Designing PRRSV Vaccine for Designing PRRSV Vaccine for Heterologous ProtectionHeterologous Protection

X.J. Meng

VA-MD College of Veterinary MedicineVirginia Tech

Blacksburg, Virginia

PRRS remains a major problem to the global swine industry

• $664 million losses/yr in the U.S. alone• Emergence of more virulent strains• Persistent infection• Heterogeneity• Co-infections with other swine agents

Emergence of new and more virulent PRRSV strains: “porcine high fever disease” caused by a highly

pathogenic PRRSV

Tian K et al. 2007

• High mortality (20-100%)• Affecting multiple organ systems

0 1 mo.

2 mos.

+/- 5 mos.

+1 yr

Exposure to PRRSV

Viremia

Total antibody response

NAbresponse

IFN producing cells

Viral load in tissues

Immuno-modulation of host immune system by PRRSV

Slide Courtesy of Dr. Fernando Osorio

Examples of co-infections: bacterial diseasesPRRSV-related diseases Effect of PRRSV infection on the related

diseaseReferences

Streptococcus suis More susceptible to septicemia and S. suis infection

Feng et al., 2001; Galina et al., 1994

Bordetella bronchiseptica More susceptible to bronchopneumonia and B. bronchiseptica infection

Brockmeier et al., 2000

Mycoplasma hyopneumoniae Potentiating effect for dual infection Thacker et al., 1999

Salmonella choleraesuis Synergistic effect for dual infection Wills et al., 2000

Pasteurella multocida Mixed reports: no interaction or predispose to P. multocida infection

Carvalho et al., 1997; Brockmeier et al., 2001;

Cooper et al., 1995Actinobacillus pleuropneumoniae

No interaction under experimental condition Pol et al., 1997

Haemophilus parasuis No interaction under experimental condition Cooper et al., 1995; Segales et al., 1999

Examples of co-infections: viral diseasesPRRSV-related diseases Effect of PRRSV infection on the

related diseaseReferences

Porcine circovirus - 2 Enhancing replication and diseases Allan et al., 2000; Harms et al., 2001; Rovira et al., 2002

Swine influenza virus Additive effect for dual infection Van Reeth et al., 2001

Porcine respiratory coronavirus

Much severe disease than single infection

Van Reeth et al., 2001

Pseudorabies virus Dual infection increased clinical signs and pneumonic lesions

Shibata et al., 2003

Classical swine fever virus No potentiation? China highly pathogenic PRRSV outbreaks?

Depner et al., 1999

Heterogeneity: a major obstacle for developing a more efficient vaccine

Murtaugh et al., 2010

Type 2 PRRSVShi et al., 2010

Current PRRSV Vaccines: successes and challenges

• MLVs:o Based on a single PRRSV straino Effective homologous protectiono Variable in success vs heterologous

strainso Safety issues: reversion ?

• Killed vaccines: o Variable in success, not very effective

Novel vaccine designs to overcome the major obstacles in PRRS control

Obstacles Potential SolutionHeterogeneity; emergence of novel strains

• Inactivated “cocktail” multivalent vaccines based on multiple strains of diverse genetic background ?

• Synthetic PRRSV based vaccines__MLVs based on shuffled chimeras representing different

genetically-divergent strains__Synthetic PRRSV vaccine with “consensus” sequence

Immune modulation • Vaccines that target dendritic cells (DCs)• Vaccines that can suppress Tregs• MLVs that contain modified glycosylation patterns

Co-infections • Multi-component vaccines against multiple swine pathogens

Molecular Breeding through DNA Shuffling

• Mimic nature’s recombination strategy but at a much accelerated rate in vitro

• Rapidly produce recombinant genes or viruses that can be screened for desired properties

• Traditional DNA shuffling• Synthetic DNA shuffling

Generation of candidate PRRSV vaccines conferring heterologous protection by traditional DNA shuffling

Viral genes from different heterologous parental strains

Random fragmentation by DNase I

Re-assembly by PCR without primers

PCR amplification with specific primers

Shuffled gene Clone into a DNA-launched PRRSV infectious clone backbone

Traditional and synthetic DNA shuffling of GP3 genes of 6 heterologous PRRSV strains

