Molecular basis of immunogenicity to botulinum neurotoxins and uses of the defined antigenic regions.

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.

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.

Clinico-immunologic aspects of botulinum toxin type B treatment of cervical dystonia.

In this multicenter study of 100 patients with cervical dystonia, we examined the immunogenicity of botulinum toxin type B (BTX-B) and correlated the clinical response with the presence of blocking antibodies (Abs) using a novel mouse protection assay. One-third of the patients who were negative for BTX-B Abs at baseline became positive for BTX-B Abs at last visit. Thus, the high antigenicity of BTX-B limits its long-term efficacy.

On the initial trigger of myasthenia gravis and suppression of the disease by antibodies against the MHC peptide region involved in the presentation of a pathogenic T-cell epitope.

Myasthenia gravis (MG) is a disabling autoimmune disease caused by autoantibodies (auto-Abs) against the self-acetylcholine receptor (AChR). Although a great deal of information is known about the molecular and cellular parameters of the disease, its initial trigger, however, is not known. To study the possibility of the involvement of microbial antigens that mimic AChR in triggering MG, we have searched the microbial proteins in the data bank for regions that are similar in structure to the regions of human (h) AChR alpha chain recognized by auto-Abs in MG patients. Hundreds of candidate structures on a large number of bacterial and viral proteins were identified. To test the feasibility of the idea, we synthesized four microbial regions similar to each of the major autodeterminants of hAChR (alpha12-27, alpha111-126, alpha122-138, alpha182-198) and investigated their ability to bind auto-Abs in MG and normal sera controls. It was found that MG sera potentially recognized a significant number of these microbial regions. The results indicate that in some MG cases, immune responses to microbial antigens may cross-react with self-antigen (in this case hAChR) and could constitute initial triggers of the disease. Although anti-AChR Abs directly contribute to the degradation of AChR at the neuromuscular junctions, autoreactive T cells provide help to B cells that synthesize anti-AChR auto-Abs. To cause MG, T cells must recognize the pathogenic epitopes in the context of MHC class II molecules related to MG. The ability to regulate AChR presentation (hence AChR-reactive T-cell activation) could form the basis of an effective strategy for the control of autoimmunity in MG by selectively inhibiting the function of the Ir gene loci linked to disease susceptibility. An animal model of MG (experimental autoimmune MG, EAMG) can be induced in C57BL/6 (B6, H-2b) mice by immunization with Torpedo californica (t) AChR. A mutant mouse of B6, B6.C-H-2bm12 (bm12), which has three amino acid changes (at residues 67, 70, and 71) in the I-A beta(b) subunit, is resistant to EAMG development. Recently, we showed that region 62-76 of I-A beta(b), which contains the above residues, is involved in the binding to a pathogenic T-cell epitope within peptide t alpha146-162. We have prepared several monoclonal antibodies (mAbs) against peptide I-A beta(b)62-76, which are highly cross-reactive with I-A(b) molecules. These mAbs inhibited in vitro the proliferation of disease-related T cells of B6 specific to tAChR peptide t alpha146-162. Passive transfer of these mAbs suppressed the occurrence of clinical EAMG, which was accompanied by lower T-cell and Ab responses to tAChR. The results indicated that blocking disease-related MHC by targeting a disease-associated region on MHC molecules could be an effective, straightforward, and feasible strategy for immunointervention in MG.

T cells of mice treated with mPEG-myasthenogenic peptide conjugate are involved in protection against EAMG by stimulating lower pathogenic antibody responses.

