Viral genetics and application to vaccine development - Dr. Jay Calvert, Zoetis, Inc, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting

1. 1
VIRAL GENETICS
AND
APPLICATION TO
VACCINE
DEVELOPMENT
JAY G CALVERT, RESEARCH DIRECTOR,
GLOBAL BIOLOGICALS RESEARCH, VMRD,
ZOETIS, KALAMAZOO, MI
NORTH AMERICAN PRRS SYMPOSIUM
DECEMBER 1-3, 2017
CHICAGO, IL

2. 2
OVERVIEW OF TODAY’S TALK
INTRO TO CONVENTIONAL MODIFIED LIVE VACCINES
THE ROLES OF VIRAL GENETICS IN VACCINE EFFICACY
SELECTING AN APPROPRIATE STARTING STRAIN
THE ATTENUATION PROCESS (PREMEDITATED
SERENDIPITY)
SUMMARY
1
2
3
4
5

3. INTRODUCTION TO
CONVENTIONAL MODIFIED
LIVE PRRS VACCINES

4. 4
More than a dozen conventional PRRS MLV vaccines are commercially licensed:
CONVENTIONAL MODIFIED LIVE VIRUS
(CELL CULTURE ATTENUATED) VACCINES
USA Europe China ROW
Ingelvac® PRRS MLV/
RespPRRS® (BIVI)
Porcilis® PRRS (MSD) JXA1-R PRRS Vaccine VP-046
BIS (Philippines)
Ingelvac® PRRS ATP
(BIVI)
Unistrain® / Amervac®
PRRS (Hipra)
TJM-F92
Rui Lan An® (Zoetis
joint-venture)
Kaketsuken (Japan)
PrimePac® PRRS +
PrimePac® PRRS RS
(Merck)
Pyrsvac-183® (Syva) HuN4-F112 BSL-PS 100 (Singapore)
Fostera® PRRS (Zoetis) Ingelvac PRRSFLEX®
EU & Ingelvac
ReproCyc® PRRS EU
(BIVI)
GDr180 Fostera® PRRS is currently
licensed in Canada, Mexico,
Philippines, Malaysia, South
Korea, Thailand, and Japan
Suvaxyn® PRRS MLV
(Zoetis)
(Market Authorization
Sep. 2017)
CH-1R
R98

5. 5
• Commercial MLV PRRS vaccines are the best solution we currently have, and are far more
efficacious than killed vaccines or experimental subunit vaccines
• MLVs mimic natural infection, which induces very robust protection against the homologous
PRRSV strain, including “sterilizing immunity” in some cases
• MLVs induce innate immunity (Natural Killer cells and interferon), as well as acquired immunity
including T-helper cells (cytokine help), cytotoxic T-cells (killing of infected cells), and B-cells
(production of neutralizing antibodies)
• MLVs are self-adjuvanting, can be delivered intramuscularly, and only need a single dose for
lifetime protection in feeder pigs (customer convenience)
CONVENTIONAL MODIFIED LIVE VIRUS (CELL
CULTURE ATTENUATED) VACCINES - BENEFITS

6. THE ROLES OF VIRAL
GENETICS IN VACCINE
EFFICACY

7. 7
Under control of the vaccine designer Not under control of the vaccine designer
Selection of parental virus strain (starting
genetics)
Genetics, health status, and nutrition of
vaccinated animals
Attenuation method and endpoint (final
vaccine genetics)
Immunological history of the herd
Formulation Environmental conditions
Label recommendations (storage, size and
number of doses, route of administration,
age at vaccination, etc.)
Actual usage of the vaccine (storage, size and
number of doses, route of administration, age at
vaccination, etc.)
Genetics, virulence, and immunogenicity of the
challenge virus(es).
Co-infections with other pathogens during
vaccination and/or challenge.
FACTORS THAT INFLUENCE VACCINE EFFICACY

8. SELECTING AN
APPROPRIATE STARTING
STRAIN

9. 9
• Selection of an appropriate parental field strain to become the vaccine is critically
important, and must happen before attenuation can begin
• It can take 5-7 years to advance from strain selection to licensed vaccine product
• Using the most recent “hot” strain as the parent of a new vaccine is a risky strategy
• New variants appear and disappear frequently
• They don’t always leave economically important descendants after 5-7 years
• Recent strains may offer relatively poor cross-protection against other recent, but distantly
related, field strains
TIMING

10. 10
Canine Parvovirus
• Example of “linear”
(stepwise) evolution
• Ancestral strains may be
replaced by newer strains
• Overall diversity
increases slowly
• Vaccine update decisions
are relatively simple
DIFFERENT PATTERNS OF VIRUS EVOLUTION

11. 11
PRRS virus
• Example of “radial” (multi-
directional) evolution
• Ancestral strains persist and
give rise to many discrete new
branches
• Over all diversity increases
exponentially
• Vaccine update decisions are
very complex
DIFFERENT PATTERNS OF VIRUS EVOLUTION
From: Dr. Mike Murtaugh - PRRS '174' - What are we learning about PRRSV
evolution and characterization of new emerging strains of PRRSV?

