Designing a multi-epitope vaccine against La Crosse virus using immunoinformatics approach

La Crosse virus (LACV) is a mosquito-borne Orthobunyavirus responsible for pediatric encephalitis, with no currently approved vaccine or targeted antiviral therapy. This study aimed to design a multi-epitope subunit vaccine targeting the conserved nucleoprotein of LACV using an integrated immunoinformatics approach. The nucleoprotein exhibited strong antigenicity and favorable physicochemical properties, including stability and hydrophilicity. Highly conserved CTL, HTL, and B-cell epitopes were initially predicted, from which high-affinity, non-allergenic, and non-toxic epitopes were selected. Population coverage analysis revealed extensive global applicability, with MHC class I and II coverage of 98.55% and 81.81%, respectively. Three vaccine constructs (V1, V2 and V3) were developed, while construct V1 demonstrated superior performance, with the highest antigenicity (0.7846) and solubility (0.709). Structural validation confirmed stability, with over 93% of residues in favored regions of the Ramachandran plot. Molecular docking with the TLR8 receptor revealed strong interactions. This was further supported by MD simulations, DCCM analysis, binding free energy calculations, and PCA, all of which confirmed stable binding between the vaccine construct V1 and TLR8, exhibiting a docking score of -1212.6. Molecular dynamics simulations further confirmed stability, with consistent RMSD, stable radius of gyration, and persistent hydrogen bonding (40-60 bonds). Codon optimization demonstrated favorable translational efficiency and confirmed the suitability of the construct V1 for heterologous expression in E. coli. Immune simulation predicted robust humoral and cellular responses, including elevated IgM, IgG, and cytokines. Overall, the proposed vaccine candidate V1 demonstrates strong immunogenic potential and structural stability, warranting further experimental validation for potential clinical application.