Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

Abstract

Highlights d Crystal structures of six VHHs that bind and neutralize BoNT/ A1 or BoNT/B1 d Three VHHs inhibit the membrane insertion or the light chain unfolding of BoNT/A1 d Three VHHs block the synaptotagmin, ganglioside, and/or lipid binding of BoNT/B1 d Rationally designed bifunctional VHH heterodimers show superior antitoxin potency

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function

“…Previous studies have demonstrated that expression of VHH heterodimeric VNAs composed of two BoNT-neutralizing VHHs can radically improve in vivo antitoxin potencies [ 21 , 25 ]. To similarly increase the potency of BoNT/E VHHs, we initially engineered and produced VHH heterodimer VNAs using the three BoNT/E-neutralizing VHHs identified in the first screen: JLE-E5, JLE-E9 and JLE-G6.…”

“…Based on the precise binding sites for JLE-E5 and JLE-E9, which bind to closely apposed epitopes on BoNT/E [ 28 ], we engineered a VNA called JLE-E9/40/JLE-E5 which was predicted to have the optimal VHH orientation and spacer length to facilitate the ability of both VHH components to bind simultaneously to their BoNT/E epitopes ( Figure 4 A,B). To test for simultaneous binding, we performed gel filtration studies ( Figure 4 C) as previously reported [ 25 ]. Co-incubation of JLE-E9/40/JLE-E5 or JLE-E5/29/JLE-E9 with a two-fold molar excess of BoNT/E produced a single peak upon gel filtration, representing the heterodimeric BoNT/E-VNA complex expected when both VHHs are bound to the same molecule ( Figure 4 C).…”

“…This and other similar catalytically inactive mutant holotoxins appear to retain most or all of the conformational form of the active holotoxins based on their excellent immunogenicity as antitoxins, the presence of a very low level of residual toxicity, and the demonstration that they possess similar receptor binding and crystal structure expected for the active toxin [ 33 , 34 ]. Indeed, we similarly employed catalytically inactivated mutants of BoNT/A and BoNT/B to obtain potent antitoxin VHHs to each serotype [ 21 , 25 ]. Of the 19 unique VHHs recognizing ciBoNTE that we identified, eight were demonstrated to possess significant BoNT/E neutralizing activity.…”

“…We and others have reported the identification of VHH-based agents that either neutralize BoNT/A or BoNT/B intoxication of cells or inhibit light chain protease domains within the neuronal cytosol [ 21 , 22 , 23 , 24 , 25 ]. Bakherad et al [ 26 ] reported a VHH that neutralized BoNT/E and partially protected against intoxication in mice.…”

“…Bakherad et al [ 26 ] reported a VHH that neutralized BoNT/E and partially protected against intoxication in mice. VHH-based neutralizing agents (VNAs) consisting of two linked neutralizing VHH components have proven highly effective in protecting mice from BoNT/A and BoNT/B intoxication when administered as proteins [ 21 , 25 ], on red blood cells [ 27 ], or by gene therapy [ 12 , 20 ], offering novel therapeutic options for botulism.…”