Humoral Immune Responses to the Live-Attenuated African Swine Fever Vaccine Candidate VNUA-ASFV-LAV2 Reveal Serological Markers for Vaccine Monitoring and DIVA Surveillance

African swine fever (ASF) is a highly contagious and often fatal disease of swine that causes substantial economic losses worldwide. Despite extensive efforts, the lack of safe and effective vaccines remains a major obstacle to ASF control. We previously de-veloped a cell-adapted live-attenuated virus (LAV) vaccine candidate, VNUA-ASFV-LAV2, through serial passage of a highly virulent genotype II ASF virus (ASFV) in cell culture. VNUA-ASFV-LAV2 demonstrated favorable safety and protec-tive efficacy against contemporary pandemic genotype II ASFV. In this study, we sys-tematically evaluated antibody responses in pigs immunized with VNUA-ASFV-LAV2. Fourteen indirect ELISAs were developed using ASFV structural, nonstructural, and previously uncharacterized proteins associated with the viral outer envelope, capsid, inner envelope, and replication functions. Serum samples collected from vaccinated and challenged pigs were used to assess the performance of these assays. The results de-monetarized that p17-, p22-, p30-, pE199L-, and pB602L-based ELISAs have strong po-tential for monitoring antibody responses in pigs vaccinated with VNUA-ASFV-LAV2. Among these assays, p22- and p30-based ELISAs enabled early detection of ASFV-specific antibodies in both vaccinated (as early as 7-14 days post vaccination) and wild-type ASFV infected pigs (17 days post infection). Notably, the pF317L-based ELISA successfully differentiated wild-type ASFV-infected pigs from VNUA-ASFV-LAV2-vaccinated pigs, demonstrating its potential as a DIVA-compatible diagnostic assay. These findings provide the first comprehensive analysis of humoral immune responses elicited by VNUA-ASFV-LAV2 in pigs and identify promising se-rological targets for vaccine monitoring and DIVA-based surveillance, supporting the future field application and commercialization of this vaccine candidate.