Identification Of Porcine Delta Coronavirus (PDCoV) Structural Protein SLA-Ⅰ Restricted T Cell Epitopes And Immunogenicity Evaluation Of Recombinant T Cell Epitopes

Porcine delta coronavirus(also known as porcine delta coronavirus,PDCoV),a member of the genus delta-coronavirus,can cause acute vomiting,diarrhoea and death in newborn piglets,posing a serious threat to the healthy development of the pig industry.To date,the antigenic epitopes of PDCoV are not completely clear,and no studies on T cell epitopes have been reported.In this study,we identified the T-cell epitopes of PDCoV structural protein and evaluated the immune effect of the recombinant peptide with multiple T-cell epitopes to provide a reference basis for the development of a new vaccine for PDCoV.The main elements of this study are as follows:1.Identification of SLA-I-restricted T-cell epitopes of PDCoV structural proteinIn order to predict the SLA-I-restricted T-cell epitopes of PDCoV structural proteins,the protein properties of four structural proteins of PDCoV(S,N,M and E)were first analysed,and then the SLA-1-restricted T-cell epitopes of PDCoV were predicted using the online website IEBD.17 peptides containing potential T-cell epitopes were screened according to the combined scores of the predicted results,including six of S proteins(S1-S6),6 of N proteins(N1-N6),3 of M proteins(M1-M3)and 2 of E proteins(E1-E2),which were sent to the company for peptide synthesis.To prepare PDCoV-sensitized lymphocytes for T-cell identification,5 piglets were orally inoculated with 4 m L of virus solution containing 106 TCID50/m L PDCoV(CHN-SC2015),and 3 piglets served as controls.Clinical signs and detoxification of the piglets were monitored after the attack to verify the effect of the attack.To identify T-cell epitopes,peripheral blood lymphocytes(PBMC)were collected from piglets at 21 days of age and identified by ELISPOT,q PCR and intracellular cytokine staining(ICS),respectively.ICS determined that all five epitopes were able to induce IFN-γ production by CD8+ T cells,among which M3 and N6 were bispecific T cell epitopes that could also induce IFN-γ production by CD4+ T cells,and the results of the three experiments indicated that the five peptides were T cell epitopes.The binding of the five T cells to common SLA-I complexes was predicted,showing that the five T cell epitopes were able to bind to a variety of SLA-I complexes.Finally,the SLA-I-restricted genetic background of the experimental piglets was analysed and the results showed a high degree of polymorphism of the SLA-I complex in the experimental piglets.2.Evaluation of the immunogenic effect of recombinant peptides with T-cell epitopes from PDCoVThe peptide "KK",a flexible linker lysosomal protein target sequence,was used to link the five identified T-cell epitopes according to the natural structure of the viral protein sequence,named "5T",and the peptide was synthesized by the company.In order to verify the specific cellular immune response induced by the immunization of "5T",10 pigs were divided into 5 experimental peptide immunization groups,4 PBS control groups and 1normal control group,and the experimental groups were immunized with 500μg/head on day 1 and 1mg/head on day 14.PBMC from piglets were collected on day 7 after the second immunization,and the ELISPOT assay showed that 5T and M1 induced 500-1000 specific IFN-γ spots in PBMC from immunized piglets,indicating that 5T induced a specific cellular immune response to M1 in piglets.To verify the protective effect of "5T" multicellular epitope recombinant peptide,5 immunized and 4 PBS control groups were orally inoculated with 1m L of PDCoV containing 108copies/m L on day 30 of the second vaccination.Histopathological HE staining and immunohistochemistry were performed.Clinical signs showed that only the PBS control P9 piglets showed significant diarrhoea on day 3,while the rest of the piglets showed normal signs.Faecal excretion showed no difference between the experimental and control groups,but tissue toxicity showed significantly lower PDCoV RNA copies in the heart,jejunum,colon and rectum of the experimental piglets compared to the control group;pathological autopsy showed that the small intestine of two piglets(P1and P3)in the immunized group was normal,but the mesenteric lymph nodes were slightly enlarged.Immunohistochemistry showed a small amount of PDCoV antigen in the ileal and jejunal epithelial cells of the immunized and control piglets.In summary,the results indicated that the 5T poly-T cell epitope peptide in this study had a partial immunoprotective effect on piglets.