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.
We have used a set of synthetic overlapping peptides encompassing the entire heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A) to map, in two mouse strains (BALB/c, H2
d, and SJL, H2
S), ...the regions on the H-chain recognized by Abs in the last bleed of non-protective anti-BoNT/A antisera and in the bleed of protective antisera immediately following it in the bleeding schedule. Although the protective antisera bound slightly higher amounts of total (IgG
+
IgM) Abs, non-protective and protective BALB/c antisera showed similar peptide-binding profiles involving peptides N6/N7, N25, C2/C3, C9/C10/C11, C15, C18, C24, C30, and C31 and, at lower amounts of bound Abs, peptides N19, C6/C7, and C28. IgG
+
IgM antibodies of the protective SJL antisera recognized peptides N5, N22, and C21, and these peptides were only slightly recognized (N22, C21) or unrecognized (N5) by the non-protective antisera. Additionally, peptides N7/N8, N25, C11, C15, and less so N27/N28 bound two-fold or more Abs from the SJL protective antisera than the non-protective antisera. The Abs bound to peptides C4 and C29 were of relatively lower affinity. Peptides C2/C3, C7, C18/C19, C24, C30, and C31 bound higher amounts of Abs in the SJL protective versus the non-protective antisera, but the differences were less than double. We also mapped the binding profiles of the IgG Abs in these sera. BALB/c and SJL had 13–36-fold higher of IgG Abs that bound to BoNT/A in the protective antisera relative to non-protective antisera. The IgG Abs in the protective antisera of each mouse haplotype bound to the same peptides that bound total Abs in the correlate antiserum. But in both mouse strains, the non-protective Abs showed little or no IgG Abs that bound to these peptides. In the SJL haplotype, the IgG response to peptide N5 was transient, appearing strongly in early protective Abs and disappearing by day 70. It is not clear whether the response to region N5 plays a role in initiating and contributing to the protective activity of the toxin in the SJL strain in the early stages but is not needed in later hyperimmune stages of the Ab response. It is concluded that the switch in BALB/c and SJL mice from non-protective to protective Abs is not associated with major changes in the epitope-recognition profiles. Although some slight differences between non-protective and protective antisera appeared in their levels of Abs that were bound by some peptides, these differences are not sufficient to explain differences in the protection properties. Protection was mostly associated with the immunoglobulin class of the antibodies. IgM antibodies were non-protective, while IgG Abs produced after the switch were protective.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The mouse protection assay (MPA), which is an in vivo assay, is currently the most widely used method for monitoring blocking antibodies (Abs) in botulinum neurotoxin (BoNT)-treated patients. In ...recent studies we found that a number of the regions on the heavy (H) subunit of BoNT/A that bind blocking mouse Abs coincided, or overlapped, with the regions that bind to mouse synaptosomes (snps). This suggested that blocking anti-BoNT/A Abs would be expected to inhibit BoNT/A binding to snps. In the present work, we analyzed sera from 58 cervical dystonia (CD) patients who had been treated with BOTOX
® (a preparation of BoNT/A serotype) for blocking Abs by MPA and by their abilities to inhibit in vitro the binding of
125I-labeled active BoNT/A or inactive toxin (toxoid) to mouse brain snps. With active
125I-labeled BoNT/A-snps binding, the MPA-positive sera (
n
=
30) displayed inhibition levels that were distinctly higher (mean
=
21.1
±
5.8) than those obtained with MPA-negative sera (
n
=
28) (mean
=
−1.3
±
3.9;
p
<
0.0001) or control sera (
n
=
19) (mean
=
−3.4
±
2.8;
p
<
0.0001). Similarly, inhibition levels by MPA-positive sera of
125I-labeled toxoid snp-binding (mean
=
48.6
±
8.7) were distinctly higher than inhibition by MPA-negative sera (mean
=
10.0
±
7.6;
p
<
0.0001) or control sera (mean
=
1.8
±
6.9;
p
<
0.0001). Thus, using labeled active toxin or toxoid, the inhibition assay correlated very well with the MPA. The inhibitory activity of the non-protective sera generally correlated with the duration of survival after toxin challenge (correlation coefficients of inhibition: active toxin
