T Cell Responses Triggered By Dendritic Cells Pulsed With VP1-VP4 Fusion Protein Of Foot-and-mouth Disease Virus

Foot-and-mouth disease (FMD) is a highly contagious disease affecting cloven-hoofed animals. The causative agent of the disease is the FMD virus (FMDV).And there is no effective control approaches.FMDV capsid contains four types of structural proteins, VP1, VP2, VP3, and VP4.And it is proved that VP1 is rich in T cell and B cell antigetic epitopes.Though VP4 is located in internal capsid and relatively conservative, VP4 is rich in T cell epitopes as well.DCs were not largely susceptible to infection by FMDV but were susceptible to culture-adapted virus. Binding specific antibodies to FMDV can not only infect DCs, resulting in loss of antigen-presenting function, but also cause the high levels of DCs’death. However, exposing moDCs to IC containing inactivated FMDV resulted in significantly increased T cell stimulation. Therefore, attention has been shifted onto the cellular immune response mechanisms to the FMDV.Dendritic cells (DCs) are the most powerful professional antigen-presenting cells in the body at present. DCs can uptake exogenous antigens through pinocytosis, receptor- mediated endocytosis and phagocytosis. DCs present antigen to T cells with different mechanisms that triger cellular immunity. Currently, how dendritic cells uptake FMDV and then initiate response of cellular immunity to FMDV remains elusive.In this study, the recombinant plasmid pUC57-VP1 was used as template and the one end of the restriction site in VP1 was altered by PCR. VP1 gene segments were inserted into the prokaryotic expression vectors, pET-32a (+), through NcoⅠand BamHⅠto construct the recombinant plasmid pET-32a-VP1. pBluescriptⅡSK(+)-VP4 and pET-32a-VP1 were digested with XhoⅠa nd BamHⅠt o construct the recombinant plasmid pET-32a-VP1-VP4. Recombinant plasmid pET-32a-VP1-VP4 were transformed into host bacteria BL21(DE3) to express. And the expression conditions were optimized. SDS-PAGE was applied to confirm the optimal protein expression protocol with identification by Western blot either by anti-His-Tag antibodies or FMDV positive serum. The experimental results showed that the pET-32a-VP1-VP4 gene was correct as identified with restricted digestion, and was consistent with the published sequence at GeneBank Foot-and-mouth disease virus O isolate O/NYOO and the homology was 100%. The proteins expressed by host bacterium BL21(DE3) were detected by SDS-PAGE. The expected VP1-VP4 proteins with molecular weight of 49 kD were abundantly translated in BL21(DE3) whith induction of IPTG at concentration of 1 mmol/L, 30℃and for 5 h. The resultant VP1-VP4 fusion protein antigens were purified through electric elution.The bone marrow-derived dendritic cells (BMDCs) which were respectively pretreated with different combinations of proteasome inhibitors, lysosomal inhibitors, inhibitors of mannose receptor, scavenger receptor inhibitors and macropinocytosis inhibitors were pulsed with FMDV VP1-VP4 fusion protein antigens and then cocultured with lymph node T cells. BMDCs and T cells were used as control. The culture supernatants were harvested after 9, 24, 48, 72 and 96 h for determining both IFN-γand IL-4 contents with ELISA. The results showed that the contents of IL-4 in each group at indicated time points were significantly lower than that of IFN-γ. Notably, the contents of IFN-γfrom coculture system treated with proteasome inhibitors at indicated time points were lower than that of group with no inhibitor administration, but higher than that of group treated with lysosomal inhibitor, indicating that the lysosomal processing of VP1-VP4 proteins in the BMDCs play an important role in the initiation of T cell responses. The contents of IFN-γin supernatants of the groups treated with inhibitor of mannose receptor were higher than that of groups with no inhibitor (except 24 h, P>0.05), indicating that the recognition of VP1-VP4 by mannose receptor was able to suppress BMDCs-triggered activation of T cell response. Obviously, the contents of IFN-γin supernatans of the groups treated with scavenger receptor inhibitor were significantly higher than that of groups with no inhibitor, which showed that the recognition of VP1-VP4 fusion proteins by scavenger receptor plays a suppressive role in BMDCs-mediated activation of T cell response. Remarkably, the contents of IFN-γin supernatants harvested from the coculture system treated with macropinocytosis inhibitor were significantly lower than those groups pretreated with inhibitor of mannose receptor, scavenger receptor inhibitor, respectively, and no inhibitor administration group as well. These data indicate that macropinocytosis is the main way of BMDCs for uptake of VP1-VP4 antigen.In conclusion, the in vitro antigen presenting assays showed that BMDCs pulsed with FMDV VP1-VP4 proteins are able to activate the T cells efficiently. Macropinocytosis is the principal pathway of BMDCs for VP1-VP4 antigen capture. Surprisingly, the recognition of VP1-VP4 both by mannose receptor and scavenger receptor plays a suppressive role in BMDCs-mediated activation of na?ve T cell response.These results not only pave the way for further study the antigen-presenting pathways in DCs for FMDV VP1-VP4 protein antigens, but also serve as theoretical guidance for the design and development of novel vaccines.