Immunogenicity And Protective Efficacy Of A Novel Cocktail Recombinant BCG Strain

Background and Objective:Mycobacterium bovis Bacille Calmette-Guerin (BCG) has been widely used as a prophylaxis vaccine against tuberculosis. It could effectively prevent children from serious tuberculosis, while its protective effect among the adult is unstable. BCG mainly induces specific CD4+Th1type immune response, but its efficacy gradually decreased with time, which only last for10to15years; on the other hand, its ability to induce the CD8+T cell response is weak, which could not prevent the reactivation of the latent tuberculosis infection. Based on the advantages and insufficient of BCG, we want to construct an enhanced recombinant BCG (rBCGs) strain named ABX by combining our previous constructed three rBCGs: rBCG::85A, rBCG::85B and rBCG::X, which were overexpressed the immunodominant antigens including Ag85A, Ag85B and HspX, respectively. The short-term protective effect and immunogenicity was evaluated and compared between the ABX and parental BCG in vaccinated C57BL/6mice. We aim to establish a possible basis for pre-clinical investigation of the enhanced ABX.Methods:1. Evaluation the protective effect of the ABX against acute M.tb H37Rv infection. The rBCG::85A, rBCG::85B and rBCG::X were equally mixed to construct the ABX. The C57BL/6mice were immunized by subcutaneous injection with either the ABX (the experimental group) or parental BCGs (the positive control group) at a dose of 106CFU per100μl. PBS was used as the negative control. Eight weeks after the immunization, the mice were challenged i.v with M.tb H37Rv (1.2×106CFU). Four weeks later, the protective effect of the ABX was evaluated by analysis of bacterial load, histopathological change and short-term survive rate.2. Analysis the cellar immune responses induced by the ABX. After immunized theC57BL/6mice twelve and thirty-two weeks, the splenocytes were collected.(1) The level of antigen specific TNF-α, IFN-γ, IL-2and IL-4secreted from splenocytes were detected by ELISA.(2) The number of antigen-specific CD4+T cells which secrete TNF-α, IFN-γ and IL-2were counted by flow cytometry (FCM) and intracellular cytokines staining (ICS).(3) The cytotoxicity of Ag85B-specific CD8+T cells was analyzed by the CFSE-labeled method in vivo.3. Evaluation the immune memory level of the ABX. The number of antigen-specific TCM (CD62Lhi CD44hi) and TEM (CD62L10CD44hi) in CD4+/CD8+T cells were measured by FCM. Further, the amount of IFN-γ production TEM and IL-2production TCM were analyzed by intracellular cytokines staining.4. Evaluation the humoral immune response of the ABX. The titers of antigen-specific IgG, IgG1and IgG2a in mice sera were detected by ELISA.Results:1. The protective effect of the ABX against M. tb acute infection: The bacterial load in the lung of the ABX immunized mice were significantly reduced (P=0.001) and0.585log lower than that in parental BCG group. There was slight histopathological change in the lung of the ABX immunized mice, only few acid-fast bacteria were found. The short-term survival rate was100%in the ABX group.2. Antigen-specific cell-mediated immune responses induced by the ABX:(1) The levels of Ag85A, Ag85B and HspX-specific TNF-α and IFN-γ produced by the immunized mice in the ABX group were increased after twelve and thirty-two weeks. The level of PPD, Ag85B and HspX-specific IL-2produced by the immunized mice in the ABX group was higher than that in the parental BCG group (p<0.05) after twelve weeks. Moreover, the induced levels of TNF-α, IFN-γ and IL-2were higher after thirty-two weeks than that after twelve weeks in the ABX group. However, the level of the antigen specific IL-4was lower than30pg/ml and there was no difference among all the groups.(2) The number of CD4+T cells which secrete TNF-α and IFN-γ in the ABX group were more than that in parental BCG group after twelve and thirty-two weeks. The number of IL-2+cells was increased with the prolonged of the immune time in the ABX group. Most of the CD4+T cells were single TNF-α+or IFN-γ+or IL-2+cells or TNF-α+-IFN-γ+double positive cells in ABX group, which were more than that in parental BCG group (p<0.05). Both the number of Ag85A specific triple positive cell and the number of Ag85B specific IFN-γ+-IL-2+double positive cells in ABX group were higher than that in parental BCG group (p<0.05)(3) The specific killing rates in rBCG::ABX group were3times than that in parental BCG group (p<0.001) after twelve and thirty-two weeks; the activity was keep at high level until thirty-two weeks.3. The immune memory level of the ABX. When stimulated with CD4antigenic peptide (Ag85A, Ag85B and HspX), the number of TCM and TEM cells in ABX group were more than that in parental BCG group after twelve and thirty-two weeks (p<0.05), and the TEM was the main memory cell in ABX group (P<0.05). When stimulated with CD8antigenic peptide, Tcm was the main memory cell in ABX group. The number of the IL-2produced Tcm cells and IFN-y produced TEM cells were more than that in parental BCG group (P<0.05).4. The humoral immune response of the ABX: The level of the antigen-specific IgG and its subtype, especially IgG2a in ABX group was higher than that in parental BCG group after thirty-two weeks (P<0.05). And the ratio of IgG2a:IgG1was greater than1, which implied that the antibody response was Thl type immunity.Conclusion:The protective effects induced by the ABX were better than the parental BCG. The ABX vaccine could induce the stable and longer-lasting antigen specific Thl-type cell mediated immune response and elicit specific multifunctiona CD4+T cells which produced either IFN-γ+or TNF-α+or IL-2+or IFN-γ+/TNF-α+. The ABX vaccine also enhanced specific CD8+T killing activity and maintained higher level of memory cells and IgG2a.These findings indicated that the ABX vaccine might be an ideal candidate to be further used in clinic to protect TB.