| Malaria is the most significant vector-borne disease in the worldwide. The vast majority of deaths occurred not only in Africa, but also in South America and South-East Asia. The number of reported clinical cases of malaria ranges between 300 to 500 million, with one to three million fatalities each year. Approximately 90% of these deaths occur in Africa, mostly in young children. Most malaria infections in Africa south of the Sahara are caused by Plasmodium falciparum, which causes severe infection and even death. As parasite resistance to antimalarial drugs become more widespread and the mosquito vector develops resistance to insecticides, there is an urgent need for a safe, effective vaccine against this pathogen, especially for children.There has been more than 20 vaccine candidate antigens entering the clinical trial, cotaining recombinant blood stage vaccine candidate PfCP-2.9 which developed by our laboratory. PfCP-2.9 is a chimeric protein consisting of the 19kDa C-terminal region of MSP1 and the domain III of AMA1 via a hinge sequence. The chimeric protein has been produced in Picha Pastoris at a high yield. Immunization of animals with PfCP-2.9 formulated with ISA720 elicited high levels of antibodies. Antiserum raised against the PfCP-2.9 can efficiently block in vitro parasite growth, but antisera against either of the two domains has no inhibition of parasite growth at the same concentration. In this study, we investigated the specificity of antibodies to PfCP-2.9 from vaccinationed volunteers, and use monoclonal antibody to PfCP-2.9 to analyse antibody function.Recombinant vaccine PfCP-2.9 is comosed of the 19-kDa portion of MSP1 and domain III of AMA-1 via a hinge sequence.MSPl-19 contains two Epidermal Growth Factor-like domain. First, we constructed and expressed the two EGF-like domains, EGF1 and EGF2. PCR method to amplified egf1 and egf2 fragments from PfCP-2.9 plasmid and cloned them into the expression vector pPIC9K. The recombinant plasmid was transferred into P.Pastoris GS115 and transfectants were selected withG418 for the clone of higher expression. Mouse anti- PfCP-2.9 sera was used for western blotting. The result showed that after induction with methanol a 7kDa protein band could be discerned, but the expression of EGF2 couldn't be detected. So egf2 fragment was fused to the Glutathione transferase(GST). The fusion gst-egf2 fragment was cloned to the expression vector pPIC9K and the expression could be detected this time. The protein in supernatant was purified by Ni-NTA affinity chromatography as six tandem histidine was designed and added to the C-terminal of the protein. The high purity purified protein EGF1 and GST-EGF2 were used for later antibody detection.Six component of PfCP-2.9: PfCP-2.9, MSPl-19, AMAl(III), EGF1, GST-EGF2, P28 were used as coating antigen to detect 52 sera of PfCP-2.9 vaccine immune volunteers(40 from immunized groups, 12 from placebo-control groups) with Enzyme-linked immunosorbent assay. The result showed that all immune group volunteers could generate specificity antibodies to PfCP-2.9, MSPl-19, AMAl(III), EGF1, GST-EGF2, but no antibodies to P28 could be detected. Among give component antigen, the antibody titers to PfCP-2.9 was the highest of average 32,475, which the highest Ab titer reached above 10~6. This 52 volunteers comes from 4 immune dose groups, the Ab titer of 4 groups has no statistically significant difference. All immune group volunteers' sera could recognize MSPl-19 & AMAl(III), but their Ab titers was far below that of anti- PfCP-2.9. The average Ab titers to MSPl-19 was 5,009, three times of that anti-AMAl(III), the highest one could reach above 2xlO4. The total Ab titer of EGF1 and EGF2 was only about half of MSPl-19, and the titers to EGF1 was about twice that of EGF2. Recombinant vaccine PfCP-2.9 could induce high level of Ab after second immunization, the Ab titers to each component was continue to arising after third immunization. Anti- PfCP-2.9 antibody in some volunteers was far higher than the total titers of MSPl-19 and AMAl(III) which can be concluded that fusion antigen has more powerful immunity than two isolated component.Based on the above experiments, we analyse the antibody subclass of the immune volunteers. As a result, IgGl was the main subclass associated withimmunization. At the same time, IgG3 and IgG4 could be detected, where as IgG2 was minor antibody response.In addition, we compared the antibody to PfCP-2.9 of clinical immune volunteers with malaria population from endemic area. From the result we learned that volunteers immunized with PfCP-2.9 could induce stronger antibody response than that living in endemic area, suggesting that vaccine immune could generate more powerful protection agaist natural infection.For further investigation, we use 10 strains anti-PfCP-2.9 monoclonal antibodies prepared by our laboratory to compete with sera of volunteers to see whether there was antibodies of same conformation as mAb in the volunteers' sera. The results showed that 6 mAbs could compete with sera of volunteers, suggesting that some antibodies that recognize the same epitope with monoclonal antibodies was induced in volunteers. Among these mAbs, 3 mAbs could inhibit the growth of parasite in vitro which indicated that there was inhibitory antibody generated in the vaccine volunteers.Summarily, recombinant blood stage vaccine PfCP-2.9 is highly immunogenic in human bodies and the level of antibody has no relationship with the immune dose. The level of antibodies induced by fusion antigen was much higher than the individual components. There was no antibodies to P28 was detected. The prevailing subclass of specific IgG induced by PfCP-2.9 was IgGl. Higher antibody level could be induced by PfCP-2.9 compared with malaria patients living in endemic area. We establish the program of competitive Elisa to detect the type of antibodies in the sera of volunteers, thus the type of antibodies in human bodies could be analysed indirectly and exoteric, in order to estimate the immune protection of malaria candidate vaccine PfCP-2.9.
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