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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/A i , 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; H C , C-terminal domain (residues 855-1296) of H chain; H N , N-terminal domain (residues 449-859) of H chain; MPA, mouse protection assay; SI, stimulation index (SI = cpm of 3 H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNT i , inactivated TeNT.

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.

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., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

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.

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-2 b ) 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.

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., (Copyright © 2017. Published by Elsevier B.V.)

Binding behaviors of the H N and the H C 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 H N and H C regions were prepared (H N 519-845 and H C 967-1296) 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 H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 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 H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have recently reported the submolecular T-cell recognition profile of the C-terminal half (H C , 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 (H N, residues 449-859) of the heavy chain by using 29 synthetic overlapping peptides encompassing the entire H N 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 H N peptides were significantly increased compared to antibody-negative patients. Influence of treatment parameters on the T-cell recognition of the H N peptides was also observed. The results were compared with those of previously identified H C region., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

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., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2011. Published by Elsevier GmbH.)

We have previously reported that botulinum neurotoxin type A (BoNT/A)-specific T-cell responses occur in a majority of patients treated with botulinum neurotoxins (BoNT). In this study, we first determined if T-cell responses against BoNT/A and tetanus toxin (TeNT) differ between cervical dystonia (CD) patients and other movement disorder cases. Secondly, we have examined in CD cases the treatment parameters that may have an effect on the T-cell responses against BoNT/A. We found that T-cell responses to BoNT/A were significantly higher in patients with CD than in those with other movement disorders. An increase in TeNT T-cell response in CD was observed when compared to un-treated controls. CD patients who were injected with BoNT/B mounted higher responses to BoNT/A than patients treated with BoNT/A only. Frequent injections (more than 2.1/year) were associated with a significantly higher T-cell response to BoNT/A in CD. T cell responses to BoNT/A did not differ between CD patients who had clinically responsive and non-responsive status at the time of enrollment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We determined the T-cell responses against botulinum neurotoxin type A (BoNT/A) and tetanus toxin (TeNT) of peripheral blood lymphocytes from 95 BoNT-treated patients and 63 non-treated control subjects. The patient group included 80 cervical dystonia and 15 other movement disorder cases. Positive T-cell responses to BoNT/A were detected in 70% of the treated patients, and in only 3% of controls. T-cell responses of BoNT-treated patients against BoNT/A did not differ between patients who were clinically responsive and those who had become non-responsive to the treatment. BoNT-treated patients gave significantly higher in vitro T-cell responses to TeNT than did the controls., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141-159), L14 (183-201) and L18 (239-257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365-383) and L29 (379-397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Recently, we determined the molecular locations on BoNT/A of the antigenic regions recognized by blocking Abs of cervical dystonia patients immunoresistant to BoNT/A treatment. In the present work we tested the possibility of reducing the levels of the Ab response against immunodominant antigenic sites on the heavy chain of BoNT/A in order to diminish immunoresistance caused by blocking Abs. Four antigenic regions on BoNT/A represented by peptides N8 (residues 547-565), N25 (785-803), C15 (1051-1069) and C31 (1275-1296) were tested for suppressing Ab responses against the correlate regions. The conjugates were synthesized with monomethoxypolyethylene glycol (mPEG) attached to the peptide N-termini. Tolerization with a given mPEG-peptide reduced the Ab levels against the correlate region and the antisera became less protective than antisera of untolerized controls that were immunized only with inactive BoNT/A. On days 31 and 52 in the immunization course mPEG-N8 was most effective and the antisera of tolerized mice were weaker and less protective relative to controls. Other mPEG-peptides were also suppressed the Ab responses to various extents. Bleeds up to 5 months showed that tolerization can be made to persist for the entire period. The results indicated that the tolerization procedure might be potentially useful for clinical applications to immunoresistant patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1-453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

