英文文献汇报.pptx

IF=2.2181.Introduction Merozoite surface protein-1 of malaria parasites,present on the surface of merozoite and playing a key role in the invasion process,is one of the leading vaccine targets for both P.falciparum and P.vivax malaria.MSP-1 occurs as a 200 kDa precursor and undergoes step-wise proteolytic processing resulting in a glycosyl-phosphatidylinositol-anchored 42 kDa protein fragment(MSP-1 42)on the surface of free merozoite.A large number of studies have shown the protective potential of MSP-1 fragments(42 and 19 kDa)of P.falci-parum and P.yoelii.DNA vaccines have emerged as a promising approach for a variety of infectious agents including malaria parasites.DNA based vaccines are particularly important for developing multistage/multiantigen vaccines for complex parasites like malaria thereby inducing specific and protective immunity against different life cycle stages(pre-erythrocytic,erythrocytic and sexual stages)of malaria.The advantages of DNA vaccines are the ease of their production and their ability to induce responses without any exogenous adjuvant,which is an absolute requirement for protein based vaccine formulations.DNA vaccines have been shown to induce strong humoral and cellular responses to malarial antigens in immunized.However,clinical trials of these candidate vaccines when used alone or in repeated homologous boosting regimens have been disappointing,with short lived low levels of induced specific T-cells responses.Sequential immunization with different plasmids/vectors known as heterologous prime-boosting appears to be a better approach and has been shown to induce enhanced and persistent levels of antibody and cell mediated immune responses against malaria.In heterologous prime-boosting strategy the priming was done with DNA followed by the booster immunization with a recombinant protein,a recombinant viral vaccine,or exposure to the live organism.In the present study we have evaluated the immunogenicity of a DNA vaccine encoding MSP-1 42 of P.vivax using prime-boosting strategy in BALB/c mice.We have also compared the immunogenicity of PvMSP-1 42 recombinant protein in BALB/c mice.2.Materials and methodsDNA vaccine constructs encoding P.vivax MSP-1 42In vitro transfection of mammalian cells with P.vivax MSP-1 42 pcDNA 3.1 plasmidMonoclonal antibodies against P.vivax MSP-1 42Localization of P.vivax MSP-1 42 by indirect immunofluorescenceSDSPAGE and immunoblottingPurification of recombinant P.vivax MSP-1 42 expressed in E.coliPurification of plasmid for immunizationImmunization studies in miceEnzyme linked immunosorbent assayDetermination of IgG subclassesCell proliferation assayDetection of cytokines IFN-,IL-2 and IL-12 by ELISA3.Results3.1.Construction of a P.vivax merozoite surface protein-1 42-pcDNA 3.1 plasmid DNA vaccine and its in vitro expression in COS1 cells3.2.Antibody responses in immune mice against P.vivax merozoite surface protein-1 42-pcDNA3.1 DNA vaccine construct and recombinant PvMSP-1 42 protein3.3.Measurement of P.vivax merozoite surface protein-1 42 specific IgG isotypes in immune mice sera3.4.In vitro T cell responses against P.vivax merozoite surface protein-1 423.5.In vitro cytokines production against P.vivax merozoite surface protein-1 424.Discussions In the present study,we have prepared a plasmid DNA construct encoding P.vivax PvMSP-1 42 antigen.The expression of PvMSP-1 42 protein was checked by in vitro transfection of COS-1 mammalian cells with PvMSP-1 42-pcDNA 3.1 plasmid DNA construct.Immunogenicity of PvMSP-1 42 DNA vaccine construct was investigated by priming the BALB/c mice either with PvMSP-1 42 plasmid DNA or with recombinant PvMSP-1 42 protein and boosting with recombinant PvMSP-1 42 protein.In the present study we have conducted DNA vaccination for P.vivax MSP-1 42 and compared the immunogenicity of DNA vaccine construct with corresponding protein vaccination in BALB/c mice.Immunization of mice with recombinant PvMSP-1 42 resulted in the induction of high titres of anti-rPvMSP-1 42 antibodies than that of DNA immunization.However,the antibody titres observed in PvMSP-1 42 plasmid DNA immunized mice were significantly higher than the mice in the control group(vector immunized).DNA vaccine plasmids encoding merozoite surface protein-1 from murine malaria has been shown to provide protection against malaria infection.The importance of heterologous prime-boost strategy in malaria infection has been demonstrated by a number of studies.In the present study,priming with P.vivax MSP-1 42 plasmid DNA and boosting with recombinant PvMSP-142 protein exhibited a significant increase in the antibody responses in DNA/Protein group compared to DNA/DNA group as it was evident on comparing the 1OD titre as well as endpoint titre values of different immunized groups.These results suggest that the priming with plasmid DNA and boosting with recombinant protein is essential for the enhancement of immune responses.However,the antibody titres in Protein/Protein group were significantly higher than DNA/Protein group.This may be attributed to the use of FCA/IFA for protein immunization which resulted in high levels of antibody in Protein/Protein group.Previous studies have shown that cell mediated immune responses,in addition to antibody responses,are required for protection against malaria.Present study on measuring T cell responses in mice after DNA immunization and protein boosting exhibited that T cell proliferation was significantly enhanced in DNA/Protein and Protein/Protein groups.Elevated levels of cytokines(IL-2,IL-12,and IFN-)and high titres of antigen specific IgG isotypes(IgG1 IgG2b IgG2a)indicated that both Th1 and Th2 subsets of T helper cells may be produced during immunization of mice in the present study.This type of responses,i.e.,the activation of both Th1 and Th2 cells,is considered ideal for a blood-stage vaccine.Our findings on DNA prime and protein boosting have also indicated that among the DNA immunized groups,priming with DNA and boosting with protein clearly elevated the levels of all the cytokines in DNA/Protein group suggesting that DNA can prime the mice but itself is not sufficient enough to enhance the immune responses.5.Conclusions In the present study,we have cloned 42 kDa fragment of P.vivax merozoite surface protein-1(PvMSP-1 42)in eukaryotic expression vector pcDNA3.1.We checked the in vitro expression of PvMSP-1 42-pcDNA3.1 in COS-1 cell line.Indirect fluorescent assay and western blotting confirmed the expression of PvMSP-1 42 in COS-1 cells.In order to study the immunogenicity of recombinant vaccine plasmid,we immunized BALB/c mice with PvMSP-1 42-pcDNA3.1 plasmid DNA and compared its immunogenicity with the purified recombinant PvMSP-1 42 protein.PvMSP-1 42 plasmid DNA vaccine construct was immunogenic in mice,but the use of plasmid DNA alone may not be sufficient to elicit substantial immune responses.In order to increase the immunogenicity of PvMSP-1 42 plasmid DNA vaccine we used heterologous prime boosting with PvMSP-1 42 recombinant protein.Our results showed that the priming with DNA followed by boosting with recombinant protein significantly enhanced the immune responses of DNA/Protein immunized mice.