Mapping of the Antibody and T Cell Recognition Profiles of the HN Domain (Residues 449–859) of the Heavy Chain of Botulinum Neurotoxin A in Two High-Responder Mouse Strains

Using a set of synthetic overlapping peptides, encompassing the entire N-terminal domain (H_N,) of the heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A), we have mapped on H_N, the regions recognized by Abs (B cells) and by T cells in two inbred mouse strains. After one BoNT/A toxoid injection, BALB/c T cells mounted a weak in vitro response to a region within overlap 687–705/701–719. The remaining peptides stimulated no detectable responses. After 3 injections, BALB/c T cells gave stronger responses to an expanded region within the overlap 687–705/701–719/715–733, peaking at 701–719. BoNT/A-primed BALB/c T cells showed substantial cross-reaction with BoNT/B but did not respond to TeNT. Unlike BALB/c T cells, BoNT/A-primed T cells of SJL cross-reacted well with both BoNT/B and with TeNT. They also recognized a lager epitope profile than the corresponding BALB/c T cells. After one injection with BoNT/A toxoid, SJL T cells responded in vitro to a number of the H_N peptides. Regionswithin peptides 617–635 and 561–579 stimulated strong in vitro responses. Several peptides (463–481, 589–607, 659–677, 729–747, 827–845, and 841–859 revoked weak-to-medium proliferative activities. Four other peptides stimulated very low but reproducible responses (SI between 2.0 and 3.0). After 3 BoNT/A injections, SJL T cells responded in vitro strongly to peptides 463–481, 561–579, 617–635, 743–761, and 841–859. There were medium or weak responses to at least 10 other peptides. The cells also responded well to the l-chain peptide 218–231. Antisera of BALB/c and SJL, obtained after 3 injections with BoNT/A toxoid, protected at very high dilutions recipient mice against LD₁₀₅ of BoNT/A. BALB/c Abs showed medium-to-high binding to peptides 533–551/547–565, 785–803, and 813–831/827–845. Four other peptides showed very low binding. The corresponding SJL Abs had high binding to the overlap 533–551/547–565/561–579, and peptides 743–761, 785–803, and 813–831. Three other peptides bound low amounts of Abs. The results indicate that the responses to each Ab or T cell epitope is under separate genetic control and that, in a given strain, the Ab and T cell recognition regions may coincide but, in addition, H_N contains regions that are recognized only by Abs or only by T cells.     

Mapping of the antibody and T cell recognition profiles of the HN domain (residues 449-859) of the heavy chain of botulinum neurotoxin A in two high-responder mouse strains

Using a set of synthetic overlapping peptides, encompassing the entire N-terminal domain (HN,) of the heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A), we have mapped on HN, the regions recognized by Abs (B cells) and by T cells in two inbred mouse strains. After one BoNT/A toxoid injection, BALB/c T cells mounted a weak in vitro response to a region within overlap 687-705/701-719. The remaining peptides stimulated no detectable responses. After 3 injections, BALB/c T cells gave stronger responses to an expanded region within the overlap 687-705/701-719/715-733, peaking at 701-719. BoNT/A-primed BALB/c T cells showed substantial cross-reaction with BoNT/B but did not respond to TeNT. Unlike BALB/c T cells, BoNT/A-primed T cells of SJL cross-reacted well with both BoNT/B and with TeNT. They also recognized a lager epitope profile than the corresponding BALB/c T cells. After one injection with BoNT/A toxoid, SJL T cells responded in vitro to a number of the HN peptides. Regions within peptides 617-635 and 561-579 stimulated strong in vitro responses. Several peptides (463-481, 589-607, 659-677, 729-747, 827-845, and 841-859 revoked weak-to-medium proliferative activities. Four other peptides stimulated very low bu reproducible responses (SI between 2.0 and 3.0). After 3 BoNT/A injections, SJL T cells responded in vitro strongly to peptides 463-481, 561-579, 617-635, 743-761, and 841-859. There were medium or weak responses to at least 10 other peptides. The cells also responded well to the L-chain peptide 218-231. Antisera of BALB/c and SJL obtained after 3 injections with BoNT/A toxoid, protected at very high dilution recipient mice against LD105 of BoNT/A. BALB/c Abs showed medium-to-high binding to peptides 533-551/547-565, 785-803, and 813-831/827-845. Four other peptides showed very low binding. The corresponding SJL Abs had high binding to the overlap 533-551/547-565/561-579, and peptides 743-761, 785-803, and 813-831. Thre other peptides bound low amounts of Abs. The results indicate that the responses teach Ab or T cell epitope is under separate genetic control and that, in a given strain the Ab and T cell recognition regions may coincide but, in addition, HN contains regions that are recognized only by Abs or only by T cells.     

