Maternally Transmitted Antibodies to Pregnancy-Associated Variant Antigens on the Surface of Erythrocytes Infected with Plasmodium falciparum: Relation to Child Susceptibility to Malaria
Michel Cot1,,
Jean Yves Le Hesran1,2,
Trine Staalsoe3,4,
Nadine Fievet1,2,
Lars Hviid3,4 and
Philippe Deloron1
1 Mother and Child Health in the Tropics, Research Institute for Development (IRD), Hôpital Tenon, Paris, France.
2 Organisation de Coordination pour la Lutte contre les Endémies en Afrique Centrale (OCEAC), Yaounde, Cameroon.
3 Centre for Medical Parasitology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.
4 Institute for Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark.
Received for publication February 18, 2002; accepted for publication August 28, 2002.
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ABSTRACT
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The consequences of pregnancy-associated malaria on a childs health have been poorly investigated. Malarial infection of the placenta seems to result in a higher susceptibility of children to the parasite during their first year of life. In 19931995, the authors investigated the role of antibodies in variable surface antigens (VSA) specific to chondroitin sulfate A (CSA)-binding parasites to assess the parasitologic status of the child. Flow cytometry was used to measure levels of antibodies to VSA from CSA-selected and -unselected parasite lines in the cord blood of 79 newborns in Ebolowa, Cameroon. These newborns were subsequently followed up for 2 years to determine the date of first occurrence of blood parasites and mean parasite density during follow-up. Maternally transmitted antibodies to VSA expressed by CSA-binding parasites, but not antibodies to any other specificity, were negatively related to time of first appearance of Plasmodium falciparum in a childs blood and were positively related to mean parasite density during the first 2 years of life. If maternal infection is thought to be the main mechanism influencing susceptibility of the newborn to malaria, antibodies to VSA may better denote maternal malaria infection during almost the entire pregnancy as opposed to placental blood smear reflecting infection during the last few months of pregnancy.
antibodies; antigens, surface; child; infant, newborn; malaria; Plasmodium falciparum; pregnancy
Abbreviations:
Abbreviations: CSA, chondroitin sulfate A; PAM, pregnancy-associated malaria; VSA, variant surface antigen; VSACSA, variant surface antigen expressed by chondroitin sulfate A-adhering parasites.
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INTRODUCTION
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In areas in which Plasmodium falciparum is endemic, pregnant women are more likely than their nonpregnant counterparts to develop malarial disease (1). One of the most striking features of pregnancy-associated malaria (PAM) is accumulation in the placenta of infected red blood cells (2) that preferentially adhere to chondroitin sulfate A (CSA) expressed by the syncytiotrophoblast (3, 4), a phenomenon mediated by variant surface antigens (VSAs) expressed on the surface of infected red blood cells (5, 6). Placental parasites bind to CSA but not to other ligands such as CD36 and ICAM-1, whereas the opposite is true for most isolates from nonpregnant women (3, 7). Expression of this particular VSA by parasites binding to CSA in the placenta is thought to be the basis for the higher susceptibility of primigravidae. Multigravid pregnant women from areas in which this parasite is endemic have high levels of antibodies to VSAs expressed by CSA-adhering parasites (VSACSA), whereas few nonpregnant women and no men have such antibodies (8, 9). These antibodies inhibit cytoadherence of placental parasites to the human syncytiotrophoblast (7, 9). This finding may explain the decreasing susceptibility to PAM with increasing parity thought to reflect acquisition of VSACSA-specific antibodies interfering with parasite adhesion to CSA (810).
