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

Abstract

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.

Figure 1. Natural Acquisition of IgG Antibodies to VAR2CSA.

(a–d) Ab levels to FV2 were determined using plasma from (a, b) primigravidae and (c, d) multigravidae in Ngali II and Yaoundé. The total number of samples evaluated per month ranged from 19–37 for Ngali II and 39–46 for Yaoundé. Ab levels were divided into 4 categories: negative (white; below cutoff: the mean +2 SD of adult males in the same site), low (grey; above cutoff, but <5000 MFI), intermediate (checkered; 5,000–10,000 MFI) and high (black; >10,000 MFI). The percentage of women within each Ab group is shown. Women with Ab levels ≥5,000 MFI were considered to have strong Ab responses. (e–f) In comparison to pregnant women, only 1/40 male who lived in the two study sites had intermediate and 6/40 had low Ab levels to FV2. Antibody cutoff values for Ngali II and Yaoundé women were 2756 and 1192 MFI, respectively.

Figure 2. Correlation between Antibody Levels to VAR2CSA and Placental Malaria at Delivery.

Anti-FV2 Ab levels (MFI ± SEM) were determined between 3 to 9 months of pregnancy in women in Ngali II (a) and Yaoundé (b) with known placental malaria status (Ngali II: PM⁻ n = 13, PM⁺ n = 14; Yaoundé: PM⁻ n = 10, PM⁺ n = 15). (a) A significant difference between PM⁻ and PM⁺ women in Ngali II was found throughout pregnancy using multilevel polynomial regression analysis (p = 0.0047). (b) PM⁻ women in Yaoundé also had higher mean Ab levels compared to PM⁺ women, although difference was not significant. (c–d) Data from PM⁻ and PM⁺ women were subdivided into primigravidae (PG) and multigravidae (MG). All PG in Ngali II and Yaoundé were PM⁺ at delivery, explaining why there are only 3 groups in the figures. Similar Ab levels were present in placental PM⁺ PG and PM⁺ MG. (e) The same plasma samples from PM⁻ and PM⁺ women in Ngali II collected at 4 to 7 months were tested for surface binding to CSA-binding IE (7G8 strain). In support of the results in Fig. 2a, higher levels of Ab was detected in women in Ngali II who were PM⁻ compared to those who were PM⁺ (p = 0.05). P values are based on multilevel polynomial regression analysis. Abbreviations: FV2, full-length VAR2CSA; PM⁻, placental malaria-negative; PM⁺, placental malaria-positive; PG, primigravidae; MG, multigravidae.

Figure 3. High Avidity Antibodies to VAR2CSA were Associated with Absence of Placental Malaria in Ngali II.

(a) The proportion (%) of Ab that remains bound to FV2 after incubation with 3M NH₄SCN (i.e., % high avidity Ab) in the plasma of primigravidae and multigravidae living in Ngali II is shown. Results represent the mean + SEM for 11 to 51 data points per bar. Throughout the course of pregnancy, the proportion of high avidity Ab to FV2 was higher in multigravid than primigravid women (p<0.0001; multilevel polynomial regression analysis). (b) The scattergram shows that the proportion of high avidity Ab to FV2 was highly variable. However, the proportion (%) of high avidity Ab in plasma of women who were PM⁻ was significantly higher than in plasma of PM⁺ women during pregnancy (PM⁻, n = 13; PM⁺, n = 14) (p = 0.0009), based on multilevel polynomial regression analysis. (c) A significantly higher proportion of high avidity Ab was present in plasma of PM⁻ women than PM⁺ women at 5–6 months (p<0.0001), but not at 3–4 and 7–8 months.