=
0.445;
p
=
0.0167; inactive toxoid
=
0.774;
p
<
0.0001). It is concluded that the snp-inhibition assay reported here is reliable, reproducible and correlates very well with the MPA. It requires much less serum (0.75% of the amount needed for the MPA) and is considerably less costly than the MPA. With either
125I-labeled active toxin or toxoid, it is possible to distinguish CD sera that have blocking Abs from those that lack such Abs. Since the results with the toxoid were as discriminating as those of the active toxin, it would not even be necessary to use active toxin in these assays.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In studies from this laboratory, we localized the regions on the H chain of botulinum neurotoxin A (BoNT/A) that are recognized by anti-BoNT/A antibodies (Abs) and block the activity of the toxin in ...vivo. These Abs were obtained from cervical dystonia patients who had been treated with BoNT/A and had become unresponsive to the treatment, as well as blocking Abs raised in mouse, horse, and chicken. We also localized the regions involved in BoNT/A binding to mouse brain synaptosomes (snp). Comparison of spatial proximities in the three-dimensional structure of the Ab-binding regions and the snp binding showed that except for one, the Ab-binding regions either coincide or overlap with the snp regions. It should be folly expected that protective Abs when bound to the toxin at sites that coincide or overlap with snp binding would prevent the toxin from binding to nerve synapse and therefore block toxin entry into the neuron. Thus, analysis of the locations of the Ab-binding and the snp-binding regions provides a molecular rationale for the ability of protecting Abs to block BoNT/A action in vivo.
The present studies were carried out in order to investigate the cross-reaction of botulinum neurotoxins (BoNTs) with human and mouse antibodies against tetanus neurotoxin (TeNT) and determine ...whether injection of BoNT into a host that has been primed with TeNT would result in boosting of the response to the injected BoNT. Human antisera against TeNT obtained from 9 individuals were found to exhibit substantial cross-reaction with BoNTs A and B. We prepared antibodies (Abs) against inactivated tetanus neurotoxin (TeNT) in outbred mice and determined the binding of these Abs to active TeNT and active botulinum neurotoxins (BoNTs) A and B. Blood samples were collected before immunization (day 0) and on days 42, 82 and 125 after the first injection. The reactions of these sera with the immunizing antigen (inactivated TeNT), active TeNT, active BoNT A and active BoNT B were determined. At a fixed dilution (1:62.5 v v), the sera contained high levels of Abs that reacted with TeNT and also with BoNTs A and B. Throughout the test period (up through day 125) and at different dilutions the cross-reactions of the antisera with BoNT B were almost twice those with BoNT A. The reactions of the antisera with the immunizing antigen (inactive TeNT) or with active TeNT were essentially equal throughout the dilution range tested (1:16-1:500 v v). To determine whether injection of BoNT A or B into a host that had been primed with TeNT resulted in boosting of the response to the priming antigen (TeNT) as well as BoNT A or B, mice were primed with TeNT and boosted 21 days later with TeNT, BoNT A or BoNT B. Appropriate controls were also employed. Blood samples were collected prior to TeNT priming (day -1) and on days 21, 32, 46 and 67 after priming. In TeNT-primed mice, BoNTs A or B boosted the anti-TeNT Ab responses slightly but had no significant boosting effect on the Ab populations that bind to BoNTs A or B. It is concluded that while Abs against TeNT cross react with BoNTs and the cross reaction with BoNT B is almost double that of BoNT A, injection of BoNTs A or B in the presence of a prior active immunity against TeNT is not very likely to make the host mount an Ab response against the injected BoNT.