Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding

Abstract

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLβ3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4–expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1–specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

Antibodies to Intercellular Adhesion Molecule 1-Binding Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLβ Are Biomarkers of Protective Immunity to Malaria in a Cohort of Young Children from Papua New Guinea

“…These results are consistent with the conservation of these specialized classes of PfEMP1 proteins across large geographic distances, since previous studies have focused on African parasites or reference isolates from diverse locations (18,21,31). The high reactivity of children's sera to the full-length DBL␤ PF11_0521 supports the notion that conserved epitopes exist that can be targeted by crossreactive and protective antibodies (18,21,32). However, the ICAM1-binding motif is located in the C-terminal part of the protein (SD3 region), and no sequence traits in the N-terminal part of the domain have been linked to ICAM1 binding.…”

“…PF11_0521 belongs to a class of group A PFEMP1 proteins with DBL␤ domains shown to bind ICAM1 and found exclusively in PfEMP1 proteins with EPCR-binding CIDR␣1 (16,32). These domains are adjacent to each other, and together, they may produce a binding phenotype often associated with sequestration leading to cerebral…”

“…Furthermore, ICAM1binding-inhibitory antibodies are common in hyperimmune adults living in areas of endemicity (19), suggesting that they may play an important role in the maintenance of clinical immunity. The mechanism of protection may be through direct inhibition of ICAM1 binding, as shown in in vitro experiments (21,32); indirectly, through prevention of binding of other domains, such as EPCR-binding CIDR␣1 (12); or synergistic antibody responses targeting multiple domains.…”

Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin

“…This is consistent with inhibition of rosetting by impairing access to the RBC-binding site. The close proximity or partial overlap of the BD20E4 epitope with the RBC-binding site is reminiscent of findings observed for the binding site of the 24E9 Fab mAb, which overlaps with the ICAM-1 binding site on the surface of the DBLβ3-D4 domain of PfEMP1-PFD1235w [ 44 ]. None of the anti-DBL1 VarO mAbs appear to bind to the region of subdomain 3 identified as the binding site of mAbs that potently disrupted FCR3S1.2 (alias IT4Var60) or R29 (alias IT4Var9) rosettes [ 45 ] located in a different region of the DBL1 surface (see Additional file 4 : Figure S3).…”

MS-Based Approaches Enable the Structural Characterization of Transcription Factor / DNA Response Element Complex

“…In HDX experiments, the exchange rate of backbone amide hydrogens, which is affected by solvent accessibility and possible involvement in hydrogen bonding, can be directly determined by MS analysis. This technique has been broadly employed to study conformational changes [19,20], protein folding [21,22] and protein-protein interactions [23,24], as well as nucleic acid-protein complexes [25][26][27][28][29][30]. Among the MS3D techniques, chemical and photo-activated cross-linking (XL) are employed to generate stable covalent bridges between contiguous functional groups, which can reveal their mutual placement in the targeted assembly [13,16].…”