A Mycobacterium tuberculosis multi-epitope DNA vaccine encoding adaptive immune antigens provokes IFNγ/Th1 immunity and confers potential protection

Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (MTB), remains a significant global health threat. However, the licensed Bacille Calmette-Guérin- (BCG) vaccine provides only limited protection in adults, underscoring the urgent need for more effective preventive strategies. Recent studies have shown that multi-epitope DNA vaccines are superior to traditional vaccines in terms of immunogenicity, safety and stability. In this study, we develop a multi-epitope DNA vaccine that contains CD8 ⁺ T-cell epitopes, CD4 ⁺ T-cell epitopes, and B-cell epitopes using bioinformatics tools. These epitopes are derived from three genome-encoded proteins, ESAT-6, Rv2660c, and RpfB, which exhibit stage-specific immunodominance in the early, resting, and convalescent stages of MTB infection. Using reverse vaccinology and computational immunomodulation, we demonstrate that the multiepitope vaccine increases antigen-specific antibody titres, activates CD8 ⁺ T and CD4 ⁺ T cells, and enhances IFN-γ secretion. <i>In vitro</i> validation studies in HEK293T cells confirm high-yield expression of multi-epitope-encoded antigens, whereas <i>in vivo</i> immunization experiments reveal significant expansion of NK cells and Th1-polarized lymphocytes, with concomitant upregulation of pro-inflammatory mediators. Collectively, these results highlight the potent activation of adaptive immunity through Th1-driven mechanisms and IFN-γ-mediated mycobacterial clearance, which are crucial for defending against MTB.