Immunogenicity And Protective Efficacy Against Murine Tuberculosis Of A Prime-boost Regimen With Bcg And A DNA Vaccine Expressing ESAT-6 And Ag85A Fusion Protein

Background and ObjectivesHeterologous prime-boost regimens utilizing BCG as a prime vaccine probably represent the best hope for the immunity measure of novel anti-tuberculosis.ESAT-6 and Ag85A have been thought as two major immundominant antigen for TB vaccine.In this study, we firstly the cloned fusion gene of ESAT-6 and Ag85A encoding expressing the fusion protein of Ag85A and ESAT-6 (r685A).Then,we constructed eukaryotic expression plasmid pcD685A and procaryotic expression plasmid pPro685A.The Immunogenicity and protective efficacy against murine tuberculosis of a prime-boost regimen with BCG and a DNA vaccine expressing ESAT-6 and Ag85A fusion protein was invertigated.Methods1. The gene fbpA and esxA encoding M. tuberculosis Ag85A and ESAT-6 protein were amplified by PCR from M. tuberculosis H37RV strain respectively. The fusion gene esxA- fbpA was generated by gene splicing with the overlap extension (GeneSOEing) method. The recombinant r685A gene was cloned into the corresponding sites of prokaryotic expression vector pProEXHTb and eukaryotic expression vector pcDNA3.1(+), resulting in recombinant plasmids named pPro685A and pcD685A, respectively.2. Transform the prokaryotic expression recombinant plasmid pPro685A into the competence Escherichia coli BL21 cells.The protein r685A was induced and purified. Protein purification was performed using a Ni-NTA purification system. These processes were examined by SDS-PAGE and verified by Western blotting. The purified product of r685A protein was further quantitated by BCA method.3. Transform the recombinant plasmid pcD685A into the competence Escherichia coli DH5αcells.Plasmid DNA was extracted and purified in scale, and the concentration was determined by spectrophotometric method.4. C57BL/6 mice were immunized with a BCG prime and pcD685A booster. IgG antibodies against r685A protein were determined by ELISA, and the expression of IFN-γand IL-10 in lung tissue was determined by qRT-PCR.5. After immunization, the mice were challenged with M.tb H37Rv strain, the protective immunity was determined by the organs bacterial loads and organs histopathological examination of infected mice.Results1. The recombinant prokaryotic plasmid pPro685A and eukaryotic plasmid pcD685A were constructed sucessfully.2. The expression of r685A was examined by SDS-PAGE and verified by Western blotting. The purified protein of r685A was congfirmed by SDS-PAGE.3. As expected, the most significant IgG-specific response to r685A protein was induced in BCG prime and pcD685A DNA booster group, when r685A protein was used for detection. IFN-γresponse increased in all groups except the vector control group. Mice vaccinated with BCG plus pcD685A induced the highest levels of IFN-γresponses in the lungs.Combination with BCG increased significantly the expression of IFN-γwhen compared with BCG or pcD685A alone. 4. The most significant reduction in bacterial load of both spleen and lung was obtained in mice vaccinated with BCG prime and pcD685A DNA booster when compared with BCG or pcD685A alone.5. After challenged with virulent M.tb H37Rv, as showen in histopathological examination, the most slight variation was in BCG prime and pcD685A DNA booster group.ConclusionThus,our study indicates that pcD685A may be an efficient booster vaccine against TB with prior BCG immunity.