title: Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.
abstract: BACKGROUND
  Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called "AntiBotABE".
  
  
  RESULTS
  In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay.
  
  
  CONCLUSIONS
  After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.
authors.affiliation: Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Unité Interaction Hôte-Pathogène, 1 Place du Général Valérie André, BP73, 91220, Brétigny-sur-Orge, CEDEX, France. arnaud.avril@irba.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Avril
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106, Braunschweig, Germany. s.miethe@tu-braunschweig.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Miethe
authors.affiliation: Institut Pasteur, Centre National de Référence des bactéries anaérobies et du botulisme, 75724, Paris, France. mpopoff@pasteur.fr.
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.affiliation: Institut Pasteur, Centre National de Référence des bactéries anaérobies et du botulisme, 75724, Paris, France. christelle.mazuet@pasteur.fr.
authors.forename: Christelle
authors.initials: C
authors.lastname: Mazuet
authors.affiliation: Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Unité Interaction Hôte-Pathogène, 1 Place du Général Valérie André, BP73, 91220, Brétigny-sur-Orge, CEDEX, France. sihamchahboun@hotmail.fr.
authors.forename: Siham
authors.initials: S
authors.lastname: Chahboun
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. christine.escargueil@nibsc.org.
authors.forename: Christine
authors.initials: C
authors.lastname: Rasetti-Escargueil
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. thea.sesardic@nibsc.org.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Sesardic
authors.affiliation: Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Unité Interaction Hôte-Pathogène, 1 Place du Général Valérie André, BP73, 91220, Brétigny-sur-Orge, CEDEX, France. pthullier@yahoo.com.
authors.forename: Philippe
authors.initials: P
authors.lastname: Thullier
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106, Braunschweig, Germany. m.hust@tu-bs.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.affiliation: Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Unité Interaction Hôte-Pathogène, 1 Place du Général Valérie André, BP73, 91220, Brétigny-sur-Orge, CEDEX, France. thibaut.pelat@biotem.fr.|BIOTEM, Parc d'activité Bièvre Dauphine 885, rue Alphonse Gourju, 38140, Apprieu, France. thibaut.pelat@biotem.fr.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Pelat
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D054045:Biological Warfare Agents; D019274:Botulinum Toxins, Type A / Q000276:immunology*; D003014:Clostridium botulinum / Q000276:immunology; D006801:Humans; D008251:Macaca; D008297:Male; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D054045:Biological Warfare Agents; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D019274:Botulinum Toxins, Type A