmed that only one study he’s seen, shows that vaccination prior to breakthrough infection reduces the incidence of long COVID, but only slightly, about 15 percent. “That means that of you have a breakthrough infection following vaccination and boosting, that you still can contract long COVID,” he said. Haseltine believes this is most relevant in the current situation where the vaccinated population seems to be equally at risk of infection as the non-vaccinated population. “That means that they are equally, that they’re basically unprotected from long COVID, but with a slight edge, like a 15 percent edge of protection,” he said. Robust protection against severe disease and death, but not long COVID Haseltine said this contrasts with more than 90 percent protection from serious disease and death that vaccines afford. “Even at the height of the pandemic, only about one to two percent at the most, of people who were infected in the United States died,” he said. “Under those criteria, greater than one to two percent of people who are infected [after] having been vaccinated are very likely to experience lifelong complications of COVID-19, such as long COVID.” He said this means brain damage, mental confusion, and fatigue. “In some ways, the fatigue part of long COVID resembles chronic fatigue syndrome,” said Haseltine.

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Implications of chronic fatigue syndrome due to COVID-19 Haseltine thinks we’re going to find that as big as chronic fatigue syndrome (CFS) is, long COVID-associated chronic fatigue will be bigger. “The rough estimate is 150 to 200 million Americans have been infected,” he said. “If you have two percent of that, that is a very large number.” Haseltine also said there are fears that this will not only stress the medical community, but also the economy. “There is some realization that long COVID symptoms are taking people out of the job market,” he said. This article originally appeared on the website Healthline and can be read online here.

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Longtime HIV patient is effectively cured after stem cell transplant Washington Post | July 27, 2022 | Article

The man is among a handful who have gone into remission after the procedure, but it is not an option for most people with the virus. A 66-year-old man with HIV is in long-term remission after receiving a transplant of blood stem cells containing a rare mutation, raising the prospect that doctors may someday be able to use gene editing to re-create the mutation and cure patients of the virus that causes AIDS, a medical team announced Wednesday. For now, the crucial virus-defeating mutation is rare, leaving the treatment unavailable to the vast majority of the 38 million patients living with HIV, including over 1.2 million in the United States. Bone marrow transplants also carry significant risk and have been used only on HIV patients who have developed cancer. The patient, who had lived more than half his life with the virus, is among a handful of people who went into remission after receiving stem cells from a donor with the rare mutation, said doctors from City of Hope, a cancer and research center in Duarte, Calif., who treated him. “This is one step in the long road to cure,” said William Haseltine, a former Harvard Medical School professor, who founded the university’s cancer and HIV/AIDS research departments. Haseltine, now chairman and president of the nonprofit think tank Access Health International, was not involved in the City of Hope case. … Haseltine said that researchers must figure out how to reach enough of the right cells inside the body. At the same time, they must ensure the treatment does not cause unwanted effects to other genes. “The message to people living with HIV is that this is a signal of hope,” said Scully of Johns Hopkins. “It is feasible. It has been

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replicated again. It’s also a signal that the scientific community is really engaged with trying to solve this puzzle.” This article originally appeared on Washington Post, and can be read in its entirety here.

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As Anthony Fauci announces retirement, experts weigh in on how history will remember him Salon | August 17, 2022 | Article

Several months after the COVID-19 pandemic arrived in the United States, Dr. Anthony Fauci shared an observation that proved to be oddly prescient. Speaking before the Aspen Ideas Festival, the director of the National Institute of Allergy and Infectious Diseases pointed out that "there is a general anti-science, anti-authority, antivaccine feeling among some people in this country — an alarmingly large percentage of people, relatively speaking." Two years have passed since Fauci uttered those remarks. Now Fauci has announced that — after a career spanning back to the 1980s and including service under seven presidents — he is going to retire. Few physicians in the United States become household names; fewer still are remembered by historians, whether for positive or negative reasons. Regardless of one's opinion of the headlinemaking public health official, "Anthony Fauci" is a name that will be uttered for centuries by future historians of United States politics in the late twentieth and early twenty-first century. Yet how history will remember Fauci is still not entirely clear. His legacy is multifaceted because it spanned different pandemics and epidemics (HIV/AIDS, COVID-19, and now monkeypox). History is still being written for many of those outbreaks, and decades may pass while data is collected and unintended consequences are inventoried. Until all of that work has happened, the full breadth of Fauci's legacy will not be etched in stone. Salon reached out to a number of experts, both historians and fellow medical professionals, to gauge how history might remember the retiring physician. Intriguingly, two responses prevailed: First, that Fauci is a ubiquitous figure, with many top minds from the public health world recalling direct interactions with him; and second, that his leading role in shaping America's response to the 1153

COVID-19 pandemic — a task almost certainly made more challenging by the politically-charged backlash to which he alluded in Aspen — saved countless lives. Whatever your opinion on Fauci's tenure, experts attested that the United States would be a very different place right now if Fauci hadn't been in power during the early years of the 2020s. "The most important priority for the National Institute of Health [NIH], and especially the National Institute for Allergies and Infectious Diseases, led by Dr. Fauci was the creation of a vaccine," said Dr. William Haseltine, the chair and president of the global health think tank Access Health International and a biologist renowned for his work in confronting the HIV/AIDS epidemic. "Nobody could have imagined it could have been done so rapidly, so fast. And so well in addition to that, not only did they develop the [COVID] vaccine — from a scientific point of view, they managed and conducted a global trial of the COVID-19 vaccine in, what I would say, is unimaginably rapid time." Haseltine pointed to the years of foundational research done to develop HIV vaccines — research that was absolutely essential to developing the COVID-19 vaccines, and which was also led by Fauci. This article originally appeared on Salon, and can be read in its entirety here.

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How To Stay Protected Against Polio Essence | August 24, 2022 | Article

Polio, short for Poliomyelitis, is a disabling and life-threatening disease that can infect a person’s spinal cord, causing paralysis. Artifacts suggest the disease has existed since ancient times. An Egyptian stele from 1850-1350 BC depicts a priest lacking muscular strength in his leg. historians believe the stele confirms the existence of the disease thousands of years before there was a name for it. By the 18th century, prominent scientists in Great Britain and Germany developed a clinical understanding of polio. Shortly thereafter, outbreaks were occurring in the U.S., with the most widespread pandemic taking place in the 1940s and 1950s. Polio is highly contagious, meaning a person can spread the virus even if they aren’t experiencing symptoms. During each year of the 1940s’ pandemic, an average of 35,000 people became disabled. Therefore, parents and guardians were extremely cautious of their child’s whereabouts into the 1950s, when a vaccine was developed. “When I was a child in the 50s, we had to avoid the swimming pool and had to be in groups of no more than three throughout the whole summer,” Dr. William Haseltine, author of My Lifelong Fight Against Disease: From Polio and AIDS to COVID-19, says to ESSENCE. … Polio throughout history To slow the spread of polio in the 1950s, towns practiced protocols similar to the ones enforced during the earliest days of the Covid-19 outbreak. Churches, schools and theaters were among the places shut down. When Dr. Jonas Salk developed the injectable Polio vaccine in 1955, (followed by Dr. Albert Sabin development of a more effective oral vaccine in 1961), global vaccination campaigns began. “The sabin vaccine was used very very widely across the world as part of the major efforts to control and eliminate polio,” Dr. Haseltine elaborated. 1155

… How polio came back In July 2022, a case of polio was detected in Rockland County, New York. A 20-year old resident contracted the virus after traveling to Hungary and Poland. Upon his arrival in the states, he was hospitalized and initially diagnosed with Acute Flaccid Myelitis. After more thorough testing, the doctors confirmed that he contracted a strain of polio found outside of the United States. The individual from Rockland County was not vaccinated, which contributed to his paralysis. Vaccinations were already low in his county to begin with: Rockland and Orange county polio vaccination rates are at 60 percent, compared to 93 percent nationwide by age 2. “There’s been a major anti-vax movement… As a result of that, a very large fraction of some populations have not been vaccinated for the past 30-40 years against polio…In some parts of the U.S., particularly, rural parts of the U.S. and not heavily urbanized populations, the vaccination rates can be around 50 percent. That means they are subject to infection and paralysis by polio,” Dr. Haseltine details. The good news is that 92.6 percent of the U.S. population is vaccinated against polio within 24 months of birth. If most of the population remains vaccinated, the number of infections will seize just as in the 70s. In New York, Dr. Haseltine says the current outbreak’s severity is “about a 3 or 4” out of 10. This article originally appeared on Essence, and can be read in its entirety here.

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How President Biden Can Meet His Pledge to End HIV by 2030 Healthcare Executive | September 19, 2022 | Article

Preexposure prophylaxis and emphasizing the U=U (undetectable = untransmittable) message are central to the efforts to end the HIV epidemic, a goal that the Biden administration picked up from the Trump administration. Late last year, President Joe Biden released a new National HIV/AIDS strategy to provide the framework and direction for the administration’s policies, research, programs and planning through 2025. The plans have the lofty goal of ending the HIV epidemic in the United States by 2030. New HIV infections have declined from their peak in the mid1980s. People with HIV who receive care and treatment are living longer, healthier lives. But there were still 18,489 deaths among people diagnosed with HIV in 2020 in the U.S., and 650 000 AIDSrelated deaths globally. Biden administration’s strategy has the milestones of reducioing new HIV infections in the U.S. by 75%by 2025 and by 90% by 2030. William Haseltine, Ph.D., president of ACCESS Health International and a former Harvard Medical School professor, noted the aim of helping communities take advantage of HIV services outside traditional healthcare settings through self-testing and mobile services is an important update to the government’s HIV/AIDS plans and something he has long advocated for. “The strategy also addresses hepatitis, substance abuse, mental health, and other STI syndemics often connected with HIV, taking a whole person approach to HIV care,” he said. “This pledge demonstrates a continued commitment to combating HIV both nationally and internationally and ending HIV transmission in the US even amongst other competing health crises such as Covid-19.” Ending the HIV epidemic in the U.S. is a common if ambitious goal for the administration, some members of Congress, and the HIV community. Obstacles loom, though. 1157

“EHE (ending the HIV epidemic) originated during the previous administration and resulted in increased federal funding for HIV testing, prevention, treatment, and the response to emerging HIV outbreaks directed at communities and states in which more than half of new HIV diagnoses occurred in 2016 and 2017,” said Connie Jorstad, associate principal at Avalere Health. Clinical advances in HIV prevention and treatment have made ending the epidemic possible, said Jorstad, who used the “U = U” coinage, which stands for undetectable = untransmittable and is supposed to reinforce the goals of HIV preexposure prophylaxis (PrEP). So far, the Biden administration has reinstated the Office of National HIV Policy, which provides the leadership in development and implementation of the National HIV/AIDS Strategy (20222025). The administration’s FY 2023 budget request included $337 million to continue funding efforts to end the epidemic, as well as funding increases for existing federal programs across HHS. The proposed funds include $165 million more for the Ryan White HIV/AIDS Program, $47 million more for HIV and hepatitis prevention activities at the Indian Health Service, and $115 million for CDC HIV prevention programs. The FY 2023 budget request also proposed a national PrEP program to expand access to and uptake of PrEP. “PrEP is a relatively recent addition to the HIV prevention toolbox but is only being used by approximately a quarter of people who would benefit,” Jorstad said. “Efforts to educate and train providers on PrEP as well as those to reduce stigma among providers can help. U=U is an important message that people living with HIV, providers, insurers, and communities need to hear.” Beyond funding, Jorstad noted addressing individual, provider, and community-based stigma and engaging community stakeholders across sectors continue to be a challenge. “Progress has been made to lessen gaps in HIV-related outcomes, but disparities exist,” she said. “Federal, state, local, and clinic-level data are being used to determine disparities and identify strategies to address them. Closing disparate gaps can require new or different approaches and will benefit from stakeholder buy-in at all levels, from community to executives.”

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This article originally appeared on Essence, and can be read online here: How President Biden Can Meet His Pledge to End HIV by 2030

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Without a nasal vaccine, the U.S. edge in fighting Covid is on the line Politico | October 15, 2022 | Article

Biden administration officials are raising concerns that the slow pace of developing a nasal vaccine for Covid-19 in the U.S. could pose a security risk as China, Iran and Russia approve their own vaccines taken through the nose or mouth. Though nasal and oral vaccines are being studied in the U.S., none are close to coming on the market because Congress hasn’t approved more money to support research and development. Big pharmaceutical companies are also not investing in these nextgeneration vaccines because they don’t see much profit potential. China already has a Covid vaccine that’s ingested through the mouth. India, Russia and Iran have authorized nasal vaccines. And while none of those have yet been proven to stop Covid transmission, officials say the U.S. could find itself at a global disadvantage, particularly if a deadlier variant emerges. “Intranasal vaccines — vaccines that are variant-resistant — those are critical tools to have in the toolbox for protecting Americans, not just for Covid but also for future pandemics and also for future biosecurity threats,” Ashish Jha, the administration’s Covid-19 response coordinator, told POLITICO. Researchers working on nasal vaccines are hopeful that they could stop virus transmission by generating immunity against it in the nose and other parts of the upper respiratory system where the coronavirus enters the body. If that bears out in clinical trials, nasal vaccines would be superior to existing mRNA vaccines, which prevent severe disease but don’t stop transmission. Officials at the National Institute of Allergy and Infectious Diseases are attuned to the danger of failing to develop such a nasal vaccine since it would protect people in case of a more contagious and deadlier coronavirus variant, said Karin Bok, the acting deputy

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director for pandemic preparedness and emergency response at the agency’s Vaccine Research Center. The center has mapped the nasal and oral Covid vaccines in development in the U.S. and abroad. It is also testing nasal versions of the Moderna vaccine and two other types of injectable Covid-19 vaccines in monkeys, Bok said. But that probably won’t lead to a nasal Covid vaccine being approved in the U.S. anytime soon because funding for clinical trials and production is lacking. Bok and Jha say the cost is high. If China were to develop a nasal vaccine capable of stopping Covid transmission, that could turn the tables on the current pandemic trajectory, which has the U.S. emerging and much of China stuck in lockdown. Even though India, Iran, China and Russia haven’t proved their non-injectable vaccines stop transmission, the potential is there, experts said. “Countries where transmission is reduced are going to be healthier, are going to have stronger economies. And the U.S. needs to catch up,” said Marty Moore, the founder and chief scientific officer of Meissa Vaccines, a small biotech company that’s trying to develop a nasal vaccine in the U.S. … Tempering expectations But some scientists doubt that a nasal vaccine will be a gamechanger. William Haseltine, a former professor at Harvard Medical School with expertise in HIV/AIDS and genomics, believes that enthusiasm should be tempered about the potential of nasal vaccines to prevent infection, given that natural nasal exposure to the virus doesn’t prevent people from getting reinfected. “Why in the world do you think that if you [spray] a vaccine up the nose … you can do any better?” he asked POLITICO. Attempts to develop a nasal version of the AstraZeneca Covid19 vaccine, the injectable version of which was widely used globally at the beginning of the vaccination campaign, experienced a setback after only a minority of participants in an early stage clinical trial showed some immune response in respiratory mucous membranes. Haseltine argued that scientists still don’t have a good understanding of nasal immunity and that government funding

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would be better directed to antiviral drugs that keep Covid-19 in check. And Bok doesn’t think any of the existing non-injectable vaccines stop Covid-19 transmission. “I would be very surprised if India or China licensed it with data proving that an intranasal vaccine is better than the ones we have,” she said. This article originally appeared on Politico, and can be read online here: Without a nasal vaccine, the U.S. edge in fighting Covid is on the line

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Promising Pfizer RSV Vaccine for Pregnancy May Protect Newborns Very Well Family | November 08, 2022 | Article

Dr. William Haseltine, Chair and President of ACCESS Health International, Inc, believes the vaccine is “badly needed and timely, given the current rising RSV infection rate. Clinical trials demonstrated that babies born to vaccinated mothers had greatly reduced rates of RSV infection.”1 Dr. Haseltine believes the best strategy to protect newborns is to protect the mother as newborns depend on maternal antibodies not their own. Older children 6 months and older could eventually be candidates for vaccination. In fact, more children were hospitalized in the vaccinated group than those who were not vaccinated. Sadly, the deaths of two children are also attributed to this initial vaccine.9 Dr. Haseltine explains the difference between the vaccine in the 1960s and the ones being worked on now. There are other vaccines, particularly geared toward older people. Drugmakers GSK, Janssen, and Bavarian Nordic are working on those shots. Dr. Haseltine says they must be administered with powerful immune stimulants for an older population that is more resistant to vaccines. Such a vaccine could save many lives. This article originally appeared on Very Well Family, and can be read online here: Promising Pfizer RSV Vaccine for Pregnancy May Protect Newborns

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Coronavirus variants are dodging antibody treatments. New lab-made options may help. Washington Post | November 18, 2022 | Article

In the evolutionary chess match between the coronavirus and humans, scientists’ next move can’t come soon enough for the millions of Americans relying on treatments known as monoclonal antibodies. These lab-made therapies are rapidly losing their healing power, forcing researchers around the world to devise new antibodies that are both more potent and more resistant to new variants. Some monoclonal antibodies have been rendered largely ineffective as the virus has mutated; others are expected to become so this winter if a wave of new omicron subvariants comes to dominate the pandemic landscape. For instance, the U.S. Food and Drug Administration recently warned that the widely used therapies Bebtelovimab and Evusheld may no longer work against some versions of the coronavirus … Driven by this urgent need, scientists are exploring new ways to tackle the problem — including antibodies that seek out fresh targets among the vulnerable parts of the virus. “I would hope that some of these will be in clinical trials soon, and then it won’t take long” to determine whether they work, said William Haseltine, a former Harvard Medical School professor who founded the university’s cancer and HIV/AIDS research departments. This article originally appeared in the Washington Post, and can be read in its entirety online here: Coronavirus variants are dodging antibody treatments. New lab-made options may help. EN

