点击显示 收起
Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Unité d'Immunologie Structurale, Centre National de la Recherche ScientifiqueUnité de Recherche Associée , Département de Biologie Structurale and Chimie, Institut Pasteur
Institut de Recherche pour le Développement, UR, Laboratoire de Parasitologie, Faculté des Sciences, Pharmaceutiques et Biologiques, Paris, France
A recombinant Duffy bindinglike (DBL) domain from a previously identified placental isolate, 732, was expressed by use of the baculovirus/insect cell system and was purified in milligram quantities. The recombinant protein binds specifically to chondroitin sulfate A (CSA) and inhibits CSA binding by placental infected erythrocytes (IEs). Polyclonal antibodies raised against the domain recognized the surfaces of live IEs from CSA-adherent clinical placental isolates. These antibodies also abrogated the in vitro binding of IEs to CSA. The 732 DBL-3 domain was specifically recognized by plasma from pregnant women but not by plasma from control subjects. In addition, the protein was, comparatively, significantly more reactive with plasma from women with infected placentas, strongly suggesting that the 732 DBL-3 domain carries preferentially IE-expressed immunogenic epitopes. High levels of plasma antibodies to the recombinant domain were associated with reduced placental parasite density. This is the first report of a recombinant DBL- domain derived from a placental isolate that shows CSA-binding properties.
The expression of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) on the surfaces of infected erythrocytes (IEs) is associated with distinct adhesive properties [1, 2]. The protein, which is encoded by var genes, binds cellular receptors such as CD36 [3], intercellular adhesion molecule1 (ICAM-1) [4], and chondroitin sulfate A (CSA) [5]. PfEMP-1 variants with the CSA-binding phenotype appear to be constantly expressed by parasites infecting pregnant women but are less commonly expressed by parasites infecting children and nonpregnant adults. The inability of the mother to control parasites in the placenta probably contributes to the pathogenesis of pregnancy-associated malaria (PAM) [6].
Several var genes have been shown to fulfil at least 1 of the criteria necessary to qualify as a PAM-causing gene, such as up-regulation in CSA-binding parasites, more frequent transcription in placental parasites than in parasites from children, surface expression, CSA binding, induction of antibodies capable of blocking CSA binding, sex-specific and parity-dependent recognition, and domain/epitope conservation in field isolates [7]. The first domain implicated in PAM was a Duffy bindinglike (DBL) domain from the var1CSA gene of the FCR3CSA laboratory strain [8] and the CS2var gene of the ItGCS2 laboratory strain [9]. These induce blocking antibodies but are not expressed in placental isolates. The leading candidate gene, var2CSA, is up-regulated in CSA-binding parasites, but it lacks DBL- domains, and surface-expressed protein remains to be demonstrated [10].
Five distinct DBL- domains that share 39%44% amino acid sequence identity, collectively termed "varPAM DBL- domain types," were previously characterized from placental isolates and were shown to bind to CSA when expressed on the surfaces of COS cells [11, 12]. These domains exhibit 44%50% and 37%44% identity with the DBL- domains from var1CSA and CS2var, respectively. The 720/734varPAM gene is very similar to the var1-like 3D7chr5var gene, which is commonly found in placental isolates [13]. The amino acid sequences in their DBL- domains are 82% identical, whereas the other varPAM DBL- domains, compared with that of 3D7chr5var, show only 35%47% identity. On the basis of the overall lower homology and its distinct domain organization, 732var was chosen for further study. Insect cellproduced 732 DBL-3 domain was analyzed functionally and immunologically, as a first step toward understanding the structural requirements for CSA interaction and to investigate the contribution of these genotypes to the pathogenesis of PAM.
PARTICIPANTS, MATERIALS, AND METHODS
Cell lines.
