نبذة مختصرة : The only available vaccine against pulmonary tuberculosis is Bacille Calmette-Guerin (BCG). As the efficacy reported of the vaccine is not up to the mark, there is an urgent need to develop improved anti-tuberculosis vaccines. Antigen 85B (Ag85B) is a very promising vaccine candidate molecule of Mycobacterium tuberculosis and interferon (IFN)-γ and has been considered the most attractive correlate of protective immunity. The aim of this study was to construct a novel recombinant BCG (rBCG) to secrete Ag85B and mouse IFN-γ under control of the Mycobacterial heat shock protein 60 (hsp60) promoter and the antigen signal sequence. Second aim of the present study is to evaluate the immune response in C57BL/6 elicted by the new rBCG. Expression of the fusion protein was readily detectable by western blotting and IFN-γ bioactivity was detected indirectly by enzyme-linked immunosorbent assay (ELISA). Compared with BCG, rBCG::Ag85B-IFN-γ was substantially more active in inducing the production of IFN-γ and tumor necrosis factor (TNF)-α from mouse splenocytes. ELISA analysis for IgG, IgG1 and IgG2c showed that rBCG::Ag85B-IFN-γ induced higher titer of Ag85B and facilitated Th1 type immune response. rBCG::Ag85B-IFN-γ also improved nitric oxide production levels and enhanced antigen-specific splenocyte proliferation. Moreover, rBCG::Ag85B-IFN-γ induced human monocytes such as THP-1 cells to enhance expression of CD80, CD86, CD40 and HLA-DR. Flow cytometry analysis confirmed that rBCG::Ag85B-IFN-γ significantly activated CD4+ T cells. Assessing combinations of IFN-γ, TNF-α and interleukin-2 at the single-cell level by multiparameter flow cytometry, we found that rBCG::Ag85B-IFN-γ improved the multifunctional T cells level in comparison to BCG. In conclusion, the present study indicates that rBCG::Ag85B-IFN-γ increases cell mediated immune response and is a potential candidate vaccine for immunotherapeutic protocols against pulmonary tuberculosis.
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