Broad Innate Immune Activation Enhances the Protective Efficacy of rBCG-LTAK63 Against Mycobacterium tuberculosis

Ana Carolina de Oliveira Carvalho¹Monalisa Martins Trentini²Dunia Rodriguez²Lázaro Moreira Marques-Neto²Paulo Henrique Santana Silveira²Nancy Starobinas²Sergio Costa Oliveira¹Luciana C C Leite²Alex Kanno^2*

• ¹University of São Paulo, São Paulo, Brazil
• ²Instituto Butantan, São Paulo, Brazil

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⁻/⁻, AIRmax and AIRminTT mice were used to evaluate inflammasome-associated responses using IL-1β as a primary readout. 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-1⁻/⁻ and Nlrp3⁻/⁻ BMDMs, indicating the contribution of additional processing pathways. Immunization of AIRmax and AIRminTT mice with BCG or rBCG-LTAK63 revealed differences in their ability to control Mtb infection. BCG only induced protection in AIRmax mice, while rBCG-LTAK63 induced protection in both genotypes. This demonstrates that protection is achieved in a diminished innate inflammatory response scenario, but its full engagement can further reduce pulmonary bacterial loads. In vivo characterization of T cell responses in these mice did not reveal a significant effect on the Th1/Th17 responses. On the other hand, a co-culture system of inflammasome-activated BMDMs and splenocytes showed that rBCG-LTAK63-primed macrophages strongly promoted CD4+ T cell activation and polarization toward Th1/Th17 responses. Together, these findings demonstrate that rBCG-LTAK63 enhances protection against TB through a broad innate activation including inflammasome-associated mechanisms.