MXPA98000191A - Novedous peptides derived from human protein 60 produced by thermal shock, for the treatment of diabetes, compositions, methods and equi - Google Patents

Abstract

Novel peptides which are epitopes of the human 60 kDa heat shock protein (hsp60) can be used for the diagnosis and treatment of insulin dependent diabetes mellitus (IDDM), pharmaceutical compositions containing said peptides and equipment for use in ID diagnosis

Description

NOVEDOUS PEPTIDES DERIVED FROM HUMAN PROTEIN 60 PRODUCED BY THERMAL SHOCK. FOR THE TREATMENT OF DIABETES: COMPOSITIONS, METHODS AND EQUIPMENT FIELD OF THE INVENTION The present invention relates to novel peptides that are epitopes of the heat shock protein of 60 KDa (hsp 60), and to pharmaceutical compositions comprising them, for the diagnosis and treatment of insulin-dependent diabetes mellitus (IDDM).

BACKGROUND OF THE INVENTION Type I diabetes, or IDDM, is an autoimmune disease caused by T cells that attack and destroy insulin-producing beta cells located in the islands of the pancreas (Castaño and Eisenbarth, 1990). The autoimmune process that culminates in IDDM begins and progresses without symptoms. The disease emerges clinically only when the cumulative loss of beta cells exceeds the ability of residual beta cells to deliver insulin. It is actually believed that the collapse of glucose homeostasis and clinical IDDM occurs only after 80 to 90 percent of the beta cells have been inactivated by the immune system. Thus, patients who are identified as suffering from IDDM are probably at an advanced stage of autoimmune destruction of their beta cells. In addition, the diagnosis of incipient, preclinical diabetes, through the detection of unological markers of beta-cell autoimmunity, can be made only after the beginning of the autoimmune process. Therefore, the therapeutic objective is to find a safe, specific and effective way to make appear an autoimmune process that is already well established in a hidden way. The inventors of the present have examined this question before studying the spontaneous development of diabetes in mice of the NOD strain, which is considered to be a faithful model of human IDDM (Castaño and Eisenbarth, 1990). NOD mice develop insulitis at approximately four weeks of age, which begins as a moderate peri-islet infiltrate and progresses to severe intra-islet inflammation. Hyperglycemia, which shows insulin insufficiency, begins in females of the colony at approximately 14-17 weeks of age. At 35-40 weeks of age, almost all the females of the NOD mice had developed severe diabetes and most die in the absence of insulin treatment. Male NOD mice have a lower incidence of disease, for reasons that are unclear. It has been shown that diabetes of NOD mice is caused by autoinological uni T cells (Bendelac and coauthors, 1987). The reactivity of T cells and autoantibodies to various antigens have been detected in human IDDM patients, as well as in NOD mice (Elias, 1994) and it is not clear whether the immunity to any individual target antigen from the possible ones is the primary cause of the disease. Beyond the question of what is the cause, it is a matter of therapy. It has been shown that the initiation of the autoimmune process in NOD mice can be prevented by subjecting the mice, before the onset of diabetes, to various manipulations, such as a restricted diet, viral infections or non-specific stimulation of the immune system (Bo man and coauthors, 1994). Diabetes can also be prevented in NOD by inducing immunological tolerance in prediabetic mice, to the desired glutamic acid rboxylase (Kaufman and coauthors, 1993, Tish and coauthors, 1993). Insulin dependent diabetes mellitus (IDDM) that develops spontaneously in female NOD mice has been associated with immunological reactivity to a variety of autoantigens (Bach, 1994). Notable among these antigens is the peptide p277 of the sequence of the heat shock protein molecule of 60 kDa (hsp60) of mammal. This corresponds to residues 437-460 of the human hsp60 molecule (Elias and coauthors, 1991, Israeli patent application No. 94241, patent publication of TCP W090 / 10449. The human p277 peptide has the following sequence: Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile-Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp (amino acids 437-460 of SEQ ID N0: 1). NOD prediabetic mice manifest spontaneous responses of the diabetogenic T cell to hsp60 and to the human (2) or mouse variants of the peptide p277 (3). The mouse and human peptides differ in an amino acid and are immunologically reactive (3) with each other. Some mouse strains, not susceptible to diabetes, such as C57BL / 6, develop transient hyperglycemia and transient insulitis when they are immunized to p277 covalently conjugated to an extra immunogenic carrier molecule (4). And mice of strain C57BL / KsJ develop spontaneous T cell responses to hsp60 and to p277 after treatment with a very low dose of beta cell streptozotocin (STZ) toxin, which induces autoimmune diabetes (5). In addition to being involved in the expression of the disease, the p277 peptide appears to be functional in the healing of the autoimmune process: the subcutaneous administration of p277 in incomplete Freund's adjuvant (IFA, mineral oil) leads to the arrest of the disease progression in young NOD mice (2) or NOD mice 12-17 weeks of age, with advanced insulitis (6, 7). Both the human variant (6, 7) and the mouse variant (3) of p277 were effective. The NOD mice transgenic for the mouse hsp60 gene in a MHC class II promoter showed a down regulation of their spontaneous T cell proliferative response to p277 and a significant proportion of the mice did not develop diabetes (8). Additionally, administration of p277 to C57BL / KsJ mice aborted the development of autoimmune diabetes in mice that had received a very low dose of STZ early; the treatment of these mice with a peptide of the GAD65 molecule was not effective (9). Peptide variants p277 in which one or both cysteine residues at positions 6 and 11 were replaced by valine residues, designated p277 (Val6), p277 (Valii) and p277 (Val6-Val11), respectively, were described in the application of corresponding Israeli patent No. 112094, and proved to be as active as p277 in the treatment of diabetes. It is an object of the present invention to provide additional peptides of human hsp60; said peptides being useful for the diagnosis and treatment of IDDM.

