Heterologous DNA-Adenovirus Prime-Boost Strategy Expressing Bluetongue Virus VP2 and VP7 Proteins Protects Against Virulent Challenge

Background/Objectives: Bluetongue virus (BTV) is an emerging arbovirus causing significant economic losses in the ruminant industry. Current vaccines offer limited cross-protection against heterologous serotypes and do not enable differentiation between infected and vaccinated animals (DIVA). Subunit-based vaccines provide a potential DIVA-compatible solution. This study aimed to develop a vaccination protocol expressing BTV structural proteins VP7 or VP2 using antibiotic-resistance-free DNA plasmids and replication-defective adenovirus vectors. Methods: We evaluated homologous DNA prime-boost and heterologous DNA prime-adenovirus boost strategies in a murine model, assessing adaptive immune responses and protection against virulent BTV challenge. Results: The heterologous DNA-adenovirus prime-boost strategy expressing both antigens conferred full protection, preventing viremia, while homologous DNA-DNA prime-boost provided only partial protection. Both VP7 and VP2 elicited cellular and humoral immune responses, but the heterologous strategy significantly enhanced anti-BTV IgG, neutralizing antibody titers, and T cell activation. CD8+ T cell responses showed the strongest correlation with viral load reduction, suggesting that immunity to VP7 could offer cross-protection against multiple BTV serotypes. Conclusion: These findings highlight the potential of heterologous DNA-adenovirus vaccination as an effective DIVA-compatible strategy for BTV control. By inducing strong and protective immune responses, this approach could improve disease surveillance and management, ultimately reducing the impact of BTV on livestock industries.