12. 12
GENETIC RELATEDNESS
Nucleotide Substitution per 100 residues
0
33.8
51015202530
ORF5_Amervac
ORF5_Olot_91
ORF5_Porcilis
ORF5_Lelystad
ORF5_Ingelvac PRRSFLEX EU
ORF5_Suvaxyn PRRS MLV
ORF5_BEL13-13V117
ORF5_GER09-613
ORF5_AUT15-Acro
ORF5_Lena
ORF5_VR2332
Mean=86.9%
Mean=85.4%
…to divergent recent field strains
may be maximized by using vaccines
based on older (ancestral) strains

13. 13
Yes and No. It’s a complex issue!
• YES. Obviously some degree of genetic relatedness is required for cross-protection
• This is because acquired immunity is based on shared epitopes (B, Tcytotoxic, Thelper)
• Homologous protection in PRRSV (100% aa identity, 100% shared epitopes) is uniformly
very good.
• Heterologous protection within PRRS-1 or PRRS-2 (80-99% aa identity, 11-90% shared
epitopes) is variable, and ranges from very good to poor (but usually detectable).
• Heterologous protection between PRRS-1 and PRRS-2 (60% aa identify, 1% shared
epitopes) is only fair or poor (but detectable with proper study design).
• Heterologous protection between distantly related Nidoviruses such as PRRSV and
PEDV (<40% aa identity, <<1% shared epitopes) is negligible/ undetectable.
DOES GENETIC SIMILARITY PREDICT CROSS-
PROTECTION?

14. 14
Yes and No. It’s a complex issue!
• NO. Factors other than genetic similarity/shared epitopes confound predictions.
• The immunogenicity of the first virus is dependent on:
• Ability to replicate to high titer (antigenic load)
• Ability to induce potent innate and acquired immune responses
• Relatively little viral interference with host function (low level of immune dysfunction)
• Health and genetics of the host animal
• Lack of interfering co-infections
• The severity of disease induced by the second virus is dependent on:
• Challenge dose and timing
• Inherent virulence of the virus
• Immune evasion / immune dysfunction mechanisms
• Age, health, and genetics of the host animal
• Co-infections with other pathogens
When existing vaccine options are more closely related to each other than they are to
the challenge strain (often the case), the small differences in ORF5 sequence identity
are useless for vaccine selection.
DOES GENETIC SIMILARITY PREDICT CROSS-
PROTECTION?

15. 15
• Green trend line added to
approximate contribution of
genetic relatedness.
• Highly Pathogenic Asian
PRRS may be below the
trend line due to remarkably
high virulence.
J.G. Calvert, J. Angulo, J.R.D. Allison, B. O'Brien, R.G. Ankenbauer (2015)
FOSTERA® PRRS VACCINE HELPS PROVIDE HETEROLOGOUS CROSS-
PROTECTION AGAINST A WIDE RANGE OF DIVERSE PORCINE
REPRODUCTIVE AND RESPIRATORY SYNDROME (PRRS) VIRUSES

16. THE ATTENUATION
PROCESS
(PREMEDITATED SERENDIPITY)

17. 17
• Field strains are very efficacious – but not safe.
• The field strain is passaged many times in a cell culture system, resulting in:
• Rapid selection for mutations in the virus that confer adaptation (fast growth) in the new cell type (and
often reduced growth in the natural cell type)
• Gradual accumulation of mutations in viral genes that defend the virus from the immune system, since
there is an absence of selection for these functions in cell culture.
• Periodically the virus is checked for safety (attenuation of disease) and efficacy. At different
passage levels the virus may be:
• Under-attenuated (still efficacious, but still not safe enough). Needs more passages!
• Over-attenuated (very safe, but no longer efficacious because it can’t grow well in the host animal).
Too many passages!
• Appropriately-attenuated (safe, and still efficacious). This is a vaccine candidate!
On a given attenuation attempt, the window of opportunity may be large or small or even negative in
size. You can’t predict which random mutations will appear, and in what order.
HOW DOES ATTENUATION WORK?

