Reverse Vaccinology and Immunoinformatics-Based Design of Serotype-Specific and Multiserotype Multi-Epitope Vaccine Candidates Against Bluetongue Virus Serotypes 1, 3, 4, and 8 Circulating in the Mediterranean Basin

Bluetongue is an economically important arboviral disease affecting domestic and wild ruminants health and well-being, caused by bluetongue virus (BTV) and transmitted by Culicoides midges. More than 30 BTV serotypes may co-circulate regionally, while current vaccines face limitations related to safety and incomplete cross-protection. Here, reverse vaccinology and immunoinformatics were used to design four serotype-specific and one multiserotype multi-epitope vaccine candidates targeting BTV-1, BTV-3, BTV-4 and BTV-8. Cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes were predicted from VP2, VP5, VP7 and NS1, and linear B-cell epitopes from VP2 and VP5. Twenty-five CTL, 24 HTL and 15 B-cell epitopes met the computational antigenicity, non-allergenicity and non-toxicity screening criteria; HTL epitopes were also predicted to induce interferon-γ. Five constructs comprising 13-25 epitopes (252-471 amino acids) were fused to bovine β-defensin 2 as a putative adjuvant. Molecular docking with bovine Toll-like receptor 4 ectodomain yielded predicted binding energies ranging from -19.6 to -10.7 kcal/mol. Molecular dynamics simulations (100 ns), immune simulation, codon optimization and in silico cloning further characterized the candidates. These computational findings support the prioritization of these constructs for experimental evaluation as potential serotype-specific and multiserotype BTV vaccine candidates.