Protection Against Tuberculosis By Vaccination With A Recombinant Bcg Strain Over-expressing HspX Protein

Background and objectiveAg85B is a protective antigen secreted by M. tb during log-phase growth in vitro, and the protective effect of recombinant BCG strain overexpressing antigen Ag85B against infection with M. tb is better than that of the parent BCG, which has been finished Phase I clinical test. Compared with Ag85B, HspX is a major protein expressed by M. tb during stationary stage in vitro or dormancy in vivo, and BCG only expresses a small amount of HspX during oxygen depletionor upon infection with macrophage. HspX could induce intensive Th1 immune response. Although it is capable of inducing Th1 responses to some important immunodominant antigens secreted by M. tb, BCG does not induce IFN-γimmune responses to HspX. We identified three recombinant BCG strains including recombinant BCG strain over-expressing antigen HspX (rBCG::X) or Ag85B (rBCG::85B), BCG strain containing the vector pMV261 (rBCG::261). To evaluate the immunogenicity and protective efficacy of these vaccines, recombinant BCG strains were used to immunize the mice.Methods1. Analyze the bio-informatics of hspX gene of M. tb sequences by NCBI BLASTn.2. Recombinant protein HspX or Ag85B was expressed from E.coli BL21 (DE3) strain containing pProHspX or pProAg85B induced by IPTG and purified by Ni-NTA technology, respectively.3. Both cytosol and supernatant proteins of three recombinant BCG strains cultures were analyzed by Western blotting with anti-Ag85B and anti-Rv2031c polyclonal antibody, respectively.4. C57BL/6 mice were immunized with rBCG::X, rBCG::85B and rBCG::261, respectively. Six and 24 weeks after immunization, immunogenicity of each recombinant BCG strain was analyzed by detecting antigen-specific antibody IgG responses and interferon-gamma (IFN-γ) assay.5. C57BL/6 and BALB/c mice were immunized with rBCG::X, rBCG::85B and rBCG::261, respectively. Six weeks after immunization, the immunized mice were infected by M. tb. Four weeks after infection, protective efficacy between different genera of mice was evaluated by bacterial load of spleen and lung and lung pathology. 18 weeks after infection, we detected the bacterial load of spleen and lung and lung pathology of C57BL/6 mice, and evaluated the long-term protective efficacy.Results1. The hspX gene were analysed by NCBI BLASTn and found seven mycobacterial strains have the same gene sequences as M.tb H37Rv strain (identities=100%), all of which belong to M. tb complex and BCG;espetially, one strain belongs to MDR-TB.2. SDS-PAGE and Western blotting of recombinant protein HspX or Ag85B showed high purity and protein activity.3. rBCG::X strain expressed high levels of both HspX protein in the cytosol and Ag85B protein in the cytosol and supernatant.4. C57BL/6 mice immunized rBCG::X produced higher antibody titres against HspX or Ag85B than rBCG::261 strain at 6 and 24 weeks (p < 0.05). IFN-γ ELISPOT assay showed the number of IFN-γsecreting cells stimulated with HspX or Ag85B in rBCG::X group was much more than that of rBCG::261 groups at 6 and 24 weeks (p < 0.05).5. Mice vaccinated with rBCG::X produced a more consistent and enduring protective effect against infection with M. tb, showing lower bacterial load in lung (p < 0.05) and less severe lung pathology, than the control mice vaccinated with rBCG::261. The protection induced by rBCG::X was slightly better than rBCG::85B group (p < 0.05).Conclusion1. The long-term protection induced by rBCG::X was associated with the more thickened cell wall and significant increases in antigen-specific IFN-γto both HspX and Ag85B proteins.2. Our research supports the hypothesis that mycobacterial antigens that induced differential immune responses between BCG-vaccinated individuals and LTBI are the key targets to develop recombinant BCG vaccines for inducing M. tb-like protective response, which suggest that latency antigens of M. tb may be promising targets for developing more effective recombinant BCG strains to protect against TB.