Update on novel experimental pig vaccine approaches - Dr. Tanja Opriessnig, The Roslin Institute, University of Edinburgh and Iowa State University, from the 2016 North American PRRS Symposium, December 3‐4, 2016, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2016-north-american-prrs-symposium

1. T. Opriessnig, P. Halbur, P. Gauger, J. Zhang, Q. Chen
D. Tian, Y. Ni, X.J. Meng, M. Tan
IOWA STATE UNIVERSITY
Department of Veterinary
Diagnostic and Production
Animal Medicine
THE UNIVERSITY of
EDINBURGH

2. PRDC
Vaccines
• A substance used to stimulate the production of antibodies and provide
immunity against one or several diseases, prepared from the causative
agent of a disease, its products, or a synthetic substitute, treated to act
as an antigen without inducing the disease
• In pigs commonly used to prevent diseases
• Cross protection problems can occur if a pathogen is genetically
diverse and multiple variants exist
Live virus vaccines
• Cell culture attenuation
• Known virulent strains
(serum therapy)
Inactivated virus vaccines
• Attenuated strains
• Virulent strains
• Multiple virulent isolates
enriched with viral antigens
Subunit vaccines expressing
selected proteins
• Different vectors
• Attenuated or live
• One or more proteins

3. PRDC
Overview
• SAVE virus attenuation approach (PRRSV)
– Collaboration with X.J. Meng’s group at Virginia Tech including Debin Tian and Yin
Yin Ni
• P-Particle subunit vaccine approach (IAV)
– Collaborations with Ming Tan at the Cincinnati Children's Hospital and Phil Gauger
at Iowa State University
– Preliminary data, more work is in progress
• Heterologous live virus exposure (PEDV)
– Collaboration with JQ Zhang and Qi Chen at Iowa State University
– Short intro: Main presentation during the CRWAD Meeting, Monday Dec 5th,10am

4. PRDC
SAVE approach - Introduction
SAVE: Synthetic attenuated virus engineering
− Using computer algorithm to re-code a given amino
acid sequence: change of codon pair bias
Codon bias: Actual encodings are biased to use some
synonymous codons more frequently than others.
Codon pair bias: some synonymous codon pairs are used
more or less frequently than expected.
E.g. Ala-Glu GCC GAA (27.7% X 29% = 8.03%)
GCA GAG (15.8% X 39.6% = 6.26%)
GCA GAG used 7 fold more often than GCC GAA
PRRSV
Virus attenuation by genome-scale
changes in codon pair bias
Coleman JR et al., Science 2008
1
Courtesy of Prof. XJ Meng

5. PRDC
• Advantages:
– Same amino acid sequence
– Same codon bias
– The attenuation is not subjected to reverse back
– Rapid
GCC GAA …………………………… GCA GAG
Ala Glu …………………………...... Ala Glu
SAVE approach - Introduction1 PRRSV
Hypothesis: Rapid attenuation of PRRSV
by applying the SAVE approach to deoptimize
codon-pairs of critical PRRSV viral genes
Courtesy of Prof. XJ Meng

6. PRDC
Analysis of swine genes
SAVE approach - Introduction1 PRRSV
Courtesy of Prof. XJ Meng

7. PRDC
SAVE approach - Introduction1 PRRSV
Principle process steps and estimated time line
Process step Duration
Isolation of the farm-specific PRRSV strain from serum or lung tissues 3-7 days
Sequencing of the ORF5 gene 2-3 days
Computer-based codon-pair deoptimization 1 day
Genetic modification of the farm-specific PRRSV strain 30 days
Sequence confirmation of the correct modification 2-3 days
Cell culture of the modified strain and growth to appropriate titers 7-15 days
Shipment of the vaccine to the farm 2 days
Total estimated time line 47-61 days

8. PRDC
Gene sequences after de-optimization
Deoptimized
gene
(length in bp)
Deoptimized
coding region
of gene
(nt position)
Number of
silent
mutations
introduced
CPB* of
original
gene
fragment
CPB of
deoptimized
gene fragment
MFE¶ of
original gene
fragment
(kcal/mol)
MFE of
deoptimized
gene fragment
(kcal/mol)
gp5 (603) 148  546 78 -0.049 -0.354 -134.8 -122.6
nsp9 (1938) 82  1938 459 0.016 -0.317 -628.7 -585.0
SAVE5 vaccine production1 PRRSV
Ni et al., 2014
Virology

