Evidence for the involvement of VAR2CSA in pregnancy-associated malaria

Abstract

In Plasmodium falciparum-endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

Figure 1.

Analysis by flow cytometry (a–d) and confocal microscopy (e–h) of intact erythrocytes infected by P. falciparum line NF54 (a, b, e, f, and i) or FCR3 (c, d, g, and h) and labeled by IgG in rabbit antisera raised against VAR2CSA DBL5-ɛ (a–d, solid red, and e–h) or VAR4 DBL5-δ (a–d, solid gray) as a control serum. Infected erythrocytes subjected to several rounds of in vitro selection for adhesion to CSA (a, c, e, g, and i) or unselected (b, d, f, and h) are shown. Confocal microscopy images (e–h) were unstained (left), stained with ethidium bromide and labeled by anti-VAR2CSA IgG (middle), or stained by anti-VAR2CSA IgG only. Confocal panel i shows the double stained parasites. The left image (red) is IgG staining with pooled plasma from pregnant women; the middle image (green) is stained with anti-VAR2CSA IgG; and the right image is an overlay of the two first images. The staining of the central spot in the confocal images without EtBr is caused by autofluorescence of hemozoin.

Figure 2.

Flow cytometry analyses of intact infected erythrocytes (a and b) labeled by IgG against VAR2CSA DBL5-ɛ (solid red) and VAR4 DBL5-δ (solid gray). Erythrocytes were infected with CSA-selected NF54 (a) or a subline of this parasite, which had been subjected to additional in vitro selection for binding to VAR2CSA DBL5-ɛ–specific IgG (b). (c and d) The recognition of the two parasite lines by plasma IgG from 10 Ghanaian term-pregnant women (pFb), 10 sympatric Ghanaian men (Mb), and 10 Danish controls (DK). For each group, the median (central line), the central 50% (box), the central 80% (bars), and the central 90% (•) of data points are shown.

Figure 3.

Colabeling of intact erythrocytes infected by P. falciparum line FCR3 by IgG in rabbit antisera raised against VAR2CSA DBL5-ɛ and by plasma pools from P. falciparum–exposed, term-pregnant women (a and c) or from sympatric men (b and d). Infected erythrocytes subjected to several rounds of in vitro selection for adhesion to CSA (a and b) or without pretreatment (c and d) are shown.

Figure 4.

Plasma levels of IgG with specificity for the DBL1-X and DBL5-ɛ domains of VAR2CSA and the DBL5-δ domain of VAR4 in 46 women (Fa) and 39 sympatric men (Ma) from an area of hyperendemic seasonal malaria transmission (a) and in 27 term-pregnant women (pFb) and 30 sympatric 39 men (Mb) from an area of hyperendemic stable malaria transmission (b). (c) Levels of GLURP-specific IgG in the same groups of malaria-exposed individuals as in a and b. Plasma levels of VAR2CSA DBL5-ɛ–specific IgG at the time of delivery in 62 pregnant women (gravidities ranging from 1 to 10) from an area of hyperendemic seasonal malaria transmission (d). For each group, the median (central line), the central 50% (box), the central 80% (bars), and the central 90% (•) of data points are shown.