Elsevier

Toxicon

Volume 56, Issue 6, November 2010, Pages 990-998
Toxicon

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

https://doi.org/10.1016/j.toxicon.2010.07.003Get rights and content

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.

Introduction

Botulinum neurotoxins (BoNT) act on the peripheral nervous system to inhibit the release of acetylcholine from pre-synaptic nerve terminals at the neuromuscular junction, causing flaccid paralysis. The holotoxins are about 150 kDa consisting of three domains that are separately responsible for neuron receptor binding, translocation and catalysis. Once internalized into motor neurons, the mode of action of these toxins involves proteolytic cleavage of SNARE proteins that play key roles in neurotransmitter release. SNARE proteolysis is mediated by the 50 kDa metalloproteinase domain of the toxin, also called the light chain (Lc). The proteases for several of the seven known botulinum serotypes, notably BoNT/A and BoNT/B, are remarkably stable once in nerve cell cytosol and intoxication can last for several months before normal function returns. Because of its extreme potency, persistence and relative ease of production, BoNTs are considered among the most serious (CDC Category A) of the current bioterrorism threats.

Antitoxin agents are available that can prevent BoNT intoxication if administered prior to the development of major symptoms. Currently there is no antidote that can reverse the symptoms of intoxication once they have occurred. As a result, botulism patients must be maintained on respirators, often for many months, before motor function eventually returns. Reversal of nerve intoxication must involve inhibition and/or elimination of the protease from intoxicated neurons. Several research teams are working to develop small molecule drugs that inhibit BoNT Lc proteases and reverse intoxication. Biomolecules that bind BoNT proteases with high affinity, particularly those that inhibit its enzyme activity, could also have value in the development of botulism therapeutic agents.

Camelids such as camels, llamas and alpacas produce a class of heavy chain only antibodies (HcAbs) that lack a light chain and thus bind antigens entirely through their VH domain. The VH domain from HcAbs is called the VHH domain. Recombinant VHHs (also called nanobodies) generally express to high levels in a soluble and functional form within microbial host systems (Arbabi Ghahroudi et al., 1997), probably because the domain naturally folds and functions independent of VL interactions. VHHs also generally have improved hydrodynamic properties and stability as compared to conventional recombinant antibodies (Dumoulin et al., 2002, van der Linden et al., 1999). Furthermore, VHHs appear to have an improved ability to bind enzyme active site pockets leading to biomolecular inhibitors of catalytic function (Lauwereys et al., 1998). Evidence is growing that VHHs are often functional as intracellular antibodies termed “intrabodies” when expressed in the reducing environment of eukaryotic cytosol (Jobling et al., 2003, Verheesen et al., 2006). The unique features of VHHs have begun to be exploited for therapeutic and other possible commercial applications (Gibbs, 2005). Here we report the identification and characterization of recombinant VHHs, prepared from alpacas immunized with BoNT/A and BoNT/B proteases, which bind the BoNT proteases with high affinity. Some of the BoNT VHHs potently inhibit BoNT protease function and remain functional when expressed within neurons.

Section snippets

Preparation of recombinant BoNT/A and BoNT/B proteases

The coding sequences of BoNT/A protease (A-Lc), encoding amino acids 1–448 of BoNT/A holotoxin, and BoNT/B protease (B-Lc), amino acids 1–442 of BoNT/B holotoxin, were synthesized employing codons optimized for expression within Escherichia coli (Midland Certified Reagent Company, Inc.). These DNAs were cloned into pET14b (Novagen) in frame with the hexahistidine coding sequence at the amino terminus. The plasmids were transformed into Rosetta-gami E. coli cells (Novagen) and induced for

Identification of alpaca VHHs with affinity for BoNT/A and BoNT/B proteases

Two alpacas were immunized with recombinant BoNT/A light chain (A-Lc) protease achieving titers exceeding 106. A VHH-display library was produced from B cells of the immunized alpacas in which the VHH coding DNA was amplified by PCR and ligated into an M13 phage display vector. Several hundred thousand independent clones were obtained, amplified and the phage was panned through multiple cycles for binding to A-Lc. After characterization of more than 100 clones recognizing A-Lc by ELISA, 36

Conclusions

Botulinum neurotoxin is a NIAID Category A Biodefense Priority Pathogen because of its extreme toxicity and the lack of available therapeutic agents. Biomolecule therapies for botulism can be envisioned that include protein agents delivered to, or expressed within intoxicated neurons that inactivate or accelerate degradation of the cytosolic BoNT protease. Such agents will require simple, high affinity, specific binding agents that recognize the intraneuronal protease responsible for the

Acknowledgements

We are grateful to Dr. David Wilson for preparing some of the A-Lc and to Dr. Randall Kincaid for providing the GST fusion proteins to A-Lc and B-Lc. We greatly appreciate the assistance of Tony Pernthaner and Sally Cole for immunizing the alpacas, titering alpaca sera and preparing alpaca cDNA, and Dr. David Maass for preparing the A-Lc VHH-display library. We thank Nick Salzameda for testing the VHHs for inhibition of B-Lc activity. Finally we thank Dr. Patrick Skelly and Dr. Saul Tzipori for

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