9. PRDC
IFA of infected MARC-145 cells
SAVE5 vaccine production
VR-2385 NEG
SAVE5 SAVE9
1 PRRSV
Ni et al., 2014
Virology

10. PRDC
In vitro expression of viral proteins
SAVE5 vaccine production1 PRRSV
Ni et al., 2014
Virology

11. PRDC
Growth kinetics on PK15-CD163 cells
SAVE5 vaccine production1 PRRSV
Ni et al., 2014
Virology

12. PRDC
Viral RNA loads in sera and lungs
SAVE5 vaccine pathogenicity study in pigs1 PRRSV
Ni et al., 2014
Virology

13. PRDC
Infectious virus titers in serum samples
SAVE5 vaccine pathogenicity study in pigs1 PRRSV
Ni et al., 2014
Virology

14. PRDC
Significantly reduced lung lesions
SAVE5 vaccine pathogenicity study in pigs1 PRRSV
Ni et al., 2014
Virology

15. PRDC
• For the first time, SAVE approach was applied to a veterinary virus, PRRSV.
• PRRSV with codon-pair deoptimized GP5 gene was successfully rescued and
displayed an attenuated phenotype both in vitro and in vivo.
• Implication for rapid vaccine development for PRRSV and other important viruses
SAVE5 vaccine pathogenicity study in pigs
Conclusions
1 PRRSV

16. PRDC
To assess the immunogenicity and protective efficacy of SAVE5
in decreasing clinical signs, lesions and viremia associated with
wild-type PRRSV challenge using a conventional pig model
SAVE5 vaccine challenge study in pigsPRRSV
1
Objective

17. PRDC
Group Number of pigs Vaccination D0 Challenge D42
R1 R2 Vaccine Route Challenge Route
NEG-CONTROL 9 Saline IM Saline Intranasal
VAC-IM-CONTROL 10 SAVE5 IM Saline Intranasal
VAC-IM-PRRSV 10 10 SAVE5 IM VR2385 Intranasal
VAC-IN-PRRSV 10 SAVE5 Intranasal VR2385 Intranasal
POS-CONTROL 10 9 Saline IM VR2385 Intranasal
SAVE5 vaccine challenge study in pigsPRRSV
1
Experimental design
Vaccination:
• 3 mL of SAVE5 at a dose of 104.5 TCID50/mL IM
into the right neck
• 3 mL of SAVE5 at a dose of 104.5 TCID50/mL
intranasally
• 3 mL of saline IM into the right neck
Challenge:
• 2 mL of VR-2385 at a dose of 106.6 TCID50/mL
intranasally
Necropsy: At D54 (12 days post challenge)
9 weeks of age3 weeks of age

18. PRDC
SAVE5 vaccine challenge study in pigsPRRSV
1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 7 14 21 28 35 42 48 54
GroupmeanELISAS/Pratios
Day post vaccination
POS-CONTROL VAC-D42-NEG VAC-D42-POS
A
A
A
A
A
A
B B B B B
B
Antibody responses after vaccination
VAC-D42-POS:
10/30 (4 IM and 6 IN)
VAC-D42-NEG:
20/30 (16 IM and 4 IN)

19. PRDC
PRRSV viremia
SAVE5 vaccine challenge study in pigsPRRSV
1
0
1
2
3
4
5
6
7
0 7 14 21 28 35 42 45 48 51 54
Log10groupmeanPRRSVRNAinserum
Day post vaccination
POS-CONTROL VAC-D42-NEG
VAC-D42-POS
A
A
BB
A
A
A
A
B
A
A
A
B B
B B B B
SAVE5 virus
VR-2385 virus

20. PRDC
Subgroups Number
of pigs
Macroscopic lung lesions
0-100%
Interstitial pneumonia PRRSV antigen4
VAC-D42-POS 10 4.2±1.6A (2.5; 0.6, 7.8) 1.0±0.3A (1; 0.3, 1.7) 3/10 (0.4±0.2)A
VAC-D42-NEG 20 15.1±2.4A (16; 10, 20.2) 2.1±0.2B (2; 1.7, 2.5) 18/20 (1.2±0.1)B
POS-CONTROL 19 27.4±5.1B (21; 16.8, 38.1) 2.1±0.2B (2; 1.5, 2.6) 19/19 (1.3±0.1)B
SAVE5 vaccine challenge study in pigsPRRSV
1
Lesions 12 days post VR-2385 challenge

