The Regions on the Light Chain of Botulinum Neurotoxin Type A Recognized by T Cells from Toxin-Treated Cervical Dystonia Patients. The Complete Human T-Cell Recognition Map of the Toxin Molecule.

We have recently mapped the in vitro proliferative responses of T cells from botulinum neurotoxin type A (BoNT/A)-treated cervical dystonia (CD) patients with overlapping peptides encompassing BoNT/A heavy chain (residues 449-1296). In the present study, we determined the recognition profiles, by peripheral blood lymphocytes (PBL) from the same set of patients, of BoNT/A light (L) chain (residues 1-453) by using 32 synthetic overlapping peptides that encompassed the entire L chain. Profiles of the T-cell responses (expressed in stimulation index, SI; Z score based on transformed SI) to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 3-13 (average 7.2) peptides/sample at Z > 3.0 level. Two peptide regions representing residues 113-131 and 225-243 were recognized by around 40% of these patients. Regarding treatment parameters, treatment history with current BOTOX® only group produced significantly lower average T-cell responses to the 32 L-chain peptides compared to treatments with mix of type A including original and current BOTOX®. Influence of other treatment parameters on T-cell recognition of the L-chain peptides was also observed. Results of the submolecular T-cell recognition of the L chain are compared to those of the H chain and the T-cell recognition profile of the entire BoNT/A molecule is discussed. Abbreviations used: BoNT/A, botulinum neurotoxin type A; BoNT/Ai, inactivated BoNT/A; BoNT/B, botulinum neurotoxin type B; CD, cervical dystonia; L chain, the light chain (residues 1-448) of BoNT/A; LNC, lymph node cells; H chain, the heavy chain (residues 449-1296) of BoNT/A; HC, C-terminal domain (residues 855-1296) of H chain; HN, N-terminal domain (residues 449-859) of H chain; MPA, mouse protection assay; SI, stimulation index (SI = cpm of 3H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNTi, inactivated TeNT.

Development of humanized scFv antibody fragment(s) that targets and blocks specific HLA alleles linked to myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease caused by sensitization of the immune system to self-antigens. We have previously shown that targeting MG-susceptible alleles can significantly inhibit proliferation of disease-specific T cells. In this work, we humanized a murine monoclonal antibody (mAb) LG11, capable of blocking MG-associated DQ beta 1 (DQB1) allele and reformatted it into single-chain fragment variable (scFv). A fully functional humanized scFv was obtained by optimizing variable domain orientations and linker lengths, along with the optimization of expression conditions and codons to suit Escherichia coli expression machinery. Characterization of humanized scFv (FL8) revealed that the reformatted scFv, despite recognizing the same epitope as the parent murine LG11 mAb, exhibited superior binding affinity (0.97 nM) compared to the LG11 mAb, towards the immunizing antigen (DQB1*0601/70-90) and was able to block the proliferation of T cells cultured from PBLs of MG-patients typed DQB1*0601. The scFv was also capable of binding a variant MG-associated allele (DQB1*0502/70-90) with moderate affinity (18.7 nM), a feature that was absent in the LG11. To our knowledge, this is the first report of humanizing a MG-associated human leukocyte antigen (HLA) scFv for preclinical studies.

Influences of HLA DRB1, DQA1 and DQB1 on T-cell recognition of epitopes and of larger regions of the botulinum neurotoxin molecule.

Previously, we have examined the proliferative responses of T-cells from 25 patients and 8 controls to 32 light chain (L1-L32) and 60 heavy chain peptides (N1-N29, C1-C31) representing the entire clostridium botulinum neurotoxin type A (BoNT/A)[OM1-OM3]. In the current work, these T-cell responses were analyzed in the context of the patients HLA-DRB1, DQA1 and DQB1 variation. There were strong associations between the DQA1*01:02 and its derived haplotypes and cumulative T-cell proliferative responses. With or without HLA based differentiation the responses showed marked correlation. Inter-epitope correlation of responses demonstrably associated with particular regions (peptides N1-N29) peaking in the region covered by of N18-29. A second region of higher correlation was observed close to the carboxyl terminal of the heavy chain. Region N15 to N29 was found to have a significantly lower Stimulation Indices when DRB1*01:01-DQA1*01:01-DQB1*05:01 was present. This pattern was also evident in the HLA analysis where DQA1*01:02 associations were collectively most significant in the N1-N13 & C16-C31 region. Responses in these regions correlated well with one another. HLA-specific correlation analysis revealed that DQA1*01:01 bearing haplotype had the strongest inter-epitope correlations despite having a generally negative association with simulation indices. Structural and immunogenic implications of these findings are discussed.

Decreased bone mineral density in experimental myasthenia gravis in C57BL/6 mice.

Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.

Injection of inactive Bordetella pertussis and complete Freund's adjuvant with Torpedo californica AChR increases the occurrence of experimental autoimmune myasthenia gravis in C57BL/6 mice.

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2b) mice by 2-3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA + iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.

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 regions of the HN domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients.