BRIEF DESCRIPTION OF THE INVENTION In a study of fragments and peptides of human shp60 molecule, it was unexpectedly found that patients with IDDM and NOD mice with IDDM responded to other hsp60 T cell epitopes, which can be used for diagnosis and IDDM therapy. These epitopes, by themselves or in combination with p277 or a variant of p277, selected from p277 (Val6), p277 (val11) and p277 (Val6 -Val * 1) can improve the efficacy of the treatment. These new peptides are identified in Table 1, TABLE 1 SYNTHETIC PEPTIDES OF Hsp60 AND ITS SEQUENCE Residues No. of amino acid sequence Peptides SEQ ID NO: 1. - (single letter code) P3 31-50 136-155 NPVEIRRGVMLAVDAV KFGADARALMLQGVDLLADA Plo? AEL p PH11 1 15,511 to -117,700 VIAELKKQSKPVTTPEEIAQ P12 166-185 EEIAQVATISANGDKEIGNI pi "RKGVITVKDGKTLNDELEII p18 195-214 255-274 286-305 LVLNRLKVGLQVVAVKAPGF PP22 QSIVPALEIANAHRKPLVIIA p20 * '3 34,466 to -336,655 GEVIVTKDDAMLLKGKGDKA P29 421-440 VTDALNATRAAVEEGIVLGG p30 436-455 IVLGGGCALLRCIPALDSLT p32 466-485 EIIKRTLKIPAMTIAKNAGV p35 511-530 VNMVEKGIIDPTKWRTALL pp3399 3 34433--336666 GKVGEVIVTKDDAM It was shown that other hsp60 peptides, including those designated p278 (corresponding to positions 458-474 of the human hsp60 sequence), pl9 (corresponding to positions 271-290 of the human hsp60 sequence) and p21 (which corresponds to positions 301-320 of the human hsp60 sequence) were not as effective. It is noteworthy that the amino terminus of p278 overlaps with the effective p277 peptide in three residues (NED) and the carboxy terminus of p278 overlaps with the effective p32 peptide in 9 residues (EIIKRTLKI). So that, the remaining 11 residues of p32 are critical (PAMTIAKNAGV). Thus, the present invention relates to the peptides identified in Table 1, and to their salts and functional derivatives. It is another object of the present invention to provide methods and equipment for the early diagnosis of IDDM, using the peptides of the invention. In the course of developing IDDM, animals express hsp60 molecules or molecules that are mutually reactive with them, which find their way into the blood and urine of animals. They also express antibodies and T cells specifically directed to such molecules. Thus, the presence of hsp60 (or molecules that are mutually reactive with it) or antibodies or T cells specific for them, in the blood or in the urine, serves as an analysis for the detection of the IDDM process, rather than The destruction of the beta cells is complete and the individual is sentenced to diabetes for life.

