Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection

Abstract

Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 (PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion.

Fig. 1. PfSEA-1 is essential for parasite growth

(A) Immunoblot analysis of D10-PfSEA1-DD. Parasites were sorbitol-synchronized at the ring stage and incubated until the schizont stage (44 to 48 hours) with or without 0.25 μM Shield-1 and probed with mouse anti–PfSEA-1 produced by DNA immunization (top panel) or rabbit anti-MSP2 (loading control, bottom panel). Lane 1, Shield-1–treated cultures; lane 2, untreated cultures; lane 3, uninfected RBCs. The top arrow indicates PfSEA-1 fused with a DD tag; the bottom arrow indicates MSP-2. (B) Replication curves for D10-PfSEA1-DD parasites. Ring-stage parasites were cultured with or without 0.25 μM Shield-1. Parasitemia was measured by flow cytometry. Each data point represents the mean of three replicates; error bars indicate SEM.

Fig. 2. Antibodies to PfSEA-1 inhibit malaria parasite growth

Polyclonal antibodies to PfSEA-1 generated by DNA (A to C) and recombinant protein (D and E) immunization, and mAbs to PfSEA-1 (F) inhibit parasite growth and invasion by 58 to 74% for three parasite strains in vitro. Mature trophozoite-stage 3D7 [(A), (D), and (F)], W2 [(B) and (E)], and D10 (C) parasites were were cultured in the presence of mouse antisera to PfSEA-1 [1:10 dilution, (A) to (E)]. Negative controls included no mouse sera and preimmune mouse sera (1:10 dilution). In (F), parasites were incubated in the presence of mAbs to PfSEA-1 CF3 or CF6 (250 μg/ml) or buffer. Parasites were cultured for 24 hours, and ring-stage parasites were enumerated by microscopy. Bars represent the mean of five independent replicates, with each replicate performed in triplicate. Error bars represent SEMs. Comparisons between pre- and postimmune mouse sera by a nonparametric Mann-Whitney U test are indicated. (A) to (C) and (F) are representative of three independent experiments. (D) and (E) are representative of five independent experiments.

Fig. 3. Immunolocalization of PfSEA-1

(A) Infected RBCs were probed with mouse anti–PfSEA-1 (green) and rabbit anti–MSP-1 (red) and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) to label parasite nuclei. PfSEA-1 is detected only in schizont-infected RBCs. Scale bar for free merozoite images, 2 μm. Scale bar for remaining images, 10 μm. (B) Schizont-infected RBCs do not label when probed with preimmune mouse sera. Scale bar, 10 μm. (C) Nonpermeabilized, unfixed schizont-infected RBCs were probed with mouse anti–PfSEA-1 (red) and rabbit anti–glycophorin A (green) and counterstained with DAPI to label parasite nuclei. PfSEA-1 colocalized with glycophorin A to the surface of schizont-infected RBCs. Scale bar, 5 μm. (D to F) Nonpermeabilized, unfixed schizont-infected RBCs were probed with mouse anti–PfSEA-1 (5-nm gold particles) and rabbit anti–glycophorin A (10-nm gold particles). PfSEA-1 localized to the schizont/parasitophorous vacuole membrane (black arrow), Maurer’s clefts (yellow arrow), and the inner leaflet of the RBC membrane (gray arrow), whereas glycophorin A was confined to the outer leaflet of the RBC membrane (white arrow). Scale bar in (D), 0.5 μm. Scale bars in (E) and (F), 0.1 μm.

Fig. 4. Antibodies to PfSEA-1 inhibit schizont egress

Antibodies to PfSEA-1 generated by DNA (A to C) and recombinant protein (D and E) immunization, as well as mAb to rPfSEA-1A (F and G), inhibit schizont egress for three parasite strains in vitro. Early schizont stage 3D7 [(A) and (D)], W2 [(B) and (E)], and D10 (C) parasites were incubated in the presence of mouse anti–PfSEA-1 (1:10 dilution). Negative controls included no mouse sera and preimmune mouse sera (1:10 dilution). In (F), 3D7 parasites were incubated in the presence of mAbs to PfSEA-1 CF3 or CF6 (at 250 μg/ml) or buffer. In (G), 3D7 parasites were incubated in the presence of increasing concentrations of mAb to PfSEA-1 CF6 or buffer. Schizonts were counted 12 hours after treatment, and the percent of initial schizonts arrested was calculated. Bars represent the mean of five independent replicates, with each replicate performed in triplicate. Error bars represent SEMs. Comparisons between pre- and postimmune mouse sera and between buffer and mAb-treated cultures by a nonparametric Mann-Whitney U test are indicated. All panels are representative of three independent experiments.

