Construction And Immunogenicity Of Gene Engineer Vaccines Agaist PRRSV And PCV2

To obtain safe and effective recombinant vaccines against PRRSV and PCV2, ORF5 and ORF3 genes of PRRSV were cloned.Recombinant DNA vaccines and recombineant fowlpox virus (rFPV) coexpressing ORF5,ORF3 of PRRSVand ORF2 of PCV2 were constructed,and the immunogenicity of DNA vaccines and recombinant fowlpox viruses against PRRSV and PCV2 was evaluated.ORF3 and ORF5 cDNA encoding GP3 and GP5 proteins of porcine reproductive and respiratory syndrome virus (PRRSV) strain Chang Chun were amplified by RT-PCR.Comparied the ORF3 and ORF5 genes with some strains in native and abroad, it was found that there was higher homology between the North American strain and strain Chang Chun, the homology at a mino acids level was 89.4% and 89.6% repectively. The homology was 96.1% and 95.5% between the strains HB-1 and Chang Chun. It indicated that the PRRSV strain Chang Chun was a branch of North American type.Four DNA vaccine plasmids, pVAX1-ORF5, pVAX1-ORF5-ORF3, pVIR-IL18- ORF5 and pVIR-IL18-ORF5-ORF3 expressing ORF5 (ORF5/ORF3) gene and pig IL-18 gene were constructed and evaluated for its ability to induce humoral and cellular immune responses in mice.All animals vaccinated with DNA vaccine plasmids developed specific anti-PRRSV antibodies.To evaluate the cellular immune function, T cell subsets was examined after immunization, the results indicated that the CD8+,CD4+,CD3+T lymphocyte percentages in the recombinant plasmids vaccinated groups were significantly higher than the pVAX1-inoculated group.One recombinant fowlpox viruses rFPV-IL18-ORF5-ORF3 containing the ORF5/ ORF3 cDNAs of PRRSV and IL18 of Swine were constructed and evaluated for its abilities to induce humoral and cellular responses in mice.All animals vaccinated with rFPV-IL18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody.To evaluate the cellular immune function,T lymphocyte percentages,the PRRSV-specific CTL cytotoxicity and number of IFN-γproducing cells were examined. the results indicated that the percentage numbers of CD8+CD3+T-lymphocytes subgroups, the cytotoxicity of specific CTL and number of IFN-γproducing cells of the rFPV-IL18-ORF5-ORF3-vaccinated group were significant higher than inactivated vaccine and wtFPV inoculated group.Two DNA vaccine plasmids,pRIES-ORF2-ORF5and pRIES-ORF2-ORF5-ORF3 expressing ORF5 (ORF5/ORF3) genes of PRRSV and ORF2 gene of PCV2 were constructed and evaluated for theirs abilities to induce humoral and cellular responses in mice.The results showed that all animals vaccinated with DNA vaccine plasmids developed specific anti-PRRSV and anti-PCV2 antibodies.To evaluate the cellular immune function, T-lymphocyte proliferation, CD4+CD3+and CD8+CD3+ T-lymphocytes subgroups, PRRSV and PCV2-specific CTL cytotoxicity and number of IFN-γproducing cells were examined were examined after immunization. The results indica-ted that the CD+CD8+ T lymphocyte percentages, PRRSV and PCV2-specific cytotoxic T lymphocyte responses and the number of IFN-γproducing cells in recombinant DNA vaccines plasmids inoculated groups were significantly higher than the inactivated vaccine-inoculated group and the pIRES-inoculated group.Two recombinant fowlpox virus,rFPV-ORF2-ORF5 and rFPV-ORF2-ORF5-ORF3 containing ORF5(ORF5/ORF3) cDNA of PRRSV and ORF2 cDNA of PCV2 was constructed and evaluated for theirs abilities to induce humoral and cellular immune responses in mice.The results showed that All animals vaccinated with rFPVs developed specific anti-PRRSV and anti-PCV2 antibodies.The results of cellular immune detection indicated that the CD8+CD3+ T lymphocyte percentages,the PRRSV and PCV2-specific cytotoxicity activity and the number of IFN-γproducing cells in the rFPV-vaccinated groups were significantly higher than the inactivated vaccine and wtFPV inoculated groups.Pigs were inoculated with two recombinant vaccines rFPV-IL18-ORF5-ORF3 and pVIR-IL18-ORF5-ORF3 alone or in combination. And the PRRSV specific antibody,T lymphoproliferation,T lymphocyte subpopulation in Peripheral blood and the production of IFN-γin pig serum were detected. Results showed that pigs immunized with recombinent vaccines developed specific humoral and cellular immune responses against the PRRSV proteins.At 28 dpi, sera from recombinent vaccines vaccine inoculated pigs showed PRRSV neutralizing activity,and continued to increase after the boost. At 14 dpi,pigs in the rFPV-IL18-ORF5-ORF3 inoculated groups showed PRRSV specific T-lymphocyte proliferation response.At 42 dpi,the stimulation index,the percentages of CD4+CD3+and CD8+CD3+T-lymphocytes and the IFN-γproduction of pVIR-IL18-ORF5-ORF3/rFPV-IL18-ORF5-ORF3 inoculated pigs were significantly higher than inactivated vaccine inoculated pigs and control groups.The results of PRRSV-PCV2 combination immunization indicated that specific anti-PRRSV and anti-PCV2 antibody in rFPV-ORF2-ORF5-ORF3 and pIRES-ORF2 -ORF5-ORF3 vaccinated pigs were significantly increased compared with the pIRES and PBS inoculated groups. At 28 dpi, sera from recombinant vaccines inoculated pigs showed PRRSV and PCV2 neutralizing activity. At 14 dpi, Pigs in the rFPV-ORF2-ORF5-ORF3 inoculated group showed a specific T-lymphocyte proliferation response, At 42 dpi, the Stimulation Index of the recombinant vaccines inoculated groups was significantly higher than inactivated vaccine inoculated group and two control groups. Peripheral blood T lymphocytes percentages,the production of IFN-γin the pigs serum were analyzed at 42 dpi.Results showed that the percentages of CD4+CD3+,CD8+CD3+T-lymphocytes and the production of IFN-γin the pigs serum of the recombinant vaccines inoculated groups were significantly higher compared with the inactivated vaccine inoculated group and two control groups.In summary, we show that mice and pigs immunized with the rFPV and DNA vaccines expressing ORF2 protein of PCV2 and GP5(GP5/GP3) of PRRSV developed a specific immune response against the PRRSV and PCV2 proteins.While the mechanisms responsible for protection against PRRSV and PCV2 infection remain unclear, our findings indicated that rFPV and DNA vaccine may be attractive vaccines for preventing diseases associated with PRRSV and PCV2 infection.Our research provides a new method for immunization against these two viral diseases.