The presence or incipience of IDDM in a patient can be diagnosed by analyzing the blood or urine of the patient for the presence of antibodies or T cells that are immunologically reactive with human hsp60, using as antigen a peptide p2 or p32 of the invention. Accordingly, the present invention provides a method for diagnosing the presence or incipience of IDDM in a patient, which comprises analyzing said patient for the presence of anti-hsp60 antibodies or a T cell that immunoreacts with hsp60, using as antigen a peptide of the present invention, such that a result indicating the positive presence of anti-hsp60 antibodies or of a T cell that immunoreacts with hsp60, indicates a high probability of the presence or the incipience of IDDM. In the method for diagnosing IDDM, the patient can be analyzed for the presence of anti-hsp60 antibodies, wherein the method of analysis may comprise a radioimmunoassay or an ELISA. The patient can also be analyzed for the presence of a T cell that immunoreacts with hsp60. In one embodiment of this aspect, the test method comprises a T cell proliferation test, comprising the steps of: (i) preparing a mononuclear cell fraction containing T cells from a blood sample obtained from the patient; (ii) adding to the mononuclear cell fraction an antigen selected from the peptide of the invention; (iii) incubating the cell fraction in the presence of the antigen for an adequate period of time and under suitable culture conditions; (iv) adding a labeled nucleotide to the culture of cells incubated from (iii), at an appropriate time before the end of the incubation period, to provide for incorporation of the labeled nucleotide into the DNA of the proliferating T cells; and (v) determining the amount of proliferating T cells, by analyzing the amount of labeled nucleotide incorporated into the T cells. In step (iv) above, the labeled nucleotide is preferably 3H-thymidine. The determination of the number of proliferating T cells is carried out by calculating the stimulus index of the T cells by common and current methods. In another embodiment of this aspect of the invention, the test method comprises a T-cell cytokine response test, wherein steps (i) to (iii) are as in the previous T-cell proliferation test, and in a fourth step (iv), the presence of cytokine is detected, such as IFN-gamma, IL-2, IL-4, IL-6, IL-10, TNFalpha or TGFbeta, secreted by the lymphocytes that respond, present in the middle; by means of common and current methods, with equipment obtainable in commerce.

