Synthetic Biology and the Future of Swine Vaccines Hiep Vu, DVM, PhD Assistant Professor University of Nebraska-Lincoln
My laboratory
My group
Nebraska Center for Virology Pregunta al ponente: porciforum.info
Viral diseases are the leading cause of losses 2013-2014 PEDV outbreak in the US $0.9 - $1.8 billion 2018-2019 ASFV outbreak in China $150 billion
Piglets died from PEDV Credit: Matt Ackerman
2019-2020 ASFV outbreak in Vietnam $2 billion
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Viruses are intra cellular parasites
“…a virus is just a piece of bad news wrapped in protein…” Peter and Jane Medawar Pregunta al ponente: porciforum.info
Protective immunity against viruses Two types of adaptive immunity • Antibodies: block virus attachment • T cells: kill virus infected cells
Antibody protection
T-cell protection
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Conventional Vaccinology Limitations •
Not all viruses can be cultivated
•
Time-consuming
•
Sometimes fail to confer heterologous protection
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Synthetic Biology “Redesign organisms for useful purposes” Key elements • Next generation sequencing • Bioinformatics • DNA synthesis • Advances in molecular technologies
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Synthetic biology for vaccine development • Subunit vaccines • Prescription vaccines • Live-attenuated vaccines • Broadly protective vaccines
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Protective antigens … viral proteins capable of eliciting a protective immunity SARS-CoV-2 Influenza virus PCV2 CSFV PEDV
S protein HA protein Capsid protein E protein S protein
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Subunit vaccines Protein-based vaccine
DNA vaccine
Subunit vaccines: contain only viral antigens that best stimulate the protective immunity
Viral vector vaccine
mRNA vaccine
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COVID-19 vaccines S protein
Adenovirus vector vaccines (Astrazeneca, J&J)
SARS-CoV-2 mRNA vaccines (Pfizer, Moderna)
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Timeline for mRNA-1273 vaccine progression 31/12/19
13/1/20
20/1/20
10/1/20
1st outbreak reported in China
14/1/20
mRNA-1273 designed
2019-nCoV sequence published
GMP production initiated
Corbett et al. 2020. Nature, Vol. 586
18/12/20 16/3/20
1st confirmed case in the US
31/1/22
FDA approved for emergency use Phase 1 clinical trial
FDA full approval
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Factors contributing to the rapid development of COVID-19 vaccines •
Protective antigen is known (e.g. viral spike protein)
•
Mechanism of protection is known (e.g. neutralizing antibody)
•
Safe and effective vaccine platforms were readily available -
mRNA - platform
-
Adenovirus vector platform
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USDA new regulations for veterinary vaccines
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Prescription Vaccines (RxV) •
Need a written prescription from veterinarians
•
Administration within the context of a VeterinarianClient-Patient Relationship
•
Manufactured using approved production platforms - A single vector or expression system (“backbone”) - Non-replicating, non-viable
•
No claims efficacy or potency
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Technologies for RxV RNA particles Alphavirus
mRNA molecule
RNA particles
Helper mRNA Providing the protein coat
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Technologies for RxV
Baculovirus expression system •
High levels of protein expression
•
Easy scale-up
•
Eukaryotic protein processing capabilities
•
The virus does not infect mammalian hosts Pregunta al ponente: porciforum.info
An example of RxV usage in the US Atypical porcine pestivirus (APPV)
E2 gene E2
E1/E2
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Another example
RxV for swine influenza virus HA
• • • •
SIV genome evolves rapidly Commercial vaccines can’t be updated fast enough HA is the viral surface protein Antibodies against HA are fully protective Anderson et al. 2016 Pregunta al ponente: porciforum.info
Another example
RxV for swine influenza virus Farm 1
Farm 2
Farm 3
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What are RxV used for? •
Viruses with high mutation rates - Influenza virus, rotavirus…
•
Viruses that can’t be cultivated in vitro - PCV3, APPV…
•
Newly emerging viruses - PEDV, porcine sapovirus, APPV…
•
Endemic viruses not interested by large companies - APPV, porcine sapovirus, porcine teschovirus Pregunta al ponente: porciforum.info
RxV cannot be used for •
Diseases under Control and/or Eradication Programs
•
