Molecular characterization and immunoinformatics-based design of a multi-epitope vaccine against Staphylococcus...

Staphylococcus nepalensis has recently been identified in clinical settings, raising questions regarding its potential role as an emerging opportunistic pathogen. However, no vaccine-based preventive strategy has yet been proposed against this bacterium. The objective of this study was to characterize a clinical isolate using PCR-based molecular identification and to design a rational multi-epitope vaccine candidate against S. nepalensis through an integrated immunoinformatics-driven reverse vaccinology approach. Species-level identification was performed by PCR amplification and sequencing of the 16S rRNA gene. Antigenic proteins lacking predicted allergenic or toxic properties were selected for epitope mapping. Predicted linear B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes were further evaluated for sequence conservancy across available strains to ensure broad-spectrum coverage. Conserved epitopes were assembled into a single construct incorporating β-defensin and a PADRE sequence to enhance immunogenic potential. Structural modeling and validation suggested acceptable stereochemical quality, with 94.8% of residues located in favored regions of the Ramachandran plot. Disulfide engineering was performed to improve structural stability of the vaccine construct. Protein-protein docking computationally predicted favorable binding interactions between the vaccine construct and human TLR2 and TLR4 receptors, with ClusPro docking scores of -1489.2 and -1198.1, respectively. Molecular dynamics simulations suggested dynamic stability of the complexes over a 100 ns trajectory. Immune simulation analyses indicated the potential activation of both humoral and cellular immune responses, including antibody production and cytokine secretion. Codon optimization and in silico cloning suggested possible expression feasibility in Escherichia coli. Collectively, this study presents a computational framework for multi-epitope vaccine design against S. nepalensis, and the findings require experimental validation to confirm biological efficacy and immunogenicity.