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    Antibodies among sera from Kenyan men and children to surface antigens expressed by CSA-binding infected erythrocytes determined by flow cytometry. (A) Binding of IgG to the surface of CS2-infected erythrocytes among a selection of sera from men and non-exposed controls; the mean of 10 (error bar shows 3 standard deviations) non-exposed controls is shown. Samples 442 and 392 were regarded as positive for specific IgG binding; samples 274 and 94 were around the threshold for classification as positive. (B) Binding of IgG to the surface of CS2-infected erythrocytes among sera from children and non-exposed controls; samples 86, 83, 9, 68, 4, 55, 30, 19, 61, and 103 were regarded as positive for specific IgG binding. All values represent mean (+ range) for duplicate assays. Sera were tested at 1/5 dilution. The cut-off level for classification as positive is represented by the broken horizontal line (mean + 3 standard deviations of non-exposed samples). IgG binding was expressed as the ratio of the mean fluorescence intensity for IEs divided by that for RBCs, to account for variation in background binding of IgG to uninfected RBCs. A relative score of 1 indicates no significant IgG binding. Samples were tested in Kilifi, Kenya, using a Coulter EpicXL flow cytometer.

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    Antibodies among sera from Papua New Guinea men and non-exposed Australian residents to surface antigens expressed by P. falciparum-infected erythrocytes determined by flow cytometry. (A) Binding of IgG among men’s samples to the surface of infected erythrocytes using the CSA-binding isolate CS2. Values represent mean for duplicate assays (error bars show range). Controls (gray bar) represent the mean (error bar shows 3 standard deviations) of sera from non-exposed donors. Multigravidas (striped bar) represent the median level (error bar shows 95% CI) of IgG binding for 38 samples tested from multigravid women from the same PNG population. The cut-off level for classification as positive is represented by the broken horizontal line (mean + 3 standard deviations of non-exposed samples). (B) Binding of IgG to the surface of infected erythrocytes using the CSA-binding isolate HCS3 compared with E8B-ICAM1, which adheres to CD36 and ICAM-1. Values represent mean for duplicate assays (error bars show range). Controls represent the mean (+ 3 standard deviations) of sera from non-exposed donors. The cut-off level for classification as positive for HCS3 is represented by the broken horizontal line. For A and B, sera were tested at 1/20 dilution, and IgG binding is expressed as the ratio of the mean fluorescence intensity for IEs divided by that for RBCs, to account for variation in background binding of IgG to uninfected RBCs. A relative score of 1 indicates no significant IgG binding. Samples were tested in Melbourne, Australia, using a Becton-Dickinson FACSCali-bur flow cytometer.

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    (A) Inhibition of adhesion of CS2 infected erythrocytes to CSA by sera from Malawian men. Values represent mean + range expressed relative to controls (sera from non-exposed donors). The median (+ 95% CI) level of inhibition by sera from 24 multi-gravid women is show for comparison (striped bar). Flow cytometry analysis of IgG binding to the surface of intact CS2 infected erythrocytes: (B) sera from a PNG adult male (gray line, unfilled plot) compared with serum from a non-exposed adult donor (black line, filled plot). Data plotted using FlowJo software.

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Antibodies among Men and Children to Placental-Binding Plasmodium falciparum-Infected Erythrocytes that Express var2csa

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  • 1 Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia; Centre for Geographic Medicine Research, Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Victoria 3050, Australia; Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

INTRODUCTION

Plasmodium falciparum-infected erythrocytes (IEs) can adhere and sequester in deep vascular beds of various organs, contributing to the pathogenesis of severe disease and complications of malaria (reviewed in Refs. 1 and 2). One of several receptors for IE adhesion is chondroitin sulfate A (CSA),3 which mediates parasite adhesion and sequestration in the placenta.4 IEs accumulating in the placenta generally adhere, at varying levels, to CSA, whereas there is little adhesion of these IEs to other receptors, such as CD36 or intercellular adhesion molecule-1 (ICAM-1).46 Adhesion to CSA is less common among isolates from nonpregnant individuals; these typically adhere to CD36, ICAM-1, and other receptors.4,5 Studies have also implicated hyaluronic acid (HA) as an additional receptor for placental adhesion7,8 and binding to non-immune immunoglobulins.9

