IgM levels to FV2 and ID1-ID2a in Cameroonian women.

<p>IgM levels to FV2 and ID1-ID2a (3D7 and FCR3) were measured in plasma collected at first smear-positive visit and the following visit approximately one month later from women living in <b>A</b>) city of Yaoundé (PG n = 17, MG n = 26) and <b>B</b>) village of Ngali II (PG n = 21, MG n = 33). No statistically significant differences in IgM levels to either ID1-ID2a or FV2 were observed between PG and MG in both locations (all p values>0.05). Since the beads were coupled at saturation, it appears that both PG and MG produced higher IgM levels to FV2 compared to ID1-ID2a 3D7 (both locations p-values <0.001). Yaoundé PG and MG produced higher IgM levels to FV2 compared to ID1-ID2a FCR3 (p<0.05). Small ♀- PG, big ♀- MG. Median MFI and IQR are plotted.</p

The Antibody Response of Pregnant Cameroonian Women to VAR2CSA ID1-ID2a, a Small Recombinant Protein Containing the CSA-Binding Site

<div><p>In pregnant women, <i>Plasmodium falciparum</i>-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women naturally acquire antibodies to ID1-ID2a and if antibodies to ID1-ID2a correlate with absence of placental malaria at delivery. Antibody levels to full-length VAR2CSA and ID1-ID2a were measured in plasma samples from 745 pregnant Cameroonian women, 144 Cameroonian men, and 66 US subjects. IgM levels and IgG avidity to ID1-ID2a were also determined. As expected, antibodies to ID1-ID2a were absent in US controls. Although pregnant Cameroonian women developed increasing levels of antibodies to full-length VAR2CSA during pregnancy, no increase in either IgM or IgG to ID1-ID2a was observed. Surprisingly, no differences in antibody levels to ID1-ID2a were detected between Cameroonian men and pregnant women. For example, in rural settings only 8–9% of males had antibodies to full-length VAR2CSA, but 90–96% had antibodies to ID1-ID2a. In addition, no significant difference in the avidity of IgG to ID1-ID2a was found between pregnant women and Cameroonian men, and no correlation between antibody levels at delivery and absence of placental malaria was found. Thus, the response to ID1-ID2a was not pregnancy specific, but predominantly against cross-reactivity epitopes, which may have been induced by other PfEMP1 antigens, malarial antigens, or microbes. Currently, ID1-ID2a is a leading vaccine candidate, since it binds to the CSA with the same affinity as the full-length molecule and elicits binding-inhibitory antibodies in animals. Further studies are needed to determine if the presence of naturally acquired cross-reactive antibodies in women living in malaria endemic countries will alter the response to ID1-ID2a following vaccination with ID1-ID2a.</p></div

Prevalence of IgG to FV2 and ID1-ID2a in non-pregnant individuals and pregnant women at delivery.

a<p>Cut-off for FV2 seropositivity was determined based on the mean + 2 SD for resident males after excluding the occasional male (n = 2 Yaoundé, n = 1 Ngali, n = 1 Simbok) with MFI greater than the mean +3 SD.</p>b<p>US controls (n = 66) were used to establish a cut-off for seropositivity to ID1-ID2a.</p>c<p>3% of women who were not seropositive for FV2 were seropositive for DBL5 FCR3, indicating they had been exposed to CSA-binding <i>P. falciparum</i> IE.</p>d<p>Mean ± SD.</p

IgG levels to N-terminal domains of FV2 and DBL5 in the city of Yaoundé.

<p>Ab levels to DBL1 (3D7 and 7G8), DBL1+2 (7G8), DBL2 FCR3 and DBL5 FCR3 in the plasma of 116 PM+ and 348 PM− multigravidae women living in the city of Yaoundé were measured at delivery (cross-sectional cohort). Both PM+ and PM− women had significantly higher levels of Ab to DBL5 compared to Ab to DBL1, DBL 1+2 and DBL 2 (all p values <0.0001). PM+ and PM− multigravidae had significantly higher levels of Ab to DBL1 3D7, DBL1 7G8, DBL1+2 and DBL5 compared to males (all p values <0.0001). Only a marginally significant differences was found in Ab levels to DBL2 between males and females (p = 0.051). No statistical differences were found in Ab levels to any of the domains between PM+ and PM− women (all p values>0.05). Open circles represent PM− and black circles represent PM+. Median and IQR are also plotted.</p

Proportion of high avidity IgG to FV2 and ID1-ID2a.

<p>A subset of women in Yaoundé and Ngali II with Ab ID1-ID2a were tested in the avidity assay. The proportion of Ab that remained bound to <b>A</b>) FV2, <b>B</b>) ID1-ID2a 3D7, and <b>C</b>) ID1-ID2a FCR3 in the presence of chaotropic salt was measured in samples collected in city of Yaoundé (n = 6 males, n = 18 women) and village of Ngali II (n = 9 males, n = 20 women). Although the mean proportion of high avidity Ab to FV2 was significantly higher in Ngali II pregnant women compared to males (*** p = 0.0003), no statistically significant differences were found in Ab avidity to ID1-ID2a (3D7 and FCR3) between males and females, both in the city and village (all p>values 0.05). ♂- males, ♀- females Median and IQR are also plotted.</p

Comparison of Ab levels between FV2 and ID1-ID2a.

