Immunogenicity And Protection Of Polyepitope DNA Vaccine, M.RCAg-1D, Against Plasmodium Falciparum By Different Immunization Methods

Human malaria infections caused by Plasmodium falciparum claim up to 3 million lives annually (especially children and pregnant women). In view of its high morbidity and motality rates and the progression of drug resistence that threatens treatment efficacy, populations living in epidemic areas are in great need of novel effective antimalaria control measures, such as an antimalaria vaccine. The unique obstacles to develop antimalarial vaccines have been Plasmodium' s complex life cycles, antigenic stage-specificity, diversity and variation for a long time. Thus, the ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. Delivery of combinations of malaria antigens can evoke enhanced immune responses and protect to a greater extent than can a single antigen alone, as well as overcome genetic restrictions in different Plasmodium strains.In the previous work, we have constructed polyepitope libraries with epitope shuffling technology, screened positive clones with high antigenicity by dot blot and obtained a positive clone named M. RCAg-1 which could induce cross-protection in mice and inhibitory antibodies against Plasmodium falicparum in rabbits.Protein vaccine is not suitable for generalization in poor countries owing to its high-cost, and a single DNA vaccine is difficult to induce satisfactory protection and obtain diversity of immune response after immunization for its low immunogenicity. So this study focuses on the immunogenicity and protection of M. RCAg-1D vaccines in different immunization methods, and the adjuvant effect of different gold beads in the gene gun vaccination.The results are shown in the following. The multiepitope DNA vaccine against Plasmodium falciparum M. RCAg-1D induced significant CD4~+ T cell responses and high titers of specific antibodies in immunized mice by gene gun, which could effectively protect immunized mice from deaths when challenged by P. yoelii. This suggests that gene gun immunization by intradermal could improve the immunogenicity of DNA vaccine significantly, which might be related to up-regulation of the transfection, expression of the exogenous gene and release of the M. RCAg-1 protein. By electroporation in vivo M. RCAg-1D gene vaccine could induce high titers of specific antibodies but with low avidity in mice which could not effectively protect immunized mice from deaths when challenged by P. yoelii. M. RCAg-1D DNA vaccine encapsued by liposome could induce significantly higher titers of specific antibodies than naked DNA vaccinated intramusculary or subcutaneous, although there are not enough protective antibodies in immunized mice by these three methods. This might be related with the same presentation pattern of gene vaccine by in vivo electroporation or by liposome capsued DNA vaccination as those by naked DNA vaccinated intramusculary and a single improvement of the transfection efficiency. Results of comparision of different gold beads as adjuvant tell us that using different gold beads can induced different levels of antibody in gene gun immunization and when using 1.0μm as the plasmid DNA vector in gene gun vaccination, M. RCAg-1D DNA vaccine could induce the highest titer of specific antibody in immunized mice, this maybe related to the effection of different gold beads to release, transfection pattern of the exogenous genes.