| Malaria is still one of the most deadly infectious disease, vaccines will be an important means to prevent infection, to reduce clinical cases and to block transmission by mosquitoes. DNA vaccine is a new form of vaccine which might provide several important advantages over current vaccines, including easiness to construction, high stability, flexibility to modulation and the ability to activate both arms of the immune mechanism. Cytokines play key roles in the process of immune response and, the plasmid type of cytokine can modulate immune response in DNA vaccination. Prime-boost vaccination, a novel strategy involving priming with DNA vaccines and, boosting with attenuated pox viruses or recombinant proteins, has resulted in the generation of unparalleled levels of specific immunity and, in some cases, afforded protection against infectious agents. AMA1 is one of the most important candidate antigens in blood-stage malaria vaccines and, antibodies to AMA1 play a key role in protection. In this study, the ectodomain of AMA1 was selected as immunogen and, three kinds of prototype vaccines were constructed, including, recombinant proteins, plasmid DNA and, recombinant MVA. The immune characteristics of each vaccine were analyzed and, mice were primed with plasmid DNA and boosted with recombinant protein or rMVA in different orders.First, nucleic acid sequence encoding ectodomain of AMAlwas amplified from P.f genome and, was cloned into prokaryotic expression vector. Series of vectors which encoding different parts of AMA1 were constructed, including pET-16b/E, pET-16b/I+IL pET-16b/L pET-30a(+)/II and pET-30a(+)/III. E.coli BL21 DE3 were transformed and induced, recombinant proteins representing subdomains of AMA1 were produced. Inclusion bodies of expressed proteins were dissolved in denaturants and purified by NTA-Ni gel and refolded under suitable conditions. BALB/c mice were immunized intra peritoneal with recombinant proteins in Freund's adjuvant, anti-sera were prepared and identified by immunofluorescence assay and immune-blotting. Activities of mice immune sera in in vitro growth inhibition of P.f parasites were determined and, the relationships between protective antibody response and the structure of recombinant proteins were analyzed. The results showed that integrality of the ectodomain of AMA1 was crucial to the induction of protective antibodies.Based on the results of protein immunizations, the intact gene fragment of AMA1 ectodomain was adopted and, DNA vaccination plasmid VR1020/E was constructed. BALB/c mice were immunized in muscles with 100 μg ofVR1020/E for two times, obvious antibody and splenocytes proliferation were induced. Cytokine expressing plasmids pcDNA3/GM-CSF and pcDNA3.1(-)/IL-4 and , bicistronic plasmid pGM-CSF/pTPA-E were constructed. Cytokine-encoding plasmids were used seperately in combination with VR1020/E to inoculate mice and, both of the antibody and cellular response were promoted remarkably by cytokine plasmids, in which, GM-CSF and EL- 12 encoding plasmids separately facilitated Th2 and Thl response.MVA is a highly attenuated vaccinia virus, recombinant MVAs had potentials in inducing immune responses to infectious agents and tumor cells. In this study, gene transfer vector pIIIdHR.ssp/E was constructed and, recombinant MVA that efficiently express AMA1 protein was selected after homologous recombination. Groups of BALB/c mice were injected intra peritoneal with 107-108 TCID50 doses of rMVA/E for three times, evident anti-AMA1 antibody and splenocytes proliferation were observed. IgG2a was dominant in antibody response indicated that Thl type of immune response was induced by the virus.After the immune characteristics of three different kinds of prototype vaccines had been studied. Groups of mice which had been primed with VR1020/E plus different cytokine encoding plasmids were boosted with rMVA/E. Antibodies induced by DNA immunization were greatly enhanced by boosting with rMVA/E, as well as IgG1 was increased , IgG2a was boosted more evidentl...
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