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

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

Experimental model recapitulates the spontaneous abortion and low birth weight outcomes characteristic of PM.

<p><i>(A)</i> Pregnant BALB/c females infected (dark bars) with <i>P. berghei</i> on gestational day (G)13 have an increased rate of abortion and decreased proportion of viable pups per litter compared to uninfected mice (light bars) by G19, day 6 of infection/control injection (D6). Proportion of viable fetuses depicted by solid bars; aborted fetuses, by striped bars. <i>(B)</i> Body weight of viable fetuses is decreased with maternal malaria infection (dark symbols) as compared to weight of fetuses from uninfected mice (light symbols). Dots are individual viable fetuses; bars represent the median of each group. 20–83 fetuses were collected per group (from 4–13 pregnant females per group). *** <i>P</i><0.001 (Mann-Whitney).</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

Placental <i>Angpt2</i> mRNA expression is increased by malaria infection (<i>P</i> = 0.0063, 2-way ANOVA).

<p><i>(A</i>) <i>Angpt1</i> and <i>(B</i>) <i>Angpt2</i> transcripts were measured by real-time quantitative PCR using cDNA templates reverse transcribed from placental RNA from pregnant mice uninfected (light bars) and infected with <i>P. berghei</i> (dark bars). Copy number was normalized to housekeeping gene expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009481#s2" target="_blank">Materials and Methods</a>. <i>(C)</i> The relative expression of <i>Angpt2</i>/<i>Angpt1</i> is also increased in placentas associated with viable fetuses of infected mice (<i>P</i> = 0.0032, 2-way ANOVA on log-transformed data). Dots are individual placentas associated with viable fetuses; bars represent the median of each group. 4–6 mice are represented per group. *<i>P</i><0.01 (Bonferonni post-test). D, day post infection/control injection; G, gestational day.</p

Plasma angiopoietin levels at delivery are dysregulated in PM and with LBW outcomes.

<p><i>(A–C)</i> Peripheral plasma and <i>(D)</i> matched placental plasma obtained at delivery of normal birth weight (NBW) or LBW infants from primigravid women with (PM+) or without (PM-) PM were measured for ANG-1 and ANG-2 by ELISA. <i>(A)</i> Mean maternal peripheral plasma ANG-1 is elevated with LBW deliveries in PM- but not PM+ women. Statistical analyses by Mann-Whitney test. <i>(B)</i> Maternal peripheral plasma ANG-2 is elevated with PM. Statistical analyses by Mann-Whitney test. <i>(C)</i> Elevated maternal peripheral plasma ANG-2/ANG-1 ratio at delivery is associated with PM (<i>P</i> = 0.0016) and LBW (<i>P</i> = 0.0406); 2-way ANOVA on log-transformed data. Statistical analyses between groups by t-test on log-transformed data. <i>(D)</i> Placental plasma ANG-2/ANG-1 ratio levels are elevated compared to peripheral plasma levels. Statistical analysis by t-test on log-transformed data. Dots represent individual women, lines represent the median of each group. Peripheral plasma, PM- NBW (n = 47), PM- LBW (n = 44), PM+ NBW (n = 51), PM+ LBW (n = 35). Placental plasma, PM- NBW (n = 41), PM- LBW (n = 34), PM+ NBW (n = 44), PM+ LBW (n = 28). * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, # <i>P</i> = 0.06, ^∧<i>P</i> = 0.486.</p