Immunogenicity assessment and epitope mapping of the ASFV proteome by profiling serum antibodies with ASFV antigen phage libraries

The continued spread and regular outbreaks of African swine fever (ASF) have severely threatened the pig-related industries, causing economic losses. African swine fever virus (ASFV) encoded more than 150 different proteins, but the biological characteristics of the majority of these proteins remain unknown. In this study, we leveraged the Phage ImmunoPrecipitation Sequencing (PhIp-Seq) platform to perform an exhaustive serological analysis of ASFV to characterize the specific reactivities of serum anti-ASFV IgG antibodies against the ASFV proteome at peptide resolution. High-resolution epitope mapping of the ASFV antigens was conducted, and a total of 29 ASFV antigens with high immunogenicity were identified, 14 of which, to the best of our knowledge, have not been previously identified as serological antigens. The immunogenicity of these 29 antigens was evaluated, and their conservation was statistically analyzed across 169 ASFV strains. We found that the uncharacterized protein DP238L is a conserved antigen that is widely hit within the population. The immunogenicity of DP238L and multi-epitope recombinant proteins was validated by immunoblotting and animal immunization trials, confirming the immunogenicity of the identified antigens and the reliability of the PhIp-Seq based epitope mapping strategy. These findings provide insights into the structures and functions of antigen proteins and identify crucial targets for ASFV detection and vaccine development.