In another aspect, the invention provides an in vivo method in which an antigen selected from the peptides of this invention is injected subcutaneously into a patient; and the occurrence of a detectable skin reaction (delayed type hypersensitivity (DTH) is observed.) The present invention also relates to means for carrying out said analyzes, as well as to equipment for carrying out such analyzes. Any of the various assays used to carry out the present invention Each kit includes all the materials necessary to carry out a single analysis, or a fixed number of analyzes.For example, such equipment for determining the presence of anti-hsp60 antibodies may contain a solid phase immobilized peptide of the invention, and a labeled antibody, capable of recognizing the non-variable region of the anti-hsp60 anti-cue to be detected, such as labeled anti-human Fab. The kit may also contain instructions for use the case and containers to hold the case materials, any conventional label or tag, such as a radio, can be used oisotope, an enzyme, a chromophore or a fluorophore. A typical radioisotope is iodine 125 or sulfur 35. Typical enzymes for that purpose include horseradish peroxidase, horseradish galactosidase and alkaline phosphatase. A team to diagnose the presence of IDDM by analyzing the resistance of anti-hsp60 antibodies, comprende: (i) an antigen selected from the peptides of the present invention; and (ii) a labeled antibody, capable of recognizing the non-variable region of the anti-hsp60 antibodies to be detected. A device for diagnosing the presence of IDDM by analyzing the presence of a T cell that immunoreacts with hsp60, will comprise: (i) an antigen selected from the peptides of the present invention; (ii) a suitable medium for the culture of lymphocytes (T cells); and (iii) a labeled nucleotide for the T cell proliferation test or a cytokine, for example, interferon-gamma analysis equipment, for the cytokine test. For the in vivo test, the kit will comprise only one peptide of the invention, in a form suitable for injection. The present invention further relates to means for preventing or treating IDDM. The vaccine with an antigenic peptide of the present invention can provide down-regulation specific to autoimmunity to the antigen and effectively create a resistance to the autoimmune process of the IDDM. The same applies with respect to the vaccine with T cells specific for such antigens, in attenuated or avirulent form, or after having been treated to improve their antigenicity, or fragments or active fractions thereof. If it is shown that the patient is already in the incipient, preclinical, IDDM stages, the injection with said antigen or T cell (or fraction) can create a down regulation of the autoimmunity for that antigen and, in this way, stop the process autoimmune disease before it causes significant and permanent damage. The peptide can also be used as a therapeutic agent to stop the autoimmune process, even after it is quite advanced, as was recently shown in the laboratory of the present inventors, with respect to the treatment of NOD mice with the p277 peptide ( Elias and Cohen, 1994). Accordingly, the present invention provides a preparation for preventing or treating insulin dependent diabetes mellitus (IDDM), comprising: (a) T cells that have developed specificity for a protein or a peptide that is immunologically reactive with a peptide of the invention; cells that have been activated by incubating them in the presence of the peptide; (b) said T cells that have been irradiated or otherwise attenuated; (c) said T cells, which have been subjected to a pressure treatment, by means of hydrostatic pressure, treatment with a chemical interlacing agent and / or treatment with a cytoskeletal interlacing agent; (d) fragments of, or surface proteins released from (a), (b) or (c); or (e) a peptide consisting essentially of the variable region of the receptor of (a), specific for that protein; or a salt, a functional derivative, a precursor or an active fraction thereof. In a preferred embodiment of the invention, the preparation comprises human T cells that have developed specificity by in vitro contact with the peptide of the invention. The present invention also provides a pharmaceutical composition for the prevention or treatment of IDDM, comprising a pharmaceutically acceptable carrier and, as an active ingredient, an effective amount of a peptide of the invention, a salt or a functional derivative thereof. The invention further relates to a method for preventing or treating IDDM, which comprises administering to a patient in need thereof, a pharmaceutical composition comprising a peptide of the invention, a salt or a functional derivative thereof, or a preparation comprising T cells that have developed specificity towards the peptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the positions of the peptides referred to herein, in the complete sequence of the human hsp60 molecule. Figure 2 shows the proliferation of NOD mouse T cells for the p2 peptides, p32, p277 (Val «- ain) and p278 of human hsp60. Figure 3 is a graph showing the T cell proliferative responses of NOD mice to the peptides. Groups of three NOD mice were immersed with mouse pl2 peptides, mouse p277, GAD-p35 and MT-p278 in IFA, each at a dose of 25 μg in IFA. The lymphatic nodes were drained 10 days later and analyzed for proliferative responses to the corresponding peptide at concentrations of 5, 10, 20 and 50 μg / ml. The stimulation at the optimal concentration of 20 μg / ml is shown. The following cpm scales were obtained in media controls: mouse pl2, mouse 881, p277, 1243, MT-p278, 698 and GAD-p35 1430. The mouse p38 peptide is a peptide derived from mouse hsp60 (556 -573), which has no sequence homology with the peptides tested and which serves as a negative control of specificity. These results are representative of the three experiments carried out. Each analysis was carried out in triplicate, for which the values of standard deviation (DN) are indicated by the bars. There was no mutual reactivity between the peptides (not shown). Figure 4 is a graph showing the effect of the administration of the peptide on diabetes. Groups of 10-20 NOD mice were treated at 10 weeks of age, with 100 μg of mouse pl2, p277, p35-GAD or MT-p278 in IFA, or with IFA only. The mice were bled monthly and followed up for the onset of hyperglycemia. In comparison with the control group, treated with IFA, the mice treated with P12 and with P277 were protected significantly, P <; 0.05. Figure 5 shows the IgGl antibody isotypes, IgG2a and IgG2b, in response to the treatment of peptides. The mice were treated as described in the legend of Figure 4. The individual samples were analyzed for antichodes to mouse P12 A, p277 B, GAD-p35 C and MT-p278 D, of the isotypes IgG1, IgG2a and IgG2b. Similar effects were obtained in two experiments. The results are presented as the absorbance at 405 nm (OD) of 10 individual mice in each group. The level of significance of the prevalence of IgG1 and IgG2b antibodies in groups A and B, compared to C and D, is P < 0.001. The differences between IgG1 and IgG2b anti-phosphate levels, in comparison with IgG2a antibodies, in groups A and B, were significant (P <0.001). There was no mutual reactivity between the anticuefos (not shown). Figure 6 shows the negative correlation between anticuefos and glucose in the blood. A group of NOD female mice was treated with pl2 (10 mice) or with IFA alone (9 mice) as described in the legend of Figure 4. The amount of anti-p2 specific anti-cough was ped (OD units of ELISA in FIG. sera, diluted 1:50, measured at 7 months of age) together with the blood glucose concentration, measured at 7 months of age. The degree of correlation between high levels of anticuefos and glucose in the blood is P < 0.002. Figure 7 is a graph showing the T cell proliferation responses (S.I.) of a patient IDDM donor to the recovery antigens, to the hsp60 protein and to the hsp60 synthetic peptides. Figure 8 is a graph showing the T cell proliferation responses (SI from a healthy donor to the recovery antigens, to hsp60 and to the hsp60 synthetic peptides.) Figure 9 is a graph showing the T cell proliferation responses. (S.I.) of another IDDM patient donor, the recovery antigen, hsp60 and hsp60 synthetic peptides Figures 10A and 10B are graphs showing the T cell proliferation (SI) responses of two human donor patients. IDDM to the synthetic peptides of hsp60.

