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

INTRODUCTION: 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 detoxifiedheat-labile toxin subunit (rBCG-LTAK63), which confers superior protection against(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.

METHODS: Bone marrow-derived macrophages (BMDMs) from wild-type (C57Bl/6),-/-,-/-,-/- and phenotypically selected AIRmax and AIRminmice 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 CD4T cell activation and polarization. Additionally, immunization of AIRmax and AIRminmice with BCG or rBCG-LTAK63 was performed to evaluate protection against Mtb.

RESULTS: 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-/- and-/- BMDMs, indicating the contribution of additional processing pathways. Co-culture of inflammasome-activated BMDMs and splenocytes showed that rBCG-LTAK63-primed macrophages strongly promoted CD4T cell activation and polarization toward Th1/Th17 responses., BCG only induced protection in AIRmax mice, while rBCG-LTAK63 induced protection in both AIRmax and AIRmingenotypes.

CONCLUSION: 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.