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An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra-erythrocytic gametocytes produced either by a laboratory-adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti-gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti-GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06-1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte-infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti-gametocyte vaccine candidates and/or biomarkers for gametocyte carriage., (© 2016 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

Introduction: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes., Methods: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum., Results: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70)., Conclusions: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Background: Naturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses., Methodology/principal Findings: Parasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-1(19) (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01)., Conclusions/significance: In a longitudinal study, anti-Pfs230 and Pfs48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses.

Background: The efficacy of amodiaquine (AQ), sulphadoxine-pyrimethamine (SP) and the combination of SP+AQ in the treatment of Cameroonian children with clinical malaria was investigated. The prevalence of molecular markers for resistance to these drugs was studied to set the baseline for surveillance of their evolution with time., Methods: Seven hundred and sixty children aged 6-59 months with uncomplicated falciparum malaria were studied in three ecologically different regions of Cameroon - Mutengene (littoral equatorial forest), Yaoundé (forest-savannah mosaic) and Garoua (guinea-savannah). Study children were randomized to receive either AQ, SP or the combination AQ+SP. Clinical outcome was classified according to WHO criteria, as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The occurrence of mutations in pfcrt, pfmdr1, dhfr and dhps genes was studied by either RFLP or dot blot techniques and the prevalence of these mutations related to parasitological and therapeutic failures., Results: After correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 70.1% and 80.9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 80.3%, 67.5% and 76.2% in Yaoundé respectively. High levels of Pfcrt 76T (87.11%) and Pfmdr1 86Y mutations (73.83%) were associated with quinoline resistance in the south compared to the north, 31.67% (76T) and 22.08% (86Y). There was a significant variation (p < 0.001) of the prevalence of the SGK haplotype between Garoua in the north (8.33%), Yaoundé (36.29%) in the savannah-forest mosaic and Mutengene (66.41%) in the South of Cameroon and a weak relation between SGK haplotype and SP failure. The 540E mutation on the dhps gene was extremely rare (0.3%) and occurred only in Mutengene while the pfmdr1 1034K and 1040D mutations were not detected in any of the three sites., Conclusion: In this study the prevalence of molecular markers for quinoline and anti-folate resistances showed high levels and differed between the south and north of Cameroon. AQ, SP and AQ+SP treatments were well tolerated but with low levels of efficacy that suggested alternative treatments were needed in Cameroon since 2005.

Background: Observations in natural Plasmodium falciparum populations after removal of failing drugs suggest that there is a fitness cost of drug resistance., Methods: To examine the effect of transient removal of drug pressure, we analyzed seasonal changes in the prevalence of chloroquine (CQ)-resistant parasite genotypes in The Gambia. Parasite isolates from 441 children presenting with uncomplicated falciparum malaria over 5 seasons (1998-2002) were linked to weekly rainfall data., Results: The prevalence of CQ-resistant parasites increased slightly over 5 years, with the 76T allele of pfcrt (odds ratio [OR] per year, 1.16; P=.03) and the 86Y allele of pfmdr1 (OR per year, 1.18; P=.02) becoming significantly more common. However, intraseasonal analysis showed that these alleles decreased in prevalence each dry season. Wild-type parasites with respect to both loci predominated as transmission began each year, with resistant parasites becoming more common as drug use increased. This pattern was seen for both pfcrt-76T (OR per week, 1.09; P=.001) and pfmdr1-86Y (OR per week, 1.07; P=.001) and could not be explained by seasonal changes in the clonal complexity of infections., Conclusions: The fitness cost of CQ resistance works against the persistence of resistant parasites through the dry season.

Immunity to the sexual stages of Plasmodium falciparum can be induced during natural infections. Characterization of this immunity may facilitate the design of a transmission-blocking vaccine (TBV). This study aimed to assess the prevalence and serological correlates of functional transmission-blocking immunity in Gambian children (aged 1-4 years old) who were P. falciparum gametocyte carriers. Serological assays showed 100% response to fixed, whole parasites but only 42% to live gametes. Responses to the antigens Pfs230 and Pfs48/45 were 54.1% and 37.3%, respectively, in an IgG1 ELISA. 14/55 sera were capable of reducing the infectivity of laboratory isolate NF54 in a standard membrane-feeding assay (SMFA). This activity was strongly correlated with IgG1 responses to Pfs48/45 (r = 0.49, P < 0.001) and to a serological reaction with epitopes of the same molecule (r = 0.38, P = 0.003). A weaker correlation was observed with IgG1 to Pfs230 (r = 0.29, P = 0.03). In direct membrane feeding assays (DMFA) with autologous isolates, sera from 4/29 children showed transmission-blocking activity. There was no correlation with serological assays and the DMFA or between the SMFA and DMFA. This may be caused by variation in sexual stage antigens and/or alternative modes of transmission-blocking immunity, both of which have implications for vaccine implementation.