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Social Media

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January 2022

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January 5 – TITLE: Difficulties Of Single Monoclonal Antibody Treatment Of SARS-CoV-2: The Sotrovimab Experience In Australia Here, I describe a recent study which tells us that resistance to singular antibody treatment can develop rapidly. This means effective therapy will require the use of two or more monoclonal antibodies. https://www.forbes.com/sites/williamhaseltine/2022/01/04/di fficulties-of-single-monoclonal-antibody-treatment-of-sars-cov-2the-sotrovimab-experience-in-australia/?sh=6fc1fca926c6 – TITLE: 3D Printed Skin? Potential New Treatment For Chronic Wounds An exciting step in the development of new treatment methods for chronic wounds is multilayer 3D printed skin created with “bioink”. Here, I describe the process. http://www.forbes.com/sites/williamhaseltine/2022/01/04/3d -printed-skin-potential-new-treatment-for-chronic-wounds/ – TITLE: Breaching The Barrier That Protects Children From Serious Covid-19-Related Disease A recent study shows evidence that natural immunity is responsible for children’s enhanced defense against disease including Covid-19. Using this knowledge we can create targeted therapies that could potentially protect both children and adults. https://www.forbes.com/sites/williamhaseltine/2022/01/04/b reaching-the-barrier-that-protects-children-from-serious-covid-19related-disease/?sh=49da213824da January 10 – TITLE: The Challenges of Treating COVID-19: Lessons from Gilead’s Remdesivir Here, I describe the pros and cons of Gilead’s monoclonal treatment Remdesivir. The race is on to manufacture and approve

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additional treatments with multidimensional approaches for fighting the pandemic. https://www.forbes.com/preview/61dc4f56d2a0bf0001728697 / – TITLE: SARS-CoV-2 Protein Orf6 Suppresses MHC-I And Recognition By Cytotoxic T Cells Scientists continue to unveil the exact mechanisms by which SARS-CoV-2 evades immune protection. Here, I describe a study of one of these mechanisms, protein Orf6. https://www.forbes.com/sites/williamhaseltine/2022/01/07/sa rs-cov-2-protein-orf6-suppresses-mhc-i-and-recognition-bycytotoxic-t-cells/?sh=65ba2d9c2dc4 – TITLE: New Potential Covid Virus Variants Of Concern Here, I describe the lineage of B.1.640, a potential variant of concern. Understanding the building blocks of each previous variant gives us the opportunity to characterize and understand the potential threat that new variants pose. https://www.forbes.com/sites/williamhaseltine/2022/01/07/n ew-potential-covid-virus-variants-of-concern/?sh=6e4102481c17 – TITLE: Hook, Line, And Sinker: Do Rockfish Hold The Secret To Living Longer? Aging is a significant risk factor for human diseases like cancer, diabetes, and neurological disorders. Here, I describe how understanding the genetic adaptations that promote longevity in some rockfish species could provide insights into human aging. https://www.forbes.com/sites/williamhaseltine/2022/01/03/h ook-line-and-sinker-do-rockfish-hold-the-secret-to-livinglonger/?sh=7f5922727a56 January 11 – TITLE: New Insights Into Lung Damage And Repair Relevant To Covid-19 Lungs that have suffered severe damage have misplaced basal cells which interrupt the healing process. Here I explain a recent study

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that shines light on this process which is the first step towards developing a therapeutic intervention. http://www.forbes.com/sites/williamhaseltine/2022/01/06/ne w-insights-into-lung-damage-and-repair-relevant-to-covid-19/ – TITLE: The Case For Third And Fourth Booster Shots Here I discuss the importance of mRNA boosters in saving lives and hospital beds. Early messaging around the booster created confusion, we need a widespread booster campaign to assist in keeping our healthcare systems functioning. https://www.forbes.com/sites/williamhaseltine/2022/01/11/t he-case-for-third-and-fourth-booster-shots/?sh=1cb9cf46795e – TITLE: Omicron: Less Virulent But Still Dangerous Here I describe several studies showing that Omicron is less virulent than previous strains. Despite this it is still dangerous and we would do well to avoid infection by continuing to social distance, wear masks, and schedule vaccinations. https://www.forbes.com/sites/williamhaseltine/2022/01/11/o micron-less-virulent-but-still-dangerous/?sh=563f4bc82ea6 January 13 – “We are basing our plan on hopes the virus will behave like we want it to, i.e.: become less and less deadly, but there’s no guarantee that that will happen” https://www.barrons.com/articles/when-will-us-covid-casesomicron-peak-51641544919 – TITLE: We Can’t Stop Reporting Covid Cases Experts calling for a shift from reporting Covid cases to simply recording hospitalizations and deaths are making a grave mistake. The more knowledge we have about this ever-evolving virus, the more power we will have to control the pandemic. https://www.forbes.com/sites/williamhaseltine/2022/01/12/w e-cant-stop-reporting-covid-cases/?sh=d8a4f8060f31 – TITLE: Time to Expand Vaccination to Infants and Toddlers and Boosters for Children 1169

Omicron has unique adaptations that have increased infection and hospitalization among children as young as infants. Here, I discuss why this means vaccine eligibility should be extended to children under five. https://www.forbes.com/sites/williamhaseltine/2022/01/12/ti me-to-expand-vaccination-to-infants-and-toddlers-and-boostersfor-children/?sh=32589fbb2c7c January 19 – TITLE: Endemic Disease Does Not Necessarily Mean Mild Disease With record cases around the world, it is clear that herd immunity is a myth and that Covid-19 is still a very serious disease with many unknown outcomes. Here, I discuss some of the things we need to prepare for. https://www.forbes.com/sites/williamhaseltine/2022/01/18/e ndemic-disease-does-not-necessarily-mean-milddisease/?sh=4bbd00bc5d76 – TITLE: Inhaled Remdesivir for Home Use for Covid Treatment Here, I outline the challenges of treating Covid with an IV infusion of remdesivir and discuss how an inhaled version could be a more effective treatment. As more antiviral drugs are produced, we must ensure universal access to vulnerable populations. https://www.forbes.com/sites/williamhaseltine/2022/01/18/in haled-remdesivir-for-home-use-for-covidtreatment/?sh=67f2e6a77883 – TITLE: New Data Suggests That 50% Of Omicron Infections In Healthy Young Men Remain Transmissible After Five Days A recent study emphasizes the importance of taking Omicron seriously. The data indicates that 50% of infections remain transmissible after five days and its potent immune suppression makes it a real threat. https://www.forbes.com/sites/williamhaseltine/2022/01/19/n ew-data-suggests-that-50-of-omicron-infections-in-healthy1170

young-men-remain-transmissible-after-fivedays/?sh=3703737c1fa9 January 21 – Here, I describe the science behind a new antiviral approach, ensovibep. As of now it has to be delivered intravenously, but it is a valuable addition to our arsenal of antiviral drugs in this pandemic. http://www.forbes.com/sites/williamhaseltine/2022/01/20/no vel-antiviral-approach-to-covid-19-treatment/ – In a recent study, researchers discovered that SARS-CoV-2 nonstructural protein 5 (NSP5) suppresses innate immunity. Here, I discuss the study and NSP5’s potential as a target for drugmakers developing treatments to debilitate SARS-CoV-2. https://www.forbes.com/sites/williamhaseltine/2022/01/19/m ajor-protease-of-sars-cov-2-acts-to-suppress-innateimmunity/?sh=56fc1ce751b5 January 27 – TITLE: Birth Of The Omicron Family: BA.1, BA.2, BA.3. Each As Different As Alpha Is From Delta. The Omicron variant is not a single strain, but a family of three, each of which we must take seriously. Here, I introduce each of these variants, how they compare to each other, and how they diverged from the Wuhan strain. https://www.forbes.com/sites/williamhaseltine/2022/01/26/bi rth-of-the-omicron-family-ba1-ba2-ba3-each-as-different-asalpha-is-from-delta/?sh=409900cd3da9 – TITLE: New Clues to Long Covid: Prolonged Inflammatory Response A recent analysis provides evidence of Long Covid’s sustained inflammation and activation of immune response for at least 8 months after initial infection. Here, I describe the analysis and discuss the seriousness of Long Covid as a medical condition. http://www.forbes.com/sites/williamhaseltine/2022/01/25/ne w-clues-to-long-covid-prolonged-inflammatory-response/ 1171

– TITLE: The Danger Of Covid-19 Monotherapy: Drug Resistance We can use past experiences of HIV and influenza antiviral drug resistance as a lesson in the dangers of monotherapy against Covid19. Here I discuss these experiences and the importance of expanding our range of targets for drug development to begin testing combination therapy options. http://www.forbes.com/sites/williamhaseltine/2022/01/24/th e-danger-of-covid-19-monotherapy-drug-resistance/ January 31 – Researchers at the University of Hong Kong are exploring a novel vaccine strategy to combat Covid-19 which combines a vaccine injection and intranasal booster. Here I describe the study and discuss the importance of boosting immunity directly through the nose and upper respiratory system. https://www.forbes.com/sites/williamhaseltine/2022/01/31/n ovel-vaccine-booster-strategy-prevents-covid-19infection/?sh=2cc5b5896056 – Two new studies suggest CBD may potentially help in the fight against Covid-19. Although they do not warrant an immediate application of CBD for treatment, they do open the door to a new area of research for anti-SARS-CoV-2 drugs. https://www.forbes.com/sites/williamhaseltine/2022/01/28/d o-cannabinoids-offer-a-treatment-for-covid-19maybe/?sh=75757b1473f1 – Here, I describe the science behind a recent paper from the University of Washington which helps us understand the properties of Omicron in molecular detail. https://www.forbes.com/sites/williamhaseltine/2022/01/28/u nderstanding-omicron-a-structure-function-tour-deforce/?sh=76a4d9db6019 –

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A new study sheds light on one of the roles SARS-CoV-2 NSP13 (nonstructural protein 13) plays in the virus' initial attack on the immune system. Here, I explain. https://www.forbes.com/sites/williamhaseltine/2022/01/28/fu nctions-of-sars-cov-2-nsp13-helicase-replication-transcription-andsuppression-of-natural-immunity/?sh=3bf578d76743 – In February 2021, a paper published in Cellular & Molecular Immunology confirmed that NSP12 suppresses the activity of a coding gene, IRF3 (interferon regulatory factor 3), key to the production of interferon. https://www.forbes.com/sites/williamhaseltine/2022/01/27/fu nctions-of-sars-cov-2-nsp12-polymerase-replication-transcriptionand-suppression-of-natural-immunity/?sh=3338e155174e

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February 2022

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February 3 Researchers at the University of Illinois have developed another promising approach to Covid-19 treatment. Here I describe their approach, a “decoy” protein that tricks SARS-CoV-2 into binding to it instead of to host cells. http://www.forbes.com/sites/williamhaseltine/2022/02/02/de coy-protein-offers-new-treatment-approach-for-covid-19/ – Omicron and its three forms are now the dominant virus worldwide. Here, I describe what we know about the transmissibility, immune escape, spike protein and more for B.1, B.2 and B.3 https://www.forbes.com/sites/williamhaseltine/2022/02/02/t he-omicron-surprise/?sh=13236b655338 February 7 - New Blurbs Here I explain why expecting mothers need to be vigilant about reducing their risk of exposure to the SARS-CoV-2 virus. This means upgrading to surgical masks or N95/KN95 masks, avoiding crowded spaces, and being fully vaccinated. https://www.forbes.com/sites/williamhaseltine/2022/02/07/c ovid-infection-during-pregnancy-what-are-therisks/?sh=1b7d3b4863b1 – Two or three doses of currently-available vaccines provide excellent protection against severe disease leading to hospitalization or death from Omicron but, their protection may only last 4-6 months. Here, I explain. https://www.forbes.com/sites/williamhaseltine/2022/02/08/g ood-news-full-vaccination-protects-against-omicronhospitalization-and-death/?sh=b148a8f7bb2a February 10 Stanford scientists have found an improved method of genome sequencing which can diagnose a patient within eight hours, a process which normally takes weeks to months. Here, I describe a

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recent study that helps explain the importance of this new technology. https://www.forbes.com/sites/williamhaseltine/2022/02/08/q uantum-leap-in-newborn-whole-genomesequencing/?sh=47f50d42206e February 15 TITLE: Losing the Sense of Smell: How Covid-19 Infection Induces Long-Lasting Symptoms "A recent study finds that the Covid-19 virus need not infect olfactory neurons to damage the sense of smell, rather damage to the neurons is indirect. This discovery may help us understand Covid induced damage to other organs and tissues such as the heart, brain, and kidney." https://www.forbes.com/sites/williamhaseltine/2022/02/14/lo sing-the-sense-of-smell-how-covid-19-infection-induces-longlasting-symptoms/?sh=36444dc33f03 February 17 From the outset of the Covid pandemic, the extent to which the virus SARS-CoV-2 might vary was seriously underestimated. Here we analyze the different ways the SARS-CoV-2 virus adapts to overcome and infect the human population and its best defenses. https://www.forbes.com/sites/williamhaseltine/2022/02/17/d o-not-underestimate-the-consequences-of-sars-cov-2-escape-theomicron-example/?sh=6efb6fc93e1e February 21 TITLE: Protection In The Present From The Deep Past A recent study describes gasdermins and their role against invading microbes in innate immunity. This study is a step forward for immunology as we uncover more antimicrobial agents that have successfully defended against viruses and diseases for eons. https://www.forbes.com/sites/williamhaseltine/2022/02/21/p rotection-in-the-present-from-the-deep-past/?sh=13fb3d6230ff – TITLE: Discovery of New HIV Variant Sends Warning for COVID Pandemic 1176

Viruses do not necessarily evolve to become milder, the selective pressure on a virus is to survive. The discovery of a new HIV variant reminds us that a coronavirus variant could emerge with the transmissibility of Omicron and the deadliness of the original SARS. https://www.scientificamerican.com/article/discovery-of-newhiv-variant-sends-warning-for-covid-pandemic/ – TITLE: The SARS-CoV-2 Genome: The Importance of the Termini As we consider the potential of future variants, we must be mindful of the structural flexibility of genomic termini as an inherent source of variation. Its relevance to viral evolution, replication and pathogenicity calls for careful tracking of the ends of the genome of SARS-CoV-2. https://www.insideprecisionmedicine.com/topics/patientcare/coronavirus/the-sars-cov-2-genome-the-importance-of-thetermini/ February 25 TITLE: Pericyte Damage: Surprising Cause of CovidRelated Myocarditis A recent study reveals the details of how SARS-CoV-2 affects pericytes, a cell type critical for the maintenance and repair of blood vessels. Spike protein binding to pericyte surface receptors causes dysfunction without infection of the cell itself. https://www.forbes.com/sites/williamhaseltine/2022/02/25/p ericyte-damage-surprising-cause-of-covid-relatedmyocarditis/?sh=4b477109de59 – TITLE: Whither the Omicron Family: BA.1, BA.1.1, BA.2, BA.2.H78Y, BA.3? Omicron has stormed onto the scene, quickly becoming one of the most dominant global variants. But, more questions remain than are answered; what should we expect from omicron moving forward? http://www.forbes.com/sites/williamhaseltine/2022/02/23/w hither-the-omicron-family-ba1-ba11-ba2-ba2h78y-ba3/

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March 2022

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March 1 - New Blurbs – TITLE: Whence And Whither Covid-19: The Many Faces Of Omicron As the Omicron family of variants spreads, a new version arises in Denmark. Here I examine how it differs from the other mutations and whether BA.2 + H78Y will be the next variant to become dominant. https://www.forbes.com/sites/williamhaseltine/2022/02/28/w hence-and-whither-covid-19-the-many-faces-ofomicron/?sh=77536854c084 – TITLE: Op-Ed: Will the next coronavirus variant escape our best immune defenses? Vaccines have raised almost the same T-cell response to all variants but a recent study has shown a decline of T-cell response to Omicron. This decline can be overcome by booster vaccination but is a warning that future variants may escape both antibodies and Tcell immunity. https://www.latimes.com/opinion/story/2022-02-27/covid19-virus-vaccines-evasion-t-cells – TITLE: Covid-19 Has Orphaned 5.2 Million Children The number of children orphaned globally by Covid-19 is estimated to have increased from approximately 2.7 million in April to 5.2 million in October 2021. We have both a moral and public health responsibility to protect and support these children. https://www.forbes.com/sites/williamhaseltine/2022/03/01/c ovid-19-has-orphaned-52-million-children/?sh=5e01d5eb66b0 – TITLE: Covid-19 Pathogenesis: Spike Protein Alone Can Damage Cells New research indicates that SARS-CoV-2 may be able to impact viral pathogenesis without entering host cells— extracellular binding of the spike protein to host ACE2 has the potential to alter both cell physiology and expression of viral proteins. http://www.forbes.com/sites/williamhaseltine/2022/03/02/co vid-19-pathogenesis-spike-protein-alone-can-damage-cells/ 1179

