Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants

Abstract

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.

FIGURE 1

mAb recognition of P. falciparum-infected erythrocytes obtained from naturally infected individuals from Benin (○) and Tanzania (●). IEs obtained from the placenta at delivery, peripheral venous blood of pregnant women, or peripheral blood of children with malaria were labeled with each of the eight mAbs and analyzed by flow cytometry. The number of mAbs showing significant labeling was recorded for each isolate. CH, children with malaria; PP, placenta; PV, peripheral venous blood.

FIGURE 2

mAb reactivity with native and recombinant VAR2CSA. Erythrocytes infected by the VAR2CSA-expressing P. falciparum parasite line FCR3-BeWo (A) or recombinant full-length constructs of P. falciparum FCR3-VAR2CSA (B) or 3D7-VAR2CSA (C) were incubated with varying concentrations of mAbs. Reactivity was tested by flow cytometry (A) or ELISA (B, C). A pool of all eight mAbs (All) and a mAb of unrelated specificity (Control) were included in the flow cytometry experiments as positive and negative controls, respectively. Each experiment was performed at least twice. Total Ab concentrations are given, and the results of a typical experiment are shown. Ab labels are as in Fig. 1.

FIGURE 3

mAb interference with the binding of recombinant full-length FCR3-VAR2CSA (FCR3-FV2) to CSPG-h. FCR3-FV2 was preincubated with the indicated concentrations of single mAbs (A) or the indicated combinations of Abs (B) before flowing FCR3-FV2 past CSPG-h coupled to the surface of a Biacore chip. Ab labels are as in Fig. 1.

FIGURE 4

mAb interference with the binding of P. falciparum IEs to CSA. The ability of the Abs to interfere with static CSA-specific adhesion of placental primary isolates (B cell supernatants at 1:5 dilution) (A) or flow adhesion of CSA-selected FCR3 IEs (purified mAbs at a final concentration of 120 µg/ml) (B) was measured. Soluble CSA was used at a concentration of 50 µg/ml. Ab labels are as in Fig. 1. Typical flow assay micrograph frames of CSA-adhering IEs after incubation in PBS (C), a pool of all Abs (D), and sCSA (E) also are shown. sCSA, soluble CSA.

FIGURE 5

Phagocytosis of opsonized P. falciparum IEs. Erythrocytes infected by EtBr-labeled P. falciparum FCR3-BeWo (A), HB3-BeWo (B), and FCR3-Ctrl (C) were opsonized with human mAbs (B cell supernatants at 1:5 dilution) either alone (PAM1.4, PAM2.8, PAM3.10, PAM4.7, PAM5.2, PAM6.1, PAM7.5, or PAM8.1), in combination (All), or with plasma from P. falciparum-exposed men, P. falciparum-exposed multigravid women, or unexposed controls and coincubated with THP-1 cells. The difference in phagocytosis in the absence (open curve) and presence of Abs (shaded curves) is shown in A. Each experiment was performed at least twice. The results of a typical experiment are shown in as the percentage of THP-1 cells that became EtBr-positive after coincubation with EtBr-labeled IEs. EM, exposed men; EMW, exposed multigravid women; UC, unexposed controls.