Relationship of binding of immunoglobulin G to Plasmodium falciparum-infected erythrocytes with parasite endemicity and antibody responses to conserved antigen in immune individuals

Abstract

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.

FIG. 1.

Examples of histograms of flow cytometry data acquisition. The two histograms show results from the acquisition of 5,000 events of a 3% P. falciparum culture after gating on FL1 > 10¹ (left panel) and on FL1 > 10² (right panel). The gating on high FL1 values (>10²) selected the mature population of the cultured parasites. The binding of human IgG to IRBC was revealed by an Ig anti-IgG conjugated to TO. The fluorescence of TO-positive IRBC was measured in the FL2 channel. Shown are the reference negative control (plain curve histogram, pool neg.), the positive control (dashed curve, pool HIS), and a strong responder from Ndiop (filled curve).

FIG. 2.

Individual results of LIR in the two villages plotted in increasing age order. Individual LIR results (LIR calculated on 5,000 events gated at >10¹) measured in 81 individual samples from Dielmo and Ndiop are plotted after sorting in increasing age order. Sampling was done simultaneously in the two villages just after the end of the rainy season. The arrows indicate the separation between results from young individuals (≤15 years old) and data from older individuals. The mean LIRs are plotted as dashed lines and are indicated for Dielmo and Ndiop.

FIG. 3.

Mean level (± standard error) of IRBC recognition measured by flow cytometry in 81 samples from each village as a function of hemoglobin phenotype. Mean levels (± standard error) of the IRBC recognition ratio (LIR) measured in 81 individuals living in Dielmo and Ndiop are plotted. Shown are the mean LIRs from individuals with the HbAA phenotype (black) and the HbAS phenotype (grey) (n = 12 for Dielmo; n = 20 for Ndiop). The dashed line corresponds to the measure of IgG binding of the positive control (pool of immune sera).