An ESAT-6-convergent prime-boost vaccination combining recombinant BCG expressing Mycobacterium marinum ESX-1 and ESAT-6/GLA-SE improves TB protection.

Although Bacille Calmette-Guérin (BCG) protects children against disseminated tuberculosis (TB), its limited efficacy against adult pulmonary TB underscores the need for improved vaccination strategies. We previously developed a recombinant BCG strain expressing the ESX-1 type VII secretion system of Mycobacterium marinum (BCG::ESX-1Mmar), which enhances immunogenicity through cytosolic immune signaling while maintaining low virulence. Here, we evaluated an ESAT-6-convergent prime-boost vaccination strategy in which mice were primed with ESX-1-competent BCG::ESX-1Mmar and subsequently boosted with Mycobacterium tuberculosis (Mtb)-derived ESAT-6 formulated with the TLR4 agonist adjuvant GLA-SE. Compared with ESAT-6/GLA-SE boosting following parental BCG priming, the BCG::ESX-1Mmar-primed regimen robustly increased antigen-specific CD4⁺ T cells localized within the lung parenchyma. This strategy markedly enhanced polyfunctional Th1 responses against ESAT-6 and PPD, exceeding those induced by BCG::ESX-1Mmar alone or the conventional BCG-prime/subunit-boost approach. Importantly, ESAT-6 boosting of recombinant BCG::ESX-1Mmar conferred superior long-term protection against hypervirulent Mtb challenge and significantly reduced pulmonary inflammation. Together, these findings demonstrate that leveraging an ESX-1-competent recombinant BCG platform for targeted ESAT-6 boosting can overcome key limitations of classical BCG vaccination and represents a promising strategy for next-generation TB immunization.