Enhancing Mycobacterial Clearance via Heterologous BCG Prime Boost Strategy Using Recombinant Mycobacterium smegmatis

Bacille Calmette–Guérin (BCG) remains the only licensed tuberculosis (TB) vaccine, yet its protective efficacy against adult pulmonary TB is limited. To overcome this, we evaluated a novel recombinant Mycobacterium smegmatis (rMs) booster vaccine expressing Ag85B and ESAT-6 via the pMyong2 vector, combined with oat-derived β-glucan adjuvant, to enhance BCG-primed immunity. BALB/c mice were primed with BCG and boosted twice with homologous BCG or varying doses of rMs with or without β-glucan. Five weeks post-booster, mice were challenged intraperitoneally with M. tuberculosis H37Ra. Systemic bacterial clearance in liver tissue, cell-mediated immunity IFN-γ, IL-17a, and antibody responses IgG, IgG2c, IgA were evaluated 3 weeks post-challenge. All rMs booster groups (rMs, high-dose rMs H, low-dose with β-glucan, rMs β) achieved complete systemic clearance of H37Ra with no detectable CFUs (colony forming unit), significantly outperforming mock-vaccinated and repeated BCG groups (** p < 0.01). Repeated BCG revaccination showed higher bacterial loads than single BCG (*** p < 0.001), confirming no booster effect. Low-dose rMs with β-glucan maintained 100% survival and total bacterial elimination despite a 50% dose reduction. Immune markers did not strictly correlate with clearance. Homologous BCG revaccination provides no additive protection, whereas pMyong2-based rMs confers robust systemic bacterial clearance. Adjuvanting with β-glucan enables dose reduction while preserving maximum efficacy, establishing rMs as a promising heterologous TB booster candidate.