An unanticipated discovery was made while examining genetics of the immune response in patients treated with botulinum neurotoxin (BoNT), which included cervical dystonia (CD) patients. Initial examination of HLA DQA1:DQB1 frequencies revealed an unexpectedly high number of DQA1*0102:DQB1*0604 homozygotes (hz) in the CD patients. We typed the BoNT-treated CD Caucasian subset for HLA-DRB1, DQA1, and DQB1 and succeeded in typing HLA-DRB1, -DQA1, and -DQB1 for 75 of the patients. Two statistical methods found the DQB1 locus associated with CD and one method found a probable association of DQB1*0604. Examination of the allele and haplotype pairing indicated that DQB1*0604 hz comprised most to all of the positive association. Other than this genotype, one other allele, DQB1*0504 contributes to the association of the DQB1 locus. These findings indicate a probable infectious and/or autoimmune component in some CD patients. However, longer distance associations within an extended and conserved DQB1*0604 bearing haplotype leave a possibility that a locus proximal to DQB1 might be involved.

Forty-four Caucasian American myasthenia gravis (MG) patients from Southeast Texas underwent high resolution HLA DQ analysis. For the majority of patients who were late onset or male, no significant associations with DQ were observed. However, associations with DQ increased in female patients and early onset patients. At the allele level, DQB1 *0503, *0604, *0502 and *0402 collectively contributed to a positive association of the DQ locus with early onset MG (EOMG), while individually failing to show significant association. At DQ level, the novel haplotype DQA1*0401:DQB1*0201 was the primary factor in the association of combined DQ loci with early onset. In addition, *0104:*0503, *0102:*0604, *0102:*0502 and *0303:*0402 collectively contributed to the positive association of the haplotype loci. DR3-DQ2.5cis, a well known risk factor for MG in Western Eurasia, was not found associated with disease in any group. For typical EOMG [early onset, no thymoma, anti-acetylcholine receptor (AChR) antibody (Ab) positive] no association with DQA1 locus was found, however DQB1*0604 demonstrated an 'uncorrected' positive association. A few DQ haplotype (DQA1:DQB1) were positively associated with typical EOMG; a positive individual association for *0401:*0201 was complimented by the contributions of *0102:*0604 and *0303:*0402 haplotypes. A small minority of patients that were atypical and EOMG had a strong genetic association with DQA1*0104:DQB1*0503, the group included an anti-MuSK Ab positive and an anti-AChR negative patient. This report finds common ground with European studies regarding MuSK association; however similarities in association for typical early onset disease resembled HLA risk factors in East Asia and Southern Europe., (© 2010 Blackwell Publishing Ltd.)

In previous studies, we showed that certain peptides of the H(N) and H(C) domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505-523/519-537/533-551/547-565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799-817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785-803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H(C) domain, the overlap C23/C24 (1163-1181/1177-1195) provided no protection. Peptide C31 (1275-1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051-1069/1065-1083) or peptides C18/C19/C20 (1093-1111/1107-1125/1121-1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.

The action of a botulinum neurotoxin (BoNT) commences by binding at the nerve terminal via its H- (heavy) chain to a cell-surface receptor, which consists of a ganglioside and a cell-surface protein. Binding enables the L-chain, a Zn2+-dependent endopeptidase, to be internalized and act intracellularly, cleaving one or more SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins required for vesicle docking and fusion, which results in reduced neurotransmitter release. Sprouts emerge at motor-nerve terminals that reestablish synaptic contact and lead to restoration of exocytosis. As the terminals recover, sprouts retreat and synaptic function is fully re-established. Neutralizing antibodies (Abs) induced by vaccination can prevent the neuronal changes produced by BoNT. Until recently, vaccines against BoNT have been based on toxins inactivated by treatment with formaldehyde (toxoids) and contain either one (monovalent) or five (pentavalent) toxoids, but formalin-based toxoids have many undesirable side effects. Availability of the gene sequences of BoNT serotypes enabled design of recombinant subunit vaccines that have included the C-terminal domain of the H chain (HC, its subdomains (HC-N and HC-C), the L- (catalytic) chain, and the L-chain expressed with the translocation domain (LCHN). Of these, the HC displays the highest protective ability. Recent vaccines have used whole toxins inactivated by three key mutations at the enzyme active site, which have been found to be very effective in mice against the correlated toxin. Immune responses to BoNTs A and B epitopes are under the hosts MHC (major histocompatibility complex) control. Anti-BoNT/A blocking Abs bind at sites that coincide or overlap with those that bind synaptosomes and to BoNT/B at sites that overlap with synaptotagmin-II and ganglioside-binding sites. Therefore, locations occupied by blocking Abs preclude the respective toxin from binding to its receptor and thus from binding to cell surface. Information on BoNT epitopes for blocking Abs, sites for binding to cell surface receptors, and T-cell epitopes that provide help to B cells making blocking Abs afford a prospect for rational design of stable synthetic vaccines. These constructs should be clinically useful for epitope-selective modulation of Ab responses to restore effective BoNT treatment in immunoresistant patients.