Experimental autoimmune myasthenia gravis (EAMG) can be induced in C57BL/6 (B6) mice by immunization with Torpedo californica acetylcholine receptor (tAChR). We had previously shown that pretreatment with a monomethoxypolyethylene glycol (mPEG) conjugate of myasthenogenic tAChR alpha-chain peptide alpha125-148 (mPEG-peptide) suppressed EAMG. In order to understand the mechanism involving T cells in the induction of this suppression, we have studied, in the present work, the in vitro responses of T cells from mPEG-peptide treated B6 mice after an initial tAChR injection to determine the early effect of mPEG-peptide treatment on these responses. Treatment with mPEG-peptide reduced the T cell responses to tAChR and several tAChR alpha-chain peptides. To further investigate the T cell helper function in vivo, we transferred T cells from B6 mice that received either mPEG-peptide or control PBS followed by two tAChR injections to non-immune B6 mice. T cell transfer from mPEG-peptide pretreated mice down regulated, in recipient mice, Ab induction (after cell transfer) and Ab production (after two tAChR injections) toward alpha-chain peptides. Treatment of B6 mice with mPEG-peptide did not alter the ability of their APC to present peptide alpha146-162 to peptide-specific B6 T cells. The results indicate that suppression of EAMG by treatment with mPEG-peptide is due to T cell involvement and not to a defect in APC function.

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.

Antigen mimicry in autoimmune disease. Can immune responses to microbial antigens that mimic acetylcholine receptor act as initial triggers of Myasthenia gravis?

Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies against self acetylcholine receptor (AChR). Although a great deal of information is known about the molecular and cellular parameters of the disease, its initial trigger is not known. In order to study the possibility of the involvement of microbial antigens that mimic AChR in triggering MG, we have searched the microbial proteins in the data bank for regions that are similar in structure to the regions of human (h) AChR alpha chain recognized by autoAbs in MG patients. Hundreds of candidate structures on a large number of bacterial and viral proteins were identified. To test the feasibility of the idea, we synthesized four microbial regions similar to each of the major autodeterminants of hAChR (alpha12-27, alpha111-126, alpha122-138, alpha182-200) and investigated their ability to bind autoAbs in MG and normal sera controls. It was found that MG sera recognized a significant number of these microbial regions. The results indicate that in some MG cases immune responses to microbial antigens may cross-react with self antigen (in this case hAChR) and could constitute initial triggers of the disease.

Structure, activity, and immune (T and B cell) recognition of botulinum neurotoxins.

Botulism, which was first reported over a century ago, is caused by botulinum neurotoxins produced by Clostridium botulinum in seven immunological serotypes (A through G). The primary structures of a number of these BoNTs have been determined and are reviewed here, together with their gene structure and synthesis. The biological actions of BoNTs, which result in their ability to block neurotransmitter release have been the subject of intensive study, and in this review we discuss the binding of BoNTs to the cell surface as well as the mechanism of their intercellular action. The ability of BoNTs to block neurotransmitter release has been exploited in therapeutic applications to reduce muscle hyperactivity for the treatment of a variety of clinical conditions associated with involuntary muscle spasm and contractions. The advantages, limitations, and risks of these applications are discussed. Certain compounds provide some limited protection against BoNT. However, more effective protection has been obtained immunologically either by passive immunity (i.e., by administration of anti-BoNT Abs) or by immunization with inactivated toxin. More recently, excellent protection has been obtained by immunization with the receptor-binding region comprising the C-terminal (residues 860 to 1296) fragment (Hc) of the heavy chain of BoNT/A. Here we review the mapping of the epitopes on the Hc region of BoNT/A that are recognized by anti-BoNT/A Abs raised in horse, human, and mouse. The epitopes on the Hc that are recognized by anti-Hc Abs and by Hc-primed T lymphocytes were mapped in two mouse strains [BALB/c (H-2d) and SJL (H-2s)]. The peptides, which contain Ab or T cell epitopes (or both) on the Hc, were used as immunogens in BALB/c and SJL mice and we identified those peptides whose Ab and/or T-cell response cross-react with Hc. Identification of these peptides is an important first step in the intricate requirements for the design of a synthetic vaccine.

Subunit interacting surfaces of human hemoglobin in solution: localization of the alpha-beta subunit interacting surfaces on the alpha-chain by a comprehensive synthetic strategy.