Insect cells (Spodoptera frugiperda 9 [Sf9]) were maintained at 27°C in TC-100 medium (Invitrogen) containing 50 g/mL gentamicine (Invitrogen), 2 g/mL glucose, yeastolate (Invitrogen), and 5% fetal calf serum (FCS; Eurobio). High Five (HF) cells (Invitrogen) were grown in Insect XPress medium (Cambrex), also at 27°C. CHO cells were grown in Ham's medium (PAA) supplemented with 10% FCS (PAA), 2 mmol/L glutamine (PAA), and 100× penicillin-streptamycin (PAA). Wild-type CHO-K1 cells express glycosaminoglycans (GAGs) on the cell surface; CHO pgsA-745 cells are defective with respect to xylosyltransferase and do not express GAGs. CHO CD36 and CHO ICAM-1 are stable transfectants thereof, expressing the corresponding receptors on a GAG-free background (CHO transfectants were gifts from T. Staalsoe, Copenhagen University Hospital).
Construction, expression, and purification of the recombinant 732 DBL-3 domain.
The 732 DBL-3 domain sequence (GenBank accession no. AF334807) was cloned from a Cameroonian placental isolate [12], and full-length 732var (GenBank accession no. AY679117) was obtained by polymerase chain reaction (PCR) with unique and degenerate primers [14]. Arginine residues at potential N-glycosylation sites (N156 and N214) were mutated to glutamine by site-directed mutagenesis (Quikchange; Stratagene). The mutated gene fused to the signal sequence plus the 17 N-terminal residues of merozoite surface protein (MSP) 1 from P. vivax, and a C-terminal hexahistidine tag (gift from S. Longacre; Institut Pasteur) was ligated into the pBlueBac4.5 Topo plasmid (Invitrogen). The resultant construct was cotransfected with Bac-N-Blue DNA (Invitrogen) into Sf9 cells, and the recombinant viral clones were confirmed by PCR.
HF cells were used for protein expression. The XPress medium, into which the recombinant protein was secreted, was harvested and dialyzed 3 times (814 h/step) against 20 mmol/L Tris (pH 7.5) and 500 mmol/L NaCl. Purification steps were performed at 4°C as rapidly as possible, to minimize protein degradation. After incubation with Talon gel (Clontech; Becton Dickinson), the protein was eluted with an imidazole gradient (0200 mmol/L), and the fractions were pooled and concentrated in a YM-10 Centricon column (Amicon; Millipore) and were further purified by gel filtration chromatography (16/60 Superdex 200; Amersham Pharmacia). The recombinant product was verified by N-terminal sequencing.
Antibody production.
One hundred micrograms of 732 DBL-3 domain in 300 L of phosphate buffer suspended with Freund's complete adjuvant (Sigma) was used for immunization. Chicken antibody was used to overcome nonspecific binding of mouse or rat antibodies to placental isolates. IgY, the functional equivalent of mammalian IgG, was purified from egg yolk by polyethylene glycol precipitation [15, 16]. Antibody specificity was tested by immunoblot with goat antichicken IgY (diluted 1 : 15,000; Sigma).
Affinity of the recombinant 732 DBL-3 domain for CSA on CHO cells.
CHO cells were seeded at a concentration of 3 × 105 cells/mL on coverslips, were fixed with 2% paraformaldehyde in PBS for 30 min at room temperature (RT), and were washed and blocked with 3% bovine serum albumin (BSA) in PBS (BSA-PBS) for 30 min at RT. Cells were incubated with 0.5 mol/L of recombinant protein (732 DBL-3 or rifin50) in BSA-PBS for 40 min at RT. Protein binding was detected by use of primary antibody (1 : 500) and then Alexa-Fluor 594conjugated secondary antibody (1 : 1000; Molecular Probes), each incubated for 30 min at 4°C. CSA, CSB, and CSC (100 g/mL each) were used to demonstrate specificity. Cell nuclei were visualized by use of 5 g/mL 4,6-diamidino-2-phenylindole hydrochloride (DAPI; Roth). The cells were mounted in MOWIOL 4-88 (Calbiochem) and were observed under 20× magnification (Leitz DMRB; Leica).
Parasite collection and cultivation.
