Clinico-immunologic aspects of botulinum toxin type B treatment of cervical dystonia.

In this multicenter study of 100 patients with cervical dystonia, we examined the immunogenicity of botulinum toxin type B (BTX-B) and correlated the clinical response with the presence of blocking antibodies (Abs) using a novel mouse protection assay. One-third of the patients who were negative for BTX-B Abs at baseline became positive for BTX-B Abs at last visit. Thus, the high antigenicity of BTX-B limits its long-term efficacy.

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species.

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/1205-1223 overlap, 1261-1279 and 1275-1296. In addition, peptides 883-901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 1149-1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amounts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869-887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093-1111, and 1275-1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Protection against alpha-bungarotoxin poisoning by immunization with synthetic toxin peptides.

The purpose of the present work was to determine the ability of BgTX peptides, corresponding to the various loops and exposed regions of alpha-bungarotoxin (BgTX) and representing regions that are recognized by B and/or T cells, to stimulate protective immunity in mice against in vivo challenge with BgTX. The BgTX LD50 values in non-immune mice or mice that had been immunized with proteins and peptides unrelated to BgTX were: Balb/c, 0.128 microgram/g; SJL, 0.156 microgram/g. Immunization of Balb/c and SJL mice with each of the synthetic peptides in its free form afforded considerable protection against BgTX poisoning. Peptides L1 (residues 3-16), L2 (residues 26-41) and C-tail (residues 66-74) of BgTX were the most protective and mice immunized with these peptides survived LD50 values that were three times higher than control mice. Immunization with an equimolar mixture of the three peptides was even more protective and these mice survived even higher challenge doses of BgTX (4.6-fold higher than LD50 of controls; i.e. protection index, PI = 4.6). An OVA conjugate carrying all three peptides, when used as an immunogen, conferred extremely high protection (PI > or = 18.1) which was almost double the protection obtained by BgTX immunization (PI = 9.7). Thus, the conjugate of the three peptides should serve as an effective vaccine against BgTX poisoning. Furthermore, these results with BgTX peptides should serve as a prototype for the design and synthesis of peptide vaccines against other members of this large family of toxins which include both long and short neurotoxins as well as cytotoxins.

Antibody and T-cell recognition of alpha-bungarotoxin and its synthetic loop-peptides.

Peptides representing the loops and surface regions of alpha-bungarotoxin (BgTX) and control peptide analogs in which these sequences were randomized were synthesized and used to map the recognition profiles of the antibodies and T-cells obtained after BgTX immunization. Also, the abilities of anti-peptide antibodies and T-cells to recognize the immunizing peptide and BgTX were determined. Three regions of BgTX were immunodominant by both rabbit and mouse anti-BgTX antibodies. These regions resided within loops L1 (residues 3-16), L2 (residues 26-41) and the C-terminal tail (residues 66-74) of the toxin. The regions recognized by BgTX-primed T-lymphocytes were mapped in five mouse strains: C57BL/6(H-2b), Balb/c (H-2d), CBA (H-2k), C3H/He (H-2k) and SJL (H-2s). The H-2b and H-2d haplotypes were high responders to BgTX, while the H-2k and H-2s were intermediate responders. The T-cell recognition profile of the peptides varied with the haplotype, consistent with Ir gene control of the responses to the individual regions. The submolecular specificities of antibodies and T-cells were compared in three of the mouse strains (C57BL/6, Balb/c and SJL). In a given mouse strain, there were regions that were strongly recognized by both antibodies and T-cells as well as regions that were predominantly recognized either by antibodies or by T-cells. The peptides were used as immunogens in their free form (i.e. without coupling to any carrier) in two of the mouse strains, Balb/c and SJL. In both mouse strains, the peptides gave strong antibody responses. Antibodies against peptide L2 showed the highest binding to intact BgTX. Antibodies against the other peptides exhibited lower binding activity to the intact toxin, and this activity was dependent on the peptide and the mouse strain. The response of peptide-primed T-cells to a given immunizing peptide was not related to whether this region was immunodominant with BgTX-primed T-cells. The ability of peptide-primed T-cells to recognize the intact toxin varied with the peptide and was dependent on the host strain. These results indicate that anti-peptide antibody and T-cell responses are also under genetic control and that their ability to cross-react with the parent toxin is not only dependent on the conformational exposure of the correlate region in intact BgTX.