Immunoinformatics-Guided Design and In Silico Evaluation of a Multi-Epitope Vaccine Against Influenza A H10N5 and H3N2 Strains Based on Hemagglutinin and Neuraminidase Proteins

Shabbir, M. Z.; Kumar, P.; Rehman, M. A. U.; Kumar, J.; Urooj, U.; Batool, S. I.; Sourav, C.; Ghazanfar, R.; Nagari, Z.; Hameed, D.; Wahid, A.; Atique, A.; Siddique, M. D.

doi:10.64898/2026.07.03.736294

bioRxiv · 10.64898/2026.07.03.736294

Immunoinformatics-Guided Design and In Silico Evaluation of a Multi-Epitope Vaccine Against Influenza A H10N5 and H3N2 Strains Based on Hemagglutinin and Neuraminidase Proteins

Shabbir, M. Z.·Kumar, P.·Rehman, M. A. U.·Kumar, J.·Urooj, U.·Batool, S. I.·Sourav, C.·Ghazanfar, R.·Nagari, Z.·Hameed, D.·Wahid, A.·Atique, A.·Siddique, M. D.

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Abstract

Influenza A viruses H3N2 and H10N5 represent, respectively, a persistently dominant seasonal pathogen and a newly documented zoonotic threat with the latter strain variants responsible for the first confirmed human fatality in January 2024, yet no vaccine platform currently addresses co-protection against both subtypes within a unified immunogen. We report here the immunoinformatics based vaccine design and multi-layered computational validation of a 419-amino-acid multi-epitope subunit vaccine construct targeting conserved hemagglutinin (HA) and neuraminidase (NA) antigens identified through multiple sequence alignment of the avian H10N5 (A/swine/Hubei/10/2008) and H3N2 human reference strain sequences to identify viral agents undergoing mammalian adaptations. Linear B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were predicted using ABCpred, BCEpred, BepiPred 2.0, NetMHCpan 2.1, and NetMHCpan 4.0, then filtered through VaxiJen 3.0, AllerTOP v2.1, and ToxinPred to retain only antigenic, non-allergenic, non-toxic candidates. The final construct, incorporating an avian {beta}-defensin N-terminal adjuvant with GPGPG, AAY, and EAAAK linkers, exhibited a molecular weight of 43.9 kDa, instability index of 31.15, and SOLPro solubility probability of 0.763. Tertiary structure modeling via I-TASSER and GalaxyRefine achieved 84.4% Ramachandran-favored residues. Molecular docking against TLR3 and TLR7 yielded binding free energies of -16.1 and -16.8 kcal/mol with picomolar dissociation constants. Molecular dynamics simulations confirmed complex stability over extended trajectories. Furthermore, codon optimization produced a Codon Adaptation Index of 1.0 for E. coli K12 expression. In silico immune simulation demonstrated robust activation of humoral and cellular immunity including elevated IgG1, IgM, IFN-{gamma}, IL-2, rapid NK cell expansion, and broad B-cell clonal diversity. These findings establish a computationally validated candidate capable of providing protection against influenza in multiple host organisms, warranting experimental advancement.

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Shabbir, M. Z., Kumar, P., Rehman, M. A. U., Kumar, J., Urooj, U., Batool, S. I., Sourav, C., Ghazanfar, R., Nagari, Z., Hameed, D., Wahid, A., Atique, A., Siddique, M. D.. 2026-07-08. Immunoinformatics-Guided Design and In Silico Evaluation of a Multi-Epitope Vaccine Against Influenza A H10N5 and H3N2 Strains Based on Hemagglutinin and Neuraminidase Proteins. https://doi.org/10.64898/2026.07.03.736294

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