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PLoS One. 2015 Oct 6;10(10):e0139905. doi: 10.1371/journal.pone.0139905. eCollection 2015.

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E.

Author information

1
Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany.
2
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, United Kingdom.
3
Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), Département de Microbiologie, Unité de biotechnologie des anticorps et des toxines, 24 avenue des Maquis du Grésivaudan, B.P. 87, 38702 La Tronche Cedex, France.
4
University of Helsinki, Faculty of Veterinary Medicine, Centre of Excellence in Microbial Food Safety Research, Department of Food Hygiene and Environmental Health, P.O. Box 66 (Agnes Sjöbergin katu 2), 00014 Helsinki University, Helsinki, Finland.
5
Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 25 avenue du Docteur Roux, 75015, Paris, France.

Abstract

BACKGROUND:

Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and 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 agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure.

RESULTS:

In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay.

CONCLUSION:

These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.

PMID:
26440796
PMCID:
PMC4595074
DOI:
10.1371/journal.pone.0139905
[Indexed for MEDLINE]
Free PMC Article

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