N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity

The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.     

恶性疟原虫海南分离株FCC1/HN有性期特异抗原Pfs48/45基因的克隆与表达

采用基因工程技术,将编码恶性疟原虫有性期特异抗原Ｐｆｓ８／４５的基因克隆到真核表达质粒ｐｃＤ－ＮＡ３,并进行ＤＮＡ序列测定,再通过磷酸钙—ＤＮＡ共沉淀转化法将重组质粒ｐｃＤＮＡ３－ｐＦＳ４８／４５导入ＨｅＬａ细胞,建立稳定分泌Ｐｆｓ４８／４５蛋白的阳性克隆株。结果显示,我国海南ＦＣＣ１／ＨＮ株Ｐｆｓ４８／４５抗原基因序列与ＮＦ５４株者高度同源,提示该基因在不同虫株间高度保守,是研制疟疾疫苗的理想靶抗原;在ＨｅＬａ细胞中表达的Ｐｆｓ４８／４５蛋白分子量约为４６／４３．５ｋＤａ双联体蛋白,其表达量占细胞培养上清蛋白总量的１８．２７％。经ＷｅｓｔｅｒｎＢｌｏｔ分析显示,表在蛋白能被配子体免疫鼠血清特异性识别,提示表达的重组蛋白Ｐｆｓ４８／４５具有免疫活性。真核表达系统ｐｃＤＮＡ３／Ｐｆｓ４８／４５／ＨｅＬａ的建立为进一步研究重组Ｐｆｓ４８／４５抗原的免疫原性和保护性奠定基础。     

Nasal immunization with a malaria transmission-blocking vaccine candidate, Pfs25, induces complete protective immunity in mice against field isolates of Plasmodium falciparum

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.     

Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230.

Antibodies to the sexual-stage surface antigens of Plasmodium falciparum, Pfs230 and Pfs48/45, can abolish the infectivity of gametes to mosquitoes; these antigens have been proposed as candidates for inclusion in a malaria transmission-blocking vaccine. One possible mechanism of antibody-mediated transmission blocking is complement-mediated gamete lysis. We have used a panel of human sera from geographically distinct regions where malaria is endemic to investigate whether this may be a mechanism of naturally acquired transmission-blocking immunity to P. falciparum. By immunoprecipitation, we have shown that antibody recognition of Pfs230 and Pfs48/45 is limited, despite universal exposure to P. falciparum gametocytes. In vitro complement-mediated lysis of P. falciparum gametes was positively associated with the presence of antibodies to Pfs230 but not with antibodies to the N-terminal region of the precursor molecule (Pfs260), which is shed from the gametocyte surface at the time of gametogenesis. Similarly, antibodies to two other gametocyte-specific proteins, Pfs48/45 and Pfg27/25, were not associated with gamete lysis. All sera which mediate gamete lysis contain immunoglobulin G1 (IgG1) and/or IgG3 antibodies to gamete surface proteins as determined by an enzyme-linked immunosorbent assay. These data suggest that Pfs230 is a major target of complement-fixing antibodies which may be important for antibody-mediated transmission-blocking immunity.     

Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum.

Antibodies to Pfs25, a cysteine-rich 25-kDa protein present on the surface of Plasmodium falciparum zygotes, can completely block the transmission of malaria parasites when mixed with infectious blood and fed to mosquitoes through a membrane feeding apparatus. Recently, a polypeptide analog, Pfs25-B, secreted from recombinant Saccharomyces cerevisiae was found to react with conformation-dependent, transmission-blocking monoclonal antibodies and to elicit transmission-blocking antibodies in experimental animals when emulsified in either Freund's or muramyl tripeptide adjuvant. In this study, Pfs25-B adsorbed to alum induced transmission-blocking antibodies in both rodents and primates. Bacterially produced Pfs25, however, did not elicit complete transmission-blocking antibodies in rodents. Furthermore, unlike monoclonal antibodies to Pfs25, which block transmission only after ookinete development, antisera to Pfs25-B adsorbed to alum appeared to block the in vivo development of zygotes to ookinetes as well.     

Susceptibility of Plasmodium falciparum cyclic AMP-dependent protein kinase and its mammalian homologue to the inhibitors

Cyclic AMP-dependent protein kinase (protein kinase A, PKA) is a key element in many cell signaling pathways. An essential role of Plasmodium falciparum PKA (PfPKA) activity was reported in the intraerythrocytic growth of the malaria parasite. However, molecular characterization of PfPKA using purified recombinant proteins has not yet been performed. Here, we report the first successful purification of the enzymatically active PKA catalytic subunit of P. falciparum (PfPKA-C) using a wheat germ cell-free expression system. Interestingly, parasite enzymatic activity was weakly inhibited as compared with the inhibition of mammalian PKA catalytic subunit (PKA-C) by the specific PKA inhibitor, H89. Furthermore, PfPKA-C was only slightly inhibited by protein kinase inhibitor (PKI). These results suggest that substrate sites of PfPKA-C may be different from those of mammalian PKA-Cs. In addition, potential PKI corresponding to malarial PKA-C would also be different from those of mammalian cells.     