DETAILED DESCRIPTION OF THE INVENTION Whenever a "peptide of the invention" or any of the individual designations, such as "peptide pl2" or "peptide p32" is mentioned in the present specification and in the claims, its salts and functional derivatives are also contemplated, always and when the biological activity of the peptide is maintained with respect to diabetes. The "salts" of the peptides of the invention contemplated by the present invention are the physiologically acceptable organic and inorganic salts. The "functional derivatives" of the peptides of the invention, as used herein, cover derivatives that can be prepared from the functional groups that occur as side chains in the residues or the N or C end groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, that is, they do not destroy the activity of the peptide or impart toxic properties to compositions containing them or adversely affect their antigenic properties. These derivatives, for example, may include the aliphatic esters of the caFoxyl groups; the amides of the caFoxyl groups produced by reaction with ammonia or with primary or secondary amines; N-acyl derivatives of free amino groups of the amino acid residues, formed by reaction with acyl portions (for example, alkanoyl or aroyl cycloalkyl groups) or O-acyl derivatives of free hydroxyl groups (for example, those of the seryl residues or threonyl), formed by reaction with acyl portions. The peptides of the invention can be used as immunogens in pharmaceutical compositions, particularly in vaccines for relieving and treating IDDM, as well as in the manner of an antigen in diagnostic compositions for diagnosing IDDM. These pharmaceutical and diagnostic compositions, which can be prepared in a manner known in the art, also form part of the present invention. The therapeutic composition according to the present invention can be administered orally or parenterally, for example, subcutaneously, intramuscularly, intravenously, intranasally or intra-rectally. The invention will now be illustrated in a non-limiting manner by means of the following examples and the appended figures.

