Dr. Tanja Opriessnig - Update on novel experimental pig vaccine approaches

Update on novel experimental pig vaccine approaches

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

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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

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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

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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

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• 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 genesCourtesy of Prof. XJ Meng

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Analysis of swine genes


Courtesy of Prof. XJ Meng

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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 daysTotal estimated time line 47-61 days

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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., 2014Virology

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IFA of infected MARC-145 cells

SAVE5 vaccine production

VR-2385 NEG

SAVE5 SAVE9

1 PRRSV


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In vitro expression of viral proteins



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Growth kinetics on PK15-CD163 cells



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Viral RNA loads in sera and lungs

SAVE5 vaccine pathogenicity study in pigs1 PRRSV


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Infectious virus titers in serum samples



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Significantly reduced lung lesions



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• 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

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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 pigsPRRSV1

Objective

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Group Number of pigs Vaccination D0 Challenge D42R1 R2 Vaccine Route Challenge Route

NEG-CONTROL 9 Saline IM Saline Intranasal

VAC-IM-CONTROL 10 SAVE5 IM Saline IntranasalVAC-IM-PRRSV 10 10 SAVE5 IM VR2385 IntranasalVAC-IN-PRRSV 10 SAVE5 Intranasal VR2385 IntranasalPOS-CONTROL 10 9 Saline IM VR2385 Intranasal


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

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0 7 14 21 28 35 42 48 540

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

POS-CONTROL VAC-D42-NEG VAC-D42-POS

Day post vaccination

Gro

up m

ean

ELIS

A S/

P ra

tios

A

A

AA

AA

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)

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PRRSV viremia


0 7 14 21 28 35 42 45 48 51 540

1

2

3

4

5

6

7

POS-CONTROL VAC-D42-NEG

VAC-D42-POS

Day post vaccination

Log

10 g

roup

mea

n PR

RSV

RNA

in s

erum

A

A

BB

A

AA

A

B

AA

A

B BB B B B

SAVE5 virus

VR-2385 virus

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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


Lesions 12 days post VR-2385 challenge

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• 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


Conclusions

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Additional information

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• 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 approachPRRSV1

Implications

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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 IAVCoughingNasal dischargeLethargyReduced appetiteFever

IAV introduction

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P-particle vaccine platform

Vaccine platform based on protruding (P) domains of norovirus (NoV)

A: NoV VLPB: 24mer P particleC: 12mer particleD: P dimerE: Linear structure of NoV VP1 with the two domains

2 IAV

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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.

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P-particle vaccine platform2 IAV

Antibody response against M2e in miceXia et al., 2011Vaccine

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Complex vaccine platform

Simple strategy to turn small, low immunogenic antigens into higher-order complexes

2 IAV

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Complex vaccine platform2 IAV

Complex P-Particle

Significantly improved immunogenicity and more effective in the mouse challenge model

Wang et al., 2014Biomaterials

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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

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Experimental design

Group Pigs Vaccination 3 + 5 weeks

Challenge7 weeks

Platform Route Challenge

EXP-PARTICLE- IAV7 Yes M2e P-particle IN pH1N1

EXP-COMPLEX-IAV8 Yes M2e Complex IN pH1N1

COM-pH1N1-IAV8 Yes pH1N1 IM pH1N1

POS-CONTROL8 Saline Saline IM pH1N1

NEG-CONTROL8 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)

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Experimental design

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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- IAV0/7 (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-IAV0/8 (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-IAV0/8 (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-CONTROL0/8 (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-CONTROL0/8 (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

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IAV RNA shedding in nasal swabs

NS dpc 1 NS dpc 2 NS dpc 3 NS dpc 4 NS dpc 5 BALF dpc 50

1

2

3

4 EXP-PARTICLE- IAV EXP-COMPLEX-IAV COM-pH1N1-IAV POS-ControlNEG-CONTROL

Gro

up m

ean

IAV

log1

0 ge

nom

ic c

opie

s/ n

asal

sw

ab o

r ml o

f BA

LF

AA

AA

A

BB

BB

A

B

A,B

B

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Macroscopic lesions

Negative Control

Positive Control

Commercial vaccine

P-Particle vaccine

Complex vaccine

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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

PRDCIntranasal 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

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Clinical signs

Diarrhea, anorexia

Acute vomiting (Source: www.petspigs.com)

Diarrhea (Source: Darin Madson)

PEDV Normal

Suckling pigsDiarrheaVomitingLethargyHigh morbidityHigh mortalityIntroduction

PEDV3

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PEDV geno-groups

G1b: North America, Europe, AsiaLow-to-moderate pathogenicity

G2b: North America, Asia, UkraineHigh pathogenicity

US and Asian vaccines

G1a: Asian vaccines

G1

G2PEDV spike gene sequencing

3

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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

PEDV3

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Experimental design

GROUPS Pig# VaccinationType Adjuvant Genogroup Route Timing

EXP-IM-PEDV 7 Experimental live Adjuplex™ G1b IM dpc -28EXP-ORAL-PEDV 8 Experimental live None G1b Orally dpc -28COM-IM-PEDV 8 Commercial

inactivatedAmphigen® G2b IM dpc -28

and -14POS-CONTROL 8 Sham None Saline IM dpc -28NEG-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

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IgG antibody levels over time

-28 -21 -14 -7 0 7 140

1

2

3

EXP-IM-PEDV EXP-ORAL-PEDV

Day post PEDV challenge

Gro

up m

ean

ELI

SA

IgG

S/P

ratio

s

A

A

A

BB

B

A,B

CB C C

B,C

3/3

1/43/8

4/4

2/8

A

A

A

BB

B

A,B

CB C C

B,C

3/3

3/3

1/43/8

4/4

2/8

A

AA

A

A

BB

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


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IgA antibody levels over time

-28 -21 -14 -7 0 7 140

1

2

3

4 EXP-IM-PEDV EXP-ORAL-PEDV COM-IM-PEDV


Gro

up m

ean

ELI

SA

IgA

S/P

ratio

s

AA

A

AA

B B B

B

A,B

B

C

5/81/8

3/4 2/4

1/4

4/4

AA

A

A

B B B

B

A,B

B

C1/4

4/4

4/4

4/4

7/8

7/88/8


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RNA shedding levels

-28 -21 -14 -7 0 1 2 3 4 5 6 7 8 9 10 11 12 130

1

2

3

4

5

6

7

8

9EXP-IM-PEDV EXP-ORAL-PEDV COM-IM-PEDV POS-CONTROLNEG-CONTROL


Gro

up m

ean

log1

0 PE

DV

geno

mic

cop

ies

in fe

cal s

wab

s

A

AA

A

A AA

A

A

A A

A,B

A,B

A,B

B

B BB,CB

BBB

BC

AB,C

A,B

A,B

A,B

C

A,B

A

AA

A

B

B,C

B B B B


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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


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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