Regions of botulinum neurotoxin A light chain recognized by human anti-toxin antibodies from cervical dystonia patients immunoresistant to toxin treatment. The antigenic structure of the active toxin recognized by human antibodies

Abstract

This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141–159), L14 (183–201) and L18 (239–257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365–383) and L29 (379–397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation.

Introduction

Botulinum neurotoxins (BoNTs) (Dickson and Shevky, 1923a, Dickson and Shevky, 1923b) block the release of acetylcholine from nerve terminals at the neuromuscular junction and cause reversible paralysis. This activity is used beneficially by employing the toxins in the treatment of a variety of clinical conditions connected with involuntary muscle spasm and contractions as well as cosmetic and other therapeutic applications (Atassi and Oshima, 1999, Becker-Wegerich et al., 2002, Binder et al., 2002, Borodic et al., 1996, Borodic et al., 2001, Gui et al., 2003, Jankovic, 2004a, Silberstein, 2001, Turton et al., 2002). But the therapeutic effects are of limited duration and periodic injections are required. In some patients, the treatment evokes the appearance of blocking antibody (Ab) and/or T cell responses against the toxin that make the patient unresponsive to further treatment (Atassi, 2004, Atassi and Oshima, 1999, Göschel et al., 1997, Jankovic, 2002, Jankovic, 2004b, Jankovic et al., 2006). Recent reports, however, have shown (Comella et al., 2004, Jankovic et al., 2003) that the immune response rate to the current BoNT/A preparation has been significantly reduced. The presence of blocking Abs in patients’ sera can be confirmed by an in vivo mouse protection assay (MPA), which determines the ability of a fixed volume of the serum to protect recipient mice against a lethal dose (LD₁₀₀) of BoNT/A.

Recently we completed mapping of the sub-molecular immune recognition profile of inactivated botulinum neurotoxin serotype A in different host species (Atassi and Dolimbek, 2004, Atassi et al., 1996, Dolimbek et al., 2010). It was not possible to employ active BoNT as the immunogen for antibody stimulation because the antigen dose required to elicit Abs would be much higher than the lethal dose of the toxin. To define the immune recognition of the active toxin in humans, we obtained sera from cervical dystonia (CD) patients treated with active BoNT/A. Cervical dystonia is a neurological movement disorder characterized by involuntary contractions of neck-muscles that cause abnormal neck movements and posture. No lasting therapy is presently available for CD that can induce permanent clinical remission, but injections of the affected muscles with minute doses of BoNT (usually type A or B) diminish the symptoms. After a number of injections with BoNT/A a small number of patients produce blocking Abs and the patients are rendered unresponsive to the treatment with the toxin (Atassi, 2004, Atassi et al., 1996, Atassi et al., 2008, Dolimbek et al., 2005, Dolimbek et al., 2007, Dolimbek et al., 2008). Antisera of these patients should enable analysis of the sub-molecular recognition of BoNT in its active form by human blocking Abs.

Active BoNT/A is a dimer of an 848-residue heavy (H) chain and a 448-reidue light (L) chain (total 1296 residues). We have previously shown that blocking Abs bind to the H-chain of the toxin and have localized the binding epitopes for blocking human Abs from CD patients (as well as Abs from other host species) (Atassi, 2004, Atassi et al., 1996, Atassi et al., 2008, Dolimbek et al., 2005, Dolimbek et al., 2007, Dolimbek et al., 2008) and the sites that bind to the cell surface receptor (Maruta et al. 2004) on the H-chain of BoNT/A. In the present work, we determined on the L-chain of BoNT/A the continuous antigenic regions recognized by human blocking anti-BoNT/A Abs. These results, together with those previously reported for the H-chain enable us here to describe of the entire antigenic structure of the toxin molecule. Localization of the regions that bind human blocking Abs is a fundamental requirement for the design of a synthetic peptide vaccine against toxin poisoning and of epitope-targeted immune modulators.