Analyses On Immunoenhancement Effect Of Porcine Gp96N For Synthetic Peptides And Subunit Vaccines Of PRRSV And Inactivated Vaccine Of PCV2

The Gp96, a member of heat shock protein (HSP) family, displays various immunological functions includingstimulating the expression of cytokines by activating the antigen presentation cells in innate immunity, and eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity, as well as acting as immunological adhuvant. In this study, we focused on the immunological effects of Gp96N on synthetic peptides and subunit vaccinesof porcine reproductive and respiratory syndrome virus (PRRSV) and inactivated vaccine of porcine circovirus type2(PCV2). Porcine Gp96N was first expressed by two expression systems of E.coliBL21/pET-32a/Gp96N and p.pastoris-X33/pPICZaA/Gp96N.Then purified Gp96was used as adjuvant in combination with the three vaccines in mice and piglets in order toanalyze the immunoenhancement effect of porcine Gp96N. Our results provide theoretical basis for the application and development of Gp96in vaccines.RNA was extracted from porcine kidney to amplify the cDNA ofGp96gene-encoded N-terminal22-370amino acids (aa) with RT-PCR.The amplified product was cloned into the expression vector pET-32a and pPICZaA, and then the recombinant plasmids were transformed into E.coli/BL21and p.pastoris-X33, respeCtively.The transformants were screened on resistance plates and named E.coliBL21/pET-32a/Gp96N and p.pastoris-X33/pPICZaA/Gp96N.The expressed protein (termed Gp96N) was purified.Two B-cell epitopes and seven T-cell epitopes on nonstructural and structural proteins of PRRSV and a Pan DR T-helper cell epitope were synthesized and mixed with the Gp96N as an adjuvant, and then immune responses were evaluated in mice and piglets. The results showed that Gp96N could enhance the humoral and cellular immune effects of PRRSV synthetic peptides. Moreover, it could significantly up-regulate the expression levels of IL-12and TNF-α and down-regulate the levels of IL-4and IL-10. Following challenge with the virulent PRRSV isolate JXwn06, the piglets vaccinated with the mixture of Gp96N and synthenic peptides presented milder clinical symptoms, lower viremia, and less pathological lesions in their lungs compared with the piglets immuned with the peptides alone although all the vaccinated piglets in each group died, suggesting the Gp96N can alleviate the clinical symptoms of challenging piglets in some extent.Two multi-epitope subunit vaccines, named as Cpl and Cp2, were designed based on the conserved B cell epitopes of PRRSV proteins.The Gp96N was used as the adjuvant. Immune responses elicited by the different combinations of Cpl/Cp2and Gp96N were examined in mice and piglets. The results indicated that the group of Cpl/Cp2-Gp96N (CG) combination could induce3-4-fold higher titers of Cpl/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups received Cpl/Cp2immunization alone or with Freund’s adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from the vaccinated mice or piglets. The production of IFN-y in mice splenocytes, TNF-a, IFN-y, and IL-12in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4was reduced by half and IL-10was decreased to an undetectable level. These results suggest that the porcine Gp96N can effectively enhance the innate and adaptive immune responses of Cpl/Cp2with a Thl-type bias. Therefore, the multi-epitope subunit vaccine Cpl/Cp2co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.Mice and piglets were immuned with different doses of Gp96N combined with inactivated vaccine of PCV2. The titer of PC V2-specific-IgG and the expression level of some cytokines in serum including IFN-y,TNF-a,IL-1β and IL-6were tested by ELISA kit. The results showed that Gp96N could significantly increase titers of IgG antibody compared to the groups immunized with the inactivated and commercial vaccines of PCV2alone. In addition, Gp96N could significantly elevate the levels of IFN-y and TNF-a, but had no effects on the levels of IL-1β and IL-6in sera of piglets.Taken together, all the findings indicate that Gp96N can enhance humoral and celluar responses of epitope peptide and subunit vaccines of PRRSV, and improve humoral immunological effect on inactivated vaccine of PCV2in some extent, suggesting that Gp96shares the potential immune effect for vaccines as an adjuvant.