Transmission-blocking Antibodies Research Articles

Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials

In previously published studies, Saccharomyces cerevisiae recombinant protein expression systems have been employed to express the malaria parasite antigen Pfs25, a candidate transmission-blocking vaccine antigen against Plasmodium falciparum malaria. However, despite having been in two Phase 1 trials, the recombinant Pfs25 so produced (previously called TBV25H) exists as a mixture of two monomeric protein conformational forms, Pfs25H-A and Pfs25H-B. In this study, we optimized the expression and purification of the two Pfs25H conformers in S. cerevisiae, and characterized their biochemical and antigenic properties, immunogenicities, and transmission-blocking activities. Pfs25H-A is apparently homogeneous, and has the correct conformation as measured by monoclonal antibody recognition. It is, however, expressed at a low yield of only 0.19 mg/l. By contrast, Pfs25H-B is produced as a heterogeneous population of molecules that do not seem to have the correct conformation. Nonetheless, both forms appear equally effective in their ability to produce transmission-blocking antibodies in mice. To address the low yield seen with S. cerevisiae, we also expressed Pfs25 in Pichia pastoris. P. pastoris is apparently superior to S. cerevisiae in producing higher yield, immunologically more potent, biologically more active Pfs25H-A.

What – if anything – is a strain?

Two worldwide variants of the Plasmodium vivax circumsporozoite protein (CSP), VK210 and VK247, induce distinct antibody responses and can co-infect individual humans and Anopheles. The prevalence of VK210 and VK247 can change on a seasonal or a year-to-year basis. Different sympatric species of Anopheles might be more susceptible to each variant. VK210 could be associated with higher parasitaemia and chloroquine resistance. This is a relatively obscure line of research, but it could provide crucial insights into a question that has long beset parasitologists: what is a strain? Or, as Shute asked, albeit rhetorically in 1958, ‘is not the word strain in regard to malaria used much too loosely?’The latest reports from Mexico 1xPlasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites. Gonzalez-Ceron, L. et al. Exp. Parasitol. 2001; 98: 152–161Crossref | PubMed | Scopus (20)See all References1 and Colombia 2xVariants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Columbian isolates. Gonzalez, J.M. et al. Mem. Inst. Oswaldo Cruz. 2001; 96: 709–712Crossref | PubMedSee all References2 present their results from experiments on local VK210 and VK247 parasites in Anopheles albimanus, which contrast in an intriguing manner. In the first instance, A. albimanus is susceptible to VK210 and near refractory to VK247 parasites (opposite to Anopheles pseudopunctipennis). VK247 survivorship can be tracked to the ookinete and early oocyst stages. In the second instance, VK210 and VK247 develop successfully in A. albimanus derived from Guatemala, but only VK247 parasites survive in A. albimanus from the Pacific Coast of Colombia (also applies to a recent report from Brazil). The VK247 produces more sporozoites than VK210. The Mexican group proposes that factors innate to the vector population mediate the local prevalence of VK210 and VK247. The Colombian group suggests that frequency-dependence in transmission-blocking antibodies or relative immunogenicity mediates prevalence. Both groups reveal a remarkable, perhaps exemplary, parasite–host–vector system.The VK210 and VK247 dichotomy includes phenotypes (entities distinguished by their apparent properties or interactions with their environment, rather than their genetic constitutions per se) at least for antibody response, which presumably comprise a collection of genotypes. These current papers suggest that there could be geographic elements to variation in the suite of other supposed traits, but probably not congruent with the recently proposed split of P. vivax into Old and New World subspecies (based on sequence data in addition to relative infectivity to A. albimanus). Thus, P. vivax now seems to be the surprise frontrunner for addressing the recurring question: is a strain a heuristic concept without a tangible biological basis, or a discrete biological entity that can be distinguished by a modern technique? Are VK210 and VK247 strains?