Pregnant women are particularly susceptible to malaria, despite substantial pre-existing immunity. During pregnancy, a combination of adhesion to specific placental receptors and immune pressure selects for novel or rare IE variants that evade existing immunity.10 Prior to, or at the commencement of the first pregnancy, individuals have lower levels of antibodies to variant surface antigens (VSA) expressed by placental isolates and CSA-binding IEs,5,1113 compared with other isolates. After exposure in pregnancy, most women develop antibodies to these variants,14,15 which are associated with reduced risk of maternal malaria and complications in some studies.16,17 Acquired antibodies target both polymorphic and conserved epitopes, and the antibody repertoire to different placental-binding variants expands with successive pregnancies.18 Adhesion to CSA is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1).1921 PfEMP1 is highly diverse and clonally variant, encoded by members of the var multigene family, and is the primary target of host antibodies to the surface of IEs.22,23 Recent studies have identified a relatively conserved var gene, var2csa, that appears to be the major gene encoding adhesion to CSA and an important target of protective immune responses.21,2426 Other CSA-binding domains have been identified, but their significance in malaria pathogenesis remains unclear.27,28

Key questions remain, however, regarding the broader significance of adhesion to CSA outside placental malaria. A proportion of clinical isolates from African children and Thai adults can adhere to CSA in vitro,5,2931 but adhesion to CSA is not typically the dominant phenotype. IEs can also adhere in vitro to CSA on cultured cerebral and pulmonary endothelial cells.32 It is presently not known whether acquisition of antibodies to CSA/placental-binding variants that typically express var2csa-PfEMP1 is truly restricted to pregnancy. Exposure and acquisition of antibodies to CSA-binding IEs prior to pregnancy may have a significant influence on the susceptibility of primigravidas, course of infection, or acquisition of immunity to maternal malaria. Here, we have evaluated the presence and specificity of antibodies among African men and children and Papua New Guinean men to determine whether there is significant exposure to placental CSA-binding isolates expressing var2csa that results in acquisition of antibodies that may have biologic significance.

MATERIALS AND METHODS

Study populations and samples.

Forty plasma samples from Kenyan men (mean age 24 years; range 16–59 years) were randomly selected from a community-based cross-sectional survey performed in the Kilifi District in 1998, and samples from children were obtained from a community-based cross-sectional survey performed in the same population in 2000 (mean age 7.0 years; range 5–8 years). This population experiences year-round malaria with substantial seasonal variation.33,34 Serum samples were collected from 31 men (mean age 30 years; range 16–49 years) attending children’s clinics at Modilon Hospital, Madang town clinic, and Yagaum Health Center, Papua New Guinea (PNG), an area that experiences year-round malaria with some seasonal variation.35 Some samples were used from men resident in Malawi (mean age 29 years; range 21–40 years), collected from parents of children presenting to medical services in Blantyre.5 For comparison, some samples were collected from multigravid women from the same populations as the men and children in each region. Informed consent was obtained from all donors. Samples were also collected from non-exposed donors resident in Melbourne, Australia, and in Oxford, U.K. Ethical approval was obtained from the Ethics Committee, Kenya Medical Research Institute, Nairobi, the Medial Research Advisory Committee, Department of Health, Port Moresby, PNG, the Clinical Research Ethics Committee, Royal Melbourne Hospital Research Foundation, Australia, the Human Research Ethics Committee of the Walter and Eliza Hall Institute, Australia, and the College of Medicine Medical Research and Ethics Committee, University of Malawi.

P. falciparum isolates and culture.

P. falciparum was cultured in vitro, as previously described,5 using human group O positive red blood cells (RBCs). CSA-binding isolates used were CS2 and HCS3, which were generated by selection for adhesion to CSA36 and also adhere to HA,8 and are genetically distinct. Both CS2 and HCS3 express var2csa as the dominant var transcript.25,37 Isolates E8B and A4 adhere to CD36 and ICAM-1 and are of the same genetic lineage as CS2.

