In Silico Development and Structural Evaluation of a Broad-Spectrum Chimeric Multi-Epitope Vaccine against Co-Infection by Human Metapneumovirus, Respiratory Syncytial Virus, and Influenza A Virus

Co-infections involving Human Metapneumovirus ( hMPV ), Respiratory Syncytial Virus ( RSV ), and Influenza A Virus ( IAV ) often exacerbate disease severity in vulnerable populations. Here, we employed a structure-based immunoinformatics approach to design a multi-epitope subunit vaccine targeting these pathogens. The construct incorporated cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes from the Fusion and Glycoprotein proteins of hMPV and RSV , and the Hemagglutinin (HA) and Matrix proteins (M2) of IAV , linked with an adjuvant and optimized spacers to enhance immunogenicity and stability. Structural modeling confirmed correct folding, and molecular docking predicted a stable interaction with Toll-Like Receptor 4 (TLR4) − 277.43 kcal/mol. Molecular dynamics simulations indicated a compact and stable complex with restricted conformational motions, while MM/GBSA analysis yielded a favorable binding free energy (–121.72 kcal/mol) dominated by electrostatic and van der Waals interactions. Immune simulations predicted strong humoral and cellular responses, including high antibody titers, IFN-γ and IL-2 production, and durable memory formation. Codon optimization achieved a codon adaptation index (CAI) of 0.98 and a GC content of 51.24%, suggesting efficient expression in Escherichia coli . These findings highlight the construct as a structurally stable, immunogenic, and expression-ready vaccine candidate warranting experimental validation against hMPV , RSV , and IAV .