A widespread reduction in Plasmodium falciparum gametocyte prevalence could reduce malaria transmission. After infection with P. falciparum, a variable proportion of people are found to be gametocytemic. We analyzed risk factors associated with gametocytemia at presentation and 7 days later. We enrolled 1,198 children in 2 antimalarial drug trials between September and December 1998. The children were assigned to 1 of 4 treatment groups: chloroquine only; pyrimethamine-sulfadoxine (PSD) only; PSD combined with 1 dose of artesunate; and PSD combined with 3 doses of artesunate. By the time of enrollment, 200 (17%) of 1,198 children were gametocyte carriers. Three independent risk factors were associated with gametocytemia at enrollment. Children with anemia were more likely to carry gametocytes, whereas children with fever (> 37.4 degrees C) or high parasite densities (> 100,000 parasites/microL) were less frequently gametocyte carriers. Children with at least 2 of the risk factors were 4 times more likely to be gametocytemic than children with < 2 risk factors (odds ratio [OR], 4.4; 95% confidence interval [CI], 2.7-7.1). Seven days after the start of treatment, 355 (37%) of 466 assessable children were found to be gametocyte carriers. Children treated with PSD alone had a significantly higher risk of being gametocytemic by Day 7 compared with children in the other 3 treatment groups. In the subgroup of children who had no detectable gametocytes on enrollment, the effect of treatment with PSD + 3 doses of artesunate was most marked. Nineteen (10%) of 198 children treated with PSD + 3 doses of artesunate became gametocytemic, in contrast to 184 (57%) of 321 children treated with PSD alone (OR, 12.7; 95% CI, 7.3-22.1). Early treatment with highly effective antimalarial therapy has the greatest chance of preventing gametocytemia. The choice of a first-line antimalarial drug for uncomplicated malaria should not only take into consideration the ablation asexual parasitemia but also the suppression of gametocytemia.

Combination therapy that includes artemisinin derivatives cures most falciparum malaria infections. Lowering transmission by reducing gametocyte infectivity would be an additional benefit. To examine the effect of such therapy on transmission, Gambian children with Plasmodium falciparum malaria were treated with standard regimens of chloroquine or pyrimethamine-sulfadoxine alone or in combination with 1 or 3 doses of artesunate. The infectivity to mosquitoes of gametocytes in peripheral blood was determined 4 or 7 days after treatment. Infection of mosquitoes was observed in all treatment groups and was positively associated with gametocyte density. The probability of transmission was lowest in those who received pyrimethamine-sulfadoxine and 3 doses of artesunate, and it was 8-fold higher in the group that received pyrimethamine-sulfadoxine alone. Artesunate reduced posttreatment infectivity dramatically but did not abolish it completely. The study raises questions about any policy to use pyrimethamine-sulfadoxine alone as the first-line treatment for malaria.

We have isolated a LAMMER-like gene from Plasmodium falciparum by vectorette technique. The gene consists of 3316 bp encoding a protein 881 amino acids with a predicted molecular mass of approximately 106.7 kDa. The encoded protein, termed PfLAMMER, is composed of two distinct domains. The N-terminal domain is not related to any previously described protein kinases and has several interesting features including multiple consensus phosphorylation sites for a range of protein kinases, a number of RS/SR dipeptides, a large proportion of charged amino acids, two putative nuclear localisation signals and 14 copies of a tetramer DKYD repeats. The C-terminal domain is characteristic of a kinase in the LAMMER family with the highest homology to the Arabidopsis thaliana AFC3 kinase. Genomic restriction analysis showed that PfLAMMER is encoded by a single copy gene in the parasite genome. A single transcript of approximately 3800 nucleotides is expressed specifically in the sexual stage, indicating that PfLAMMER may be important in regulating the processes of sexual differentiation of the parasite.

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.