By using synthetic overlapping peptides encompassing the entire alpha-chain of adult human hemoglobin (HbA), we have mapped on the alpha-chain the regions responsible for its binding to the beta-chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides alpha81-95, alpha101-115, alpha111-125, and alpha131-141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide alpha31-45, which in the crystal had the highest number of contact residues of all the alpha-chain peptides, did not bind the beta-chain in solution. Similarly, peptide alpha91-105, with seven contact residues in the crystal, showed low binding with the beta-chain in solution. On the other hand, peptides alpha41-55 and alpha121-135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide alpha121-135 had the highest binding activity of the alpha-chain peptides. These studies and our previous findings, which localized on the beta-chain the regions that bind to the alpha-chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.

In vitro inhibition of human malignant brain tumour cell line proliferation by anti-urokinase-type plasminogen activator monoclonal antibodies.

A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.

Anti-urokinase-type plasminogen activator monoclonal antibodies inhibit the proliferation of human breast cancer cell lines in vitro.

The levels of several tumor-associated proteases, including plasminogen activators (PA), are elevated in many malignant tumors compared to their benign tumor counterparts. Extracellular matrix degradation mediated by PA may facilitate tumor cell invasion and metastasis. In this study, the anti-proliferative activities of anti-urokinase-type plasminogen activator monoclonal antibodies (anti-UK MAbs) against human breast cancer cell lines were tested. Immunofluorescence studies localized urokinase (UK) on the surfaces of breast cancer cells. Inhibition studies showed that anti-UK MAb concentrations exerted 50% inhibition of 3H-thymidine uptake by human breast cancer cell lines; CRL-1500 and CRL-1504 were 5.6 x 10(-9)-1.82 x 10(-13) and 3.16 x 10(-10)-3.54 x 10(-12) M, respectively. Anti-UK MAbs exhibited little effect (10-20%) on normal human lymphocyte and liver cell lines. Dye exclusion indicated that anti-UK MAbs had a potent cytolytic effect on human breast cancer cells. Taken together, these results demonstrated the potential of anti-UK MAbs to be a valuable reagent for cancer immunotherapy and anti-metastatic therapy.

B-cell activation in vitro by helper T cells specific to a protein region that is recognized both by T cells and by antibodies.

This laboratory had previously mapped the regions of T and B cell recognition on sperm whale myoglobin (Mb). Mb has five regions (E1-E5) that are recognized by both T cells and B cells (i.e. antibodies, Abs) and an additional region (E6) that is recognized exclusively by T cells (i.e., TE6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2d haplotype that TE6 cells preferentially activated Mb-primed B cells (BMb) that made Abs against sites within E3 and E4 on the same protein. In the present work, we established, from Mb-primed SJL mice, an E4-specific T cell line (TE4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic BMb cells in vitro to produce anti-Mb Abs that recognized each of the antigenic sites within regions E1, E2, E3, E4 and E5. We confirmed the ability of TE4 to activate B cells that produce Abs against each of these regions by allowing TE4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of TE4 cells was dependent on the in vitro concentration of the challenge Ag (intact Mb or peptide E4). Thus, T cells against an epitope may provide help restricted to B cells that make Abs against selected antigenic sites or they may activate B cells that make Abs against all the antigenic sites of a protein. This might depend on the site-specificity of the T cell and/or on the host.

Human glioma associated intermediate filament proteins: over-expression, co-localization and cross-reactivity.

The identification of human brain tumor-associated markers could facilitate the development of new diagnostic and therapeutic strategies for these malignancies. The type III intermediate filament proteins (IFPs): vimentin, desmin and glial fibrillary acidic protein (GFAP) were studied in human glioma tissue extracts, in sera from glioma patients and in low passage glioma cell lines prepared from primary cultures of freshly dissected tumors. Radioimmunoassay (RIA) studies, using anti-GFAP, anti-desmin and anti-vimentin mAbs, showed high levels of these proteins in glioma extracts. Binding studies with authentic IFPs indicated the absence of circulating antibodies against these proteins in the sera of glioma patients. On the other hand, these sera showed high levels of vimentin. Binding studies with these antibodies using RIAs and western immunoblotting, showed that while anti-GFAF mAbs were specific to GFAP, anti-desmin mAb cross-reacted completely with GFAP, anti-vimentin mAb cross-reacted substantially with desmin and GFAP. Immunofluorescence staining of frozen sections revealed high levels of neurofilaments in gliomas and strikingly low levels in normal brain tissue. Double immunofluorescence staining showed co-occurrence of all three IFPs in the same filaments. This suggests either co-expression or cross-reactivity of these proteins due to their high degree of homology. Thus, caution should be exercised in the use and interpretation of immunohistochemical data using antibodies to IFs.