Antibody assays.

Samples were tested for IgG binding to the surface of pigmented trophozoite stage IEs using flow cytometry.15,38 Cells were sequentially incubated with test serum or plasma diluted 1/20 or 1/5, goat or rabbit anti-human IgG (Fc-specific, 1:100; Dako Corp., Copenhagen, Denmark), and FITC-conjugated anti-goat or anti-rabbit Ig (1:50; Dako), or Alexa-Fluor 488 conjugated anti-rabbit Ig (1:500; Molecular Probes, Inc., Carlsbad, CA), with ethidium bromide 10 μg/mL. PNG and Malawi samples were tested in Melbourne, Australia, using a Becton-Dickinson FACSCalibur. Kenyan samples were tested in Kilifi, Kenya, using a Coulter EpicXL (Coulter Corp., Hialeah, FL). Flow cytometry data was analyzed and plotted using FlowJo software (Tree Star, Inc., Ashland, OR). For each sample, IgG binding was expressed as the ratio of the mean fluorescence intensity for IEs divided by that for RBCs, to account for variation in background binding of IgG to uninfected RBCs. A relative score of 1 indicates no significant IgG binding. Samples were regarded as positive if relative IgG binding was greater than the mean + 3 SD of non-exposed control sera. Samples were coded and tested blindly.

Inhibition of adhesion to CSA by serum or plasma samples was performed, as described,15,18 using pigmented-trophozoite IEs at 4–5% parasitemia and 0.5% hematocrit. Parasites from in vitro culture were pre-incubated with human serum at 1/5 dilution for 45 minutes, 37°C, before testing for adhesion to immobilized CSA.

RESULTS

Antibodies to CSA-binding P. falciparum isolates among men and children.

Serum samples from men resident in malaria-endemic regions of Kenya and PNG and samples from children in Kenya were investigated for the presence of antibodies to placental-type P. falciparum isolates that adhere to CSA. The isolates used were CS2 and HCS3, which have been extensively studied with regard to their adhesive and antigenic properties and PfEMP1/var expression.8,15,18,25,37 Both isolates express var2csa and are recognized by acquired antibodies in a sex- and parity-associated manner typical of placental isolates.8,18

Testing Kenyan men’s samples (N = 40) at 1/20 dilution identified several with reactivity against CS2 and HCS3 IEs. In comparison to control samples, 13.6% were positive with CS2 and 34.4% were positive with HCS3 IEs, but all were at low levels (lower than IgG binding of the same samples to non-CSA-binding isolate A4; data not shown). IgG to CSA-binding IEs was therefore further evaluated by retesting 22 samples at 1/5 dilution using CS2 IEs (Figure 1A); equal numbers of high and low responders were selected. Two of 22 samples were clearly positive, and two were borderline positives. Positive samples came from younger donors. As a group, the mean level of IgG binding of men’s sera to CSA-binding IEs was significantly lower than the mean binding of sera from multigravid women in the same population (P < 0.01; Mann-Whitney U-test), and the level of IgG binding of individual positive samples was lower than the median level of IgG binding by samples from multigravidas.

Among serum from Kenyan children, 13 of 103 (12.6%) samples were positive to CS2 tested at 1/20 dilution. However, no samples were strongly positive for IgG binding compared with samples from pregnant women. Reactivity against HCS3 IEs was generally low (tested at 1/20 dilution), and samples from children were not examined further with this isolate. Selections of positive (N = 10) and negative (N = 10) samples were retested against CS2 at 1/5 dilution to confirm the presence of specific IgG to CSA-binding IEs. Ten samples were positive for IgG; seven samples were clearly positive compared with samples from non-exposed donors; and five were near the cut-off level for classification as positive (Figure 1B).