Antibody responses to the malaria vaccine SPf66 and to its constituent peptides were measured over a period of 2 years in Gambian children who had been immunized with SPf66 or with a control vaccine (inactivated polio vaccine). Three hundred and six of 308 children (99%) who had received three doses of SPf66 vaccine had antibodies to SPf66 at a level above that found in European controls who had not been exposed to malaria. Responses to the constituent peptides derived from 35.1, 55.1 and 83.1-kDa proteins were found in 88%, 97% and 97% of children, respectively; 26% had an antibody response to the NANP repeat peptide of circumsporozoite protein which is also included in the SPf66 vaccine. A response to SPf66 was found in 22% of children who had received the control vaccine. Antibody responses to NANP, 35.1, 55.1 and 83.1-kDa peptide were found in 3%, 33%, 49% and 33% of these children. Overall, no significant correlation was found between the level of anti-SPf66 antibody at the beginning of the malaria transmission season following vaccination and the subsequent risk of malaria. However, further analysis showed that among the control children who had acquired antibodies to SPf66 as a result of natural exposure to malaria, those with high levels of anti-SPf66 were less at risk of malaria, perhaps reflecting their greater previous exposure and thus immunity. In contrast, among children who had received three doses of SPf66, those with high antibody levels were at greater risk of have malaria during the subsequent malaria transmission season.

We investigated the relationship between selected host haematological and parasitological parameters and the density and infectivity of Plasmodium falciparum gametocytes. 143 individuals (age range 1-62 years) attending an outpatient clinic in Farafenni, The Gambia, who had peripheral blood gametocytaemia were recruited (mean gametocyte density 123.7/microl, range 5-17,000/microl). Of the parameters measured, packed cell volume (PCV), reticulocyte count (RetC) and lymphocyte count (LyC) were significantly associated with gametocyte density (r = - 0.17, P < 0.05; r = 0.21, P < 0.01; r = 0.18, P < 0.05, respectively). Data from membrane feeding experiments in which 15 or more mosquitoes were dissected showed that 60.7% (53/87) of gametocyte carriers infected one or more mosquitoes. Gametocyte density was strongly correlated with transmission success (TS) (r = 0.3, P < 0.005) and, in successful infections, with both mosquito prevalence (MP) (r = 0.36, P < 0.005) and mean oocyst burden (MOB) (r = 0.65, P < 0.0001). None of the other factors measured were significantly associated with any of these indices in bivariate analysis. Regression modelling showed that both gametocyte density and PCV were positively associated with gametocyte carrier infectivity to mosquitoes (LRchi2 = 100.7 and 47.2, respectively) and, in successful infections, with MOB (beta = 0.16, t = 4.9, P < 0.001; beta = 0.02, t = 2.3, P < 0.05, respectively). The positive association with PCV suggests that blood meal quality influences infection probably as a nutritional requirement, however, as this effect was most apparent at high gametocyte densities, its epidemiological significance is questionable. Though the haematological parameters associated with gametocyte density are a direct consequence of asexual infection, they may also represent an adaptive mechanism for optimization of sexual stage development.

SPf66, a synthetic peptide Plasmodium falciparum vaccine, did not protect young Gambian children against clinical attacks of malaria. Nevertheless, Gambian children who had been vaccinated with SPf66 and who were parasitaemic at the end of the first malaria transmission season after vaccination had significantly fewer detectable P. falciparum genotypes than control children, as determined by polymerase chain reaction analysis of 3 polymorphic loci--the msp1 block 2 repeat region, the msp2 repeat region, and the R11 region of the glutamate-rich protein gene (glurp). Geometric mean numbers of genotypes were 1.66 vs. 1.87, 1.95 vs. 2.43, and 1.21 vs. 1.50 for msp1, msp2 and glurp, respectively (P = 0.31, P = 0.04 and P < 0.01). Differences between groups became a little more marked for msp1 and msp2 when children with symptomatic malaria were excluded. No significant difference was found between parasites obtained from SPf66-vaccinated or control children in the prevalences of amino acid alleles at positions 44 and 47 in the 11 amino acid sequence of the merozoite surface protein 1 molecule, which is present in SPf66. The reduction in the number of genotypes observed could not be explained by a difference in parasite densities between SPf66-vaccinated and control children, as geometric mean parasite densities were almost identical in the 2 groups. These observations suggest that SPf66 vaccine may have induced an immune response which reduced the incidence of new infections in immunized children or accelerated the rate of clearance of parasites of individual genotypes. However, no reduction in the prevalence or density of parasitaemia was recorded in SPf66-vaccinated children, suggesting the existence of some kind of density-dependent mechanism for controlling low levels of malaria parasitaemia.

