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

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

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

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

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

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

Timing of fetal exposure to maternal immune complexes (IC).

<p>Peripheral plasma samples of 77/242 mothers had IC containing MSP1 or MSP2. Cord plasma IC data of the 77 corresponding newborns were analyzed for materno-fetal IC transfer. (<b>A</b>) The percentage of cord blood samples with IC increased with GA, especially after 31 wks. (<b>B</b>) Average levels of Pf-specific IC in cord plasma. P values were calculated using Kruskal-Wallis test with post-test for trend: *p<0.01 and **p<0.001. The 20–27 wks group was excluded in the analysis because of few (<3) data points.</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

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

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