Hsp16.3ΔC4, a C-terminal truncated mutant, confers strong cellular and humoral immunity against pulmonary tuberculosis

Despite its widespread use, the Bacillus Calmette-Guérin (BCG) vaccine offers limited and variable protection against adult pulmonary tuberculosis, highlighting the urgent need for next-generation vaccines that can induce durable protective immunity. We report here the development and protective efficacy of a C-terminal truncated Hsp16.31-144 variant of Mycobacterium tuberculosis. Subcutaneous immunization with Hsp16.31-140/Hsp16.3ΔC4 significantly reduced pulmonary bacterial burden and pathology by enhancing both cellular and humoral responses. Notably, co-administration of Hsp16.3ΔC4 with BCG synergistically enhanced Th1 immunity through IFN-γ and IL-2 production, concomitantly eliciting superior activated memory T and memory B cell responses. Adoptive transfer experiments further demonstrated that CD4⁺ T cells, but not CD8⁺ T cells, were the primary effectors responsible for conferring protection against the pathogen. Together, these findings establish Hsp16.3ΔC4 as a promising subunit vaccine candidate that, alone or in combination with BCG, promotes robust antigen-specific Th1 and memory responses, thereby enhancing long-term protection against pulmonary tuberculosis.