Designing a multi-epitope vaccine against Leishmania major with broad population coverage and innate immune activation through immunoinformatic approaches.

Leishmaniasis remains a major neglected tropical disease, particularly in endemic regions such as South Asia, where effective and broadly protective vaccines are still lacking. In this study, we employed an immunoinformatics-driven strategy to design a multi-epitope vaccine (MEV) candidate against Leishmania based on a locally circulating strain, L. major RS-01 from Pakistan. Whole-genome sequencing and comparative proteomic analysis have revealed a high degree of similarity between the Pakistani isolate RS-01 and the L. major Friedlin reference strain. Epitope mining was performed from the reference strain using the Immune Epitope Database IEDB focusing on epitopes conserved in the proteins of the local strain RS-01. Three immunologically relevant proteins (leishmanolysin GP63, elongation factor Tu, and a proteasomal ATPase AAA family member) were selected for inclusion in the vaccine design. A total of eight highly antigenic and conserved T-cell epitopes comprising both MHC class I and class II binders were shortlisted based on antigenicity, immunogenicity, and population coverage. Multiple MEV constructs were designed using different linker arrangements and structural configurations. Population coverage analysis demonstrated broad global coverage. Among the designed constructs, construct C5 was selected as the final vaccine candidate based on its superior epitope diversity, balanced MHC class I and II coverage, and favorable immune simulation profile, which collectively support potential activation of both humoral and cellular immune responses. Molecular docking revealed various interactions between the vaccine construct C5 and the innate immune receptors TLR4/TLR5. This shows the potential of construct C5 for enhanced innate immune recognition.