Antibodies to Plasmodium vivax transmission-blocking vaccine candidate antigens Pvs25 and Pvs28 do not show synergism

Transmission-blocking vaccines against malaria parasites target molecules expressed by sexual stage parasites to elicit antibodies that prevent the infection of the mosquito vector. Pvs25 and Pvs28, expressed on the surface of ookinetes, are potential candidates for such a vaccine and induce antibodies that block the infectivity of Plasmodium vivax in immunized animals. To improve the ability to induce transmission-blocking antibodies, Pvs25 and Pvs28 were produced as a single fusion protein by the yeast Saccharomyces cerevisiae. Mice immunized with a low dose of the chimeric molecule (Pvs25–28) developed higher antibody responses compared with mice immunized with either Pvs25 or Pvs28. In membrane feeding assays, both anti-Pvs25–28 and anti-Pvs25 antisera had similarly potent transmission-blocking activities (and both were much greater than anti-Pvs28). Furthermore, serum from mice simultaneously immunized with both Pvs25 and Pvs28, or serum mixtures of anti-Pvs25 alone and anti-Pvs28 alone did not enhance the efficacy over anti-Pvs25 serum alone, demonstrating that there is no synergism in the ability to block transmission of P. vivax between anti-Pvs25 and anti-Pvs28 antibodies.

Role of chicken serum in inhibiting Leucocytozoon caulleryi development in Culicoides arakawae infected by membrane-feeding of infective blood meals

Leucocytozoon caulleryi, the most important pathogen of leucocytozoonsis in several Asian countries, is transmitted by Culicoides arakawae. Although the vector blood meal factors that influence the infectivity of L. caulleryi remain poorly understood, the factors in infected chicken serum remain vital. C. arakawae blood-fed through a membrane were used in this experiment to examine the influence of serum factors on L. caulleryi development. Experimental results indicate that C. arakawae were successfully blood-fed through a chicken-egg-shell membrane and the serum factors in infected chicks significantly affected L. caulleryi sporogony. The inhibition effect of serum factors calculated from sporozoite averages was 53.6% +/- 2.2%. The serum factors, including transmission-blocking antibody, require further detailed study.

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 Parasitology97, 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.

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.

Camels, Cues and Cure-alls on the Net

In October last year an innocent enquiry from a graduate who had heard a talk from a scientist at Genentech about `a new vaccine for malaria' led to pages-long exchanges of views and expressions of strong feelings about the merits of transmission-blocking vaccines. Geoff Skillman (University of California at San Francisco, USA) started it: `when a mosquito carrying malaria takes a blood meal [from people carrying specific antibodies] the antibodies bind to and kill one of the stages of malaria which is unique to the mosquito ... the system is already working in their mouse model for malaria ... the mosquitos get cured of malaria and thus can't pass it along.' Patrick Rozmajzl (University of Mississippi, USA) thought while `such “altruistic” vaccines are certainly interesting ... because they do not protect the person vaccinated (only preventing future transmission) it becomes a difficult sell for people in endemic regions.' Allan Saul (Queensland Institute of Medical Research, Australia) objected to this `bad press', which he regarded as misinformed and damaging: `by being vaccinated you [eg. a father of a subsistence farming household in the Philippines] stand to save yourself a heap of money and time by preventing the rest of your family from becoming infected ... Who would not want to protect their family from the suffering of malaria? Is this “altruism”? ... continual harping on a supposedly altruistic vaccine which supposedly cannot be sold, not only makes it more difficult to get the support for researching a potentially valuable addition to malaria control measures, but may become a self-fulfilling prophecy.' Rob Anderson (University of Aarhus, Denmark) was delighted to encounter debate about controversial issues, instead of the usual stream of `what should I do to keep from getting malaria? I have heard that burning my socks in the tent each night really works ... The transmission-blocking antibody strategy appeals (I think!) because it incorporates fiddling with the immune system, probably a little bit of gene splicing and the assumption that if the targets are stages of malaria not found in humans, for example, the ookinete, there should be no complications for the human host ... if the transmission blocking antibody proponents can confront the issue of evolution of malaria strains unsusceptible to the vaccines and can commit to the long term strategy of constantly developing new vaccines to keep a step ahead, the idea has merit.' But he wanted to emphasize that appropriate control in one area is not necessarily so elsewhere and `“malaria control specialists” need to be entomologists, parasitologists, clinicians, sociologists, engineers and ecologists all wrapped into one package' and `the often disingenuous hyping of biotechnology as a cure-all for future malaria control' should be stopped. When Patrick Rozmajzl complained about `electronic drubbing' Allan Saul explained: he was attacking the message and not the messenger.

