Atique, A. | Search bioRxiv

bioRxiv2026

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

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.

bioinformatics↗

bioRxiv2025

LAP Drives Monocyte-Mediated and Extracellular Translocation of Listeria monocytogenes Across the Placental Barrier

Listeria monocytogenes (Lm) is a major cause of fetal infection, yet the mechanisms by which it traverses the placental barrier remain incompletely defined. We established and validated a physiologically relevant in vitro human placental co-culture barrier model using BeWo trophoblasts and primary human placental vascular endothelial cells (HPVECs), which exhibited robust barrier integrity and organized tight junctions. Using this model, we demonstrate that Lm exploits a Trojan-horse transmigration mechanism via infected monocytes. Lm-infected THP-1 monocytes displayed a [~]4.8-fold higher monocyte transmigration and [~]8.12-fold greater intracellular bacterial delivery across the placental barrier compared with non-pathogenic Listeria innocua (Li). Monocyte transmigration was associated with claudin-1 and occludin disruption, increased paracellular permeability, and localization of infected monocytes at junctional breach sites. Monocyte-mediated placental traversal required the Lm virulence factors Listeria adhesion protein (LAP) and internalin B (InlB), as lap- and{Delta} inlB mutants exhibited Li-like transmigration and an [~]90% reduction in intracellular bacterial delivery, despite normal monocyte uptake. Lm-infected monocytes induced a [~]1.86-fold increase in sVCAM-1 secretion, which was LAP- and InlB-dependent, consistent with pro-transmigration endothelial phenotype. In contrast, extracellular traversal required LAP and internalin A (InlA), as lap- and{Delta} inlA mutants exhibited severe defects comparable to Li, whereas{Delta} inlB and{Delta} hly were minimally impaired. Exposure of monocytes to extracellular Lm further amplified transmigration and barrier permeability. Together, these data define two distinct placental invasion routes: a monocyte-associated LAP/InlB-dependent pathway and an extracellular LAP/InlA-dependent pathway, identifying LAP as a critical noncanonical virulence factor in placental infection and mechanisms of vertical transmission.

microbiology↗