Supplementary file 1_A multi-epitope vaccine incorporating adhesin-derived antigens protects against Mycobacterium tuberculosis infection and dissemination.pdf

IntroductionAdhesion to host cells is the first and essential step in Mycobacterium tuberculosis (M. tuberculosis) infection. Among adhesion molecules, the PGRS domain of PE_PGRS33 plays a critical role in invasion but is dominated by B cell epitopes and lacks sufficient T cell epitopes, restricting its capacity to induce a balanced immune response. MethodsTo overcome this limitation, we employed an integrative reverse vaccinology pipeline combining computational prediction and experimental validation. Helper and cytotoxic T lymphocyte epitopes were incorporated from multiple M. tuberculosis adhesins as well as other virulence-associated proteins, and adjuvant sequences were systematically evaluated in silico. ResultsAmong three multi-epitope constructs, the Toll-like receptor 2 (TLR2)-agonist and pan HLA DR-binding epitope (PADRE)-adjuvanted vaccine (TLR2-vaccine) emerged as the most promising candidate. In murine models, TLR2-vaccine induced strong antigen-specific antibody and IFN-γ responses, significantly reduced bacterial loads following H37Ra challenge, and effectively prevented extrapulmonary dissemination. DiscussionThese findings highlight the potential of adhesin-inclusive multi-epitope vaccines to elicit both humoral and cellular immunity and demonstrate how computational vaccinology can accelerate the development of targeted interventions against tuberculosis.