Vaccination of natural reservoir hosts with recombinant lipidated OspA induces a transmission-blocking immunity against Lyme disease spirochaetes associated with high levels of LA-2 equivalent antibodies

As observed in humans, immune responses in naturally infected reservoir hosts of Borrelia burgdorferi sensu lato rarely target the outer surface proteins (Osp) A and B of Lyme disease spirochaetes. The absence of protective immunity in such hosts following tick-borne infection allows them to play an effective role in the maintenance of Lyme borreliosis in nature. Therefore, the question was addressed whether one of the most prominent natural reservoir host species of B. burgdorferi s.l. in Europe, the yellow-necked mouse ( Apodemus flavicollis), may lack the ability to elicit transmission-blocking antibodies to Lyme borreliosis spirochaetes. Yellow-necked mice were immunized with a recombinant lipidated OspA from B. burgdorferi sensustricto or with high numbers of UV-irradiated whole spirochaetes. All immunized mice, but not untreated controls, developed polyclonal humoral immune responses to OspA (31 kDa). Serum antibodies of animals vaccinated with the recombinant OspA contained high levels of antibody to an epitope of OspA, defined by the monoclonal antibody LA-2, whereas only low levels of LA-2 equivalent antibodies could be detected in sera from animals immunized with killed spirochaetes. Ixodes ricinus ticks infected with B. burgdorferi s.s. lost their spirochaete load after feeding on animals with high levels of LA-2 equivalent antibody; ticks feeding on animals which had only low or undetectable serum levels of LA-2 equivalent antibodies retained their spirochaete infection. Furthermore, animals with high levels of LA-2 equivalent antibody were protected against spirochaete infection. Our study shows that natural mouse reservoir hosts are highly competent to generate transmission-blocking antibodies after vaccination with a lipidated recombinant OspA and indicates that antibodies to the LA-2 epitope play a key role in the destruction of B. burgdorferi s.s. within feeding Ixodes ricinus ticks.

Two antigens on zygotes and ookinetes of Plasmodium yoelii and Plasmodium berghei that are distinct targets of transmission-blocking immunity.

We have developed transmission-blocking monoclonal antibodies (MAbs) against Plasmodium yoelii 21-kDa (Pys21) and 28-kDa (Pys25) ookinete surface proteins. These MAbs block infectivity of P. yoelii to Anopheles stephensi. One MAb, 14, cross-reacted by Western blotting with a 28-kDa surface protein (Pbs25) of P. berghei ookinetes and blocked oocyst development, as assayed by direct mosquito feeds on passively immunized P. berghei-infected mice. In total, we have identified two ookinete surface proteins in P. yoelii, one of which is also present in P. berghei. The transmission-blocking activity of the anti-Pys25 MAb 4 was complete and more potent than that of the anti-Pys21 MAb 2. Moreover, Fab fragments of MAb 4 had transmission-blocking activity in mice. In comparison, Fab fragments of MAb 2 did not have detectable transmission-blocking effect, although F(ab')2 did. Furthermore, MAb 2 and MAb 4 appeared to block the in vitro formation and development of zygotes as well.

Characterization of twoPlasmodium vivaxgenes cloned by screening with transmission-blocking monoclonal antibodies

The cloning and characterization of two Plasmodium vivax genes is described. Both genes have been isolated through screening P. vivax expression libraries with transmission-blocking monoclonal antibodies (mAb). However, because one mAb recognized a linear epitope and the other a reduction-sensitive, conformationally dependant epitope, the method of library screening and the two genes obtained were markedly different. The methods employed, as well as the resulting genes, are compared within the context of the development of a P. vivax transmission-blocking vaccine.

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb c mice with BmrPbs21 induced high anti-BmrPbs21 and anti-ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti-EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5–97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.

Detection of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of the gametocyte antigen Pfs2400 in malaria patients in Brazil.

