Screening And Identification Of T-cell Peptide Epitopes For African Swine Fever Virus

African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly pathogenic and contagious animal disease that has caused huge economic losses to the swine industry.Until today,there is no commercial vaccine has been developed to effectively prevent ASF.Humoral immune responses mediated by neutralizing antibodies have limited efficacy in preventing infection by virulent ASFV strains,while cellular immune responses induced by low virulence strains play a unique role in antiviral defense and viral clearance.The Major Histocompatibility Complex(MHC),also known as the Swine Leukocyte Antigen(SLA)in pigs,is involved in the presentation of viral antigen peptides and activation of cellular immunity through interaction with T-cell receptors.The association between SLA molecular subtypes and T-cell immune responses has been well-established.Therefore,the identification of effective T-cell epitopes for ASFV is of great significance for understanding the immune mechanisms against ASFV and the development of ASF control strategies.This study aimed to screen and identify T-cell epitopes of ASFV and preliminarily analyzed the SLA subtypes of the Duroc-landrace-yorkshire commercial crossbred pigs.Based on the ASFV T-cell epitopes and protein antigenicity prediction of these three major subtypes,eukaryotic expression plasmids for the selected proteins were designed and transfected into PK-15 cells,MAE method with citrate-phosphate buffer was used to screen T-cell epitopes.Additionally,PAM cells were infected by ASFV and a similar screening process was performed to identify T-cell epitopes.To rapidly analyze the genomic sequences of ASFV and evaluate the conservation of the identified epitopes,a non-separation cell culture method for whole-genome sequencing of ASFV was developed.The validity of the identified T-cell epitopes was further confirmed through comparisons with the results of epitope predictions,docking analysis between T-cell epitopes and SLA molecules,and preliminary identification of non-covalent interactions between T-cell epitope proteins and ligands of SLA molecules.The results revealed the distribution of SLA subtypes in 226 serum or tissue samples from the Duroc-landrace-yorkshire commercial crossbred pigs,with SLA-1*04:01,SLA-2*04:01,and SLA-3*04:01 being the dominant haplotypes,accounting for 26.63%(49/184),61.29%(38/62),and 45.00%(18/40)of the SLA class I molecules,respectively.Based on the prediction of ASFV protein antigenicity,p14 and p72 demonstrated strong immunogenicity.The eukaryotic expression plasmids for these two proteins were designed,and after transfection into PK-15 cells,three T-cell epitopes,including KVRPHTGTPTL,ALKWPI,and KKIGIKL,were identified through MAE screening with citrate-phosphate buffer.Similarly,four T-cell epitopes,IIEYIPNSV,LENIRF,HVIKDGDYTI,and KTLNITCEQMSAIL,were identified through MAE screening of PAM cells infected by ASFV.Ten ASFV-positive pig serum or anticoagulant samples were successfully sequenced using the developed non-separation cell culture method for whole-genome sequencing of ASFV.The conservation analysis of the seven epitopes indicated that KVRPHTGTPTL,ALKWPI,KKIGIKL,IIEYIPNSV,LENIRF,and KTLNITCEQMSAIL,exhibited high conservation across 58 viral strains.By comparing the results of peptides screening and prediction,six peptides could match two or more SLA molecules,indicating good antigenicity of the T cell epitopes selected.Based on the docking analysis results of T cell epitopes and SLA molecules,five peptides including KVRPHTGTPTL,KKIGIKL,IIEYIPNSV,LENIRF,KTLNITCEQMSAIL could bind in the groove of SLA molecules and be presented on the surface of cells.Analyses of non-covalent interaction between T cell epitopes and SLA protein ligands showed that all five peptides contained amino acids that interacted with SLA molecules,and the interaction distances were less than 5 ?.In summary,this study analyzed the distribution of SLA subtypes in the Duroc-landrace-yorkshire commercial crossbred pigs and predicted T-cell epitopes for ASFV based on major SLA subtypes.Seven T-cell epitope peptides were identified with MAE method by transfecting cells with eukaryotic expression plasmids or infecting cells with ASFV.A non-separation cell culture method for whole-genome sequencing of ASFV was established to evaluate the conservation of genes and epitopes.The biological characteristics of the identified T-cell epitopes,including KVRPHTGTPTL,KKIGIKL,IIEYIPNSV,LENIRF,and KTLNITCEQMSAIL,were preliminarily characterized,providing support for further studies on the cellular immune mechanisms against ASFV and the development of ASFV vaccines.