The Preparation And Different Immunoprotection Mechanism Study For Random Tandem Polyepitope Candidate Vaccine Against Plasmodium Falciparum, M. RCAg-1

Malaria is one of the most infectious diseases to human health in the world. There are an estimated 500 million cases and up to 1 million deaths from malaria each year,which mainly are under 5 years of age and pregnant women. 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.In the previous work, we have successfully constructed polyepitope Plasmodium falciparum malaria vaccines M.RCAg-1 by random tandem with epitope shuffling technology platform,11 B cell and Th cell epitopes seclected from 8 Plasmodium falciparum antigens as the study subject according to immune type involved in blood-stage infection against Plasmodium. At present, the experiments of immunogenicity and protection in vitro and vivo in immunized mice, rabbits and rhesus monkeys have been observed, which confirmed its good immunogenicity and protective effect in vivo, and the vaccine is an potential malaria candidate vaccine.Based on the national standard of biological products, the present work study on the screen of engineering bacteria expressing polyeitope vaccine M.RCAg-1 recombination protein, the establishment of the technique for highly effective protein expression and purification, the evaluation of the effect of three adjuvants acceptable in human clinical experiment formulation with purified M.RCAg-1 protein on the humoral immune response and CD4+ T cell response of inbred strain mice and New Zealand white rabbits, and the effect of antibody on Plasmodium falciparum growth in vitro. The potential reason of different inhibition effect of recombinant protein expressing in different victors immune rabbits sera on Plasmodium falciparum growth in vitro were also analyzed. The results are as follows:1. the imunogenicity and protection of recombinant antigen protein through pDS-ex-Ekase system expression is best, therefore, the bacterial strain of BL21 (DE3)-M.RCAg-1/pDS-ex-Ekase protein expression is determined as engineering bacterial strain. Also the optimal pattern of the expression and purification of M.RCAg-1 protein is determined. The recombinant protein could be obtained stably and over 97% purity which could the need of following experiment of vaccine related preclinical evaluation;2. Three kind of recombinant proteins were expressed in pDS-ex-Ekase and pDS-ex-PPase carrier system and pBV220 temperature controlling promoter system, the three proteins have almost the same amino acid sequence, the same immunogenicity in animal models, however, the obviously different immunoprotections. When New Zealand white rabbit immunized by antigen of pDS-ex-Ekase carrier system, the sera antibody inhibited the growth of Plasmodium falciparum in vitro almost 100%, However below 50% when immunized by antigens of the other two carrier systems.3. The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors, analyzed by bioinformatics and chromatographic technique, which demonstrated the change of protein molecule spaces conformation, and the obviously change of some epitope locations.4. The statistics analysis of antibody titer induced by every epitope peptid which was contained in three antigens with almost the same sequences showed the key factors of antigen immunoprotection in Humoral immune reaction and adjuvanticity T cell immune reaction. The key factors were epitope percentage and average antibody titer.The present study determined the stable and highly effective preparation and purification of M.RCAg-1 in prokaryotic expression systems, and explored the condition of semi-works production according to national biological product examination and management standard which can meet industrialization production. The study of construction in different expression systems showed the space conformation of protein molecule is crucial to immunoprotection effect of artificial polyepitope antigen. Factors influencing space conformation involved not only the peptide sequence of reading frame but also the peptide sequence in binding site between expression carrier and insertion element, these factors changed the amount ofα-helix in peptide, the epitope location in molecule, and the protein stability by influence of protein fold style in artificial antigen. The present study also demonstrated, to polyepitope artificial antigen which induced humoral immune reaction and adjuvanticity T cell immune reaction, the key factors influencing antigen immunoprotection were epitope percentage and average antibody titer which induced highly antibody level. The results could be important to the design of new and optimal erythrocytic stage polyepitope malaria vaccine.