EXAMPLES MATERIALS AND METHODS (i) Mice Female internal crossbred mice, strain NOD / lt, were supplied by the Animal Breeding Center of the Weizmann Institute of Sciences, Rehovot, Israel, or by the Jackson Laboratory, Bar Harbor, ME, E.U.A. These mice spontaneously develop autoimmune diabetes at 14 to 17 weeks of age, which mimics IDDM in humans. (ii) Antigens The peptides were synthesized in the Organic Chemistry Department of the Weizmann Institute of Sciences, using a multiple peptide synthesizer, automatic (Abimed, model AMS 422; Langenfeld, Germany), following the company's protocols, for the synthesis of N-alpha-fluorenyl ethoxyFonyl (Fmoc). The crude products were purified by reverse phase HPLC on a semipreparatory C8 column (LichrosoF RP-8, 7 mm, 250 x 10 mm, Merck, Darmstadt, Germany). Elution of the peptides was obtained by linear gradients established between 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in 75% acetonitrile in water (volume / volume). The purity of the individual peptide products was determined by analytical reverse phase HPLC, and by amino acid analysis. The peptide MT-p278 comes from the sequence of hsp60 microbacteriana (431-447). Peptide p277 has replaced positions 6 and 11 with valine (V) in place of the cysteine (C) of the natural sequence. The replacement of the two residues C by V greatly increases the stability of the peptide, without affecting its immunological activity: the peptide with V replacement is mutually completely reactive with the natural peptide by means of analysis of the T cell and the antiquase . Whenever it is not specified, we want to refer to the human sequence. The mouse pl2 and mouse p38 peptides are derived from the mouse hsp60 molecule and correspond to their sequences 168-188, 437-460 and 556-573, respectively. The GAD-p35 peptide comes from the GAD65 molecule (524-543). The amino acid sequences of all the peptides used herein are shown in table 2. Sequence of amino acid sequence Peptide SEQ ID NO: .-- (single letter code) P3 1 (31-50) KFGADARALMLQGVDLLADA plO 1 (136-155) NPVEIRRGVMLAVDAVIAEL pll 1 (151-170) VIAELKKQSKPVTTPEEIAQ pl2 1 (166-185) EEIAQVATISANGDKEIGNI pl4 1 (195-214) RKGVITVKDGKTLNDELEII pl8 1 (255-274) QSIVPALEIANAHRKPLVIIA p20 1 (286-305) LVLNRLKVGLQ VAVKAPGF p24 1 (346-365) GEVIVTKDDAMLLKGKGDKA p29 1 (421-440) VTDALNATRAAVEEGIVLGG p30 1 (436-455) IVLGGGCALLRCIPALDSLT p32 1 (466-485) EIIKRTLKIPAMTIAKNAGV p35 1 (511-530) VNMVEKGIIDPTKWRTALL p39 1 ( 343-366) GKVGEVIVTKDDAM PL9 1 (271-290) p21 LVIIAEDVDGEALSTLVLNR 1 (301-320) p278 1 KAPGFGDNRKNQLKDMAIAT (458-474) NEDQKIGIEIIKRTLKI p277 (Val) 2 VLGGGVALLRVIPALDSLTPANED mouse pl2 3 EEIAQVATISANGDKDIGNI MT-p278 4 EGDEATGANIVKVALEA p35-GAD 5 SRLSKVAPVIKARMMEYGTT mouse p38 6 PGMGAMGGMGGGMGGGMF (iii) Proliferation of T cell to peptides Mice: Nine-week-old NOD mice or mice of other strains were immunized in the plants of the front legs with 0.1 ml of an emulsion containing 25 μg of peptide in adyuv. complete before Freund (CFA; Difco, Detroit, MI, E. U. A.), mixed with an equal volume of phosphate buffered saline (PBS). The popliteal lymph nodes were removed, which were then drained and the lymphocyte suspensions were tested in triplicate cultures, in terms of proliferation in the presence of various peptides (5 μg / ml) using the C3H] -thymidine incoforation, as described (Elias and co-authors, 1991). The results are shown as the stimulus index (EI): the ratio of the average cpm in the presence of the test peptide, with respect to the average cpm of the control cultures, without the peptide. The norm errors were always less than 10% of the means. (iv) Treatment and follow-up: 100 mg of peptides were eulogized in PBS with an equal volume of IFA and injected subcutaneously into 10-week-old NOD females, as described (Elias and Cohen, 1995). Control mice received an equal volume of PBS emulsified in IFA. Mice were monitored monthly for blood glucose, without fasting, at 10 a.m., using the blood glucose sensor SMediSense Inc., Waltham, MA). It was considered that mice with blood glucose greater than 11.1 mmol / L were diabetic; this glucose concentration was greater than three standard deviations above the mean concentration of glucose in the blood, measured in non-diabetic mice (Elias and Cohen, 1995). Histological examination of the islands of the pancreas was carried out on sections stained with hematoxylin and eosin. The sections were independently marked by two observers, who did not know the identity of the groups. The chi square test was used to determine the statistical differences between the various treatments. (v) Anticuefos in serum The mice were bled monthly to detect exposed to the antiquake. ELISA analysis was performed as described (Elias and coauthors, 1991). Briefly, flat bottom MaxisoF plates (Nunc, Roskilde, Denmark) were coated for the detection of anti-peptide anticuefos, with 100 ml / concavity of peptide in PBS, at a concentration of 10 mg / ml for 2 hours, to the room temperature, and then incubated overnight at 4 ° C. After incubation with the peptide the plates were washed and blocked for two hours at 37 ° C with 7% BSA (Sigma) in PBS. The sera were diluted 1:50, then added for two hours at 37 ° C, and then incubated for two hours with 100 ml per concavity of goat anti-mouse IgG (specific for gamma chain Fc), conjugated with phosphatase alkaline (Jackson, Philadelphia, PA, USA) After washing, the plates were incubated with the substrate, diethanolamine (Sigma) and read using a reagent ELISA at 405 nm.

EXAMPLE 1 MAP FORMATION OF EPITOPES hSP60 IN NOD MICE The immunogenicity of the peptides pl2, p32, p277 (Val * -Valí1) of hsp60 and p278 was tested in NOD mice, immunizing the mice with the peptides emulsified in CFA, in the plants of the front legs, and analyzing the proliferation responses of the lymph node cells that drain, after 10 days, as described in section iii (a) above. As shown in Figure 2, peptides p277 (Val6-Val11), pl2 and p32 were strongly immunogenic, whereas p278 was not immunogenic.

