Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate.

Gozar, M. M. G., Muratova, O., Keister, D. B., Kensil, C. R., Price, V. L., and Kaslow, D. C. 2001. Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate. Experimental Parasitology 97, 61-69. The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice. Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits. Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21. As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations. Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28. The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28.

A snake venom phospholipase A(2) blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface.

Oocyst formation is a critical stage in the development of the malaria parasite in the mosquito. We have discovered that the phospholipase A(2) (PLA2) from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus) inhibits oocyst formation when added to infected chicken blood and fed to mosquitoes. A similar transmission-blocking activity was demonstrated for PLA2s from the venom of other snakes and from the honeybee. This effect is seen both with the avian malaria parasite Plasmodium gallinaceum and with the human parasite Plasmodium falciparum developing in their respective mosquito hosts. The inhibition occurs even in the presence of an irreversible inhibitor of the active site of PLA2, indicating that the hydrolytic activity of the enzyme is not required for the antiparasitic effect. Inhibition is also seen when the enzyme is fed to mosquitoes together with ookinetes, suggesting that the inhibition occurs after ookinete maturation. PLA2 has no direct effect on the parasite. However, pretreatment of midguts with PLA2 (catalytically active or inactive) dramatically lowers the level of ookinete/midgut association in vitro. It appears, therefore, that PLA2 is acting by associating with the midgut surface and preventing ookinete attachment to this surface. Thus, PLA2 is an excellent candidate for expression in transgenic mosquitoes as a means of inhibiting the transmission of malaria.

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.

Differential ability of specific regions of Plasmodium falciparum sexual-stage antigen, Pfs230, to induce malaria transmission-blocking immunity.

Antibodies raised against an Escherichia coli-produced recombinant protein encoding a 76-kDa section (region C) of malaria transmission-blocking vaccine candidate, Pfs230, have previously been shown to significantly reduce the ability of Plasmodium falciparum parasites to infect mosquitoes (71.2-89.8%). To further define the region of the Pfs230 required for transmission-blocking activity, four recombinant proteins each encoding a section of region C (Pfs230 amino acids 443-1132) were produced using the same E. coli expression system and tested for immunogenicity in mice: (i) r230/MBP.C5' encodes the first half of region C (amino acids 443-791, six cysteines); (ii) r230/MBP.CM1 encodes only cysteine motif (CM) 1 (amino acids 583-913, eight cysteines); (iii) r230/MBP.C1.6 (amino acids 453-913, eight cysteines) also includes all of CM1; and (iv) r230/MBP.C2 encodes only CM2 (amino acids 914-1268, 11 cysteines). All the recombinant proteins induced antibodies that recognized parasite-produced Pfs230, but the titre of the Pfs230 specific-antibodies generated varied, C = C1.6 = C5' > CM1 > CM2. Two recombinants, r230/MBP.C5' and r230/MBP.C1.6, induced antibody titres that were equivalent to or greater than the titre generated by r230/MBP.C. However, in contrast to r230/MBP.C, none of the recombinants induced antibodies that effectively blocked parasite infectivity to mosquitoes. This suggests that the inclusion of amino acids 914-1132 is important for the production of the transmission-blocking epitope present in region C.

Gametocyte infectivity by direct mosquito feeds in an area of seasonal malaria transmission: implications for Bancoumana, Mali as a transmission-blocking vaccine site.

Infectivity of gametocytemic volunteers living in Bancoumana, a village 60 km from Bamako, Mali, was determined by direct feeds of laboratory-reared Anopheles gambiae s. l. Gametocytemic adolescents (10-18 years old) were as infectious to mosquitoes as younger volunteers and appear to be a more suitable population for testing transmission-blocking efficacy as compared with adults (> 18 years old). To begin to validate the membrane-feeding assay, sera collected from these same volunteers were subjected to a standard membrane-feeding assay. The data suggest that areas with intense but seasonal transmission might be feasible sites for testing transmission-blocking vaccines because of the high gametocytemic rates, high mosquito infectivity rates, and lack of pre-existing humoral-mediated transmission-blocking activity. The differences observed between field-based direct mosquito feeds and laboratory-based membrane feeding assays suggests that caution be used in interpreting Phase I study results in which laboratory-based membrane-feeding assays are used as a surrogate for vaccine efficacy.

Adherence of erythrocytes during exflagellation of Plasmodium falciparum microgametes is dependent on erythrocyte surface sialic acid and glycophorins.

