Immunoprotection Of Recombinant Treponema Pallidum Adhesins In A Murine Model

Objective:To observe the specific immune response levels and protective effect in C57BL/6 mice induced by immunization alone or in combination of recombinant Treponema pallidum(Tp)adhesins rTp0751,rTp0136~N and rTp0435,and to provide basis for screening candidate molecules of syphilis vaccine and establishing a new animal model of syphilis vaccine.Methods:The prokaryotic expression vectors pET-30a-Tp0136~N,pET-30a-Tp0435,and pET-30a-Tp0751were induced to express the recombinant proteins.C57BL/6 mice were randomly divided into 5groups(group A:PBS;group B:rTp0136~N,group C:rTp0435;group D:rTp0751;group E:rTp0136~N+rTp0435+rTp0751)and immunized with purified proteins mixed with Freund's adjuvant for 3 times at 2-week intervals.The tail vein blood was collected and serum was separated before immunization at week 2,week 4,and week 6 after primary immunization.The levels of specific IgG in sera were measured by ELISA.Mice were sacrificed 2 weeks after the last immunization,and Flow Cytometry was performed to detect the levels of intracellular IL-4and IFN-?in mice spleen cells.Mice were challenged subcutaneously,intragenitally(percutaneously in the corpus cavernosa)and by gavage three weeks after the last immunization.42 days post challenge,T.pallidum burden was evaluated using real-time quantitative PCR(qPCR)to measure Tp-DNA concentrations in the heart,spleen and brain of mice.Results:SDS-PAGE analysis of recombinant Tp0136~N,Tp0435 and Tp0751showed obvious bands at the expected positions.The titers of specific IgG in sera in all immunized mice gradually increased to significantly higher level as compared to the PBS control mice(P<0.01),which reached its peak at week 6 after primary immunization(2 weeks after the last immunization).There was no significant difference between the single immunization groups and the combined immunization group.The levels of IFN-?in CD4~+cells in all immunized mice were significantly higher than those in the PBS control mice(P<0.01),with higher level in single immunization groups than the combined immunization group(P<0.05).The levels of IFN-?in CD8~+cells in all immunized mice were significantly higher than those in the PBS control mice(P<0.01),but there was no significant difference between the single immunization groups and the combined immunization group.The levels of IL-4 in CD4~+cells in all immunized mice and control mice were very low.Treponemal burden in spleen and heart extracts revealed lower DNA concentrations in all single(except for Tp0435)immunized and combined immunized mice compared to the control group(P<0.05),there was no significant difference between the single immunization groups and the combined immunization group.Lower DNA concentrations in rTp0136~N and rTp0751 immunization group were observed than compared to the control group(P<0.05),but there was no significant difference between rTp0435,combined immunization immunization group and the control group.Conclusions:1.The recombinant proteins Tp0136~N,Tp0435,Tp0751 and Tp0136~N,Tp0435,Tp0751 combined immunization can effectively induce humoral and cellular immune responses in mice.2.Recombinant proteins Tp0136~N,Tp0751 and Tp0136~N,Tp0435,Tp0751 combined immunization can inhibit T.pallidum dissemination in mice.However,combined immunization did not show more effective immunoprotection than single immunization.