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MAbs. 2015;7(6):1161-77. doi: 10.1080/19420862.2015.1082016. Epub 2015 Sep 18.

Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

Author information

1
a Division of Bacteriology; National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency ; Hertfordshire UK.
2
b Département des Maladies Infectieuses ; Unité Interaction Hôte-Pathogène; Institut de Recherche Biomédicale des Armées (IRBA) ; Brétigny-sur-Orge , France.
3
c Institut für Biochemie, Biotechnologie und Bioinformatik; Technische Universität Braunschweig ; Braunschweig , Germany.
4
d Unité des Bactéries anaérobies et Toxines; Institut Pasteur ; Paris , France.

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.

KEYWORDS:

botulinum neurotoxins, BoNT/B, VAMP2, in vitro endopeptidase, ex vivo mouse phrenic nerve-hemidiaphragm, neutralization assays, scFv-Fc, mouse paralysis, synergistic protection macaque

PMID:
26381852
PMCID:
PMC4966489
DOI:
10.1080/19420862.2015.1082016
[Indexed for MEDLINE]
Free PMC Article

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