Sera from donors exposed to malaria were tested for their ability to block the transmission of isolates from Cameroonian Plasmodium falciparum gametocyte carriers. Sera were selected from amongst Cameroonian and Gambian donors who had positive antibody reactivity against the surface of activated gametes and against epitopes of Pfs 48/45 (a potential transmission-blocking vaccine candidate antigen). Aliquots of washed blood from gametocyte carriers were resuspended in test and control sera and fed to An. gambiae mosquitoes via a membrane feeder. Comparisons of the prevalence and intensity of infections is dissected mosquitoes showed variations in the ability of sera to block the transmission of the different isolates. Sera were identified that had little or no blocking effect on the transmission of isolates unless the isolate was poorly infectious. Some sera completely blocked the transmission of some isolates whilst having little or no effect on others. The observed variation in transmission-modulating activity may have implications for the development of a transmission-blocking vaccine.

In 1994, 630 Gambian infants were immunized with three doses of the synthetic polypeptide malaria vaccine SPf66 or with a control vaccine. No significant protection against first or total attacks of malaria was observed among the children who received SPf66. However, the period of follow-up was short. Thus, 532 children were followed for a second malaria transmission season during which 291 episodes of malaria were detected. Protective efficacies of SPf66 against first attacks of malaria and against all attacks of malaria were 8% [95% CI-20%, 30%] and 2% [95% CI-26% 24%] respectively. SPf66 did not provide any significant degree of protection to Gambian infants during a second year of follow-up.

In individual donors which have never been exposed to malaria parasites, the CD4+ T cell precursor frequencies for tetanus toxoid (TT) and Plasmodium falciparum responses are similar (range 1:850-1:4800). Limiting dilution cultures set up in response to P. falciparum trophozoites can be re-stimulated with the same stage of the parasite or TT and respond with similar frequencies. A substantial overlap in the responses to different agents was confirmed in suicide selection experiments where cells responding to malaria parasite, TT or influenza virus antigens were deleted using the cell cycle inhibitor cytosine arabinoside (Ara-C). The responses of the remaining cells to P. falciparum were almost completely abrogated and only weak responses were observed to different recall antigens (0.2-21% of untreated control). Little or no effect was observed on the responses to superantigen or mitogen. Furthermore, in contrast to superantigen, the observed responses to TT and Plasmodium were polyclonal, the blastoid cells generated reacting with a range of anti-TCR Vbeta antibodies with little preferential usage.

Human peripheral blood T-cells mount vigorous proliferative responses to asexual stage parasites of P. falciparum regardless of whether the donor has been exposed to the parasite. Here using highly purified P. falciparum gametocytes we show that the same is also true for this stage of the parasite. Gametocytes, like immature trophozoites, preferentially activate CD4+ T cells gamma delta T-cell activation, commonly observed in response to mature schizonts or supernatants from asexual-stage cultures, does not occur. Furthermore, the CD4+ T-cell receptor variable region (TCRV beta) usage to those stimulated by asexual parasites and there is no preferential usage of TCRV beta elements. The CD4+ T-cell precursor frequencies for gametocyte and asexual trophozoite responses are remarkably similar. 'Cross-reactivity' of gametocytes and asexual stage responses was confirmed by selective depletion of cells responding to particular stages of the parasite using the cytostatic drug cytosine arabinoside (Ara-C). These results suggest that the CD4+ T-cell responses from malaria non-exposed donors are common to gametocytes and asexual trophozoites.

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM) were used to treat Gambian children with uncomplicated falciparum malaria in a randomized drug trial. Blood samples were taken immediately before treatment (day 0), and at day 7 and day 28 after treatment. Samples from those parasitologically positive at day 7 following treatment ('early positives') and those positive at day 28 but negative at day 7 ('late positives') have been studied by PCR followed by restriction enzyme digestion to determine the allelic status of the pfmdr 1 locus at the codon-86 position (asparagine or tyrosine), previously associated with resistance to CQ. A significantly higher prevalence of the tyr-86 allele was observed in samples taken immediately before treatment (day 0) in the early positives group when compared with the late positives group. This suggests the tyr-86 allele contributes to drug resistance in the early positives group. This association remained significant for both CQ and AM groups, implying a common genetic basis of resistance. Predominance of the allele at day 7 is consistent with a strong selection in the first week following treatment. In the late positives group, a significantly higher prevalence of the tyr-86 allele was observed in the samples at day 28 when compared with those at day 0, suggestive of selection during the period day 7 to day 28. Differences were observed in the extent of this selection in the CQ and AM groups. The samples were genotyped at 3 unlinked polymorphic loci. These analyses suggested that most parasites observed at day 7 were probably recrudescences whereas most of those at day 28 were reinfections.