Children born to a malaria-immune mother have a reduced sensitivity to malaria, both clinically and parasitologically, during the first months of life. Such a situation is thought to be related to passage across the placenta of specific antibodies from the mother to the fetus (11). Conversely, the consequences of PAM on a childs health during the first years of life have been poorly investigated. We previously reported that malarial infection of the placenta results in a higher susceptibility of children to the parasite during their first year of life. This finding did not appear to be related to maternally transmitted antibodies, because levels of antibody to asexual blood stages of P. falciparum were similar in both groups of children (12, 13). In the present study, we demonstrated that VSACSA-specific antibodies also cross the placental barrier and are present in cord blood. Moreover, we found that the levels of these antibodies at birth were related to the clinical and parasitologic characteristics of the child during the first year of life.
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MATERIALS AND METHODS
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Subjects
Between January and June 1993, we enrolled 197 women sequentially presenting for delivery at two hospital maternity units (Enongal and Ekombitié) in Ebolowa, a town of 35,000 inhabitants located 160 km south of Yaoundé, Cameroon (refer to Le Hesran et al. (12) for a complete description of the study design). In this rain-forest area, P. falciparum malaria is hyperendemic, with perennial transmission.
After maternal informed consent was obtained, umbilical cord blood samples were drawn as well as maternal peripheral and placental blood for parasitologic examination. Newborns were followed up for 2 years. For the present study, we selected 79 mother/cord blood pairs from samples, corresponding to children for whom follow-up was satisfactory. A team member went to an infants home every week, asked the mother about the health of her child, and measured the axillary temperature, preparing a blood smear to detect malaria attack only in case of fever, defined as an axillary temperature higher than 37.5°C, the presence of Plasmodium in the blood, and the absence of clinical signs that may denote a different pathologic infection. All children presenting with malaria attacks were administered a standard chloroquine treatment (25 mg/kg of body weight). In addition, a blood smear was obtained for each child (whether febrile or not) every month to assess asymptomatic parasite carrying.
The study was approved by the ethics and scientific committees of the Cameroon Ministry of Public Health. In addition, the principles outlined in the Declaration of Helsinki were followed by the physicians in charge of field work.
Malaria parasites
Two different parasite lines were used in this study. The Palo Alto (PA), long-term, in-vitroadapted parasite line, genotypically identical to the FCR3 strain, was a gift from Jürg Gysin (Université de la Méditerranée, Marseille, France). The 2H3 strain was originally collected in Daraweesh, eastern Sudan, from a woman aged 21 years presenting with uncomplicated P. falciparum malaria in November 1996. For both of these nonselected isolates, CSA-adhering sublines were established by 4 x panning on CSA-coated Falcon Petri dishes, as described previously (8). Experiments on these sublines were always completed within 10 days of the last panning. The identity of all lines was checked regularly by polymerase chain reaction of the msp1, msp2, and GLURP loci (14). Four additional unselected parasites lines were also tested: Z5 (from an asymptomatic isolate from Sudan) and E2015, E2045, and E2064 (from Ghanaian children with clinical malaria).
Hematologic measurements
At delivery, a whole blood cell count and hemoglobin content measurement on a mothers blood were carried out by using automated methods with a Coulter Counter (Coultronics France SA, Margency, France). Malarial parasites were searched for on Giemsa-stained thick smears from cord, the mothers, and placental blood and were counted against 1,000 leukocytes. Monthly thick smears collected during follow-up of the child were treated similarly.
Antibody levels measurement
Plasma levels of VSA-specific immunoglobulin G were measured by flow cytometry, as described previously (15). Briefly, red blood cells infected by late-developmental-stage parasites (late trophozoites and schizonts) were purified from the cultures by magnet-activated cell sorting (15), were stained with ethidium bromide, and were sequentially labeled with human test plasma, goat antihuman immunoglobulin G (Dako, Glostrup, Denmark), and fluorescein isothiocyanateconjugated rabbit antigoat immunoglobulin (Dako). Samples were analyzed on a Coulter EPICS XL-MCL flow cytometer (Coulter Electronics, Luton, United Kingdom). Antibody labeling on the infected red blood cells was quantified as mean fluorescence index (16).