March 7 – TITLE: Covid-19 Damage To The Male Reproductive Tract Animal studies in monkeys and hamsters show that Covid-19 can inflict serious damage to the male reproductive organs. This includes low sperm count, erectile dysfunction, and decreased testosterone. Preliminary evidence suggests vaccination may prevent or limit damage. http://www.forbes.com/sites/williamhaseltine/2022/03/07/co vid-19-damage-to-the-male-reproductive-tract/ – “With Omicron, it’s clear that two doses aren’t enough, and three doses are needed if you are going to have any protection at all.” https://www.medicalnewstoday.com/articles/4th-shots-andcovid-19-boosters-why-and-when-best-to-get-it – TITLE: Antibody-Activated Endothelial Cells Increase the Risk of Blood Clots with Covid-19 A recent study analyzed blood samples from nearly 250 individuals hospitalized for Covid-19. They revealed that antiphospholipid antibodies can activate endothelial blood vessel cells, risking widespread blood clots associated with severe Covid19. https://www.forbes.com/sites/williamhaseltine/2022/03/04/a ntibody-activated-endothelial-cells-increase-the-risk-of-bloodclots-with-covid-19/ – TITLE: Covid Ping-Pong: Human To Deer, Deer To Human A newly discovered strain of SARS-CoV-2 in Canadian deer may signal a novel route for future emerging Covid variants. A reminder that there may be many other lingering variants in both humans and animals. https://www.forbes.com/sites/williamhaseltine/2022/03/04/c ovid-ping-pong-human-to-deer-deer-tohuman/?sh=c7312812dd63 – 1180

TITLE: Covid-19 Infection Increases Heart Disease Risk, Even In Mild Cases Here I describe a new study which has shown that individuals with Covid have a 4.5% chance of serious damage to the heart and lungs post-infection. Leading me to recommend a cardiovascular workup within 12 months of Covid infection. https://www.forbes.com/sites/williamhaseltine/2022/03/07/c ovid-19-infection-increases-heart-disease-risk-even-in-mildcases/?sh=517e537220ec March 10 - New Blurbs – TITLE: A New Process Of SARS-CoV-2 Variation Uncovered: Intragenomic Recombination Roberto Partarca and I define yet another means by which coronaviruses including SARS-CoV-2 genomes vary: Intragenomic recombination involving duplications and insertion of 5 prime sequences throughout the genome. https://www.forbes.com/sites/williamhaseltine/2022/03/10/anew-process-of-sars-cov-2-variation-uncovered-intragenomicrecombination/?sh=3849d9d9c8cd – TITLE: Stripping Covid's Camouflage SARS-CoV-2 is equipped with various tricks to bypass the host's immune system. Here I examine a recent study which discusses one such trick and demonstrates the power of a novel approach to antiviral drug development. https://www.forbes.com/sites/williamhaseltine/2022/03/08/st ripping-covids-camouflage/?sh=747cd3781f75 March 11 - Quote

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“We have learned that the cold-causing coronavirus designated 229E undergoes antigenic drift similar to that of influenza.” A quote from my book, Variants!, which we recently released the 4th edition of. If you are looking for answers to your questions about variants of concern, vaccine evasion, and reinfection it is available now on Amazon: https://www.amazon.com/gp/product/B08WKPXCKD/ref=dbs_ a_def_rwt_bibl_vppi_i1 March 14 - Video In this video, I answer the questions of a young student about the universe. https://youtu.be/CyFmLp0GjwQ March 16 - New Blurbs – TITLE: Covid-19: Long Term Brain Injury Recent studies report that as many as 25% of those infected with any severity level of Covid-19 may experience long-term brain damage. These observations raise troubling issues for the medical system and society which we must begin to understand and plan for. 1182

https://www.forbes.com/sites/williamhaseltine/2022/03/14/c ovid-19-long-term-brain-injury/ – TITLE: An Ancient Form Of Immunity Protects Many Against Covid-19 Today Two recent studies explain another aspect of the natural (innate) immune system. They suggest that an ancient family of immunosuppressive agents called lectins may help prevent Covid-19 infection and limit the virus's ability to replicate and spread. https://www.forbes.com/sites/williamhaseltine/2022/03/15/a n-ancient-form-of-immunity-protects-many-against-covid-19today/?sh=49615ee66239 – “The biggest surprise has been that countries with the most developed healthcare systems have not fared well, that's including most of Europe and the United States…” https://america.cgtn.com/2022/03/16/the-heat-europe-rollsback-precautions-as-covid-19-cases-rise March 18 – Quote

“The consequence of endemic and pandemic Covid-19 is monumental for our lives and our economy.” 1183

A quote from my book, Variants!, which we recently released the 4th edition of. If you are looking for answers to your questions about variants of concern, vaccine evasion, and reinfection it is available now on Amazon: https://www.amazon.com/gp/product/B08WKPXCKD/ref=dbs_ a_def_rwt_bibl_vppi_i1 March 21 - New Blurbs TITLE: A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain. This study documents significant cortical gray matter loss, equivalent to nearly 10 years of aging. https://www.forbes.com/sites/williamhaseltine/2022/03/21/acase-of-shrunken-brains-how-covid-19-may-damage-braincells/?sh=59d101c470ff – TITLE: An Omicron-Omicron Recombinant—BA.4 A recent study outlines the ability of SARS-CoV-2 to combine parts of previous strains, creating new variants. Here, I describe a recombinant variant given the preliminary designation Omicron BA. 4 and discuss how variant recombinants may be the next chapter for the pandemic. https://www.forbes.com/sites/williamhaseltine/2022/03/16/a n-omicron-omicron-recombinant-ba4/?sh=4ca7aef759b0 March 22 - Natural Immunity Drugs that bolster innate immunity, or natural immunity, are the next frontier of pandemic control, not just for Covid-19 but existing and future infectious diseases. Read my latest book https://www.amazon.com/gp/product/0578386291/ March 23 - Letter

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I recently spoke with some High School classes in Oregon. I wanted to share some of the letters they wrote with insights from our talk. March 24 - New Blurbs There is a new class of SARS-CoV-2 that the scientific community contends may become a problem, recombinants. Here I investigate a new strain of SARS-CoV-2 that combines portions of previous variants of concern Delta and Omicron—the recombinant Deltacron. https://www.forbes.com/sites/williamhaseltine/2022/03/22/e n-garde-for-sars-cov-2-chimeras-recombinants/?sh=2f830ab85673 – Covid-19 affects many organs throughout the body, including those of our gastrointestinal tract. This is the introduction to a series on the immune processes that protect our gut in the face of SARSCoV-2 and other infections. http://www.forbes.com/sites/williamhaseltine/2022/03/24/in nate-and-adaptive-immunity-in-the-human-intestine/ – 1185

Omicron has come with a number of unpleasant surprises. Here, I address Omicron's evasion of monoclonal antibody neutralization and how we may be able to overcome this viral trait. https://www.forbes.com/sites/williamhaseltine/2022/03/23/a ntibodies-team-up-against-omicron/?sh=ba86926be0ef March 28 - New Blurbs – TITLE: A Primary Defense Against SARS-CoV-2: Defensins Recent research suggests that a family of proteins called defensins are a crucial tool in our body’s defense against SARS-CoV-2 and may explain why the intestine seems to be especially protected from Covid-19. https://www.forbes.com/sites/williamhaseltine/2022/03/26/aprimary-defense-against-sars-cov-2-defensins/?sh=3a99edc429b5 – TITLE: How Your Gut Protects You from Myriads Of Microbes. Covid-19 affects many organs throughout the body, including those of our gastrointestinal tract. This is part two of a series on the immune processes that protect our gut in the face of SARS-CoV-2 and other infections. http://www.forbes.com/sites/williamhaseltine/2022/03/25/ho w-your-gut-protects-you-from-myriads-of-microbes/ – A recent report from the Journal of Nuclear Medicine reviews the likely mechanisms underlying structural damage to the brain caused by Covid-19. Researchers are confident that for a majority of people these deficits will not be permanent or unresponsive to treatment. https://www.forbes.com/sites/williamhaseltine/2022/03/28/i maging-the-brain-what-brain-scans-reveal-about-theconsequences-of-covid-19/?sh=3e37ffbd20ea March 29 –

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Link: http://www.forbes.com/sites/williamhaseltine/2022/03/29/innate -lymphoid-cells-ilcs-guardians-against-infection/ Social Media Blurb: This is part three of a series on the immune processes that protect our gut in the face of SARS-CoV-2 and other infections. Here, we give an overview of innate lymphoid cells (ILCs), a recently discovered family of innate immune cells. Our guts have a tough job as a barrier allowing in the good and keeping out the bad. The recently-discovered innate lymphoid cells play an important role in our immune processes, including protecting our gut in the face of SARS-CoV-2 and other infections. http://www.forbes.com/sites/williamhaseltine/2022/03/29/in nate-lymphoid-cells-ilcs-guardians-against-infection/ – Link: https://www.forbes.com/sites/williamhaseltine/2022/03/29/newand-effective-monoclonal-antibody-treatment-for-ebola-on-thehorizon/?sh=52b908a25d20 Social Media Blurb: A pair of newly discovered monoclonal antibodies offer badly needed hope for the treatment of multiple strains of the Ebola Virus. They may serve as a model for the design of monoclonal antibodies for other diseases including Covid-19. What will be the cause of the next global pandemic? Ebola can't be ruled out. Fortunately, a pair of newly discovered monoclonal antibodies offer badly needed hope for the treatment of multiple Ebola strains. https://www.forbes.com/sites/williamhaseltine/2022/03/29/n ew-and-effective-monoclonal-antibody-treatment-for-ebola-onthe-horizon/?sh=52b908a25d20 March 31 Original Social Media Blurb: Covid-19 affects many organs throughout the body, including those of our gastrointestinal tract. Here, we describe how group 3 innate lymphoid cells (ILC3s) help protect our gut from SARS-CoV-2 infection. Laura’s approved and posted blurb: Covid-19 affects not only the lungs–it is also very much a virus of the gut, causing diarrhea, nausea, vomiting. Here I discuss the role of the innate lymphoid cells in protecting us against SARS-CoV-2 infection in our gut. 1187

http://www.forbes.com/sites/williamhaseltine/2022/03/31/hi de-and-seek-how-group-3-innate-lymphoid-cells-help-cloak-ace2receptors-from-sars-cov-2/ Quote

“...learn from nature, not from the laboratory or from textbooks — your job is to discover what is happening in nature, not to confirm your own ideas.” A quote from my book, Variants!, which we recently released the 4th edition of. If you are looking for answers to your questions about variants of concern, vaccine evasion, and reinfection it is available now on Amazon: https://www.amazon.com/gp/product/B08WKPXCKD/ref=dbs_ a_def_rwt_bibl_vppi_i1 Blurbs: Posted Original Social Media Blurb: Covid-19 affects many organs throughout the body, including those of our gastrointestinal tract. This is part four of a series on the immune processes that protect our gut in the face of SARS-CoV-2 and other infections. Laura’s approved and posted: Here I explain what we have learned about immune cells in our gut having memory—recognizing 1188

pathogens previously encountered. This trait of the innate lymphoid cells helps protect us from SARS-CoV-2 and other infections. http://www.forbes.com/sites/williamhaseltine/2022/03/30/gr oup-3-innate-lymphoid-cells-ilc3s-and-trained-immunity-fasterand-stronger/

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April 1 Here I explain what we have learned about immune cells in our gut having memory—recognizing pathogens previously encountered. This trait of the innate lymphoid cells helps protect us from SARS-CoV-2 and other infections. http://www.forbes.com/sites/williamhaseltine/2022/03/30/gr oup-3-innate-lymphoid-cells-ilc3s-and-trained-immunity-fasterand-stronger/ April 2 - Natural Immunity (POSTED) How does the body defend itself against the Covid-19 virus, and how can drugs assist in that fight? The answer to both questions lies with our first line of defense, natural or innate immunity. For more, read my latest book https://www.amazon.com/gp/product/0578386291/ April 4 Title: The Artful Dodger SARS-CoV-2: Evading Immunity By Inhibition Of Interferon (POSTED) Our body’s alarm systems trigger our defenses against novel microbes. But SARS-CoV-2 is crafty at evading these innate defenses, and over time has evolved to become resistant to interferons, cells that play a central role in our innate immunity. https://www.forbes.com/sites/williamhaseltine/2022/04/01/t he-artful-dodger-sars-cov-2-evading-immunity-by-inhibition-ofinterferon/?sh=76892da35cff – Title: Coronaviruses Can Recombine With Cellular And Heterologous Virus Genes To Create Unexpected Variants (POSTED) SARS-CoV-2 continues to evolve. The virus’s ability to recombine with variants of itself begs the question, Can it incorporate genes from other viruses, leading to new variants with increased transmissibility, immune evasion, or virulence? https://www.forbes.com/sites/williamhaseltine/2022/04/01/c oronaviruses-can-recombine-with-cellular-and-heterologousviruses-to-create-unexpected-variants/?sh=641fbffe5786 1191

April 5 Video In this video, I talk about the most important advice I have for any young person interested in pursuing a life in science. https://youtu.be/VrxhZlm5HGc – Title: Quantitative Markers for Covid-19 Brain Injury Here is the final blurb for LinkedIn and Facebook. Many people suffering from long-covid experience fatigue, brain fog, and other neurological symptoms. Lack of specific diagnostic markers means health systems may fail to treat them seriously. Discovery of diagnostic markers of brain inflammation in the spinal fluid & blood of post-covid patients suffering neurologic symptoms may help people access the care they need. Here’s the Twitter version (shorter by necessity) Lack of specific diagnostic markers for neurological symptoms in people suffering from long-covid means health systems may fail to treat them seriously. Discovery of markers of brain inflammation in spinal fluid & blood may help people get care they need. https://www.forbes.com/sites/williamhaseltine/2022/04/04/q uantitative-markers-for-covid-19-brain-injury/?sh=7883df3a2793 April 6 Title: Two More Members Of The Omicron Family To Keep An Eye On Social Media Blurb: As Omicron continues to spread, new variants will continue to arise. Here we describe two more Omicron family strains to keep an eye on. https://www.forbes.com/sites/williamhaseltine/2022/04/06/t wo-more-members-of-the-omicron-family-to-keep-an-eyeon/?sh=1e53d8184fea April 7 Title: Innate Lymphoid Cells And Covid-19 Severity: Chicken Or The Egg? Social Media Blurb: New research suggests that decreased levels of innate lymphoid cells (ILCs) in age may contribute to the increased risk of developing severe Covid-19. Low ILC levels may also be behind the worse disease outcomes seen in males. 1192

https://www.forbes.com/sites/williamhaseltine/2022/04/07/in nate-lymphoid-cells-and-covid-19-severity-chicken-or-theegg/?sh=470d42039f6c Title: Head in the Clouds: Living with Covid-19 Social Media: “Living with Covid” will not look the same for everyone. Some are already experiencing noticeable impairments in memory, executive function and attention. Guiding care for these individuals needs to be a priority as this pandemic transitions into an endemic. https://www.forbes.com/sites/williamhaseltine/2022/04/07/h ead-in-the-clouds-living-with-covid-19/?sh=4fa1b67a7540 April 10 - Letter

I recently spoke with some High School classes in Oregon. I wanted to share some of the letters they wrote with insights from our talk. April 11 1193

Two More Members Of The Omicron Family To Keep An Eye On As Omicron continues to spread, new variants will continue to arise. Here we describe two more Omicron family strains to keep an eye on. https://www.forbes.com/sites/williamhaseltine/2022/04/06/t wo-more-members-of-the-omicron-family-to-keep-an-eyeon/?sh=1e53d8184fea – New research suggests that decreased levels of innate lymphoid cells (ILCs) in age may contribute to the increased risk of developing severe Covid-19. Low ILC levels may also be behind the worse disease outcomes seen in males. https://www.forbes.com/sites/williamhaseltine/2022/04/07/in nate-lymphoid-cells-and-covid-19-severity-chicken-or-theegg/?sh=470d42039f6c – “Living with Covid” will not look the same for everyone. Some are already experiencing noticeable impairments in memory, executive function and attention. Guiding care for these individuals needs to be a priority as this pandemic transitions into an endemic. https://www.forbes.com/sites/williamhaseltine/2022/04/07/h ead-in-the-clouds-living-with-covid-19/?sh=4fa1b67a7540 April 12 Title: S100s: A Blunderbuss Approach To SARS-CoV-2 Defense Social Media: Scientists have recently discovered that a family of proteins called S100s may simultaneously protect us from SARSCoV-2 whilst amplifying the severity of SARS-CoV-2 symptoms. Here, we provide an in-depth look at the complex relationship between S100s and SARS-CoV-2. http://www.forbes.com/sites/williamhaseltine/2022/04/09/s1 00s-a-blunderbuss-approach-to-sars-cov-2-defense/ – Title: In the Eye of the Storm: How Covid-19 Impacts the Eye Social Media Blurb: While Covid-19 is commonly associated with infection of the lungs, heart and other vital organs, a growing 1194

body of evidence suggests that infection can also impact the eye. Thanks to a recent study we may now have a better picture of how SARS-CoV-2 infects the retina. https://www.forbes.com/sites/williamhaseltine/2022/04/11/in -the-eye-of-the-storm-how-covid-19-impacts-theeye/?sh=c2cea535781c – Title: Two Emerging Viral Adversaries—Nipah And Hendra Virus—May Soon Meet Their Match Social Media Blurb: Nipah and Hendra Virus are two dangerous emerging pathogens that may cause serious damage if left unchecked. Here we analyze antibody candidates that may impede these threats. https://www.forbes.com/sites/williamhaseltine/2022/04/11/t wo-emerging-viral-adversaries-nipah-and-hendra-virus-may-soonmeet-their-match/ April 13 Title: Remdesivir resistance reported upon treatment of an immunocompromised patient Social Media Blurb: One of the questions that arises with any single drug treatment of an RNA virus is the level of resistance. Remdesivir has been approved as a stand-alone therapy for the treatment and prevention of COVID-19. https://www.forbes.com/sites/williamhaseltine/2022/04/12/re mdesivir-resistance-reported-upon-treatment-of-animmunocompromised-patient/?sh=e0450cc1130d April 14 Title: New Analysis Shows Fluvoxamine Has The Potential To Reduce Covid-19 Hospitalizations By More Than 90% Social Media Blurb: There is an ever-expanding need for SARSCoV-2 antivirals. A recent analysis suggests fluvoxamine may greatly reduce the risk of hospitalization in Covid patients. https://www.forbes.com/sites/williamhaseltine/2022/04/13/n ew-analysis-shows-fluvoxamine-has-the-potential-to-reducecovid-19-hospitalizations-by-more-than-90/?sh=65ff6e432ce3 – Title: ‘Test-to-treat’ could transform the pandemic 1195

