title: Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm.
abstract: Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::beta-lactamase fusion activating a prodrug.
authors.affiliation: Department of Cellular and Molecular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Benoit
authors.initials: B
authors.lastname: Stijlemans
authors.forename: Katja
authors.initials: K
authors.lastname: Conrath
authors.forename: Virna
authors.initials: V
authors.lastname: Cortez-Retamozo
authors.forename: Hoang
authors.initials: H
authors.lastname: Van Xong
authors.forename: Lode
authors.initials: L
authors.lastname: Wyns
authors.forename: Peter
authors.initials: P
authors.lastname: Senter
authors.forename: Hilde
authors.initials: H
authors.lastname: Revets
authors.forename: Patrick
authors.initials: P
authors.lastname: De Baetselier
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Stefan
authors.initials: S
authors.lastname: Magez
mesh_terms: D000818:Animals; D000940:Antigenic Variation; D000941:Antigens / Q000737:chemistry; D000953:Antigens, Protozoan / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D003208:Concanavalin A / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005434:Flow Cytometry; D015723:Gene Library; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D011355:Prodrugs; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014345:Trypanosoma / Q000276:immunology / Q000378:metabolism*; D014352:Trypanosomiasis / Q000188:drug therapy; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000941:Antigens; D000953:Antigens, Protozoan; D002241:Carbohydrates; D000939:Epitopes; D019151:Peptide Library; D011355:Prodrugs; D014643:Variant Surface Glycoproteins, Trypanosoma; D003208:Concanavalin A; D001618:beta-Lactamases