Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A 1 and A 2 by targeting their heavy chain

BMC Biotechnology

Arnaud Avril⁵  Sebastian Miethe³  Michel R. Popoff¹  Christelle Mazuet¹  Siham Chahboun⁵  Christine Rasetti-Escargueil²  Dorothea Sesardic²  Philippe Thullier⁵  Michael Hust³  Thibaut Pelat⁴ 

[1] Institut Pasteur, Centre National de Référence des bactéries anaérobies et du botulisme, Paris, 75724, France

[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, UK

[3] Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, Braunschweig, 38106, Germany

[4] BIOTEM, Parc d’activité Bièvre Dauphine 885, rue Alphonse Gourju, Apprieu, 38140, France

[5] Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Unité Interaction Hôte-Pathogène, 1 Place du Général Valérie André, BP73, Brétigny-sur-Orge, 91220, CEDEX, France

关键词: Biological warfare agents;    Macaques;    AntiBotABE;    Clostridium botulinum;    Non-human primates;    Neutralizing antibodies;    scFv;    Recombinant antibodies;    Botulinum neurotoxin;   

Others  :  1228165
DOI  :  10.1186/s12896-015-0206-0

 received in 2014-12-10, accepted in 2015-09-11,  发布年份 2015

【 摘 要 】

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/A ₁(BoNT/A ₁ -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/A ₁efficiently and two cross-neutralized BoNT/A ₁and BoNT/A ₂subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A ₁and BoNT/A ₂ . The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.

【 授权许可】

   
2015 Avril et al.

【 图 表 】

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