rBCG-LTAK63 Enhances Protection Against Tuberculosis by Inducing Autophagy and Circadian Gene Regulation

Lázaro Moreira Marques-Neto¹Monalisa Martins Trentini¹Ana Carolina Ramos Moreno¹Silas F. Eto²Ana Carolina de Oliveira Carvalho¹Almiro Neto³Murilo Sena Amaral⁴André Guilherme Costa-Martins^5,6André Nicolau Aquime Gonçalves^6,7Ana Marisa ana.tavassi@butantan.gov.br²Alex Kanno¹Aldo Tagliabue⁸Diana Boraschi⁹Sergio Verjovski-Almeida^10,4Helder Nakaya^11,7Leonardo Paiva Farias³Pablo Ivan Pereira Ramos¹²Luciana C C Leite^1*

• ¹Instituto Butantan, Laboratório de Desenvolvimento de Vacinas, São Paulo, Brazil
• ²Instituto Butantan, Center of Excellence in New Target Discovery (CENTD), São Paulo, Brazil
• ³Instituto Gonçalo Moniz, Laboratório de Medicina e Saúde Pública de Precisão (MeSP2), Fundação Oswaldo Cruz (FIOCRUZ-BA), Salvador, Brazil
• ⁴Instituto Butantan, Laboratório de Ciclo Celular, São Paulo, Brazil
• ⁵Universidade de Sao Paulo Departamento de Analises Clinicas e Toxicologicas, São Paulo, Brazil
• ⁶Institute for Technological Research, Micromanufacturing Laboratory, São Paulo, Brazil
• ⁷Hospital Israelita Albert Einstein, São Paulo, Brazil
• ⁸Shenzhen Institutes of Advanced Technology (SIAT), Shenzhen, China
• ⁹Shenzhen University of Advanced Technology (SUAT), Shenzhen, China
• ¹⁰Universidade de São Paulo, Instituto de Química, Departamento de Bioquímica, São Paulo, Brazil
• ¹¹University of São Paulo. Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, São Paulo, Brazil
• ¹²Instituto Gonçalo Moniz, Centro de Integração de Dados e Conhecimentos para Saúde (CIDACS), Fundação Oswaldo Cruz (FIOCRUZ-BA), Salvador, Brazil

Tuberculosis (TB) remains a global public health challenge, with the current BCG vaccine providing limited efficacy in adults, and available treatments being lengthy and debilitating. To overcome these challenges, we have previously developed a recombinant BCG strain expressing the detoxified E. coli Heat-Labile Toxin (LTAK63), providing increased protection in mouse models and reduced lung pathology. Here, using systems biology and RNA sequencing of lung tissues in a murine model, we uncover the molecular mechanisms underlying rBCG-LTAK63's increased protection. Immunization triggered early activation of cAMP-related pathways, leading to hypoxia, autophagy, and circadian rhythm gene regulation. These processes were associated with an enhanced innate immunity and promoted long-lasting Th1/Th17 adaptive responses. Upon challenge, mice immunized with rBCG-LTAK63 exhibited an earlier onset of interferon-gamma response, reduced bacterial burden, and improved lung histopathology. Notably, circadian rhythm regulation was directly linked to a controlled inflammatory response and reduced migration of infection-susceptible cells, resulting in decreased immunopathology. Our findings demonstrate that rBCG-LTAK63 orchestrates protection through the integration of metabolic and temporal immune pathways. This work provides mechanistic insights into how rational vaccine design can reprogram host immunity to enhance protection and reduce pathology, supporting rBCG-LTAK63 as a promising next-generation TB vaccine candidate.