Mapping of the regions on the heavy chain of botulinum neurotoxin A (BoNT/A) recognized by antibodies of cervical dystonia patients with immunoresistance to BoNT/A

The purpose of this work was to map the entire recognition profile of the H chain of botulinum neurotoxin A (BoNT/A) by Abs in sera that have protective anti-BoNT/A Abs by the mouse protection assay (MPA) from cervical dystonia (CD) patients who had been treated with botulinum neurotoxin, serotype A (BOTOX). In previous studies we found that human anti-tetanus neurotoxin (TeNT) Abs cross-react with BoNT/A and BoNT/B. In the present work we devised an assay procedure for measuring specific anti-BoNT/A Abs in human sera by absorbing out or inhibiting the anti-TeNT Abs with TeNT before analyzing the sera for the anti-BoNT/A Abs. The sera were obtained from 28 CD patients who had become unresponsive to treatment with BoNT/A and the sera were found to protect mice against a lethal dose of BoNT/A. For localization of the Ab-binding regions on the H chain we employed a set of sixty, 19-residue synthetic peptides (except for peptide C31 which was 22 residues) that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by five residues. The pattern of Ab recognition varied from patient to patient, but a very limited set of peptides were recognized by most of the patients. These were, in decreasing amounts of Ab binding, peptide N25 (H chain residues 785-803), C9/C10 (967-985/981-999), C31 (1275-1296), C15 (1051-1069), C20 (1121-1139), N16 (659-677), N22 (743-761), and N4 (491-509). But not every serum recognized all these peptides. The finding that the binding profile was not the same for all the patients is consistent with previous observations that immune responses to protein antigens are under genetic control and that the response to each epitope within a protein is under separate genetic control. Except for the region within C9/C10, the other regions either coincided (N16 and C31), or overlapped (N4, N22, N25, C15 and C20), with the recently mapped synaptosomes (snps)-binding regions on the H chain. The molecular and clinical implications of these findings are discussed.     

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

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

Reduction of antibody response against botulinum neurotoxin A by synthetic monomethoxypolyethylene glycol-peptide conjugates

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.     

Submolecular recognition profiles in two mouse strains of non-protective and protective antibodies against botulinum neurotoxin A

We have used a set of synthetic overlapping peptides encompassing the entire heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A) to map, in two mouse strains (BALB/c, H2^d, and SJL, H2^S), the regions on the H-chain recognized by Abs in the last bleed of non-protective anti-BoNT/A antisera and in the bleed of protective antisera immediately following it in the bleeding schedule. Although the protective antisera bound slightly higher amounts of total (IgG + IgM) Abs, non-protective and protective BALB/c antisera showed similar peptide-binding profiles involving peptides N6/N7, N25, C2/C3, C9/C10/C11, C15, C18, C24, C30, and C31 and, at lower amounts of bound Abs, peptides N19, C6/C7, and C28. IgG + IgM antibodies of the protective SJL antisera recognized peptides N5, N22, and C21, and these peptides were only slightly recognized (N22, C21) or unrecognized (N5) by the non-protective antisera. Additionally, peptides N7/N8, N25, C11, C15, and less so N27/N28 bound two-fold or more Abs from the SJL protective antisera than the non-protective antisera. The Abs bound to peptides C4 and C29 were of relatively lower affinity. Peptides C2/C3, C7, C18/C19, C24, C30, and C31 bound higher amounts of Abs in the SJL protective versus the non-protective antisera, but the differences were less than double. We also mapped the binding profiles of the IgG Abs in these sera. BALB/c and SJL had 13–36-fold higher of IgG Abs that bound to BoNT/A in the protective antisera relative to non-protective antisera. The IgG Abs in the protective antisera of each mouse haplotype bound to the same peptides that bound total Abs in the correlate antiserum. But in both mouse strains, the non-protective Abs showed little or no IgG Abs that bound to these peptides. In the SJL haplotype, the IgG response to peptide N5 was transient, appearing strongly in early protective Abs and disappearing by day 70. It is not clear whether the response to region N5 plays a role in initiating and contributing to the protective activity of the toxin in the SJL strain in the early stages but is not needed in later hyperimmune stages of the Ab response. It is concluded that the switch in BALB/c and SJL mice from non-protective to protective Abs is not associated with major changes in the epitope-recognition profiles. Although some slight differences between non-protective and protective antisera appeared in their levels of Abs that were bound by some peptides, these differences are not sufficient to explain differences in the protection properties. Protection was mostly associated with the immunoglobulin class of the antibodies. IgM antibodies were non-protective, while IgG Abs produced after the switch were protective.     