18. 18
SCREENING FOR VACCINE CANDIDATES
Expansion
on PAM
Adaptation to
BHK21-C12-26
Cloning
X 3
Further adaptation
Increase titers
Further attenuation
Safety &
Efficacy in
young pigs
Unsafe
Not efficacious
Low titer
Candidate !
Candidate !
Not efficacious
Not efficacious
Low titer

19. 19
• Multiple parallel attenuations are recommended, since every attempt yields a different result.
• However, careful selection of the starting virus strain and the cell line(s) used for attenuation can limit the
spectrum of possible outcomes, and work in your favor.
• To the best of our knowledge, all commercially licensed live PRRS vaccines (except two) were attenuated on a
particular monkey kidney cell line, MA-104, or cell lines derived from MA-104.
• We now know why MA-104 and derivative cell lines like MARC-145 were unique among common cell lines in their ability
to grow (some) PRRS viruses
• Due to a rare mutation, MA-104 cells unexpectedly express the CD163 protein, which is the primary receptor for all
PRRS viruses (discovered at Zoetis in 2004). CD163 is normally expressed on the surface of alveolar macrophages.
• What are the exceptions? Which are the only two commercially licensed live PRRS vaccines not attenuated on
this monkey kidney cell line?
HOW DOES ATTENUATION WORK?

20. 20
FOSTERA® PRRS AND SUVAXYN ® PRRS MLV
ARE ATTENUATED ON NOVEL ENGINEERED
CELL LINES EXPRESSING PORCINE CD163

21. 21
TWO ATTENUATION METHODS IN USE TODAY
Attenuation on monkey kidney cells
expressing monkey CD163
Attenuation on (non-monkey) cells
expressing porcine CD163
Examples: More than a dozen commercial
PRRS vaccines globally
Examples: (1) Fostera® PRRS
(2) Suvaxyn® PRRS MLV
Attenuation phenotype: Strong selective
pressure for the virus to adapt to better
recognize monkey CD163. Partial loss of
affinity for porcine CD163.
Attenuation phenotype: No need for the virus
to adapt to a different CD163. Safety
achieved by accumulation of mutations in
other genes.
In the vaccinated pig: Strong selective
pressure for the virus to adapt back to better
recognize porcine CD163.
In the vaccinated pig: No need for the virus
to adapt to a different CD163. Stable
attenuation phenotype.
Label Claims: None of these vaccines has
earned a claim for use in nursing pigs as
young as 1 day of age.
Label Claims: Both have demonstrated
safety and efficacy in 1 day old nursing pigs,
earning the corresponding USDA/EMA label
claims.

22. SUMMARY

23. 23
• For now, modified live vaccines are by far the most effective tools we have for PRRS control.
• It is not cost-effective (or necessary) to attempt to develop and register a new commercial MLV
from each new virulent field strain. The process takes 5-7 years and costs millions of dollars.
• Because of the radial pattern of PRRSV evolution, maximum cross-protection may be provided
using older ancestral strains.
• At a high level, there is a clear relationship between the degree of genetic homology between two
viruses and their ability to cross-protect against each other. This is a consequence of the number
of shared (conserved) antigenic epitopes.
• At the ground level, using ORF5 sequences to compare a current field strain to a limited number
of vaccine strains, none of which closely resembles the field strain, is fruitless. There are too
many other factors in play that confound the genetic predictions of cross-protection.
CONCLUSIONS I

24. 24
• Attenuation of virulence is accomplished by serial passage on a permissive cell line, leading to
increased safety and manufacturability, while maintaining immunogenicity.
• The attenuation process is generally stopped when the vaccine has become safe enough to pass
standard tests. Additional cell passages risk loss of efficacy (over-attenuation).
• Although the random accumulation of mutations during attenuation is unpredictable, strong
selection pressures imposed by using different cell lines can influence the spectrum of possible
outcomes. In some cases, the “safety stop” is predictable.
• An alternative to monkey cell attenuation now exists, which does not require the vaccine virus to
adapt to prefer monkey CD163 over porcine CD163. Such vaccines have novel properties,
including enhanced safety and efficacy in pre-weaning pigs as young as one day old.
CONCLUSIONS II

25. THANK YOU FOR YOUR
ATTENTION
QUESTIONS?