Social Media Blurb: The Biden administration’s “test-to-treat” initiative has the potential to be one of the most significant advances in health care delivery in recent years. Yet we must ensure that logistical challenges and issues of equitable access don’t hamper the potential of this groundbreaking initiative. https://thehill.com/opinion/healthcare/3266378-test-to-treatcould-transform-the-pandemic/ April 15 Title: SARS-CoV-2 Actively Infects And Kills Lymphoid Cells Social Media Blurb: A hallmark of serious SARS-CoV-2 infection is lymphopenia. Recent work attributes lymphopenia at least in part to SARS-CoV-2 infection and killing of lymphocytes through an ACE2-independent entry route. https://www.forbes.com/sites/williamhaseltine/2022/04/14/sa rs-cov-2-actively-infects-and-kills-lymphoidcells/?sh=42627d7786b8 – To protect vulnerable populations from severe Covid-19, we need drugs that bolster our first line of bodily defense, natural or innate immunity. Read my latest book, Natural Immunity and Covid-19 on Amazon https://www.amazon.com/gp/product/0578386291/ April 19 Title: Six Ways Fluvoxamine May Act To Prevent Severe Covid-19 Social Media Blurb: Fluvoxamine has the potential to reduce the chance of progression to severe Covid-19. Here we describe the mechanisms that enable this ability. https://www.forbes.com/sites/williamhaseltine/2022/04/18/si x-ways-fluvoxamine-may-act-to-prevent-severe-covid19/?sh=5486d53f219b April 21 Title: New Members Of The Omicron Family Of Viruses: BA.2.12.1, BA.4, And BA.5 1196

Social Media Blurb: New members of the Omicron family continue to emerge. Here we describe descendants of the highly transmissible BA.2 tree. https://www.forbes.com/sites/williamhaseltine/2022/04/20/n ew-members-of-the-omicron-family-of-viruses-ba2121-ba4-andba5/?sh=5ce13afb7ff8 – Title: Moral Injury Is Similar in Healthcare Workers and Veterans Social Media Blurb: A recent study found similar rates of moral injury between veterans and Covid-19 healthcare workers. Systematic and institutional change is needed to prioritize the mental health of our healthcare workers. https://www.psychologytoday.com/us/blog/best-practices-inhealth/202204/moral-injury-is-similar-in-healthcare-workers-andveterans April 22 The natural or innate immune system is what defends the body against new threats from the microbial world. For the next stage of Covid-19 pandemic control, we need drugs that strengthen these natural defenses. Read my latest book to learn more https://www.amazon.com/gp/product/0578386291/ – Title: SARS-CoV-2 Infection Of Monocytes Triggers Inflammation Social Media Blurb: Hyper-inflammation is a hallmark of severe Covid-19. New research shines a light onto one of the potential causes: infection of monocytes and macrophages. http://www.forbes.com/sites/williamhaseltine/2022/04/21/sar s-cov-2-infection-of-monocytes-triggers-inflammation/ – Title: STING: It Takes Two To Tango Social Media Blurb: A new discovery uncovered a previously hidden binding site on a key immune protein known as STING that may enhance protection against infection. Knowing when to activate or inhibit this protein may be the key to controlling the widespread inflammatory consequences of Covid-19.

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https://www.forbes.com/sites/williamhaseltine/2022/04/21/st ing-it-takes-two-to-tango/?sh=296a678b5da9 – Social Media Blurb: The latest Omicron variant, BA.2.12.1, is on the rise in the American Northeast. Here I describe what may be the next catalyst for infections in the US and worldwide. https://www.forbes.com/sites/williamhaseltine/2022/04/22/s urvival-of-the-fittest-the-rise-of-ba2121/?sh=4fa4d69f413d – An excerpt from my recent interview on "The Heat" discussing mask mandates. https://youtu.be/-3qNOQeqWJY

April 25 - Letter

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I recently spoke with some High School classes in Oregon. I wanted to share some of the letters they wrote with insights from our talk. April 26 Some would argue that it is time to stop reporting Covid cases and move on, disregarding those that continue to be affected by the disease. To understand why this is a mistake, read my latest book: http://ow.ly/MVtz50ISTcG

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April 28 The rise of Omicron and Covid-19, in general, has forced an acceleration of new antiviral therapies and strategies. To recognize the breadth of antiviral research, read my latest book: http://ow.ly/A6NM50ISTrC

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We need to utilize the lessons of 35 years of HIV research and create combinations of powerful, long-acting drugs that target a broad range of functions to fill the gaps in our current strategy for long-term Covid-19 protection. Learn more in my latest book https://www.amazon.com/gp/product/0578386291/

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May 2 Montelukast, an asthma drug, can bind to and block a crucial SARS-CoV-2 protein. This discovery sets the stage for a new class of antiviral drugs, designed to target and interfere with the virus' immunosuppressive tactics. http://www.forbes.com/sites/williamhaseltine/2022/05/02/ast hma-medication-points-the-way-to-drugs-to-prevent-and-treatcovid-19-nsp1-a-novel-target/ A new Covid-19 monoclonal antibody, 35B5, has the potential to neutralize all current and future SARS-CoV-2 variant strains. https://www.forbes.com/sites/williamhaseltine/2022/04/29/3 5b5-a-potent-broadly-neutralizing-monoclonal-antibody-effectiveagainst-all-known-variants/?sh=7bd9d211b265 May 4, 2022 - all posted Here we analyze some of the structural properties of Omicron that enable efficient immune escape. https://www.forbes.com/sites/williamhaseltine/2022/05/03/struct ural-analysis-of-the-omicron-spike-unveils-houdini-like-immuneescape/?sh=6538228724be For some time now, it’s been established that the primary route of SARS-CoV-2 transmission is airborne. Research shows that FarUVC light inactivates the virus in a way that is harmless to us. https://www.forbes.com/sites/williamhaseltine/2022/05/02/farultraviolet-light-is-another-way-to-keep-our-public-spacessafe/?sh=6ad69a571d6b Groundbreaking research paves the way in the development of vaccines for respiratory syncytial virus (RSV). Here we summarize the key breakthroughs that opened the door. https://www.forbes.com/sites/williamhaseltine/2022/05/03/newresearch-provides-hope-in-the-search-for-a-respiratory-syncytialvirus-vaccinepart-ii/?sh=3ea0745b7687 May 6 Unique changes in the Omicron family of variant Spike protein structure enable its enhanced viral characteristics. https://www.forbes.com/sites/williamhaseltine/2022/05/06/omic 1203

ron-transmission-and-immune-evasion-displayed-by-the-omicronspikes-unique-structure/?sh=1fcc831c3acd May 7 A new study demonstrates that unvaccinated people threaten the safety of the vaccinated even when SARS-Cov-2 vaccination rates are high. https://www.forbes.com/sites/williamhaseltine/2022/05/06/st udy-shows-unvaccinated-people-are-at-increased-risk-of-infectingthe-vaccinated/?sh=77462a1478eb May 9 The kidneys are one of the major targets for Covid-19 infection, which corresponds with significant complications. Thanks to a recent study from Duke University we are now closer to understanding how and why SARS-CoV-2 disrupts kidney function by directly infecting podocytes. https://www.forbes.com/sites/williamhaseltine/2022/05/09/ta rgets-for-infection-how-sars-cov-2-damages-thekidneys/?sh=6c4418fa6bd6 Lupus is a life-threatening, chronic autoimmune disease. The exact causes have remained poorly understood. New research pinpoints genetic mutations to toll-like receptor 7 (TLR7) as one direct culprit. TLR7 may also be behind the autoimmune symptoms seen in SARS-CoV-2 infections. http://www.forbes.com/sites/williamhaseltine/2022/05/09/sci entists-discover-genetic-cause-of-lupus-findings-may-helpresearch-on-long-covid/ May 10 Part III of the series on respiratory syncytial virus vaccines provides hopeful timelines for the introduction of safe and effective RSV vaccines in the near future. https://www.forbes.com/sites/williamhaseltine/2022/05/10/n ew-research-provides-hope-in-the-search-for-a-respiratorysyncytial-virus-vaccinepart-iii/?sh=9194d3c20289 May 18 1204

Why are the kidneys so vulnerable to viral infection? This second part of our two-part series takes a deeper look at how the expression of CD147 and ACE-2 receptors mediates the infection of a specialized group of kidney cells: podocytes. https://www.forbes.com/sites/williamhaseltine/2022/05/17/adynamic-duo-how-ace-2-and-cd147-mediate-covid-19-infectionin-the-kidneys/?sh=649695671b9d May 19 The Food and Drug Administration denied an emergency use authorization for fluvoxamine as a SARS-CoV-2 antiviral. Here we talk about why. https://www.forbes.com/sites/williamhaseltine/2022/05/19/fd a-turns-down-fluvoxamine-emergency-use-authorizationrequest/?sh=52dade002c81 May 23 Cases of vector-borne disease are on the rise, and this includes the rare but potentially deadly Powassan virus. Causing severe neurological symptoms, this virus is transmitted by ticks and can be acquired in as little as 15 minutes of feeding. https://www.forbes.com/sites/williamhaseltine/2022/05/23/t he-growing-threat-of-tick-borne-disease-part-i-powassanvirus/?sh=da1dfc830d81 May 25: Scheduled

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As we learn more about long Covid, I will be sharing resources via my COVID-19 Resources website where I curate the latest news to help bring attention to the most important or timely information pertaining to COVID-19. → https://covid19resources.org/ BOSTON HERALD: https://www.bostonherald.com/2022/05/04/covid-study-nearly13-of-hospitalized-patients-had-serious-neurologic-symptoms/ May 27: scheduled

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COVID-19 infection rates are up to one million daily. If you do get sick, the antiviral drug Paxlovid is available and can reduce symptoms. Bear in mind that some physicians report more than 40% of patients experience Paxlovid Rebound. Others report much, much lower figures.You can learn more about rebound here. https://www.statnews.com/2022/05/24/paxlovid-reboundhas-covid-researchers-looking-for-theories/ May 29

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If you aren’t sure how to find trustworthy information about everything from vaccines to what we’re learning about variants, transmission, policy decisions, and more, visit this website. Updated each week, and curated by William A. Haseltine. https://covid19resources.org/ May 31: scheduled I believe third and fourth doses of the Covid-19 vaccine are vital to prevent severe disease and death in the face of Omicron and future variants. To find out why, read my latest book. AMAZON: https://www.amazon.com/Omicron-Pandemic-Endemic-FutureCovid-19-ebook/dp/B09WNC13ZF/

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Recent study suggests that viral mRNA from SARS-CoV-2 may be found in the stools of patients diagnosed with Covid-19, even after respiratory symptoms subside. Findings suggest Covid-19 likely leads to prolonged gastrointestinal (GI) infections. http://www.forbes.com/sites/williamhaseltine/2022/05/31/ex pelling-covid-it-takes-longer-than-you-may-think/

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June 2022

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June 1: - scheduled

We are learning more about how Covid-19 affects kids. This article in Immunity reviews what we understand and suggests a few reasons why we see the different outcomes that we do. https://www.cell.com/immunity/fulltext/S10747613(22)000437?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretri eve%2Fpii%2FS1074761322000437%3Fshowall%3Dtrue June 2 Poxviruses are back, and it is no surprise. Researchers have long predicted that halting smallpox vaccinations could enable the emergence of new virulent strains of this and other infectious poxviruses. https://www.forbes.com/sites/williamhaseltine/2022/06/01/t he-vaccinia-virus-that-hopped-from-rabbits-tohares/?sh=63ff998e997f June 3: Scheduled

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Monkeypox is in the news. This article by Katelyn Jetelina is a nice summary of what we know so far. Stay tuned. https://yourlocalepidemiologist.substack.com/p/monkeypox101-unansweredquestions?token=eyJ1c2VyX2lkIjo1MzU3NTIsInBvc3RfaWQiOj Q4MDgyMTc5LCJfIjoibkdpRkIiLCJpYXQiOjE2NTMzOTk5N zgsImV4cCI6MTY1MzQwMzU3OCwiaXNzIjoicHViLTI4MTIx OSIsInN1YiI6InBvc3QtcmVhY3Rpb24ifQ.fOaQYUCk1AiPm66 GYaauzWo1ehzCXKp9IncvcZeeJy0&s=r June 5: SCHEDULED

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If you are a physician, or perhaps you think you may suffer from long covid, you can learn more about what we’re learning about the effects of covid-19 on the autonomic nervous system in this webinar by Dysautonomia International. https://vimeo.com/442593497 June 6 With poxviruses now back on the radar, we must consider the outstanding threat of possible human pathogens in much greater detail. It is critically important to understand not only how poxviruses jump from one species to another but also how they evolve across generations. https://www.forbes.com/sites/williamhaseltine/2022/06/06/ra bbitpox-the-story-of-a-specialized-killer/?sh=1dfd6332da54 In addition to the spread in Europe, there is a new outbreak of the Crimean-Congo Hemorrhagic Fever in Iraq. With a high fatality rate and widespread range across multiple continents, it is time to develop the means to prevent and treat this tick-borne disease. https://www.forbes.com/sites/williamhaseltine/2022/06/03/ti ck-borne-disease-part-ii-crimean-congo-hemorrhagicfever/?sh=3df3acac7a61 June 8

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A multimodal approach to controlling the Covid-19 pandemic remains our best defense. Learn more about why classical herd immunity does not apply to SARS-CoV-2: https://academic.oup.com/jid/advancearticle/doi/10.1093/infdis/jiac109/6561438?login=false June 10: SCHEDULED A new study found that less than 10 percent of children who contracted Covid-19 in 2020 or early 2021 developed neutralizing antibody titers against the Omicron variant of SARSCoV-2. https://www.forbes.com/sites/williamhaseltine/2022/06/09/st udy-finds-previous-covid-19-infection-doesnt-protect-childrenfrom-omicron/?sh=7c151c871176

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In this webinar, Dysautonomia International explores what we know about dysautonomia symptoms in long haul Covid-19. https://vimeo.com/442593497 June 11 SCHEDULED In an Op-Ed for Project Syndicate, I argue that drug developers should take a lesson from HIV research and focus on developing combinations of small-molecule antiviral drugs to prevent and treat Covid-19. We need to find powerful ways to eliminate the SARS-CoV-2 virus. https://www.project-syndicate.org/commentary/covid-19vaccines-not-enough-need-new-antivirals-by-william-a-haseltine2022-06?utm_source=facebook&utm_medium=organicsocial&utm_campaign=page-posts-june22&barrier=accesspaylog June 12

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Each week, I select three articles about Covid-19 that represent the latest research, important policy, or trends in the global pandemic and our efforts to win this battle. https://covid19resources.org/ SCHEDULED: Prion diseases are rare, deadly neurodegenerative brain diseases that result from misfolding protein. Recent study encourages the development of antibodies to PrPC to prevent its conversion to PrPSc. If successful, their unique approach illuminates treatment for prion and other like diseases. Twitter version: Prion diseases are rare, deadly neurodegenerative brain diseases resulting from misfolding protein. Recent study encourages development of antibodies to PrPC to prevent its conversion to PrPSc. If successful, this could illuminate treatments. https://www.forbes.com/sites/williamhaseltine/2022/06/09/u nraveling-hope-for-prion-disease-and-other-progressiveneurodegenerative-diseases/?sh=37a652453471 SCHEDULED: 1216

We must heed the lessons from delayed diagnosis for other illnesses such as Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and not repeat the same mistakes when diagnosing Long Covid patients. https://thehill.com/opinion/healthcare/3518812-this-is-howwe-should-be-diagnosing-long-covid/ June 13 Posted @ 8:30am PST Recent scientific advances using a distant model organism—the fruit fly—may offer a new window into how brain damage could be repaired in humans. https://www.forbes.com/sites/williamhaseltine/2022/07/11/re search-on-flies-provides-hope-for-brain-repair/?sh=1c0df9d23e67 SCHEDULED: New research highlights the potential for Paxlovid resistance, demonstrating that our Covid-19 drug development efforts should focus on combinations of smallmolecule antiviral drugs that have proved effective at preventing and treating HIV. https://www.forbes.com/sites/williamhaseltine/2022/06/09/p axlovid-resistance-challenges-andopportunities/?sh=3e09b49665bc June 14 With every purchase of Omicron: From Pandemic to Endemic, receive access to the living eBook, as well as online access to Variants! The Shape Shifting Challenge of Covid-19. Get your copy here: https://www.amazon.com/Omicron-Pandemic-EndemicFuture-Covid-19-ebook/dp/B09WNC13ZF/

