Evaluation Of Cell-mediated Protective Immunity To Hypoxanthine Guanine Xanthine Phosphoribosyl Transferase (HGXPRT) Of Plasmodium Falciparum

Malaria remains one of the most devastating infectious diseases in the world. Due to emergence of the drug resistance of the parasite and the insecticide resistance of Anopheles mosquito, it becomes more difficult to control malaria. So it is very important to develop effective malaria vaccine to control it.Malaria vaccine to blood stage of parasite plays an important role in reducing morbidity and mortality of malaria. In early stage, research of protective immunity mechanism of blood stage mainly focuses on humoral mediated immunity. Actually, parasites of blood stage can induce protective antibody and these antibodies can completely inhibit grow of parasite in vitro. Many animal experiments also showed that antibodies play an important role in eliminating parasite of blood stage. Because erythrocyte surface lack MHC molecule, CTL can not eliminating parasite. But some experiments showed that B cell KO mice can produce protective immunity to parasite after been immunized. Although this immunity was unable to eliminate infection, they were able to control parasite load very effectively. Obviously, the protective immunity was mediated by cell immunity. In addition, CD4~+T cell could adoptively transfer protection against malaria. All results showed that both humoral mediated immunity and CD4~+T cell mediated cell immunity play important roles in immunologic mechanism of blood stage of parasite.Recently, candidate antigens of malaria vaccine to blood stage were identified in succession. But most of them induced protective humoral immunity. Makobongo generated CD4~+T cell lines to fractions of native antigens from the blood stages of the rodent parasite, and identified protective fraction-specific T cells through adoptive transfer and challenge infection experiments. Then hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) was identified as the target antigen to protective CD4~+T cells through N-terminal sequence. HGXPRT is a key enzyme in purine nucleotides salvage route, which can synthesize purine nucleotides by catalyzing reaction of 5-phospho-a-D-rbosyl pyrophosphate (5-PRPP) and purine.Mammal can synthesize purine nucleotides not only through salvage route but also through de novo route. However, plasmodium is auxotrophic for purines and only relies on the purine salvage pathway for the synthesis of its purine nucleotides. So HGXPRT plays a very important role in plasmodium and become a potential target for chemotherapy. So it was necessary to made recombinant HGXPRT and established the recombinant HGXPRT specific T cell lines. We should make sure that the protective immunity of cell mediated immunity and identified protective epitopes of T cell in these bases.We have already expressed and purified recombinant HGXPRT of plasmodium falciparum by use of Pichia pastoris. Compared with the theoretic molecular weight, 26.2KD, SDS-PAGE showed that the molecular of recombinant HGXPRT weight was about 25KD. It was also shown that the recombinant HGXPRT can react with serum of patients coming from epidemic area. BALB/ca mice were immunized s.c. on the hind footpads with HGXPRT emulsified in complete Freund's adjuvant (CFA). At 4 and 6 weeks, booster immunizations were given s.c. on the abdomen and i.p., respectively, by using same amount of antigen emulsified in incomplete Freund's adjuvant (IFA). BSA and PBS mixed with adjuvant were used as control groups. Ten days after the last booster the mice were challenged i.v. with P. yoelii parasitized RBC. From the next day of infection, we detected the parasitemia levels every day. Results showed that mice of control groups died after challenged infection 20 days, but 50% mice immunized by HGXPRT survived and healing, and peak value of parasitemia was lower than mice of control groups. The results of ELISA showed that the antibody titers produced by the mice received three administration rose continuously and the level of antibody reached to over 105.In order to study the cell mediated immunity (CMI) of HGXPRT, we established the HGXPRT specific T cell lines. BALB/c mice were immunized s.c. at double upper limbs, double footpads and peritoneum by recombinant HGXPRT protein. After 7-10 days later, armpit, inguinal and popliteal lymph nodes were removed, pooled, and single cell suspensions prepared. Cells were cultured 7 days containing 5,Mg/mlrecombinant HGXPRT. (This stage served as stimulation period for T cells). Then, lymph cells were suspended in fresh complete RPMI 1640 containing lOU/ml recombinant mouse IL-2 and irradiated syngeneic splenocytes in the absence of antigen. (This stage served as a rest period for T cells). After 7 days, cells were re-stimulated with recombinant HGXPRT protein in the presence of recombinant mouse IL-2 and irradiated spleen cells. T cell lines were maintained by repetitive stimulation/rest/stimulation cycles. As a control group, BSA specific T cells were established by the same methods as above. Lymphocytes proliferation assay showed that mean cpm (10082.67) was higher than mean cpm (6205) of HGXPRT sensitized spleen and lymph node cells. It was showed that HGXPRT specific T cell lines had been established through stimulation-rest-stimulation cycles.In order to indicate the immunoprotection effects of HGXPRT specific T cells, we made adoptive transfer experiments. 15 nude mice were divided into three groups, group of HGXPRT specific T cells, group of BSA specific T cells and group of PBS. Mice were administered i.v. with 107cells/mouse and 100/d PBS/mouse. Mice were challenged i.v. with 105P. yoelii PRBC 24h after cell transfer. From the next day of infection, we detected the parasitemia levels every day. Results showed that percentage of parasitemia of control groups rose quickly. Peak value of parasitemia of BSA group was 78.09% at 14th day and of PBS group was 82.44% at 7th day after challenge infection. But percentage of parasitemia of HGXPRT group rose gently, Peak value of parasitemia was 68.74% at 28th day. Results of survival time of three groups showed that the No2 and Nol, 3, 4 mouse of BSA group died at 8th and 9th days 3, and then No5 mouse died at 15th day after challenge infection. But lives of mice of HGXPRT group were prolonged clearly. Mouse start died at 22nd day and all died at 35th day. All above results showed that HGXPRT specific T cells can control parasite load at lower level, especially in early stage of adoptive transfer immunity and lives of mice could be prolonged.Summarily, we expressed the recombinant HGXPRT of plasmodium falciparum and established the recombinant HGXPRT specific T cell lines through the method of stimulation/rest cycles. It could prolong the lives of nude mice after adoptivelytransfer the antigen specified T cells to them when the mice were challenged with plasmodium yoelii infection. BALB/c mice acquired immunoprotection after immunized with recombinant HGXPRT protein. All results showed that recombinant HGXPRT can induce protection T cell immunity. It may be an ideal component that specific epitopes of CD4+T cell identified in nonhomology region of parasite compared with humans or rodents linked to or mixed with PfCP-2.9 malaria vaccine molecules. Consequently, it will improve the immunoprotection effect and broad-spectrum of malaria vaccine.