Identification Of SLA-I-restricted T-cell Epitopes Within The M Protein Of Porcine Reproductive And Respiratory Syndrome Virus

PRRSV can induce immunosuppressive respiratory disease and resulte in severe morbidity,secondary microbial infections and PRDC.The development of efficacious PRRSV vaccines is a challenge owing to the virus mutates rapidly and many vaccines perform poorly against heterologous challenge.The non-glycosylated membrane protein(M protein)encoded by ORF6 is a highly,conserved structural protein which shares high homology among PRRSV strains.It is shown to play a crucial part not only in virus assembly and budding but also in viral neutralization.Additionally,PRRSV-2 M protein demonstrates better capability of inducing T cell proliferation and IFN-? synthesis in immunized pigs.Accordingly,M protein is considered as a vital candidate target in cell-mediated immunity and efficacious vaccine.T-cells functions are essential for clearance of PRRSV infection and regulation of immune responses.The identification of SLA I-restricted T-cell epitopes is important for analysis of the involvement of CD8+ T cells in PRRSV infection and vaccine development.In this study,nine T-cell immunodominant epitopes from PRRSV-2 M protein were evaluated in ELISPOT assay with peripheral blood mononuclear cells(PBMCs)isolated from the Large White pig experimentally-infected with PRRSV JXA1-R strain.Eight immunodominant epitopes were confirmed that they were capable of eliciting significant IFN-?secreting,and the PBMCs from the Large White pigs showed individual variability in response to immunodominant epitopes.To predict potential T-cell epitopes from eight confirmed T-cell immunodominant epitopes via using bioinformatic tools,swine leukocyte antigen(SLA)class I alleles in the experimentally-infected Large White pigs were cloned and sequenced,and fourteen SLA class I genes were determined.Synthesizing the results of ELISPOT and SLA genotyping,fourteen SLA class I genes and their restricted peptides were predicted in silico.Then,42 p ET-peptide-SLA-I-?2m complexes that consisted of predicted restricted peptides,extracellular region of the SLA class I molecules and ?2m were constructed and tested via ELISA-based method for preliminary discriminating potential T-cell epitopes.Four potential epitopes and the SLA molecules they binding to were identified.And these four peptides were evaluated using in vitro complex refolding assays.ELISPOT results confirmed that these 9-mer potential epitopes were efficient in stimulating IFN-? secretion in specific SLA genetic background.Moreover,three epitopes were confirmed that they were highly conserved in prevailing strains.Collectively,such epitopes provided new insight into epitope identification and could lay a solid foundation for further researches on cellular immunity and T cell-based vaccines in swine.