Minako Oshima

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

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

Immune recognition of botulinum neurotoxin type A: Regions recognized by T cells and antibodies against the protective HC fragment (residues 855–1296) of the toxin

Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum. Recent studies have shown that immunization with a C-terminal fragment [H(C), residues 855-1296] of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity. The present work was carried out in order to map the molecular and cellular immunological recognition of H(C). We have previously described the synthesis of 31 overlapping peptides encompassing the entire H(C)-fragment of BoNT/A. These peptides were employed in this study to localize the continuous regions recognized by T cells and by antibodies (Abs) generated in two mouse strains against H(C). T cells from SJL that had been primed with H(C) gave a strong proliferative response to challenge in vitro with each of the six peptides spanning residues 897-985 and a lower response to peptide 1051- 1069. While H(C)-primed T cells of BALB/c recognized three regions residing within residues 939-957, 1009-1027 and 1135-1153 (strong). Recognition regions by Abs in SJL or BALB/c anti-H(C) antisera essentially overlapped. However, the level of Abs bound to each region differed between the two strains. These common or similar recognition regions by the two strains were: 855-915 (SJL) or 855-901 (BALB/c); 939-957; 967-1013 (BALB/c) or 981-1013 (SJL); 1051-1069; 1079-1111 (BALB/c) or 1093-1125 (SJL); 1177-1195; and 1275-1296. In addition, BALB/c recognized region 1135-1153. Some of these regions show considerable sequence similarity in BoNT types B and E and, therefore, H(C) of these two BoNTs might offer protection against the correlate clostridial toxins.

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc. A mixture of peptides that contained T cell and Ab epitopes was effective in both strains in eliciting T cells and Abs that cross-reacted with Hc. This mixture form gave a quicker rise (after two injections) in cross-reactive (with Hc) Ab titer as compared to other peptide mixtures or the individual peptides, and sustained in BALB/c a high Ab titer upon further booster injections. Some of the regions that elicited crossreactive immunity to Hc have sequence similarity to other clostridial toxins, suggesting that one or more of these synthetic peptides might provide cross-protection against those toxins.

Profile of the regions of acetylcholine receptor α chain recognized by T-lymphocytes and by antibodies in eamg-susceptible and non-susceptible mouse strains after different periods of immunization with the receptor

C57BL/6 (B6) mice develop a neuromuscular disease, experimental autoimmune myasthenia gravis (EAMG), after two or more immunizations with Torpedo californica acetylcholine receptor (AChR). To determine whether EAMG is related to recognition of particular region(s) on the main extracellular domain of the alpha chain (residues alpha 1-210) in prolonged immunization, we have examined the differences in the antibody and T cell recognition profiles of B6 and SJL (a strain that does not develop EAMG) mice after different periods and a number of immunizations with Torpedo AChR. In a given strain, antibodies and T cells recognized immunodominant regions, which may coincide or may be uniquely B cell or T cell determinants. Both B6 and SJL exhibited similar antibody recognition profiles after the second and through the fourth immunizations with AChR. Major differences between the two strains were found in their T cell recognition of regions in the second part (residues 100-210) of the main extracellular domain of the alpha chain. T cells of SJL recognized consistently only one region (111-126) within this part of the alpha chain, whereas in B6, T cell recognition of three peptides (111-126, 146-162 and 182-198) and next neighbor regions to them persisted throughout the period. Of these three peptides, 146-162 was an immunodominant peptide unique to B6, as the other two peptides (111-126 and 182-198) were also recognized by either T cells or antibodies in SJL. To study the role of the T cells recognizing region 146-162 in EAMG, a T cell line was generated against this region and the cells transferred into B6 mice followed by one Torpedo AChR injection. Enhancement of antibody production toward alpha chain peptides was observed as an influence of T cell transfer compared to profiles at 1 week. In addition, one out of three mice examined showed signs of EAMG. These results suggest the importance of T cells recognizing residues 146-162 in EAMG. It is concluded that the presence of persistent T cell responses to the second half (residues (100-210) of the main extracellular domain of the alpha chain is associated with the development of EAMG in B6 mice, while absence of these responses in SJL mice may enable them to escape the disease. The preservation of the immunodominance of peptide 146-162 in the T cell recognition of B6 is probably most important for the pathogenesis of EAMG in this strain.

