| [Objective]BCG, a widely used attenuated live vaccine with short period of protection, could not provide effective protection for latent tuberculosis infected people (LBTI) and adult TB. Therefore, it was urgently needed to develop new types of vaccines targeting not infected or LTBI with BCG vaccinated.Based on the pathogenesis and stage-specific of antigens, we chose and constructed several antigens with technique of gene engineering from different stages during the growth period of M. tuberculosis. WBIA was utilized to compare the difference of levels of IFN-y, and to identify whether candidate antigens could be recognized by cell immune system of M.tb infected Chinese population, which is helpful in screening predominant antigens expressed in different stages after M.tb infected and could develop new vaccines. Then, considered the protective immunogenicity and clinical trials of different adjuvant, we constructed a novel adjuvant MTO. Finally, a fusion protein A1D4containing multi-stage antigens was constructed, and delivered with MTO. The immunogenicity and protective effect of subunit vaccine were confirmed through C57BL/6mice model.[Methods]1. Prokaryotic expression and purification of target proteins. Prokaryotic expression vectors contained the predominant antigens expressing during the exponential growth phase (Ag85B) and latent infection (Rvl813, Rv2660c, Rv2623, HspX), also the fusion protein A1D4based on these antigens, were constructed and confirmed by SDS-PAGE and Western-blotting.2. Evaluated the recognition of proteins with M.tb infected Chinese population. Firstly, Clinical diagnosis standards were performed to differentiate active pulmonary TB patients, latent TB infections and healthy contacts from all subjects. In order to avoid influence of BCG vaccination, blood samples from area of high TB prevalence were gathered, and TB infected or uninfected subjects were identified by rCM-WBIA standard which was established by our past work. Blood samples from different subjects were stimulated with individual protein, and the levels of IFN-y were detected and analyzed by Human IFN-γ ELIS A kit.3. Construction and immunization of subunit vaccine. Subunit vaccine A1D4/MTO was combined with100μl MTO and100μl A1D4protein (0.2μg/μl). C57BL/6mice were immunized (s.c.) with A1D4/MTO repeated for three times with3weeks intervals. PBS group (negative control), MTO group and BCG group (positive group) were set as different controls.4. Nine weeks after immunized, serum and splenocytes were collected to analyze the immunogenicity of subunit vaccine.(1) Evaluation of humoral immune response. AlD4-specific IgG, IgGl and IgG2a titers were detected by ELISA.(2) Splenocytes were incubated with RPMI1640(negative control), PPD (positive control) and A1D4protein to stimulate the secretion of cytokines, including:IFN-y, TNF-a and IL-2. The concentration of cytokines in culture supernatants were detected by ELISA.(3) A1D4specific CD4+T and CD8+T lymphocytes which secret for single or multi cytokines were analyzed by flow cytometry and ICS. PPD served as positive control and RPMI1640as negative control.5、Protective effect of subunit vaccine. After the repeated immunized with subunit vaccine A1D4/MTO for9weeks, mice were challenged with1.2×106CFU M.tb H37Rv. Four weeks after infection, mice were sacrificed. Survival rate and bacterial loads of the lungs and spleens were analyzed.[Results]1. Expression and confirmation of proteins. The purities and biological activities of A1D4and individual proteins were confirmed by SDS-PAGE and Western-blotting. 2. Selected antigens could be well recognized by M.tb infected Chinese population. Each individual protein could induce statistically higher levels of IFN-γ in rCM-WBIA positive group after stimulation (P<0.05). By using WBIA, it is clear that A1D4protein could induce statistically higher levels of IFN-γ in rCM-WBIA (+) group after stimulation (P<0.05). Moreover, the concentration of IFN-γ stimulated by A1D4is statistically higher than that of individual protein.3. A1D4/MTO induced higher levels of A1D4-specific IgG, IgG2a and IgG1antibodies, and the ratio of IgG2a/IgG1indicated that the subunit vaccine mainly induced the Th1-type cell-mediated immune response.4. A1D4/MTO induced antigen-specific cell mediated immune response.(1) A1D4/MTO induced higher levels of A1D4specific IFN-γ, TNF-α and IL-2.(2) The total IFN-γ+CD4+and IFN-γ+CD8+T cells in A1D4/MTO group were highest among all groups, especially significantly higher than PBS and MTO groups (P<0.05), no matter stimulated with PPD or A1D4. The total TNF-α+CD4+T cells after PPD stimulation, also the total TNF-α+CD4+and CD8+T cells after A1D4stimulation in A1D4/MTO group were significantly higher than PBS and MTO groups (P<0.05), respectively. Further analyzed the multifunctional T cells in different groups. A1D4-specific IFN-γ and IFN-γ+IL-2+production CD4+T cells in A1D4/MTO group were significantly higher than MTO and PBS group (P<0.05), respectively. Moreover, IFN-γ+, IFN-γ+IL-2+, IFN-γ+TNF-α+and IFN-γ+TNF-α+IL-2+production CD8+T cells in A1D4/MTO group were statistically higher than PBS group (P<0.05). IFN-γ+and IFN-γ+TNF-α+production CD8+T cells were even higher than BCG group (P<0.05).5. Immunology protection:The bacterial load of A1D4/MTO group was lower than PBS group for both lung and spleen organ (P<0.001). The degree of lesion in the lungs of A1D4/MTO group was lower than PBS control and MTO group, and few acid-fast positive bacterial could be found. Survival rate of A1D4/MTO immunized mice reached83.33%.6. Characteristic of adjuvant MTO. MTO indicated certain protective efficacy than PBS group with lower bacterial load than PBS group. The degree of lesion in the lungs of MTO group was similar to that of PBS group. Survival rate of MTO immunized mice reached83.33%. MTO immunized mice induce higher level of PPD or A1D4specific TNF-a and IL-2than PBS group. Moreover, PPD-specific total IFN-γ+CD8+T cells, A1D4-specific total IFN-γ+or TNF-α+CD4+and CD8+T cells were statistically higher than PBS group. Also, A1D4-specific IFN-γ+production CD8+T cells in MTO group were also statistically higher than PBS group.[Conclusion]Based on the predominant antigens expressed during different stage after M. tb infected, which could be recognized by M.tb infected Chinese population, a subunit vaccine named A1D4/MTO for Mycobacterium tuberculosis was constructed. A1D4/MTO could provide significant protective efficacy against acute infection of M.tb, although it is not as good as BCG. The protective effect mainly attributed to the A1D4-specific CD4+Thl-type cell immune response, especially the increasing of A1D4-specific IFN-γ+IL-2+production CD4+T cells and IFN-γ+, IFN-γ+IL-2+, IFN-γ+TNF-α+, IFN-γ+IL-2+TNF-α+CD8+T cells. Moreover, the increase of A1D4-specific IFN-γ+, IFN-y+TNF-α+production CD4+T cells and IFN-γ+, IFN-y+TNF-α+CD8+T cells indicated that this vaccine could make up the shortcoming of BCG. This study had built a stable fundament to further evaluate the efficacy of A1D4/MTO utilized as a boost vaccine after BCG primed in mice model. |
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