Statistical analysis
We analyzed successively the effect of each antibody (against selected or unselected parasites) and other covariates on two main factors: 1) length of time in weeks between birth and the first appearance of parasitemia (either symptomatic (i.e., accompanied by fever) or asymptomatic) and 2) mean parasite density in the blood during follow-up (four classes: 0, no parasitemia; 1, 11,000 parasites/mm3; 2, 1,0015,000 parasites/mm3; and 3, >5 000 parasites/mm3). Only those data for children with more than four blood samples during follow-up were kept in the analysis.
We first selected the most informative variables related to malaria in newborns by conducting univariate analysis (Pearsons chi-square or one-way analysis of variance). We then performed multiple linear regression on length of time until the first parasitemia (procedure 2R; BMDP Statistical Software, Inc., Los Angeles, California) and polychotomous logistic regression on the parasite density (procedure PR; BMDP Statistical Software) for each antibody.
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RESULTS
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The 79 subjects selected were compared with the remainder of the cohort on the basis of sex, placental infection, birth weight, and parity of the mother. Table 1 shows that no difference was found between the two groups regarding any of these variables.
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TABLE 1. Characteristics of children from the main cohort and from the selected mother/cord blood pairs, Ebolowa, Cameroon, 19931995
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Among the 79 women followed, mean age was 22.6 years (extreme values, 13 and 39); 36 of them (45.6 percent) were primigravidae. Twenty-four (30.4 percent) of all placentas were found infected with P. falciparum at delivery. With regard to the children, the mean number of blood smears was 14 (extreme values, 4 and 31). Mean length of time until first parasitemia was 52 weeks (standard error, 3.9), and mean parasite density (arithmetic mean) was 4,588 parasites/mm3 of blood (standard error, 707). Twenty-nine children were found to be affected with at least one malaria attack (extreme values, 1 and 4), discovered during either monthly parasitologic surveys (18 subjects) or weekly follow-up because they presented with fever (11 subjects). The distribution of the proportion of positive blood smears and clinical malaria episodes among followed-up children is given in figure 1.

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FIGURE 1. Distribution of the frequency of the presence of Plasmodium falciparum parasites in thick blood smears collected from 79 children followed from birth until 2 years of age in Ebolowa, Cameroon, 19931995.
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VSAs expressed by P. falciparum-unselected lines and by CSA-selected lines (VSACSA) were recognized by antibodies in plasma-obtained cord blood from the newborns. After all isolates were tested, levels of these antibodies correlated highly with the level achieved in the corresponding mothers peripheral blood (all r > 0.72, all p < 0.001; data not shown).
The effect of different maternal or child characteristics on the occurrence of clinical episodes and on the delay or mean density of parasitemias was tested by using Pearsons chi-square analysis (four classes of parasite densities) or analysis of variance (delay in weeks). Therefore, age, parity, hematocrit, and peripheral parasitemia of the mother; and infection of the placenta by P. falciparum, birth weight, and district of residence (likely to influence the entomologic transmission of malaria; refer to Le Hesran et al. (12)) of the newborn were tested. None of these factors was significantly linked with occurrence of clinical episodes, delay, or mean density of parasitemias (all p > 0.15 except for placental infection, p = 0.18, p = 0.10, and p = 0.06, respectively). Finally, we included placental infectionand also parity of the mother and district of residence, although they were not significant but highly susceptible to influencing the parasitologic status of the mother and childin the multivariate models.
We then successively tested the effect of each antibody (PACSA and 2H3CSA, pregnancy specific; and PA, 2H3, Z5, E2015, E2045, and E2064, nonspecific) on the delay before first parasitemia (linear regression), mean parasite density, and occurrence of clinical episodes (analysis of variance). As shown in figure 2 and table 2, only PACSA and 2H3CSA were significantly linked with the first two variables. Furthermore, levels of these two antibodies increased regularly with parasite densities. Regarding clinical episodes, relations with PACSA and 2H3CSA were nearly significant (p = 0.08 and p = 0.06, respectively). None of the remaining antibodies was linked to this variable.

