| TGEV and PEDV are a member of the Coronaviridae family, induces acute, high-contact intestinal infectious diseases. PED and TGE lead to significantly economic losses and there were no effective drugs to treat TGE and PED. Therefore, development of a safe, effective vaccine has important significance. We constructed the recombinant attenuated salmonella vaccines SL7207 (pVAXD-PS1-TS), SL7207 (pVAXD-PS1), SL7207(pVAXD-TS) and evalusted the immunogenicity of SL7207 (pVAXD-PS1-TS) in mouse model. In this study, we furtherly study the immunogenicity and immune dynamic regulation of SL7207 (pVAXD-PS1-TS) in piglets. Humoral immunity, cllular immunity, mucosal immunity and neutralization assay were evaluated. Provide the scientific basis for evaluation of the vaccines, and provide a reference for future study.Strain recovery and extracte plasmid according to the manufacturer’s instructions of plasmid Mini Kit I(OMEGA). By enzyme digestion and PCR, the recombinant SL7207(pVAXD-PS1-TS), SL7207(pVAXD-PS1) and SL7207(pVAXD-TS) can amplify and enzyme the corresponding stripe, the results showed that the recombinant plasmid was correct. Then cultured the strains and prepared the oral vaccines.15 piglets were randomly divided into five groups, each group had three piglets, including the immune group SL7207(pVAXD-PS1-TS), control group SL7207 (pVAXD-PS1),SL7207(pVAXD-TS), SL7207 (pVAXD) and PBS containing 5% NaHCO3.20 dpi piglets were oral immunization with the vaccines at dosage of 1.6×1011CFU,10ml per piglet, and booster immunization with 2.0×1011CFU after two weeks later. After immunization, no clinical changes and visible lesions were observed. Blood and fecal mucin were collected at weeks 0,2,4,6,8 after the primary immunization. The levels of serum IgG, fecal IgA, serum IFN-y and serum IL-4 levels in immunized piglets were analyzed with ELISA kits(R&D) according to the manufacturer’s instructions. Meanwhile, proliferation of T lymphocytes (MTT)and neutralizing antibodies were be tested.Humoral immunity and mucosal immunity results showed that the specific antib- ody in immune groups SL7207(pVAXD-PS1-TS) and control group SL7207(pVAXD-PS1), SL7207(pVAXD-TS) began to increase at 2 weeks and serum IgG antibody, fecal IgA antibody can be detected at 4 weeks, and reached a peak at 6 weeks; at 8 weeks, the antibodies levels had decreased. Neutralizing antibodies showed that SL7207(pVAXD-PS1-TS), SL7207(pVAXD-PS1) and SL7207(pVAXD-TS) can stimulate neutralizing antibodies PEDV neutralization antibody titer of SL7207 (pVAXD-PS1-TS) is 10-1.5 (1:37.17) and it was slightly lower than SL7207(pVAXD-PS1) 10-1.7 (1:50.25); TGEV neutralization antibody titer of SL7207 (pVAXD-PS1-TS) is 10-1.5 (1:31.64) and it was slightly lower than SL7207(pVAXD-PS1)10-1.58 (1:38.02).Cellular immunity results showed that IFN-γ and IL-4 in all piglets immunized with SL7207(pVAXD-PS1-TS), SL7207(pVAXD-PS1) and SL7207(pVAXD-TS) can be detected and had a significantly higher than SL7207 (pVAXD) and PBS at 6 weeks (p<0.01). Meanwhile, IFN-y and IL-4 in SL7207 (pVAXD-PS1-TS)were higher than SL7207(pVAXD-PS1) (p<0.05).T lymphocyte proliferation showed that the level of T lymphocyte proliferation began to increase at 2 weeks and reached the peak at 6 weeks, and the proliferation in piglets immunized with SL7207(pVAXD-PS1-TS), SL7207(pVAXD-TS), SL7207 (pVAXD-PS1) were highly significant (p< 0.01) relative to PBS and SL7207(pVAXD). At 6 weeks, proliferation of T lymphocyte from piglets immunized with SL7207(pVAXD-PS1-TS) was higher than SL7207(pVAXD-TS) and SL7207(pVAXD-PS1). Therefore, the SL7207(pVAXD-PS1-TS) showed the advantage in inducing host-specific cellular immune response than single gene.In summary, the results showed that SL7207(pVAXD-PS1-TS) had a good immunogenicity in piglets. It can stimulate comprehensive immune response include-ing the humoral, mucosal and cellular immunity. The most significant advantage of the vaccine was that it can simultaneously induce immune responses against TGEV and PEDV in piglets. Besides, SL7207(pVAXD-PS1-TS) had more advantages than single-gene vaccine in inducing host-specific cellular immune response. |
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