title: Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.
abstract: Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which suggest that they may be well tolerated in potential clinical development.
authors.affiliation: a Division of Bacteriology; National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency ; Hertfordshire UK.
authors.forename: Christine
authors.initials: C
authors.lastname: Rasetti-Escargueil
authors.affiliation: b Département des Maladies Infectieuses ; Unité Interaction Hôte-Pathogène; Institut de Recherche Biomédicale des Armées (IRBA) ; Brétigny-sur-Orge , France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Avril
authors.affiliation: b Département des Maladies Infectieuses ; Unité Interaction Hôte-Pathogène; Institut de Recherche Biomédicale des Armées (IRBA) ; Brétigny-sur-Orge , France.
authors.forename: Siham
authors.initials: S
authors.lastname: Chahboun
authors.affiliation: a Division of Bacteriology; National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency ; Hertfordshire UK.
authors.forename: Rob
authors.initials: R
authors.lastname: Tierney
authors.affiliation: a Division of Bacteriology; National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency ; Hertfordshire UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Bak
authors.affiliation: c Institut für Biochemie, Biotechnologie und Bioinformatik; Technische Universität Braunschweig ; Braunschweig , Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Miethe
authors.affiliation: d Unité des Bactéries anaérobies et Toxines; Institut Pasteur ; Paris , France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Mazuet
authors.affiliation: d Unité des Bactéries anaérobies et Toxines; Institut Pasteur ; Paris , France.
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.affiliation: b Département des Maladies Infectieuses ; Unité Interaction Hôte-Pathogène; Institut de Recherche Biomédicale des Armées (IRBA) ; Brétigny-sur-Orge , France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Thullier
authors.affiliation: c Institut für Biochemie, Biotechnologie und Bioinformatik; Technische Universität Braunschweig ; Braunschweig , Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.affiliation: b Département des Maladies Infectieuses ; Unité Interaction Hôte-Pathogène; Institut de Recherche Biomédicale des Armées (IRBA) ; Brétigny-sur-Orge , France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Pelat
authors.affiliation: a Division of Bacteriology; National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency ; Hertfordshire UK.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Sesardic
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000008:administration & dosage / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000276:immunology*; D001906:Botulism / Q000276:immunology* / Q000382:microbiology / Q000517:prevention & control; D003013:Clostridium / Q000187:drug effects / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D006801:Humans; D007114:Immunization / Q000379:methods; D008252:Macaca fascicularis; D051379:Mice; D008992:Monkey Diseases / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D010243:Paralysis / Q000276:immunology / Q000517:prevention & control; D019151:Peptide Library; D010791:Phrenic Nerve / Q000187:drug effects / Q000276:immunology; D057127:Single-Chain Antibodies / Q000008:administration & dosage / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D019151:Peptide Library; D057127:Single-Chain Antibodies; C096323:rimabotulinumtoxinB; D019274:Botulinum Toxins, Type A
keywords: botulinum neurotoxins, BoNT/B, VAMP2, in vitro endopeptidase, ex vivo mouse phrenic nerve-hemidiaphragm, neutralization assays, scFv-Fc, mouse paralysis, synergistic protection macaque