Cyclin-dependent kinases (Cdks) play a central role in the regulation of the eukaryotic cell cycle. A novel gene encoding a Cdk-like protein, Pfmrk, has been isolated from the human malaria parasite Plasmodium falciparum. The gene has no introns and comprises an open reading frame encoding a protein of 324 amino acids with a predicted molecular mass of 38 kDa. Database searches revealed a striking similarity to the Cdk subfamily with the highest similarity to human MO15 (Cdk7). The overall sequence of Pfmrk shares 62% similarity and 46% identity with human MO15, in comparison to the 49-58% similarity and 34-43% identity with other human Cdks. Pfmrk contains two unique inserts: one consisting of 5 amino acids just before the cyclin-binding motif and the other composed of 13 amino acids within the T-loop equivalent region. Southern blots of genomic DNA digests and chromosomal separations showed that Pfmrk is a single-copy gene conserved between several parasite strains and is located on chromosome 10. A 2500-nucleotide transcript of this gene is expressed predominantly in the sexual blood stages (gametocytes), suggesting that Pfmrk may be involved in sexual stage development.

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.

Naturally acquired humoral immune responses to Pfs16, an integral membrane protein expressed in Plasmodium falciparum gametocytes and sporozoites, were investigated in The Gambia. A high prevalence of antibodies to this molecule was detected by peptide ELISA. Ninety-three per cent of the people taking part in a survey at the end of the rainy season (November) had serum antibodies to one or more synthetic peptides spanning the sequence: 88% reacted with one particular peptide sequence (IMLIILSGIVGFKVK) whereas only one out of ten non-Gambians (taking anti-malarial prophylaxis with no history of infection) reacted with the peptide. Epitope mapping with mouse MoAbs has shown that this peptide is located on a part of the molecule differing from immunodominant regions of the molecule identified in a previous study in Papua New Guinea.

Plasmodium falciparum trophozoite-infected erythrocytes adhere to the amelanotic melanoma C32 cell line in vitro. Here we demonstrate for the first time that immature gametocyte-infected erythrocytes also adhere to C32 cells, albeit at lower levels than trophozoites. However, anti-CD36 and anti-intercellular adhesion molecule 1 antibodies inhibit asexual and gametocyte adhesion by comparable percentages, suggesting a common dependency for binding to these cellular receptors.

SPf66 malaria vaccine is a synthetic protein with aminoacid sequences derived from pre-erythrocytic and asexual blood-stage proteins of Plasmodium falciparum. SPf66 was found to have a 31% protective efficacy in an area of intensive malaria transmission in Tanzanian children, 1-5 years old. We report a randomised, double-blind, placebo-controlled trial of SPf66 against clinical P falciparum malaria in Gambian infants. 630 children, aged 6-11 months at time of the first dose, received three doses of SPf66 or injected polio vaccine (IPV). Morbidity was monitored during the following rainy season by means of active and passive case detection. Cross-sectional surveys were carried out at the beginning and at the end of the rainy season. An episode of clinical malaria was defined as fever (> or = 37.5 degrees C) and a parasite density of 6000/microL or more. Analysis of efficacy was done on 547 children (316 SPf66/231 IPV). No differences in mortality or in health centre admissions were found between the two groups of children. 347 clinical episodes of malaria were detected during the three and a half months of surveillance. SPf66 vaccine was associated with a protective efficacy against the first or only clinical episode of 8% (95% CI -18 to 29, p = 0.50) and against the overall incidence of clinical episodes of malaria of 3% (95% CI -24 to 24, p = 0.81). No significant differences in parasite rates or in any other index of malaria were found between the two groups of children. The findings of this study differ from previous reports on SPf66 efficacy from South America and from Tanzania. In The Gambia, protection against clinical attacks of malaria during the rainy season after immunisation in children 6-11 months old at time of the first dose was not achieved.

