Influence of intermittent preventive treatment on antibodies to VAR2CSA in pregnant Cameroonian women

Intermittent preventive treatment (IPT) and insecticide-treated bed nets are the standard of care for preventing malaria in pregnant women. Since these preventive measures reduce exposure to malaria, their influence on the antibody (Ab) response to the parasite antigen VAR2CSA was evaluated in pregnant Cameroonian women exposed to holoendemic malaria. Ab levels to full-length VAR2CSA (FV2), variants of the six Duffy binding like (DBL) domains, and proportion of high avidity Ab to FV2 were measured longitudinally in 92 women before and 147 women after IPT. As predicted, reduced exposure interfered with acquisition of Ab in primigravidae, with 71% primigravidae being seronegative to FV2 at delivery. Use of IPT for > 13 weeks by multigravidae resulted in 26% of women being seronegative at delivery and a significant reduction in Ab levels to FV2, DBL5, DBL6, proportion of high avidity Ab to FV2, and number of variants recognized. Thus, in women using IPT important immune responses were not acquired by primigravidae and reduced in a portion of multigravidae, especially women with one to two previous pregnancies. Longitudinal data from individual multigravidae on IPT suggest that lower Ab levels most likely resulted from lack of boosting of the VAR2CSA response and not from a short-lived Ab response

High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4)SCN) was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+)) and those without (PM(-)) at delivery. Results showed that PM(-) women had significantly higher Ab levels (p = 0.0047) and proportion of high avidity Ab (p = 0.0009) than PM(+) women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI) and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9) and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0) reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria

Timing of the human prenatal antibody response to <i>Plasmodium falciparum</i> antigens

<div><p><i>Plasmodium falciparum</i> (Pf)-specific T- and B-cell responses may be present at birth; however, when during fetal development antibodies are produced is unknown. Accordingly, cord blood samples from 232 preterm (20–37 weeks of gestation) and 450 term (≥37 weeks) babies were screened for IgM to Pf blood-stage antigens MSP1, MSP2, AMA1, EBA175 and RESA. Overall, 25% [95% CI = 22–28%] of the 682 newborns were positive for IgM to ≥1 Pf antigens with the earliest response occurring at 22 weeks. Interestingly, the odds of being positive for cord blood Pf IgM decreased with gestational age (adjusted OR [95% CI] at 20–31 weeks = 2.55 [1.14–5.85] and at 32–36 weeks = 1.97 [0.92–4.29], with ≥37 weeks as reference); however, preterm and term newborns had similar levels of Pf IgM and recognized a comparable breadth of antigens. Having cord blood Pf IgM was associated with placental malaria (adjusted OR [95% CI] = 2.37 [1.25–4.54]). To determine if <i>in utero</i> exposure occurred via transplacental transfer of Pf-IgG immune complexes (IC), IC containing MSP1 and MSP2 were measured in plasma of 242 mother-newborn pairs. Among newborns of IC-positive mothers (77/242), the proportion of cord samples with Pf IC increased with gestational age but was not associated with Pf IgM, suggesting that fetal B cells early in gestation had not been primed by IC. Finally, when cord mononuclear cells from 64 term newborns were cultured <i>in vitro</i>, only 11% (7/64) of supernatants had Pf IgM; whereas, 95% (61/64) contained secreted Pf IgG. These data suggest fetal B cells are capable of making <i>Pf</i>-specific IgM from early in the second trimester and undergo isotype switching to IgG towards term.</p></div

Model for the timing of fetal exposure and Ab responses to Pf Ags.

<p>The model is an estimate of the likelihood of fetal exposure, and fetal Ab response, to Pf through the second and third trimesters of gestation. On the horizontal gray scales, dark shade = high; and light shade = low. Infected erythrocytes can sequester in the placenta from ~12 wks of gestation when the IVS are formed. The incidence of maternal malaria and the density of Pf parasites in the IVS are high during the second trimester, thus increasing the risk of exposure to Pf in utero. At this time of gestation, the fetus is already capable of making Pf-specific IgM. As gestation advances, fetal B cells receive secondary exposures to Pf Ags and class-switching to Pf IgG production occurs. The proportion of fetuses with Pf IgM drops rapidly at term while the proportion of Pf IgG responders increases to almost 95% at 40 weeks.</p

Timing of the prenatal Pf IgM response.

<p>(<b>A</b>) The percent of newborns (n = 682) positive for IgM to Pf Ags was stratified by gestational age category. The proportion of IgM+ newborns decreased with gestational age, Chi-square test for trend: MSP1 (i.e., positive for IgM to the 3D7 or FVO variant of MSP1), p = 0.008; MSP2 (3D7 or FC27), p = 0.0006; AMA1, ns; EBA175, ns; RESA, p = 0.002; and ≥1 Pf Ag, p = 0.001. For clarity of figures, confidence intervals of proportions are provided in the text but not on the figure. (<b>B</b>) Amount of IgM Ab present for newborns who were Ab positive for the specified antigen. Average IgM levels did not differ significantly with GA for any of the antigens (Kruskal-Wallis).</p

Descriptive characteristics of the 682 Cameroonian preterm and term newborns whose samples were used for plasma antibody studies.

<p>Descriptive characteristics of the 682 Cameroonian preterm and term newborns whose samples were used for plasma antibody studies.</p

Breadth of Pf IgM response in different gestational age groups.

<p>The 169 Cameroonian newborns who were cord IgM+ to ≥ 1 Pf Ag were included, i.e., n = 75 preterm [n = 14 (20–27 weeks), n = 19 (28–31 weeks), n = 42 (32–36 weeks)] and n = 95 term. For each GA group, the bars show the proportion of Ab-positive newborns who produced IgM recognizing 1, 2, 3, 4 and 5 Pf blood-stage Ags. Whiskers represent the 95% confidence interval of proportions. There were no significant (ns) differences in responders to multiple antigens across GA groups.</p

Influence of gestational age and placental malaria on production of Pf IgM to MSP1 and MSP2 <i>in utero</i>.

<p>Influence of gestational age and placental malaria on production of Pf IgM to MSP1 and MSP2 <i>in utero</i>.</p