Reportable Animal Diseases
•
Select Agents
•
Foreign Animal Diseases (FADs)
•
Diseases not endemic to the United States
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Some thoughts • • • • •
RxV is essentially a subunit vaccine It is safe and relatively easy to develop It is tested for safety and purity but no claim on efficacy It will be more popular in the near future It offers a faster reaction to emerging diseases
However • RxV can be produced only if protective antigens are known • Many viruses with unknown protective antigens (e.x. ASFV) Pregunta al ponente: porciforum.info
Live-attenuated vaccines Removal of a virulence gene
Virulent virus
Live-attenuated virus Pregunta al ponente: porciforum.info
Attenuation of ASFV by removal of virulence genes
Bottleneck • Need to know virulence genes • Need a robust cell line to cultivate the virus Pregunta al ponente: porciforum.info
Live-attenuated vaccines Codon deoptimization approach PRRSV
Limiting amino acids Pregunta al ponente: porciforum.info
Live-attenuated vaccines Codon deoptimization approach Amino acids are coded by multiple codons
Codon for Leucine
Some are used more frequent than the others Protein expression levels can be manipulated •
Optimization: change to frequently used codons
•
De-optimization: change to rarely used codons
Triplet
Frequency/thousand
CUU
11.9
CUC
10.2
CUA
4.2
CUG
48.4
UUA
6.7
UUG
10.7
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Attenuation of PRRSV by codon deoptimization • Modifications at many positions Reduced risk of reversion to virulence
• Vaccine strains can be generated faster However • Engineered virus is considered GMO Difficult to license Pregunta al ponente: porciforum.info
Expanding vaccine heterologous protection An example of PRRSV
• • •
Vaccines have been available since 1994 Viral genome is highly variable Protective antigens are not known Pregunta al ponente: porciforum.info
Design of a consensus PRRSV genome Consensus concept
Hypothesis: A vaccine based on the consensus PRRSV genome would confer broad protection Pregunta al ponente: porciforum.info
Generation of the synthetic PRRSV strain
b
Synthesis of the PRRSV-CON genome
Construction of a cDNA clone
ΦT7
D
(3,318 nt)
B
C (4,404 nt)
(3,259 nt)
A (4,511 nt)
T7
cDNA clone
In vitro transcription to produce RNA transcript
AAAAAAA
Transfection of the vRNA into cells Pregunta al ponente: porciforum.info
Replication in MARC-145 cells Growth kinetics in MACR-145
PRRSV-CON
TCID50 per mL (log10)
7
FL12
6 5 4 3
FL12 PRRSV-CON
2 1 0
12
24
48
72
96
120
Hours p.i.
(Vu et al. J Virol 2015 Vol 89, 12070-12083)
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Cross-protection experiments Groups 1 2 3
Immunized with PBS PRRSV-CON FL12
Challenged with MN184 Or 16244B
FL12
PRRSV-CON
Parameters of protection • Levels of virus in blood • Levels of virus in tissues
MN184 16244B
Inoculation Acclimation
-7
Challenge
Necropsy
Recover from primary infection
0
Days
52
67
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Cross-protection experiments Exp 1: Challenge with MN184 isolate Virus in blood FL12
Virus in tissue
Copy # per mL (log10)
7 6 5 4 3 2 1 0
0
1
4
7
10
15
PRRSV-CON
p<0.0001
PRRSV-CON Copy # per µg of total RNA (log10)
PBS
8
FL12
Days p.i.
(Vu et al. J Virol 2015 Vol 89, 12070-12083)
7
p=0.0004
6
MN184
p=0.0062
5 4
16244B
3 2 1 0
PBS
FL12
PRRSV-CON
Imunization groups
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Cross-protection experiments Exp 2: Challenge with 16266B isolate Virus in blood PBS
8
FL12
Virus in tissue
PRRSV-CON
6 5 4 3 2 1 -2
1
4
7
11
PRRSV-CON
p<0.0001
Copy # per 1 µg total RNA (log10)
Copy # per mL (log10)
7
0
FL12
p<0.0001
7 6
MN184 p<0.0001
5
16244B
4 3 2 1 0
14
Days Post-challenge
(Vu et al. J Virol 2015 Vol 89, 12070-12083)
PBS
FL12
PRRSV-CON
Immunization groups
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Summary Advances in molecular technologies have made it possible to redesign viruses for useful purposes. Synthetic biology can be deployed for rapid development of safe and effective antiviral vaccine • Subunit vaccines • Prescription vaccines • Live-attenuated vaccines • Broadly protective vaccines
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Acknowledgement Collaborators
Dr. Fernando Osorio Dr. Asit Pattnaik Dr. Fangrui Ma Dr. Alan Doster Dr. David Steffen
Students and post-docs Jayesh Chaudhari Sushmita Kumari Kassandra Durazo The Nguyen Hung Luong Bikash Sahoo
Lorena Bustamante Raquel Leme Haiyan Sun Kay Kipmstom-Burkgren
Funding
National Pork Board • 13-155 • 14-200 • 15-159
USDA-NIFA-AFRI • 2016-67015-24922 • 2018-67015-28294 • 2020-67015-31414 Pregunta al ponente: porciforum.info
Thank you for your attention!!!