Broad innate immune activation enhances the protective efficacy of rBCG-LTAK63 against Mycobacterium tuberculosis

Tuberculosis (TB) continues to be one of the leading infectious causes of mortality world-wide, while the Bacillus Calmette-Guérin (BCG) vaccine provides variable protection. To address this limitation, our group developed a recombinant BCG strain expressing a detoxified Escherichia coli heat-labile toxin subunit (rBCG-LTAK63), which confers superior protection against Mycobacterium tuberculosis (Mtb). However, the mechanisms underlying this enhanced efficacy remain to be better characterized. Here, we investigated the capacity of rBCG-LTAK63 to enhance inflammasome-associated innate immune responses and its impact on T cell activation and protection against Mtb.

Bone marrow-derived macrophages (BMDMs) from wild-type (C57Bl/6), Casp-1-/-, Nlrp3-/-, Aim-2-/- and phenotypically selected AIRmax and AIRmin^TT mice were used to evaluate inflammasome-associated responses using IL-1β as a primary readout. A co-culture system of inflammasome-activated BMDMs and splenocytes was employed to assess CD4⁺ T cell activation and polarization. Additionally, immunization of AIRmax and AIRmin^TT mice with BCG or rBCG-LTAK63 was performed to evaluate protection against Mtb.

rBCG-LTAK63 induced significantly higher IL-1β production than parental BCG. IL-1β production was largely ASC-associated and predominantly dependent on the NLRP3/caspase-1 axis; however, residual IL-1β production was still detected in Casp-/- and Nlrp3-/- BMDMs, indicating the contribution of additional processing pathways. Co-culture of inflammasome-activated BMDMs and splenocytes showed that rBCG-LTAK63-primed macrophages strongly promoted CD4⁺ T cell activation and polarization toward Th1/Th17 responses. In vivo, BCG only induced protection in AIRmax mice, while rBCG-LTAK63 induced protection in both AIRmax and AIRmin^TT genotypes.

This demonstrates that protection is achieved in a diminished innate inflammatory response scenario, but its full engagement can further reduce pulmonary bacterial loads. Together, these findings demonstrate that rBCG-LTAK63 enhances protection against TB through a broad innate activation including inflammasome-associated mechanisms.