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) > El (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_E9 cells activated strongly E4-, E3- or El-, 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.     

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., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) 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 (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb 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 T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 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.     

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., T_E6) and to which no Abs are detectable. The responses to the site are each under separate genetic control. Recently, we showed in an H-2^d haplotype that T_E6 cells preferentially activated Mb-primed B cells (B_Mb) 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 (T_E4) by passage in vitro with synthetic peptide E4. At relatively low numbers, these T cells activated syngeneic B_Mb 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 T_E4 to activate B cells that produce Abs against each of these regions by allowing T_E4 to activate in vitro syngeneic B cells that had been primed with E1, E2, E3, E4 or E5. The helper activity of T_E4 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.     

T cell recognition of myoglobin. Localization of the sites stimulating T cell proliferative responses by synthetic overlapping peptides encompassing the entire molecule

A comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein has previously been introduced by this laboratory. The strategy consists of studying the immunochemical activity of a series of consecutive synthetic peptides that encompass the entire protein chain and that are uniform in size and in overlap at their N- and C-terminals with neighbouring peptides. By application of this strategy to sperm whale myoglobin, we have been able to delineate the continuous sites of T cell recognition of myoglobin in three high responder mouse strains. Thirteen 17-residue peptides that encompass the entire myoglobin chain and overlap by five residues at both ends were synthesized, purified and characterized. The peptides were examined in vitro for their ability to stimulate lymph node cells from myoglobin-primed DBA/2 (H-2d), BALB/c (H-2d) and SJL (H-2s) mice as well as long-term cultures of myoglobin-specific T cells. Several regions of the molecule (T sites) were found to stimulate myoglobin-primed lymph node cells and myoglobin-specific longterm T cell cultures. This strategy has enabled the localization of the full profile of dominant sites of T cell recognition in myoglobin for these mouse strains. Of these T sites, one region, residues 107-125, was clearly immunodominant in these strains and was found to coincide with the antigenic (i.e. antibody binding) site 4 of myoglobin. Also, other regions stimulated T cells and appeared to coincide with previously known antigenic sites. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites which are recognized exclusively by T cells and to which no detectable antibody response is directed.     

Immune recognition of botulinum neurotoxin B: antibody-binding regions on the heavy chain of the toxin

The purpose of this work was to map the continuous regions recognized by human, horse and mouse anti-botulinum neurotoxin B (BoNT/B) antibodies (Abs). We synthesized a panel of sixty 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and together encompassed the entire heavy chain of BoNT/B (H/B, residues 442-1291). Abs from the three host species recognized similar, but not identical, peptides. There were also peptides recognized by two or only by one host species. Where a peptide was recognized by Abs of more than one host species, these Abs were at different levels among the species. Human, horse and mouse Abs bound, although in different amounts, to regions within peptides 736-754, 778-796, 848-866, 932-950, 974-992, 1058-1076 and 1128-1146. Human and horse Abs bound to peptides 890-908 and 1170-1188. Human and mouse Abs recognized peptides 470-488/484-502 overlap, 638-656, 722-740, 862-880, 1030-1048, 1072-1090, 1240-1258 and 1268-1291. We concluded that the antigenic regions localized with the three antisera are quite similar, exhibiting in some cases a small shift to the left or to the right. This is consistent with what is known about protein immune recognition. In the three-dimensional structure, the regions recognized on H/B by anti-BoNT/B Abs occupied surface locations and analysis revealed no correlation between these surface locations and surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. A region that bound mouse Abs overlapped with a recently defined site on BoNT/B that binds to mouse and rat synaptotagmin II, thus providing a molecular explanation for the blocking (protecting) activity of these Abs. The regions thus localized afford candidates for incorporation into a synthetic vaccine design.     