Several samples from PNG men, tested at 1/20, were also clearly positive for antibodies to CS2 and HCS3 (Figure 2, A and B, respectively). The relative level of IgG binding of individual positive men’s samples was similar to that observed for many multigravid women. The median value for IgG binding for multigravid samples was 7.1 (range 2–36) using isolate CS2, and the values for the most highly reactive men’s samples were 6.0 for CS2 (Figure 2A) and 8.0 for HCS3 (Figure 2B). However, as a group, the median level of IgG binding to CS2 by samples from men was lower than median level of IgG binding for samples from multigravidas from the same population (P < 0.01, Mann-Whitney U-test; data not shown). IgG binding of the same samples to CS2 and HCS3 was not significantly correlated, and IgG binding was greater to HCS3 IEs (P < 0.001; Mann-Whitney U-test). Some sera were clearly positive to CS2 or HCS3 IEs, rather than positive to both, or differed substantially in the level of binding to the two isolates, reflecting the specificity of IgG binding measured. Analysis of flow cytometry data showed that among positive samples the majority of IEs were labeled (data not shown). There was no association between concurrent infection and antibody levels; there was a trend toward an inverse correlation between age and antibody binding (P = 0.086 for HCS3; P = 0.185 for CS2). All men’s samples were positive against recombinant 3D7 AMA1, by ELISA (data not shown), indicating that all donors had substantial malaria exposure. Some samples from Malawian men also tested positive for IgG to CS2 and HCS3 measured by flow cytometry (data not shown). A selection of sera from Malawian men was available for assays of inhibition of parasite adhesion to CSA (Figure 3A). Four of 23 (17.4%) sera tested inhibited adhesion of CS2 IEs to CSA by > 50% (relative to non-exposed serum controls), and six (26.1%) were inhibited by > 25%, suggesting that acquired antibodies against CSA-binding IEs present among men are biologically relevant; by comparison, the median level of inhibition by 24 samples from multigravid women in the same population was 77.4%. In agglutination assays,15 2 of 37 sera and 1 of 16 men’s samples were positive against CS2 and HCS3, respectively. The sample positive against HCS3 did not agglutinate CS2.

Evaluating antibody specificity.

Several approaches were used to evaluate the specificity and relevance of antibodies measured. To confirm the specificity of the IgG binding observed, we tested samples from PNG men against isolate E8B-ICAM1, which adheres to CD36 and ICAM1 and does not express var2csa8,25; results strongly suggest that antibodies were variant-specific. Several men were positive for IgG binding to E8B-ICAM1, including samples that lacked antibodies to CS2 or HCS3 (Figure 2B and data not shown). Some samples were strongly positive for antibodies to E8B-ICAM1 but weakly reactive or negative with CS2 or HCS3 IEs, or vice-versa. Furthermore, among PNG men’s samples, the level of relative IgG binding considered positive against CS2 and HCS3 was similar to the level seen for samples positive to E8B-ICAM1 (Figure 2B). Overall IgG binding to E8B-ICAM1 was higher than to CS2 (P < 0.001; Mann-Whitney U-test), but not to HCS3, among PNG men’s samples. Similarly, among Kenyan samples, IgG reactivity to isolate A4, which adheres to CD36 and ICAM-1, did not relate to reactivity against CS2 or HCS3 IEs (data not shown), confirming the specificity of antibodies measured. Antibody reactivity to IEs was several-fold higher than that for uninfected erythrocytes.

Antibody measurements against CS2 and HCS3 were not explained by antibodies to a minor non-CSA-binding subpopulation of IEs. Phenotypes of CS2 and HCS3 were confirmed during the present studies, and isolates were reselected for adhesion to CSA before being used in antibody assays. Examination of plots of flow cytometry data demonstrated that, among positive samples for both men and children, the majority of IEs were labeled and the increase in fluorescence represented labeling of the whole cell population rather just a subset of cells (an example is shown in Figure 3B). P. falciparum isolate CS2 in particular has a very stable phenotype that shows little or no change in phenotype over several months in continuous culture (J.G. Beeson, unpublished observations).

