Invasion-inhibitory antibodies inhibit proteolytic processing of apical membrane antigen 1 of Plasmodium falciparum merozoites

Abstract

Apical membrane antigen 1 (AMA-1) is a promising vaccine candidate for Plasmodium falciparum malaria. Antibodies against AMA-1 of P. falciparum (PfAMA-1) interrupt merozoite invasion into RBCs. Initially localized within the apical complex, PfAMA-1 is proteolytically processed and redistributed circumferentially on merozoites at about the time of their release and invasion into RBCs. An 83-kDa precursor form of PfAMA-1 is processed to 66-kDa and then to 48- and 44-kDa products. We show that, even at low concentrations, IgG antibodies against correctly folded recombinant PfAMA-1 cross-linked and trapped the 52-, 48-, and 44-kDa proteolytic products on merozoites. These products are normally shed into the culture medium. At higher concentrations antibodies inhibited invasion into RBCs and caused a reduction in the amount of 44- and 48-kDa products, both on merozoites and in the culture medium. A corresponding increase also occurred in the amount of the 66- and 52-kDa forms detected on the merozoites. These antibodies also prevented circumferential redistribution of AMA-1. In contrast, monovalent invasion-inhibitory Fab fragments caused accumulation of 66- and 52-kDa forms, with no cross-linking, trapping, or prevention of redistribution. Antibodies at low concentrations can be used as trapping agents for intermediate and soluble forms of AMA-1 and are useful for studying proteolytic processing of AMA-1. With this technique, it was confirmed that protease inhibitor chymostatin and Ca2+ chelators can inhibit the breakdown of the 66-kDa form. We propose that antibodies to AMA-1 capable of inhibiting erythrocyte invasion act by disrupting proteolytic processing of AMA-1.

Fig. 1.

(A) Processing assay with immune serum pool at two dilutions. Each lane represents merozoites released from ≈1.4 × 10⁵ schizonts: lane 1, preimmune (1:10 dilution); lanes 2 and 3, postimmune at 1:10 and 1:2,500 dilutions, respectively; lane 4, soluble AMA-1 fragments immunoprecipitated from culture supernatant (representative of ≈5 × 10⁶ rupturing schizonts, assuming 100% recovery). (B) Processing assay samples corresponding to lanes 3 and 4 of A run under reduced conditions and immunostained with biotin-labeled IgG against reduced and alkylated AMA-1 protein. (C) Immunoprecipitation from culture supernatants of the processing assay containing 1:10 preimmune (lane 1), 1:10 postimmune (lane 2), and 1:2,500 postimmune serum (lane 3). PfAMA-1-specific bands and molecular mass marker positions (Multimark, Invitrogen) are shown with arrows.

Fig. 3.

Effect of anti-AMA-1 antibodies on AMA-1 processing during schizont maturation and merozoite release. The processing assay was performed with 1:10 dilution of preimmune or postimmune pools. Samples were drawn at five times (T1–T5 corresponding to 0–90% rupture; see Results). To rule out immunoprecipitation from culture supernatant, postimmune serum was added to a preimmune serum-containing well (at T5, keeping the final dilution 1:10), and incubation continued at 37°C for another 30 min (lane a, postimmune sample at T6; lane b, preimmune control incubated with the postimmune serum at T6). Lane c corresponds to the sample in lane a analyzed for reactivity to AMA-1-specific mAb 4G2dc1.

Fig. 2.

Processing assay showing dose–response of an individual inhibitory rabbit serum on AMA-1 processing. Final serum dilutions used in the assay were 1:2,400, 1:810, 1:270, 1:90, 1:30, and 1:20.

Fig. 4.

(A) Processing assay showing the effects of IgG against reduced and alkylated AMA-1 (IgG-R/A) and refolded AMA-1 (IgG-Ref) proteins. IgG concentrations in lanes 1–4 were 0.00035, 0.0035, 0.035, and 0.35 mg/ml, respectively. (B) Processing assay showing the effect of anti-AMA-1 Fab fragments. Fab fragment concentrations in lanes 1–5 were 0.000014, 0.00014, 0.0014, 0.014, and 0.14 mg/ml, respectively. C indicates media control. A parallel assay with trapping antibodies (1:2,500 postimmune pool) is also shown.

Fig. 5.

Double immunofluorescence image, with a dual-cube filter, of free merozoites released in the presence of preimmune (Left, 1:10 dilution) or postimmune (Right, 1:10 dilution) serum. The preimmune sample was incubated with 1:10 postimmune sera for 1.5 h on ice after rupture. Slides were acetone-fixed, and AMA-1 was visualized by staining with FITC-conjugated anti-rabbit (green fluorescence), and the merozoite surface was demarcated by staining with P. falciparum MSP-1-specific mAb 5.2 and anti-mouse phycoerythrin (red fluorescence). (Insets) Enlarged view of a single merozoite.

Fig. 6.

(a) Processing assay in the presence of protease inhibitors. (b) Identical assay performed in the presence of trapping antibodies (1:2,500 AMA-1 immune serum pool). (A) Lane 1, PMSF (200 μM); lane 2, TLCK (100 μM); lane 3, TPCK (100 μM); lane 4, leupeptin (100 μM); lane 5, chymostatin (100 μM); lane 6, antipain (100 μM); lane 7, E64 (10 μM); lane 8, pepstatin (5 μM); lane 9, 1,10-phenanthroline (1 mM); lane 10, EDTA (1 mM); lane 11, EGTA (1 mM); lane 12, ethanol control; lane 13, DMSO control; lane 14, PBS control. (B) Dose–response of chymostatin, EDTA, and EGTA on AMA-1 processing. Concentrations of inhibitors used from lanes 1–4 were: chymostatin, 100, 50, 25, and 12.5 μM; EDTA and EGTA, 2, 1, 0.5, and 0.25 mM, respectively; lane c, DMSO control; lane c′, PBS control. (C) Processing assay showing the effect of 1 mM MgCl₂ or 1 mM CaCl₂ added to reverse the EDTA and EGTA (1 mM each) mediated processing inhibition. Lane 1, EDTA; lane 2, EGTA; lane c, PBS control.

Fig. 7.

Proposed model of AMA-1 processing. The PfAMA-1 molecule is represented by a prosequence (open oval), three subdomains based on the predicted disulfide bond structure (see ref. 29) (dark domain I-, medium domain II-, and light domain III-colored circles), transmembrane domain (clear rectangle), and cytoplasmic domain (gray rectangle). Putative membrane-bound sheddases are represented by filled triangles. PfAMA-1₄₈ and PfAMA-1₄₄ represent the soluble forms of AMA-1. PfAMA-1₄₄ is represented in its native conformation with a disulfide bond (—SS—) connecting the 44-kDa form to its proteolytic fragment (see ref. 11). Arrows indicate the substrate (start) and products (end) of each putative enzymatic step. Dashed arrow represents translocation of PfAMA-1₆₆ from the apical complex to the surface (apical end). Our data suggest that PfAMA-1 processing on merozoites includes a 52-kDa form, which is either a normal intermediate or a product of anomalous processing that may further be processed to PfAMA-1₄₈₊₄₄. In this hypothetical model, proteolytic steps 1 and 2 are expected to be chymostatin-sensitive, whereas steps 2 and 3 are EGTA-sensitive. All three proteolytic steps and the redistribution of AMA-1 appear to be sensitive to anti-AMA-1 antibodies. (Left) Normal processing, translocation, and redistribution of AMA-1. (Right) Antibody-mediated processing inhibition, cross-linking, and trapping of AMA-1 fragments.