Immunotherapeutic efficacy of recombinant<i>Mycobacterium smegmatis</i>expressing Ag85B–ESAT6 fusion protein against persistent tuberculosis infection in mice

The application of immunotherapy in combination with chemotherapy is considered an effective treatment strategy against persistent Mycobacterium tuberculosis (Mtb) infection. In this study, we constructed a novel recombinant Mycobacterium smegmatis (rMS) strain that expresses Ag85B and ESAT6 fusion protein (AE–rMS). Immunization of C57BL/6 mice with AE–rMS generated mainly Th1-type immune responses by strongly stimulating IFN-γ- and IL-2-producing splenocytes and increasing antigen-specific cytotoxic T lymphocyte (CTL) activity. To test the immunotherapeutic efficacy of AE–rMS, a persistent tuberculosis infection (PTBI) model was established via tail-vein injection of C57BL/6 mice with 1 × 104 colony forming units (CFU) of Mtb strain H37Rv in combination with concurrent chemotherapy drugs isoniazid (INH) and pyrazinamide (PZA). PTBI mice immunized with AE–rMS showed high levels of IFN-γ secreted by splenocytes and decreased bacteria loads in lung. Treatment with only the anti-tuberculosis (anti-TB) drugs RFP and INH (RI), decreased bacteria loads to low levels, with the Th1-type immune response further attenuated. Moreover, AE–rMS, when combined with RI treatment, further reduced the bacteria load as well as the pathological tissue damage in lung. Together, these results demonstrated the essential roles of AE–rMS-induced Th1-type responses, providing an effective treatment strategy by combining AE–rMS and RI for persistent TB.