A pilot safety and immunogenicity trial of the malaria vaccine SPf66 has been undertaken in 150 Gambian infants. No significant systemic side effects were recorded but modest local reactions were seen after the administration of a third 1.0 mg dose. SPf66 produced in Colombia was more immunogenic than SPf66 produced in the USA and a 1.0 mg dose of each vaccine gave higher antibody levels than a 0.5 mg dose. However, antibody levels fell rapidly after administration of the third dose of vaccine and showed little change over the following malaria transmission season. The incidence of clinical malaria was higher among children who received SPf66 than among children who received inactivated polio vaccine, the effect being most marked among children who received 1.0 mg Colombian SPf66. As the trial was not designed to measure the effect of SPf66 on morbidity from malaria, the significance of this finding is uncertain.

A gene, Pf77, transcribed in the sexual stages of Plasmodium falciparum was isolated from a genomic expression library with a polyclonal antibody raised to gametocyte proteins. The entire coding region was obtained from a series of overlapping genomic and cDNA clones (from gametocyte RNA). A single open reading frame is present with no introns and no tandem repeat sequences. A Pf77 probe hybridised to a single transcript present in RNA prepared from purified gametocytes and could not be detected in RNA prepared from asexual blood stages. In situ hybridisation studies confirmed that the expression of Pf77 mRNA is sexual-stage-specific and in addition, showed that Pf77 mRNA is present only in female gametocytes during the vertebrate stages of the parasite's development.

We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytosomes and the resulting food vacuoles in the parasite cytoplasm.

Many factors determine the virulence of a malaria infection. These include host innate resistance mechanisms and, with Plasmodium falciparum, the ability to cytoadhere to endothelial cells, form rosettes, and induce release of cytokines. The effect on virulence of acquired immune responses can be determined by Class I and Class II MHC-antigens; levels of immunological responsiveness may be determined too in other ways. The structure of parasite surface antigens and their great diversity modulate the immune response and influence parasite survival and hence virulence, and transmission to the vector.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

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.

Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non-immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum-immune adults measured as lymphoproliferation, production of interferon-gamma, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection.

We have examined immune responses to a cultured Plasmodium falciparum gametocyte lysate and to an affinity-purified preparation of the 48/45-kDa gamete surface Ag in a group of 30 malaria immune individuals and in 24 Europeans with no previous exposure to malaria. Cellular responses were assessed in vitro by lymphoproliferation and production of IFN-gamma; antigamete antibodies were detected by immunofluorescence, Western blotting, and competitive ELISA. Cells from all the malaria immune donors responded to the gametocyte lysate in both assays while cells from nonimmune donors gave only weak proliferative responses. Antigamete antibodies were detected in the serum of all the immune donors but not in serum from nonimmunes. Nonimmune donors were completely unresponsive to the purified 48/45-kDa surface Ag while cells from 40% of immune donors responded by either proliferation or IFN-gamma production. Only 3 of 30 immune donors had detectable antibodies to the 48/45-kDa Ag. Class II HLA type was determined for 27 of the immune donors but no relationship between HLA-DR or -DQ and responsiveness to the 48/45-kDa Ag was discerned. The possible reasons for limited recognition of this gamete surface Ag are discussed.

The in vitro polyclonal proliferative responses of peripheral blood mononuclear cells to whole blood stage parasites or fractionated antigens from the human malaria parasite Plasmodium falciparum were studied. Cells from healthy laboratory donors who had never been exposed to malaria antigens in vivo consistently proliferated to P. falciparum antigens, as did cord blood mononuclear cells. This response was only observed in sheep rosette-positive cells in the presence of adherent cells and was inhibited by NH4Cl, indicating a requirement for antigen processing. The proliferative response was strongest at day 6 and was dependent on the presence of cells expressing high levels of CD45 180-kD isomer (UCHL1 monoclonal antibody), a marker for activated or memory cells, but not for CD45R (SN130 monoclonal antibody) a marker for naive or unprimed T cells. This suggests a similarity to the recall response to tuberculin antigen. These results suggest that the proliferative response to malaria antigens observed previously and described as a nonspecific mitogenic response may be a cross-reactive response to epitopes shared between P. falciparum and other common immunogens. This would explain the establishment of T cell clones to malaria antigens from such donors, but might suggest that the epitopes to which such clones are specific may be of questionable protective or diagnostic use.

