Inducing Protective Immunity By Combined Vaccination Based On Merozoite Surface Protein 1 And Apical Membrance Antigen 1 Of Plasmodium Falciparum

Malaria is one of the widespread tropical infectious disease in the world, especially Plasmodium falciparum. In recent years, a novel approach to control the spread of malaria was needed urgently for the increasing drug resistance of plasmodium and its intermediary agent-anopheline. An efficient vaccine will be an important means to prevent infection, to reduce clinical cases and to block transmission by mosquitoes.Life cycle of plasmodium is very complex ,the erythrocytic phase of plasmodium is the major stage to cause the disease. In this stage, the Plasmodium falciparum make a neuter generation in blood and lots of RBC are destroied at the same time. And the major stage of mosquito- hemophagia spread is just in the erythrocytic phase, too. So the significance is obviously if we obtain a erythrocytic phase vaccine to reduce clinical cases, to delay the course of disease and to control the spread of malaria.Besides blood stage,liver stage is also very important to prevent infection of sporozoite into liver cells.Until now,people believe thatmulti- stage,multi-antigen vaccine is the best version of vaccine against malaria.DNA vaccine is a safe and effective vaccine which might express protein continuiously in a low level and has the ability to activate both arms of the immune mechanism. Recombinant MVA mainly active the cellular immune response and protein vaccine mainly is the humoral immune response. The combined immunization of DNA vaccine and MVA can induce a high cellular immune response. With the infected rodent animal model, priming with DNA vaccines and, boosting with MVA or recombinant proteins,so called "prime-boost" stragety, has resulted in the generation of unparalleled levels of specific immunity and, in some cases, afforded protection against infectious agents.Apical membrane antigen 1(AMA1) and merozoit surface protein 1 (MSP 1) are the important candidate antigens of erythrocytic phase vaccine. AMA1 is a membrane surface protein on the merozoite apical membrane of plasmodium and make an important contribution to relocalization of merozoite in the infective process. MSP1 is a membrane protein on the surface of merozoite and play a key role in the process of merozoite invision host cell. For this reason we select the ectodomain of AM A1 and full-length gene fragment of MSP1 as the target antigen, and have prepared DNA vector, recombined modified vccinia virus Ankara(rMVA) and recombined protein, respectively. AMA1 and MSP1 are also expressed in the liver stage, as we know CD8+ Tcell response to malaria is critical for protection against malaria infection in the liver.This research is based on the good results of combination vaccination by using the DNA vaccine VR1020/E which contain Plasmodium falciparumapical membrane antigen 1(AMA1) ectodomain, recombinant modified Vaccinia virus Ankara and protein vaccine, that have been done by Li Xun. First, the DNA vaccine VR1020/190.3 was constructed by using the full gene sequence of the merozoite surface protein 1 (MSP-1) of P. falciparum 3D7 strain, then, BALB/c mice were immunized with VR1020/190.3 alone or with VR1020/190.3 plus cytokine plasmid pcDNA3/GM-CSF,and then, boosted with rMVA/190.3, that was construced by assistant-proffessor Miao jun at the Heidelberg university . ELISA resultes indicated, the DNA vaccine that encoding full-length gene fragment of MSP 1 can induce antibody response and plasmid pcDNA3/ GM -CSF could help VR1020/190.3 to produce much more IgGl. Antibody response to MSP1 were greatly enhanced after boosting with rMVA/190.3 , their IgG levels respectively increased 53 to 10 times. All the resultes above shown that boosting with MVA can induce obviously improved the immune response that elicited by DNA vaccine .And these were according with the immunity resultes of AMA1 .Based on these ,mixing the prototype vaccine: DNA plasmid (VR1020/190.3 and VR1020/E), recombinant rMVA(rMVA/E and rMVA/190.3) and protein vaccine(E and d-GX190H) as the DNA vaccine(D),virus vaccine(V),protein vaccine(P) were used in prime-boost vaccination strategy.The combination immunity group: D3-V means mice were immunized with DNA constructs for 3 times and then boosted with rMVA once; D3-P is DNA primed immunization 3 times boosted with protein vaccine; V-P is rMVA primed immunization boosted with protein vaccine; P-V is protein vaccine primed immunization boosted with rMVA; P3 is protein vaccine immunization 3 times. The results of ELISA show that P3 group got the highest immune response,...