The Specificity Or Not On Parasite-Specific CD4+T Cells Against Infection Of Plasmodium

Background:Malaria remains one of the most devastating infectious diseases in the world.People have tried all their efforts to control this disease for more than a century,but the outcome isn't ideal.The World Malaria Report 2005 represents that Malaria kills over a million people each year and about 3.2 billion people living in 107 countries or territories are at risk-mostly children.It is hard to evaluate the impairment and the impact that malaria has contributed to the human health and economy.Finding a safe and effective way to control malaria is an important study which scientists have struggled for more than sixty years.In the past decades,medicines such as Quinine have made great contribution to control malaria,but the morbidity of malaria still ascended sharply in recent years.And it is obviously due to the emergence of the drug resistance of the parasite and the insecticide resistance of Anopheles mosquito.Researchers generally think that the development of an effective and inexpensive vaccine is an important way to control this disease.History also has proved that the most effective and economic way to control infection diseases is vaccination.At present,the number of the vaccine that was used in developed countries is 12-13 and in developing counties is 6-7.This vaccines save millions lives every year.If the vaccines for preventing diseases such as AIDS,tuberculosis and malaria have been developed successfully,then millions lives would also been saved annually.So the countries put unprecedented attention and support in the study of vaccine for malaria.However the progress toward an efficacious vaccine for malaria has been slow after half of century,due to the complex life cycle.And the development of vaccines still is one of the most urgent and the hardest challenge which that medical studies face.The life cycle of plasmodium can been divided into liver stage and blood stage which provided the foundations for vaccine strategies aimed at liver stage forms and blood stage forms.In early stage,the research of malaria vaccine mainly focuses on the development of live stage.But the blood stage of parasite plays an important role in morbidity and mortality of malaria.So the current research of malaria vaccine mainly focuses on blood stage.In the development of vaccine on the blood stage,we have identified and cloned many effective candidate antigens of malaria vaccine to blood stage which can induce protective immunity. Vaccines incorporating MSP-1(merozoite surface protein 1),MSP-2,RESA (ring-infected erythrocyte surface antigen),and AMA-1(apical membrane antigen 1) elicit a reduction in parasite density,but have induced only strain-specific immunity in clinical trials.But these polymorphic and clonally variable antigens have complicated vaccine development.In addition,experiment proved that the protective immunity that induced by these vaccines depended on the existing of high-titer antibody,and the generation of these antibodies relied on the mediating of cell immunity.To gain really effective malaria vaccine,it is not only depended on identifying and cloning antigens which can induce protective immunity,but also relied on determining and evaluating the mechanism of these protective immunity.It is very complex in the parasite immunity mechanism.Early the research of protective immunity mechanism of blood stage mainly focuses on humoral mediated immunity.Recently some dataes from animal mode and epidemiology show that CD4+ T cells mediated cell immunity plays important role in immunologic mechanism against parasite of blood stage.The rate of malaria infection increased significantly when we depleted CD4+ T cells from these mice.At the same time,the rate descended sharply when we adopted Parasite-specific CD4+ T Cells to these mice.After homologous parasite challenge,the B cell KO or defect mice those had received the T cells were able to control parasite load very effectively,although this immunity was unable to completely eliminate infection,so we supposed that CD4+ T cells could control malaria infection independently. And activated CD4+ T cells could help B cells to produce antibody.Vaccine-induced immunity to MSPI-19 was largely high titer antibody dependent,at the same time the ability of B cells to continue producing antibody needed the aid of CD4+ T cells.But how the parasite-specific CD4+ T cells present antigen and how to kill pathogen remain unclear.Matured erythrocytes do not express MHCâ… andâ…¡molecules.RBCs can not express parasite-encoded antigens on their surface.Parasite antigens must be phagocytized by macrophages and expressed them to the specific CD4+ T cells after infected RBCs broke into pieces.The process of killing pathogen begins when the T cells bind to the specific antigenic determinant;the activated T cells produce cytokines or help B cells to produce antibody which kill the target cell.We don't know whether the process of specific CD4+ T cells killing the target cell is specific or not.We generated T cell lines of all the species of Plasmodium in this experiment.Mice were challenged i.v.with P.yoelii pRBC or P.berghei pRBC 24h after P.yoelii-specific CD4+ T cells transfered.If the process of specific CD4+ T cells killing the target cell was specific,only the P.yoelii could be killed.If the infection rate of P.berghei could also be controled,it might show that the process of specific CD4+ T cells kill the target cell wasn't specific.At the same time,we transferred green fluorescent protein(GFP) to plasmodium berghei by molecular cloning technique.Then mice were challenged i.v.with P.yoelii and fluorescently-labeled P.berghei 24h after P.yoelii -specific CD4+ T cells transfer.If the fluorescently-labeled P.berghei was killed,the result could also show that the process of specific CD4+ T cells kill plasmodium wasn't specific.It may provide us with the theory that whether we could select limited antigen to control different species of Plasmodium.Objective:Mice were challenged i.v.with different species of plasmodium 24h after specific CD4+ T cells transfer.We compared the difference rate of infection RBCs by using statistics analysis.Then we judge whether the process of specific CD4+ T Cells kill plasmodium is specificity or not.Methods:1,Comparing the proliferation by lymphocytes proliferation assay;2,Comparing the plasmodium infection rate between the mice those had received P.yoelii specific T cells and the control mice that received OVA-specific T cells.These mice were all challenged with plasmodium berghei.The parasitemia was monitored by examining DiffQuick-stained blood smears under an oil immersion lens;3,Fluorescence microscope.The P.yoelii and fluorescently-labeled P.berghei were measured by fluorescence microscope when mice were challenged with these two species of plasmodium after the P.yoelii specific T cells transfer.And the control mice received OVA-specific T cells.Results:1:Generation of Parasite-specific CD4+ T Cell Lines;2:Lymphocytes proliferation assay showed that P.berghei pRBC could induce the P.yoelii-specific T cells proliferation.In addition P.yoelii pRBC could also induce the P. berghei-specific T cells proliferation;3:Mice were challenged with different species of plasmodium after they received the same parasite-specific CD4+ T Cell.Those mice that had homologous parasite challenged got immunoprotection.Peak value of parasitemia of those mice that were challenged with different plasmodium lowered than the control mice that received OVA-specific T cells, and lives of those mice were prolonged than the control group;4:The parasitemia of the P.yoelii and fluorescently-labeled P.berghei were measured by fluorescence microscope when mice were challenged with these two species of plasmodium after the P.yoelii-specific CD4+ T cells transfer.The P.yoelii-specific CD4+ T cells not only controlled the P.yoelii but also inhibited the growth of P.berghei.Conclusions:We generated parasite-specific CD4+ T cell Lines in vitro.The result of lymphocytes proliferation assay and parasite challenged after T cell lines transfer showed that the process of specific CD4+ T Cells kill plasmodium is unspecificity.It provides us with the theory that we could select limited antigen to control different species of Plasmodium...