Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases

Abstract

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (KD near 1 nM) and include potent inhibitors of BoNT/A protease activity (Ki near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

“…Blood was obtained for lymphocyte preparation 7 days after the fifth immunization, and RNA was prepared using the RNeasy kit (Qiagen, Valencia, CA). Two VHH-display phage libraries were prepared as described previously (22,23) except that the forward and reverse primers used to amplify the VHH coding region repertoire contained Not1 and Asc1 sites, which were used for ligation into the JSC vector for gene III phage display. The first library (JIG-2) was from peripheral blood lymphocytes of one immunized alpaca and contained ϳ1 ϫ 10 7 independent clones, whereas the second library (JKF-1) was generated from a pool of peripheral blood lymphocytes of the other two alpacas and contained ϳ3 ϫ 10 7 independent clones.…”

“…Anti-PA VHH Identification and Preparation-Phage library panning, phage recovery, and clone fingerprinting were performed as previously described (11,22,23) with the following variations. The first panning process utilized the JIG-2 VHHdisplay library and employed purified PA83 or PA63 coated onto Nunc Immunotubes at 10 g/ml for the first low stringency pan and then 1 g/ml for the second high stringency pan.…”

“…At least one VHH from each homology group was selected for protein expression. Expression and purification of VHHs in Escherichia coli as recombinant thioredoxin fusion proteins containing hexahistidine were performed as previously described (23). VHH heterodimers were engineered to be linked by a 15-amino acid flexible spacer ((GGGGS) 3 ).…”

A Single VHH-Based Toxin-Neutralizing Agent and an Effector Antibody Protect Mice against Challenge with Shiga Toxins 1 and 2

“…M19473.1 and EF441614.1) were amplified by PCR and ligated into pET-25B in frame with C-terminal His tags, and plasmids were confirmed by sequencing. Expression and purification of recombinant Stx subunits were performed essentially as previously described for VHH expression (33). The purified proteins were dialyzed against PBS, sterilized using 0.22-m filter, and stored at Ϫ70°C.…”

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

“…Nanobodies are the smallest antigen-binding fragments (~12-15 kDa) of naturally occurring heavy-chain-only antibodies (VHH) present in camelids (Hamers-Casterman et al 1993;Muyldermans et al 2001) and they have been exploited as inhibitors for BoNTs (Conway et al 2010;Dong et al 2010;Mukherjee et al 2012;Tremblay et al 2010). VHH provided additional sites to facilitate a more compact crystal packing Lam et al 2009), which significantly improved the diffraction quality of crystals to 2.7 Å resolution.…”