Design of peptide vaccine candidate for Epizootic Hemorrhagic Disease Virus using an Immunoinformatics based approach

Abstract

Epizootic Hemorrhagic Disease Virus (EHDV) has recently emerged as a significant pathogen affecting domestic cattle, leading to severe clinical disease and economic losses worldwide. Traditionally associated with wildlife, EHDV outbreaks in cattle are increasing, with EHDV-2 identified as the most virulent serotype responsible for high mortality rates. The absence of a commercially available vaccine for cattle or deer, coupled with the limitations and safety concerns of experimental vaccines, underscores the need for a targeted and effective vaccine strategy. In this study, a multi-epitope peptide vaccine was designed with the employment of various immunoinformatics tools. Conserved regions of the EHDV-2 VP2 protein were first selected and then screened to identify antigenic peptides capable of eliciting T-cell and B-cell responses. Selected epitopes showed strong binding affinity to both BoLA class I and II alleles, indicating potential activation of CD8⁺ and CD4⁺ T-cell-mediated immunity in cattle. Three peptides containing multiple epitopes were assembled into two constructs: one consisting solely of the peptides, and another incorporating β-defensin-2, a bovine TLR4 agonist, as an adjuvant to enhance innate immune activation. Structural modeling and docking confirmed proper folding and receptor engagement for both constructs, with the adjuvant-containing construct exhibiting stronger and more stable binding to the TLR4/MD-2 complex. Molecular dynamics simulations further demonstrated that the adjuvanted construct maintained lower RMSD, reduced residue flexibility, and greater compactness, supporting its structural stability. Collectively, these findings suggest that the designed multi-epitope vaccine construct, particularly with the βdefensin-2 adjuvant, holds significant promise for stimulating both adaptive and innate immunity in cattle against EHDV-2. Experimental validation will be essential to confirm its immunogenicity and protective efficacy. Keywords: Epizootic Hemorrhagic Disease Virus (EHDV), immunoinformatics, epitope mapping, multi-epitope peptide vaccine, immune receptor interaction