Mycobacterium tuberculosis antigen-containing exosomes reinforce BCG vaccine efficacy by augmenting long-term protection and memory response against experimental tuberculosis in BALB-C mice

Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>M.tb.</i>), affects one-third of humanity. Despite the availability of effective drug regimens, complete eradication of <i>M.tb.</i> remains challenging due to prolonged treatment duration. Additionally, MDR-TB and co-infection with HIV further exacerbate disease severity. The Bacille Calmette-Guérin (BCG) vaccine has shown inconsistent efficacy due to the absence of Th-1 antigens. Hence, there is a critical need for either a novel vaccine candidate or an efficient booster to enhance BCG's prophylactic efficacy. In this study, in-house prepared <i>M.tb.</i>-infected alveolar macrophage-derived exosomes (Rv-Exo) and ESAT-6-containing exosomes (ESAT-6 Exo) were characterized based on size, purity, and pathogen-associated molecular patterns (PAMPs), and their epitope mapping was also performed. These <i>M.tb.</i> protein-containing exosomes (MPEs) were utilized for immunization, either alone or as a booster to BCG, and evaluated in BALB/c mice against experimental <i>M.tb.</i> challenge. Our results demonstrate that ESAT-6 Exo and Rv-Exo, either alone or as a BCG booster, enhanced Th-1-biased immune responses by activating CD4⁺ and CD8⁺ T cells, increasing memory T-cell populations, and significantly reducing the <i>M.tb.</i> burden in the lungs, spleen, and lymph nodes of infected mice. These findings highlight the potential of MPE as a promising strategy against TB especially in the BCG-vaccinated population.