T cell responses in EAMG-susceptible and non-susceptible mouse strains after immunization with overlapping peptides encompassing the extracellular part of Torpedo californica acetylcholine receptor alpha chain. Implication to role in myasthenia gravis of autoimmune T-cell responses against receptor degradation products.

To study the role in myasthenia gravis (MG) of peptides resulting from acetylcholine receptor (AChR) degradation, we examined the ability of AChR peptides to induce T cell responses that are capable of cross-reacting with intact AChR. The studies were carried out in an experimental autoimmune MG (EAMG)-susceptible mouse strain [C57BL/6 (B6)] as well as in two non-susceptible strains [B6.C-H-2bm12 (bm12) and C3H/He]. A set of overlapping peptides encompassing the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica (t) AChR were used, individually or in equimolar mixtures, as immunogens. In B6, immunization with peptides alpha45-60, alpha111-126, alpha146-162 and alpha182-198 gave T cells that responded in vitro to the correlate immunizing peptide. Only the T cells against the latter three peptides cross-reacted with tAChR. Peptide alpha146-162 exhibited the highest in vitro reaction with the immunizing peptide and cross-reaction with tAChR. T cells obtained by immunization of B6 with an equimolar mixture of the peptides responded in vitro to peptides alpha111-126, alpha146-162 and alpha182-198 and cross-reacted very strongly with tAChR. In bm12 and C3H/He, a number of peptides evoked, when used individually as immunogens, strong or moderate T cell responses that recognized in vitro the correlate immunizing peptide but cross-reacted poorly with tAChR. Immunization with the mixture of the peptides gave T cells that recognized several peptides in each strain butdid not cross-react with alpha146-162 or tAChR. The results indicate that the ability to recognize alpha146-162 or AChR by T cells against peptides resulting from receptor degradation can account for the susceptibility to, and aggravation of, MG in B6.

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.

Intersite helper function of T cells specific for a protein epitope that is not recognized by antibodies.

Humoral responses to a protein require T-B cell communication for B cell activation by T cells. Previous studies from this laboratory have mapped the T and B cell recognition sites (epitopes) on sperm-whale myoglobin (Mb) and several other proteins. It was found that, five of six regions on Mb recognized by T cells are also recognized by B cells (i.e. antibodies). There is, however, one region (E6) residing within Mb residues 61-77, that is recognized only by T cells and to which no antibody (Ab) responses are detectable. To investigate the function of this exclusive T cell epitope, we established, from E6-primed BALB/c mice, an E6-specific T cell line (T(e6)) which comprised Th2-type cells. These T cells provided help in vitro to B cells from Mb-primed BALB/c mice and activated them to produce anti-Mb Abs of the IgM (58.2%) and IgG (41.8%) isotypes. The helper activity of T(e6) cells was dependent on the concentration of the challenging Ag (intact Mb or peptide E6) in culture. Action of soluble factors released from E6-activated T(e6) cells on B(mb) cells led to low production of anti-Mb Abs, suggesting that activation of the B cells was more dependent on their contact with T cells. Mapping of the epitope recognition of the anti-Mb Abs produced in vitro by B(mb) cells on activation by T(e6) revealed that this activation was not general to all antigenic regions recognized by anti-Mb Abs in BALB/c mice. E6-specific T cells caused in vitro activation and differentiation of B(mb) cells into plasma cells that secreted anti-Mb Abs directed, in decreasing order, against the following Mb regions: E4 (107-120) > E3 (87 - 100) > E1 (10 - 22). Little or no Ab responses could be detected against peptides E2 (50 - 62), E5 (141 - 153) and E6 (61 - 77). With B cells of peptide-primed BALB/c mice, T(e6) cells activated strongly E4-, E3- or E1, and only very slightly E2- or E6-, primed B cells to secrete Abs against the correlate peptide, but failed completely to activate E5-primed B cells. The results show that a protein T cell epitope, to which no Abs are detectable, plays an active role in B cell responses against other epitopes within the same protein.

