Immunoinformatics based designing of a broad-spectrum multi-epitope vaccine against co-infection of 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 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.