Antibody responses to surface antigens of Plasmodium falciparum gametocyte-infected erythrocytes and their relation to gametocytaemia.

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.

Malaria vaccines and the new malaria agenda.

The development of an effective malaria vaccine has taken many decades, but there is now a good chance that the first malaria vaccine will be licensed within the next few years. However, this vaccine (RTS,S) will not be fully effective, and more efficacious, second-generation vaccines will be needed. Good progress is being made in the development of potential vaccines directed at each of the three main stages of the parasite's life cycle, with a variety of different approaches, but many challenges remain, e.g. overcoming the problem of polymorphism in many key parasite antigens. It is likely vaccines that are effective enough to block transmission, and thus contribute to increasing drives towards malaria elimination, will need to contain antigens from different stages of the parasite's life cycle.

Parasite infectivity and immunity to Plasmodium falciparum gametocytes in Gambian children.

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.

Primary structure and sexual stage-specific expression of a LAMMER protein kinase of Plasmodium falciparum.

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.

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells.

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.

Estimates of the infectious reservoir of Plasmodium falciparum malaria in The Gambia and in Tanzania.

Separate studies carried out in Farafenni, The Gambia and Ifakara, Tanzania in 1990-94 provided comparative data on population age structure, population gametocyte prevalences and gametocyte carrier infectivity. The percentage of the population estimated to be infective to mosquitoes was 5.5% and 3.8% in The Gambia and Tanzania, respectively. The age groups 1-4 years, 5-9 years, 10-19 years and 20 years or more comprised 17.5%, 21.7%, 22.2% and 37.9%, respectively, of the infectious population in The Gambia; the corresponding figures for Tanzania were 30.9%, 25.2%, 15.7% and 28.1%. These figures are in broad agreement with those from other published studies which estimated the infectious reservoir directly and suggest that adults contribute significantly to the infectious reservoir of malaria, particularly in areas of intense seasonal transmission. Control measures aimed at reduction of transmission may have only a limited effect in areas of moderate seasonal transmission if directed only at children.

Serological responses of Gambian children to immunization with the malaria vaccine SPf66.

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.

Host haematological factors influencing the transmission of Plasmodium falciparum gametocytes to Anopheles gambiae s.s. mosquitoes.

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.

Transmission-blocking effects of sera from malaria-exposed individuals on Plasmodium falciparum isolates from gametocyte carriers.

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.

An efficacy trial of the malaria vaccine SPf66 in Gambian infants--second year of follow-up.

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.

Polyclonal T-cell responses to Plasmodium falciparum gametocytes in malaria nonexposed donors.

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.

Modulation of protein kinase C activity in Plasmodium falciparum-infected erythrocytes.

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces many morphological and biochemical changes in the host cell. Host serine/threonine protein kinases could be involved in some of these processes. The aim of this study was to determine the effect of infection on red blood cell protein kinase C (PKC) and establish the importance of this enzyme in parasite growth and sexual stage differentiation. Phorbol myristate acetate (PMA)-induced translocation of erythrocyte PKC activity is impaired in erythrocytes enriched for mature asexual stage infected cells. Western blotting shows that this is due to a relative reduction in membrane PKC protein levels rather than inhibition of enzyme activity and analysis of PKC activity isolated from whole cell lysates by DE52 chromatography suggests that total activatable PKC levels are lower in infected erythrocytes. A reduction in PMA-induced activation is also observed in PKC assays performed in situ. Downregulation of erythrocyte PKC by overnight incubation with PMA before infection causes a significant decrease in the rate of the asexual growth, suggesting that the enzyme, although lost later in infection, may be important in the earlier development of the parasite. By contrast, the lack of PKC had no effect on the production of sexual stage parasites.

Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine.

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.

Evidence for CD4+ T cell responses common to Plasmodium falciparum and recall antigens.

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.

Follow-up of Gambian children recruited to a pilot safety and immunogenicity study of the malaria vaccine SPf66.

A pilot safety and immunogenicity trial of the malaria vaccine SPf66 was undertaken in The Gambia in 1993. One hundred and fifty infants aged 6-11 months were immunized with either 0.5 mg or 1.0 mg of SPf66 produced either in Colombia or in the USA or with a control vaccine. Children who received SPf66 experienced more clinical attacks of malaria than did children in the control group during the first period of surveillance and the difference in incidence between children who had received high dose Colombian vaccine and the control children was statistically significant at the 5% level. During the 1995 malaria transmission season, 127 children from the original cohort of 150 were observed. During 18 weeks of intensive surveillance, the incidence of clinical malaria was again higher among children who had received SPf66 than among children who had received inactivated polio vaccine (6.23 vs 4.89 clinical attacks per 1000 days at risk), the effect being most marked among children who were in the high dose groups, but differences between groups were now no longer statistically significant.

PIP: 150 human subjects aged 6-11 months were involved in a pilot safety and immunogenicity trial of the malaria vaccine SPf66 conducted in The Gambia in 1993. The infants were immunized with either 0.5 mg or 1.0 mg of the vaccine produced in either Colombia or the US, or with a control vaccine. Children who received SPf66 experienced more clinical attacks of malaria than did children in the control group during the first period of surveillance, with the difference in incidence between children who had received high dose Colombian vaccine and the control children being statistically significant. 127 children from the original cohort of 150 were observed during the 1995 malaria transmission season. During 18 weeks of intensive surveillance, the incidence of clinical malaria was again higher among children who had received SPf66 than among children who had received inactivated polio vaccine. The effect was most marked among children in the high dose groups, although the intergroup differences were statistically insignificant. The SPf66 vaccine may have induced an immune response which made the immunized children more susceptible to malaria. It is also possible that the increased susceptibility to malaria among children who received SPf66 was a chance event following the randomization process. No enhancement of either disease frequency or severity was found in a much larger efficacy trial of Colombian SPf66 conducted among Gambian children during a 2-year follow-up period.