Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells

Abstract

Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate molecule for inclusion in a human malaria vaccine and is strongly conserved in the genus. We have investigated its function in merozoite invasion by incubating Plasmodium knowlesi merozoites with red cells in the presence of a previously described rat monoclonal antibody (MAb R31C2) raised against an invasion-inhibitory epitope of P. knowlesi AMA-1 and then fixing the material for ultrastructural analysis. We have found that the random, initial, long-range (12 nm) contact between merozoites and red cells occurs normally in the presence of the antibody, showing that AMA-1 plays no part in this stage of attachment. Instead, inhibited merozoites fail to reorientate, so they do not bring their apices to bear on the red cell surface and do not make close junctional apical contact. We conclude that AMA-1 may be directly responsible for reorientation or that the molecule may initiate the junctional contact, which is then presumably dependent on Duffy binding proteins for its completion.

FIG. 1.

General survey of normal invasion by P. knowlesi 50 min after addition of merozoites to fresh RBC, showing a number of ring stages. Scale bar, 5 μm.

FIG. 2.

General survey of a merozoite-RBC culture incubated for 50 min in the presence of inhibitory antibody R31C2, showing attachment of merozoites and absence of ring stages. RBC infected with mature parasites were seen. These derive from the first generation, used to prepare the merozoite inoculum. Scale bar, 5 μm.

FIG. 3.

Examples of merozoites attached to RBC by their nonapical surfaces at 10 min of incubation in the presence of inhibitory antibody R31C2. Note a small membranous vacuole present in the RBC (arrow), indicating transient apical contact by a merozoite. The RBC is partially wrapped around the lower merozoite, but there is no close junctional attachment. Scale bar, 1 μm.

FIG. 4.

Another example of nonapical attachment in the presence of inhibitory antibody (10 min). Again, although the lower merozoite gives the superficial appearance of undergoing invasion, there is no close junction, and the RBC is partially wrapped around the merozoite, as also seen in the early stages of normal invasion (see Fig. 5). Scale bar, 1 μm.

FIG. 5.

An example of early merozoite-RBC interaction in normal invasion (1 min) in which the RBC is wrapped around the merozoite without forming a close junctional contact. Scale bar, 2 μm.

FIG. 6.

Distant merozoite-RBC attachment in the presence of inhibitory antibody R31C2 (10 min of incubation), the merozoite adhering to the RBC membrane by means of the filamentous merozoite coat (FC), as shown in detail (Fig. 7). Scale bar, 1 μm.

FIG. 7.

Distant merozoite-RBC attachment in the presence of inhibitory antibody R31C2 (10 min of incubation), the merozoite adhering to the RBC membrane by means of the filamentous merozoite coat (FC). Scale bar, 0.5 μm.

FIG. 8.

An example of close junctional (CJ) attachment between the apex of a merozoite and an RBC during normal invasion, with the formation of a membranous vacuole (MV) in the RBC at the merozoite apex. Scale bar, 0.5 μm.