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We localized the BoNT regions that bind blocking Abs from 28 BoNT/A- and 30 BoNT/B-treated dystonia patients who became unresponsive to correlate toxin. The sera protected mice against LD100 of ...correlate BoNT. We analyzed Ab binding to BoNT/A- and BoNT/B-H-chain peptide panels, each being 60, 19-residue peptides overlapping by 5 residues and covering the entire correlate H chain. Abs bound to a limited set of peptides but levels varied with patient, consistent with responses to each epitope being under separate MHC control. BoNT/B-treated patients had higher anti-toxin Ab levels and bound more H regions (at least 11) than BoNT/A-treated patients (5 regions). The epitopes were on surface areas that did not correlate with surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. Some epitopes within the two toxins display substantial homology and occupy analogous 3-D locations, occasionally showing a small shift relative to one another, consistent with recognition adjustments due to structural differences between the two BoNTs. Blocking Abs bound to BoNT/A at sites that coincide or overlap with synaptosome-binding, thus preventing its binding and blocking its entry into the neuron. On BoNT/B, a mouse-Ab binding region overlapped with a site that binds to mouse and rat synaptotagmin II, thus explaining Abs blocking action.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides ...that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of 125I-labeled BoNT/A to mouse brain snps was studied. The binding of 125I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H(N) domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799-817, 659-677, 729-747, 533-551, 701-719, and 757-775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463-481, 505-523, 519-537, 603-621 and 645-663. The remaining 18 H(N) peptides had little or no inhibitory activity. In the H(C) domain, peptides 1065-1083, 1163-1181 and 1275-1296 had the highest inhibitory activities (between 25% and 29%), followed (10-12% inhibitory activity) by peptides 1107-1125, 1191-1209 and 1233-1251. Two other peptides, 1079-1097 and 1177-1195, had very low (5.8% and 4.9%) inhibitory activities. The remaining 23 H(C) peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of HN, 5 of H(C) or all 11 of H(N) and H(C) revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H(C) domain. But some of the regions reside in the HN domain and might play a role in the translocation event.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
The purpose of this work was to map the entire recognition profile of the H chain of botulinum neurotoxin A (BoNT/A) by Abs in sera that have protective anti-BoNT/A Abs by the mouse protection assay ...(MPA) from cervical dystonia (CD) patients who had been treated with botulinum neurotoxin, serotype A (BOTOX
®). In previous studies we found that human anti-tetanus neurotoxin (TeNT) Abs cross-react with BoNT/A and BoNT/B. In the present work we devised an assay procedure for measuring specific anti-BoNT/A Abs in human sera by absorbing out or inhibiting the anti-TeNT Abs with TeNT before analyzing the sera for the anti-BoNT/A Abs. The sera were obtained from 28 CD patients who had become unresponsive to treatment with BoNT/A and the sera were found to protect mice against a lethal dose of BoNT/A. For localization of the Ab-binding regions on the H chain we employed a set of sixty, 19-residue synthetic peptides (except for peptide C31 which was 22 residues) that encompassed the entire H chain sequence 449–1296 and overlapped consecutively by five residues. The pattern of Ab recognition varied from patient to patient, but a very limited set of peptides were recognized by most of the patients. These were, in decreasing amounts of Ab binding, peptide N25 (H chain residues 785–803), C9/C10 (967–985/981–999), C31 (1275–1296), C15 (1051–1069), C20 (1121–1139), N16 (659–677), N22 (743–761), and N4 (491–509). But not every serum recognized all these peptides. The finding that the binding profile was not the same for all the patients is consistent with previous observations that immune responses to protein antigens are under genetic control and that the response to each epitope within a protein is under separate genetic control. Except for the region within C9/C10, the other regions either coincided (N16 and C31), or overlapped (N4, N22, N25, C15 and C20), with the recently mapped synaptosomes (snps)-binding regions on the H chain. The molecular and clinical implications of these findings are discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