Experiments were conducted after informed consent was received from participants, in accordance with the human experimentation guidelines of the institutions involved; the study was approved by the ethics committees of the institutions. Placental parasites of Gabonese origin were extracted from blood collected into EDTA monovettes without the use of needles from incisions made in the placenta (maternal side) [17]. Parasites of Senegalese origin were collected by flushing the placentas of O blood group women with 0.1% sodium heparin in PBS [18]. Placental thick-blood smears were analyzed microscopically. Parasites were used directly in flow cytometry or were frozen after 2 cycles for further culture. P. falciparum isolates were grown at 37°C at 5% hematocrit in RPMI 1640 medium containing 200 mmol/L glutamine (PAA), 10% human serum (AB+; PAA), and 5% Albumax II (Gibco). The CSA-adherent population of the isolates was enriched by use of CSA immobilized on plastic petri dishes, as described elsewhere [5]. Midtrophozoite stage cultures of Gb337, VIP43, Gb03, and FCR3 were harvested (parasitemia of >2%) and were enriched in MACS columns (Miltenyi Biotec) for further downstream applications.
Static in vitro inhibition assay.
CSA was immobilized on plates, and IE binding was inhibited [19]. Inhibitory activities of polyclonal antibody or human plasma (diluted at concentrations of 1 : 25, 1 : 50, 1 : 100, and 1 : 200) were determined by preincubating IEs (5% hematocrit; 2% parasitemia) for 1 h at 37°C with antibody. BSA (5%) and CSC (100 g/mL each in PBS) served as controls for CSA specificity. To investigate the effect of the recombinant protein, the above assay was modified. IEs (3 × 107) were pipetted into a 1-cm circle that was drawn by use of a DakoCytomation pen (Dako Denmark) and that contained 100 L of 732 DBL-3 domain (120 mol/L). The parasite-protein mix was left for 1 h at RT. Rifin50 protein, which has no known role in CSA binding, was the control for protein specificity. CSA binding is expressed as percentages, comparing the number of bound IEs per millimeter in the test sample (with protein or antibody) with the number of bound IEs per millimeter in the reference sample (with neither protein nor antibody).
Immunofluorescence assay.
The CSA-enriched and the isogenic nonCSA-enriched strains were tested in parallel. Nuclear DNA staining was visualized by incubation with DAPI (4 g/mL) for 30 min at RT. IEs were stained with anti732 DBL-3 domain antibodies (1 : 100) and were counterstained with rabbit antichicken IgY conjugated to Alexa-Fluor 488 (6 g/mL; Molecular Probes); each step was for 30 min at 4°C. In control experiments, IEs were treated with 0.1 mg/mL trypsin at 37°C for 10 min before staining. Cells were resuspended with 10 L of 2% FCS-PBS and were counted under 40× magnification (Axioskop 2 plus; Zeiss).
Plasma sample collection and patient data.
The pregnant-women cohort study was conducted in Thiadiaye, Senegal, where malaria is seasonally transmitted (15 infectious mosquito bites/person/year) during the rainy season, from September 2001 to May 2002. The cohort comprised 257 women, whose mean ± SD age was 26.1 ± 6.5 years. In the cohort were 60 primiparous, 49 secundiparous, and 148 multiparous women; 66 and 41 women presented with P. falciparum parasites in the placental blood and the peripheral blood, respectively, when examined microscopically. Samples were collected at delivery, were coded, and were tested in a blinded fashion. Control plasma samples were collected from 17 Senegalese adults living in the same area as the pregnant women (9 men and 8 nulligravid women) and from 55 nonexposed European adults.
ELISAs.
ELISAs were used to analyze human responses to the purified recombinant 732 DBL-3 domain [20]. Plates were coated with 2.5 g/mL concentrations of antigen. Wells were incubated with 100 L of human plasma (1 : 200), followed by horseradish peroxidaseconjugated antihuman IgG (1 : 15,000). The optical density (OD) was obtained by subtracting the average OD of duplicate wells from that of the corresponding blank wells. Values were converted into arbitrary units (AUs), as follows [21]:
Participants with an antibody response were defined as those with an AU value >2 SDs above the mean AU value of the 55 negative control plasma samples (i.e., those from the nonexposed European adults).
