Study On Immunogenicity Of Plasmodium Falciparum Antigen Pf332

Plasmodium falciparum malaria is one of the most important infectious diseases. With as many as 500 million clinical cases annually, malaria accounts for the death of over one million people every year. One of the main hurdles in the combat against malaria is the emergence of drug-resistant P. falciparum strains and insecticide-resist- ant mosquitoes, which dramatically reduces the efficiency of conventional treatment and mosquito prevention. Therefore, a vaccine that averts or reduces infection and minimizes morbidity and mortality would be an efficient tool in malaria control and preventive programs.Blood-stage vaccines aim at reducing the overall parasite burden and the associated morbidity. The main targets of such a vaccine are P. falciparum antigens expressed on the surface of merozoites and infected red blood cells (iRBC). Adding complexity however, surface antigens often undergo antigenic variation or are highly polymorphic between different parasite strains.The success of a vaccine directed against these highly variable antigens would therefore depend on its ability to elicit a broad-range of cross-reactive antibodies.In recent years, the availability of genomic sequences has provided a powerful platform for rational antigen identification and in particular the identification of antigens with low variability but functional importance. One such antigen is Pf332, a blood-stage protein that can be found in association with the RBC membrane in late-stage trophozoites and schizonts. Pf332 is the largest protein identified in P. falciparum and it is present in all parasite strains studied so far. The Pf332 antigen contains a conserved Duffy-binding like (DBL)-domain homologous to the DBL-domains of the erythrocyte-binding ligand (EBL) family of invasion proteins, indicating that Pf332 may exert functions related to the EBLs .We have recently found that affinity-purified naturally acquired human antibodies targeting the N-terminally located DBL-domain of Pf332 display a prominent invasion inhibitory effect in various parasite strains.On this basis , the immunogenicity of the Duffy-binding like(DBL)-domain of the Pf332 has been investigated in our study.Firstly,the DBL fragment was subcloned into pGEX-4T-1 vector by PCR and transformed into BL21 (DE3) with the plasmid of laboratory's strain DBL-pQE(M15) as template. The His- and GST- tagged recombinant proteins were expressed by engineered bacteria and purified using affinity chromatography. The His-tagged Pf332-DBL was subsequently used for immunizations and the GST-tagged protein for detection of anti-Pf332-DBL specific antibodies.The recombinant proteins were confirmed by Wesrern blot with anti-GST and anti-His monoclonal antibody,mouse anti-Pf332-DBL antibody as the first antibody.The concentration of DBL-His and DBL-GST were respectively 400μg/mL and 1000μg/mL.Secondly, 12 Pf332-specfic monoclonal antibodies, which were all IgG1 subtype, were obtained through by immunization BALB/c mice. The specificity of the mAbs were further characterized by: 1) Western blot with both recombinant Pf332- DBL-GST and the native Pf332 proteins;2) Indirected fluorescent assay (IFA) with fixed infected RBCs;3) Peptide scan by ELISA with 9 25mer tandem oligo-peptides of the DBL domain used for the immunization. The data confirmed that all mAbs specifically recognized the protein.Thirdly, the function of the mAbs was characterized with an invasion/growth inhibition assay by addition of the mAbs to P. falciparum culture separately. Two mAbs showed significant anti-parasite activity in a dose dependent manner. Importantly, the activity of the mAbs was significantly stronger than the purified human hyper-immune IgG which was originally used for anti-malaria treatment. Thus we have development a panel of antibodies with significant anti-parasite effect and the data strongly indicated the Pf332 molecule plays a vital role in the development of P. falciparum inside human body.Fourthly, the immunogenicity of Pf332-DBL was investigated systematically through immunization of 4 animals including BALB/c mice, C57 mice, Wistar rats and New Zealand white rabbits in combination with four different adjuvants including complete Freund's adjuvant/incomplete Freund's adjuvant, Montanide ISA 720, aluminum hydroxide, levamisole with PBS as a control group. The dynamic antibody responses of each animal after the immunization were investigated and compared among the adjuvants groups by indirect ELISA. The DBL-His protein was found to be immunogenic in all immunized animal groups, although the antibody responses varied between different adjuvant groups. In BALB/c mice, the ISA720 formulation produced the highest antibody titer followed by aluminum adjuvant; in C57 mice, the ISA720 formulations produced the highest antibody titer followed by Freund's adjuvant group; in Winstar rats, the ISA720 formulation produced the highest antibody titer followed by Freund's adjuvant; in New Zealand white rabbits, the aluminum adjuvant formulation produced the highest antibody titer followed by Freund's adjuvant. The adjuvant effect of levamisole in combination with DBL-His was weak or absent in all animals tested. Since antibody subclass that induced after immunization is an indicator of the relative contribution of Th2- versus Th1-type immune responses, the ratios of major subclass IgG1/IgG2a were determined among different adjuvant groups in mice and rats. In mice and rats, the production of IgG1 versus IgG2a is widely interpreted as a reflection of Th2- or Th1 reactivity. The result showed that, both in mice or rats, IgG1 were significantly generated in alum adjuvant group. Further, The dynamic changes and longevity of antibodies specific to the Pf332-DBL antigen was monitored for 17 weeks after the first immunization by indirect ELISA among different adjuvant groups. All animals except BALB/c and New Zealand white rabbits immunized with levamisole formulations, had a significant amount of antibodies after the forth immunization compared to pre-immune animals, and could maintain a certain degree of antibody titer 17 weeks after the first immunization. To analyze the specificity of the Pf332-DBL antibodies detected in the immunized animals, Western blot analyses were carried out with both recombinant DBL-GST and native Pf332 antigen expressed by the P.falciparum, and the parasite cultured in vitro were indentified by indirect immunofluorescence with immuned serum ,the result showed that antibodies from all immunized animal groups recognized the Pf332 proteins.In conclusion, the present study has found that Pf332-DBL played an important role in the P. falciparum merozoite invasion, and the parts from 1 to 25 and from 76 to 100 amino acids were the key regions of the molecule related to erythrocyte invasion. Pf332-DBL is immunogenic and could stimulate the immune system to generate strong immune response. The Pf332-DBL domain is a potential anti-malaria vaccine candidate.