An Inhibitory Antibody Blocks Interactions between Components of the Malarial Invasion Machinery

Abstract

Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1.

Binding ofPlasmodiummerozoite proteins RON2 and AMA1 triggers commitment to invasion

“…mAbs 1F9 and 4G2 have been shown to inhibit invasion, and this result suggests that invasion is not only blocked by interfering with the binding of AMA1 and RON2 but may also dislodge the bound complex. As the epitope recognized by mAb 4G2 lies adjacent to the hydrophobic pocket in the flexible domain II loop (21,22), these data also suggest that the inhibitory activity of mAb 4G2 could be mediated by stabilizing the domain II loop in an unfavorable conformation for RON2 binding.…”

“…Previously, Collins et al (22) showed that a mutation in the hydrophobic pocket (Y251A) of AMA1 prevented the binding of AMA1 to a complex of RON proteins in vitro. However, the specific RON protein that binds this pocket was not identified.…”

“…and Tg identified a hydrophobic pocket formed by two PAN domains (18)(19)(20). Monoclonal antibodies (mAbs) that bind near (mAb 4G2) or in (mAb 1F9) the PfAMA1 hydrophobic pocket interfere with merozoite invasion of RBCs (21)(22)(23). Studies in Tg and Pf showed that in parasite detergent extracts, AMA1 coimmunoprecipitated with a complex of proteins that are members of a family found in the rhoptry neck (RONs; refs.…”

Applications of 19F-NMR in Fragment-Based Drug Discovery

“…Several monoclonal antibodies targeting AMA1 block the interaction with the RON complex and thereby inhibit parasite invasion [40,43,44]. Likewise, several peptides derived from phage-display or from AMA1's binding partner in the RON complex, RON2, are inhibitory [45], establishing the AMA1-RON interaction as a potential therapeutic target [39,46].…”

Using Population Genetics to Guide Malaria Vaccine Design

“…AMA1 then relocates to the parasite surface [75] where further processing during invasion leads to shedding of two fragments of 44 and 48 kDa whilst the 22kDa cytoplasmic tail remains in the membrane and is carried into the invaded erythrocyte [76,77]. AMA1 is now known to function in formation of the tight junction through interactions with rhoptry neck proteins [78,79]. This molecule is also expressed in sporozoites where it is involved in hepatocyte invasion [80].…”