Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates

Abstract

Background: Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA) that can mediate adhesion to chondroitin sulfate A (CSA) in vitro. IgG antibodies with specificity for the VSA expressed by these parasites (VSAPAM) are associated with protection from maternal anaemia, prematurity and low birth weight, which is the greatest risk factor for death in the first month of life.

Methods: In this study, the development of anti-VSAPAM antibodies in a group of 151 women who presented to the maternity ward of Albert Schweitzer Hospital in Lambaréné, Gabon for delivery was analysed using flow cytometry assays. Plasma samples from placenta infected primiparous women were also investigated for their capacity to inhibit parasite binding to CSA in vitro.

Results: In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSAPAM-specific IgG compared to women who had no malaria infections at delivery. Placental isolates of Gabonese and Senegalese origin tested on plasma samples from Gabon showed parity dependency and gender specificity patterns. There was a significant correlation of plasma reactivity as measured by flow cytometry between different placental isolates. In the plasma of infected primiparous women, VSAPAM-specific IgG measured by flow cytometry could be correlated with anti-adhesion antibodies measured by the inhibition of CSA binding.

Conclusion: Recognition of placental parasites shows a parity- and sex- dependent pattern, like that previously observed in laboratory strains selected to bind to CSA. Placental infections at delivery in primiparous women appear to be sufficient to induce functional antibodies which can both recognize the surface of the infected erythrocytes as well as block their binding to CSA. The correlation between serum reactivities of placental field isolates from different geographic locations and collected at different times is indicative of the conserved nature of the antigen(s) mediating PAM.

Figure 1

Parity dependence of recognition of variant surface antigens on four placental parasite isolates by VSA_PAM-specific IgG in plasma obtained from 151 women at delivery from an area of hyperendemic malaria transmission (Lambaréné, Gabon). Parasite isolate and the corresponding statistical significance of the correlation between parity and mean fluorescence intensity are shown. The fit line using least squares regression is shown for each relationship.

Figure 2

Relationship of anti-VSA levels between different placental isolates. Anti-VSA levels were measured as mean fluorescence intensity (MFI). Spearman rank correlation coefficients together with their strengths are given for each parasite pair. The fit line using least squares regression is shown for each comparison.

Figure 3

Effect of placental infections as determined by thick blood smears on the level of anti-VSA antibodies. The MFIs of plasma were obtained and compared between women who presented with positive and negative placentas in (A) primiparous women and (B) multiparous women (≥ 2). The same comparisons was made between positive and negative placentas in (C) pauciparous women and (D) multiparous women (≥ 3). The data showed here were performed on the parasite Gb337. P values for each comparison are indicated in the graphs and the grand means of all MFI values for each group are shown as dashed lines. The center line in each diamond shows the group mean, and the vertical spans of the diamond show the 95% confidence interval.

Figure 4

Comparison of antibody levels in surface recognition and CSA biding. CSA adhesion activities (■) and the corresponding anti-VSA levels (□) measured as mean fluorescent intensities of plasma obtained from primiparous women presented with infected placentas are shown. High CSA binding also stands for low anti-CSA adhesion activity. Plasma samples from a semi-immune nulligravid woman and a male served as controls.