Serological Analysis And High-throughput Epitope Identification Of African Swine Fever Virus

African Swine Fever(ASF)is a highly contagious disease caused by the African Swine Fever Virus(ASFV),which poses a severe threat to the swine industry in our country.The complexity of ASFV and limitations in understanding of its protein structure,function,and immune response mechanisms have hindered the development of ASF vaccines,which greatly restrict our ability to prevent and control ASF.Therefore,it is urgent to carry out research on the pathogenic characteristics and vaccine development related to ASF.Epitopes,as the smallest structural unit that induces immune responses,play a crucial role in understanding the entire immune response process and improving our knowledge of virus protein structure and function.They are important research targets for epitope vaccine development,monoclonal antibody preparation,and diagnostic reagent development,providing the key foundation for solving numerous disease problems.The identification and validation of effective antigen epitopes are essential for the prevention and control of diseases,and it is necessary to systematically explore and deeply study the ASFV antigen epitopes.The Virscan technique,based on phage display library,offers a high-throughput serological analysis approach for exploring the antibody response spectrum in target sera.It enables the precise,rapid,systematic,and high-throughput screening and identification of epitopes at a vast scale,allowing the investigation of immunological responses across numerous individuals.Bacteriophage T7 can effectively stimulate antigen-specific immune responses in vivo,with high stability and safety,as well as ease of large-scale production and low cost,making them a promising tool for vaccine research and development.The present study comprises three main aspects:1)Construction of a Swine-Virscan T7 phage display library that exhibits the pig virus proteome,which was truncated to 56 amino acids(AA)with overlapping adjacent segments of 28 amino acids.Utilizing Phage Immuno Precipitation Sequencing(PhIPSeq)on a cohort of 54 ASF-positive sera and 46 negative control sera,we successfully identified a collection of 113 antigenic epitopes that displayed affinity with at least 3ASF-positive sera.Subsequently,a subset of these epitopes underwent validation using Dot-ELISA.Combining ASF nucleic acid detection and PhIP-Seq results,ASF-positive sera samples were classified into acute infection,subacute infection,and convalescent periods,and the antibody reactivity profiles of different groups were analyzed,and it was found that there were significant differences in the breadth of the antibody reactivity profiles in different infection periods.2)To further determine the ASFV antigenic epitope boundaries,a library was designed and synthesized with truncations of 20 amino acids and overlapping adjacent segments of 15 amino acids using the ASFV China HLJ/18 strain proteome as a reference.This library was displayed on the T7 phage surface to construct the ASFV-20mer-Virscan library,which was subjected to PhIP-Seq with the ASF-positive sera mentioned above.Through the joint analysis of Swine-Virscan and ASFV-20 merVirscan results,we successfully identified a set of 35 highly reliable ASFV antigens.Additionally,a high-resolution epitope map was created to visually represent the precise locations of these antigens.To further enhance our understanding,a subset of these antigens had their protein structures simulated using AlphaFold 2,with antigenic domains carefully annotated.3)By biopanning of ASF convalescent sera against the Swine-Virscan library,a sub-library with affinity for the convalescent sera was obtained.Analysis of this sublibrary identified 17 ASFV epitopes,which from 10 primary source proteins.Following unbiased amplification,this sub-library was employed for immunization in individual pigs.Notably,the results revealed that the post-immunization sera exhibited binding capabilities to 8 significant ASF antigens,such as p54 and p30,displaying a similar antibody response profile to ASF convalescent sera.In summary,this study comprehensively and deeply analyzed the antibody reactivity profile of ASF-positive sera at the epitope level.To some extent,it fills the gaps in understanding ASFV infection and humoral immune response.Moreover,key antigenic proteins that are rarely studied but commonly induce antibodies in convalescent sera,such as DP238 L,were discovered.And individuals with a similar antibody response profile to ASF convalescent sera were obtained through phage library immunization.These findings provide new ideas and experimental evidence for ASF vaccine development.