Pan-genome-guided reverse vaccinology identifies a conserved lipoprotein-based multi-epitope vaccine candidate against Staphylococcus haemolyticus

Abstract

Staphylococcus haemolyticus is an emerging opportunistic pathogen linked to bloodstream infections, device-associated infections, and other nosocomial complications, highlighting the need for alternative preventive measures. In this study, 36 complete S. haemolyticus genomes were analyzed using a pan-genome-guided reverse vaccinology pipeline. Pan-genome analysis supported an open architecture, and subtractive proteomics of 1,837 non-redundant core proteins identified two virulence-associated candidates. A conserved, predicted surface-accessible lipoprotein was prioritized and used to select B-cell, MHC class I, and MHC class II epitopes for construction of a 328-amino-acid multi-epitope vaccine (MEV) construct incorporating an HBD-3 adjuvant and appropriate linkers. The construct showed favorable predicted physicochemical and immunological properties, including high antigenicity, non-allergenicity, and broad HLA population coverage. AlphaFold2 indicated substantial uncertainty in the linker-rich epitope regions, although refinement improved local stereochemical quality. Docking and normal mode analyses suggested structurally plausible interactions with TLR2/TLR1 and TLR4–MD-2. A 100 ns explicit-solvent molecular dynamics simulation of the selected TLR2/TLR1–MEV complex indicated maintenance of overall association and compactness despite considerable MEV mobility. Preliminary MM/GBSA analysis provided complementary energetic support for the modeled interaction. Codon optimization and in silico cloning further supported potential expression in Escherichia coli . Overall, these computational findings prioritize a conserved lipoprotein-derived vaccine candidate against S. haemolyticus , although experimental validation is required to confirm expression, folding, safety, immunogenicity, and protective efficacy.