Antibody responses to botulinum neurotoxin type A of toxin-treated spastic equinus children with cerebral palsy: A randomized clinical trial comparing two injection schedules.

We have conducted a 26-month-long comparative study involving young patients (2-6years old) with a clinical diagnosis of spastic equinus secondary to cerebral palsy who have been treated with BoNT/A (BOTOX®, Allergan) tri-annually or annually. Serum samples were obtained to determine the presence or absence of blocking antibodies (Abs) by a mouse protection assay (MPA) and levels of anti-BoNT/A Abs by radioimmune assay (RIA). HLA DQ alleles were typed using blood samples to determine the possible association of certain HLA type(s) with the disease or with the Ab status. Blocking Abs were detected in only two out of 18 serum samples of the tri-annual group, but none were found in 20 samples of the annual group. The MPA-positive serum samples gave in RIA significantly higher anti-BoNT/A Ab-binding levels than the MPA-negative samples. On the other hand, when two MPA-positive sample data were excluded, serum samples from tri-annual and annual groups showed similar anti-BoNT/A Ab levels. Linkage of the disorder with a particular HLA DQA1 and DQB1 allele types was not observed due to the small sample size. However, by combining results with other studies on BoNT/A-treated Caucasian patients with cervical dystonia (CD), we found that, among Caucasian patients treated with BoNT/A, DQA1*01:02 and DQB1*06:04 were higher in Ab-positive than in Ab-negative patients. The genetic linkage was on the threshold of corrected significance.

Submolecular recognition of the C-terminal domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients.

We determined the T-cell proliferative responses of the peripheral blood lymphocytes (PBL) from 25 botulinum neurotoxin (BoNT)-treated patients to 31 overlapping synthetic peptides encompassing the C-terminal half (residues 855-1296) of BoNT/A heavy chain. Responses of PBL to HC peptides varied among patients. Samples from 14 patients treated solely with BoNT/A recognized 2-13 (average 6.4) peptides/sample at Z>3.0 level. Six peptide regions representing residues 855-873, 1023-1041, 1051-1069, 1093-1111, 1135-1153 and 1247-1265 were frequently recognized by 36-57% of these PBLs. Influence of treatment parameters on T-cell recognition of the peptides was also investigated.

A Highly Specific Monoclonal Antibody for Botulinum Neurotoxin Type A-Cleaved SNAP25.

Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A's location, distribution, and movement within a cell. Due to BoNT/A's high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.

Regions recognized on the light chain of botulinum neurotoxin type A by T lymphocytes of SJL and BALB/c mice primed with inactivated toxin.

Lymph node cells (LNC) from SJL (H-2(s)) and BALB/c (H-2(d)) mice primed once with inactivated botulinum neurotoxin type A (BoNT/A) were examined for their T-cell responses to each of 32 synthetic overlapping peptides (19 residues each, L1-L32) that encompass the entire L chain (residues 1-448) of BoNT/A. LNC of SJL gave strong responses to 6 regions on, L2 (residues 15-23), L10/11/12 (127-173), L19 (253-271) and L21 (281-299), and moderate to weak responses to L9 (113-131), L14/15 (183-215) and L27 (365-383). In BALB/c, LNC gave a substantial T-cell response only against peptide L12 (residues 155-173), and responded very weakly to 9 other peptides. The results were compared with the recognition profiles determined previously in these two strains after multiple BoNT/A injections. Overall responses to the L-chain peptides of T cells in later profiles were found to be somewhat weakened in SJL and stayed essentially at a similar level in BALB/c, although responses to BoNT/A increased. In SJL, response to L10 (127-145) remained the highest in the later profile. Strong responses against L12 (155-173) observed in both strains at early stage were reduced to an insignificant level. Cross-reactivity to tetanus neurotoxin by BoNT/A-specific T cells was observed in SJL but not in BALB/c. Design of an effective synthetic peptide vaccine will require incorporation of both T cell- and Ab-recognition elements of the BoNT molecule. Significance and possible implications of these results on BoNT/A-specific T-cell responses of BoNT-treated patients are discussed.