We have recently reported the submolecular T-cell recognition profile of the C-terminal half (HC, residues 855-1296) of the heavy (H) chain of botulinum neurotoxin type A (BoNT/A) with peripheral blood lymphocytes (PBL) from 25 BoNT-treated cervical dystonia (CD) patients. In the current study, we describe the mapping of the T-cell responses of the patients to the N-terminal half (HN, residues 449-859) of the heavy chain by using 29 synthetic overlapping peptides encompassing the entire HN domain of BoNT/A. The profiles of the T-cell responses to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 1-9 (average 3.7) peptides/sample at Z>3.0 level. Three peptide regions representing residues 631-649, 659-677 and 743-761 were frequently recognized by 29-64% of the patients. In patients with positive anti-BoNT/A antibody responses the overall positive T cell responses to the HN peptides were significantly increased compared to antibody-negative patients. Influence of treatment parameters on the T-cell recognition of the HN peptides was also observed. The results were compared with those of previously identified HC region.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

Binding behaviors of the HN and the HC domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A HN and HC regions were prepared (HN519-845 and HC967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of HN519-845 to Neuro 2a cells without affecting HC967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967-1296 and HN519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both HC967-1296 and HN519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967-1296 and HN519-845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967-1296 than on the binding of HN519-845. Results indicate that while both the HN and HC domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.

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.

MHC Genes Linked to Autoimmune Disease.

Autoimmune diseases (ADs), or autoinflammatoiy diseases, are growing in complexity as diagnoses improve and many factors escalate disease risk. Considerable genetic similarity is found among ADs, and they are frequently associated with major histocompatibility complex (MHC) genes. However, a given disease may be associated with more than one human leukocyte antigen (HLA) allotype, and a given HLA may be associated with more than one AD. The associations of non-MHC genes with AD present an additional problem, and the situation is further complicated by the role that other factors, such as age, diet, therapeutic drugs, and regional influences, play in disease. This review discusses some of the genetics and biochemistry of HLA-linked AD and inflammation, covering some of the best-studied examples and summarizing indicators for class I- and II-mediated disease. However, the scope of this review limits a detailed discussion of all known ADs.

Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin.

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519-845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519-845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519-593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain.

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.

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.

Non-MHC genes linked to autoimmune disease.

The genetic traits that result in autoimmune diseases represent complicating factors in explicating the molecular and cellular elements of autoimmune responses and how these responses can be overcome or manipulated. This article focuses on the major non-major histocompatibility complex genes that have been found to be linked to autoimmune diseases. A given gene may associate with a number of autoimmune diseases and, conversely, a given disease may link to a number of common autoimmune disease (AD) genes. Collaboration and interaction among genes and the number of diseases that develop and the extensive risk factors shared among ADs further complicate the outcome. This article describes the various relationships between gene regions associated with multiple ADs and the complexity of those relationships.

Inhibition in vivo of the activity of botulinum neurotoxin A by small molecules selected by virtual screening.

To search for small molecular size inhibitors of botulinum neurotoxin A (BoNT/A) endopeptidase activity, we have screened the NCI library containing about 1 million structures against the substrate binding pocket of BoNT/A. Virtual screening (VS) was performed with the software Glide (Grid-based ligand docking energetics) and the findings were confirmed by AutoDock. Ten compounds were found that had favorable energetic and glide criteria and 5 of these compounds were selected for their ability to protect mice in vivo against a lethal dose of BoNT/A. Each compound was incubated at different molar excesses with a lethal dose of the toxin and then the mixture injected intravenously into mice. At 4690 M excess, compounds NSC94520 and NSC99639 protected all (100%) the mice from lethal toxicity. Compounds NSC48461 and NSC627733 gave 75% protection. Compound NSC348884 showed the least inhibition of toxicity allowing only a fraction (25%) of the mice to survive challenge with a lethal dose; and in the case of the mice that did not survive there was a considerable delay of mortality. At 2400 M excess compounds NSC94520 remained fully protective while and NSC99639 afforded 75% protection and at 1200 M excess each of these two compounds gave 50% protection. The two compounds gave no protection at 600 or less molar excess. When each compound was administered intravenously at 4690 M excess at different times (from 1 h to 6 h) after the intravenous injection of the active toxin, none of the compounds was able to protect the animals from toxicity. The findings show the value of VS in identifying potential inhibitors of the toxin for further development and improvement.

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.

T-cell recognition of acetylcholine receptor provides a reliable means for monitoring autoimmunity to acetylcholine receptor in antibody-negative myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disease usually associated with autoantibodies (auto-Abs) against nicotinic acetylcholine receptor (AChR). Some MG patients appear negative for anti-AChR Abs (seronegative), and a fraction of these have auto-Abs against muscle-specific kinase. The remaining patients, although displaying MG symptoms, show no detectable auto-Abs. We describe here a possible association of a rare human leukocyte antigen (HLA)-DQ type and AChR Ab-negative MG. We also found that the majority of seronegative patients exhibit an anti-AChR autoimmune T lymphocyte response. We investigated the existence of AChR-reactive T cells in peripheral blood lymphocytes from seronegative patients by their proliferative responses against a mixture of 18 overlapping synthetic peptides encompassing the extracellular part of human AChR α-chain. Of the 10 samples, eight exhibited positive T-cell proliferative responses against the peptide mixtures. The proliferative assay was equally efficient using a mixture of eight peptides frequently recognized by MG T cells. This T-cell proliferative assay should provide a reliable method for monitoring seronegative MG patients.