Immunoinformatics based designing of a broad-spectrum multi-epitope vaccine against co-infection of human metapneumovirus, respiratory syncytial virus, and influenza A virus

Abstract

Abstract 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 two epitopes each for cytotoxic T lymphocytes (CTLs), helper T lymphocytes (HTLs), and B cells, derived from the fusion proteins of hMPV and RSV , as well as the neuraminidase protein of IAV . These epitopes were linked with an adjuvant and optimized spacers to enhance immunogenicity and structural 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 .