Reduction of established antibody responses against botulinum neurotoxin A by synthetic monomethoxypolyethylene glycol peptide conjugates.

In cervical dystonia, injection of botulinum neurotoxin (BoNT) A or B into affected neck muscle reduces symptoms but may elicit anti-toxin antibodies (Abs) that block responsiveness to treatment. Previously, we localized the BoNT/A and BoNT/B sites that bind mouse or human blocking Abs. We also reported that site-specific auto-Abs can be suppressed by a monomethoxypolyethylene glycol (mPEG)-epitope conjugate. So we elicited here anti-toxin Abs in outbred mice by immunization with sublethal-suboptimal doses of active BoNT/A and determined the efficacy of selected mPEG-epitopes in reducing established anti-BoNT/A Abs. We tested in outbred mice four synthetic mPEG-N(α)-epitopes [N8 (residues 547-565), N25 (785-803), C15 (1051-1069), C31 (1275-1296)] of BoNT/A in tolerance against ongoing anti-toxin Abs. After short immunizations, tolerization with an mPEG-peptide reduced Abs to correlate peptide and caused varying Ab reductions to the other 3 peptides. Anti-N8 Abs were unaffected by mPEG-N25 tolerization, but mPEG-N8 and mPEG-N25 caused drop in anti-BoNT/A Abs. After long immunization with BoNT/A, tolerization with mPEG-N8 lessened anti-N8 Abs. Anti-C15 Abs decreased by tolerization with mPEG-C15 or any other mPEG-peptide. Anti-N25 Abs were not altered by mPEG-N25, but decreased after tolerization with mPEG-C15. Anti-C31 Abs disappeared on day 474 by tolerization with mPEG-C31 or mPEG-N8, mPEG-N25 or mPEG-C15. When an Ab response returns, a decrease can be re-established by re-administering the correlate mPEG-peptide. The method may be beneficial for extending BoNT treatment in immunoresistant patients.

Fusion of Golgi-derived vesicles mediated by SNAP-25 is essential for sympathetic neuron outgrowth but relatively insensitive to botulinum neurotoxins in vitro.

Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of Mr  = 25 000 (SNAP-25) in neurites exposed to botulinum neurotoxin type A (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was ~ 1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein 2 (SV2) was found to be colocalized with VAMP 1-3, but not with VAMP 7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.

Selective inhibition of meningeal nociceptors by botulinum neurotoxin type A: therapeutic implications for migraine and other pains.

BACKGROUND: Meningeal and other trigeminal nociceptors are thought to play important roles in the initiation of migraine headache. Currently, the only approved peripherally administered chronic migraine prophylactic drug is onabotulinumtoxinA. The purpose of this study was to determine how botulinum neurotoxin type A (BoNT-A) affects naïve and sensitized meningeal nociceptors. MATERIAL AND METHODS: Using electrophysiological techniques, we identified 43 C- and 36 Aδ-meningeal nociceptors, and measured their spontaneous and evoked firing before and after BoNT-A administration to intracranial dura and extracranial suture-receptive fields. RESULTS: As a rule, BoNT-A inhibited C- but not Aδ-meningeal nociceptors. When applied to nonsensitized C-units, BoNT-A inhibited responses to mechanical stimulation of the dura with suprathreshold forces. When applied to sensitized units, BoNT-A reversed mechanical hypersensitivity. When applied before sensitization, BoNT-A prevented development of mechanical hypersensitivity. When applied extracranially to suture branches of intracranial meningeal nociceptors, BoNT-A inhibited the mechanical responsiveness of the suture branch but not dural axon. In contrast, BoNT-A did not inhibit C-unit responses to mechanical stimulation of the dura with threshold forces, or their spontaneous activity. DISCUSSION: The study provides evidence for the ability of BoNT-A to inhibit mechanical nociception in peripheral trigeminovascular neurons. These findings suggest that BoNT-A interferes with neuronal surface expression of high-threshold mechanosensitive ion channels linked preferentially to mechanical pain by preventing their fusion into the nerve terminal membrane.

