• A novel multi-epitope DNA vaccine targeting ESAT-6, Rv2660c, and RpfB induces robust IFN-γ/Th1 immune responses, enhancing both humoral and cellular immunity against Mycobacterium tuberculosis.
• Reverse vaccinology and immunoinformatics enabled precise selection of CD8+ T, CD4+ T, and B-cell epitopes, demonstrating the power of computational design in vaccine development.
• In vitro and in vivo validation confirmed high antigen expression and significant expansion of NK cells and Th1-polarized lymphocytes, with upregulation of pro-inflammatory mediators.
• This vaccine represents a promising candidate for improving TB prevention, addressing the limitations of BCG and advancing toward clinical translation.