Abstract
Abstract
Eyach virus is a tick-borne pathogen associated with neurological complications resembling encephalitis, and its increasing emergence highlights a growing public health concern. The absence of specific antiviral therapies and limited surveillance data emphasize the urgent need for effective preventive strategies such as vaccine development. This study presents an immunoinformatics-driven design and in silico evaluation of a multi-epitope vaccine candidate targeting Eyach virus. Structural proteins VP5 and VP7 were analyzed to identify highly antigenic, non-allergenic, and non-toxic B-cell, CTL, and HTL epitopes. The selected epitopes demonstrated broad global population coverage of 97.94%, indicating wide immunogenic applicability. These epitopes were assembled into a 256 amino acid vaccine construct using suitable linkers and β-defensin-3 as an adjuvant. Physicochemical properties analysis revealed a stable, hydrophilic, and soluble protein profile. Structural modeling and refinement confirmed high stereochemical quality, with 96.3% of residues located in favored regions. Molecular docking analysis indicated favorable predicted interactions between the vaccine construct and TLR3 and TLR4. Molecular dynamics simulations further confirmed the structural stability, compactness, and consistent behavior of the complexes. In addition, MM/GBSA analysis revealed favorable binding free energies, with the vaccine–TLR3 complex exhibiting a stronger binding free energy of − 203.29 kcal/mol. Immune simulation predicted robust humoral and cellular immune responses, including elevated immunoglobulin levels, cytokine production, and memory cell formation following a three-dose regimen. Overall, the findings suggest that the proposed multi-epitope vaccine is a promising candidate against Eyach virus; however, experimental validation is required to confirm its safety and efficacy.