Synaptotagmin II and gangliosides bind independently with botulinum neurotoxin B but each restrains the other.

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37-63) or human SytII (hSytII 34-60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.

Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A).

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

The rat Digit Abduction Score (DAS) assay: a physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis.

Botulinum neurotoxins (BoNT) are approved for a number of therapeutic indications, including blepharospasm, cervical dystonia and hyperhidrosis, and have also shown efficacy in a variety of pain disorders. The potency of any given BoNT preparation can be routinely assessed by using the Digit Abduction Score (DAS) assay, which measures the local muscle weakening efficacy of BoNT following injection into mouse hindlimb muscle. While most studies have employed mice to assess BoNT efficacy in the DAS, few have utilized rats. In this study, we applied the DAS assay to a rat model and compared the potency of IM-BOTOX(®) (onabotulinumtoxinA) injections between two separate hind limb muscles, gastrocnemius and tibialis anterior (TA). The results demonstrated that the DAS assay can be performed on rats with similar criteria and parameters as for mice. Moreover in the rat, BoNT can be injected into either the gastrocnemius or TA muscle to elicit similar DAS scoring responses. Interestingly, onabotulinumtoxinA potency in the rat DAS was ∼3-fold higher following TA injections than gastrocnemius injections. Additionally, our data showed that the durational kinetics of onabotulinumtoxinA in the rat DAS are approximately twice as long as in the mouse DAS. These results position the rat DAS as a more flexible model for examining the mechanisms of BoNT diffusion and muscle paralysis, while mouse DAS can be used for physiological screening of BoNT because of the potential for higher throughput. Overall, these data confirm the utility of the DAS assay for characterizing the physiological potency of BoNT and related compounds.

Therapeutic effectiveness of botulinum neurotoxin A: potent blockade of autonomic transmission by targeted cleavage of only the pertinent SNAP-25.

In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.

Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B.

Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.

Antibody and T cell recognition of the light chain of botulinum neurotoxin A in two high-responder mouse strains.

We investigated in two inbred mouse strains the submolecular recognition of botulinum neurotoxin type A (BoNT/A) by Abs (B cells) and by T lymphocytes. For mapping, we employed a set of overlapping synthetic peptides that encompassed the entire light (L) chain of BoNT/A. After 3 BoNT/A toxoid injections, BALB/c T cells responded in vitro to challenge by peptides L18 (residues 239-257), L23 (309-327), L27 (365-383), L29 (393-411), or L31 (421-439) and more weakly to peptides L3 (29-47), L9/L10 (113-145), L15 (197-215), L17 (225-243), or L26 (351-369). The other peptides stimulated little or no T cell responses. SJL mice mounted, after 3 BoNT/A injections, stronger T cell responses that were medium-to-strong to peptides L2/L3 (15-47), L10/L11 (127-159), L19 (253-271), or L23 (309-327) and low to peptides L17 (225-243), L21 (281-299), L27 (365-383), or L30/L31 (407-439). After 3 BoNT/A injections, BALB/c and SJL antisera protected mice against lethal BoNT/A doses, but displayed restricted epitope profiles compared to outbred (ICR) mice Abs. BALB/c Abs displayed medium-to-high binding to peptides L4/L5 (43-75), L10/L11 (127-159), L18 (239-257) or L27 (365-383). SJL Abs were high to peptides L4/L5 (43-75), L14 (183-201), L16 (211-229), or L18/L19 (239-271), and medium to peptides L10 (127-145), L11 (141-159), L12 (155-173) or L29 (393-411). The other peptides had little or no binding. Responses to each T cell or Ab epitope were under separate genetic control. T and B (antibody) cell recognition regions may coincide, but there were also regions recognized only by Abs or by T cells.

