title: High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.
abstract: The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussels, Brussels, Belgium. bstijlem@vub.ac.be
authors.forename: Benoît
authors.initials: B
authors.lastname: Stijlemans
authors.forename: Guy
authors.initials: G
authors.lastname: Caljon
authors.forename: Senthil Kumar A
authors.initials: SK
authors.lastname: Natesan
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Katja
authors.initials: K
authors.lastname: Conrath
authors.forename: David
authors.initials: D
authors.lastname: Pérez-Morga
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
authors.forename: Alexandros
authors.initials: A
authors.lastname: Nikolaou
authors.forename: Lea
authors.initials: L
authors.lastname: Brys
authors.forename: Etienne
authors.initials: E
authors.lastname: Pays
authors.forename: Stefan
authors.initials: S
authors.lastname: Magez
authors.forename: Mark C
authors.initials: MC
authors.lastname: Field
authors.forename: Patrick
authors.initials: P
authors.lastname: De Baetselier
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D000915:Antibody Affinity; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053758:Nanoparticles; D014344:Trypanocidal Agents / Q000494:pharmacology / Q000627:therapeutic use; D014346:Trypanosoma brucei brucei / Q000276:immunology* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D014353:Trypanosomiasis, African / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D014344:Trypanocidal Agents; D014643:Variant Surface Glycoproteins, Trypanosoma