Construction And Immunogenicity Of Plasmodium Falciparum Chimeric Protein 5(PfCP-5)

Malaria remains one of the most significant vector-borne diseases in the worldwide. The number of reported clinical cases of malaria ranges between 300 to 500 million each year, among of them 3 million are caused into death by Plasmodium falciparum and most of death are children under 5 years. As parasite resistance to antimalarial drugs become more widespread and the mosquito vector develops resistance to insecticides, it becomes more difficult to control malaria. So, development of an effective malaria vaccine would be a new important alternative approach.Sporozoites of Plasmodium falciparum enter people through biting of the infected mosquito. Then they migrate hepatocytes and develop into schizonts within the liver. The schizonts rupture and the meroziotes invade the erythrocyte and develop and replicate in erythrocyte. So it is an important link in inhibition of the blood-stage Plasmodium through blooking of invading of the merozoites and it is a main target of blood-stage vaccines. Many of research indicate that the receptor-ligand mediate the process of invading. The proteins in merozoite surface would possible be mediate the process as ligands, So the proteins are target antigens of blood-stage malaria vaccine. The purpose of this research is to investigate the function of immunoprotection of Merozoite Surface Protein (MSP) 3, 4 and 5 of Plasmodium falciparum. MSP3 is a exo-secreted protein and associated with the merozoite surface,the mechanism of anti-MSP3 antibody is ADCI-antibody dependent cellular inhibition. The ADCI assay requires the cooperation of antibodies with monocyte. Its effective functional fragments are MSP3b(184-210), MSP3c(203-230), MSP3d(211-252). The epitopes of its B and T helper cells have been identified in each peptide. Anti–MSP3b, anti–MSP3c and anti–MSP3d affinity-purified antibodies were found to exert a strong monocyte-mediated anti-parasitesitic activity in vitro. The passive transfer of anti–MSP3b antibodies resulted in the clearance of parasites. The passive transfer of anti–MSP3d antibodies resulted in a decrease of parasitemia. Further validated the functional anti-parasite activity of naturally occurring antibodies against the 69-aa region covered by peptides MSP3b, MSP3c and MSP3d. MSP4 is anchored to the merozoite membrane by a GPI moiety. MSP4 contains a single epidermal growth factor (EGF) like domain at the C terminus of the protein. There is hghly homology between MSP4 and MSP5 in EGF-like domain in C terminal and antibodies to the two EGF-like domain show some inhibition in vitro.The functional domains of these three MSPs, the EGF-like domain in C terminus of MSP4 (MSP4D,204-248 residues),the 69(184-252) residues of MSP3 and the EGF-like domain in C terminus of MSP5 (MSP5D,208-251 residues) were constructed as a fusion protein designated as PfCP-5 (Plasmodium falciparum Chimeric Protein 5). The DNA sequences of each domain of Plasmodium falciparum 3D7 strain were obatained from GenBank. The fusion gene was fused with MSP4D-MSP3-69-MSP5D. The DNA sequence of the fusion gene was redesigned using Pichia pastoris codon usage and removed of potential glycosylation sites. For the purpose of cloning, the 3'terminus of the gene was added with XhoI restriction endonucleases site and the 5'terminal was added with EcoRI. To facilitate purification of recombinant protein, a 6-His TAG was added at its C-terminus. The resdesigned sequence of the gene was synthesized and was ligated into the vector pPIC9. Then the target fragment containing signal sequence was ligated into the expression vector, pPIC9K. The linearized recombinant pPIC9K digested with BglⅡwas electro-transformated into Pichia pastoris. The clones selected by G418 was picked and induced by ethanol. Through analysis by SDS-PAGE and Western blotting, the gene was shown to be expressed in this system successfully. The expressed protein was purified by Ni-NTA. To obtain a recombinant protein of MSP3, the gene was expressed with a fusion protein of the GST-MSP3 in E.coli. The protein was purified through Glutathione Sepherose 4B.Both New Zealand rabbits and BALB/c mice were used for immunization experiments. 