Mode of Action of Invasion-Inhibitory Antibodies Directed against Apical Membrane Antigen 1 of Plasmodium falciparum

Abstract

Antibodies against apical membrane antigen 1 (AMA-1) of Plasmodium falciparum inhibit merozoite invasion into erythrocytes. Invasion-inhibitory polyclonal AMA-1 antibodies inhibit secondary proteolytic processing and surface redistribution of AMA-1 on merozoites. We present evidence supporting inhibition of processing and redistribution as probable causes of inhibition of invasion by polyclonal antibodies. Polyclonal anti-AMA-1 was much more inhibitory than monoclonal antibody (MAb) 4G2dc1 in an invasion assay. Although both polyclonal and monoclonal immunoglobulin G (IgG) inhibited secondary processing of the 66-kDa form of AMA-1, only polyclonal IgG caused its anomalous processing, inhibited its redistribution, and cross-linked soluble forms of AMA-1 on merozoites. Moreover, Fab fragments of polyclonal IgG that fail to cross-link did not show the enhancement of inhibitory effect over intact IgG, as observed in the case of Fab fragments of MAb 4G2dc1. We propose that although blocking of biologically important sites is a common direct mode of action of anti-AMA-1 antibodies, blocking of AMA-1 secondary processing and redistribution are additional indirect inhibitory mechanisms by which polyclonal IgG inhibits invasion. We also report a processing inhibition assay that uses a C-terminal AMA-1-specific MAb, 28G2dc1, to detect merozoite-bound remnants of processing (ϳ20 kDa from normal processing to 48 and 44 kDa and ϳ10 kDa from anomalous processing to a 52-kDa soluble form of AMA-1). The ratio of intensity of 10-kDa bands to the sum of 10-and 20-kDa bands was positively correlated with inhibition of invasion by polyclonal antibodies. This assay may serve as an important immunochemical correlate for inhibition of invasion.

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

“…Purified Fab fragments of mAb 4G2 were considerably more potent in invasion-inhibition assays, relative to that of the intact antibody. Similar results were reported by Dutta et al who examined the invasion-inhibitory activity of impure preparations of 4G2 Fab (43). This result is intriguingly reminiscent of similar findings reported in an early study of invasion-inhibition by a mAb specific for AMA1 of the simian malaria parasite P. knowlesi; in that work, the authors speculated that the enhanced potency of the Fab fragments might be a result of charge effects, removal of the Fc perhaps reducing electrostatic repulsion between the antibody and the negatively charged merozoite surface (61,62).…”

“…However, some antibodies that prevent invasion also interfere with shedding, raising the possibility that shedding is essential for invasion. Its inhibition may represent one mechanism by which anti-AMA1 antibodies exert invasion-inhibitory activity (43,44).…”

Phase I dose escalation safety and immunogenicity trial of Plasmodium falciparum apical membrane protein (AMA-1) FMP2.1, adjuvanted with AS02A, in malaria-naïve adults at the Walter Reed Army Institute of Research

“…We have described an immuno-chemical correlate of anti-AMA-1 antibodies that measures the ability of these antibodies to inhibit the natural proteolytic processing of PfAMA-1 on the merozoite surface [29,30]. We conducted a parallel GIA and PIA on sera collected 2 weeks post third immunization.…”

“…This antibody was a kind gift of Dr. Alan W. Thomas, Biomedical Primate Research Center, Rijswijk, The Netherlands. The PIA ratio was calculated as band intensities of the 10-kDa/(10-kDa + 20-kDa) AMA-1 specific bands on the Western blot [29]. The GIA plate contained 80 L of 0.5% parasitemia schizonts + normal human RBC at 4% hematocrit and 20 L of test serum in triplicates.…”

“…Immunization of rabbits with a pre-clinical lot of FMP2.1 formulated with AS02A-induced functional antibodies with strong growth inhibitory and processing inhibition activity in in vitro assays [29,30]. Evaluation of clinical grade (cGMP, current Good Manufacturing Practice) FMP2.1 formulated with AS02A in a standard rhesus monkey model demonstrated that the vaccine was safe, minimally reactogenic, and able to induce potent cellular and humoral immune responses, including growth inhibitory antibodies against homologous P. falciparum 3D7 parasites (Stewart, unpublished).…”