Integrated immunoinformatics-guided development and validation of an immunodominant multi-epitope vaccine candidate against lumpy skin disease virus

Lumpy skin disease virus (LSDV), a transboundary Capripoxvirus , continues to threaten livestock productivity worldwide. Existing live-attenuated and inactivated vaccines are limited by safety concerns, incomplete protection, and poor compatibility with DIVA-based surveillance. Using an immunoinformatics workflow, we engineered and preliminarily validated a 14.3 kDa multi-epitope subunit vaccine based on the envelope protein P32 and surface protein A27L. Cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were identified and rigorously filtered for antigenicity, non-allergenicity, non-toxicity, host non-homology, and sequence conservancy. Selected epitopes were assembled into a chimeric construct with the RS09 adjuvant and optimized linkers. The final vaccine comprised two CTL, two HTL, and two B-cell epitopes and showed favorable predicted properties, including high antigenicity (0.90), low instability (24.32), and good solubility (0.87), with no allergenic or toxic signatures. Structural validation supported a high-quality model, with an ERRAT score of 99% and >91% of residues in favored Ramachandran regions. The construct showed the strongest binding to bovine TLR9 (ΔG = −16.2 kcal/mol; Kd = 3.5×10 –12 ), with stable interaction confirmed by 100 ns molecular dynamics simulations. Immune simulation predicted strong humoral and cellular responses, marked by elevated IgM, IgG1, IgG2, IFN-γ, and IL-2, consistent with a Th1-biased immune profile. Codon optimization and in silico cloning supported bacterial expression, and recombinant production was confirmed by SDS-PAGE and Western blot analysis, which showed a distinct band at approximately 14.3 kDa, providing experimental support for construct feasibility. Together, these findings identify a rationally designed and experimentally supported multi-epitope vaccine candidate against LSDV and establish a promising platform for safer, targeted, and potentially DIVA-compatible next-generation capripoxvirus vaccines.