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June 15

Spillover events–zoonosis–have probably triggered every viral pandemic that’s occurred since the start of the twentieth century. What’s more, an August 2021 analysis of disease outbreaks over the past four centuries indicates that the yearly probability of pandemics 1218

could increase several-fold in the coming decades, largely because of human-induced environmental changes An infographic published recently in Nature shows the growing threat of spillover events worldwide. NATURE: https://www.nature.com/articles/d41586-02201373-z TWITTER: Zoonosis has probably triggered every viral pandemic since 1900. Analysis of disease outbreaks over the past 4 centuries indicates pandemics could increase in coming decades. Growing threat of spillover events worldwide: NATURE: https://www.nature.com/articles/d41586-02201373-z June 16 Scheduled This is Part I in a series on the enteroviruses that appear to cause a polio-like neurological disease, Acute Flaccid Myelitis. Reports of this life-threatening condition have increased within the US and elsewhere, and viruses from the same family as the poliovirus are implicated. https://www.forbes.com/sites/williamhaseltine/2022/06/10/t here-may-be-a-new-polio-epidemic-on-its-wayif-so-what-wecan-do/?sh=4ea067b15ebc June 17

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Read Maria’s experience with long Covid, and if someone you know is suffering 1 -2 months after being infected, please share her words. https://crookedtimber.org/2020/05/18/indefinitely-ill-postcovid-fatigue/ June 18 SCHEDULED: SARS-CoV-2 makes use of a variety of accessory proteins to evade and suppress our immune response. New research on the accessory protein ORF7a suggests it may help the virus gain easier entry into our cells. https://www.forbes.com/sites/williamhaseltine/2022/06/11/at tack-and-counterattack-how-sars-cov-2-blocks-our-naturalimmune-defenses/?sh=7bcc92b14ce7 June 19:

The CDC reports 1 of every 5 people aged 18-64 who suffered from Covid-19 has experienced at least one post-acute condition. This article in Modern Healthcare offers more data on the 1220

prevalence of Long Covid and some of the challenges providers face.. https://www.modernhealthcare.com/patient-care/long-covidposes-unique-challenges-providers-seeking-answers June 20: Scheduled For the past two and half years, Covid-19 introduced a significant risk to travel. Here we discuss how to mitigate this risk. https://www.forbes.com/sites/williamhaseltine/2022/06/14/is -it-safe-to-fly-the-national-academy-of-sciences-engineering-andmedicine-weighs-in/?sh=565cb3a94c32 June 21 Understanding the dynamics of airflow on aircraft may save you from a Covid infection during your upcoming summer vacation. https://www.forbes.com/sites/williamhaseltine/2022/06/16/d ont-you-wish-you-didnt-need-to-breathe-someone-elses-exhaledair-while-flying/?sh=84273081ff21 June 22

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In the early days of the pandemic, we didn’t know much about how the virus spread. 2+ years in, we know so much more. I suggest… keep washing your hands, but you can relax when it comes to doing things like wiping down your groceries. https://www.usnews.com/news/health-news/articles/202205-04/covid-transmission-1-000-times-more-likely-from-air-vssurfaces-study June 23 SARS-CoV-2 has a variety of accessory proteins that help it evade and suppress our immune system. Recent findings suggest that the accessory protein ORF7a helps suppress our immune cells, making it harder for the virus to be detected. http://www.forbes.com/sites/williamhaseltine/2022/06/16/fly ing-under-the-radar-how-sars-cov-2-orf7a-contributes-toimmune-evasion-and-inflammation/ June 24

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Bottom line: After Paxlovid, a return of symptoms and test positivity may occur. Use antigen tests to determine when to exit isolation, even if you’re feeling better. In other words, test on Days 2, 3, and 4 after treatment to ensure no rebound. A good rule of thumb is that if you have a positive antigen test, you’re contagious. The FDA and Pfizer have also confirmed that those who relapse are eligible for re-treatment. This will be particularly important for the highest risk individuals, including those who are medically fragile, or those with severe rebounding symptoms. TWITTER: Bottom line: After Paxlovid, a return of symptoms and test positivity may occur. Use antigen tests to determine when to exit isolation (days 2,3,4). A good rule of thumb is that if you have a positive antigen test, you’re contagious. Posted: That means that if you have a breakthrough infection following vaccination and boosting, you still can contract long COVID” I spoke to Healthline about what we know about Long Covid. https://www.healthline.com/health-news/worried-aboutlong-covid-you-may-be-at-less-risk-if-youve-had-omicron-variant June 25 Even if we develop highly effective preventive drugs for Covid19, we need to be working with social scientists to ensure community uptake and adherence to medication regimes. https://www.forbes.com/sites/williamhaseltine/2022/06/17/m edical-science-must-go-hand-in-hand-with-social-science-foreffective-control-of-covid-19-and-otherpandemics/?sh=6de6b67e3f40 With Alzheimer’s disease, DNA mutations occur in brain cells at a much faster rate than normal. Thanks to a recent study from researchers at Brigham Women’s Hospital and Boston Children’s Hospital we may be one step closer to understanding why this happens https://www.forbes.com/sites/williamhaseltine/2022/06/23/st udy-uncovers-a-new-way-to-think-about-alzheimersdisease/?sh=319437ac3eba. 1223

June 26

If you aren’t sure how to find trustworthy information about everything from vaccines to what we’re learning about variants, transmission, policy decisions, and more, visit this website. Updated each week, and curated by William A. Haseltine. https://covid19resources.org/ Monitoring the CO2 around you may help you avoid high-risk Covid settings. https://www.forbes.com/sites/williamhaseltine/2022/06/23/c arbon-dioxide-levels-may-predict-covid-risk-in-your-immediatesurroundings/?sh=20c7e3f5108b June 27 Although there are no clinical tests to identify Long Covid at present, recent research points to several potential biomarker candidates. https://www.forbes.com/sites/williamhaseltine/2022/06/23/sa rs-cov-2-spike-a-potential-biomarker-for-longcovid/?sh=6b0fedb671a6 1224

June 28 The Omicron variants are among the most infectious viruses in the past one hundred years. To learn more about how these variants came to be, read my latest book: AMAZON: https://www.amazon.com/Omicron-PandemicEndemic-Future-Covid-19-ebook/dp/B09WNC13ZF/

New studies suggest that pregnant women who catch Covid-19 are more likely to suffer from an early miscarriage and their children more likely to receive a neurodevelopmental diagnosis. Maternal vaccination can help minimize complications and adverse outcomes. http://www.forbes.com/sites/williamhaseltine/2022/06/27/co vid-19-during-pregnancy-increased-risk-of-preterm-delivery-andinfant-neurodevelopmental-issues/ With the resurgence of polio cases and the emergence of new enteroviruses also causing paralytic conditions, we must use what we have learned from the success and challenges of poliovirus vaccines to mount a formidable defence against both novel and familiar viral threats.

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https://www.forbes.com/sites/williamhaseltine/2022/06/28/t here-may-be-a-new-polio-epidemic-on-its-wayif-so-what-wecan-do-part-iii/?sh=11598fc41fb1 June 29

Recent studies suggest 30% more Covid-19 cases than the official numbers report. Why? This article in The Guardian offers several possible answers. It remains true that without accurate case numbers, it will continue to be difficult to contain this pandemic. https://www.theguardian.com/world/2022/jun/01/us-covidsurge-cases-rate June 30 Where do variants come from? New variants may arise in longterm infections of immunocompromised patients. https://www.forbes.com/sites/williamhaseltine/2022/06/30/o rigin-of-virus-variation-real-time-evolution-of-sars-cov-2-in-animmunocompromised-patient/?sh=27eb376d349c New data from the National Center for Health Statistics reveals Long Covid is more common than previously believed. 1226

https://www.forbes.com/sites/williamhaseltine/2022/06/30/h ow-common-is-long-covid-more-common-than-youdthink/?sh=5e7100ffd276

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July 2022

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July 1

As we all grope for understanding, several points are now crystal clear. Here are some of my conclusions about the current state of the pandemic. July 3

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If you or someone you care about is suffering from post-covid symptoms, this article reminds how important it is to listen, and to believe their experience. https://www.jenniferdiamondbayarea.com/writing/covid-19long-haulers-and-the-burden-of-doubt July 6

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This study published in The European Heart Journal found that across the globe, there were fewer hospitalisations, diagnostic and interventional procedures, and outpatient consultations due to the pandemic, with the collateral damage being higher in lower income countries. https://academic.oup.com/eurheartj/advancearticle/doi/10.1093/eurheartj/ehac227/6594507 Donna Tetreault's new book The C.A.S.T.L.E Method is a must-read for all parents and grandparents. https://www.amazon.com/CASTLE-Method-DonnaTetreault/dp/1641706643/ref=sr_1_1?dchild=1&keywords=the% 20castle%20method%20donna%20tetreault&qid=1631058790&sr= 8-1

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The Many Faces of Omicron As Omicron continues to evolve, here we analyze virological evolution in terms of new monoclonal antibody treatments. https://www.forbes.com/sites/williamhaseltine/2022/07/06/t he-many-faces-of-omicron/?sh=28fe8f544fc6 July 8 NYC will be the first to dispense the antiviral drug Paxlovid instantly, free of charge, at mobile testing units. Yet we need to address confusion around the drug among healthcare providers and patients https://www.forbes.com/sites/williamhaseltine/2022/07/08/n yc-to-offer-instant-access-to-paxlovid-at-mobile-testing-sites-butmessaging-around-the-drug-needs-to-improve/?sh=1eb6fed571ab July 10

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July 11

As Long Covid continues to affect an increasingly large portion of the population, people are once again scrambling for answers about risk, symptoms, diagnoses, and treatment. I am hopeful that with the help of our new book–A Family Guide to Long Covid– you and your loved ones are able to get the straightforward answers you need. Available NOW on Amazon. https://www.amazon.com/dp/B0B64PZ5MH The BA.2.75 variant is the latest in a long line of Omicron variants. It is a dark horse candidate to be the worst one yet. https://www.forbes.com/sites/williamhaseltine/2022/07/11/b a275-a-dark-horse-in-the-covid-pandemic/?sh=6062a9de5985 July 12 A calling card of Omicron is its ability to evade natural immunity, vaccine protection, and monoclonal antibody treatments. To discover more about Omicron’s unique pathogenicity, read my latest book. AMAZON: https://www.amazon.com/OmicronPandemic-Endemic-Future-Covid-19-ebook/dp/B09WNC13ZF/

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July 13

Though limited in scope, a new study in Israel found that in the Omicron wave, fewer children had complications from Multisystem Inflammatory Syndrome than in the previous waves of 1234

the Covid-19 pandemic. Read the study in JAMA: https://jamanetwork.com/journals/jama/fullarticle/2792718 The existing rift in US health equality will disproportionately affect certain racial and ethnic communities suffering with Long Covid. https://www.forbes.com/sites/williamhaseltine/2022/07/12/longcovid-and-its-unequal-burden/?sh=6a6bbd342102 Here we examine a new monoclonal antibody candidate that neutralizes Omicron BA.1 and BA.2, as well as all variants that came before. https://www.forbes.com/sites/williamhaseltine/2022/07/13/p asteur-institute-scientists-discover-sars-cov-2-broadly-neutralizingantibody/?sh=1295579e48ab Forbes Post scheduled @12:15pm PST https://www.forbes.com/sites/williamhaseltine/2022/07/13/m ore-danger-ahead-with-ba5-covid-19-reinfection-doubles-therisk-for-death-blood-clots-and-lung-damage/?sh=5bb10ead3cec July 14 Thanks to the work of scientists at Johns Hopkins University, a partially paralyzed man was able to feed himself for the first time in thirty years using just his mind and a pair of robotic arms. https://www.forbes.com/sites/williamhaseltine/2022/07/14/r obotic-arms-allow-partially-paralyzed-man-to-feedhimself/?sh=21590d4c2194 A recent study reinforces the hard truth about Covid-19 vaccines: they play a critical role in preventing hospitalizations and deaths, but provide little protection against Long Covid. https://www.forbes.com/sites/williamhaseltine/2022/07/14/s hould-you-get-vaccinated-for-covid-19-yes-will-it-protect-youfrom-long-covid-probably-not/?sh=3506e84b7d8e Most SARS-CoV-2 infections are cleared within a week or two, but it is not always the case. Immunocompromised individuals are 1235

susceptible to month- or even year-long infections, known as chronic infections. A growing body of research suggests they may underlie the emergence of new variants. http://www.forbes.com/sites/williamhaseltine/2022/07/14/arechronic-infections-to-blame-for-sars-cov-2-variants/ July 15

The World Health Organization defined Long Covid in October of 2021: “Post Covid-19 condition occurs in individuals with a history of probable or confirmed SARS CoV-2 infection, usually 3 months from the onset of Covid-19 with symptoms that last for at least 2 months and cannot be explained by an alternative diagnosis. Common symptoms include fatigue, shortness of breath, cognitive dysfunction but also others and generally have an impact on everyday functioning. Symptoms may be new-onset following initial recovery from an acute Covid-19 episode or persist from the initial illness. Symptoms may also fluctuate or relapse over time.” The WHO did not restrict their definition to confirmed infections only. Unequal access to Covid-19 testing, particularly at the beginning of the pandemic, entails that many people with Long Covid do not have serological proof of SARS-CoV-2 infection. If 1236

you’d like to learn more about Long Covid, I’ve just released a new book, The Family Guide to Long Covid: Questions & Answers. Available on Amazon. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/ July 17

How is Long Covid different from Covid-19? Covid-19 is caused by the active replication of SARS-CoV-2 virus in the body, while Long Covid develops once replication during the acute phase of infection has ceased. Covid-19 is easy to diagnose with rapid antigen or PCR tests. There is no one test to diagnose Long Covid and the symptoms are wide-ranging. The symptoms of a Covid-19 infection are temporary, normally lasting anywhere from a few days to one to two weeks. In contrast, Long Covid symptoms can last for months or years. If you’d like to learn more about Long Covid, I’ve just released a new book, The Family Guide to Long Covid: Questions & Answers. Available on Amazon.

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https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/ July 19 One possible explanation for the origin of Omicron is crossinfection between humans and animals, like housepets and rats. To find out how cross-infection works, read my latest book. AMAZON: https://www.amazon.com/Omicron-PandemicEndemic-Future-Covid-19-ebook/dp/B09WNC13ZF/

July 20

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A recent pre-print study used physics-based modeling to study the effect of mask-wearing on infection. The upshot: Masks work. Read it here: https://www.medrxiv.org/content/10.1101/2022.07.05.22277221 v1.full.pdf July 21 POSTED @ 8:45am PST Link: https://www.forbes.com/sites/williamhaseltine/2022/07/20/newsars-cov-2-variant-ba275-evades-all-approved-monoclonalantibody-therapies/?sh=7740823049ea Social Media Blurb: The latest in the Omicron family of variants, BA.2.75, is more resistant to monoclonal antibodies than its predecessors, posing problems for treating Covid-19. SCHEDULED: A new study shows that mild Covid inflicts paradoxical brain damage: detected by imaging but not by neuropsychological assessment. https://www.forbes.com/sites/williamhaseltine/2022/07/20/m ild-covid-inflicts-paradoxical-brain-damage-detected-by-imagingbut-not-by-neuropsychological-assessment/?sh=4499ecf7309a 1239

POSTED @ 12:35pm PST http://www.forbes.com/sites/williamhaseltine/2022/07/21/ca n-inflammation-lead-to-cognitive-issues-past-research-says-yeswith-implications-for-covid-19/ Social Media Blurb: Reports of cognitive issues following a bout of Covid-19 are now quite common. In some cases, these symptoms may persist for months or years as part of Long Covid. Past research on cancer and aging may provide a valuable starting point on the journey for answers. July 22

Research suggests that the symptoms experienced by Long Covid patients could be the result of up to four different syndromes: permanent damage to vital organs; post-intensive-care syndrome; post-viral fatigue syndrome; and continued Covid-19 syndrome. Long Covid may be the result of low levels of complete or partial viral persistence. It might also arise due to an autoimmune response caused by the initial Covid-19 infection. Long Covid may also result from chronic inflammation triggered by the initial infection.

