T Lymphocyte Response Triggered By Dendritic Cells Pulsed With VP1 Antigen Of FMDV

Foot-and-mouth disease (FMD) is a highly acute vesicular and contagious viral disease of cloven-hoofed animals caused by FMD virus (FMDV). FMDV capsid contains 60 copies of each of four structural proteins, VP1, VP2, VP3, and VP4. Studies have shown that the structural protein VP1 on the surface of the virus particles in the form of protrusions. VP1 is rich in B and T epitopes and it plays a major role in inducing FMDV neutralizing antibody and in the immune response. Dendritic cell(DC) is the most powerful professional antigen presenting cells. DC captured antigen through phagocytic receptors or pattern recognition receptors and presented them to T cells. Meanwhile, DC can regulate the body's immune response to pathogens, but it is unclear the role of anti-FMDV infection.MoDC were not largely susceptible to infection by integrin-binding FMDV but were susceptible to culture-adapted virus. Binding specific antibodies to integrin-binding FMDV at neutralizing or subneutralizing IgG concentrations significantly enhanced infection via CD32 (FcyR). Infection of moDC by the FMDV IC was productive and associated with high levels of cell death. Infected moDC were unable to efficiently stimulate FMDV-specific CD4+memory T cells, but exposing moDC to IC containing inactivated FMDV resulted in significantly increased T cell stimulation. Activated T cells can differentiate into Thl subset with the stimulation of IL-12. Thl cells are able to mediate cellular immune response and promote opsonised-antibodies(IgG), which clear the infection of intracellular pathogens. In the role of IL-4, Th cells is conducive to Th2 cell differentiation, which mediate humoral immune response to produce neutralizing antibodies to clear the pathogen in the peripheral circulation. Currently, how dendritic cells initiate T cell response to FMDV remains elusive.In this study, VP1 of FMDV was purified from prokaryotic expression system. And DC pulsed with VP1 after purifiction were cocultured with T cells. Detection the IFN-y and IL-4 levels of supernatants was performed with ELISA to reveal the antigen presentation mechanism of DC presented VP1 antigen of FMDV.Firstly, the recombinant plasmid pUC57-VP1 and the prokaryotic expression vector pET-32a(+) were digested with restriction endonucleases NcoI-HF and Xhol, respectively. Then, VP1 gene and the prokaryotic expression vector pET-32a(+) were connected through T4 DNA Ligase. The resulting connection was transformed into E.coli DH5a. Recombinant plasmids were transformed into E.coli BL21(DE3). Positive clone, a recombinant plasmid named as pET32a(+)-VP1, the interesting genes were identified by restriction analysis and DNA sequencing. The homology of the sequences of VP1 gene among foot-and-mouth disease virus O isolate O/NYOO of FMDVs registered in GenBank was compared(AY333431.1). It was found that the pUC57-VP1 gene shared highest homology with foot-and-mouth disease virus O isolate O/NYOO strain (100%), indicating that recombinant expression plasmid pET32a (+)-VP1 was successfully constructed. In order to identify whether the recombinant expression plasmid pET32a (+)-VP1 can express in E.coli, recombinant expression was induced in bacteria pET32a (+)-VP1. Meanwhile, the empty vector pET-32a(+) was used as a negative control. The results of SDS-PAGE and Western-blotting showed that there was an obvious protein band of 43 kDa, and the empty vector had no specific band in the corresponding position. In order to verify whether the DC pulsed with FMDV VP1 protein are able to activate T cells effectively, DC pulsed with VP1 antigen of FMDV were cocultured with lymph node T cells. Negative control was performed by coculturing lymph node T cells with DC, and the DC alone as an empty control. The levels of IFN-y and IL-4 in supernatants at different time points were determined with ELISA. The results showed that the DC pulsed with VPl antigen of FMDV activated the T cells efficiently. These data pave the smooth way for VP1 antigen of FMDV presentation of DC and imply new strategy for novel vaccine development.