Construction Of Recombinant Swinepox Viruses Expressing Multi-Epitope Peptide For Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failure in sows, and respiratory disease in pigs and becomes the causative agent of the most important infectious disease affecting the porcine herds worldwide. Commercial vaccines are mostly inactivated vaccines. Some modified live vaccines are also available. An improvement of vaccination strategies against PRRSV is required, as current vaccines have limited efficacy. Never Commercially available vaccines are only partially effective in protection against PRRSV. Epitope-based vaccines and vaccines based on recombinant virus genomes (virus vectored vaccines) represent alternative and complementary view. We will combine the two vaccine design strategies to generate novel vaccines against PRRSV.To characterize neutralizing epitopes, we selected phages from a12-mer phage display library using anti-PRRSV neutralizing monoclonal antibodyAl, A2and A7. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated. The two representative sequences of mimotopes corresponding mAbs were determined. Mimotope named P1was recognized by neutralizing MAb A1. Another mimotope named P2was recognized by neutralizing MAb A2and A7. SRHDHIH motif in mimotope P2was found at the position from aa156to161in N-terminal ectodomain of GP3, whereas no significant consensus sequences were found in the mimotope P1.Epitope-based vaccination is a promising means to develop the novel vaccine gainst porcine reproductive and respiratory syndrome virus (PRRSV) infection. The nucleotide sequence of the one mimotope P2and GP5liner epitpoe linked in tandem with spacers formed the321bp designed gene HP. The gene was synthesized and cloned into pET32a. The expressed protein, designated rHP, was produced by IPTG-induced pET32a-HP is transformed E.coli BL21cells, and analyzed by SDS-PAGE. The protein showed an apparent molecular weight of30kDa. Western blot analysis showed that rHP protein expressed by BL21cells transformed with pET32a-HP reacted with the anti-PRRSVV mAbs A7. The multi-mimotope protein rHP promises to be a novel epitope-based vaccine candidate for PRRSV.To develop a safer, more efficacious vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) infection, we exploited a new recombinant swinepox virus vector system constructed in our lab,which could efficiently expressed exogenous gene with eGFP as the marker. After homologous recombination with wild SPV genome, the gene of multi-epitope peptide HP was inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-HP) was isolated after10passages and verified by PCR, western blotting and immunofluorescence assays. The HP gene was identified in the recombinant SPV and the expression of multi-epitope peptide HP was obsvered in PK15infected with rSPV-HP. High level of anti-HP antibodies was observed in mice immunized with rSPV-hp, and titer of PRRSV-specific neutralizing antibodies was1:60(10-1.78). These results suggested that the recombinant rSPV-HP could be a promising candidate for the vaccine development against PRRSV infections.