Four antigens of Plasmodium falciparum have so far been identified as targets of transmission-blocking antibodies; three of them (Pfs 230, 48/45) are detectable in gametocytes and expressed on gametes, the fourth (Pfs 25) appears only after fertilization. Epitope analyses of each antigen were made with competitive immunoassays, and the extent of antigenic diversity determined amongst numerous isolates of P. falciparum. There was minimal variation within one of the two epitopes on Pfs 230 both of which induce transmission-blocking antibodies. The epitopes on Pfs 25 to which blocking monoclonal antibodies respond showed a variability amongst different isolates by immunofluorescence which was unexpected in view of sequence data on the molecule. Five epitope regions have been identified on Pfs 48/45 and antibodies to them interact in a complex manner. Antigenic diversity affecting these epitopes was minimal. In P. vivax malaria much greater polymorphism was seen amongst gamete surface antigens. Natural P. falciparum infections induce antibody responses to gametocyte/gamete surface antigens that will suppress infectivity to mosquitoes but these responses may involve reactivity with any of a series of different epitopes, interactions between antibodies, and may be sequential. In P. vivax infections antibody to the sexual stage antigens may suppress or enhance transmission depending on the antibody level. Cytokine production induced by sexual stage antigens may also modulate transmission, by rendering gametocytes non-infective. Experimental studies showed marked MHC-restriction of immune responses to gamete antigens (but not to the Pfs 25 zygote antigen); the evidence from studies in humans is less convincing. Antibody responses to the sexual stage antigens seem to be more frequent in persons who have experienced only one or a few attacks of malaria as opposed to those who have been exposed frequently. Some form of down-regulation may therefore be occurring.

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit the development of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10(-8) M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.

The multiplication of two strains of Plasmodium falciparum in culture, as measured by [3H]hypoxanthine incorporation, was inhibited in a dose-dependent manner by granule proteins secreted by purified eosinophils obtained from patients with the hypereosinophilic syndrome. Morphological examination revealed the presence of abnormal parasites inside erythrocytes, indicating that they were killed in situ, and the later stages of the developmental cycle were found to be most susceptible to these toxic effects. A monoclonal antibody against eosinophil cationic protein partially blocked the inhibitory effect, suggesting that it was caused by more than one of the eosinophil granule proteins. Thus some of the antimalarial effects of molecules such as the tumor necrosis factor, which activates eosinophils, may be mediated through the enhanced production of eosinophil secretion products.

The antibody response to an epitope on gamete antigens of Plasmodium falciparum in persons naturally exposed to malaria has been investigated by competitive enzyme-linked immunosorbent assay. The assay detects antibodies to an epitope on the 48/45-kilodalton (kDa) gamete surface antigen by competition with horseradish peroxidase-labeled monoclonal antibody IIC5-B10. Five sera previously shown to immunoprecipitate the 230- and 48/45-kDa antigens significantly inhibited IIC5-B10 binding to an average of 24.2% of control. The one serum which precipitated only the 48/45-kDa antigen did not inhibit IIC5-B10 binding. For 26 sera which were negative by immunoprecipitation, mean binding in the assay was 112.7% of control (pooled London nonimmune sera). Recognition of both 230-kDa and 48/45-kDa antigens was associated with a titer of 1:9 or greater (reciprocal geometric mean titer, 27.6) for inhibition to more than 2 standard deviations from the mean of the negative sera. The results show that the IIC5-B10 binding site is a naturally immunogenic epitope recognized by the majority of persons who had antibodies to the 48/45-kDa protein. An additional finding was enhancement of binding of IIC5-B10 to an average of 154.4% of control by five sera which recognized only the 230-kDa antigen, presumably due to conformational alteration of the gamete antigen complex.

Freshly isolated human peripheral blood monocytes inhibited the growth of blood-stage asexual Plasmodium falciparum parasites in vitro. The monocytes contained intracellular parasite pigment and a few whole parasites, but the remaining parasites reinvaded fresh red cells successfully and were morphologically normal. Anti-parasitic activity of these macrophages was not significantly enhanced by treatment with recombinant tumour necrosis factor alpha, recombinant gamma-interferon or lymphoblastoid alpha-interferon. Catalase had no effect on this parasite inhibition, suggesting a hydrogen peroxide independent mechanism. Anti-parasitic activity was, however, enhanced by prior maturation of the monocytes. Monocytes matured for 6 days caused 100% killing of parasites. In contrast to identical concentrations of freshly isolated monocytes the parasites incubated with these matured macrophages showed intraerythrocytic death similar to the crisis forms seen in vivo. gamma-interferon present either during the assay or as a pretreatment had no significant enhancing effect on the killing, although cytotoxicity to tumour cell lines was enhanced. Conditioned medium from macrophages showed only moderate parasite inhibition.

