Identification Of CD8~+ Cytotoxic T Lymphocyte Epitopes Of Porcine Reproductive And Respiratory Syndrome Virus Matrix Protein In Balb/c Mice

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases affecting the swine industry worldwide, characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. It was difficult to take prevention and control measures, effectively, due to the factors including variation and recombination, its characteristic of antibody dependent enhancement. Moreover, it can attack and destroy the host's immune system, thus result in complication and viremia, etc. Although live PRRS vaccines can provide protection against homologous challenge, the genetic diversity of field PRRSV isolates is very high, and vaccine effect against heterologous challenge may be limited. Also, live PRRS vaccines have been observed to revert to virulence, and the killed vaccines have so far proved less effective. So many researchers have devoted to the new vaccine research and development, for elimination of this disease, but the performance is not so good. At present, we still lack systematically understand of the immunological mechanism of PRRS. Many researchers believed that humoral immunity plays a major role in the prevention and control of this disease for a long time.This view of point play the positive role in the study of humoral immunity. However, the cellular immune mechanisms of this disease still remains unclear, this has led to our lack of a comprehensive understanding of immune mechanisms about PRRS. Cell-mediated immunity is very important for viral control and clearance. Once an infection is established, cellular immune responses are equally important in host defense. Cytotoxic T lymphocytes, or CTLs, are generated by immune activation of cytotoxic T cells. These effector cells have lytic capability and are critical in the recognition and elimination of altered self-cells. Cytotoxic T lymphocyte is a key issue in controlling virus, cancer and transplantation antigen. MHC I restricted CTLs specific for the virus can eliminate virus-infected self-cells and thus eliminate potential sources of new virus. Of course, both humoral and cell-mediated immune reponses to pathogens are very important, they take the different responsibility respectively for different antigen. PRRSV can induce both humoral and cell-mediated immunity has been confirmed. Some recent research results indicated that cell-mediated immunity may play more important role for clearance of PRRSV. These results may provide the theoretical foundation of our study. PRRSV has three major structural proteins: GP5, M, N. M protein is matrix protein of PRRSV and its amino acid sequence is highly conservative among different isolates of PRRSV. Furthermore, the M protein has very good immunogenicity and associate with protection against PRRSV infection .M protein can induce strong antigen specific cytotoxic CD8 T cells as well as neutralization antibody.The antigen peptides loaded on the MHC I molecules recognized by cytotoxic T lymphocyte (CTL) cells are usually called CTL epitopes. CD8+ CTL play a pivotal role in both virus elimination and induction of immunopathology. Only after the recognition of CTL epitopes presented on the right MHC molecue of antigen presenting cells the effector T cells can be activated. Hence, identification of CTL epitopes is crucial in understanding the rules of T cell activation and designing of synthetic vaccines. So far, there are no research reports about the identification of CTL epitopes of PRRSV. In this study, we indetified two CTL epitopes of PRRSV Ch-1a strain matrix protein in Balb/c mice. Firstly, the M and Ubiquitin fusion gene (U-M) was generated by SOE PCR, after that we constructed the eukaryotic plasmid expressing M and U-M gene respectively. The expression of fusion U-M and M protein were verified by transient transfection in BHK-21 cells followed by indirect immuno-fluorescence assay. Secondly, we generated a recombinant vaccinia virus expressing the M gene of PRRSV named rWR-PRRSV-M by homologous recombination. Thirdly, Balb/c mice were immunized with DNA priming and recombinant vaccinia virus boosting strategy. Fourthly, we synthesized 27 CTL epitopes of PRRSV M protein bases on bioinformatics analysis. Finally, after final immunization mice were sacrificed and their spleen cells were stimulated by the presumptive CTL epitopes. Two epitopes which are capble of stimulating the IFN-γproduction in mice CD8 T cells were selected out by flow cytometry and ELISPOT assay: H-2Kd restricted K93FITSRCRL and H-2Dd restricted F57GYMTFVHF.In this study, we identified two PRRSV M protein CD8+ T cells epitopes, K93FITSRCRL and F57GYMTFVHF. These results could be expected to make some contributions to the further study and provide better understanding of cell-mediated immune responses of PRRS. Thus, the identification of CTL epitopes may pave a way and offercertain convenience to the prevention and control of PRRS.