Immunogenicity And Protection Of Polyepitope Artificial Antigen, M.RCAg-1, Against Plasmodium Falciparum In Animal Models

Malaria is one of the most infectious diseases to human health in the world. There are an estimated 500 million cases and up to 3 million deaths from malaria each year. The mortality levels are greatest in sub-Saharan Africa, where children under 5 years of age and pregnant women account for ninety percent of all deaths due to malaria. The emergence and spread of drug-resistant parasites and insecticide-resistant Anopheles mosquito vectors make prevention and treatment of malaria a big problem in the world. Plasmodium's complex life cycles, antigenic stage-specificity, diversity and variation, have been the unique obstacles to develop antimalarial vaccines for a long time. Thus, the development of a multiple antigens and epitopes vaccine against Plasmodium falciparum to elicit relative immune responses at different stages has become a major hotspot in the development of malaria vaccines. However, the investigation of polyepitope vaccines is still focused on the artificially synthesized polypeptides or polyepitope gene vaccines with single format. The former is high-cost and the latter induces low immunogenicity. It is difficult for both of them to induce satisfactory protection and obtain diversity of immune responses after immunization.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 VR312(renamed as M.RCAg-1 later)which could induce cross-protection in mice and inhibitory antibodies against Plasmodium falicparum in rabbits.Based on the previous work, this study centers on the immunogenicity and protection of M.RCAg-1 vaccines in animal models and the epidemiological analysis of M.RCAg-1 protein as well as the standardization of flow cytometry for detecting parasitemia with nucleic acid dye. The results are as follows:1. The patient serum from different epidemic area could significantly recognize the recombinant protein, M.RCAg-1 which suggested that M.RCAg-1 antigen could probably surmount the immune evasion due to the difference of the strains.2. The recognization between serum from residents in Cameroon and M.RCAg-1 protein is significantly positively correlated with ages of residents which suggested that the mortality would be reduced if the children in epidemic areas were inoculated M.RCAg-1 vaccine in early childhood.3. M.RCAg-1 protein vaccine induced significant CD4~+ T cell responses and high titers of specific antibodies in immunized mice, rabbits and rhesus monkeys which could effectively inhibit in vitro development of Plasmodium falciparum. This might be related to the optimized conformation of M.RCAg-1 protein.4. M.RCAg-1 gene vaccine could induce high titers of specific antibodies but with low avidity in mice and rabbits which could not effectively inhibit the in vitro growth of Plasmodium falciparum. In rhesus monkeys, DNA prime-protein boost regimen could induce high levels of antibodies which could not significantly inhibit the in vitro growth of Plasmodium falciparum. This might be related with the presentation pattern of gene vaccine through which produced antibodies with different ratios and avidity from those by protein immunization.5. The results of cellular immunity in mice immunized with the recombinant protein suggested that the cells with the same specificity could develop into the different T cells subsets which depended on the antigen conformation and the cytokines milieu.6. The flow cytometry with hydroethidine could objectively and quickly detect the parasitemia in the culture as well as evaluate the efficacy of the synchronization for the parasites.