The susceptibility of the human malaria parasite, Plasmodium falciparum, to killing in vitro by macrophage secretory products was investigated. The effect of O2 radicals and tumor necrosis factor on parasite viability was assessed both morphologically and by following the uptake of [3H]hypoxanthine. H2O2 produced by the interaction of glucose and glucose oxidase was found to reduce viability; this effect was reversed by the addition of exogenous catalase. Further studies indicated that the catalase level within the erythrocyte was not altered upon parasite invasion. O2 radicals produced during the xanthine-xanthine oxidase interaction also killed P. falciparum. The addition of various O2 radical scavengers (including catalase) did not reverse this effect; therefore, it was not possible to determine which of the O2 radicals were involved in the killing process. Samples from three different sources containing tumor necrosis factor, a nonspecific soluble mediator derived from Mycobacterium bovis BCG-activated macrophages treated with endotoxin, also killed the parasite. There was no evidence that tumor necrosis factor or the products of the xanthine-xanthine oxidase interaction caused damage to the erythrocyte membrane that could be implicated as an important aspect of the killing process. These findings all strongly suggest that such macrophage products play an important role in immunity to malaria.

The multiplication of Plasmodium falciparum in culture, as measured by [3H]hypoxanthine incorporation, was inhibited in a dose-dependent manner by rabbit tumor necrosis serum. The regimen by which tumor necrosis serum is produced caused significant increases in the levels of triglycerides and lipid peroxides, with the latter being indicated by the level of malondialdehyde in the serum. When tumor necrosis serum was depleted of lipoproteins by aerosil (fumed silica), no parasiticidal activity remained, and when it was separated by ultracentrifugation, more than 70% of the parasiticidal activity was found in the lipoprotein fraction. This suggests that lipid peroxides may account for most of the parasiticidal activity in tumor necrosis serum but that a nonlipid toxic factor may also be present.

Immunity to the sexual stages of Plasmodium falciparum can be induced during natural infections. Characterization of this immunity may facilitate the design of a transmission-blocking vaccine (TBV). This study aimed to assess the prevalence and serological correlates of functional transmission-blocking immunity in Gambian children (aged 1–4 years old) who were P. falciparum gametocyte carriers. Serological assays showed 100% response to fixed, whole parasites but only 42% to live gametes. Responses to the antigens Pfs230 and Pfs48/45 were 54.1% and 37.3%, respectively, in an IgG1 ELISA. 14/55 sera were capable of reducing the infectivity of laboratory isolate NF54 in a standard membrane-feeding assay (SMFA). This activity was strongly correlated with IgG1 responses to Pfs48/ 45 (r = 0.49, P < 0.001) and to a serological reaction with epitopes of the same molecule (r = 0.38, P = 0.003). A weaker correlation was observed with IgG1 to Pfs230 (r = 0.29, P = 0.03). In direct membrane feeding assays (DMFA) with autologous isolates, sera from 4/29 children showed transmission-blocking activity. There was no correlation with serological assays and the DMFA or between the SMFA and DMFA. This may be caused by variation in sexual stage antigens and/or alternative modes of transmission-blocking immunity, both of which have implications for vaccine implementation. [ABSTRACT FROM AUTHOR]

Antibody titres of children and adults living in a malaria endemic region were measured by. the indirect fluorescent antibody technique, using fluorescein-conjugated monospecific immunoglobulin antisera. The antigenicity of Plasmodium falciparum trophozoites was compared with that of schizonts obtained by in vitro culture of infected blood. Malarial antibody was detected with antisera to IgG, IgM and, at low levels, to IgA but not with antisera to IgD or IgE. Higher titres were obtained with schizonts as the source of antigen than with trophozoites, Adults in whom IgM antibodies were detected had a mean IgM concentration significantly higher than that for individuals who were IgM antibody negative, and there was a positive correlation between IgG fluorescent antibody titres and total IgG in the children examined.. Serial plasma samples from children with acute P. falciparum infections were examined to determine changes in immunoglobulin concentrations, antibody titres and the levels of serum antigens as a result of the infection. [ABSTRACT FROM AUTHOR]

Investigates the presence of Plasmodium falciparum gametocytaemia among adult populations in Gambia. Role of the immune system in gametocyte production of asymptomatic adults; Prevalence of gametocytaemia; Detection of asexual malarial parasites.