VAR2CSA Serology to Detect Plasmodium falciparum Transmission Patterns in Pregnancy

Abstract

Pregnant women constitute a promising sentinel group for continuous monitoring of malaria transmission. To identify antibody signatures of recent Plasmodium falciparum exposure during pregnancy, we dissected IgG responses against VAR2CSA, the parasite antigen that mediates placental sequestration. We used a multiplex peptide-based suspension array in 2,354 samples from pregnant women from Mozambique, Benin, Kenya, Gabon, Tanzania, and Spain. Two VAR2CSA peptides of limited polymorphism were immunogenic and targeted by IgG responses readily boosted during infection and with estimated half-lives of <2 years. Seroprevalence against these peptides reflected declines and rebounds of transmission in southern Mozambique during 2004-2012, reduced exposure associated with use of preventive measures during pregnancy, and local clusters of transmission that were missed by detection of P. falciparum infections. These data suggest that VAR2CSA serology can provide a useful adjunct for the fine-scale estimation of the malaria burden among pregnant women over time and space.

Keywords: Benin; Gabon; Kenya; Malaria; Mozambique; Plasmodium falciparum; Spain; Tanzania; VAR2CSA; exposure; immunity; parasites; pregnancy; serology; transmission.

Figure 1

Plasmodium falciparum VAR2CSA IgG in malaria-exposed and -nonexposed pregnant women. A) nMFI measured in pregnant women from Mozambique and Spain. Red dashed line represents the mean nMFI from bovine serum albumin + 3 SDs. B) Seroprevalence among pregnant women from Spain (blue) and Mozambique (black). Asterisks indicate antigens recognized by pregnant women from Mozambique at levels above IgG against bovine serum albumin + 3 SDs and above levels in pregnant women from Spain (q-value <0.05 by Simes procedure) and those antigens poorly recognized by pregnant women from Spain (seroprevalence <5%). C, D) Prevalence of P. falciparum infection in peripheral and placental blood by quantitative PCR (C) and nMFIs (D) among pregnant women from Mozambique delivering in 2004–2005 and 2010–2012. Red lines represent the geometric mean and T-bars the 95% CI. Asterisks indicate antigens recognized by IgG whose levels dropped between 2004 and 2012, as assessed by linear regression adjusted by intermittent preventive treatment during pregnancy, parity, age, and HIV status (q-value <0.05 by Simes procedure). nMFI, normalized median fluorescent intensity.

Figure 2

IgG responses during pregnancy against selected VAR2CSA antigens and polymorphism in target sequences in serologic study of Plasmodium falciparum in pregnant women. A) P. falciparum prevalence by quantitative PCR (qPCR) in 239 pregnant women from Mozambique at recruitment, second administration of IPTp, and delivery. Cumulative prevalence at delivery refers to peripheral or placental infection detected by microscopy, qPCR, or histology at any time point. B) Ratio of nMFIs at delivery in women from Mozambique infected during pregnancy compared with uninfected women. Error bars indicate 95% CIs. C) Seroprevalence at delivery, showing the cumulative prevalence of infection during pregnancy (red dashed line) and the prevalence at delivery by qPCR (light blue line). D) Ratio of nMFIs at delivery in multigravid compared with primigravid women, adjusted by IPTp, parity, age, and HIV status. Error bars indicate 95% CIs. E) IgG dynamics during pregnancy with estimates of time to double (T_2x) and half-life (T_1/2) obtained from linear mixed-effect regression model. Red points represent P. falciparum infection, dark gray lines the seropositivity cutoff, red lines the fitted-estimation, and dashed lines the 95% CI. F) Space-feeling representation of DBL1X-ID1 showing p5 (blue) and p8 (red). G) Logo representation of p5 and p8 sequences obtained from 50 P. falciparum isolates (20 from Mozambique, 10 from Benin, 10 from Gabon, and 10 from Kenya). IPTp, intermittent preventive treatment during pregnancy; nMFI, normalized median fluorescent intensity.

Figure 3

IgG seroprevalence against VAR2CSA selected antigens according to study period, country, anemia status and intermittent preventive treatment group in the serological study of Plasmodium falciparum in pregnant women. A) Pregnant women from Mozambique delivering during different periods. B) HIV-uninfected pregnant women from Benin, Gabon, Mozambique, and Tanzania. C) HIV-infected pregnant women from Kenya and Mozambique. D) Nonanemic (NA) and anemic (A) pregnant women. E) HIV-uninfected pregnant women receiving mefloquine (MQ) or sulfadoxine/pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp). F) HIV-infected pregnant women receiving MQ or placebo (PL) as IPTp. Maternal microscopic infection was defined by the presence of P. falciparum parasites in peripheral blood or in placenta on microscopic or histologic examination, respectively. Maternal quantitative PCR (qPCR)–positive infection was defined by a positive result on qPCR of peripheral or placental blood. P-values were obtained from multivariate regression models adjusted for HIV, parity, age, IPTp, and country when applicable. T-bars represent 95% CIs. *Crude p<0.05; **adjusted p<0.05. B, Benin; G, Gabon; K, Kenya; M, Mozambique; OM, optical microscopy; T, Tanzania.

Figure 4

Geographic patterns of Plasmodium falciparum infection and IgG seropositivity in pregnant women living in southern Mozambique. Geographic distribution of seropositive pregnant women (HIV-uninfected and HIV-infected) living in Manhiça District, Mozambique, who delivered during 2010–2012 and for whom microscopy, quantitative PCR (qPCR), and spatial geocoordinates were available. Distribution shows pregnant women with and without P. falciparum infection at delivery, either in peripheral or in placental blood, detected by microscopy or histology (A) or qPCR (B), as well as AMA1 (C), MSP1₁₉ (D), p5 (E), p8 (F), and p5+8 (G) seropositive and seronegative pregnant women at delivery. Grey dots indicate seronegative women, blue dots indicate seropositive women, red dots indicate seropositive women selected by the hotspot cluster algorithm; red circles indicate the most likely hotspot (continuous line p<0.05, dashed line p>0.05). Maps were generated by using OpenStreetMap (https://www.openstreetmap.org). Hotspot NE, not estimated because of low/high prevalence of seroresponders.