title: 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.
authors: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
authors: Frank
authors: F
authors: Lennartz
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark;
authors: Anja
authors: A
authors: Bengtsson
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark;
authors: Rebecca W
authors: RW
authors: Olsen
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark;
authors: Louise
authors: L
authors: Joergensen
authors: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom;
authors: Alan
authors: A
authors: Brown
authors: Institut de Biologie Structurale, Grenoble F-38044, France;
authors: Louise
authors: L
authors: Remy
authors: Institute of Microbiology, Academy of Sciences of the Czech Republic, 117 20 Prague, Czech Republic; and Faculty of Science, Charles University in Prague, 116 36 Prague, Czech Republic.
authors: Petr
authors: P
authors: Man
authors: Institut de Biologie Structurale, Grenoble F-38044, France;
authors: Eric
authors: E
authors: Forest
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark;
authors: Lea K
authors: LK
authors: Barfod
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark;
authors: Yvonne
authors: Y
authors: Adams
authors: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; matthew.higgins@bioch.ox.ac.uk atrj@sund.ku.dk.
authors: Matthew K
authors: MK
authors: Higgins
authors: Department of Immunology and Microbiology, Centre for Medical Parasitology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 1014, Denmark; Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen 2100, Denmark; matthew.higgins@bioch.ox.ac.uk atrj@sund.ku.dk.
authors: Anja T R
authors: http://orcid.org/0000-0002-4004-7554
authors: AT
authors: Jensen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology*; D002448:Cell Adhesion; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism / Q000469:parasitology; D000939:Epitopes / Q000276:immunology; D004910:Erythrocyte Membrane / Q000276:immunology; D004912:Erythrocytes / Q000469:parasitology; D006825:Hybridomas; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology*; D016778:Malaria, Falciparum / Q000276:immunology / Q000469:parasitology; D051379:Mice; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000276:immunology*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D015800:Protozoan Proteins; C085731:erythrocyte membrane protein 1, Plasmodium falciparum; D018799:Intercellular Adhesion Molecule-1