Antibodies to PfsEGXP, an Early Gametocyte-Enriched Phosphoprotein, Predict Decreased Plasmodium falciparum Gametocyte Density in Humans

Abstract

Background: Antigametocyte-specific immune responses may regulate Plasmodium falciparum gametocyte density, providing the rationale for pursuing transmission-blocking vaccines (TBVs) that target gametocytes in the human host.

Methods: To identify novel antigametocyte TBV antigens, we interrogated the gametocyte proteome with our whole proteome differential screening method using plasma from a treatment-reinfection study conducted in western Kenya. At the start of the high-transmission season, 144 males (12-35 years) were enrolled and treated with quinine and doxycycline, peripheral venous blood samples were obtained, volunteers were observed, and weekly blood films were obtained for 18 weeks to quantify gametocytemia. Using plasma pooled from individuals with low versus high gametocyte carriage, we differentially screened a P falciparum gametocyte stage complementary deoxyribonucleic acid expression library.

Results: We identified 8 parasite genes uniquely recognized by gametocyte-resistant but not by gametocyte-susceptible individuals. Antibodies to one of these antigens, PfsEGXP, predicted lower gametocytemia measured over the 18-week transmission season (P = .021). When analyzed dichotomously, anti-PfsEGXP responders had 31% lower gametocyte density over 18 weeks of follow-up, compared with nonresponders (P = .04).

Conclusions: PfsEGXP is one of the first reported gametocyte-specific target of antibodies that predict decreased gametocyte density in humans and supports our novel TBV antigen discovery platform.

Figure 1.

Antibody response (total immunoglobulin G) to crude asexual and stage-specific sexual stage red blood cell (RBC) lysates in plasma from (n = 15) malaria-naive North American controls (light gray bars), gametocytemia/resistant (black bars), and gametocytemia/susceptible (gray bars) as determined by enzyme-linked immunosorbent assay. Each bar represents the mean of duplicate wells. Error bars = standard error of the mean. Abbreviations: iRBC, infected RBC lysate; O.D., optical density; uRBC, uninfected RBC lysate.

Figure 2.

Differential recognition of recombinant malarial proteins by immunoglobulin G (IgG) antibodies in plasma from malaria-naive North American controls (light gray bars, n = 15) and plasma pooled from resistant individuals (black bars, n = 10) and susceptible individuals (gray bars, n = 10) (Table 1). In this multiplexed bead-based assay, recombinant antigens were coupled to microspheres, probed with plasma, and incubated in biotinylated total IgG-specific secondary reagents. After incubation with streptavidin-phycoerythrin, bound antibody was analyzed on a BioPlex 100 multiplexed analyzer. Bovine serum albumin (negative control) reactivity subtracted from all values: 10219 = Pf3D7_1021900 aa1082–1653, 14662 = Pf3D7_1466200 aa1379–1687 (pfs355-A), 9246 = Pf3D7_0924600 aa1–409, 5069 aa1–195 = Pf3D7_0506900 aa1–195, 5069 aa101–195 = Pf3D7_0506900 aa101–195, and 411 = Pf3D7_0411000 aa294–843. Bars represent the mean of duplicate wells performed in triplicate (n = 3; error bars = standard error of the mean). Abbreviation: aa, amino acids.

Figure 3.

Repeated measures analysis of posttreatment blood films collected over 18 weeks of follow-up. (A) Antibody responses to rPfsEGXP-A modeled as a binary variable (responder defined as ≥ mean + 2 standard deviation of malaria-naive North American controls) or (B) as tertiles. Analysis is restricted to study participants who became gametocytemic at any time point over the follow-up period and is adjusted for age and asexual parasitemia. (C) Cox proportional hazard analysis of time to patent gametocytemia in rPfsEGXP-A responders versus nonresponders. Bars in A and B represent least square means adjusted for confounders (error bars = standard error of the mean). Abbreviations: IgG, immunoglobulin G; P falciparum, Plasmodium falciparum.

Figure 4.

Immunolocalization of PfsEGXP in methanol-fixed, Plasmodium falciparum NF54 strain early ring-like (day 1) to Stage III (day 6) gametocytes. Tightly synchronized gametocyte stage parasites were probed with rabbit affinity purified polyclonal anti-rPfsEGXP-A (1:100) (red) prepared by recombinant protein immunization, mouse anti-ring-infected erythrocyte surface antigen (RESA; 1:100), and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to stain parasite nuclei. Immunofluorescence images are representative of experiments performed at least 3 times. Scale bar = 5 μm. Abbreviation: p.i., postinvasion.