Human T-cell responses to botulinum neurotoxin Proliferative responses in vitro of lymphocytes from botulinum neurotoxin A-treated movement disorder patients

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.

Association with HLA DQ of early onset myasthenia gravis in Southeast Texas region of the United States.

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.

Comparison of Peptide-Coating Conditions in Solid Phase Plate Assays for Detection of Anti-Peptide Antibodies

Mice were immunized with 14 free (i.e. not conjugated to any carrier) synthetic peptides representing the entire human hemoglobin alpha-chain. Antibodies against each peptide were determined using solid phase radioimmunoassay, both with free peptides and peptides coupled to a protein carrier as the coating antigen. It has been demonstrated that large improvements in the ability to detect anti-peptide antibodies were achieved in some cases by precoating the assay wells with free peptides and in other cases by precoating with peptide-protein conjugates. Sodium carbonate buffer, pH 9.6, had a favorable effect on the coating of two of the free peptides when compared with phosphate-buffered saline, pH 7.2. The assay with the plates coated with optimum peptide form (free peptide or peptide-protein conjugate) was superior in the detection of antibody binding to 9 of the peptides when compared with the assay using chemically activated plates. The results suggest that the appropriate form (conjugated or free) and conditions for immobilizing small peptides to plastic supports are not universal but will have to be determined for each test peptide.

Association of HLA Class II alleles and haplotypes with cervical dystonia: HLA DR13-DQ6 (DQB1*0604) homozygotes are at greatly increased risk of cervical dystonia in Caucasian Americans

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.

Subtle differences in HLA DQ haplotype-associated presentation of AChR α-chain peptides may suffice to mediate myasthenia gravis

The HLA DQA1 and DQB1 alleles were determined on a set of 24 myasthenia gravis patients that had previously been examined for their T-cell proliferative responses to the 18 overlapping peptides representing the extracellular domain of hAChR α-chain. Patient responses according to assumed cis or trans haplotypes were significantly higher in most cases relative to normal controls. Comparisons of in vitro peptide-stimulated T-cell responses of patient pairs which had DQA1:DQB1 in common displayed responses in tighter distribution relative to comparisons in which patient pairs did not share the same DQA1:DQB1 haplotype. Similar haplotypes, such as DQA1*0102:DQB1*0602 and DQA1*0102:DQB1*0604, tended to exhibit similar responses and were grouped according to this similarity. Modified F-test and Students T-test analyses on DQ isoform bearing groups revealed that high responses to peptide α34–49 were associated with A1*0102:B1*0602/0604, A1*0301:B1*0302 and A1*0401/0303:B1*0301. Peptide α146–162 showed higher responses in A1*0301:B1*0302 group and moderate responses in A1*0401/0303:B1*0301 groups. Differences in the age of disease onset relative to DQ haplotypes were also observed. Groups of A1*0301:B1*0302, A1*0501:B1*0201 and A1*0102:B1*0604 showed earlier ages of disease onset relative to those of A1*0102:B1*0602 or A1*0505:B1*0301.

Suppression of experimental myasthenia gravis by monoclonal antibodies against MHC peptide region involved in presentation of a pathogenic T-cell epitope

We have prepared monoclonal antibodies (mAbs) against an antigen-binding region of I-A, region 62-76 of I-Abeta(b), which is involved in the T-cell participation in the pathogenesis of EAMG. The mAbs reacted with its parent molecules and inhibited the proliferation of disease-related T-cells. Passive transfer of these mAbs suppressed the occurrence of clinical EAMG, which was accompanied by decreased T-cell and Ab responses to tAChR. The results indicated that blocking the function of disease-related MHC by targeting a disease-associated region on MHC molecules could be an effective, straightforward and feasible strategy for immunointervention in MG.