Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells

Abstract

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

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

“…We report here the use of recent crystal structure and mechanistic data for two BoNT/E-neutralizing VHHs, JLE-E5 and JLE-E9 [28], and the use of these data to predict and then validate the mechanisms by which they neutralize intoxication of neurons. To test these VHHs as heterodimeric VNAs we prepared four different constructs, each containing two of the original three BoNT/E-neutralizing VHHs from Table 1 (JLE-E5, JLE-E9 and JLE-G6).…”

“…Three BoNT/E-neutralizing VHHs (JLE-E5, JLE-E9 and JLE-G6) were identified among the VHHs selected on coated ciBoNTE (Table 1). In co-crystallization studies, JLE-E5 and JLE-E9 have now been studied for their mechanisms of neutralization and both were shown to neutralize intoxication by inhibiting BoNT/E endosomal membrane association [28]. JLE-G6 binds to the H C E receptor binding domain of BoNT/E, and thus likely neutralizes the toxin by inhibiting its ability to bind its neuronal receptors, SV2 and/or gangliosides [25].…”

“…We next produced a fourth 'designer' VNA by incorporating new structural information [28] predicted to result in enhanced neutralization properties [25]. Prior studies showed that creating bivalent VNAs that could bind simultaneously to the target is an efficient strategy to increase affinity and potency.…”

“…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 4A,B). To test for simultaneous binding, we performed gel filtration studies (Figure 4C) as previously reported [25].…”

“…VHHs were characterized for the ability to neutralize BoNT/E intoxication of neurons and/or to inhibit LC/E protease cleavage of Synaptosomal-Associated Protein, 25kDa (SNAP-25). To test their in vivo potential as antitoxins, several VHH heterodimeric VNAs were prepared and compared for their BoNT/E antitoxin potencies in mice, including one 'designer' VNA consisting of two closely apposed VHHs linked for simultaneous binding to BoNT/E based on crystal structure data [28].…”