Pfs25蛋白单克隆抗体的制备及双抗体夹心ELISA的建立

目的:制备恶性疟原虫子孢子囊表面膜蛋白Pfs25的单克隆抗体( mAb),建立检测Pfs25蛋白的双抗体夹心ELISA方法.方法:纯化毕赤酵母表达的重组Pfs25蛋白,并免疫BALB/c小鼠,采用骨髓瘤细胞Sp2/0与免疫BALB/c鼠脾细胞杂交的细胞融合技术,通过间接ELISA检测获得分泌抗Pfs25抗体的阳性杂交瘤细胞株,通过免疫F1鼠诱生腹水,纯化腹水,并进行mAb的各项生物学鉴定.辣根过氧化物酶(HRP)标记纯化后的抗体,以4B7为包被抗体,1B4为酶标抗体,建立了双抗体夹心ELISA法.结果:获得3株抗Pfs25的杂交瘤细胞株,其中2株有良好的稳定性和特异性.并建立了双抗体夹心ELISA检测法,检测有效范围在0.07～1 mg/mL,其检测灵敏度为41.6 ng/mL.结论:成功制备抗Pfs25蛋白的单克隆抗体,并建立了一种可用于Pfs25蛋白检测的双抗体夹心ELISA法,为Pfs25蛋白制备传播阻断型疟疾疫苗奠定了基础.     

Functional analysis of Plasmodium falciparum apical membrane antigen 1 utilizing interspecies domains

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of P. falciparum (PfAMA1) with that of P. chabaudi (PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against Escherichia coli expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal pro region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.     

Effect of CpG oligodeoxynucleotides on the immunogenicity of Pfs25, a Plasmodium falciparum transmission-blocking vaccine antigen

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes of Plasmodium falciparum, completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission of P. falciparum parasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.     

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.     

Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium berghei parasites expressing the target antigen Pfs25

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.     

Enhanced immunity to Plasmodium falciparum circumsporozoite protein (PfCSP) by using Salmonella enterica serovar Typhi expressing PfCSP and a PfCSP-encoding DNA vaccine in a heterologous prime-boost strategy

Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.     

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.     

Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies

Immunological intervention, in addition to vector control and malaria chemotherapy, will be needed to stop the resurgence of malaria, a disease with a devastating impact on the health of 300 to 500 million people annually. We have pursued a vaccination strategy, based on DNA immunization in mice with genes encoding two antigens present on the sexual stages of Plasmodium falciparum, Pfs25 and Pfg27, to induce biologically important antibodies that can block development of the parasite in the Anopheles mosquito and thus transmission of the disease. DNA encoding Pfs25 when administered by the intramuscular route, either alone or with DNA encoding Pfg27, had the most potent transmission-blocking effects, resulting in up to a 97% decrease in oocyst numbers in mosquito midguts and a 75% decrease in rate of infection. Immunization with DNA encoding a Pfg27-Pfs25 fusion protein was less effective and DNA encoding Pfg27 elicited antibodies in sera that had only modest effects on the infectivity of the parasite. These results show for the first time that DNA vaccination can result in potent transmission-blocking antibodies in mice and suggest that the Pfs25 gene should be included as part of a multicomponent DNA vaccine.     

Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites

A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immune enhancer. After DNA immunization, antigen-specific gamma interferon (IFN-gamma) responses were detected by ELISPOT in 15/31 volunteers to multiple class I- and/or class II-restricted T-cell epitopes derived from all five antigens. Responses to multiple epitopes (相似文献   

Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

Production of Pfs25, a Plasmodium falciparum transmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed in Escherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes of P. falciparum and demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund''s adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 μg/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes, Anopheles gambiae and Anopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 in E. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation of E. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials.     

Immunogenicity of Plasmodium falciparum sexual stage antigens: implications for the design of a transmission blocking vaccine.

Immunogenicity of sexual stage antigens and boosting of transmission blocking antibodies following a natural infection are two critical factors in the design of an effective, subunit vaccine to block the transmission of malaria from man to mosquito. Immunogenicity and boosting are both T cell-dependent. Antigens, such as the 230-kDa, the 48/45-kDa, and the 40/10-kDa, expressed early in the extracellular forms of the sexual stage of Plasmodium falciparum, have limited immunogenicity in humans and in mice. In contrast, Pfs25, expressed predominantly in zygotes and ookinetes, has widespread immunogenicity in mice. Pfs25 expressed by a recombinant vaccinia virus (vSIDK) is also widely immunogenic in mice, and induces transmission blocking antibodies following multiple inoculations with vSIDK. The implications of these immunogenicity data are discussed relative to the design of an effective transmission blocking vaccine.     

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.     

A region of Plasmodium falciparum antigen Pfs25 that is the target of highly potent transmission-blocking antibodies

Each of the four epidermal growth factor (EGF)-like domains of the Plasmodium falciparum sexual-stage antigen Pfs25 has been individually expressed as a yeast-secreted recombinant protein (yEGF1 through yEGF4). All four are recognized by the immune sera of animals and humans vaccinated with TBV25H (the corresponding yeast-secreted full-length recombinant form of Pfs25), with antibody titers to yEGF1 and yEGF2 weakly correlating with the ability of the sera to block the transmission of parasites to the mosquito host. All four proteins are poorly immunogenic in mice vaccinated with aluminum hydroxide-absorbed formulations. However, all four successfully primed the mice to mount an effective secondary antibody response after a single boost with TBV25H. Sera from mice vaccinated with yEGF2-TBV25H completely block the development of oocysts in mosquito midguts in membrane-feeding assays. Further, of the four proteins, only the depletion of antibodies to yEGF2 from the sera of rabbits vaccinated with TBV25H consistently abolished the ability of those sera to block oocyst development. Thus, antibodies to the second EGF-like domain of Pfs25 appear to mediate a very potent blocking activity, even at low titers. Vaccination strategies that target antibody response towards this domain may improve the efficacy of future transmission-blocking vaccines.     

A plant-produced Pfs230 vaccine candidate blocks transmission of Plasmodium falciparum

Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.