Antibodies against a synthetic peptide designed to mimic a surface area of the H chain of botulinum neurotoxin A.

A surface-simulation peptide, SQMIN[GG]TTNI[G]NSIS[G]RDTH[G]NLES, (SS-peptide) was synthesized that described the spatial interrelationships of 21 residues on the surface of botulinum neurotoxin type A (BoNT/A). The glycine residues in brackets were spacers between surface segments of BoNT/A. The SS-peptide did not contain an antigenic or a synaptosome (snps)-binding site of BoNT/A and it did not bind anti-BoNT/A antibodies (Abs) or inhibit toxin binding to synaptosomes. Antibodies prepared by immunization with the free peptide or with peptide-ovalbumin (OVA) conjugate did not protect mice in vivo against a lethal dose of the toxin. Early Abs (day 52) against free SS-peptide recognized the peptide and showed a small cross-reaction with native toxin, but later Abs (day 115) exhibited a higher cross-reaction with to active toxin. Similarly, early Abs (day 52) against peptide-OVA conjugate displayed a low cross-reaction with native toxin, but the cross-reaction also increased in later bleeds (day 115). Both, the free peptide or its OVA conjugate, elicited predominantly IgG Abs that in the course of immunization were increasingly more capable of binding to a peptide conformation resembling the shape of the surface area on the native BoNT/A. The Abs were able to detect the conformational changes of the toxoid. This demonstrates that Abs could be prepared essentially against a peptide that mimics a surface area and such Abs could recognize and bind to the correlate surface area on the native protein. The area selected could, but need not, be an antigenic site when the native protein is used as an immunogen. The ability to make Abs against protein surface areas that are mimicked by surface-simulation synthesis provides versatile and valuable tools for analytical, therapeutic, clinical and diagnostic applications.

Location of the synaptosome-binding regions on botulinum neurotoxin B.

The regions of botulinum neurotoxin B (BoNT/B) involved in binding to mouse brain synaptosomes (snps) were localized. Sixty 19-residue overlapping peptides (peptide C31 consisted of 24 residues) encompassing BoNT/B H chain (residues 442-1291) were synthesized and used to inhibit binding of (125)I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both H(N) and H(C) domains of neurotoxin. At 37 °C, inhibitory activities on H(N) resided, in decreasing order, in peptides 638-656 (26.7%), 596-614 (18.2%), 512-530 (13.9%), 778-796 (13.8%), and 526-544 (11.6%). On H(C), activity resided in decreasing order in peptides 1170-1188 (44.6%), 1128-1146 (21.6%), 1184-1202 (18.6%), 1156-1174 (13.0%), 946-964 (11.8%), 1114-1132 (11.2%), 1100-1118 (6.2%), 876-894 (6.1%), 1268-1291 (4.6%), and 1226-1244 (4.3%). The 45 remaining H(N) and H(C) peptides had no activity. At 4 °C, peptide C24 (1170-1188) remained quite active (inhibiting, 31.2%), while activities of peptides N15, C21, and C25 were little under 10%. The snp-binding regions contained sites that bind synaptotagmin II and gangliosides. Despite the low degree of sequence homology, BoNT/B and BoNT/A display significant structural homology and appeared to bind in part to the same snp-binding regions. Binding of each labeled toxin to snps was inhibited ~50% by the other toxin, 70-72% by its correlate H(C), and by the H(C) of the other toxin [29% (BoNT/A by H(C) of B) or 32% (BoNT/B by H(C) of A)]. In the three-dimensional structure of BoNT/B, the greater part of H(C), one H(N) face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the H(C) head and employs regions on one H(N) face and the belt, reserving flexibility for the belt's unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking antibody (Ab)-binding regions explaining how such Abs prevent BoNT/B toxicity.

Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission.

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.