<p><b>A–C</b>) Ab levels to FV2, DBL3, DBL5 and ID1-ID2a FCR3 were measured in plasma of 116 PM+ and 348 PM- pregnant women living in city of Yaoundé. In addition, comparisons were made in Ab levels to FV2 and ID1-ID2a in <b>D</b>) males in Yaoundé, <b>E</b>) males in the village of Ngali II, and <b>F</b>) males in Simbok village. Positive correlations were found for all comparisons; Pearson's correlation coefficients (r) are reported.</p

IgG levels to ID1-ID2a in Cameroonian women during pregnancy.

<p>Ab levels to FV2 (<b>A, D</b>), ID1-ID2a 3D7 (<b>B, E</b>), and ID1-ID2a FCR3 (<b>C, F</b>) were measured in plasma of pregnant women living in the city of Yaoundé (<b>A-C</b>) and village of Ngali II (<b>D–F</b>). Samples were randomly selected within a trimester so only one data point per women per trimester was included. Number of samples per trimester ranged from: <b>A–C</b>: Primigravidae (PG) n = 33–39, multigravidae (MG) n = 63–74; <b>D–F</b>: PG n = 13–15, MG n = 33–68. To determine if Ab levels increased during pregnancy, data for women with samples collected in the first, second, and third trimesters (City: PG n = 32, MG n = 53; Village: PG n = 7, MG n = 17) were assessed using the Friedman's test. As expected, Ab levels to FV2 increased during pregnancy in the village (Fig. <b>1B</b>: PG: p = 0.008, MG p = 0.001). Although a similar trend was observed in the city, the increase was not significant, probably due to low transmission (<b>Fig. 1A</b>). Likewise, higher Ab levels to FV2 were observed in PG and MG compared to males in the city (all p values <0.001) and village (all p values <0.0001). In contrast, Ab levels to ID1-ID2a (both strains) did not increase during pregnancy in either location (p>0.05) and no significant differences were found in Ab levels to ID1-ID2a (3D7 or FCR3) between males and females in the two sites (all p values>0.05) (Fig. 1B–1F). Although statistically significant higher Ab levels to ID1-ID2a 3D7 were found in MG in the village compared to males (Fig. <b>E</b>, p<0.05), the trend was not found with the FCR3 strain. ♂- males, small ♀- PG, big ♀- MG; I, II, III trim  =  first, second and third trimester, respectively. Median MFI and Inter-Quartile Range (IQR) are plotted.</p

IgG levels to ID1-ID2a in different cohorts.

<p>IgG levels to FV2 and ID1-ID2a (3D7 and FCR3) were measured in plasma collected at delivery from women living in <b>A</b>) city of Yaoundé (n = 35 males, n = 33 PG, n = 63 MG), <b>B</b>) village of Ngali II (n = 58 males, n = 15 PG, n = 68 MG), <b>C</b>) Simbok village (n = 51 males, n = 102 women, and <b>D</b>) in a cross-sectional cohort of MG in Yaoundé (PM+n = 116, PM− n = 348), as well as, US pregnant (n = 42) and non-pregnant subjects (n = 24). Major findings included: 1) significantly higher Ab levels to ID1-ID2a (both strains) in Cameroonian males (city and village) compared to US pregnant women (p<0.001); 2) similar IgG levels to FV2 in US pregnant and non-pregnant controls and males in Yaoundé; but, higher levels in Cameroonian males living in the village (p<0.0001), suggesting repeated infection induced Ab to FV2 in some males; 3) no significant differences in IgG to ID1-ID2a in Cameroonian males and females living in the city and village (both strains p> values 0.05); and 4) no significant differences in IgG to ID1-ID2a FCR3 between PM+ and PM- MG in the city (<b>D</b>: p = 0.46). Horizontal bars represent median, first and third quartiles, and vertical bars represent Inter-Quartile Range (IQR). □- US non-pregnant (US NP), ▪ US pregnant women (US PW), ♂- Cameroonian males (M), small ♀- Cameroonian PG, big ♀- Cameroonian MG. Median MFI and IQR are plotted.</p

Maternal sEng at delivery is increased with placental malaria in primigravidae and not multigravidae.

<p>sEng was measured in peripheral plasma samples of Malawian women at delivery. Placental malaria (PM+) was defined as positive by placental blood smear microscopy. Bars represent medians. 2-way ANOVA on log-transformed data: gravidity, p<0.0001. *, p<0.05; **, p<0.01; ***, p<0.001 by Bonferonni post-test.</p

Overview of study populations from Cameroon and Malawi: timing of malaria infections during pregnancy in Cameroon and summary of adverse birth outcomes from Malawi.

<p>Women who participated in the longitudinal study in Cameroon were followed throughout gestation, at up to 7 antenatal visits and at delivery. Peripheral <i>P. falciparum</i> malaria infection was assessed by microscopy of peripheral blood smear at each visit; MIP+, blood-smear positive. Women who participated in the cross-sectional study in Malawi were enrolled at delivery. Placental malaria (PM) was determined by microscopy of placental blood smear. Adverse birth outcomes were defined as normal birth weight (NBW, ≥2500 g) or low birth weight (LBW, <2500 g) with or without growth restriction (small for gestational age (SGA), less than 10^th centile according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024985#pone.0024985-Landis1" target="_blank">[26]</a>) or preterm delivery (PTD, <37 weeks of gestation).</p