EXAMPLE 2 NOD MICE TREATMENT WITH p277 (Val-Val **). Pl20 P32 To test whether the peptides pl2 and p32 can block the progression of diabetes such as p277 (Val6-Val11), peptides p277 (Val6-Val11), pl2 or p32 (100 μg in 0.1 cc of IFA emulsion) were administered subcutaneously to groups of NOD / Lt female mice of 10-12 weeks of age, from the Jackson Laboratory, Bar-Ha rbor, ME, USA Diabetes was tested, determined as persistent blood glucose levels of more than 11.1 mmol / l, at 25 weeks of age. Control mice were not treated or treated with p278.

As shown in Table 1, p277 (Val6-Val11), pl2 and p32 were effective in the treatment of diabetes, with the incidence of diabetes in untreated mice or in mice treated with p278, 90%; whereas mice treated with p277 (Val6-Val1: 1), p2 and p32 show an incidence of 10%, 20% and 30%, respectively. On the other hand, the control peptide p278 had no therapeutic effect.

TABLE 3 THERAPEUTIC EFFECT OF PEPTIDES OF hsp60 DiabeMortanNo. from Peptide tes (%) dad (%) receptors none 90 50 100 p278 90 45 100 p277 (Val6 - Val11) 10 * 5 * 100 pl2 20 * 10 * 10 p32 50 * 25 * 20 * P < 0.05 It is possible that a combination of two or three epitope peptides of hsp60 is more effective than a single peptide, since more T cell populations will be affected by the therapy.

EXAMPLE 3 PATIENTS WITH NEWLY DIAGNOSED IDM SHOW PROLIFERATION REPLIES OF CELL T TO hsp60. p277 (Val * -Val ***). Pl2 Y p32 To determine the T cell responses to the various hsp60 peptides, peripheral blood lymphocytes from patients who had just been diagnosed with IDDM (2 weeks-4 months) were tested in a proliferation assay. 10 to 20 ml of blood was drawn into a sterile tube containing heparin as an anticoagulant and diluted in PBS 1: 2. Peripheral mononuclear cells (PBMC) were isolated by centrifuging the blood on a layer of lymphoprol. The PBMC were tested for proliferation, in triplicate, in the presence of the various antigens (10 μg / ml) for six days, using the incorporation of [3 H] -thymidine as a measure of proliferation. The tested antigens were human hsp60 or the peptides p277 (Val6-Val11), pl2, p32 of hsp60 and the control peptide p278. The T-cell proliferation response is illustrated as the stimulus index (EI): the relationship between the thymidine-induced incoforation stimulated by the peptide and the inciproration of background thymidine (without added antigen) by the T cells. The results are summarized below in table 4.

TABLE 4 Pacien (IE) of T cell proliferation response No. hsp60 P277 (V) P12 P32 P278 1 5 5..66 4 4..55 4.0 1.1 0.5 2 7 7..55 5 5..00 4.5 1.3 0.8 3 8 8..00 5 5..66 1.2 7.1 0.7 4 3 3..44 1 1..00 1.9 2.9 0.9 11..22 11..11 1.7 1.0 ND 6 66..77 11..33 5.2 4.5 ND 7 1100..33 33..99 1.2 6.0 ND 8 11..33 11..22 1.5 1.1 ND It is considered that a stimulus index (EI) of more than 2.0 is a positive response. ND = not determined; p277 (V) = p277 (Val * -Val11). It can be seen that most of the patients responded to hsp60 (6/8) and that the six that responded to hsp60 also responded to at least one of the three hsp60 peptides: p2, p32 or p277 (Val * -Val11) . In such a manner, a response to the group of peptides can serve to characterize the individuals that respond to the entire hsp60 molecule.

