Isolation and characterization of a highly specific monoclonal antibody targeting the botulinum neurotoxin type E exposed SNAP-25 neoepitope

Abstract

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. Specific detection and quantification of BoNT/E-cleaved SNAP-25 neoepitope is essential for diagnosis of BoNT/E intoxication as well as for characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. Immunized rabbits developed a specific and robust polyclonal antibody response, whereas immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. Sequence alignment of the isolated clones revealed high similarity between both heavy and light chains, with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled sensitive detection of the cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. This novel antibody can be further used to develop an in vitro cell-based assay to diagnose BoNT/E intoxication and to characterize antitoxin preparations, thus eliminating the use of animals in the standard mouse bioassay.

Novel botulinum neurotoxin-A tibial nerve perineural injection to alleviate overactive bladder symptoms in male rats

“…The OAB symptoms were, however surprisingly, alleviated after BoNT-A perineural injection, implicating the presence of alternative comparable pathways. Using biological markers for botulinum toxin, including C-fos and SNAP-25 cleavage, several previous studies have attempted to confirm the routes for the movement of the toxin (Nugent et al 2018 ; Mechaly et al 2021 , 2022 ). Specifically, evaluating the expression of SNAP-25 on the ventral root of nerve roots L4–S4 may provide clarity to determine the efficacy of the distal injection site.…”

A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins

“…ELISA was performed in 96-well plates (Maxisorp, Nunclon) precoated with 100 ng/well of a recombinant rabbithuman chimera monoclonal antibody (MAb) that specifically recognizes BoNT/E-cleaved SNAP-25 (Mechaly et al, 2021). This antibody was prepared by immunizing NZW rabbits with a KLH-conjugated peptide comprising eight amino acids (Thr173 -Arg180) of the SNAP-25 sequence preceding the BoNT/E cleavage site (between Arg180 and Ile181) and generating MAbs from lymphatic organ-derived B cells in a phage display expression system (Mechaly et al, 2018;Mechaly et al, 2019).…”