Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain

Abstract

BackgroundBotulism 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”.ResultsIn 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.ConclusionsAfter 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0206-0) contains supplementary material, which is available to authorized users.

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

“…The reactivity of the phage eluted after each round against BoNT/A1 was assessed in phage-ELISA. The signal increasing 9-fold between round 1 and 3 confirmed the enrichment in scFvs highly reactive against the target [ 73 ].…”

“…For anti-BoNT/E-HC antibodies, a titer of 1/600,000 against BoNT/E3 holotoxin was observed. Finally, for anti-BoNT/E-LC antibodies, a titer of 1/600,000 against BoNT/E3 holotoxin was observed [ 51 , 73 , 74 , 75 ].…”

“…Immune-libraries were generated as previously described [ 1 , 2 , 4 , 65 , 71 , 72 , 73 ]. Briefly, the DNA encoding the Fd fragments and the κLC were separately sub-cloned in pGEM ® -T vector and then sequentially cloned in the pHAL14 or pHAL32 phagemid vector.…”

“…This library was packaged with M13K07 or Hyperphage [ 51 ]. The immune-library directed against BoNT/A1-HC consisted of 5.45 × 10 7 unique clones and was finally packaged in M13K07 helper-phage [ 73 ].…”

“…The affinity of the scFv A1HC38 was measured at 1.9 nM with Kon = 6.34 × 10 4 M −1 S −1 and Koff = 1.2 × 10 −4 S −1 . The twenty-four scFvs (37.5% of all non-redundant and non-recombined scFvs) presenting affinities better than 10 nM were selected for the neutralization capacity assessments [ 73 ].…”