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