DISCUSSION

Our data demonstrate the acquisition of antibodies to placental-type CSA-binding P. falciparum IEs outside pregnancy. This observation was confirmed in three different malaria-exposed populations. Consistent with this finding, prior studies have found CSA-binding IEs among children and nonpregnant adults with acute illness.5,2931 Of further relevance, increased levels of expression of var2csa have been reported among infections in some children and nonpregnant adults.39,40 Our results suggest that this level of exposure is sufficient to induce antibodies outside of pregnancy. However, the prevalence and levels of antibodies to these isolates in men and children was low overall, compared with pregnant women, reflecting reduced exposure.

Antibodies to VSA appear to wane quickly following resolution of infection,41 which may also explain why antibodies to CSA-binding parasites were typically at low levels or were undetectable. Uncommon phenotypes would give intermittent, occasional exposure, and the likelihood of detecting antibodies would be low. It is possible that other individuals tested here had a capacity to respond to VSA of CSA-binding placental-type variants, or have had significant exposure, but antibodies were below the limit of detection in the assays at the time of sample collection. It remains possible that CSA-binding isolates in nonpregnant hosts are not the same as placental CSA-binding variants but express epitopes shared with var2csa and induce cross-reactive antibodies. However, available data suggests that var2csa is the major, or only, gene expressed that encodes CSA-binding domains.26,42 PfEMP1, encoded by var2csa, is likely to be the major target of antibodies against the surface of CSA-binding isolates used here, but there may be other important targets. Presently, var2csa-PfEMP1 is the only protein identified that has confirmed expression on the surface of CSA-binding IEs and shows antibody reactivity that appears to be largely gender and parity dependent,24 similar to antibody reactivity against the intact CSA-binding or placental IEs. Substantial data points to PfEMP1 as the primary target of acquired antibodies to the surface of IEs.22,23,43 Other surface antigens include rifin44,45 and surfin46 proteins, but their relevance to placental malaria has not been addressed. Further studies are needed to define the prime targets of acquired antibodies and quantify their contribution to total surface reactivity. Tools to measure antigen-specific responses would be invaluable for further investigation of antibody responses to VSA. Results from recent studies using parasite lines with suppressed var gene expression generated by transfection suggest that IgG binding to PfEMP1 accounts for at least 65% of the total reactivity to the surface of IEs.47 Application of these approaches to studies with CSA and placental-binding parasites may allow a clearer determination of the importance of var2csa-PfEMP1 as a target of acquired antibodies.

Several observations confirmed the specificity of antibodies detected in our assays. Antibodies among men and children did not represent IgG binding to subpopulations of non-CSA-binding IEs. CS2 and HCS3 were generated by repeated selection and are highly homogeneous isolates, showing very little or no adhesion to other known receptors, and express a single dominant PfEMP1 type18 and var2csa as the single dominant var transcript.25,37 Using CSA-coated beads to quantify the proportion of IEs that can adhere, we found that > 95% of CS2 IEs used in our antibody assays bind to CSA (J.G. Beeson, unpublished observations). Inhibition of adhesion to CSA by some samples further confirmed the presence of antibodies to CSA-binding IEs among men and suggested they are biologically relevant. Among PNG men, the level of IgG binding to CSA-binding IEs was similar to the level observed for samples that were positive against E8B-ICAM-1, further suggesting IgG levels were biologically significant. Interestingly, antibodies appeared more prevalent to CS2 than HCS3 in Kenya, whereas the opposite was observed in PNG. Reactivity of sera against CSA-binding IEs was specific and could not be simply explained by nonspecific broadly reactive antibodies to the IE surface; IgG binding did not correlate with antibodies to non-CSA-binding isolates. Reactivity of some samples was specific for CS2 compared with HCS3, or vice versa. We have recently examined the isolate-specificity of antibody reactivity against CSA-binding and placental isolates and established that CS2 and HCS3 IEs express distinct antigenic determinants and acquired antibodies among pregnant women largely target polymorphic epitopes.18 Recent studies have demonstrated a high degree of sequence diversity in var2csa between different isolates.48 The isolate-specific nature of antibodies to CS2 and HCS3 may be explained by polymorphism in var2csa.18 However, it remains to be established that var2csa is the major target of antibodies to the surface of CSA-binding IEs, and it is possible that there are other differences between the isolates in the expression of surface antigens. A lack of antibodies to CS2 or HCS3 IEs did not reflect a lack of exposure to malaria, as negative sera were positive in other malaria-specific assays.

