Pulmonary delivery of antigen-enhanced BCG overcomes safety barriers in immunocompromised hosts and protects against TB in the absence of adaptive immunity

The low efficacy of Bacille Calmette-Guérin (BCG) in preventing pulmonary tuberculosis (TB) underscores the need for improved TB vaccines. Recombinant BCG (rBCG) strains secreting the virulence-associated effector molecule ESAT-6 from Mycobacterium tuberculosis ( Mtb ) markedly improve efficacy and immunogenicity in animal models of TB but have been considered unsuitable for clinical translation due to safety concerns identified in intravenous SCID mouse models. Here, we demonstrate that pulmonary delivery fundamentally reshapes the safety and protective efficacy of the ESAT-6-secreting rBCG strains, BCG::RD1 and BCG::ESAT6-PE25SS. In sharp contrast to intravenous delivery, pulmonary administration was markedly better tolerated, improved survival, and reduced systemic dissemination and brain pathology of severely immunocompromised mice. Strikingly, pulmonary rBCG vaccination also conferred superior protection against aerosol Mtb challenge in wild-type, type 2 diabetic, and adaptive immunity-deficient Rag1 ^⁻/⁻ and Rag2 ^⁻/⁻ Il2rg ^⁻/⁻ mice, with BCG::RD1 showing the strongest adaptive immunity-independent protection. Mechanistically, pulmonary rBCG vaccination promoted lung innate immune activation, expansion of myeloid-biased progenitors in the bone marrow, and enhanced antimycobacterial activity of macrophages, consistent with trained innate immunity. Collectively, these findings reveal that pulmonary vaccination largely overcomes safety concerns of ESAT-6-secreting rBCG strains and provide preclinical evidence for a viable strategy to improve protection against TB in immunocompromised individuals.