Development and Immune Efficacy Evaluation of Two Live Triple-Gene-Deleted Vaccine Candidates Against Bovine Herpesvirus Type 1

Simple SummaryBovine herpesvirus type 1 (BoHV-1) causes severe respiratory disease and reproductive disorders in cattle, leading to major economic losses worldwide. Current vaccines in China have limitations, as they cannot distinguish vaccinated animals from infected ones. In this study, we developed two new live vaccines by removing three viral genes (rBoHV-∆gG-∆gE/gI and rBoHV-∆TK-∆gE/gI). We tested their safety and effectiveness in rabbits. The results show that both vaccines were safe and induced strong immune responses. After exposure to the virus, vaccinated rabbits showed no fever, significantly reduced virus shedding, and less lung damage. Notably, the rBoHV-∆TK-∆gE/gI vaccine worked best at a high dose, while the rBoHV-∆gG-∆gE/gI vaccine was effective even at a low dose. These findings suggest that our new vaccines could help control BoHV-1 infections and support eradication programs by allowing differentiation between vaccinated and naturally infected animals.Bovine herpesvirus type 1 (BoHV-1) causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses worldwide. Current inactivated vaccines in China fail to induce robust cellular immunity and cannot differentiate infected from vaccinated animals (DIVA). To address these limitations, we constructed two triple-gene-deleted BoHV-1 vaccine candidates, ∆T3 (∆TK-∆gE/gI) and ∆g3 (∆gG-∆gE/gI), using Red/ET two-step recombination technology based on an infectious BAC clone. Their safety and immunogenicity were evaluated in a rabbit model. Both deletion mutants exhibited growth kinetics similar to the wild-type strain but with significantly reduced viral titers and plaque areas. The triple-gene deletion mutants showed superior safety compared with single-gene deletion mutants, with minimal clinical signs and low viral shedding. Immunization with ∆T3 and ∆g3 induced robustgB-specific and neutralizing antibody responses. Following challenge with the virulent BoHV-1 BC01 strain, vaccinated rabbits maintained normal body temperatures, showed significantly reduced viral shedding (approximately 100-fold), and exhibited milder pulmonary lesions. These findings demonstrate that ∆T3 high-dose and ∆g3 low-dose regimens are promising DIVA-compatible vaccine candidates, offering an effective strategy for IBR control and eradication programs.