21. PRDC
• The SAVE approach can effectively attenuate a PRRSV
strain
• Additional work needs to be done to further improve SAVE5
vaccine efficacy
• Ability to utilize the SAVE technology to rapidly produce,
safe and efficacious, farm-specific PRRSV vaccines
– Very practical
– Could have a major impact on reducing the major economic
losses associated with PRRSV
SAVE5 vaccine challenge study in pigsPRRSV
1
Conclusions

22. PRDC
SAVE5 vaccine challenge study in pigsPRRSV
1
Additional information

23. PRDC
• Requires significantly less time compared to the traditional
cell culture attenuation
• Attenuates the virus without altering the antigenicity of the
virus protein on the virion
– The protein sequence of the SAVE5 ORF5 is identical to the
wild-type PRRSV ORF5
• Potential drawback is over-attenuation which may affect the
ability of the virus to replicate in the host
SAVE5 approachPRRSV
1
Implications

24. PRDC
Statement of the problem
• Populations of IAV’s found in pigs are currently
very diverse
• Vaccination, while often used, is often not
effective
– Cross-protection against genetically different IAV
strains is limited
2 IAV
http://sciencenordic.com/sick-pigs-give-
insight-swine-flu (Mette Valheim)
Clinical Signs of IAV
Coughing
Nasal discharge
Lethargy
Reduced appetite
Fever
IAV introduction

25. PRDC
P-particle vaccine platform
Vaccine platform based on protruding
(P) domains of norovirus (NoV)
A: NoV VLP
B: 24mer P particle
C: 12mer particle
D: P dimer
E: Linear structure of NoV VP1 with the two
domains
2 IAV

26. PRDC
IAV M2e epitope2 IAV
Possible target for an universal IAV vaccine
– 24 amino acid residues in length
– Integral membrane protein of IAV
– Low in copy numbers on the virus particle
• Abundantly expressed on the surface of IAV-
infected cells
– M2e domains are thought of being highly
conserved among IAV strains
From: Schlütter J. 2011. Prevention: Vaccine for all seasons. Nature 480(7376):S6-8.

27. PRDC
P-particle vaccine platform2 IAV
Antibody response against M2e in miceXia et al., 2011
Vaccine

28. PRDC
Complex vaccine platform
Simple strategy to turn small, low immunogenic
antigens into higher-order complexes
2 IAV

29. PRDC
Complex vaccine platform2 IAV
Complex P-Particle
Significantly improved immunogenicity and more
effective in the mouse challenge model
Wang et al., 2014
Biomaterials

30. PRDC
pH1N1 pig challenge study2 IAV
Objective
To compare a commercial inactivated pandemic (p)
H1N1 vaccine and the two novel subunit vaccines,
using the IAV M2e epitope as antigen, in a growing pig
challenge model

31. PRDC
pH1N1 pig challenge study2 IAV
Experimental design
Group Pigs Vaccination
3 + 5 weeks
Challenge
7 weeks
Platform Route Challenge
EXP-PARTICLE- IAV
7 Yes M2e P-particle IN pH1N1
EXP-COMPLEX-IAV
8 Yes M2e Complex IN pH1N1
COM-pH1N1-IAV
8 Yes pH1N1 IM pH1N1
POS-CONTROL
8 Saline Saline IM pH1N1
NEG-CONTROL
8 Saline Saline IM Saline
Challenge:
• pH1N1 isolate A/California/04/2009
• IN and intracheally, 2×105 TCID50/mL
For experimental vaccination:
• Mucosal atomization device
• PolyI:C adjuvant (Sigma-Aldrich)