6-8 weeks aged BALB/c mice were divided into five groups according to five different adjuvants, i.e. ISA720 adjuvant, Freund's adjuvant, combination of aluminum hydroxide AL(OH)3/CpG Oligonucleotide (CpG1826) adjuvants, ISA70MVG and ISA 206 adjuvant. Each mouse was immunized subcutaneously with 20μg protein for three times and two weeks interval. After 8 days of each immunization, the serum was prepared from the vein of the tail. The antibody titters and the IgG subtypes were detected by ELISA. The results are shown as the following. (1) the antibody titers of all immunized animals varied from 1/20×104. to 1/76.5×104. Among them, the animals from group of AL(OH)3 /CpG adjuvants generated highest level of antibodies with titers of 1/76.5×104, then the Freund's adjuvant group1/ 48.9×104, and the ISA720 adjuvant 1/55.2×104. However, animals in ISA70MVG and ISA206 adjuvant group gave much lower level of titers with 1/25.3×104 and 1/20.9×104 respectively. The difference among the three groups of AL(OH)3/CpG Oligonucleotide (CpG1826) adjuvants group, the Freund's adjuvant group and the ISA720 adjuvant group was not significant (p>0.05), however the antibody titers of the three groups was 3.0, 2.18 and 1.93 folds comparing with ISA70MVG group, and was 3.66,2.64 and 2.33 folds comparing with ISA206 adjuvant group.(2) Moreover, IFA was performed to detect the recognition between the immune sera and the parasites. The IFA result showed positive reactions IFA positve reactions of AL(OH)3/CpG Oligonucleotide (CpG1826) adjuvants group, the Freund's adjuvant group, the ISA720 adjuvant group were strong relatively,however, the IFA positive reactions of ISA70MVG and ISA 206 adjuvant group were weak relatively.(3)The titers of antibody subtypes were also detected: the titer of IgG1 is 106, which is most predominate, followed by IgG2a , then IgG2b and IgG3,which is corresponding and the titer was about 104.The New Zealand rabbits (male, three rabbits per group) were grouped into ISA720 adjuvant group, Freund's adjuvant group, aluminum hydroxide AL(OH)3/CpG Oligonucleotide (CpG2007) adjuvants group, each of them were immunized subcutaneously three times at three week intervals with 100μg protein. After 8 days of each immunization, the sera obtained were detected the antibody titers by ELISA. The antibody titers were: ISA720 adjuvant group 102×104,Freund's adjuvant group 1/72.7×104,aluminum hydroxide AL(OH)3/CpG Oligonucleotide (CpG2007) adjuvants group 1/25.2×104,The difference among three groups was significant (p<0.01). Moreover, the immunogenicity in BALB/c mice of another fusion protein. GST-MSP3-69 expressed in E coli was also detected by ELISA. The antibody titers of three different adjuvant groups in BALB/c mice were 1/138×104 for ISA720 adjuvant group,1/116×104 for Freund's adjuvant group and 1/72.5×104 for aluminum hydroxide AL(OH)3/CpG Oligonucleotide (CpG1826).Anti-rabbit sera and anti-mouse sera were purified through Sepherose 4B-PfCP-5 and sepherose 4B GST-MSP3-69 affinity purification colume. Anti-PfCP-5 IgG was added to the culture of HN/HCC1 strain of Plasmodium falciparum to detect the effect of growth inhibition in vitro. The validity of anti-rabbit sera and the purified specific IgG(final concentration 1.25mg/ml) was not found in vitro, but when monocytes were added together with anti-PfCP-5 and anti-GST-MSP3-69 IgG ,the specific growth inhibition index was 68% and 51.5% respectivly.In summary, the PfCP-5 gene was constructed and the chimeric protein PfCP-5 was expressed in Pichia pastoris. And the individual component of PfCP-5, msp3-69 gene was constructed and the protein of MSP3-69 fusioned with GST was expressed in E coli. The results of the animal immunization studies showed that the immunogenicity of the two proteins was ideal. The aluminum hydroxide AL(OH)3/CpG Oligonucleotide adjuvants could stimulate a strong response in mice. The anti-sera could recognize the nature protein of Plasmodium falciparum cultured in vitro. This indicated the antibodes elicited could recognize the conformational epitopes of nature protein. The direct growth inhibition of Plasmodium falciparum in vitro of anti-sera and the affinity purified anti-PfCP-5 IgG (1.25mg/ml) was not found. While the monocytes were added with the affinity purified specific anti-PfCP-5 IgG and the fragment MSP3-69 to the parasite cultures, the specific growth inhibitory index was 68% and 51.5% respectively.These data provide foundation to the post-assay, affinity purification of anti-human IgG and ADCI assay,and inserting PfCP-5 or MSP3-69 to PfCP-2.9, which was on the clinical trial, to improve its protection and immune efficiency.