Fig. 5. Vaccination with rPbSEA-1A protects mice from challenge with P. berghei ANKA

(A) Expression and purification of rPbSEA-1A from E. coli soluble lysates. Lane 1, nickel chelate chromatography of soluble E. coli lysate; lane 2, hydrophobic interaction chromatography of lane 1; lane 3, anion exchange chromatography of lane 2. (B) Antibody response of mice vaccinated with rPbSEA-1A in three vaccine trials. After vaccination, mice generated robust anti–rPbSEA-1A IgG responses in each trial. Bars represent mean fluorescence; error bars represent SEMs. White bars represent anti–rPbSEA-1A IgG responses in mice immunized with rPbSEA-1A, gray bars represent anti-BSA IgG responses in mice immunized with rPbSEA-1A, black bars represent anti–rPbSEA-1A IgG responses in mice immunized with adjuvant alone, and bricked bars represent anti-BSA IgG responses in mice immunized with adjuvant alone. (C) Vaccine trial 1: BALB/c mice vaccinated ip with TiterMax (n = 11) and challenged ip with 10⁶ P. berghei ANKA–infected RBCs had 2.25-fold higher parasitemia on day 7 after challenge (P = 0.0002) as compared to mice vaccinated with rPbSEA-1A (n = 10). (D) Vaccine trial 2: C57BL/6 mice vaccinated sc with TiterMax (n = 10) and challenged with 200 P. berghei ANKA sporozoites iv had 1.52-fold higher parasitemia on day 7 after challenge (P = 0.002) as compared to mice vaccinated with rPbSEA-1A (n = 10). (E) Vaccine trial 3: BALB/c mice vaccinated ip with TiterMax (n = 10) and challenged ip with 10⁴ P. berghei ANKA–infected RBCs had 3.05-fold higher parasitemia on day 7 after challenge (P = 0.001) as compared to mice vaccinated with rPbSEA-1A (n = 10). (F and G) Vaccine trial 4: BALB/c mice vaccinated ip with TiterMax (n = 9) and challenged ip with 10⁴ P. berghei ANKA–infected RBCs had 3.9-fold higher parasitemia on day 7 after challenge (P = 0.0009) as compared to mice vaccinated with rPbSEA-1A in TiterMax. The median survival of rPbSEA-1A vaccinated mice was 1.8-fold longer (P < 0.0001) as compared to that of adjuvant-only control mice (n = 9). (H and I) Vaccine trial 5: Antisera (rPbSEA-1A or adjuvant alone) or normal mouse sera were passively transferred (0.5 ml, ip) into naïve BALB/c mice (n = 5 mice per group) on day −1 and day +1. On day 0, mice were challenged ip with 10⁴ P. berghei ANKA–infected RBCs. Control mice had 2.7-fold higher parasitemia on day 7 after challenge (P = 0.009), compared to anti-rPbSEA-1A treated mice. The median survival of rPbSEA-1A vaccinated mice was 2.0-fold longer than that of controls (P < 0.0001).

Fig. 6. Antibodies to rPfSEA-1A predict reduced malaria severity and parasitemia

(A) Incidence of severe malaria and death in Tanzanian children aged 1.5 to 3.5 years during intervals with detectable and undetectable antibodies to PfSEA-1 (1688 and 23,806 weeks, respectively). No cases of severe malaria or death occurred during intervals with detectable antibodies to rPfSEA-1A. Error bars represent 95% CI, adjusted for repeated measures. (B) IgG antibodies to rPfSEA-1A predict decreased P. falciparum parasitemia. We enrolled n = 138 Kenyan males aged 12 to 35 years at the start of a high-transmission season, treated them with quinine and doxycycline, and followed them for 18 weeks with weekly blood smears. We collected serum 2 weeks after treatment and measured IgG antibody levels to rPfSEA-1A. In GEE repeated-measures models, individuals with detectable IgG antibodies to rPfSEA-1A had 50% lower parasite densities over 18 weeks of followup as compared to individuals with no detectable IgG antibodies to rPfSEA-1A, after adjusting for potential confounders (P < 0.04). Columns depict least-square mean parasitemia; error bars depict SEM.