32. PRDC
pH1N1 pig challenge study2 IAV
Experimental design

33. PRDC
pH1N1 pig challenge study2 IAV
Antibody responses
Group -28 (Serum) 0 (Serum) 5 (Serum) 5 (BALF)
NP ELISA NP ELISA pH1N1 HI NP ELISA pH1N1 HI NP ELISA
EXP-PARTICLE- IAV
0/7 (0.96±0.02)A,3 1/7 (0.94±0.06)AB 0/7 (3.1±1.9)A 0/7 (0.80±0.02)A 0/7 (3.8±1.5)A 0/7 (1.20±0.13)A
EXP-COMPLEX-IAV
0/8 (1.00±0.01)A 0/8 (0.93±0.02)AB 0/8 (1.8±2.0)A 0/8 (0.83±0.03)A 0/8 (3.4±1.9)A 0/8 (1.29±0.12)A
COM-pH1N1-IAV
0/8 (0.95±0.02)A 1/8 (0.74±0.08)A 8/8 (8.3±1.1)B 7/8 (0.42±0.05)B 8/8 (8.5±1.0)B 0/8 (0.88±0.05)A
POS-CONTROL
0/8 (0.96±0.02)A 0/8 (0.94±0.04)AB 0/8 (1.5±1.8)A 0/8 (0.84±0.03)A 0/8 (4.2±1.5)A 1/8 (1.10±0.17)A
NEG-CONTROL
0/8 (0.94±0.01)A 0/8 (0.95±0.02)B 0/8 (2.2±2.0)A 0/8 (0.94±0.02)A 0/8 (2.1±1.8)A 0/8 (0.94±0.05)A

34. PRDC
pH1N1 pig challenge study2 IAV
IAV RNA shedding in nasal swabs
0
1
2
3
4
5
NS dpc 1 NS dpc 2 NS dpc 3 NS dpc 4 NS dpc 5 BALF dpc 5
GroupmeanIAVlog10genomiccopies/nasal
swabormlofBALF
EXP-PARTICLE- IAV EXP-COMPLEX-IAV COM-pH1N1-IAV
POS-Control NEG-CONTROL
A
A
A
A
A
B
B
B
B
A
B
A,B
B

35. PRDC
pH1N1 pig challenge study2 IAV
Macroscopic lesions
Negative
Control
Positive
Control
Commercial
vaccine
P-Particle
vaccine
Complex
vaccine

36. PRDC
The commercial pH1N1-specific vaccine effectively protected
pigs against homologous challenge
• Reduced clinical signs, virus shedding in nasal secretions and
oral fluids and reduced macroscopic and microscopic lesions
• Further highlights the importance using IAV type-specific
vaccines in pigs
2 IAV
pH1N1 pig challenge study
Conclusion 1

37. PRDC
Intranasal vaccination with experimental M2e epitope-based subunit vaccines
did not protect the pigs against pH1N1 challenge
• M2e contains only 24 amino acids and represents one small epitope on IAV
• M2e-specific immune response likely only blocks the ion channel activity
required for efficient viral un-coating during IAV invasion
• Dose:
– Small size of the M2e epitope: accounting for only 4-7% of the experimental
vaccines
– Previous mouse trials: 15-30 µg of the subunit vaccines 3 x
– This pig trial: 50 µg of the same subunit vaccines 2 x
• Vaccine doses, protein concentration and administration route need to be
further investigated and better adjusted from usage in mice to usage in pigs
2 IAV
pH1N1 pig challenge study
Conclusion 2

38. PRDC
Clinical signs
Diarrhea, anorexia
Acute vomiting (Source: www.petspigs.com)
Diarrhea (Source: Darin Madson)
PEDV Normal
Suckling pigs
Diarrhea
Vomiting
Lethargy
High morbidity
High mortality
Introduction
PEDV
3

39. PRDC
PEDV geno-groups
G1b: North America, Europe, Asia
Low-to-moderate pathogenicity
G2b: North America, Asia, Ukraine
High pathogenicity
US and Asian vaccines
G1a: Asian vaccines
G1
G2
PEDV spike gene
sequencing
3

40. PRDC
G1a exposure to protect against G2b
Assumption: PEDV strains from the G1b cluster (Spike-gene-based phylogeny)
are less pathogenic compared to the G2b cluster
To determine the ability of an experimental G1b-based live vaccine
and a commercial G2b–based inactivated vaccine to protect growing
pigs against G2b challenge
Objective
PEDV
3

