Performing Department
(N/A)
Non Technical Summary
Diseases of swine are caused by the PRRS virus (PRRSV). PRRSV continues to distress the global pork industry by causing economic losses estimated at $664 million every year due to reproductive failure, fatal respiratory disease, and subsequent secondary bacterial infection.Swine influenza virus (SIV) causes acute respiratory diseases in breeding and nursery pigs and can predispose the infected animals to secondary viral and bacterial infections. In addition to the adverse economic impact on pig producers, SIV is also a zoonotic pathogen detrimental to humans. Coinfectionof pigs with PRRSV, porcine circovirus type 2 (PCV2), and SIV on the same farms also plays a critical role in the porcine respiratory disease complex and is frequently reported.Since the late 1990s, killed and modified live virus (MLV) PRRSV vaccines based on attenuated European or North American PRRSV strains have been commercialized and used to control PRRS. The MLV-PRRS vaccine has safety issues due to the persistence of the vaccine virus in the host and increased mutation rates. Consequently, vaccine-like virulent PRRSV strains have emerged and caused vaccine failures instead of protecting the pigs. Likewise, currently available inactivated swine influenza vaccines are ineffective in controlling SIV in pigs due to the strain variability.Previously, we developed a quadruple gene-deleted pseudorabies virus (PRVqmv) vaccine vector platform in which envelope glycoprotein gE, US9, gG, and thymidine kinase (TK) genes are deleted. A chimeric PCV2 capsid protein (Cap) was also inserted, resulting in the PRVqmv-Cap.In this project, our goals are to engineer the PRVqmv further, i) to incorporate the PRSSV envelope proteins glycoproteins (gp2.gp3,gp4, and gp5) and the matrix protein, and test its vaccine protective efficacy against a virulent PRRSV challenge strain and ii) to incorporate the SIV envelope glycoproteins H1N1 and H3N2, and test its protective efficacy against the two different (H1N1 and H3N2 strains) virulent influenza challenge viruses.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important global diseases of swine caused by PRRS virus (PRRSV). PRRSV continues to distress the global pork industry by causing economic losses estimated at $664 million every year due to reproductive failure, fatal respiratory disease and subsequent secondary bacterial infection.Swine influenza virus (SIV) causes acute respiratory diseases in breeding and nursery pigs and can predispose the infected animals to secondary viral and bacterial infections. In addition to the adverse economic impact on pig producers, SIV is also a zoonotic pathogen detrimental to humans. Coinfections of pigs with PRRSV, porcine circovirus type 2 (PCV2), and SIV on the same farms also play a critical role in the porcine respiratory disease complex and are frequently reported.Since the late 1990s, killed and modified live virus (MLV) PRRSV vaccines based on attenuated European or North American PRRSV strains have been commercialized and used to control PRRS. The MLV-PRRS vaccine has safety issues due to the persistence of the vaccine virus in the host and increased mutation rates. Consequently, vaccine-like virulent PRRSV strains have emerged and caused vaccine failures instead of protecting the pigs. Likewise, currently available inactivated swine influenza vaccines are ineffective in controlling SIV in pigs due to the strain variability.Previously, we developed a quadruple gene-deleted pseudorabies virus (PRVqmv) vaccine vector platform in which envelope glycoprotein gE, US9, gG, and thymidine kinase (TK) genes are deleted. A chimeric PCV2 capsid protein (Cap) was also inserted, resulting in the PRVqmv-Cap.In this project, we have four specific objectives.Construct two versions (prototype 1 and 2) of the chimeric PRRSV, gPs(2a-3-4) and gP5-M, as two polyprotein coding genes and incorporate them into the PRVqmv-Cap vector genome in two different loci separately.Construct chimeric SIV H1N1 and H3N2-GMCSF (H3N2+) gene cassettes to code for two polyprotein-coding genes and incorporate them in the PRVqmv-Cap vector genome. Evaluate the safety, stability, intranasal replication, and protective efficacy of the two prototype vaccine versions, PRVqmv-Cap-PRRSV (prototype I) and PRVqmv-Cap-PRRSVmut (prototype II) in pigs.Evaluate the safety, stability, intranasal replication, and protective efficacy of the PRVqmv-Cap Sub-H1N1- H3N2-GMCSF(+)-in pigs against the SIV H1N1 and SIV H3N2 challenge.Objectives 1 and 2 will be performed at the laboratory of Dr. Chowdhury (PD), School of Veterinary Medicine, LSU. Objectives 3 and 4 will be conducted by Dr. Gourapura (Co-PD) at the Center for Food Animal Health, The Ohio State University.
Project Methods
Objective 1.Two chimeric polyprotein coding genes encoding PRRSV envelope proteins (glycoproteins gP2a-gP3-gP4 and gP5-M) will be synthesized commercially. Our vaccine design strategy includes self-cleavable P2A peptides (P2A, E2A, F2A, and T2A) to express multiple PRRSV proteins as polyproteins and using a single promoter and PolyA sequences. Synthesized chimeric polyprotein coding expression cassettes will be incorporated in the gE-Us9 and gG deletion loci of the PRVqmv- -Cap genome, respectively. The resulting PRVqmv-Subunit PCV-PRRSV vaccine virus will be verified by nucleotide sequencing. Additionally, immunoblotting with proper protein and Tag-specific antibodies will verify appropriate molecular mass and post-translational processing of the chimeric subunit PRRSV proteins expressed in the backbone of the PRVqmv vector.Objective 2.As in objective 1, two chimeric genes, SIV-H1N1 and H3N2-GMCSF, will be synthesized and incorporated into the PRVqmv-Cap into the gG-deletion and gE/Us9-deletion loci, respectively. The resulting PRVqmv-Subunit SIV vaccine virus will be verified by nucleotide sequencing. Additionally, chimeric subunit SIV polyproteins, expressed individually upon proteolytic cleavage as above in the PRVqmv-Subunit SIV vaccine virus-infected cells, will be analyzed by immunoblotting with appropriate protein- and Tag-specific antibodies for their appropriate molecular mass and post-translational processing.Objective 3.Immunization/challenge experiments will be performed in pigs to evaluate the protective efficacy of the PRVqmv-Cap-Sub-PRRSV vaccines generated in Objective 1 against virulent PRRSV challenge. Protection will be determined by the absence of clinical disease, lack of viremia, lung pathology, and viral load in tissues following the challenge of vaccinated pigs. In addition, the induction of protective levels of PRRSV-specific neutralizing antibody titers and PRRSV antigen-specific cellular immune responses will be analyzed upon primary and booster vaccination. At the successful completion of this objective, we expect to have a safe and effective, protective PRVqmv vectored vaccine that would be protective not