PARTNERSHIP: NOVEL PSEUDORABIES VIRUS (PRV) VECTORED SUBUNIT VACCINE AGAINST PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME AND SWINE INFLUENZA VIRUSES

<b>Project Methods</b><br> 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