Molecular recognition of botulinum neurotoxin B heavy chain by human antibodies from cervical dystonia patients that develop immunoresistance to toxin treatment

We determined the entire profile of the continuous antigenic regions recognized by blocking antibodies (Abs) in sera from 30 BoNT/B-treated cervical dystonia (CD) patients who developed unresponsiveness to treatment. The sera protected mice against a lethal dose of BoNT/B. We analyzed Ab binding to a panel of 60 synthetic 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and encompassed the entire BoNT/B heavy (H) chain (residues 442–1291). Most Abs recognized a limited set of peptides but the pattern and Ab levels bound varied with the patient, consistent with genetic control of immune responses and with responses to each epitope being separately controlled. Abs were bound by peptides (in decreasing order): C1 (residues 848–866), C10 (974–992), C16 (1058–1076), C14 (1030–1048), N15 (638–656), N21/N22 (722–740/736–754), N24/N25 (764–782/778–796) and N29 (834–852). Peptides N3/N4 (470–488/484–502), N27 (806–824), C2 (862–880), C4 (890–908), C6/C7 (918–936/932–950), C17 (1072–1090), C24 (1170–1188), C29 (1240–1258) and C31 (1268–1291) exhibited low Ab binding. The remaining peptides bound little or no Abs. Of the 30 antisera, 28 (93.3%) had Abs that bound to peptides C1, C10, C14 or C16, and 27 (90.0%) bound to peptide N22. No peptide was recognized by all the antisera, but peptide combinations N24 + C1, N22 + N24 + C1, N24 + C1 + C10, C10 + C14 + C16, N22 + N24 + C1 + C10, C1 + C10 + C14 + C16 or N22 + N24 + C1 + C10 + C14 bound blocking Abs in 30 (100%) antisera. BoNT/B-treated CD patients had higher Ab levels and bound to more epitopes (at least 11) than did BoNT/A-treated patients (5 regions). The regions recognized by anti-BoNT/B Abs occupied surface areas that displayed no correlation to surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. These regions afford candidates for epitope-specific manipulation of anti-toxin immune responses.