Construction And Screening For The Second Generation Of Polyepitope Chimeric Vaccine Against Plasmodium Falciparum

Malaria is one of the most severe infectious diseases in the world,there are 243 million cases and 0.86million deaths caused by malaria in 2008,and Plasmodium falciparum is the most deadly species. Recent years,though marked progresses have been obtained in"Malaria Control",there is still an urgent demand of preventive and therapeutic vaccine.To overcome the problem of highly antigenic variation and stage specincity in P.falciparum,sub-unit or polyepitope chimeric vaccine is a promising strategy.In previous study,we generated a novel approach named"epitope shuffling".Using the approach,we constructed a polyepitope gene library with high diversity of epitope combination,and from which our first generation of polyeptitope vaccine designated M.RCAg-1 with high immunogenicity and in vitro antipalasite efficacy was obtained.However,the antigen's further clinical study had been hampered by the redundancy of 43 amino acids from vector at N terminal and 6×His tag,though which could be cut off by Enterokinase with high cost.Based on the previous work,we have made several improvements,which includes:(1)15 B cell and Th cell epitopes from 12 main antigens of P.falciparum blood stage have been selected,(2)definite spacer sequences have been introduced between adjacent epitopes,(3)two parasite-derived fusion sequences FN and FC as flanking sequences have been fixed at N and C terminals of polyepitope antigen,(4)to meet the"national standard of DNA recombinant products",no vector peptides or tags are contained in the sequence.The second generation of polyepitope chimeric vaccine have been constructed and screened.Meanwhile,by comparative analysis,we have explored the relationship among epitope composition,second strueture,immunogenicity and in vitro antiparasite efficacy of the polyepitope antigens.Moreover,high efficient immunoaffinity purification of polyepitope antigens by IgY specific to FC has been investigated.The results are as follows:1. The second generation of polyepitope gene libraly with high immunogenicity and diversity of epitope combination had been successfully constructed.Besides,the immune serum from mice could recognize the native proteins of P.falciparum cultured in lab with high diversity. 2.The chimeric antigen named D10 with high immunogenicity was screened from the gene library. The results of GIA using total and specific IgG from rabbit were 47% and 67%,which were both higher than 44% of the result fiom positive control MSP1 antibody.On the other hand,D10 could be recognized by 67% of 72 samples of patients' sera from epidemic area and the recognization was significantly negatively correlated with parasitemia of patients,which suggested that D10 could be correlated with the protection against P.falciparum.Based on these results,antigen D10, full sequences derived from P.falciparum,is worthy of further development.3."Coil-Helix-Coil-Helix"structure existed in the secondary structures of polyepitope antigens could reflect the immunogenicity.And the"4 successive Th cell epitope"structure contained polyepitope antigens had higher in vitro antiparasite efficacy of antibodies than others.4. Inhibiting and enhancing antibodies had been proved to be existed in the total antibodies against polyepitope fragments of chimeric antigens,which would probably affect the in vitro antiparasite efficacy of total antibodies by synergism mechanism.5. High efficiency of chimeric antigens' purification by immunoaffinity chromatography with polyclonal IgY antibodies specific to C-terminal fusion polypeptides had been confirmed.6. Hydroethidine-based flow cytometry,as a relative quantitative method,could be applied in the analysis of parasitemia and life cycle in the culture of P.falciparum,and the working concentration of Hydroethidine had been optimized with 10μg/ml.In conclusion,the second generation of polyepitope chimeric antigen D10 had been constructed and screened by the improved"epitope shuffling",which would be worthy of further development.And the present results indicated that N and C terminal fusions could be used as"endogenous tag"for the identification and immunoaffinity purification of the antigens.Besides,the assumption had been confirmed that polyepitope fragments could impact the immunogenicity and in vitro antiparasite efficacy of the chimeric antigens.These results would be valuable for the design and optimization of various kinds of polyepitope chimeric vaccines.