The glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for antibody-dependent monocyte-mediated inhibition of parasite growth in vitro

Abstract

Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.

FIG. 1

Purification and reactivity of anti-R0 and anti-R2 antibodies in ELISAs. (A) Schematic representation of the purification of anti-R0 and anti-R2 antibodies. Numbers represent IgG fractions listed in Table 1. Run-through and eluates are indicated by thin and thick arrows, respectively. (B and C) Results obtained with different fractions in ELISA. (B) Binding of IgG fractions to R0; (C) binding of IgG fractions to R2-coated plates. IgG concentration is plotted on a semilogarithmic scale.

FIG. 2

Analysis of antibody specificities in fraction 1 (A) and fraction 2 (B). The fractions were each diluted 640-fold and preincubated with the recombinant antigens R0 (open symbols) of R2 (filled symbols) at the indicated concentration before addition to wells coated with R0 (squares) or R2 (triangles). The key indicates the competing antigen (solution) and the solid-phase antigen (ELISA) used for each curve. Please note that the curves do not originate from the same point on the ordinate since competing antigen was already added at this point.

FIG. 3

Immunoprecipitation analysis of P. falciparum proteins. Parasites were grown and labelled as previously described (21). Labelled polypeptides were subjected to immunoprecipitation with fraction 2 (lane 1, culture supernatant; lane 2, cell pellet), fraction 1 (lane 3, culture supernatant; lane 4, cell pellet), or purified IgG from Danish donors never exposed to malaria (lane 5, culture supernatant; lane 6, cell pellet). The sizes (in kilodaltons) of molecular mass markers are indicated on the right.

FIG. 4

ADCI assay. (A) Shown are the means of the SGI (calculated as described in Materials and Methods) obtained with the following antibodies: fraction 1 (135 μg/ml) (number of independent experiments [n = 1) and fraction 2 (135 μg/ml) (n = 3); IgG (200 μg/ml) from normal French blood donors (n = 2), IgG (200 μg/ml) from hyperimmune African blood donors (n = 6), and a 1/10 solution in RPMI 1640 of the antibodies affinity purified on peptides GL5 (n = 3) and GL9 (n = 3) as described in Materials and Methods. Human affinity-purified antibodies on MSP-3b (n = 3) and RESA (n = 3), diluted 1/10 in RPMI 1640, were included as positive and negative controls, respectively (23). (B) Dose dependency of anti-GLURP antibodies in ADCI experiments. Shown is the SGI plotted versus the IgG concentration for fraction 1 (n = 3) and fraction 2 (n = 2). (C) Comparison of ADCI effects of the fractions 2 to 5. Shown are the means of the SGI of purified IgG adjusted to 45 μg/ml (fractions 2, 3, and 4) and 200 μg/ml (fraction 5 and normal French IgG). The experiments with fraction 2 and normal French IgG were performed twice.