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TWITTER VERSION: Long Covid could be the result permanent damage to vital organs; post-intensive-care syndrome; post-viral fatigue syndrome; and continued Covid-19 syndrome; viral persistence; or an autoimmune response or chronic inflammation triggered by the initial infection. July 24

Each week, I select three articles about Covid-19 that represent the latest research, important policy, or trends in the global pandemic and our efforts to win this battle. https://covid19resources.org/ July 25 The road to diagnosis for many Long Covid patients is often challenging. Thankfully, there are steps that all patients can take to advocate for a correct diagnosis. https://www.nydailynews.com/opinion/ny-oped-advocatelong-covid-20220721-cazjz2zk6zcv3iohh6w4rravlu-story.html With cases surging and hospitalizations rising in the US, we must not repeat past messaging mistakes and give the public the tools they need to protect themselves. https://thehill.com/opinion/healthcare/3571207-learn-frompast-pandemic-mistakes-and-authorize-second-boosters-for-all/ 1241

July 26

Anywhere from 5–50% of people infected with Covid—of all ages, including mild infections—will develop Long Covid. That’s why I am so glad to be able to offer our new resource: A Family Guide to Long Covid, Questions and Answers. I am hopeful it will address many of the questions you may have if you or someone you love is suffering from Long Covid. AMAZON: https://www.amazon.com/Family-Guide-LongCovid-Questions-ebook/dp/B0B64PZ5MH/ July 27

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After two years of isolation, we’re starting to see odd behavior in viruses, such as high instances of flu and RSV outside of its normal winter season, and kids showing lower levels of antibodies to many common viruses thanks to their “infection honeymoon.” Read more here about the effects of the Covid-19 pandemic on other viruses: https://www.statnews.com/2022/05/25/viruses-thatwere-on-hiatus-during-covid-are-back-and-behaving-inunexpected-ways/ Twitter version: After 2 yrs of isolation, we’re starting to see odd behavior in viruses: high instances of flu + RSV outside normal winter season; kids showing lower levels of antibodies to many common viruses thanks to their “infection honeymoon.” Read more: https://www.statnews.com/2022/05/25/viruses-that-were-onhiatus-during-covid-are-back-and-behaving-in-unexpected-ways/ I spoke to @washingtonpost reporter @majohnso (Mark Johnson) about an HIV patient who is in long-term remission after receiving a transplant of blood stem cells containing a rare mutation and what this means for the future of HIV treatment. https://www.washingtonpost.com/science/2022/07/27/hivremission-stem-cell-transplant-city-of-hope/ July 28 SCHEDULED: A new antibody, SP1-77, strongly neutralizes all SARS-CoV-2 variants, including Omicron BA.4/5. https://www.forbes.com/sites/williamhaseltine/2022/07/26/p rogress-in-the-search-for-broadly-neutralizing-monoclonalantibodies-iii/?sh=b9b4adb4e7f7 SCHEDULED: A recent paper published by the Eindhoven University of Technology details an improved method for growing miniature functional kidneys. https://www.forbes.com/sites/williamhaseltine/2022/07/26/n ew-wave-materials-help-create-mini-functionalkidneys/?sh=73ded1eb149b July 29

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When does Long Covid begin? The majority of people recover from the acute phase of SARSCoV-2 infection within two to three weeks. If symptoms persist for more than four weeks after initial infection, a person can be classified as having Long Covid. These symptoms may continue to present for months or even years. Long Covid can also manifest in those who did not develop symptoms during Covid-19 infection. It is estimated that up to one in five Covid-19 patients with no symptoms end up presenting at least one Long Covid symptom.12 In such cases, symptoms usually appear within three months of recovery from acute infection.We do not know if there is an upper limit for the onset of Long Covid. If you or a loved one is experiencing a sudden onset of symptoms that mirror those commonly associated with Long Covid, even if it has been weeks or months since initial infection, don’t rule out the possibility from the get go. July 31

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Learn more about Long Covid in my new book: A Family Guide to Long Covid, Questions and Answers. Available now on AMAZON: https://www.amazon.com/Family-Guide-Long-Covid-Questionsebook/dp/B0B64PZ5MH/

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August 2022

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Aug 1 The ZCB11 antibody demonstrates strong neutralization of all major variants, adding itself to the growing list of broadly neutralizing monoclonal treatments. https://www.forbes.com/sites/williamhaseltine/2022/07/28/p rogress-in-the-search-for-broadly-neutralizing-monoclonalantibodies-iv/?sh=5c8d83966a27 Scientists at Northwestern University have engineered a new pain-relieving device that could serve as a replacement for opioids or other highly addictive medications. https://www.forbes.com/sites/williamhaseltine/2022/07/29/acooling-implantable-device-for-pain-relief/?sh=6eafdf831c04 Aug 2: 9am Scheduled: A review of current COVID-19 research explores the complex relationship between biological sex and its influence on SARSCoV-2 immune responses. https://www.forbes.com/sites/williamhaseltine/2022/07/29/c ovid-19-gender-and-immune-response-whats-therelationship/?sh=5f9dc8d66a27 Scheduled (1-3pm) Two more antibodies, COV44-62 and COV44-79, broadly neutralize a wide range of SARS-CoV-2 variants. https://www.forbes.com/sites/williamhaseltine/2022/0 8/01/progress-in-the-search-for-broadly-neutralizingmonoclonal-antibodies-v/?sh=931cfd03624b Aug 3:

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A recent study published in Nature Medicine found 62 symptoms associated with SARS-CoV-2 infection beyond 12 weeks. Read more about what they found, including risk factors for developing Long Covid. https://www.nature.com/articles/s41591-022-01909-w Aug 4: Scheduled 7am and other times, per platform: Even mild Covid-19 may worsen cognitive health. How? By triggering a strong inflammatory response that makes its way to the central nervous system, leading to overabundant activation of immune cells in the brain. http://www.forbes.com/sites/williamhaseltine/2022/08/01/ev en-mild-covid-19-may-cause-lasting-brain-fog-part-1/ Scheduled 3-5 pm: For many people, an encounter with Covid-19 brings lingering cognitive symptoms. A new study describes why and how this might be happening. https://www.forbes.com/sites/williamhaseltine/2022/08/02/e ven-mild-covid-19-may-cause-lasting-brain-fog-part2/?sh=300e7a686188 Aug 5:

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Sex hormones and sex differences in response to vaccines may illuminate why Covid-19 outcomes vary between men and women. https://www.forbes.com/sites/williamhaseltine/2022/08/04/c ovid-19-gender-and-immune-response-whats-the-relationshippart-two/?sh=7ba4645e3226 Aug 8: Covid-19 can trigger body-wide inflammation that eventually makes its way to the brain, impairing cognitive function. Research suggests that other viral diseases may share the same mechanism of damage. http://www.forbes.com/sites/williamhaseltine/2022/08/05/ev en-mild-covid-19-may-cause-lasting-brain-fog-part-3/ Aug 9:

The more severe your symptoms during Covid-19 infection, the more likely you will develop Long Covid. However, it can also manifest even if you did not develop symptoms while you had Covid-19: up to one in five asymptomatic people end up having at least one Long Covid symptom. You can get more questions answered in my newest book, The Family Guide to Long Covid. Available on Amazon. 1249

https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/ Loss of smell may predict persistent cognitive impairment after Covid-19 recovery, suggest results presented at the Alzheimer's Association International Conference. https://www.forbes.com/sites/williamhaseltine/2022/08/09/lo ss-of-smell-linked-to-long-term-covid-cognitiveimpairment/?sh=5292bf47540d Aug 11: Using CRISPR technology, scientists at U.C. Berkeley have discovered a naturally occurring protein in the lungs that may have the ability to inhibit SARS-CoV-2 infections. https://www.forbes.com/sites/williamhaseltine/2022/08/11/a dvanced-technology-crispr-shows-that-mucus-is-your-bodys-firstline-of-defense-against-viruses/?sh=27d1168d4797 Aug 12:

Research on Long Covid must be inclusive in order for us to develop the right approach to this disease. Effective drug development, policy decisions, and treatment guidelines are all negatively impacted from bias in data collection. We must ensure inclusivity along with reducing barriers to participating in research studies. Aug 14: 1250

The Patient-Led Research Collaborative collected data from people suffering from Long Covid. One question asked was whether or not they had shared their experience on social media. Responses were fairly split between yes and no. Learn more about the Patient Resource Collaborative here https://patientresearchcovid19.com/research/report-1/ Aug 15: Influenza A viruses continue to cause seasonal outbreaks year in and year out. Developing a broadly neutralizing vaccine effective against all subtypes has proven challenging. Here, I explain why this is. http://www.forbes.com/sites/williamhaseltine/2022/08/12/ge tting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part1/ Aug 16:

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What is Long Covid? What are the symptoms, and treatments? Who can get it? What we know is that Long Covid is a disabling post-viral illness, and researchers are still working toward a consensus of understanding. In the meantime, we hope that our new book, The Family Guide to Long Covid: Questions and Answers, will help spread awareness. It works through many of the most common questions about Long Covid and is being constantly updated with new information. AMAZON: https://www.amazon.com/Family-Guide-LongCovid-Questions-ebook/dp/B0B64PZ5MH/ Aug 17:

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Easily accessible, accurate health data is necessary to understand clinical information in real-time, prevent medical errors, and make public-health decisions. But as COVID-19 and monkeypox have shown, the US is falling short. https://www.project-syndicate.org/commentary/publichealth-data-lack-of-standardization-is-a-big-problem-by-william-ahaseltine-2022-08 Aug 18: Bostwana’s remarkable achievement in reducing HIV transmission should be replicated around the world. https://www.forbes.com/sites/williamhaseltine/2022/08/17/a n-end-to-hiv-in-botswana-why-cant-we-accomplish-the-same-inthe-us/?sh=78035ce08024 A new study from Cardiff University shows that a SARS-CoV2 spike protein mutation appears invisible to CD8 T cells, thus dodging immune responses from previous infection and vaccines. https://www.forbes.com/sites/williamhaseltine/2022/08/17/w ill-covid-19-vaccines-continue-to-protect-us-from-hospitalizationand-death/?sh=4c6382f14f77 Aug 19:

Learn more: https://www.williamhaseltine.com/ Every virus has its preferred niche — a particular host or demographic it is more inclined to infect and a particular set of strategies and tactics it uses to achieve this. A virus develops a niche 1253

over millions of years, all the while adapting and mutating in response to host immune defenses. The longer a […] https://www.insideprecisionmedicine.com/topics/patie nt-care/coronavirus/controlling-covid-19-what-can-welearn-from-previous-pandemics/ Aug 20: Although decent, our current influenza vaccines are not as effective as they ought to be. Part of the issue may have to do with the production process. Are recent changes enough to improve effectiveness? http://www.forbes.com/sites/williamhaseltine/2022/08/18/ge tting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part2/ Aug 21:

Visit our Covid-19 Resources website for articles about Covid19 that represent the latest research, important policy, or trends in the global pandemic and our efforts to win this battle. https://covid19resources.org/ Aug 22:

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I recently spoke to MIC about the current polio epidemic in the UK and US, why it is spreading, how it affects us, and what we can do to prevent it. https://www.mic.com/life/why-is-polio-spreading A group of scientists at Harvard University has developed a new method to grow artificial hearts from scratch. This study serves as a significant stepping stone toward developing artificial hearts that are fully functional. https://www.forbes.com/sites/williamhaseltine/2022/08/20/h ow-to-mend-a-broken-heart/?sh=7bf7d7e326cd Aug 24: I spoke to @Salon about Dr. Fauci's legacy. From early on in his career, Tony a very active and involved as a leader, both as a researcher and as a global leader for control of pandemic diseases. https://www.salon.com/2022/08/23/fauci-retirement-legacy/ Aug 26: Just as each face must be perfectly aligned to solve a Rubik's Cube, each protein in SARS-CoV-2 must be ideally mutated in order to create a highly transmissible variant. https://www.forbes.com/sites/williamhaseltine/2022/08/25/s olving-the-question-of-covid-variant-increased-fitness-is-likedeciphering-a-rubiks-cube/?sh=187f1f496f56 Aug 29: A study investigates how, despite lacking one of two important components of adaptive immunity, immunocompromised patients are able to suppress SARS-CoV-2 infection through T cell immune responses. https://www.forbes.com/sites/williamhaseltine/2022/08/26/t he-heavy-lifting-of-t-cells-in-immunocompromised-covid-19patients/?sh=64513a5978d1 mRNA vaccine technology rose to prominence during the Covid-19 pandemic. But the same system can be used to combat other viruses, with the promise of quicker turnaround and cheaper 1255

manufacturing. Will the next influenza vaccine be an mRNA vaccine? http://www.forbes.com/sites/williamhaseltine/2022/08/29/ge tting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part3/ BA.5, the leading variant in the United States, contains intrinsic virological properties that give it an edge over other variants. https://www.forbes.com/sites/williamhaseltine/2022/08/29/in creased-disease-potential-of-covid-variant-ba5-currentlycirculating-in-the-united-states/?sh=342628194b9d

In an Op-Ed for @ProSyn, I wrote about how Unclear, opaque public-health messaging has been a major problem throughout the COVID-19 pandemic. To regain the public's trust, health officials must start learning from their mistakes and stop confusing credibility with infallibility. Link: https://www.project-syndicate.org/commentary/publichealth-messaging-disinformation-age-by-william-a-haseltine-202208 Aug 30:

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That’s why I am so glad to be able to offer our new resource: A Family Guide to Long Covid, Questions and Answers. I am hopeful it will address many of the questions you may have if you or someone you love is suffering from Long Covid. AMAZON: https://www.amazon.com/Family-Guide-LongCovid-Questions-ebook/dp/B0B64PZ5MH/ Aug 31: ANIMATED:

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Learn more about a recent breakthrough on the road to developing a much sought-after broadly neutralizing vaccine against all influenza A viruses: https://www.williamhaseltine.com/getting-a-grip-oninfluenza-the-pursuit-of-a-universal-vaccine-part-1/

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September 2022

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Sept 1: A study from the Imperial College of London suggests that people with Covid-19 should isolate themselves for a minimum of five days and safely exit depending on rapid antigen test results. https://www.forbes.com/sites/williamhaseltine/2022/09/01/fi ve-days-is-too-short-study-says-to-end-covid-19-isolation-basedon-rapid-tests-results-instead/?sh=65a2b8c6ca1c These latest broadly neutralizing monoclonal antibodies not only overcome major Omicron variants, but many other related coronaviruses as well. https://www.forbes.com/sites/williamhaseltine/2022/09/01/p rogress-in-the-search-for-broadly-neutralizing-monoclonalantibodies-vi/?sh=46ec3c3732b1 Sept 2:

Learn more: https://www.williamhaseltine.com/even-mild-covid-19-maycause-lasting-brain-fog-part-1/ Sept 3: 1260

A new report finds Long Covid is keeping as many as 4 million people out of work. We need better treatment and prevention, wider access to disability insurance, expanded sick leave, and employer accommodations to reduce the economic burden of Long Covid. https://www.forbes.com/sites/williamhaseltine/2022/09/02/lo ng-covid-is-keeping-millions-of-people-out-ofwork/?sh=8ee092e52cc8 Sept 6: Tuesday: The P2G3 antibody is the latest in a long line of potential SARSCoV-2 monoclonal antibody treatments to neutralize a broad range of viral variants. https://www.forbes.com/sites/williamhaseltine/2022/09/06/p rogress-in-the-search-for-broadly-neutralizing-monoclonalantibodies-vii/?sh=79522733bd4f Sept 7:

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If you aren’t sure how to find trustworthy information about everything from vaccines to what we’re learning about variants, transmission, policy decisions, and more, visit this website. Curated by William A. Haseltine. https://covid19resources.org/ Sept 8: Mucosal vaccines have received a lot of attention in recent years. How are they different from your usual flu vaccine, and are they more effective? https://www.forbes.com/sites/williamhaseltine/2022/09/07/g etting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part4/?sh=3f7065011676 Sept 9:

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#covid #covid_19 #longcovid #longcovidrecovery #longcovidsymptoms #longcovidawareness #longcovidkids #longcovidresearch #longcovidsupport Sept 11:

Whether a Long Covid patient receives long-term disability coverage depends on their ability to provide medical evidence of their condition—a requirement some simply can’t meet. Even those who can should expect a waiting period that might stretch on for months, as the Social Security Administration works through the backlog of claims that built up when the agency had to cease its inperson operations.