41. PRDC
Experimental design
GROUPS Pig# Vaccination
Type Adjuvant Genogroup Route Timing
EXP-IM-PEDV 7 Experimental live Adjuplex™ G1b IM dpc -28
EXP-ORAL-PEDV 8 Experimental live None G1b Orally dpc -28
COM-IM-PEDV 8 Commercial
inactivated
Amphigen® G2b IM dpc -28
and -14
POS-CONTROL 8 Sham None Saline IM dpc -28
NEG-CONTROL 8 Sham None Saline Orally dpc -28
Vaccination:
• Exp IM: 2.4 mL of G1b 14-20697, 7th passage, 5 × 104 TCID50 /mL
• Exp oral: 10 mL of of G1b 14-20697, 7th passage, 6.8 ×103 TCID50/mLl
• COM IM: 2 mL Porcine Epidemic Diarrhea Vaccine (Zoetis), G2b PEDV
PEDV
G1a exposure to protect against G2b3

42. PRDC
IgG antibody levels over time
0
1
2
3
-28 -21 -14 -7 0 7 14
GroupmeanELISAIgGS/Pratios
Day post PEDV challenge
EXP-IM-PEDV EXP-ORAL-PEDV
A
A
A
B
B
B
A,B
CB C C
B,C
3/3
1/4
3/8
4/4
2/8
A
A
A
B
B
B
A,B
CB C C
B,C
3/3
3/3
1/4
3/8
4/4
2/8
A
A
A
A
A
B
B
B
A,B
CB C C
B,C
3/3
1/4
4/4
3/8
8/8
8/8
2/8 6/8
7/8
7/8
3/4 3/4
PEDV
G1a exposure to protect against G2b3

43. PRDC
IgA antibody levels over time
0
1
2
3
4
-28 -21 -14 -7 0 7 14
GroupmeanELISAIgAS/Pratios
Day post PEDV challenge
EXP-IM-PEDV EXP-ORAL-PEDV COM-IM-PEDV
POS-CONTROL NEG-CONTROL
A
A
A
A
A
B B B
B
A,B
B
C
5/8
1/8
3/4
2/4
1/4
4/4
A
A
A
A
B B B
B
A,B
B
C
1/4
4/4
4/4
4/4
7/8
7/8
8/8
PEDV
G1a exposure to protect against G2b3

44. PRDC
RNA shedding levels
0
1
2
3
4
5
6
7
8
9
-28 -21 -14 -7 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Groupmeanlog10PEDVgenomiccopiesinfecalswabs
Day post PEDV challenge
EXP-IM-PEDV EXP-ORAL-PEDV COM-IM-PEDV POS-CONTROL NEG-CONTROL
A
A
A
A
A A
A
A
A
A A
A,B
A,B
A,B
B
B
BB,C
B
B
BB
BC
A
B,C
A,
B
A,B
A,B
C
A,B
A
AA
A
B
B,C
B B B B
PEDV
G1a exposure to protect against G2b3

45. PRDC
Conclusions
• Commercial inactivated IM G2b-based PEDV vaccine
– Low IgA response (serum)
– High IgG response (serum)
– Protected pigs against homologous G2b challenge
• The experimental IM G1b-based live virus vaccine
– Did not replicate in the pig and was not protective
• The experimental ORAL G1b-based vaccine
– Induced a high IgA response (serum and feces)
– Moderate IgG response (serum)
– Virus shedding pattern mimicked that of the POS-CONTROL group
– Limited protection
• A genotype specific humoral and/or cellular immune response
may be important for PEDV protection
PEDV
G1a exposure to protect against G2b3

46. PRDC
Acknowledgements
Iowa State University
• Mingxi Guo
• Alessandra Castro
• Eve Fontanella
• Kelsey Oakley
• Huigang Shen
• Patrick Halbur
• Phil Gauger
• Jianqiang Zhang
• Qi Chen
The Roslin Institute
• Priscilla Gerber
• Jin Cui
• Luigi Marongiu
• Marta Campillo
Funding:
• Iowa Livestock Health Advisory Council (ILHAC)
• BBSRC Institute Strategic Programme Grants BB/J004324/1 and BBS/E/D/20241864
Virginia Polytechnic Institute and
State University
• Debin Tian
• Yin-Yin Ni
• XJ Meng
Cincinnati Children’s
Hospital
• Ming Tan