Sera collected from 164 individuals who had clinical Plasmodium falciparum malaria and came from several areas of Brazil where malaria is endemic were tested for the presence of anti-gametocyte antibodies. Antibodies directed against P. falciparum gametocytes were detected, by IFAT, in the sera of 67.1% of these patients. The prevalence of these antibodies was significantly higher in patients who had undergone multiple attacks of malaria than in those who were experiencing their first attack at the time of serum collection. Although circulating gametocytes were detected in 22% of the patients at this time, there was no difference in the percentages of IFAT positivity between apparent gametocyte 'carriers' and 'non-carriers'. All sera were also tested by ELISA, using a dimer of the nonamer peptide [PEE(L/V)VEEV(I/V)]2, which represents a tandem consensus repeat of the P. falciparum gametocyte antigen, Pfs2400, a target of transmission-blocking antibodies. ELISA demonstrated that 32.9% of the patients had antibodies that reacted with this peptide. Positive ELISA reactions were significantly more frequent amongst the sera of patients who had had multiple malaria attacks than in those undergoing their first malaria episode; positivity was lower in the gametocyte 'carriers' than in their 'non-carriers'. These results demonstrate that anti-gametocyte antibodies, which have already been shown to have potential transmission-blocking activity, are naturally elicited in Brazilian patients, the highest rates of seropositivity occurring after multiple malaria attacks.

Predicted disulfide-bonded structures for three uniquely related proteins of Plasmodium falciparum, Pfs230, [formula omitted] and Pf12

Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important andidate candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs48 45 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics. Although having similarities to the ‘cystine knot’ described in other proteins, the arrangement proposed here appears to be unique among described structures.

Plasmodium falciparum: A Comparison of the Activity of Pfs230-Specific Antibodies in an Assay of Transmission-Blocking Immunity and Specific Competition ELISAs

The activity of monoclonal antibodies (mAbs) that specifically recognize the Plasmodium falciparum sexual stage-specific protein Pfs230 was analyzed. All mAbs reacted with the surface of extracellular sexual forms of the parasite in a suspension immunofluorescence antibody reaction and precipitated the Pfs230 protein from an NP-40 extract of surface radioiodinated macrogametes/zygotes. Only mAb that bound complement blocked transmission, whereas mAb that did not bind complement but competed with the complement binding mAb for binding to the same epitope did not block transmission. These mAbs were used to develop Pfs230-specific competition ELISAs to analyze epitope diversity and to analyze the binding characteristics of anti-Pfs230 antibodies in human serum. Transmission-blocking (TB) antibodies in test/field sera competed in the competition ELISA for binding with epitope-specific, labeled mAbs against Pfs230. At least five different epitope regions could be defined with the competition ELISAs. All 46 sera from gametocyte carriers immunoprecipitated the Pfs230 molecule, while 19 of these sera blocked transmission in the bioassay. Five of the transmission-blocking and one of the nonblocking sera competed with monoclonal antibodies. A method comparison analysis was used to determine agreement between reactions in a competitive ELISA and the TB activity examined in the bioassay. The index of agreement κ between outcomes of the bioassay and ELISA was fair to poor (κ = 0.25) but since its range includes values below 0 the relation between the data obtained by the bioassay and the competition ELISA can be explained by chance alone. The serological data did not reveal a correlation between immunoprecipitation of Pfs230 and TB activity.

Mapping of two overlapping linear epitopes in Pfg27 recognized by Plasmodium falciparum transmission-blocking monoclonal antibodies

We have reported previously the production of Plasmodium falciparum transmission-blocking monoclonal antibodies (mAb) recognizing a reduction-insensitive cross-reacting epitope in the gametocyte antigen Pfg27 and the gamete surface antigens Pfs230 and Pfs4845. In this study, the amino acid sequence of this epitope in Pfg27 was determined. First, the epitope was localized near the N terminus of the protein by probing recombinant overlapping fragments spanning Pfg27 with transmission-blocking mAb in immunoblot experiments. The amino acid sequence of the epitope was then determined by using overlapping synthetic peptides spanning the smallest immunoreactive recombinant fragment in an ELISA. The sequence KPLDKFGNIYDYHYEH (amino acids 10–25 in the Pfg27 sequence) was shown to contain two overlapping epitopes recognized by transmission-blocking mAb. Comparison of the sequence of the gene encoding Pfg27 in seven different P. falciparum strains demonstrated that these sequential epitopes are totally conserved. Immunization of mice with synthetic peptides derived from Pfg27, conjugated with keyhole limpet hemocyanin (KLH) and formulated in Freund's adjuvant or alum, resulted in the production of antibodies capable of recognizing the peptides as well as the native Pfg27.

Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

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.

Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement-fixing.

We have studied the properties of 16 newly derived monoclonal antibodies (MoAbs) against Pfs230, a gamete surface protein of Plasmodium falciparum and a target of transmission-blocking antibodies. All 16 MoAbs recognized Pfs230 by immunoprecipitation from non-ionic detergent extracts of the protein radio-labelled with 125Iodine. The MoAbs also recognized this protein on Western blots under non-reducing conditions but none of them recognized the protein under reducing conditions. Using an immunoradiometric assay the MoAbs appear to define nine different epitope regions. The MoAbs were tested for their ability to lyse extra-cellular female gametes of P. falciparum isolate 3D7. Eight of the MoAbs, all of isotype IgG2a, mediated complement-dependent lysis of the gametes; seven of the MoAbs, all isotype IgG1, failed to lyse the gametes in the presence of active complement. The eight complement-fixing MoAbs mediated almost total suppression of infectivity of gametocytes of P. falciparum 3D7 to mosquitoes; where tested this suppression was mainly complement-dependent. The seven non-complement-fixing MoAbs had no significant effect on the infectivity of gametocytes of P. falciparum 3D7 to mosquitoes. When tested by immunofluorescence the target epitopes of all the MoAbs were conserved in each of the five different isolates of P. falciparum which were tested.

Characterization of the effector mechanisms of a transmission-blocking antibody upon differentiation of Plasmodium berghei gametocytes into ookinetes in vitro.

The transmission-blocking monoclonal antibody 13.1, which recognizes the ookinete surface antigen Pbs21 of Plasmodium berghei, and an IgG2a isotype control antibody 26.37 were purified by caprylic acid and ammonium sulphate precipitation. Fab fragments were prepared by papain digestion. IgG but not Fab from antibody 13.1 reduced ookinete formation by P. berghei in culture by as much as 94% at a concentration of 100 micrograms/ml. There was little difference in antibody efficacy in the range 6.25-400 micrograms/ml in this assay. The parasite was most sensitive to antibody activity in the first 6-9 h of culture, i.e. the gamete/zygote and early retort stages. Peripheral blood leucocytes (PBL) were essential to achieve maximal inhibition by mAb 13.1 (activity was abrogated totally if PBL were removed). Together the data suggest that one of the mechanisms of action of this antibody is antibody-mediated PBL killing. Phagocytosis of parasites was noted in these experiments in all cultures. We have not attempted in this study to distinguish between Fc-mediated opsonization, as opposed to antibody-dependent cellular cytotoxicity.

Crystallization, sequence and preliminary crystallographic data for transmission-blocking anti-malaria Fab 4B7 with cyclic peptides from the Pfs25 protein of P. falciparum.

X-ray quality crystals of an Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide-complexed forms. Initially, the intact immunoglobulin was crystallized because cleavage with pepsin or papain did not produce a homogeneous product. Further proteolytic trials with elastase produced a suitable Fab fragment from which crystals have been obtained, both for the free Fab and in complex with cyclic peptides and in the presence of linear peptides. While linear peptides bind to MAb 4B7, cyclic peptides modeled on a predicted beta-hairpin loop of the third EGF-like domain of Pfs25 bind better and readily co-crystallize with the Fab. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino-acid sequence for the complete Fab deduced. This work explores the use of glycerol as an additive and the modification of the peptide sequence outside the epitope for improving in the crystallization. Data sets have been collected from crystals of several Fab-peptide complexes and from uncomplexed Fab to resolutions ranging from 2.4 to 3.3 A. The packing arrangements of several crystal forms have been determined by molecular replacement, and refinement of their three-dimensional structures is in progress. The three-dimensional structure of this Fab complexed with the various peptides will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the malaria parasite.