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
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.
Section snippets
Botulinum neurotoxin, synthetic toxin peptides
The synthesis, purification and characterization of the L-chain peptides used here (Table 1) have recently been reported (Dolimbek et al. 2010). Active botulinum neurotoxin (strain A Hall) and formaldehyde-inactivated BoNT/A (toxoid) were obtained from Metabiologics (Madison, WI, USA). The formaldehyde was removed from the toxoid by dialysis against 0.1 M sodium phosphate buffer/0.05 M NaCl, pH 7.4 in Spectrapor membrane with 3500-molecular weight cut-off (Spectrum Medical Industries, Los
Correction for broad binding activity of peptide L18
Antisera were obtained from 28 MPA-positive immunoresistant patients to determine their binding activity to each of the 32 synthetic peptides. In the initial assays we found that the binding activity of peptide L18 was unexpectedly elevated and was in fact significantly higher than the total anti-BoNT/A Abs determined in the same assay on a given antiserum. This perplexing finding suggested to us the likelihood that peptide L18 may have some homology to regions of pathogens and microorganisms
Discussion
We have employed in the current study a comprehensive synthetic peptide strategy (Kazim and Atassi, 1980, Kazim and Atassi, 1982), which we had also used (Dolimbek et al. 2007) to map the antigenic regions on the H-subunit of BoNT/A that bind blocking Abs from the same CD patients studied here. To enable mapping of the T cell responses, which have minimum peptide size requirement (Atassi et al., 1987, Bixler et al., 1986), we employed here a set of 32, 19-residue peptides that encompassed the
Acknowledgements
The work was supported by an unrestricted grant from Allergan and by the Welch Foundation (Grant No. Q007) due to the award to M.Z. Atassi of the Robert A. Welch Chair of Chemistry.
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