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FIGURE 2. Correlation between levels of antibodies against variant surface antigens expressed by chondroitin sulfate A (CSA)-selected parasites (top) and against variant surface antigens expressed by unselected parental lines (Palo Alto (PA) and 2H3, bottom) measured by flow cytometry as mean fluorescence index (MFI) in the cord blood of 79 newborns from Ebolowa, Cameroon, 1993, and time (in weeks) of first occurrence of Plasmodium falciparum parasites in blood.
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TABLE 2. Plasma levels of antibodies recognizing CSA*-selected and -unselected Plasmodium falciparum-infected red blood cells in newborns from Ebolowa, Cameroon, 19931995
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Finally, we performed multivariate analyses by including selected covariates (placental infection, parity of the mother, and area of residence (which has been reported to be related to level of malaria transmission) for each antibody on the delay before first parasitemia (multilinear regression), mean parasite density (polychotomous logistic regression), and clinical episodes (multilinear regression). Similar to the findings from univariate analysis, the two pregnancy-specific antibodies appeared to be strongly negatively correlated with delay before first parasitemia (multilinear regression, p = 0.005 for PACSA and p = 0.0005 for 2H3CSA). Among nonspecific antibodies, only E2015 was significantly linked with the delay (p = 0.01). Polychotomous regression demonstrated an association of these two antibodies with mean parasite density, whereas no association was found with nonspecific antibodies (table 3). Interestingly, the values of adjusted odds ratios between these two antibodies and parasite density increased with density (PACSA: odds ratios = 4.2, 15, and 9.2 for low, medium, and high densities, respectively; 2H3CSA: odds ratios = 9.7, 9.5, and 21 for low, medium, and high densities, respectively; table 3). Placental infection (although almost significant, p = 0.08) was not correlated with either of these variables (delay or parasite density). The same tendency was observed with the occurrence of clinical episodes, although statistical significance was not reached for any of the PACSA and 2H3CSA antibodies (p = 0.08 and p = 0.06, respectively). No association was found with nonspecific antibodies.
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TABLE 3. Results of polychotomic regression of parasite density (four classes of density) on the two pregnancy-associated antibodies, 2H3CSA and PACSA, and other covariates, Ebolowa, Cameroon, 19931995*
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DISCUSSION
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We measured the levels of antibodies to various specificities in the cord blood of 79 newborns from Cameroon, who were subsequently followed up over a 2-year period. Maternally transmitted anti-PAM antibodies, but not antibodies to any other specificity, were negatively related to length of time until the first appearance of P. falciparum parasites in the peripheral blood and were positively related to mean parasite density during the first 2 years of life. In addition, no specific association between anti-PAM antibodies and occurrence of malaria attacks was found. These results could indicate that, with regard to the role of anti-PAM antibodies, such clinical events can be considered a subset of all parasitemias observed during follow-up.
It is unlikely that anti-PAM antibodies present in serum play an active role in increasing susceptibility of a child to P. falciparum malarial infection. Indeed, such antibodies have been shown to react specifically with P. falciparum parasites that cytoadhere in the placenta. Such a parasite population is present in only pregnant woman and usually is not encountered in nonpregnant hosts, including children (7). Moreover, none of the antibodies of other specificity was related, either positively or negatively, with the parasitologic status of the child (12). Similarly, it has been reported that antibody levels to P. falciparum schizonts and Pf155 are not associated with resistance to malarial infection (12, 17); this lack of association reinforces the significance of the observed relation between anti-PAM antibody levels at birth and subsequent susceptibility to malarial infection. It has been shown that anti-PAM antibodies are acquired at ±20 weeks of gestation in primigravidae (but 8 weeks earlier in multigravidae) and are linked to placental infection (9, 16, 18). Such anti-PAM antibodies may indeed be considered a marker for risk of P. falciparum infection during pregnancy. Therefore, the presence of anti-PAM antibodies in cord blood (and in peripheral blood of the mother) may merely reflect the past occurrence of a P. falciparum infection during pregnancy of the mother. In that respect, these antibodies are likely to indicate that the past infection occurred during (almost) the entire pregnancy, as opposed to placental blood smears (or even placenta histology) that are more likely to reflect infection during the last few months of pregnancy.