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

We have recently mapped the in vitro proliferative responses of T cells from botulinum neurotoxin type A (BoNT/A)-treated cervical dystonia (CD) patients with overlapping peptides encompassing BoNT/A heavy chain (residues 449–1296). In the present study, we determined the recognition profiles, by peripheral blood lymphocytes (PBL) from the same set of patients, of BoNT/A light (L) chain (residues 1–453) by using 32 synthetic overlapping peptides that encompassed the entire L chain. Profiles of the T-cell responses (expressed in stimulation index, SI; Z score based on transformed SI) to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 3–13 (average 7.2) peptides/sample at Z > 3.0 level. Two peptide regions representing residues 113–131 and 225–243 were recognized by around 40% of these patients. Regarding treatment parameters, treatment history with current BOTOX^® only group produced significantly lower average T-cell responses to the 32 L-chain peptides compared to treatments with mix of type A including original and current BOTOX^®. Influence of other treatment parameters on T-cell recognition of the L-chain peptides was also observed. Results of the submolecular T-cell recognition of the L chain are compared to those of the H chain and the T-cell recognition profile of the entire BoNT/A molecule is discussed.

Abbreviations used: BoNT/A, botulinum neurotoxin type A; BoNT/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 ³H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNT_i, inactivated TeNT.     

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

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

Induction of experimental allergic encephalomyelitis by myelin proteolipid-protein-specific T cell clones and synthetic peptides.

Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. SJL(H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 (HSLGKWLGHPDKF) develop acute experimental allergic encephalomyelitis (EAE). In the present study a T cell line and 4 clones were derived from SJL/J mice following immunization with this synthetic peptide. Severe clinical and histological EAE could be induced by adoptive transfer of the peptide-specific T cell line and 3 of 4 T cell clones. The T cell line/clones all responded strongly to PLP peptide 139-151 in in vitro proliferative assays. However, two different reactivity patterns emerged when truncated PLP peptides 141-150 and 141-149 were tested, suggesting that more than 1 epitope may be present within the PLP 139-151 determinant. To evaluate the encephalitogenic potential of the truncated peptides, we compared the ability of 2 truncated PLP peptides to induce EAE in vivo and proliferative responses in vitro. Immunization with PLP peptide 141-150 induced acute EAE in about 70% of mice tested, but PLP peptide 141-149 induced a comparatively mild form of EAE in 4 out of 9 mice tested. Lymph node cells from mice immunized with these peptides showed in vitro proliferative responses to each of the peptides, but the response to peptide 139-151 was always strongest. These combined in vivo and in vitro data further define the epitopes involved in PLP-induced EAE in SJL mice. Furthermore, the availability of multiple PLP-specific T cell clones will enable us to study the diversity of the T cell repertoire to PLP.     

Engineering of immunogenic peptides by co-linear synthesis of determinants recognized by B and T cells

The potential of synthetic peptides as vaccines is restricted by their frequent lack of immunogenicity. As with haptens, coupling to a carrier protein is usually required to provide T cell help to anti-peptide antibody-producing B cells. In spite of their short length, a few natural or synthetic peptides are immunogenic: they all include both a determinant recognized by B cells and a proven or putative determinant recognized by T cells. We speculated that it should be possible to induce immunogenicity in peptide haptens by the inclusion of a well characterized determinant recognized by T cells. We thus synthesized two peptides, corresponding to different regions of the major protein VP6 of bovine rotavirus, co-linearly linked to a peptide of influenza virus hemagglutinin which had been shown to induce T helper cells in BALB/c mice. Both peptides induced anti-rotavirus antibodies and were more immunogenic than the corresponding bovine serum albumin-conjugated peptides.     

T cell recognition of ragweed allergen Ra3: localization of the full T cell recognition profile by synthetic overlapping peptides representing the entire protein chain

In the preceding report, we described the systematic localization of the full profile of the continuous antigenic sites on ragweed allergen, Ra3, with antibodies from three different host species using a comprehensive strategy, previously introduced by this laboratory. The strategy consists of studying the immunochemical activity of a series of consecutive synthetic overlapping peptides, of uniform size and overlaps, which encompass the entire protein chain. This study reports the localization of the continuous regions on Ra3 that are recognized by T cells from mice immunized with Ra3. The 10 overlapping peptides encompassing the entire Ra3 molecule were examined in vitro for their ability to stimulate lymph node cells from Ra3-primed BALB/c (H-2d), C3H/He (H-2k) SWR (H-2q) and SJL (H-2s) mice. Several regions of the molecule (T sites) were found to stimulate Ra3-primed lymph node cells. This strategy has enabled the localization of the full submolecular profile of T cell recognition of the Ra3 molecule by these mouse strains. Three of the regions recognized by T cells coincided with regions recognized by antibodies (i.e. B cells). It is noteworthy that in addition to sites recognized by both T and B cells the protein has at least one site which is recognized exclusively by T cells and to which no detectable antibody response is directed.     

Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course,induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: Preliminary analysis of MOG T cell epitopes

Myelin basic protein (MBP) and proteolipid protein (PLP), the most abundant proteins of central nervous system (CNS) myelin, have been extensively studied as possible primary target antigens in multiple sclerosis (MS), a primary demyelinating autoimmune disease of the CNS. However, there is increasing evidence to suggest that autoimmune reactivity against the quantitatively minor myelin component, myelin oligodendrocyte glycoprotein (MOG), can also play a role in the pathogenicity of MS. We recently demonstrated a predominant response to MOG by peripheral blood lymphocytes from patients with MS tested for their reactivity against various myelin antigens, including MBP and PLP. To ascertain whether or not T cell reactivity to MOG in MS is a potentially pathogenic response, we have tested the ability of synthetic MOG peptides (pMOG) representing potential T cell epitopes, to induce neurological disease in mice. Both strains of mice tested (SJL/J and PL/J mice) were able to mount a primary T cell response to some of the five MOG peptides synthesized, pMOG 1–21, 35–55, 67–87, 104–117 and 202–218. T cell lines could be raised in both strains to pMOG 35–55 and 67–87, but epitope definition revealed that each strain recognized a different minimal epitope within these two peptides. T cell lines to pMOG 1–21 and 202–218 could also be raised in SJL/J and PL/J mice, respectively. T cell reactivity to pMOG 104–117 was not observed in either mouse strain. None of the peptides tested induced detectable clinical signs in SJL/J mice. In contrast, an MS-like chronic relasping-remitting disease could be induced in PL/J mice with pMOG 35–55. The disease presented with a delayed onset and with clinical signs which differed significantly in their progression and expression from the typical ascending paralysis of experimental autoimmune encephalomyelitis induced with other myelin components, such as MBP and PLP. Histological examination of CNS tissue from mice injected with pMOG 35–55 revealed only mild neuropathological signs with few inflammatory foci in brain and spinal cord. Some myelin splitting and edema were detected upon electron microscopic examination in the spinal cord and cerebellum. Transfer of pMOG 35–55 reactive T cells into naive PL/J mice resulted in pathological changes characterized by inflammatory foci in the brain and spinal cord. This passively induced disease was clinically silent, as was also reported for Lewis rats injected with T cells specific for the same MOG peptide. These data, which demonstrate unequivocally the encephalitogenic activity of MOG, support our contention that MOG may be as important as MBP or PLP in disease pathogenesis and could be a primary target antigen in autoimmune diseases of the CNS.     

Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody

Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.     

T(h)1 but not T(h)0 cell help is efficient to induce cytotoxic T lymphocytes by immunization with short synthetic peptides

Immunization of BALB/c mice with peptide HVSGHRMAWDMMMNWA, encompassing residues 121-135 from hepatitis C virus E1 protein, induced CD4(+) T(h)1 cells as well as a long-lasting CD8(+) cytotoxic T lymphocyte (CTL) response in vivo when the peptide was administered s.c. with or without incomplete Freund's adjuvant. Using truncated peptides from this sequence it was shown that the determinant recognized by cytotoxic T cells was encompassed by residues SGHRMAWDM. Deletion of residues from the N-terminus or the C-terminus of the wild-type peptide abrogated its helper character. When Val122 of the wild peptide was replaced by Ala, the ability to induce a cytotoxic response was lost concomitantly with the loss of the T(h)1 pattern of cytokine production. Interestingly, the Ala-modified peptide, when co-immunized with a peptide encompassing residues 323-329 from ovalbumin (OVA), which is able to induce a T(h)1 response in BALB/c mice, restored the capacity of the modified peptide to induce CTL. However, co-immunization of the Ala-modified peptide with a peptide encompassing residues 106-118 from sperm whale myoglobin, which induces a T(h)0 cytokine profile in BALB/c mice, was much less efficient than the OVA peptide to restore CTL induction. These results demonstrate that CTL induction with a short synthetic peptide requires that this peptide contains domains recognized by T(c) cells as well as by T(h)1 cells. For those peptides that do not contain this type of T(h) domain, competent T cell help can be provided by co-immunization with a distinct peptide that is able to stimulate a T(h)1 response.