Construction Of A Series Of Mutants Of PfCP-2.9 Malaria Vaccine Candidate And Their Epitopes And Immunological Analysis

Malaria remains to be a severe tropical disease. It is reported by World Health Organization that it caused more than 247 million cases around the world and about 1 million death in 2006, most of which were in children under five years of age in Sub-Saharan Africa. With the emergence and spread of insecticide-resistant mosquitoes and drug-resistant parasites, there is an urgent need for the development of a safe and effective vaccine to prevent or even to eradicate the disease.In a previous study, plasmodium falciparum chimeric protein-2.9 (PfCP-2.9) was constructed by fusing the C terminal 19kDa frangment (MSP1-19) of merozoite surface protein 1(MSP1) with apical membrane antigen-1 (AMA-1) domain III (AMA-1 (III)) through a 28-mer peptide. In animal studies, the chimeric protein was proved to enhance the immunogenicity, and in vitro growth inhibitory activities. Two separate Phase I clinical trials have also demonstrated that the vaccine candidate was safe, tolerable and immunogenic. In order to investigate the enhancement of the immunological mechanism, by which the vaccine exerts function, we performed an epitope mapping of PfCP-2.9 by Western Blot and enzyme-linked immunosorbent assays (ELISA).In this study, we designed and created 17 mutants of PfCP-2.9 with single amino acid substitution. Nine of them were mutated on the AMA-1 (III) component, and the rest eight were made on the MSP 1-19 component. The mutated amino acids selected on AMA-1 (III) component are those highly conserved in the Plasmodium or involved in the formation of the epitope recognized by mAbF8.12.19, which was not inhibitory but involved in cross-reactivities across species. The eight aminio acid substitutions on MSP1-19 component were those proved to be involved in the formation of blocking epitopes or those surface-exposed on the crystal structure of PfMSPl-19 and putatively involved in the inhibitory or blocking epitopes. The above mutations were made by the method of overlap extention polymerase chain reaction (PCR), which generate the upstream or downstream fragments through the first round of PCR using the forward universial primer and a reverse mutant primer or the reverse universial primer and a forward mutant primer, respectively; and then combine the purified upstream and downstream fragments with the forward and reverse universial primes by the second round of PCR.The mutant genes were subsequently ligased into the expression vector of pPIC9K and sequenced. The vector with the correct target sequence was further transformed into the Pichia pastoris expression system to secrete the mutated proteins in the supernatant. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis, it was confirmed that all the 17 mutant genes were sucdessfully expressed and the mutant proteins could be recognized by the rabbit immune sera. These results provided bases for the investigation into the epitopes of PfCP-2.9.The epitope mapping was carried out by Western Blot and ELISA, using the above mutant proteins as well as a serial of mAbs, which were prepared and characterized in our previous work or obtained from other labs. The results we obtained may have the following implications:(i) An inhibitory epitope recognized by mAb7G was found, and the epitope is closely related with three substitutions (M62:Phe491→Ala, M82:Glu511→Gln and M84:Arg513->Lys), because any of them could reduce the binding of the mAb. In addition, all of the three amino acids are in close proximity to each other in conformation on the C terminal unstructured region of AMA-1(Ⅲ). (ii) M62 and M82 could further reduce the binding of mAbG 11.12, while M62 could reduce the binding of mAbW9.10. Therefore, there is possibility that the two mAbs compete with mAb7G at the position of M62 and M82 and at the position of M62, separately. (iii) Some information about blocking epitopes was also obtained. There are four definitive amino acids (Asn15, Glu27, Leu31 and Glu43) that were involved in the formation of blocking epitopes on PfMSPl-19. It was reported that mutation of these four amino acids abolished the binding of blocking mAbs of mAb7.5, mAb2.2, mAb1E1 and mAb111.4, seperately. In this study, it was confirmed that the epitope recognized by mAb1E1 was the same as that identified on the PfMSP1-19 molecule. (iv) The amino acid of Asn15 was found to be of critical importance, since the substitution of this residue to Arg on PfCP-2.9 reduced the binding of most mAbs.To investigate the recognition differences of sera from malaria infected individuals between PfCP-2.9 and its mutant proteins, we collected 96 sera samples from patients infected with Plasmodium falciparum. The results showed that 91 out of the 96 (94.8%) sera samples recognized PfCP-2.9, which indicated that anti-PfCP-2.9 specific antibodies could be produced during natural infections.74 positive sera samples were futher selected to compare the recognition between PfCP-2.9 and the mutant proteins. And the results showed that most of the mutant proteins could be recognized by the selected sera. However, apparent differences were found between PfCP-2.9 and the mutant proteins with substitutions on the MSP1-19 component (especially the mutations on blocking epitopes of PfMSP1-19), which implied that blocking antibodies dominate these sera samples. These results provide evidence for future vaccines eliminating blocking epitopes.To investigate the immunogenicity of the mutant proteins, we performed animal studies in New Zealand White rabbits with Pichia expressed and purified PfCP-2.9 as well as three mutant proteins with substitution on blocking epitopes. The analysis from the rabbit immue sera showed that there were no significant differences between PfCP-2.9 and mutatated proteins, either in the ELISA titers (p>0.05) or in the in vitro growth inhibiton assay (GIA) by sera, as well as total IgG (p>0.05).Briefly, in this study, we created a panel of 17 mutant proteins of PfCP-2.9 with single amino acid substitution. With the mAbs against PfCP-2.9 prepared in previous work, we have mapped some epitopes of the vaccine candidate by Western Blot and ELISA, and raised the possible biological function of these epitopes. Recognition of the mutant proteins by sera from natural infections was also investigated, and the results showed that the binding of some mutant proteins was altered by the sera samples, compared with the binding of the wild-type protein. The data obtained may prove to be valuable and helpful for designing more effective malaria vaccines against blood-stage parasites in future studies.