ESAT-6-derived lipopeptide-based mucosal vaccine elicits distinct humoral immune responses concomitant with a reduction in mycobacterium infection.

Respiratory mycobacterial infections remain a major global health threat, yet current vaccine strategies, which focus primarily on cell-mediated immunity, have met with limited success. We previously demonstrated that a vaccine composed of five lipopeptides (LPs) encoding immunodominant ESAT-6 epitopes elicits protective cellular immune responses against Mycobacterium tuberculosis infection. In this study, we enhanced the ESAT-LPs vaccine with a novel mucosal immune modulator, heat-killed Caulobacter crescentus (HKCC), and examined both cellular and humoral immunity in a mouse model (BALB/c) of M. avium (Mav) infection. Our findings revealed that incorporating HKCC significantly increased cellular responses and mucosal IgA and systemic IgG titers in pre- and post-exposure models, respectively. These elevated antibody levels correlated with reduced mycobacterial burden in the lungs, spleens, and livers and with improved lung pathology. Notably, serum samples from vaccinated mice markedly reduced the intracellular Mav burden and activated antimicrobial mechanisms in an ex vivo bone marrow-derived macrophage infection model. Heat-inactivated serum samples also reduced intracellular Mav burden, suggesting a complement-independent intracellular bactericidal function of the tested serum samples. Collectively, the ESAT-LPs-HKCC mucosal vaccine induces a robust, distinct humoral response and a remarkable reduction in bacterial load in both prophylactic and therapeutic settings, offering a promising new strategy for combating mycobacterial infections.