We localized the BoNT regions that bind blocking Abs from 28 BoNT/A- and 30 BoNT/B-treated dystonia patients who became unresponsive to, and whose sera protected mice against LD100 of, the correlate BoNT. We analyzed Ab binding to BoNT/A- and BoNT/B-peptide panels, each of which consisted of 60, 19-residue peptides that overlapped consecutively by 5 residues and covered the entire H chain of the correlate toxin. 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 equivalent 3-D locations, occasionally showing a small shift relative to one another, consistent with recognition adjustments accommodating structural differences between the two BoNTs. Blocking Abs bound to BoNT/A at sites that coincided or overlapped with those involved in synaptosome-binding, thus preventing its binding and blocking its entry into the neuron. On BoNT/B, Ab-binding regions overlapped with the sites that bind to mouse and rat synaptotagmin II or to ganglioside, thereby explaining Ab blocking of BoNT/B action.

We determined the entire profile of the continuous antigenic regions recognized by blocking antibodies (Abs) in sera from 30BoNT/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.

Autoimmunity is mediated by a variety of mechanisms, molecular and cellular events, and responses. Predisposition to a given autoimmune response requires the requisite allele(s) that controls antigen presentation by antigen-presenting cells for T cell recognition. Some autoimmune responses emerge following infection by a pathogen, whose protein(s) possess structural similarities in some of its epitopes to regions on proteins of the host. Thus, antibodies evoked against a pathogen might cross-react with a self-protein and act as autoantibodies, and the involved autoantigen then provides a source for persistent stimulation. Proteins to which the immune system is ordinarily self-tolerant might, if altered, elicit autoimmune responses. Ways in which self-proteins can be altered include mutations and altered expression, posttranslational modification, covalent modifications, denaturation, native disorder or misfolding. Sequestered proteins normally sheltered from immune recognition become immunogenic and targets of immune effector functions, once exposed to the immune system. Other alterations can occur because of disruption in the levels or activity of regulatory proteins. These include certain alleles of the cytotoxic T lymphocyte-associated antigen-4 gene (possibly a nonspecific exacerbating molecule of disease risk in several autoimmune diseases), the lymphoid protein tyrosine phosphatase nonreceptor type 22 gene (associated with type 1 diabetes and other autoimmune diseases), TNF-alpha (involved in chronic inflammation, autoimmunity and malignancies) and the FOXP3 gene (expressed by CD4+C25+ regulatory T cells), whose mutations can cause immune dysregulation, polyendocrinopathy and X-linked inheritance syndromes of systemic autoimmunity. An autoimmune response can also arise from natural antibodies or autoantibodies that occur independently of known immunization and are able to bind to microbial antigens, altered proteins as well as self-antigens. Natural autoantibodies possess in general a low intrinsic affinity for antigen, but can function as templates for the generation of pathogenic autoantibodies, that emerge through a process of clonal selection entailing somatic hypermutation and class switch DNA recombination, as driven by antigen.