Polyepitope, Artificial Antigen DNA Vaccine Against Plasmodium Falciparum Created And Optimized By Molecular Breeding Technology

Malaria is one of the most infectious diseases to human health in the global crisis. It causes approximately 2.5 million deaths, and 300 to 500 million cases of infection per year, since the resistances and tolerances of its parasites and mosquitoes to chemical medicines or insecticides are increased. P. falciparum is the greatest one of pathogen species causes morbidity and mortality, and its complex life cycles, antigenic stage-specificity, diversity and variation, have been the unique obstacle to develop antimalarial vaccines for a long time. Thus, the development of a multiple antigens and epitopes vaccine against P. falciparum to elicit relative immune response at different stage has become a major hotspot in antimalarial vaccine study, according to the characteristic of immune response at each life stage of parasite. However, in the polyepitope vaccine aspect, it is still focused on the artificially synthesized polypeptides or polyepitope gene with single format. For the former is high-cost and the latter is low-immunogenicity, it is too difficult to reach the satisfactory protection results or to obtain immune response diversity after immunization. In order to deal with the shortcoming of polyepitope DNA vaccine, we have developed a method named "epitope shuffling technology" to optimize the polyepitope DNA vaccine by use of the random recombinant principle of molecular breeding technology or DNA shuffling.In this study, we chose 14 B-cell and Th-cell epitopes of antigens mainly from the blood stage of P. falciparum, based on the type of immune response against the blood-stage parasite infection. In the process of epitope shuffling established, we have brought forward and attempted to use the PCR-assemble technology (PAT) and isocaudamer random recombinant technique (IRRT) successively, and finally constructed five sub-libraries (named L1, L2, L3, L4 and L5 according to gene length from small to large) with different polyepitope gene length by the means of IRRT. Each polyepitope library was shown the higher diversity of polyepitope genes based on the PCR-SSCP analysis. With the polyepitope library immunization, we have proved that the length of polyepitope gene affects the immunogenicity of DNA vaccine; the sub-libraries L4 with about 2.0kb gene size and L3 with about 1.2kb size were achieved the best level of immune response, and cross protection in the rodent animal model after library immunization. It is fully demonstrated that the different gene length of polyepitope artificial antigen is significantly influenced on the immunogenicity of DNA vaccine, and that the polyepitope-library immunization can elicit good immune response, and it is feasible to screen the optimal chimeric polyepitope gene from the library.Three polyepitope genes ES312, ES391 and ES452 with high immunogenicity were screened from the duplicated express library of sub-libraries L3 and L4 in prokaryote, by use of the high throughput immunochemical selection with serial dilution of sera pool from polyepitope library immunization. The experimental results in vivo indicated that the immunogenic levels of three polyepitope gene vaccines were 100-200 times higher than that of other gene vaccine with the appropriate length, and the results detected by ELISA and immunoprecipitation assay showed higher diversity of specific antibodies were induced, under the same express level of each gene. And it is also presented that there are higher ability of the three-polyepitope genes to induce the CD4 cell in vivo. In the rodent animal model, polyepitope gene ES312 immunization has obtained 100％cross protection; and in the experiments of P. falciparum growth inhibition assay in vitro, the efficiency of ES312 against malaria growth detected by flow cytometry is up to 95.8％. The results of gene sequencing further indicated that the epitope assemble formats or the second structure of protein among the three-polyepitope genes was all possessed consensus and conservative structure. All these data is enough to present that the epitope assemble formats and the space structures of full protein determine the immunogenicity of polyepitope gene vaccine.Through the genetic immunization experiment of polyepitope artificial antigen in vivo, we have demonstrated that one of three polyepitope genes—ES312, with a certain tandem format, could elicit higher specific antibody response. At the same time, we found that mainly cytokine IFN-γderived by Th1 cell was expressed after the gene prime immunization, and higher level of Th2 cell response was induced followed by the second boost. And it is the immune response of host that vaccinated mice can resistant the P. yoelii parasite infection at the blood stage. From the results of ES312-immunized mice challenged after CD4 or CD8 lymphocyte transient blocking, we also found that the ability of host to inhibit the malaria parasite growth was increased. All these seem to further demonstrate that to induce the specific CD4 cell and humoral immune response is critical to prevent the malaria blood-stage infection, and that specific structure of polyepitope gene assembled is vital factor to induce this immune response in host.In addition, in order to assess the protective effect of a blood-stage vaccine against malaria in vitro with more accurately, here we have optimized the method to detect the parasitemia of parasitized red blood cells by flow cytometry, and brought forward an improved formula to calculate the efficiency of malaria growth inhibition.The results in this study indicated that the epitope shuffling method developed by us exploits a new way for construction and optimization of DNA vaccine with polyepitope artificial antigen. And these data confirmed our assumption that space conformation of artificial antigen determines the immunogenicity and immune protection of vaccine. The novel strategy of polyepitope vaccine construction is contributed not only to antimalarial vaccine, but also to other vaccine against virus, bacterial, protozoan infectious disease, as well as the gene therapy against tumor, allergy and neonatal tolerance.