Comparison Of Immune Response To Plasmodium Falciparum Chimeric Protein 2.9 (PfCP-2.9) In Vaccinated Volunteers With That In Naturally Infected Individuals

Malaria, transmitted by mosquitoes and one of the world's most common and serious tropical diseases, results in approximately one million deaths every year -- the majority of which occur in the most resource-poor countries. Half of the world's population is at risk of acquiring malaria. Most malaria infections in Africa south of the Sahara are caused by Plasmodium falciparum, which causes severe infection and even death. Young children and pregnant women are at the highest risk of malaria infection and mortality. Many children experience initial malaria infection during their first two years of life, when they have not yet developed sufficient immunity, making these early years particularly dangerous. Malaria is a leading cause of death for children worldwide; it is estimated that a child dies from malaria ever 30 seconds. 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.Although the knowledge of the parasite's biological behaviors is indefinite, significant progress has been made in the development of malaria vaccine during the last 30 years. According to the stages of the parasite's life cycle and some main conserved antigens, which play an important role in multiple or alternative steps during the invasion process, researchers have developed a series of vaccine against malaria, such as CSP vaccine, MSP1 vaccine, pfs25 vaccine, SPf66 synthetic vaccine, recombinant blood stage vaccine candidate PfCP-2.9 which developed by our laboratory and so on. 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 parasite growth in vitro. In this study, we investigated the specificity of antibodies against PfCP-2.9 from vaccinationed volunteers, analysed the correlation between the immune reponse and Human Lymphocyte Antigen, used monoclonal antibody againsst PfCP-2.9 to analyse antibody function and used chromatographic fractionated specificity antibodies to analyse antibody efficacy by Growth Inhibition Assay.Recombinant vaccine PfCP-2.9 is composed of the 19-kDa portion of MSP1 and domain III of AMA1 via a hinge sequence.MSP1-19 contains two Epidermal Growth Factor-like domain. For investigation of the specificity of immune response to PfCP-2.9 and the region of immune protection, we have also constructed and expressed the MSP1-19, AMA1 (III) fragment and the two EGF-like domains, EGF1 and EGF2.To investigate the immunogenicity of the recombinant vaccine PfCP-2.9, full length and the components of PfCP-2.9: PfCP-2.9, MSP1-19, AMA1(III) were used as coating antigen to detect 68 sera of PfCP-2.9 vaccine immune volunteers(48 from immunized groups, 20 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, MSP1-19 and AMA1 (III). Among the give component antigens, the antibody titer to PfCP-2.9 was the highest of average 13,931, which the highest Ab titer reached above 106. The 68 volunteers came from 3 immune dose groups (5ug, 20ug, and 50ug), and the Ab titer of 50ug group is the highest and the difference has statistically significant (P<0.01). All immune groups volunteers'sera could recognize MSP1-19 & AMA1 (III), but their Ab titers were far below that of anti- PfCP-2.9. The average Ab titer against MSP1-19 was 5,534, three times of that of anti-AMA1 (III), the highest one could reach above 5×104. The volunteers were divided into 2 schedule groups and were vaccined three times. Schedule A was administated on days 0, 60 and 180. Schedule B was administated on days 0, 90 and 180. The Ab titers of 2 schedule groups had no statistically significant difference. Anti- PfCP-2.9 antibody in some volunteers was far higher than the sum of the titers of MSP1-19 plus AMA1 (III) which can be concluded that fusion antigen has more powerful immunogenicity than two isolated components. The anti-PfCP-2.9 antibody response was likely suppressed by HLA-DR6 alleles, which was analysed by enzyme-linked immunosorbent assay and correlation analysis.To investigate whether or not the anti-PfCP-2.9 antibody was able to recognise the native parasites of Plasmodium falciparum, sera from the vaccined volunteers were tested by IFA against the FCC1/HN strain of P.f. The result showed that the sera from the vaccined volunteers could recognise the native parasites. Moreover, sera from the vaccined voluteers were testd by Western-Blot against the native protein extracted from the FCC1/HN strain of P.f. The result showed that the sera from the vaccined volunteers could recognise the native MSP1 and AMA1 Bands. It was indicated that the conformational structure of the fusion protein was approximate to that of the native MSP1 and AMA1.Based on the above experiments, we analysed the antibody subclass of the immune volunteers. As a result, IgG1 was the main subclass associated with immunization. At the same time, IgG3 and IgG4 could be detected, whereas IgG2 was minor antibody response. The average Ab titer of 50ug group was the highest one among three immune dose groups (5ug, 20ug, and 50ug) and the Ab level of IgG4 was also much more higher than that of the other dose groups (P=0.017 versus 5ug group; P=0.007 versus 20ug group).In addition, we compared the antibody against 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. In malaria population from endemic area, the antibody titers of re-infected patients against PfCP-2.9, MSP1-19 and AMA1 (III) is much higher than the antibody titers of primary infected patients. The antibody titers of children patients against PfCP-2.9, MSP1-19 and AMA1 (III) were a little higher than the antibody titers of adult patients but the difference has not statistically significant. IgG1 was the main antibody subclass of re-infected patients. And IgG3 was following. IgG4 could be detected, and IgG2 could also be detected slightly.For further investigation, we used 14 strains of anti-PfCP-2.9 monoclonal antibodies prepared by our laboratory to compete with sera of volunteers to see whether there were antibodies against the same conformational epitopes in the volunteers'sera as those in the mAb. The results showed that 9 mAbs could compete with sera of volunteers, suggesting that some antibodies that recognize the same epitope with monoclonal antibodies was induced in the volunteers. Among these mAbs, 5 mAbs could inhibit the growth of parasite in vitro which indicated that there should be inhibitory antibody generated in the vaccine volunteers. We also used the anti-PfCP-2.9 monoclonal antibodies to compete with sera of immune rabbits. The results showed that all mAbs could compete with sera of immune rabbits and most of the competition was intensive, suggesting that the epitopes recognized by immune rabbits'antibodies were much more than the epitopes recognized by volunteers.To investigate the GIA activities of antibody specific for the PfCP-2.9 and its components, the antibodies specific for PfCP-2.9, MSP1-19, AMA1 (III), EGF1 and EGF2 were purified from the sera of volunteers and malaria patients by affinity chromatography. And the GIA activities of the antibodies was measured by Growth Inhibition Assay. The results showed that all the antibodies inhibited parasite growth at various level except that the antibodies specific for EGF2 domain that did not inhibit the parasite growth at all In comparison, the antibodies isolated from natural infection individuals were slightly higher than that from the vaccinated volunteers. In addition, The antibodies to MSP1-19 showed the highest inhibitory activity, suggesting that the MSP1-19 be a major domain for protective.In summary, recombinant blood stage vaccine PfCP-2.9 is highly immunogenic in human bodies and the level of antibody has relationship with the immune dose. The level of antibodies induced by fusion antigen was much higher than the individual components. The IFA and Western-Blot test showed the conformational structure of the fusion protein was approximate to that of the native MSP1 and AMA1. The prevailing subclass of specific IgG induced by PfCP-2.9 was IgG1. IgG4 might be main composition which resulted in the higher antibody titer of 50ug group. Higher antibody level could be induced by PfCP-2.9 compared with malaria patients living in endemic area. In order to estimate the immune protection of malaria candidate vaccine PfCP-2.9, we used 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. The result of GIA showed that specificity antibodies from sera of volunteers didn't have the same level of GIA activity as that of the immune animal specificity antibodies. The investigation of the different immunologic mechanism would be continued.