Comparison Of Protective Responses Induced By Recombinant BCG Strains Overexpressing Antigen Ag85A Or Ag85B Against Experimental Mycobacterium Tuberculosis Infection In Mice

BackgroundVaccines are the most cost-effective medical intervention known to prevent TB. Bacillus Calmette-Guérin (BCG) is the only vaccine used against TB worldwide, but it has variable protective efficacies for adult. It is urgently needly to develop more effective and vaccine against TB.Recombinant Bacillus Calmette-Guerin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention. Ag85A and Ag85B have the stranger immunogenicity to be the promising target antigen for TB vaccines. We found that the different immune reaction could be induced by the mice immuned DNA vaccine encoding Ag85A or Ag85B protein. In this study, in order to gain further insight into the relationship between CMI and the long-term protective of BCG, two rBCG strains overexpressing Mycobacterium tuberculosis immunodominant antigens Ag85A (rBCG::85A) and Ag85B (rBCG::85B), respectively, were constructed, and we investigated the protective efficiency induced by rBCG in the mouse model, and has laid the foundation for the exposition cell-mediated immunity(CMI) mechanism of the recombinant BCG vaccine's long-term protection.Objectives1. To construct rBCG (rBCG::85A and rBCG::85B) strains;2. To determine the protective immunity induced by rBCG;3. To investigate the protective efficiency of immune response induced by rBCG in mouse model immuned rBCG and then challenged with H37Rv.Methods1. The recombinant shuttle expressing plasmids of pMV85A and pMV85B, were constructed. Recombinant plasmids amplified in E. coli DH5αwere transformed into BCG by electroporation. The expressing of the proteins in rBCG was measured by Western blotting;2. rBCG::85A and rBCG::85B strains were inoculated to C57BL/6 mice with injection. The humoral immune response and cellular immune response were detected by ELISA, FCAS and ELISPOT.3. After immunization, the BALB/c and C57BL/6 mice were challenged with M. tuberculosis H37Rv strain, the protective immunity of two rBCG was determined by the organs bacterial loads and organs histopathological examination of infected mice at different time-points.Results1. Three rBCG strains were obtained by transformation of BCG with the recombinant plasmids. The overexpression of proteins in both culture supernatants and cell lysates were analyzed by Western blotting. Ag85A protein was mainly found in the culture supernatant of rBCG::85A strain as a secreted protein and Ag85B protein was also confirmed to be present mainly in the culture supernatant of rBCG::85B strain.2. The humoral immune response and cellular immune response were observed in mice model. High titer of Ag85A and Ag85B specific IgG antibody were detected by ELISA. The mouse splenocytes immunized with rBCG were separated and cultured at 6 weeks and 24 weeks, when stimulated with Ag85B or Ag85A, the number of IFN-γsecreted cells increased significantly in both rBCG::85B and rBCG::85A groups. FACS showed that CD4+/CD8+ ratio of the lung increased in rBCG::85B, but decreased in rBCG::85A, and the ratio of the spleen increased in rBCG::85A, but a decline in rBCG::85B.3. The organs bacterial loads showed, both rBCG::85B and rBCG::85A inhibited the growth of M. tuberculosis more significantly in the lung and spleen than rBCG::261 at 4 weeks after challenge; and rBCG::85B vaccine reduced the bacterial load in the lung more significantly than rBCG::85A at 18 weeks after the challenge infection, at the same time, we concluded that the rBCG groups could produced more significant protective efficiency than BCG, as comfirmed by histopathological examination. Conclusions1. rBCG::85A and rBCG::85B are overexpressing Mycobacterium tuberculosis immunodominant antigens Ag85A and Ag85B, respectively. Long term protection was induced by both rBCG strains but the stronger protection was provided by rBCG overexpressing Ag85B protein.2. Our results indicate that systemic in vitro antigen-specific IFN-γcorrelated with the long term protection induced by rBCG strains and that the more enduring protection might be related with the early CD4+ T responses in the lung.