We previously reported that children born to placenta-infected mothers were more likely to develop malarial infection between 4 and 6 months of age and that parasite prevalence rates were higher in these children from 5 to 8 months of age. This difference was not related to maternally transmitted antibodies, because total and anti-P. falciparum schizont-specific immunoglobulin-G antibody levels were similar in children born to placenta-infected mothers and to placenta-noninfected mothers. Thus, malarial infection of the placenta was considered to result in a higher susceptibility of children to the parasite (13). This finding is in agreement with the data presented here, that offspring presenting with anti-PAM antibodies, and consequently whose mothers experienced a malaria placental infection during pregnancy, are more susceptible to malarial infection during the first few months of life.
Several hypotheses may account for the relation between anti-PAM antibodies and parasitologic status of the child. Firstly, the risk of malarial infection may differ between persons according to several ecologic factors, most shared by the mother and her offspring. Because anti-P. falciparum schizont antibody levels were similar in both groups of children, as well as for the mother at time of delivery (data not shown), it is likely that the risk of malaria was similar in both groups of children. Furthermore, in our sample, number of parasitemias and time until first parasitemia were not different for children living in the center of the city and those living in the peripheral districts, where transmission of malaria is supposedly higher. Alternatively, women who had high levels of VSACSA antibodies are much more likely to have presented with placental parasitemia at the time of delivery. If the blood of the newborn is contaminated by placental blood from the mother, the offspring should be more likely to show an early P. falciparum infection than those whose levels of antibodies are low. However, in most children, parasites were first detected weeks after birth, much later than congenital malaria is usually diagnosed. Moreover, the relation between antibodies and outcome variables persisted in the multivariate analysis, demonstrating that this effect is independent of the placental infection of the mother at time of delivery.
Lastly, in utero exposure to malaria antigens may induce immunologic tolerance (19) and potentially modify future susceptibility to malarial infection and/or disease. The impact of in utero exposure to other parasites, including Trypanosoma cruzi (20) and filariasis (21), has already been reported to increase susceptibility. Conversely, this exposure can prime the immune system and improve defenses against future infections (22). Because cord blood lymphocytes may be induced to differentiate into effector cells producing predominantly Th1 or Th2 cytokines (23), malaria might direct the functional capacity of fetal T cells to respond to further infection. Although the mechanism involved is not specified, the current study shows that offspring presenting with anti-PAM antibodies, probably demonstrating that the mother presented with PAM, are more susceptible to malarial infection during the first 2 years of life. A better understanding of the mechanisms underlying the immune responses to P. falciparum in newborns and children may have important implications for development of control strategies.
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ACKNOWLEDGMENTS
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This work was supported by grants from the French Ministry of Research and Technology (grant 92S0034), the French Ministry of Cooperation and Development, the Francophone Agency for High School and Research (AUPELF/UREF), and the European Community, Quality of Life and Management of Living Resources Programme (grant QLK2-CT-2001-01302).
The authors thank the medical and nursing staff of the Enongal and Ekombitié hospitals for their collaboration. They are grateful to the families of Ebolowa and the home visitors for their active participation in the project. Finally, this study would not have been possible without the support of national and regional authorities.
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NOTES
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Correspondence to Dr. Michel Cot, IRD UR010, Faculté de Pharmacie, Laboratoire de Parasitologie, 4 Avenue de lObservatoire 75270, Paris Cedex 06 France (e-mail: Michel.cot{at}ird.fr). 
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