Traditional shuffling

Synthetic shuffling

Chimeric viruses with shuffled GP3 genes from 6 different PRRSV strains are infectious

Traditional shuffling

Synthetic shuffling

GP3 shuffling did not impair the replication ability of chimeric viruses in vitro

A GP3-shuffled chimeric virus (GP3TS22) induced cross-neutralizing activities against

heterologous PRRSV

DNA shuffling of GP4 or M gene of 6 strains

Chimeric viruses with shuffled GP4 (GP4TS14) or M (MTS57) induce cross-neutralizing antibodies

against heterologous PRRSV strains

71b

FV

MN184BVR2385 VR2430 NADC20

FL12 JXA1FV

2a3

45

6FV-SPDS

2a3

45

62b

5a

2a3

4 6FV-SPDS-FV5

34 6FV-SPDS-FV25

2a3

4 62b

FV-SPDS-VR2

2a3

4 6FV-SPDS-VR5

120

624

87 546

66 468

93 183 267 324

ORF1a1b

2a3

45

672b

5’UTR 3’UTR5a

A “mosaic PRRSV” with all the structural genes shuffled from 6 heterologous strains as a vaccine

Tian et al., 2015

FV-SPDS-VR2 chimera with shuffled multiple structural genes confers heterologous protection

Tian et al., 2015

In vivo targeting of shuffled PRRSV antigen through DC-SIGN to DCs to elicit antigen-

specific T cells immunity in pigs• C-type lectin receptors (CLRs) such as DC-SIGN

are endocytic receptors expressed on DCs which capture pathogen-derived glycoproteins and internalize them for efficient antigen presentation.

• When specifically targeted through CLR antibodies, they enhance Th1 and CD8 T cell immunity

Porcine dendritic cells internalize DC428 Mab through pDC-SIGN neck domain

(A) (B)

(C) (D)

Subramaniam et al., 2015

Porcine DCs internalize recombinant mouse-porcine chimeric DC428 antibody carrying shuffled PRRSV antigen

(A)

(B) (C)

Subramaniam et al., 2015

Frequency of antigen-specific CD4 T cells immune responses induced by pDC-SIGN-targeted PRRSV antigen

(A) (B)

(C) (D)

Subramaniam et al., 2015

Frequency of antigen-specific T cells in CD4+CD8+ T cell sub-population in vaccinated pigs

(A) (B)

(C) (D)

Synthetic PRRSV with desired diversity as a vaccine for heterologous protection• Similar in principle to “synthetic DNA shuffling”

approach• 59 type 2 PRRSV full-length genomes representing

4 subtypes• Generation of a centralized, consensus PRRSV

genome “PRRSV-CON”• PRRSV-CON is infectious and induces excellent

heterologous protectionH. Vu et al., J Virol. 2015

“PRRSV-Con” conferred cross-protection against PRRSV strain 16244B

Hiep L. X. Vu et al. J. Virol. 2015;89:12070-12083

Conclusion• DNA shuffling offers an opportunity for rational

design of PRRSV MLV vaccines that confer heterologous protection

• Shuffled PRRSV chimeric antigen, when targeted through DC-SIGN directly to DCs, elicited antigen-specific T cell immunity in pigs

_Implication: enhancing both humoral and CMI immune responses?

Acknowledgements•Meng Lab – PRRSV team:–D. Tian, S. Subramaniam, L. Zhou, Y.Y. Ni, Q. Cao, C. Overend, P. Pineyro, N. Catanzaro, S.P. Kenney, C.L. Heffron, Y.W. Huang, K.F. Key

•Collaborators:–Zoetis Inc: J. Calvert, D. Pearce–Univ Edinburgh: T. Opriessnig–ISU: P.G. Halbur–VT faculty: T. LeRoith, T. Cecere, C. Zhang

•Others (providing key materials):–K. Faaberg; K. Lager; F. Osorio; A. Pattnaik; F. Leung; Y. Fang; KJ Yoon; H.C. Yang

AcknowledgementsZoetis Inc

USDA-NIFAUSDA-NIFA-2011-67012-30719

USDA-NIFA-2011-67015-30165 USDA-NIFA-2013-67015-21342

American Society for Virology-2016 Annual MeetingJune 18-22, 2016, Virginia Tech, Blacksburg, VA