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The difficulty of qualifying for disability benefits without evident documentation, such as a positive Covid-19 test result or laboratory scans, is borne by many sufferers of post-infectious illnesses. Lack of research, training, and specialization in rare chronic diseases entails that some patients might not receive an accurate diagnosis for years. Even if their symptoms are severe enough to seriously limit or obstruct their ability to work, obtaining disability coverage remains an uphill battle. Some Long Covid patients were approved based on extensive documentation of health visits or inability to work. Legislation qualifying Long Covid as a disability has also advanced outside the United States. In June 2022, a Scottish court ruled in favor of a Long Covid patient seeking compensation from their former employer on the grounds of disability discrimination. Sept 12: The MO1 antibody strongly and broadly neutralizes SARSCoV-2 variants, including the latest versions of Omicron - BA.5 and BA.2.75. https://www.forbes.com/sites/williamhaseltine/2022/09/12/p rogress-in-the-search-for-broadly-neutralizing-monoclonalantibodies-viii/?sh=65c7ceec3cb7 Sept 13:

What is Long Covid? How is it different from a Covid infection? What are the symptoms? Who is affected? How do I talk to my doctor about Long Covid? How can I advocate to get the care I need? In our book, we answer these questions and more in an accessible question and answer style. Available now: 1264

https://www.amazon.com/Family-Guide-Long-Covid-Questionsebook/dp/B0B64PZ5MH/ Sept 14: https://www.canva.com/design/DAFKWWAHH1I/_jkSGFG DYhcY7EHVXjTNw/edit?utm_content=DAFKWWAHH1I&utm_campaign=desig nshare&utm_medium=link2&utm_source=sharebutton

Learn more about the road to the universal flu vaccine: https://www.williamhaseltine.com/getting-a-grip-oninfluenza-the-pursuit-of-a-universal-vaccine-part-2/ Americans spend significantly more on medical costs than their peers, yet still have lower life expectancy, higher rates of chronic disease and maternal mortality, and fewer doctors per capita. America’s underinvestment in public health is a major reason. https://www.project-syndicate.org/commentary/us-publichealth-funding-fixing-chronic-underinvestment-by-william-ahaseltine-2022-09?barrier=accesspaylog Mucosal vaccines have received a lot of attention in recent years. How are they different from your usual flu vaccine, and are they more effective? 1265

http://www.forbes.com/sites/williamhaseltine/2022/09/07/ge tting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part4/ Sept 15: Many are pinning their hopes on nasal vaccines becoming the tool that finally halts transmission of the Covid-19 virus. I do not share the same unbridled enthusiasm that nasal vaccines will provide long-lasting protection from infection and protection from all variants https://www.forbes.com/sites/williamhaseltine/2022/09/14/n asal-vaccines-may-not-be-the-game-changer-we-think-theyare/?sh=218b84752900 Molecular Jujitsu: How SARS-CoV-2 co-opts host defense https://www.forbes.com/sites/williamhaseltine/2022/09/14/m olecular-jujitsu-sars-cov-2-co-opts-hostdefense/?sh=793a628c187d Sept 16:

Get all your Long Covid questions answered with The Family Guide to Long Covid. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/ 1266

* #covid #covid_19 #longcovid #longcovidrecovery #longcovidsymptoms #longcovidawareness #longcovidkids #longcovidresearch #longcovidsupport Sept 18:

Our newest book shares best practices for how digital finance tools can be used by insurers, healthcare providers and startups to help low-and middle-income communities afford and access healthcare globally. Available now: https://www.amazon.com/dp/B0BCX1GWW7 Sept 19: We draw attention to a variant, BA.4.6, which has the potential to trigger the next wave. https://www.forbes.com/sites/williamhaseltine/2022/09/16/w ill-the-ba46-variant-drive-the-next-wave-of-the-covid-19pandemic/?sh=10fe75507573 Recent discoveries in Alzheimer's research have thrown into question fundamental assumptions about the disease and its origins. 1267

In the first installment of this series, we offer a brief overview of Alzheimer's disease. https://www.forbes.com/sites/williamhaseltine/2022/09/16/re imagining-alzheimers-part-1/?sh=3e910f8d6291 Flu vaccines need to be readministered every year to keep pace with viral mutations. "Universal vaccines" could change this, providing us with a much broader immunity that remains effective even in the face of constant variation. How do they work? http://www.forbes.com/sites/williamhaseltine/2022/09/19/ge tting-a-grip-on-influenza-the-pursuit-of-a-universal-vaccine-part5/ Sept 21:

This website presents the latest research, important policy, or trends in the global pandemic and our efforts to win the Covid-19 battle. https://covid19resources.org/ Sept 22: Research published in the journal Proteomics demonstrates how SARS-CoV-2 protein Mpro suppresses host immune responses by cleaving host proteins. https://www.forbes.com/sites/williamhaseltine/2022/09/21/sa rs-cov-2-main-protease-suppresses-innate-immunity-by-cleaving1268

proteins-required-for-interferon-induction-andinflammation/?sh=347ed81b2136 Sept 23:

(Video) Text for Facebook and LinkedIn: Who will benefit from getting a Covid booster? Simply put: everyone. The key is when to get the booster. If you very recently contracted Covid-19 it’s wise to wait 3 to 4 months before getting your booster. While CDC guidance says you can get a bivalent booster immediately after recovering from an infection, a booster doesn’t add much benefit within 2 months of infection. If you recently had a booster, wait 5-6 months before getting another one. For children under the age of 12, it’s important to get them up to date on their booster shots as soon as possible so they are fully protected. Text for Twitter: Who will benefit from getting a Covid booster? Simply put: everyone. The key is when to get the booster. Sept 24: A new member of the Omicron family, BA.2.75.2, shows potential as the next major variant of the Covid-19 pandemic.

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https://www.forbes.com/sites/williamhaseltine/2022/09/22/w aiting-in-the-wings-a-potential-new-variant-of-sars-cov2/?sh=54f149093960 Sept 25: Animated slides (follow link to watch animation….I’ve included the individual slides below as static images to help you identify the post. Video is in Bill’s social media folder in our drive and also in Canva https://www.canva.com/design/DAFLwZBsV9Q/w_aHLytg8 4FSomKZxexdsw/watch?utm_content=DAFLwZBsV9Q&utm_ca mpaign=designshare&utm_medium=link&utm_source=publishsha relink

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Text to go with it: Learn more about how new health financing solutions can help low-and middle-income communities afford and access healthcare globally. https://www.williamhaseltine.com/fintechforhealth/ Sept 26: Flu vaccines need to be readministered every year to keep pace with viral mutations. "Universal vaccines" could change this. Here, 1271

we describe a recent success in the development of a universal influenza A vaccine. http://www.forbes.com/sites/williamhaseltine/2022/09/26/isa-universal-influenza-vaccine--one-shot-for-all-strains--on-thehorizon/ As we continue to face ongoing infectious disease threats, we need to build resilient health systems that are equipped to face both public health emergencies and ongoing population health challenges. https://thehill.com/opinion/healthcare/3659664-we-needresilient-health-systems-to-address-the-dual-crisis-of-infectiousand-chronic-diseases/ Sept 27:

Text for LinkedIn and Facebook: As we enter flu season, a lot of vaccines are now readily available. You may be asking yourself: should I get another Covid booster? Do I also need a flu shot? Should I be worried about Monkeypox? Can I get them at the same time? It’s easy to feel overwhelmed. My recommendation: Everyone will benefit from a Covid booster, regardless of whether you've been infected by Omicron or not. You can get the flu vaccine (and other routine vaccines) and Covid booster at the same visit. If you are needing both, make sure to get them before the end of October – especially the flu shot. If 1272

you are 50 or older, you should also have the Shingrix (shingles vaccine) whether you’ve already had shingles or not. The CDC recommends spacing the COVID-19 booster with Jynneos (Monkeypox Vaccine), especially in young men, 4 weeks apart. Text for Twitter: You can get the flu vaccine and Covid booster at the same visit. If you are needing both, make sure to get them before the end of October. If you are 50 or older, you should have the Shingrix (shingles vaccine) whether you’ve already had shingles or not. The CDC recommends spacing the COVID-19 booster with Jynneos (Monkeypox Vaccine), especially in young men, 4 weeks apart. Sept 28:

The CDC says you can end isolation at Day 5 of a Covid-19 infection if you are fever free for 24 hours, or if your symptoms have improved. But recent data suggests this may be unwise. It is safer to remain in isolation until you have had two consecutive days of a negative result on a rapid antigen test. Read more: https://www.williamhaseltine.com/five-days-istoo-short-study-says-to-end-covid-19-isolation-based-on-rapidtests-results-instead/

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Sept 29: I spoke to Healthcare Executive about how Biden's Pledge to End HIV by 2030 takes a "whole person approach" and how I have advocated for more self-testing HIV options. https://www.managedhealthcareexecutive.com/view/howpresident-biden-can-meet-his-pledge-to-end-hiv-by-2030 Sept 30:

Long Covid has impacted millions of people in the United States and around the world. Whether your Long Covid consists of a lingering cough or extensive immunodeficiency, understanding the widespread nature of Long Covid will inform a conversation with others. If someone is ignorant of the issue, inform them of your struggles and the struggles of many. If they continue to not understand, know that you did your best and your experience is valid. One way to better understand Long Covid, whether as a person with Long Covid or someone hoping to learn more, is to connect with Long Covid advocacy groups, which are becoming much more common as we learn more. Akin to support groups for other major disabilities, these groups connect those that have Long Covid, provide reading materials for those wishing to learn more, and advocate publicly for the policy rights of persons with Long Covid. 1274

You can also learn more from my book The Family Guide to Long Covid. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/

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October 2022

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Oct 1: A new CDC study shows that isolating in separate rooms significantly reduced the odds of SARS-CoV-2 transmission between household members. https://www.forbes.com/sites/williamhaseltine/2022/09/29/cdcstudy-says-isolating-in-a-separate-room-is-most-effective-againsthousehold-transmission-of-sars-cov-2/?sh=5cbf08c63d51 # As the SARS-CoV-2 virus continues to evolve, each emerging variant is gaining advantages over its predecessor. https://www.forbes.com/sites/williamhaseltine/2022/09/30/c ovid-virus-accelerates-with-each-new-variant/?sh=1f88728e6f29 Oct 2:

ACCESS Health International has spent over three years researching new health financing solutions, working with startups, hosting roundtables and cross sector special interest groups across Asia in countries such as Bangladesh, China, India, Vietnam,

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Malaysia, and Nepal. Our book shares what we’ve learned. Available now: https://www.amazon.com/dp/B0BCX1GWW7 Oct 3: Like influenza, SARS-CoV-2 is an expert at mutating and evading prior immunity. This includes vaccine-induced immunity. New research suggests that expanding the target area of our vaccines to include the virus' nucleocapsid protein may offer stronger and broader protection. http://www.forbes.com/sites/williamhaseltine/2022/10/03/tw o-are-better-than-one-expanding-our-covid-19-vaccine-antigens/ # A new study has found that flushing your nose twice daily with a mild saline solution shortly after testing positive for Covid-19 can drastically decrease your chances of hospitalization and death in higher-risk patients https://www.forbes.com/sites/williamhaseltine/2022/10/03/sa line-nasal-irrigation-after-covid-19-diagnosis-reduceshospitalization/?sh=677dc3f9323c # Recent discoveries in Alzheimer's disease research have thrown into question fundamental assumptions about the disease and its origins. Here, I discuss the primary risk factor of Alzheimer's and how it may cause cognitive decline http://www.forbes.com/sites/williamhaseltine/2022/10/03/rei magining-alzheimers-part-2-breaking-the-barrier/ # This series explores a recent medical technology which promises to revolutionize treatment of cancer and other conditions: CAR T therapy. https://www.forbes.com/sites/williamhaseltine/2022/10/03/fr om-lymphoma-to-lupus-and-beyond-the-remarkable-research-ofcar-t-therapy/?sh=22e669173c44 Oct 4:

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Is it safe to get the flu vaccine and the Covid booster at the same time? Absolutely! The flu vaccine can be administered at the same time as a Covid bivalent booster and should be received before the end of October. If you are 65 and older, make sure you receive a higher-dose flu vaccine. Oct 6: Recently discovered monoclonal antibodies present an opportunity to create variant-independent treatments and preventions for Covid-19. https://www.forbes.com/sites/williamhaseltine/2022/10/05/b roadly-neutralizing-monoclonal-antibodies-for-covid-19treatment-prevention-and-vaccine-design/?sh=96e27284603f # 1279

Messenger RNA (mRNA) vaccines have been crucial to containing and controlling the ongoing Covid-19 pandemic. Advances in self-amplifying RNA technology promise to make them even more cost effective and even quicker to produce. http://www.forbes.com/sites/williamhaseltine/2022/10/06/su percharging-mrna-vaccines-with-self-amplifying-rna-technology/ Oct 7:

I will be moderating two expert panels at the @metabesity virtual conference Oct 11 on building resilient health systems, and how lessons from Covid-19 can prepare us to address the ongoing epidemic of chronic diseases. Join me and @HelenClarkNZ @sandrogalea, @G_Q_Daley, Dr. Margaret Hamburg & Jeffrey Lehman. Register: https://www.metabesity2022.org/ #

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The CDC recommends spacing the COVID-19 booster with Jynneos (Monkeypox vaccine), especially in young men, 4 weeks apart. If there is a need for Jynneos in terms of an outbreak, don’t wait! But consider delaying the booster after Jynneos. Oct 9: 1281

ANIMATES SLIDES https://www.canva.com/design/DAFLwb3vvDY/5qZCSDU5 JMsc8s6jgkfdA/watch?utm_content=DAFLwb3vvDY&utm_campaign =designshare&utm_medium=link&utm_source=publishsharelink

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This week I will be moderating two expert panels at the @metabesity virtual conference Oct 11 on building resilient health systems, and how lessons from Covid-19 can prepare us to address the ongoing epidemic of chronic diseases. Join me and @HelenClarkNZ @sandrogalea, @G_Q_Daley, Dr. Margaret Hamburg & Jeffrey Lehman. You can register for this virtual conference here: https://www.metabesity2022.org/ Oct 11:

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If you are 50 or older, you should have the Shingrix (Shingles Vaccine) whether you’ve already had shingles or not. You should also consider getting the Shingrix vaccine if you've had the Zostavax vaccine in the past, or if you don't know whether you've had chickenpox. Shingrix is an inactive vaccine so you can receive it alongside other inactive or live vaccines such as a Covid booster or flu vaccine. Oct 14: 1285

A Family Guide to Long Covid: Questions and Answers covers everything you need to know about Long Covid from symptoms and diagnosis to treatment and how different populations are affected. It serves as a resource that patients can use to advocate for their care and a guide for those who want to understand, prepare, and protect their families and friends. Available now: https://www.amazon.com/Family-Guide-Long-Covid-Questionsebook/dp/B0B64PZ5MH/ Oct 15: India's first home-grown Covid-19 mRNA vaccine uses selfamplifying RNA technology, can be stored in a conventional refrigerator instead of special freezers, and doesn't require a needle. It marks a shift towards next-generation mRNA vaccines. http://www.forbes.com/sites/williamhaseltine/2022/10/14/sel f-amplifying-mrna-vaccine-receives-eua-nod-from-indianregulators/ # This second installment of the CAR T series details the recent and effective use of CAR T therapy to treat B cell cancers.

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https://www.forbes.com/sites/williamhaseltine/2022/10/14/t he-remarkable-research-of-car-t-therapy-part-ii-b-cellcancers/?sh=5b5d6d7b2ef4 Oct 16:

Our newest book identifies how digital financial technologies can enable new solutions for healthcare costs including alternative credit scoring, access to digital wallets and mobile money accounts, microinsurance, and crowdfunding. Available now: https://www.amazon.com/dp/B0BCX1GWW7 Oct. 17: Adolescents and young children experiencing trouble breathing and exercise intolerance post covid may benefit from a trip to the doctor. https://www.forbes.com/sites/williamhaseltine/2022/10/15/childr en-experiencing-respiratory-trouble-post-covid-19-improve-withthe-use-of-an-inhaler-and-exercise-regimen/?sh=105587b28152 # 1287

Thanks to new techniques in regenerative medicine we are now closer to a future where your own cells can be used to restore the sense of vision. https://www.forbes.com/sites/williamhaseltine/2022/10/15/p rogress-in-the-search-to-reverse-age-related-visionloss/?sh=6e59f2df613a # In part 3 of my series on Alzheimer's, I explore the different variants of APOE-- the primary gene associated with Alzheimer's disease. https://www.forbes.com/sites/williamhaseltine/2022/10/17/re imagining-alzheimers-part-3-the-apoe-story-in-alzheimers-andother-diseases/ Oct. 19: Each week, I republish my articles from Forbes on my website to make them available to anyone who doesn’t have a subscription. So far this month we have covered topics ranging from Alzheimers to expanding Covid-19 vaccine antigens. To get caught up, visit williamhaseltine.com/writings

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Oct. 21: Fatigue lasting weeks or even months affects approximately half of patients with Covid-19. If you are persistently and excessively tired even after resting or having a good sleep, you are likely suffering from Long Covid Fatigue. Get all your Long Covid questions answered with The Family Guide to Long Covid. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/

Oct. 22: New research published in the New England Journal of Medicine confirms a new therapeutic target for multiple myeloma CAR T therapy. https://www.forbes.com/sites/williamhaseltine/2022/10/21/c ar-t-therapy-for-drug-resistant-multiplemyeloma/?sh=407b6264640d # Improving air management can help fight Covid-19 1289

https://www.forbes.com/sites/williamhaseltine/2022/10/21/h ow-enhanced-ventilation-and-air-filtration-can-fight-covid19/?sh=bceffed38263 # Researchers have developed a "naked" self-amplifying RNA vaccine optimized for injection into the skin, rather than the muscle. http://www.forbes.com/sites/williamhaseltine/2022/10/21/int roducing-a-naked-self-amplifying-rna-vaccine-candidate/ Oct. 23: With more than 100 million globally pushed into poverty every year because of healthcare costs, innovative solutions to health financing are sorely needed. A new book published by ACCESS Health International shares best practices for how digital finance tools can help low-and middle-income communities afford and access healthcare around the globe. https://www.williamhaseltine.com/fintechforhealth/

Oct. 24: Monoclonal antibodies derived from macaque monkeys may be a new tool to fight against moderate to severe Covid-19.

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https://www.forbes.com/sites/williamhaseltine/2022/10/21/b roadly-neutralizing-sars-cov-2-antibodies-from-immunizedmacaque-monkeys/?sh=77e03a9c186f # Cautious optimism about the use of a new anti-amyloid fibril antibody for the treatment of early-stage Alzheimer's. http://www.forbes.com/sites/williamhaseltine/2022/10/22/rei magining-alzheimers-part-4-cautious-optimism-for-a-newalzheimers-disease-treatment/ Oct. 26: Visit our Covid-19 Resources website for articles about Covid19 that represent the latest research, important policy, or trends in the global pandemic and our efforts to win this battle. https://covid19resources.org/

Oct. 27: New classes of antibodies may be one of our best tools to overcome new immune evasive variants. 1291

https://www.forbes.com/sites/williamhaseltine/2022/10/26/as -protection-from-current-covid-19-monoclonal-antibodytreatments-fades-the-discovery-of-a-new-class-of-antibodiesbrings-hope/?sh=20f39b9e5dd7 # New data from the CDC shows that U.S. life expectancy dropped by a total of 2.7 years between 2019 and 2021 to 76.1 years, the lowest number since 1996. https://www.forbes.com/sites/williamhaseltine/2022/10/27/t he-dramatic-cost-of-the-covid-19-pandemic-a-historical-drop-inlife-expectancy/?sh=12ec820570da Oct. 28: While there are treatments for Long Covid, it’s not a one-sizefits all. Treatment plans are as varied and individualized as the symptoms of Long Covid. The best treatment is a holistic approach. Get all your Long Covid questions answered with The Family Guide to Long Covid. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/

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Oct. 29: Strength training has some distinct advantages when resuming an exercise routine after a Covid-19 infection. https://www.forbes.com/sites/williamhaseltine/2022/10/27/h ow-strength-training-can-help-post-covidrecovery/?sh=12f4c2b5ec85 Oct. 30: Specific types of challenges to affordability and access arise along the healthcare journey. In our latest book, Fintech for Health, we have identified five common attributes of healthcare costs that have prevented many people in Asia from accessing timely and quality healthcare services. https://www.williamhaseltine.com/fintechforhealth/

Oct. 31: High-calorie foods look, feel and taste good because they tap into our carnal desires to seek pleasure. Like sex or drugs, these foods target areas in our brain that keep us wanting more, even after our stomachs are full.

