Leishmania major remains a significant public health threat, yet vaccine development has been hampered by fragmented antigen selection approaches and limited systematic proteome-wide analysis. In this study, we developed a comprehensive immunoinformatics pipeline to systematically evaluate the entire L. major proteome (8038 proteins) using standardized selection criteria: subcellular localization (extracellular/membrane), antigenicity (VaxiJen ≥ 0.5), allergenicity assessment (AllerTOP/AlgPred consensus), signal peptide presence (SignalP ≥ 0.7), transmembrane topology (TMHs < 2), optimal sequence length (100–500 aa), host homology exclusion (≤ 30% identity), and cross-species conservation analysis. This rigorous screening identified nine potential vaccine targets, including eight novel candidates and the well-known established antigen GP46 (Q4Q6B6) that passed all filtering criteria. Remarkably, four candidates (E9AFZ7, Q4Q0L6, Q4Q0X3, Q4QC59) remain annotated as "uncharacterized proteins" in UniProt, representing completely unexplored therapeutic targets. All nine candidates exhibited optimal vaccine properties: membrane/extracellular localization, high antigenicity (VaxiJen 0.52–0.89), non-allergenicity, signal peptide presence, and absence of human homologs. To validate our pipeline's stringency, we retrospectively analyzed twelve historically proposed L. major vaccine candidates using identical criteria. Strikingly, 67% (8/12) failed our selection standards: eight due to concerning host homology (> 30% human identity) and four due to predicted allergenicity, with only GP46 meeting all requirements. Cross-species conservation analysis demonstrated broad Leishmania species coverage for our novel candidates, supporting their potential as universal vaccine targets. This study represents a paradigm shift from focusing on individual known antigens to employing systematic proteome-wide discovery, delivering a standardized framework for Leishmania vaccine candidate selection. The identification of nine candidates including GP46 antigen, including four uncharacterized proteins, significantly expands the therapeutic pipeline while our validation approach establishes quality-control standards essential for advancing neglected tropical disease vaccine development.
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