High Throughput Functional Assays of the Variant Antigen PfEMP1 Reveal a Single Domain in the 3D7 Plasmodium falciparum Genome that Binds ICAM1 with High Affinity and Is Targeted by Naturally Acquired Neutralizing Antibodies

Abstract

Plasmodium falciparum–infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLβC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLβC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2βC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2βC2_{PF11_0521} best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLβC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLβC2 domain. DBL2βC2_{PF11_0521} binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2βC2_{PF11_0521} and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses. Plasmodium falciparum exports the protein PfEMP1 to the surface of parasitized erythrocytes for roles in immunoevasion and adhesion. The size and structural complexity of this diverse protein family have limited earlier studies of PfEMP1 biology to low throughput and semi-quantitative approaches. We developed a high throughput quantitative assay of PfEMP1 adhesion and used it to analyze structural features of domains that bind the putative cerebral receptor ICAM1, as well as to survey the acquisition of functional antibodies in malaria-exposed children and adults. In studies of the PfEMP1 repertoire of clone 3D7 parasites, a single specific domain bound ICAM1 strongly. PfEMP1 domains that bind ICAM1 strongly have conserved features, including conserved amino acids within otherwise highly variant flexible loops of the protein. While neutralizing antibodies against the PfEMP1–ICAM1 interaction were uncommon in Tanzanian children, such antibodies were common in east African adults, possibly explaining why immune adults rarely carry ICAM1-binding parasites. This high throughput platform will significantly accelerate studies of PfEMP1 binding domains and the corresponding antibody responses involved in protective immunity.