Targets Of Transmission-blocking Antibodies Research Articles

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies.

Plasmodium falciparum malaria continues to evade control efforts, utilizing highly specialized sexual-stages to transmit infection between the human host and mosquito vector. In a vaccination model, antibodies directed to sexual-stage antigens, when ingested in the mosquito blood meal, can inhibit parasite growth in the midgut and consequently arrest transmission. Despite multiple datasets for the Plasmodium sexual-stage transcriptome and proteome, there have been no rational screens to identify candidate antigens for transmission-blocking vaccine (TBV) development. This study characterizes 12 proteins from across the P. falciparum sexual-stages as possible TBV targets. Recombinant proteins are heterologously expressed as full-length ectodomains in a mammalian HEK293 cell system. The proteins recapitulate native parasite epitopes as assessed by indirect fluorescence assay and a proportion exhibits immunoreactivity when tested against sera from individuals living in malaria-endemic Burkina Faso and Mali. Purified IgG generated to the mosquito-stage parasite antigen enolase demonstrates moderate inhibition of parasite development in the mosquito midgut by the ex vivo standard membrane feeding assay. The findings support the use of rational screens and comparative functional assessments in identifying proteins of the P. falciparum transmission pathway and establishing a robust pre-clinical TBV pipeline.

EGF domain II of protein Pb28 from Plasmodium berghei interacts with monoclonal transmission blocking antibody 13.1

Development of a vaccine against malaria is a major global health concern. The P28 proteins expressed on the surface of ookinetes of Plasmodium are the targets of transmission blocking antibodies. Injection of P28 proteins in vertebrate hosts induces antibodies that inhibit oocyst formation, blocking transmission of the parasite from mosquitos to human hosts. P28 proteins are crucial for parasite protection inside the mosquito midgut. Despite their importance, structural details of P28 family members have not been available to date. The purpose of this study was to structurally characterise a member of the P28 family, viz. Pb28 protein from Plasmodium berghei, and to study the interaction of Pb28 protein with the scFv (single chain variable fragment) of TBmAb (transmission blocking monoclonal antibody) 13.1 which blocks malaria transmission effectively. Pb28 protein and the TBmAb 13.1 scFv were modelled separately. To decipher the antigen-antibody interaction, ZDOCK and RDOCK programs were used. Our results suggest that, as compared to the template Pvs25, Pb28 protein has four EGF (epidermal growth factor)-like domains arranged in a triangular form with maximum root mean square deviations (RMSDs) present in the loop regions of EGF domains II and III. With the help of docking we were able to show that the B loop of EGF domain II of Pb28 protein interacts with the scFv of TBmAb 13.1. The predicted probable complex of Pb28 protein and 13.1 TBmAb suggests a mechanism for transmission blocking and may help in designing vaccine candidates in the absence of experimentally determined structures of these proteins.

Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission-blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1.5 or 15 microg Pvs25H in 0.1 or 0.5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane-feeding experiments using P. vivax-infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 microg Pvs25H/0.5 mL ISA 720 combination in the second trial.

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.

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.

Human antibody responses to Pfs 230, a sexual stage‐specific surface antigen of Plasmodium falciparum: non‐responsiveness is a stable phenotype but does not appear to be genetically regulated

The 230 kD gamete surface protein of the malaria parasite Plasmodium falciparum (Pfs 230) is a target of transmission blocking antibodies. Anti-Pfs 230 antibodies are induced following natural infection with malaria but are not found in all P. falciparum-exposed individuals. In this study we have shown that approximately 40% of malaria-exposed Gambians do not make antibodies to the native Pfs 230 molecule. This phenotype is remarkably stable over time and does not appear to be related to age, malaria exposure or major histocompatibility complex genotype. Comparison of antibody responses in twins indicates that the anti-Pfs 230 response is not strictly genetically controlled, but a high degree of concordance within both dizygous and monozygous twin pairs suggests that factors associated with exposure to malaria in childhood may be important in determining the subsequent immune response.

Pgs28 belongs to a family of epidermal growth factor-like antigens that are targets of malaria transmission-blocking antibodies.

Although Pgs28, a 28-kD surface protein of Plasmodium gallinaceum oökinetes, was previously thought not to be a target of transmission-blocking antibodies, we found that polyclonal antisera to Pgs28 completely blocked parasite infectivity to Aedes aegypti mosquitoes. Antisera raised against reduced Pgs28 were less effective in blocking transmission than were antisera to nonreduced Pgs28; thus, the target epitope(s) of transmission-blocking antibodies appears to be conformation dependent. In stage-specific assays, polyclonal antisera impaired the in vitro transformation of zygotes to mature oökinetes, as well as the in vivo development of mature oökinetes to oöcysts. Using microsequence of immunoaffinity-purified Pgs28, we cloned the 666-bp open reading frame of the Pgs28 gene. The deduced amino acid sequence of Pgs28 is strikingly similar to that of a P. gallinaceum zygote surface protein, Pgs25, and its P. falciparum analogue, Pfs25. Pgs28, like Pgs25 and Pfs25, has a presumptive secretory signal sequence, followed by four epidermal growth factor-like domains, and a terminal hydrophobic region.