Flow cytometry.
Antibodies (IgG) to variant surface antigens (VSAs) were measured by flow cytometry [22] with 7 isolates. IEs from the placentas of blood type O women (105 IEs/L; 40% parasitemia; 2.5% hematocrit) were labeled with ethidium bromide (0.1 mg/mL) in the presence of test plasma samples (1 : 20) in 96-well round-bottom plates. Cells were sequentially incubated with goat antihuman IgG (1 : 250; Dako) and fluorescein isothiocyanateconjugated rabbit antigoat IgG (1 : 100; Dako) and were analyzed by use of a FACScalibur flow cytometer (Becton Dickinson). For each sample, the level of IgG that recognized VSAs was expressed as the median fluorescence intensity (MFI) of the labeled IE, gated according to ethidium bromide fluorescence. For each isolate, the threshold for positivity was defined as 2 SDs above the mean MFI obtained with plasma samples from 30 nulligravid women from Senegal.
Statistical analysis.
Differences between groups were tested by Student's unpaired t test. Correlations were examined by the Pearson test. The relationship between placental parasite density (after log transformation) and antibody levels was assessed by a multiple linear regression model that included parity. The significance limit was P < .05. Statistical analysis was performed by use of Statview (version 5.0; SAS Institute).
RESULTS
Comparison of the 732var gene with known 3D7var genes.
The domain organization of the 732var gene (figure 1A) is close to that of the type 15 var gene [23], differing only by an additional DBL-4 domain before the transmembrane region of 732var. The 732 DBL-3 gene shares 41%51% amino acid sequence identity with annotated 3D7 DBL- domains (PlasmoDB). This is comparable to the observed identity between the 5 varPAM genes we have analyzed (39%55%) [11, 12], which were cloned with degenerate UNIEBP5 and UNIEBP3 primers [24]. These partial varPAM DBL- genes covered the domain sufficiently to retain CSA-binding properties when expressed on the surfaces of CHO cells, showing that the binding site is contained, at least in significant part, within this region [12].
The purified protein was observed in a denaturing gel as 1 major band and 2 very weak bands in the presence of reducing agent; however, only 1 band was observed under nonreducing conditions (figure 2A). The weak bands indicate that the protein was slightly cleaved but remained covalently linked by cysteine bridges in the nonreduced form. Yields of up to 7 mg/L of culture medium were obtained.
Affinity of the recombinant 732 DBL-3 domain for CSA.
The conformational integrity of the recombinant 732 DBL- domain was examined by assessment of its ability to bind to CHO-K1 cells expressing CSA. The protein adhered to CHO-K1 cells (figure 3, top row); by contrast, it showed virtually no binding either to CHO pgsA-745 cells (second row), to CHO cells expressing CD36 (third row), or to CHO cells expressing ICAM-1 (fourth row). Protein-CSA interaction was competed out by CSA but not by CSB or CSC (data not shown). When an unrelated soluble control protein (rifin50) was used, no binding was observed (data not shown). Therefore, the recombinant 732 DBL-3 domain interacts specifically with CSA.
Recognition of the recombinant 732 DBL-3 domain by human immune plasma.
In immunoblots, the recombinant 732 DBL-3 domain was recognized by a pool of plasma obtained from pregnant women and was not recognized by a pool of plasma obtained from men (figure 2B). This seropositivity was further characterized by ELISA, to evaluate antibody responses during natural malaria.
Plasma samples from the pregnant women showed sex-specific recognition, with a mean level that was significantly higher than those for plasma samples from the control groups of nonexposed European adults, malaria-exposed nulligravid women, and malaria-exposed men (P < .0001) (figure 6). To examine whether antibody recognition was placental-infection specific, the pregnant women were categorized by whether their placentas were infected (n = 66) or not infected (n = 178) at delivery. Levels of antibodies to the recombinant protein in the plasma samples from the women with infected placentas were higher than those in the plasma samples from the women with noninfected placentas (table 1). This recognition appears to be specific to placental infections only, because no such difference was observed when the women were categorized by whether their peripheral blood was infected.
