Construction and Evaluation of Quadruple‐Gene‐Deleted Pseudorabies Virus Platforms for ASFV Antigen Delivery

African Swine Fever (ASF) is a highly lethal viral disease in swine. The emergence and rapid spread of African Swine Fever virus (ASFV) in China, since 2018 have caused significant economic losses to the pig farming industry. The complexity of ASFV has impeded the development of effective vaccines, and with no commercial vaccines currently available in China, highlighting the urgent need for safe and efficacious vaccine candidates. In this study, we utilized a highly immunogenic quadruple-gene-deleted recombinant pseudorabies virus (PRV) strain (rPRV SX-10ΔUL24/TK/gI/gE) as a vector to construct two recombinant viral strains expressing ASFV p54, p72, CD2v, and pp62 proteins using the HDR-CRISPR/Cas9 system. These strains, rPRV-p54+p72 and rPRV-CD2v+pp62, demonstrated stable genetic characteristics and efficiently expressed and delivered heterologous proteins while maintaining biological properties similar to their parental strain. Safety evaluation revealed that both recombinant strains exhibited favorable safety profiles in immunized mice and piglets. Furthermore, the strains induced robust humoral and cellular immune responses, as evidenced by specific antibody enzyme-linked immunosorbent assay (ELISA), lymphocyte proliferation assays, and analysis of CD3+, CD4+, and CD8+ T lymphocytes. These findings suggest that rPRV-p54+p72 and rPRV-CD2v+pp62 are promising bivalent vaccine candidates for protecting against both PRV and ASFV infections.