Our findings are consistent with reports that antibody levels and antibody prevalence against placental-binding isolates expressing var2csa is lower overall among men than multi-gravidae. However, antibodies to these variants are acquired, to some extent, outside pregnancy. Prior studies have not reported a detailed analysis of antibodies among men or examined antibodies among children. Our findings are consistent with the hypothesis that the occurrence in natural infections of minor subpopulations of IEs that express var2csa allows this placental-binding variant to be expanded in the pregnant host as the placental vasculature develops. This infrequent, low-level exposure would lead to a low prevalence of antibodies. Parasitemic thresholds for induction of antibodies may not be reached in all instances but are clearly reached in some individuals. It is unclear why isolates expressing var2csa are not more common in nonpregnant individuals. It is possible that these variants do not sequester efficiently due to limited receptor availability in vascular beds and therefore do not become the predominant phenotype.10 Further studies among children that examine adhesion to CSA and expression of var2csa by isolates and the nature and specificity of antibodies induced would help address these issues.

The finding that some children and men have acquired antibodies to these variants suggests that a proportion of women will have acquired some antibody response to these P. falciparum variants at first pregnancy. This may influence susceptibility to malaria or the course and/or complications of infection; antibody levels may reach functional thresholds more rapidly upon re-exposure and boosting during pregnancy. These may be important considerations in longitudinal studies of immunity to maternal malaria and evaluating immune responses to malaria during pregnancy and in studies on relevant B-cell memory responses. Although antibodies to CSA-binding IEs were detected among men and children, the levels and/or prevalence of antibodies are insufficient to protect most pregnancies because pregnant women, as a group, have a higher risk of malaria than corresponding nonpregnant adults. Further studies that examine antibodies among children, men, and women prior to first pregnancy against placental isolates would be informative. The suggestion from our data that CSA or placental-binding variants expressing var2csa or genes with similar epitopes are not restricted to pregnancy may also have broader implications for understanding the pathogenesis and biology of P. falciparum malaria. The occurrence of CSA-binding variants in nonpregnant individuals may contribute to the maintenance of var2csa in the genome, and antibodies in nonpregnant individuals may also contribute to immune pressure promoting polymorphisms in var2csa. It is possible that a vaccine against CSA-binding IEs, proposed for maternal malaria, may also have some protective benefit outside pregnancy.

Figure 1.
Figure 1.

Antibodies among sera from Kenyan men and children to surface antigens expressed by CSA-binding infected erythrocytes determined by flow cytometry. (A) Binding of IgG to the surface of CS2-infected erythrocytes among a selection of sera from men and non-exposed controls; the mean of 10 (error bar shows 3 standard deviations) non-exposed controls is shown. Samples 442 and 392 were regarded as positive for specific IgG binding; samples 274 and 94 were around the threshold for classification as positive. (B) Binding of IgG to the surface of CS2-infected erythrocytes among sera from children and non-exposed controls; samples 86, 83, 9, 68, 4, 55, 30, 19, 61, and 103 were regarded as positive for specific IgG binding. All values represent mean (+ range) for duplicate assays. Sera were tested at 1/5 dilution. The cut-off level for classification as positive is represented by the broken horizontal line (mean + 3 standard deviations of non-exposed samples). IgG binding was expressed as the ratio of the mean fluorescence intensity for IEs divided by that for RBCs, to account for variation in background binding of IgG to uninfected RBCs. A relative score of 1 indicates no significant IgG binding. Samples were tested in Kilifi, Kenya, using a Coulter EpicXL flow cytometer.

Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.22

Figure 2.
Figure 2.