Control of parasite density in the placenta.
The most significant finding of the present study is that the levels of antibodies to the recombinant 732 DBL-3 domain correlated negatively with parasite density in the placenta (r = -0.320; P = .04) (figure 7B). This correlation was not present when parasites in the peripheral blood were analyzed. To investigate the combined influence of placental parasite density and parity on antibody levels, these 2 variables were included in a multiple linear regression model. Parity was included because, although other studies have shown a strong association between this parameter and PAM, our study indicated only a borderline correlation with antibody levels. This analysis confirmed that only placental parasite density was correlated with the levels of antibodies to the recombinant protein (P = .04).
DISCUSSION
The first understanding of PAM came from the observation that placental isolates bound to CSA [5] and that the parasite ligand implicated in CSA interaction belonged to the DBL- domain subtype [8, 9]. Further work on DBL- domains from placental isolates supported this observation [12]. Interestingly, the sequences for these domains were also conserved in placental parasites that had been isolated several years apart and that were from different geographic locations [11].
The cloned and purified DBL-3 domain from the 732var gene has been shown here to be correctly folded and functional, on the basis of its ability to bind to CSA expressed on CHO cells. Antibodies raised against the recombinant protein stained the surfaces of placental isolates and laboratory strains that had been selected for their CSA-binding properties. Therefore, CSA-binding parasites probably display surface antigens that share common conserved epitopes with the 732 DBL-3 domain. Furthermore, not only was the recombinant protein able to compete with IEs for CSA binding, but 732 DBL-3 domainspecific antibodies were also capable of inhibiting the binding of IEs to CSA. Thus, we conclude that the recombinant protein expressed in insect cells resembles the domain expressed on the surfaces of IEs and possesses a functionally active CSA-binding site.
By flow cytometry, antibodies in plasma from pregnant women recognized CSA-adherent IEs in a sex- and parity-dependent manner. These responses were measured with human-plasma antibodies that recognized the entire repertoire of VSAs present on IEs. Recognition correlated with protection of mothers against PAM [6] as well as other against clinical consequences of PAM, such as maternal anemia, low-birth-weight babies, and prematurity [2628]. To obtain further insight into VSA reactivities, the recognition of individual domains of PfEMP-1 has been investigated. Domains such as DBL-1, cysteine-rich interdomain region (CIDR), and DBL-2 (cloned from the CSA-selected parasite line 2O2CSA) do not show sex- or parity-specific antibody recognition, which is in line with observations that do not implicate these domains in PAM [29].
Our study extends investigations that were based on a single PfEMP-1 domain and show, for the first time, a link with immune control of placental parasitemia. We have demonstrated that antibodies in human plasma that react with the 732 DBL-3 domain are correlated with decreased parasite density in malarial parasiteinfected placentas; moreover, the protein domain exhibits the characteristics of a pregnancy-specific antigen, being specifically recognized by plasma from pregnant women and, in particular, by plasma from women with infected placentas. The levels of antibodies recognizing the antigen by ELISA correlated with the levels of antibodies reacting with VSAs expressed by 7 different fresh placental isolates. On the basis of these observations, we suggest that antibodies in human plasma that recognize the 732 DBL-3 domain form a subset of the antibodies against IE that could affect placental parasite replication. Involvement of these antibodies in the control of placental parasitemia raises hope for a DBL- domainbased vaccine against PAM.
To date, all var genes that have been implicated in PAM belong to different var gene types with different domain orders. The recombinantly expressed DBL-5 domain of the unique var2CSA gene was recently found to be recognized by human plasma in a sex- and parity-dependent manner, and the level of antibodies to the DBL-5 domain was reported to correlate with birth weight [30]. An as-yet unidentified 13.214.0-kb transcript [31, 32] and 4 novel variants identified by proteomics analysis [33] have also been published.