Transmission blocking immunity may provide clues that antimalarial immunity is largely T-independent

Humans infected with malaria produce antibodies against the sexual stages of the parasite (Mendis et al., 1987; Graves et ai., 1988), as they do against pre-erythrocytic (Nardin et al., 1979) and asexual blood stages (Cohen et al., 1961). These antibodies are evoked by gametocytes which are precursors of the sexual stages that are presented to the host immune system during a blood infection. The antibodies take effect in the midgut of a mosquito when it takes a blood meal from an infected person, where they react either with surface antigens of male and female gametes that are formed in the mosquito midgut and prevent fertilization, or with postfertilization stages such as the zygote, and prevent their further development (reviewed by Carter et ai., 1988). A potent vaccine-induced immunity against these sexual stages could reduce malar'~. ~:ansmission in endemic areas (De Zoysa et ai., i988, i99i). Parasite molecules that are targets of transmissionblocking antibodies have been defined in both of the major human malaria parasites P. falciparum (Carter et al., 1988; Rener et al., 1983; Vermulen et al., 1985) and P. vivax (Premawansa et al., 1990), and the genes coding for two of them, Pfs25, an ookinete antigen of P. falciparum (Kaslow et al., 1988) and GAM-1 of P. vivax (personal communication, P.H. David) have been cloned. The Pfs25 ookinete antigen has been successfully expressed in the vaccinia virus, and immunization of mice with recombinant vaccinia has been . . . . . . s a t ~ , to induce antibodies which blocked the infectivity of P. falciparum gametocytes to mosquitoes in membrane feeding experiments (Kaslow et al., 1991). Thus transmission blocking malaria vaccines may soon be ready for clinical testing.

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito.

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.

Two apparently nonrepeated epitopes on gametes of Plasmodium falciparum are targets of transmission-blocking antibodies

One-site and two-site immunoradiometric assays have been developed against an antigen on gametocytes of Plasmodium falciparum, using monoclonal antibodies (Mabs) which block transmission of the parasites to mosquitoes. Three such Mabs have been studied, each of which immunoprecipitates a complex of three gamete surface proteins of apparent Mr 260,000, 59,000, and 53,000 from Triton X-100 extracts of the parasites. The assays showed that the Mabs recognized one or the other of two distinct, nonrepeated epitopes on the target antigen(s). In the one-site assay certain combinations of two Mabs interacted at appropriate concentrations to enhance binding of the Mabs to the antigen. The same combinations of Mabs synergize to suppress infectivity of gametocytes to mosquitoes.

Effects of transmission-blocking monoclonal antibodies on different isolates of Plasmodium falciparum.

The strain diversity in Plasmodium falciparum has been studied with respect to gamete surface antigens which are the targets of transmission-blocking antibodies. Of 12 isolates tested, 11 were positive by immunofluorescence with the three monoclonal antibodies studied. The exception was a Liberian isolate, two clones of which were found to react with only one of the three monoclonal antibodies. Antibodies IIC5-B10 and IA3-B8, which previously have been shown to act synergistically to block infectivity of 7G8, a Brazilian clone of P. falciparum, acted in an exactly similar way with another Brazilian isolate, It.D12, and an isolate from Thailand. In the presence of complement either IA3-B8 or a third antibody, IID2-A10, strongly suppressed infectivity of It.D12 as well as 7G8, but neither isolate was strongly suppressed by IIC5-B10. IA3-B8 and IID2-A10 did not react by immunofluorescence or immunoprecipitation with gametes of L.E5; IIC5-B10 reacted positively with L.E5 gametes in these tests. In the absence of complement, the combination of IA3-B8 and IIC5-B10 did not suppress infectivity of L.E5 to mosquitoes. In contrast to its effect on gametocytes of other isolates, IIC5-B10 in the presence of complement strongly suppressed infectivity of L.E5 to mosquitoes. These results imply that IA3-B8 and IIC5-B10 react with two structurally distinct epitopes on the surface of gametes of P. falciparum and that the properties of both epitopes on gametes of L.E5 differ from those on gametes of the other isolates tested.

A surface protein expressed during the transformation of zygotes of Plasmodium gallinaceum is a target of transmission-blocking antibodies.

Antibodies against gametes of malarial parasites (Plasmodium spp.) have previously been shown to block infectivity of the parasites to mosquitoes by preventing fertilization of the parasites in the insect midgut. These antibodies did not have any effect on the development of fertilized parasites. We now report that a surface protein of Mr 26,000 synthesized by zygotes of P. gallinaceum is the target of antibodies which block infectivity of the fertilized parasites to mosquitoes. Identification of this target antigen offers a new stage of the parasite against which a malaria transmission-blocking vaccine could be developed.