Characterization Of A Novel Plasmodium Falciparum Antigen PfMAg-1

Malaria is one of the most important infectious diseases in the world. There are 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 the Plasmodium falciparum. The spreading of drug-resistant parasites and insecticide-resistant Anopheles mosquito vectors make the prevention and treatment of malaria a big problem in the world. Plasmodium's complex life cycles, antigenic stage-specificity, diversity and variation, are the obstacles to develop malarial vaccines. Erythrocyte stage parasite is responsible for the clinical syndromes of malaria, leading to death of the patients. Therefore, to develop a safe and effective intra-erythrocyte stage vaccine against P. falciparum will reduce mortality rate of the disease. However, progress towards a successful human malaria vaccine has been slow, largely due to lack of effective antigens that induce protective immunity in humans. To identify and characterize novel protective antigens of erythrocyte stage P. falciparum is still the major task for effective malaria vaccine development.The pfmag-1 gene is a new erythrocyte stage vaccine candidate recently identified from our group with a protective monoclonal antibody M26-32. It is a 75.59% AT rich gene and the full length ORF is 1938bp, encoding a 589aa polypeptide with a predicted molecular mass of 70.2kDa. The C-terminal of the protein contains 14 copies of a deca-amino-acid repeat. The dominant repeat unit was [QTDEIKND(H/N)I]. PfMAg-1 has only one gene locus on the chromosome 4th of P. falciparum and contains nine exons and eight introns. The expression of PfMAg-1 was detectable only during the late stage of the parasite developing circle within the erythrocyte by Western Blotting assy.Polymorphism in PfMAg-1 genomic sequence was assessed with 8 P. falciparum strains by sequencing the 5' terminal end (including exon 1, intron 1 and exon 2) and the 3' terminalend (the whole exon 9 sequence), respectively. The results indicated that the 5' terminal of this gene was extremely conservative, while 3' terminal at peptide level displayed some differences in the repeat of the tandem deca-amino-acid, which may reflect an active evolution with this genome region.By Genebank Blast, the PfMAg-1 peptide homologous were found from Plasmodium vivax, berghei, yoelii yoelii and chabaudi. None of the homologous covers both the repeated and unrepeated region of the PfMAg-1 molecule. A protective yoelii protein consents to the C-terminal sequence of PfMAg-1 in 40%. In addition, we found the PfMAg-1 may under an enzymic process by being cut into two parts, a C-terminal tandem repeats and an unrepeated region at the N terminal of it.Since the expression of the whole pfmag-1 coding sequence in both prokaryotic and eukaryotic expression systems was failed, we selected three predicted high immunogenicity regions from the gene with the software Lasergene 7.0, and named them as pfmag-1-n , pfmag-1-m and pfmag-1-c, which were than cloned and expressed with either a His or a GST tag in the E. coli. system and the recombinant proteins were used to immunize rabbits. The IgGs prepared from the antiserum of rabbits immunized with PfMAg-1-N, PfMAg-1-M, or PfMAg-1-C, could inhibit the growth of P.f. parasites, 3D7 strain, in vitro. Inhibition rate of anti- PfMAg-1-C IgG was 73% at the concentration of 2mg/ml, which was the most effective results among the three IgGs being used. Those data suggested that the C-terminal region could be used as a B cell epitope in malaria vaccine development.In Western Blotting, both the anti-PfMAg-1-C polyclonal antibody and anti-PfMAg-1-N monoclonal antibodies could recognize a 70KDa band and a 46KDa band from protein extracts of the stage mixture parasited erythrocytes, while only the 46KDa band was detected after mature schizonts were enriched. This indicates a cleave event happened within PfMAg-1 during the mature procedure of the parasite.After treating with saponin, the anti-PfMAg-1-C IgG could label to the membrane of trophozoites within parasite infected erythrocytes, and the anti-PfMAg-1-N monoclonal antibody, X6-2C, showed a similar fluorescence pattern on merozoites with antiserum against PfMSP-119 or anti-PfMSP-4 in immunofluorescence assay. With immunoelectron microscopy, the mAb X6-2C recognized proteins distributing inside the cytoplasm of merozoites.A PfMAg-1 homologues in plasmodium berghei ANKA strain was found by BLAST program, which share 75% similarity with PfMAg-1 except the C-terminal tandem repeats of it. The cDNA sequence (Gene ID: XP678342.1) was obtained from the GeneBank of Plasmodium berghei ANKA genomic databases, with which we designed a pair of primers and did the RT-PCR to get a cDNA fragment of the gene from the parasites. The expanded cDNA 3' fragment of the gene was obtained by 3'-RACE experiment. The corrected pbmag-1 cDNA fragment, which we name as pbmag-1, was 1341bp in length , A/T rich (73%), and cloned into a prokaryotic expressional vector and transformed into the BL21-(DE3)-RIL strain of Escherichia coli. The recombinant protein of PbMAg-1 was successfully expressed in the form of inclusive body with an optimized strategy of the codons. We found that a successful expression of plasmodium gene in heterogenetic system can be attained if the metabolism of the host has been depressed to a low level. The recombinant protein of PbMAg-1 was used to immunize mice and the antiserum was prepared for Western Blot. The antibodies could specifically recognized a 64KDa band from the protein extracts of Plasmodium berghei infected mice erythrocytes.After immunizing BALB/c mice with the PbMAg-1 recombinant protein for three times, we challenged the mice with Plasmodium berghei ANKA strain, but did not obtain protection in the animal model. No protection was observed when using the sera of immunized mice to do the experiments of passive immunization. Those results suggest that there were no protective antibodies induced by PbMAg-1, which may because of that the PbMAg-1 molecule lacks the C-terminal tandem repeats as in PfMAg-1 and also because of that the inclusion body destroyed the conformation necessary for raising the protective antibodies.For the first time we successfully use the reagent, OptiPrep, to enrich muture stage parasite, and demonstrate that the toxicity to parasite is much less than the reagent, percoll.In summary, we acquired a novel plasmodium falcipurum candidate antigen, PfMAg-1, the protective epitope were mainly located in C terminal deca-amino-acid repeat domain. PfMAg-1 is worth to be investigate further to it's C-terminal domain B-cell epitope.