CSA-binding function has now been attributed to several DBL domains, such as DBL-, -, and -X, as well as to CIDR-. Gamain et al. have shown that a 67-aa segment located in the C-terminal region of the FCR3CSA DBL-3 domain binds to CSA; accordingly, they have suggested that it contains the minimal CSA-binding motif [34]. This region has significant sequence identity with the equivalent segments of the IT-R29var and 3D7chr5var DBL- domains, which also bind to CSA. Furthermore, the var2CSA DBL-2X and -6 domains also bind to CSA, and regions on these domains show some homology to the segment [35]. However, this minimal binding motif is not found in the DBL-5 domain, which is associated with sex- and parity-specific recognition [30]. The significance of this stretch of amino acids remains unclear, because such stretches are also present on DBL- domains that do not bind to CSA [34]. The 5 varPAM DBL- domains from placental isolates that we had examined were truncated and lacked the apparent CSA-binding motif but were, nonetheless, capable of binding to CSA. These results would indicate the presence of additional CSA-binding sites within the central two-thirds of the DBL- domain. The 732 DBL-3 domain is the only varPAM CSA-binding domain that we have completely sequenced to date; however, sequence identity with the 67-aa CSA-binding segment from FCR3CSA DBL-3 is only 44%, which is not significantly different from the 42% identity for the whole of the domain. The structural correlate of CSA binding may, therefore, not be unique, and there is a clear need to define the structural characteristics of varPAM DBL- domains and their interaction with CSA ligands. This will provide better insight into their role in PAM and their capacity to induce a protective immune response.
Acknowledgments
We thank all of the individuals who participated in this study; Allioune Gaye (Centre de Santé Roi Baudouin, Guediawaye) and Sokhna Sow (Hpital de Thiadiaye), for their support in sample collection; and Jean Yves Le Hesran (Institut de Recherche pour le Développement), for the follow-up of pregnant women.
References
1. Chen Q, Fernandez V, Sundstrom A, et al. Developmental selection of var gene expression in Plasmodium falciparum. Nature 1998; 394:3925. First citation in article
2. Scherf A, Hernandez-Rivas R, Buffet P, et al. Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J 1998; 17:541826. First citation in article
3. Ockenhouse CF, Tandon NN, Magowan C, Jamieson GA, Chulay JD. Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor. Science 1989; 243:146971. First citation in article
4. Berendt AR, Simmons DL, Tansey J, Newbold CI, Marsh K. Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature 1989; 341:579. First citation in article
5. Rogerson SJ, Chaiyaroj SC, Ng K, Reeder JC, Brown GV. Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J Exp Med 1995; 182:1520. First citation in article
6. Fried M, Nosten F, Brockman A, Brabin BJ, Duffy PE. Maternal antibodies block malaria. Nature 1998; 395:8512. First citation in article
7. Rowe JA, Kyes SA. The role of Plasmodium falciparum var genes in malaria in pregnancy. Mol Microbiol 2004; 53:10119. First citation in article
8. Buffet PA, Gamain B, Scheidig C, et al. Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: a receptor for human placental infection. Proc Natl Acad Sci USA 1999; 96:127438. First citation in article
9. Reeder JC, Cowman AF, Davern KM, et al. The adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A is mediated by P. falciparum erythrocyte membrane protein 1. Proc Natl Acad Sci USA 1999; 96:5198202. First citation in article
10. Salanti A, Staalsoe T, Lavstsen T, et al. Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol Microbiol 2003; 49:17991. First citation in article
11. Khattab A, Kremsner PG, Klinkert MQ. Common surface-antigen var genes of limited diversity expressed by Plasmodium falciparum placental isolates separated by time and space. J Infect Dis 2003; 187:47783. First citation in article
12. Khattab A, Kun J, Deloron P, Kremsner PG, Klinkert MQ. Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A. J Infect Dis 2001; 183:11659. First citation in article
13. Rowe JA, Kyes SA, Rogerson SJ, Babiker HA, Raza A. Identification of a conserved Plasmodium falciparum var gene implicated in malaria in pregnancy. J Infect Dis 2002; 185:120711. First citation in article