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https://www.forbes.com/sites/williamhaseltine/2022/10/29/sa lt-fat-and-sugar-how-americans-became-addicted-toeating/?sh=708adffa6b8f # A new UK study shows that being infected with Covid-19 is linked to an increased risk of poor cardiovascular health and death. https://www.forbes.com/sites/williamhaseltine/2022/10/31/c ovid-19-infection-linked-with-poor-cardiovascular-outcomes-anddeath/?sh=78a1a06379fd

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November 2022

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Nov. 1: CAR T cell therapy successfully induced remissions for five lupus patients, according to a recent study. https://www.forbes.com/sites/williamhaseltine/2022/11/01/c ar-t-therapy-from-cancer-to-autoimmune-disease-the-lupusexample/?sh=26959d473811 Nov. 3: Here we describe a "naked" self-amplifying RNA vaccine that, unlike conventional mRNA vaccines, does not require a lipid nanoparticle formulation or pseudouridine substitutions. Preliminary results suggest the vaccine induces robust CD8+ T cell responses and is effective in prime/boost regimens. http://www.forbes.com/sites/williamhaseltine/2022/11/02/na ked-self-amplifying-rna-vaccine-shows-promise-as-a-boosterinducing-strong-cytotoxic-t-cell-responses/ ## The discovery of a new monoclonal antibody cocktail could neutralize the Lassa Virus, which currently afflicts between 300,000 and 500,000 people per year in West Africa. https://www.forbes.com/sites/williamhaseltine/2022/11/03/n ew-monoclonal-antibody-cocktail-neutralizes-lassavirus/?sh=61737b7c605d Nov. 4: The best way to prevent Long Covid is to not be infected with Covid-19. Preventative measures include social distancing, maskwearing (especially in crowded spaces), and scheduling your vaccine as soon as possible. By reducing the risk of Covid-19 infection, vaccines also reduce the risk of Long Covid. Studies also show that vaccinated individuals are less likely to experience Long Covid symptoms that impact their daily functioning. Get all your Long Covid questions answered with The Family Guide to Long Covid. https://www.amazon.com/Family-Guide-Long-CovidQuestions-ebook/dp/B0B64PZ5MH/

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Nov. 5: The discovery of a new monoclonal antibody cocktail could neutralize the Lassa Virus, which currently afflicts between 300,000 and 500,000 people per year in West Africa. https://www.forbes.com/sites/williamhaseltine/2022/11/04/st udy-finds-that-regular-physical-activity-enhances-vaccineeffectiveness-against-covid-19/?sh=43e4092c3053 Nov. 6: A person’s health and financial journey are a unique function of their circumstances. Based on our observations through the Fintech for Health program and informed by our research on case studies, interviews, and discussions with fintech and healthcare organizations around the world, we have developed typical “personas” representing everyday people who experience health financing challenges. Fintech for Health now available on Amazon: https://www.amazon.com/dp/B0BCX1GWW7 Video: https://www.canva.com/design/DAFQQXJHyjM/aBQBPUUOn 3C1yRWQb7r_9g/edit?utm_content=DAFQQXJHyjM&utm_ca 1297

mpaign=designshare&utm_medium=link2&utm_source=sharebutt on Nov. 7: New research shows that female healthcare providers were disproportionately affected by moral distress during the Covid-19 pandemic compared to their male counterparts. https://www.psychologytoday.com/us/blog/best-practices-inhealth/202211/female-healthcare-workers-have-faced-heightenedmoral-distress Nov. 8: Each week, I republish my articles from Forbes on my website to make them available to read for free. For current listings, visit: williamhaseltine.com/writings

Nov. 9: Stay up to date with the latest research regarding Covid 19 and the efforts being made to win this battle on the Covid-19 Resources https://covid19resources.org/

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Nov. 10: A new paper on CAR T therapy is doubly exciting, combining the work of CAR T therapy and mRNA technology to treat diseases beyond cancer. https://www.forbes.com/sites/williamhaseltine/2022/11/09/c ar-t-therapy-for-cardiac-fibrosis-a-newmethod/?sh=7b539b0e1994 # Until the development of long-lasting, “universal” coronavirus vaccines, our best strategy for protection against infection, hospitalization, and death is staying up to date with our booster shots. http://www.forbes.com/sites/williamhaseltine/2022/11/10/th e-best-reason-to-keep-up-to-date-with-boosters-covid-19protection-from-infection-hospitalization-and-death-all-waneover-time/ Nov. 11: How do we develop effective (and accessible) treatments for Long Covid? It requires both more awareness and recognition of Long Covid. We need more funding for studies that meaningfully

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engage patients and that build on lessons from previous research on post-infectious illnesses.

Nov. 13: ACCESS Health International has spent over three years researching new health financing solutions, working with startups, hosting roundtables and cross sector special interest groups across Asia. Our latest book, Fintech for Health identifies how digital financial technologies can enable new solutions for healthcare costs. Now available on Amazon: https://www.amazon.com/dp/B0BCX1GWW7

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Nov. 14: In the last installment of this series, I discussed a promising treatment for Alzheimer’s disease called lecanemab. Here, I describe a recent setback for lecanemab’s clinical trials and introduce other potential treatments for Alzheimer’s that are currently underway. https://www.forbes.com/sites/williamhaseltine/2022/11/12/re imagining-alzheimers-part-5-setback-for-potential-alzheimerstreatment/?sh=157b825c7dda ## A new monoclonal antibody treatment for malaria may soon aid the millions impacted by the disease annually. https://www.forbes.com/sites/williamhaseltine/2022/11/14/n ew-monoclonal-antibody-for-treatment-ofmalaria/?sh=5ec8a2793305 Nov. 15: What are the treatments for Long Covid? How do I talk to my doctor about my symptoms? Can kids get Long Covid? Get all your questions answered in A Family Guide to Long Covid. https://www.amazon.com/gp/product/B0B64PZ5MH/ref=dbs_a _def_rwt_hsch_vapi_tkin_p1_i2

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Nov. 17: Preliminary results suggest a revolutionary cancer treatment— CAR T therapy—can be improved and controlled with a molecular switch. https://www.forbes.com/sites/williamhaseltine/2022/11/15/re searchers-control-cancer-treatment-with-new-innovation-car-tswitchblade/?sh=23ff34977882 Nov. 18: Though it isn't as common as it is in adults, children can develop Long Covid symptoms. Fatigue and brain fog are the most commonly reported symptoms.

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Nov. 20: The best protection against reinfection is using preventative measures—wearing masks, handwashing, and getting vaccines and boosters. Get all your Long Covid questions answered with A Family Guide to Long Covid, available on Amazon: https://www.amazon.com/gp/product/B0B64PZ5MH/ref=dbs_a _def_rwt_hsch_vapi_tkin_p1_i2

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Nov. 22: I was recently interviewed by @majohnso from @washingtonpost about the discovery of a new class of monoclonal antibodies that may circumvent Omicron variants by binding at alternative conserved sites to the receptor-binding domain that rarely mutates. https://www.washingtonpost.com/health/2022/11/21/monoc lonal-antibodies-covid-variants/ Nov. 23: CRISPR technology may simplify and enhance CAR T cancer treatment. https://www.forbes.com/sites/williamhaseltine/2022/11/23/cr ispr-technology-to-simplify-and-enhance-car-t-cancertreatment/?sh=20e016513b24 Nov. 24:

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Happy Thanksgiving! As you dig into your dinner, a time when many of us overeat, you may well ask: Will I regret it tomorrow when I check my weight? A new study suggests that it may depend on whether you have this gene variant. https://www.forbes.com/sites/williamhaseltine/2022/11/23/d o-you-have-the-thin-gene/?sh=4b5651907c82 Nov. 26 An uncommon monoclonal antibody treatment for Zika Virus may be on the horizon in the wake of new research on the pathogen. https://www.forbes.com/sites/williamhaseltine/2022/11/23/h ope-for-a-new-treatment-on-the-horizon-for-zikavirus/?sh=4dbf93415357 Nov. 28: http://www.forbes.com/sites/williamhaseltine/2022/11/29/th e-n-protein-a-new-target-for-anti-covid-drugs/ FDA-approved drug inhibits SARS-CoV-2 nucleocapsid phosphorylation and viral replication.

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December 2022

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Dec. 1: Why do children experience milder symptoms on average compared to adults? Do children experience Long Covid, and what makes them different? What is MIS-C and what is its prevalence? I explore recent research to answer these questions. https://www.forbes.com/sites/williamhaseltine/2022/11/30/c ovid-19-and-children-what-we-know/?sh=678912e83a33 # A recent study on Crimean-Congo Hemorrhagic Fever Virus may pave the way for a new monoclonal antibody treatment in the near future. https://www.forbes.com/sites/williamhaseltine/2022/12/01/cr imean-congo-hemorrhagic-fever-virus-monoclonal-antibodies-awork-in-progress/?sh=5498f1b766a9 Dec. 5: https://www.forbes.com/sites/williamhaseltine/2022/12/05/re imagining-alzheimers-part-6-the-many-effects-of-the-apoe4variant/?sh=7f0c8e3e6584 A novel aspect of the APOE4 genetic predisposition to Alzheimer’s is uncovered and leads to the potential for new diagnostic methods and treatment # Symptomatic Covid-19 infection is associated with the risk of developing or worsening overactive bladder. https://www.forbes.com/sites/williamhaseltine/2022/12/05/sy mptomatic-covid-19-infection-is-associated-with-an-increasedrisk-of-overactive-bladder-symptoms/?sh=167fc1147857 Dec. 8: Early clinical results reveal the potential of wielding CRISPR gene-editing technology to improve CAR T therapy, a recent innovation in cancer treatment. https://www.forbes.com/sites/williamhaseltine/2022/12/06/teami ng-up-two-biotech-winners-to-fight-cancer-crispr-and-car-t/ #

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In a world where fat is added to nearly every meal, the challenge many Americans face is how to cap their daily fat intake. Now, in a recent study, investigators argue that restricting fat cravings may be outside our conscious control. https://www.forbes.com/sites/williamhaseltine/2022/12/07/t he-fat-truth-you-feel-it-in-your-gut/?sh=52fae8e8ca6e # The antiviral drug Paxlovid has the potential to reduce the mortality rate associated with Covid-19 significantly, but is underused by those who would benefit most. https://www.forbes.com/sites/williamhaseltine/2022/12/06/c ovid-treatments-like-paxlovid-are-beingunderused/?sh=2adaa3435835 # A new study reveals the integral role of spike conformation in neutralizing activity in vaccines, paving the way for advancements in the near future. https://www.forbes.com/sites/williamhaseltine/2022/12/07/w hy-the-covid-vaccines-work-and-how-to-make-thembetter/?sh=4e8c2eb7d5c3 Dec. 9: Studies show that vaccinated individuals are less likely to experience Long Covid symptoms that impact their daily lives. Stay up to date with all your vaccinations and boosters.

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Dec. 10: A new research review has analyzed six potential causes for one of the most common yet elusive long Covid symptoms: Brain Fog. https://www.psychologytoday.com/us/blog/best-practices-inhealth/202212/6-potential-causes-brain-fog-in-covid-19-patients # A growing body of research suggests giving the immune system more time to "learn" about a vaccine antigen improves immune memory, helping to keep us safe during reinfection. http://www.forbes.com/sites/williamhaseltine/2022/12/07/ex tended-antigen-availability-improves-vaccine-protection/ Dec. 11: In a new e-book published by Access Health International, we share best practices for how digital finance tools can be used by insurers, healthcare providers and startups to help low-and middleincome communities afford and access healthcare globally. https://www.amazon.com/Fintech-Health-SejalMistry/dp/B0BCSGPDJM 1309

Dec. 13: Omicron: From Pandemic to Endemic, gives a detailed analysis of how the Omicron variant evolved to evade our best defenses, and provides a roadmap for how we might protect ourselves against future variants. Now available on Amazon. https://www.amazon.com/Omicron-Pandemic-EndemicFuture-Covid-19/dp/0578281635

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Dec. 14: Visit our Covid-19 Resources website for articles about Covid19 that represent the latest research, important policy, and trends in the global pandemic. https://covid19resources.org/

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Dec. 16: Studies estimate up to half of children infected with Covid-19 have either very mild or no symptoms at all. That means children are most likely to spread Covid-19 without knowing it because they can be contagious despite having no or very few symptoms.

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Dec. 18: The World Health Organization (WHO) estimates that more than 100 million people around the world are pushed into poverty every year because of healthcare expenditures. How can we address common healthcare challenges? A recent e-book ‘Fintech for Health’ explores innovative solutions to health financing. Now available on Amazon. https://www.amazon.com/Fintech-HealthSejal-Mistry/dp/B0BCSGPDJM

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Dec. 20: ACCESS Health International has published a new book Fintech for Health which shares best practices for how digital finance tools can be used by insurers, healthcare providers and startups to help low-and middle-income communities afford and access healthcare globally. Now available as an e-reader on Amazon. https://www.amazon.com/Fintech-Health-SejalMistry/dp/B0BCSGPDJM

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# This is the second story on how a novel aspect of the APOE4 genetic predisposition to Alzheimer’s was recently uncovered by a team at MIT and may lead to the potential for new diagnostic methods and treatments. http://www.forbes.com/sites/williamhaseltine/2022/12/17/rei magining-alzheimers-part-7-cholesterol-abnormalities-maycontribute-to-alzheimers-disease/ Dec. 21: One study progresses towards a cure for rheumatoid arthritis by using CAR T therapy, a treatment originally designed to fight cancer. https://www.forbes.com/sites/williamhaseltine/2022/12/19/c ar-t-therapy-to-treat-and-cure-rheumatoidarthritis/?sh=50d553d4bdd6 # The Covid-19 vaccines have prevented more than 18 million additional hospitalizations and more than 3 million additional deaths 1315

in the United States alone but their success was only possible because of sustained investments in public health. https://www.forbes.com/sites/williamhaseltine/2022/12/19/s uccesses-like-the-covid-19-vaccines-come-from-long-terminvestments-in-public-health/?sh=4f48c5b54304 Dec. 22: A startling new structure of the SARS-CoV-2 spike protein sheds light on the critical first step of infection, fusion of the virus and cell membranes. http://www.forbes.com/sites/williamhaseltine/2022/12/21/ne w-insights-into-the-postfusion-structure-of-sars-cov-2-spikeprotein/ Dec. 23: While Long Covid doesn’t discriminate, seniors are more likely to face long-term health complications due to a Covid-19 infection.

Dec. 28:

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We have curated the latest research regarding Covid-19 so you can stay up to date on the most recent research, news and policy: https://covid19resources.org/

Dec. 29: A new study in mice suggests that CAR T therapy may provide a new way to treat and potentially cure multiple sclerosis. https://www.forbes.com/sites/williamhaseltine/2022/12/21/c ar-t-therapy-a-promising-new-therapy-for-multiplesclerosis/?sh=3960b1f4c059 # https://www.forbes.com/sites/williamhaseltine/2022/12/22/n ew-monoclonal-antibody-fully-approved-for-the-treatment-ofcovid-19/?sh=56c87b5d3045 The Food and Drug Administration has approved Tocilizumab for use against moderate to severe Covid-19 symptoms. Dec. 30:

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A new study led by Boston University School of Public Health researchers suggests that the reported Covid-19 death toll in Africa is substantially higher than official records indicate. https://www.forbes.com/sites/williamhaseltine/2022/12/28/af rican-morgue-data-reveals-a-more-significant-covid-19-death-tollthan-official-reporting/?sh=637eca7730c8 # The FDA recently approved the drug Tocilizumab for use in hospitalized adults with severe Covid. Here we take a closer look at the data behind the approval. https://www.forbes.com/sites/williamhaseltine/2022/12/28/h ow-recently-approved-tocilizumab-treats-covid19/?sh=40e0f6763f2c Dec. 31: If you are 65 and older and experience a Covid-19 infection, be sure to monitor your symptoms in the weeks to come. If they don’t start to go away, contact your physician.

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Acknowledgements

would first like to thank the many people who have influenced my thinking on COVID-19, from my colleagues in classrooms, laboratories and offices worldwide, to the family and friends I speak with each day. I am especially grateful to my colleagues at ACCESS Health International for their daily efforts and especially to Koloman Rath and Anna Dirksen for their support as we updated this edition of the book. I would also like to extend my thanks to Brian Stauffer, who designed the cover of this book and its companion book, My Lifelong Fight Against Disease: From Polio and AIDS to COVID-19. Finally, I thank my wife, Maria Eugenia, for her love and support and my children, stepchildren and grandchildren: Mara, Alexander, Karina, Manuela, Camila, Pedro Agustin, Enrique Matias, and Carlos Eduardo.

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