Plasmodium falciparum: membrane feeding assays and competition ELISAs for the measurement of transmission reduction in sera from Cameroon.

The effect of natural malaria transmission-blocking factors in the blood of Plasmodium falciparum gametocyte carriers was assessed in two types of functional bioassays. In the direct membrane feeding assay (DMFA), a comparison is made between the infectivity of gametocytes from a naturally infected gametocyte carrier in the presence of autologous plasma and the infectivity in the presence of replacement plasma from nonimmune donors. In the standard membrane feeder assay (SMFA), cultured NF54 gametocytes are used to measure the capacity of endemic sera to block transmission. In the DMFA, 18 out of 48 sera (37.5%) from Cameroonian gametocyte carriers reduced transmission significantly, while in the SMFA 22 out of 48 sera (45.8%) produced transmission reduction. There was a positive correlation between both assays (r + 0.41, P < 0.05). Antibodies against epitopes of transmission-blocking target antigens Pfs48/45 and Pfs230 were measured in competition ELISAs and compared with the results of DMFA and SMFA. Serological reactivity in competition ELISAs against three epitopes of Pfs48/45 was significantly higher in the group of transmission-reducing sera in both the DMFA and the SMFA, especially for epitope III. No significant difference was found for Pfs230 antibodies (epitope I). Sensitivity of the serological assays was approximately 60%, with a specificity of around 70%. Serological tests cannot replace the functional bioassay in field situations as yet, but can contribute in the selection of sera for SMFA evaluation.

Transmission blockade of Plasmodium falciparum malaria by anti-Pfs230-specific antibodies is isotype dependent.

By use of the parental hybridoma cell line 63F2A2 that produces specific antibodies of immunoglobulin isotype G1 (IgG1; 63F2A2.1) against Pfs230, we attempted to enrich for the synthesis of the downstream switch variant IgG2b and IgG2a monoclonal antibodies (MAbs) of the hybridoma cell line (63F2A2.2b and 63F2A2.2a, respectively). The parental IgG1 did not reduce the Plasmodium falciparum transmission in a bioassay irrespective of the presence of complement. MAbs 63F2A2.2b and 63F2A2.2a were effective in reducing the infectivity of P. falciparum parasites to Anopheles gambiae mosquitoes in membrane-feeding experiments. A transmission reduction of 91% was accomplished by the 63F2A2.2b switch variant, and a reduction of greater than 99% was accomplished by the 63F2A2.2a switch variant, but only in the presence of active human complement. Subsequently, the transmission-reducing effect of MAb 63F2A2.2b or 63F2A2.2a was confirmed in vitro by the rapid lysis of newly formed macrogametes or zygotes in the presence of active complement. MAb 63F2A2.1 did not lyse the newly formed macrogametes or zygotes irrespective of the presence of complement.

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 kappa between outcomes of the bioassay and ELISA was fair to poor (kappa = 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.

A comparison of transmission-blocking activity with reactivity in a Plasmodium falciparum 48/45-kD molecule-specific competition enzyme-linked immunosorbent assay.

Monoclonal antibodies (MAbs) 32F1 and 32F3 react with two independent epitopes of a protein doublet with molecular weights of 48 and 45 kilodaltons (kD) expressed on the surface of Plasmodium falciparum (Pfs48/45) macrogametes and zygotes; only 32F3 blocks transmission. These MAbs were used to develop a Pfs48/45-specific competition enzyme-linked immunosorbent assay (ELISA) using 32F1 to capture antigen and labeled 32F3 for quantification and analysis of the contribution of antibodies in human serum to transmission-blocking activity. A comparison analysis was used to determine agreement of competition ELISA titers and transmission-blocking activity as observed in the bioassay in three groups of serum samples: 37 from European travelers with previous exposure to malaria, 56 from gametocyte carriers, and 66 from schoolchildren from a malaria-endemic area in Cameroon. The index of agreement between outcomes of the ELISA and transmission-blocking assay in gametocyte carriers and in travelers was specifically defined as fair-to-moderate; in schoolchildren the agreement was not significant. The combined analysis of all sera showed a significant and fair-to-moderate agreement between the results of the competition ELISA and the transmission-blocking assay, with a relative specificity of 94% (of 105 cases negative in the transmission-blocking assay, 99 were also negative in the competition ELISA) and a relative sensitivity of 44% (of 54 cases positive in the transmission-blocking assay, 24 were also positive in the competition ELISA). This study shows that a positive C48/45-ELISA is indicative for transmission-blocking activity in the mosquito assay, while a negative result does not exclude transmission-blocking activity.

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 immunity as observed in a feeder system and serological reactivity to Pfs 48/45 and Pfs230 in field sera.

Monoclonal antibodies (mAbs) and human sera from gametocyte carriers were applied in the bio-assay to test for their transmission-blocking capacity. Competition ELISA's have been developed for the detection of natural transmission blocking antibodies. Approximately 55% of the sera blocking in the bio-assay gave positive results in these competition ELISA's.

Transmission blocking immunity as observed in a feeder system and serological reactivity to Pfs 48/45 and Pfs230 in field sera

Monoclonal antibodies (mAbs) and human sera from gametocyte carriers were applied in the bio-assay to test for their transmission-blocking capacity. Competition ELISA's have been developed for the detection of natural transmission blocking antibodies. Approximately 55 of the sera blocking in the bio-assay gave positive results in these competition ELISA's.

Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA.

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures.

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens (Mr 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2-3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [3H]palmitic acid covalently. The iso-electric point (pI) of these proteins was assessed as being 6.0 +/- 0.1 (for both 48 and 45 kDa) and 5.6 +/- 0.1 (for 25 kDa).