14. Khattab A. The molecular basis of pathogenesis of Plasmodium falciparum malaria during pregnancy [PhD thesis]. Tübingen, Germany: Eberhard-Karls-Universitt, 2003. First citation in article
15. Polson A, von Wechmar MB, van Regenmortel MH. Isolation of viral IgY antibodies from yolks of immunized hens. Immunol Commun 1980; 9:47593. First citation in article
16. Polson A, von Wechmar MB, Fazakerley G. Antibodies to proteins from yolk of immunized hens. Immunol Commun 1980; 9:495514. First citation in article
17. Mayengue PI, Rieth H, Khattab A, et al. Submicroscopic Plasmodium falciparum infections and multiplicity of infection in matched peripheral, placental and umbilical cord blood samples from Gabonese women. Trop Med Int Health 2004; 9:94958. First citation in article
18. Moore JM, Nahlen B, Ofulla AV, et al. A simple perfusion technique for isolation of maternal intervillous blood mononuclear cells from human placentae. J Immunol Methods 1997; 209:93104. First citation in article
19. Khattab A, Reinhardt C, Staalsoe T, et al. Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates. Malar J 2004; 3:21. First citation in article
20. Abdel-Latif MS, Khattab A, Lindenthal C, Kremsner PG, Klinkert MQ. Recognition of variant rifin antigens by human antibodies induced during natural Plasmodium falciparum infections. Infect Immun 2002; 70:701321. First citation in article
21. Rasheed FN, Bulmer JN, De Francisco A, et al. Relationships between maternal malaria and malarial immune responses in mothers and neonates. Parasite Immunol 1995; 17:110. First citation in article
22. Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry 1999; 35:32936. First citation in article
23. Gardner MJ, Hall N, Fung E, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002; 419:498511. First citation in article
24. Peterson DS, Miller LH, Wellems TE. Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins. Proc Natl Acad Sci USA 1995; 92:71004. First citation in article
25. Scholander C, Treutiger CJ, Hultenby K, Wahlgren M. Novel fibrillar structure confers adhesive property to malaria-infected erythrocytes. Nat Med 1996; 2:2048. First citation in article
26. O'Neil-Dunne I, Achur RN, Agbor-Enoh ST, et al. Gravidity-dependent production of antibodies that inhibit binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate proteoglycan during pregnancy. Infect Immun 2001; 69:748792. First citation in article
27. Duffy PE, Fried M. Antibodies that inhibit Plasmodium falciparum adhesion to chondroitin sulfate A are associated with increased birth weight and the gestational age of newborns. Infect Immun 2003; 71:66203. First citation in article
28. Staalsoe T, Shulman CE, Bulmer JN, Kawuondo K, Marsh K, Hviid L. Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria. Lancet 2004; 363:2839. First citation in article
29. Jensen AT, Zornig HD, Buhmann C, et al. Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria. Infect Immun 2003; 71:41936. First citation in article
30. Salanti A, Dahlbck M, Turner L, et al. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J Exp Med 2004; 200:1197203. First citation in article
31. Duffy MF, Brown GV, Basuki W, et al. Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype. Mol Microbiol 2002; 43:128593. First citation in article
32. Kyes SA, Christodoulou Z, Raza A, et al. A well-conserved Plasmodium falciparum var gene shows an unusual stage-specific transcript pattern. Mol Microbiol 2003; 48:133948. First citation in article
33. Fried M, Wendler JP, Mutabingwa TK, Duffy PE. Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites. Proteomics 2004; 4:108693. First citation in article
34. Gamain B, Smith JD, Avril M, et al. Identification of a 67-amino-acid region of the Plasmodium falciparum variant surface antigen that binds chondroitin sulphate A and elicits antibodies reactive with the surface of placental isolates. Mol Microbiol 2004; 53:44555. First citation in article
35. Gamain B, Trimnell AR, Scheidig C, Scherf A, Miller LH, Smith JD. Identification of multiple chondroitin sulfate A (CSA)binding domains in the var2CSA gene transcribed in CSA-binding parasites. J Infect Dis 2005; 191:10103. First citation in article