Construction Of A Neotype Transgenic Attenuated Malaria Vaccine And Its Antimalarial Mechanism

Malaria is a parasitic disease with long history, posing a serious threat to human health and social development. According to the data offered by World Heath Organization (WHO),243 million cases of Plasmodium infection were reported and about 863 thousand patients died of malaria in 2008, of which the majorities were younger than 5 years old. It has achieved greatly in the control of malaria. However, the diagnosis and the treatment are still difficult due to drug-resistance of Plasmodium and the resistance of vectors to pesticide. Generally, the safe, cheap and effective vaccine is considered as the key strategy in controlling and eliminating malaria. So WHO, United Nations Development Programe, World Bank etc. have listed the development of vaccine against malaria as one of three prior vaccine projects.Malaria infection starts from the biting of the female anopheles, by which sporozoites of Plasmodium with saliva are injected into the hosts’blood circulation. Then the sporozoites enter hepatic cells and are turned into schizonts by schizogony. Mature schizonts cause the rupture of hepatic cells and are released into the blood. These schizonts experience another schizogony in erythrocyte. They cause erythrocyte fragmentation after several multiplication cycles, and then lead to the occurrence of clinical symptoms, such as periodically fever and anemia. So sporozoite is an important upstream stat in infection process. The effective immunity of sporozoite vaccine can block the entering of sporozoite into hepatic cells, terminate the development of further infection, prevent and treat the malaria effectively.Vaccines against malaria have been developed for more than 40 years, and the research experienced whole Plasmodium vaccine period (killed vaccine and live radiation-attenuated vaccine), genetic engineering subunit vaccine and chemosynthesis polypeptide vaccine period, nucleic acid and pathogen vector vaccine period etc. However, tests show that these vaccines are not safe or effective as expected due to low specificity of immunological response, low titer specific antibodies, short duration of immunity, etc. Thus, increasing the safety and strengthening the immunological ability and prolonging the reaction time are essential for vaccine research.Mueller and their colleagues found that sporozoites of Plasmodium with UIS3 gene knockout were attenuated. After infecting animal models, these sporozoites stayed in hepatocells not in erythrocytes. They can be used as attenuated vaccine, and have immuno-protective effect on mice with malaria. In this study, we knocked UIS3 gene out in Plasmodium berghei (P.b) by gene targeting technique. In order to gain a good immunity, we transferred B-lymphocyte chemoattractant(BLC) gene into the attenuated P.b plasmid, and then transferred the new plasmid into sporozoites by transgenic technique. Thus, we obtained a new strain, UIS3-/BLC+, which is attenuated and can strengthen immunological ability.BLC is a specific chemokine of B cells. It can stimulate B-lymphocyte receptor-1 expression, guide the migration and homing of B cells, promote immune response by attracting B cells and CD4+T cells. Modification by BLC gene can enhance both specific humoral immunity and cellullar immunologic response to Plasmodium.According to BLC sequence of mice from genetic library, the primer was designed. BLC cDNA was amplified by PR-PCR. Then the amplified cDNA was inserted into the plasmid of Plasmodium with UIS3 knockout linked by restriction endonuclease and T4 ligase. This recombinant plasmid contains Pyrimethamine-resistant gene and report gene of green fluorescent protein, and can be expressed in transformant. After screening, digestion and sequencing, the recombinant plasmid was transfected into eukaryocyte in vitro. Analysis of agarose gel electrophoresis showed that the molecular weight of cDNA and the digestion result of recombinant plasmid were the same as expected. DNA sequencing also confirmed the open-reading frame of target gene. It proved from both mRNA and protein levels that BLC gene can be expressed in eukaryocyte. Before transformation, Plasmodium was cultured in vitro for short term. While during mature schizont period, schizont was separated by a density gradient mediator, Nycoprep, for transformation by electroporation. After digesting recombinant plasmid and transfecting into Plasmodium, a new strain was screened (pUIS3-/BLC+). After transfection by recombinant plasmid, Plasmodium was injected into mice via caudal vein, and Pyrimethamine was also injected intraperitoneally for screening. The positive transformant was obtained after twice screening, and displayed green fluorescence under fluorescence microscope. PCR test also revealed recombinant plasmid, confirming the integration of plasmid and Plasmodium gene.Selecting Plasmodium with pUIS3-/BLC+ as the vaccine, and mice infected by Plasmodium as the model, we focused on the anti-malaria mechanism by molecular biological, immunological and histological methods in this study. The results show that Plasmodium with pUIS3-/BLC+ can grow in hepatocyte, but it can’t develop into erythrocytic stage. So it is attenuated with gene knockout. Comparing with immune reaction induced by sporozoite with UIS3-, mice with pUIS3-/BLC+ can produce effective humoral immune reaction and cellular immune reaction, with significantly higher titer and longer duration of antibody, and stronger T cell respones. Anti-malaria test in vivo and in vitro all prove that sporozoite with pUIS3-/BLC+ is more capable to depress the growth and transformation of Plasmodium.This study shows that sporozoite of Plasmodium with gene knockout and transferred immunoenhancing gene provides good model for development of vaccine against malaria. It is promising in clinical application and is worthy further research.