Antibodies among sera from Papua New Guinea men and non-exposed Australian residents to surface antigens expressed by P. falciparum-infected erythrocytes determined by flow cytometry. (A) Binding of IgG among men’s samples to the surface of infected erythrocytes using the CSA-binding isolate CS2. Values represent mean for duplicate assays (error bars show range). Controls (gray bar) represent the mean (error bar shows 3 standard deviations) of sera from non-exposed donors. Multigravidas (striped bar) represent the median level (error bar shows 95% CI) of IgG binding for 38 samples tested from multigravid women from the same PNG population. The cut-off level for classification as positive is represented by the broken horizontal line (mean + 3 standard deviations of non-exposed samples). (B) Binding of IgG to the surface of infected erythrocytes using the CSA-binding isolate HCS3 compared with E8B-ICAM1, which adheres to CD36 and ICAM-1. Values represent mean for duplicate assays (error bars show range). Controls represent the mean (+ 3 standard deviations) of sera from non-exposed donors. The cut-off level for classification as positive for HCS3 is represented by the broken horizontal line. For A and B, sera were tested at 1/20 dilution, and IgG binding is expressed as the ratio of the mean fluorescence intensity for IEs divided by that for RBCs, to account for variation in background binding of IgG to uninfected RBCs. A relative score of 1 indicates no significant IgG binding. Samples were tested in Melbourne, Australia, using a Becton-Dickinson FACSCali-bur flow cytometer.

Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.22

Figure 3.
Figure 3.

(A) Inhibition of adhesion of CS2 infected erythrocytes to CSA by sera from Malawian men. Values represent mean + range expressed relative to controls (sera from non-exposed donors). The median (+ 95% CI) level of inhibition by sera from 24 multi-gravid women is show for comparison (striped bar). Flow cytometry analysis of IgG binding to the surface of intact CS2 infected erythrocytes: (B) sera from a PNG adult male (gray line, unfilled plot) compared with serum from a non-exposed adult donor (black line, filled plot). Data plotted using FlowJo software.

Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.22

*

Address correspondence to James G. Beeson, Institute of Medical Research, Victoria 3050, Australia. E-mail: beeson@wehi.edu.au

Authors’ addresses: James Beeson, Kristina Persson, and Joanne Chesson, Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia, Telephone: +61-3-9345-2555, Fax: +61-3-9347-0852, E-mail: beeson@wehi.edu.au. Francis Ndungu, Sophie Uyoga, Thomas Williams, and Kevin Marsh, Centre for Geographic Medicine Research, Coast, Kenya Medical Research Institute, Kilifi, Kenya, Telephone: +254-0415-25043, Fax: +254-0415-22390. Greg Kelly, Sandra Hallamore, and Graham Brown, Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Victoria 3050, Australia, Telephone: +61-3-8344-5478, Fax: +61-3-9347-1863. John Reeder, Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea, Telephone: +675-852-2909, Fax: +675-852-3289.

Acknowledgments: We thank Brett Lowe and Moses Mosobo for help with coordinating laboratory studies in Kenya; Andrew Raiko and Alfred Cortes for assistance with sample collection in PNG; Terri Taylor and Malcolm Molyneux for providing samples from Malawian men; and Stephen Rogerson for helpful discussions. We are indebted to all individuals who participated in the studies at each site. Human erythrocytes and sera for in vitro culture were provided by the Australian Red Cross Blood Service, Melbourne, Australia. This paper is published with the permission of the Director of KEMRI.

Financial support: Funding was provided by the National Health and Medical Research Council of Australia (Neil Hamilton Fairley Fellowship and Career Development Award to J.G.B.; program grant to G.V.B.); The Wellcome Trust, U.K. (program grant to K.M.; fellowship to T.N.W.); Miller Fellowship of the Walter and Eliza Hall Institute (J.G.B.); Teggerstiftelsen, Maud and Birger Gustavssons Stiftelse, and a Wenner-Gren Fellowship, Sweden (to K.E.M.P.); Greensborough and Watsonia sub-branches of the Returned Services League, Australia, and Australian Nursing Solutions (G.L.K.).

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Author Notes

Reprint requests: James Beeson, Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia, Telephone: +61-3-9345-2555, Fax: +61-3-9347-0852, E-mail: beeson@wehi.edu.au.
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