Abstract

Background and Aim: Lumpy skin disease (LSD) is a severe transboundary viral infection in cattle, caused by the LSD virus (LSDV), leading to economic losses in the livestock industry. Conventional live-attenuated vaccines face limitations such as strain recombination, incomplete protection, and adverse effects. Therefore, safer and more targeted vaccine strategies are urgently needed. This study aimed to design, simulate, and express a novel multi-epitope vaccine (MEV) candidate against LSDV using a computational immunoinformatic pipeline.

Materials and Methods: Four immunogenic LSDV proteins – P35, A4L, A33R, and L1R – were selected based on their structural and antigenic significance. B- and T-cell epitopes were predicted and filtered using antigenicity, allergenicity, and toxicity criteria. Selected epitopes were linked using specific linkers and an adjuvant to construct an MEV. Molecular docking was performed with bovine toll-like receptors (TLRs), and stability was evaluated through molecular dynamic simulations (GROMACS and iMODS). Codon optimization and heterologous expression of the construct were performed in Escherichia coli using the pET-28a(+) vector. Expression was checked through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.

Results: A total of 23 epitopes from the four LSDV proteins were incorporated into a 514 amino acid-long vaccine construct. The designed construct demonstrated high antigenicity, non-allergenicity, solubility, and favorable physicochemical properties. Docking with bovine TLR4 revealed stable binding with significant interaction residues. Molecular dynamics confirmed structural stability over 50 ns simulations. The recombinant construct was successfully expressed as a ~59 kDa His-tagged protein in E. coli, confirmed by SDS-PAGE and Western blotting.

Conclusion: This study demonstrates a comprehensive computational and experimental workflow for developing a multi-epitope subunit vaccine against LSDV. The MEV candidate shows strong immunogenic potential, structural stability, and recombinant expression feasibility, offering a promising alternative to traditional vaccines. Further in vivo evaluation is warranted to assess protective efficacy.

Keywords: Escherichia coli, immunoinformatics, lumpy skin disease virus, molecular docking, multi-epitope vaccine, recombinant protein expression, toll-like receptors.