Malaria male gametocytes within a newly ingested infected blood meal in the mosquito midgut emerge from erythrocytes and extrude approximately eight flagellar microgametes in a process termed exflagellation. In culture, and in blood removed from infected patients, emerging microgametes avidly adhere to neighboring uninfected and infected erythrocytes, as well as to emerged female macrogametes, creating "exflagellation centers". The mechanism of erythrocyte adherence is not known nor has it been determined for what purpose microgametes may bind to erythrocytes. The proposition of a function underlying erythrocyte adherence is supported by the observation of species-specificity in adhesion: microgametes of the human malaria Plasmodium falciparum can bind human erythrocytes but not chicken erythrocytes, whereas avian host Plasmodium gallinaceum microgametes bind chicken but not human erythrocytes. In this study we developed a binding assay in which normal, enzyme-treated, variant or null erythrocytes are identified by a cell surface fluorescent label and assayed for adherence to exflagellating microgametes. Neuraminidase, trypsin or ficin treatment of human erythrocytes eliminated their ability to adhere to Plasmodium falciparum microgametes, suggesting a role of sialic acid and one or more glycophorins in the binding to a putative gamete receptor. Using nulls lacking glycophorin A [En(a-)], glycophorin B (S-s-U-) or a combination of glycophorin A and B (Mk/Mk) we showed that erythrocytes lacking glycophorin B retain the ability to bind but a lack of glycophorin A reduced adherence by exflagellating microgametes. We propose that either the sialic acid moiety of glycophorins, predominantly glycophorin A, or a more complex interaction involving the glycophorin peptide backbone, is the erythrocyte receptor for adhesion to microgametes.

A developmental defect in Plasmodium falciparum male gametogenesis.

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha-tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.

Quantification of Plasmodium falciparum sporozoites by ribosomal RNA detection.

We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed.

Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes.

Six regions of malaria transmission-blocking target antigen, Pfs230, encoding 80% of the 363-kDa protein, were expressed as recombinant proteins in E. coli as fusions with maltose-binding protein (MBP). Antisera generated against amylose-purified recombinant Pfs230/MBP fusion proteins (r230/MBP.A-r230/MBP.F) all recognized the 360-kDa form of parasite-produced Pfs230 by immunoblot. However, only antisera against the four carboxy regions (C-F) of Pfs230 and not the two amino regions (A and B) recognized the 310-kDa form of Pfs230, the form expressed on the surface of gametes. The data suggest that the 310-kDa form of Pfs230 arises from the cleavage of 50 kDa from the amino terminus of the 360-kDa form. Furthermore, antisera against r230/MBP.C bound to the surface of intact gametes and significantly reduced (by 71.2-89.8% (rank sum analysis, P < 0.01)) the infectivity of P. falciparum parasites to mosquitoes. This is the first report of a recombinant form of a P. falciparum gametocyte protein capable of inducing antisera that reduce malaria parasite infectivity to mosquitoes.

Synthetic peptides from P. falciparum sexual stage 25-kDa protein induce antibodies that react with the native protein: the role of IL-2 and conformational structure on immunogenicity of Pfs25.

To identify B-cell epitopes of the Plasmodium falciparum 25-kDa ookinete protein, Pfs25, 41 overlapping synthetic peptides spanning the entire length of the protein were used individually to immunize CAF1 (F1 hybrid of BALB/c female and A/J male) mice. Antipeptide sera were tested for reactivity to live intact zygote/early ookinete (post-fertilization stage) by immunofluorescence, and by Western blot analysis under nonreducing and reducing conditions, immunoprecipitation of 35S-cysteine-labeled antigen, and ELISA using a vaccinia recombinant Pfs25 antigen. Fourteen B-cell epitopes were identified. These peptides were immunogenic only when administered with high-dose recombinant interleukin-2. Antibodies to 11 peptides recognized only the native conformational structure, one peptide induced antibodies that recognized both reduced and native protein, and two other peptides, after primary immunization, made antibodies to denatured Pfs25, but after boosting the antibodies reacted to both denatured and native Pfs25. Anti-sera to peptides in the first (peptide 7) and fourth (peptide 34) epidermal growth factor-like domains of Pfs25 reacted most strongly with zygotes/ookinetes by immunofluorescence assay. The antibodies elicited by immunization with peptide 34 suppressed infectivity of the parasite to mosquitoes. We further observed that the secondary structure of Pfs25 may be important for immunogenicity because monoclonal antibodies (MAbs) 1C7 and 1D2, both transmission-blocking MAbs, protected enzyme cleavage sites in Pfs25 from proteolysis, suggesting that discontinuous segments of Pfs25 may come together to form immunogenic epitopic sites. Thus, definition of B- and T-cell epitopes may be required to construct a Pfs25 vaccine for optimum immunogenicity.

Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys.

BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity. MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively. To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A. nancymai and A. vociferans. After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P. falciparum. RESULTS: All three control monkeys required treatment by Day 19. Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment. In contrast, both of the A. nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection. One of the two yMSP1(19)-vaccinated A. vociferans had a prolonged prepatent period of > 28 days before requiring treatment. No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period. Sera from the two A. nancymai that self-cured had no detectable effect on in vitro invasion. CONCLUSIONS: Vaccination of A. nancymai with yMSP1(19) induced protective immune responses. The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion. Our data indicate that vaccine trials with the highly adapted FVO strain of P. falciparum can be tested in A. nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.

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.

Alpha-tubulin II is a male-specific protein in Plasmodium falciparum.

The tubulin gene family in Plasmodium falciparum consists of one beta-tubulin and two alpha-tubulin genes (alpha-tubulin I and II). We present here data indicating that alpha-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4-5) through mature (day 10-11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites. mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-alpha- and anti-beta-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant alpha-tubulin II protein obtained using the PCR-amplified alpha-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of alpha-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite. alpha-Tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus.

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.

Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies, on gametes of different isolates of Plasmodium falciparum.

We have studied the properties of epitopes on Plasmodium falciparum gamete surface protein Pfs 48/45, a target antigen of malaria transmission blocking antibodies. Using a two site immunoradiometric assay we have defined three spacially separate, non-repeated, epitope regions on the peptides representing this antigen. Epitope region I is a target of monoclonal antibodies (MoAbs) which strongly suppress infectivity of gametocytes of P. falciparum to mosquitoes; the effect is complement independent and is mediated as effectively by the monovalent Fab fragments as by intact MoAb. Epitope region II consists of two spacially close subregions, IIa and IIb; variant forms of epitopes IIa and IIb occurred in different isolates of P. falciparum. Epitope region III also showed slight structural modification between isolates. MoAbs against regions II or III were relatively ineffective in suppressing gametocyte infectivity compared to MoAbs against region I. However, certain combinations of MoAbs against regions II and III together acted synergistically to suppress infectivity to mosquitoes. All these epitopes failed to react with MoAb when the antigen was presented in reduced form. A fourth epitope, however, was identified which reacted strongly with MoAb when the antigen was presented in reduced form. The MoAb against this epitope had no effect on the infectivity of gametocytes of P. falciparum to mosquitoes.

Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum.

Malaria is initiated by the inoculation of a susceptible host with sporozoites from an infected mosquito. The sporozoites enter hepatocytes and develop for a period as exoerythrocyte or hepatic stage parasites. Vaccination with irradiated sporozoites can provide protective immunity and a recent study shows that this can also be conferred by immunization with a recombinant salmonella expressing only the circumsporozoite protein that normally covers the sporozoites. Protection against infection is likely to be mediated by cytotoxic CD8+ cells, as depletion of CD8+ T cells in a sporozoite-immunized animal can completely abrogate immunity. Here we demonstrate directly the existence of CD8+ cytotoxic T lymphocytes (CTL) that recognize the circumsporozoite protein. B10.BR mice immunized with sporozoites or with recombinant vaccinia virus expressing the CS protein of Plasmodium falciparum contain CTL that specifically kill L cell fibroblasts transfected with the gene encoding the same CS protein. The peptide epitope from the CS protein that is recognized by CTL from this strain of mice is from a variant region of the protein.

A vaccine candidate from the sexual stage of human malaria that contains EGF-like domains.

Malaria vaccines are being developed against different stages in the parasite's life cycle, each increasing the opportunity to control malaria in its diverse settings. Sporozoite vaccines are designed to prevent mosquito-induced infection; first generation recombinant or synthetic peptide vaccines have been tested in humans. Asexual erythrocytic stage vaccines, developed to prevent or reduce the severity of disease, have been tested in animals and in humans. A third strategy is to produce sexual stage vaccines that would induce antibodies which would prevent infection of mosquitoes when ingested in a bloodmeal containing sexual stage parasites. Although not directly protective, the sexual stage vaccine combined with a sporozoite or asexual stage vaccine (protective component) could prolong the useful life of the protective component by reducing transmission of resistant vaccine-induced mutants. In areas of low endemnicity, the sexual stage vaccine could reduce transmission below the critical threshold required to maintain the infected population, thereby assisting in the control or eradication of malaria. Transmission of Plasmodium falciparum, the major human malaria, can be blocked by monoclonal antibodies against three sexual stage-specific antigens. We have cloned the gene encoding the surface protein of relative molecular mass Mr 25,000 (25K; Pfs25), expressed on zygotes and ookinetes of P. falciparum. The deduced amino-acid sequence consists of a signal sequence, a hydrophobic C-terminus, and four tandem epidermal growth factor EGF-like domains.