Localization of the regions on the C-terminal domain of the heavy chain of botulinum A recognized by T lymphocytes and by antibodies after immunization of mice with pentavalent toxoid.

We have mapped the regions recognized by T and/or B cells (Abs) on the C-terminal domain (Hc) of the heavy chain of botulinum neurotoxin serotype A (BoNT/A) after immunization of two inbred mouse strains with pentavalent toxoid (BoNTs A, B, C, D and E). Using a set of synthetic overlapping peptides, encompassing the entire Hc domain (residues 855-1296), we demonstrated that T cells of Balb/c (H-2d) mice, primed with one injection of toxoid, recognized two major regions within residues 897-915 and 939-957. After multiple inoculations with toxoid, T cells of Balb/c expanded their recognition ability and responded very well to challenge with peptide 1261-1279 and moderately to stimulation with peptide 1149-1167. Unlike Balb/c T cells, those of toxoid-primed SJL (H-2s) mice exhibited a more complex profile and responded to challenge with a large number of overlapping peptides. After one toxoid injection, however, three peptides, 897-915, 939-957/953-971 overlap and 1051-1069, were the most potent T cells stimulators. After three toxoid injections, peptides 897-915 and 1051-1069 remained immunodominant while the third region was shifted upstream to 925-943/939-957 overlap. The immunodominant epitope within peptide 897-915 was recognized exclusively by T cells, since no Abs were detected against this region. The Ab binding profiles of the two mouse strains were quite similar, showing only small quantitative differences. Both, Balb/c and SJL anti-toxoid Abs displayed strong binding mainly to peptide 1177-1195, followed by peptides 869-887/883-901 overlap and 1275-1296. In addition, a significant amount of Balb/c anti-toxoid Abs was bound to peptide 1135-1153. Unlike Balb/c Abs, that interacted weakly with peptides 995-1013 and 1051-1069, the anti-toxoid Abs of SJL mice exhibited strong binding toward both peptides. The results showed that, in a given strain, the regions recognized by anti-toxoid Abs and T cells may coincide or may be uniquely B or T cell determinants.

Autoimmune responses against acetylcholine receptor: T and B cell collaboration and manipulation by synthetic peptides.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are induced by antibodies (Abs) against self acetylcholine receptor (AChR). We have mapped the T and B cell epitopes on AChR alpha subunit in human MG and in EAMG-susceptible (C57BL/6, B6) and nonsusceptible mouse strains. A T-cell epitope within residues alpha 146-162 (P14) of Torpedo californica (t)AChR plays an important role in EAMG pathogenesis of the auto Ab-induced disease. P14-specific T cell (P14Th) lines from tAChR-primed B6 mice activated, in vivo and in vitro, tAChR-primed B cells that secreted anti-AChR Abs directed against four other regions on the tAChR alpha-chain, but not against P14 itself. P14Th cells are pathogenic because they help B cells that make Abs against a conserved tAChR region (t alpha 122-150) involved in ACh binding. These Abs cross-react with region alpha 122-150 of mouse (m)AChR, thereby disrupting its normal physiological function. Thus, a T cell epitope not recognized by Abs plays an active role in B cell responses against other epitopes on the protein. We have found that in B6, the MHC region 62-76 of I-A beta(b) is involved in the presentation of P14 to T cells. Anti-peptide Abs, prepared in BALB/c, were found to inhibit in vitro the proliferation of P14-specific T-cells. Furthermore, this MHC peptide elicited Abs in B6 mice and we are investigating whether immunization of B6 with this peptide, before priming with tAChR, would suppress in vivo the T-cell response to the epitope in P14. Thus, these preliminary results would suggest that immunization with the MHC peptide might be employed for control of the autoimmune disease.

Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) 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.