EXAMPLE 4 PROLIFERATIVE RESPONSES OF CELL T Spontaneous T-cell responses were detected in prediabetic NOD mice to the mouse p277 peptide (Elias and coauthors, 1991; Birk and coauthors, 1996) and to larger fragments of the mouse hsp60 molecule containing the mouse p277 sequence (Birk and co-authors, nineteen ninety six). To detect other T cell epitopes in the mouse hsp60 molecule, NOD mice were immersed in peptide assemblies that overlap the hsp60 sequence and all mice were found to strongly respond to mouse p277 and mouse pl2 (Figure 3). Other immunogenic peptides for NOD mice are peptide MT-p278 (residues 458-474 in the hsp60 microbacterial molecule) and GAD-p35 (residues 524-543 in the GAD65 molecule). Figure 3 shows that MT-p278 is as strongly immunogenic as mouse p277 and mouse pl2; GAD-p35 is also immunogenic, but less so. A longitudinal study of female NOD mice at 3-16 weeks showed no spontaneous responses to mouse pl2 in their spleens (not shown), although responses were seen to mouse p277 and to mouse whole hsp60. In this way, four immunogenic peptides: pl2 and p277 of the mouse hsp60 molecule; GAD-p35 of the GAD65 molecule associated with diabetes and MT-p278, a foreign immunogen, spontaneous responses were detected only to mouse p277 and to MT-p278.

THE TREATMENT WITH THE PEPTIDE With a protocol that proved effective with mouse p277 (Elias and coauthors, 1991; Elias and Cohen, 1994; Elias and Cohen, 1995), groups of 10-week NOD female mice were treated with a single subcutaneous injection of each peptide. (100 mg), emulsified in IFA. The mice were observed for the development of diabetes until 8 months of age. Figure 4 shows that both mouse p277 and mouse pl2 were effective peptides to inhibit the development of diabetes (P <0.05). In contrast, the treatment with the peptides MT-p278 or GAD-p35 was not different from the treatment with IFA alone. A total of three experiments showed essentially the same results.

THE ANTIBODIES Successful treatment of STZ-induced diabetes with the p277 mouse peptide was associated with the appearance of antipeptide anticuefos predominantly of the IgG1 and IgG2b isotypes (Elias and Cohen, 1996). NOD mice treated with peptide were therefore examined for their serum anti-convolutions. Figure 5 shows that the two peptides effective to stop diabetes, mouse pl2 and mouse p277, were also effective to induce strong antibody titers of the IgG1 and IgG2b isotypes (P <0.001). Anti-IgG1 and IgG2b anti-cotuene titers were also significantly higher than anti-IgG2a titers in these groups (p <0.001). Mice treated with peptides MT-p278 or GAD-p35 did not respond as strongly; none of the mice treated with GAD-p35, made specific IgGl antibodies, and only two of the ten mice treated with MT-p278 made anti-IgGl isotype antibodies. Mice treated with MT-p278 or GAD-p35 made significantly lower titers of IgG2b antibodies (P <0.001). Thus, the effectiveness to inhibit diabetes was associated with the induction of an antiquase response, mainly of the IgG1 and IgG2b isotypes. The relationship between the effective therapeutic response and the anti-cotuex titer was confirmed by a comparison of the concentration of glucose in the blood, with the concentration of anticuefos in individual mice, at 7 months of age. Figure 6 shows that mice with higher anti-mouse anti-drug pl2 titers tended to have lower concentrations of glucose in the blood; conversely, mice treated with mouse pl2, which had little antibody to mouse pl2, tended to have high blood glucose content (P <0.002).

DISCUSSION The results presented in this example indicate that the P12 peptide of the mouse hsp60 molecule, like peptide p277, can be effective to treat mice about to enter declared hyperglycemia. In contrast to p277, no spontaneous T cell proliferation responses were observed to pl2 in the spleens of rediabetic NOD p mice. Thus, a spontaneous anti-peptide proliferation response, detectable in the periphery, is not a requirement for a peptide to be effective in blocking the autoimmune diabetic procedures. The discovery that peptide p277 is not the only hsp60 peptide that can modulate diabetes in NOD is important. It was conceivable that the involvement of hsp60 in NOD diabetes could be effected by a similarity between the peptide p277 of hsp60 and a certain unknown molecule, more specific for beta cells (Cohen, 1991). However, it is highly unlikely that two different segments of hsp60: p277 and pl2 could reproduce segments of the proposed, but unknown, beta-cell molecule. The effectiveness of pl2 in the treatment of peptides supports the conclusion that the hsp60-like molecule, functional in diabetes, is the hsp60 itself (Birk and coauthors, 1996). The fact that neither the peptide MT-p278 nor the peptide GAD-p35 can stop the development of diabetes indicates that not any auto-antigen nor any antigen of spontaneous proliferation of T cell can be used to abort the autoimmune process. Interestingly, MT-p278 failed to induce high titers of anti-convolutions or protect, despite the fact that the peptide is strongly in univogen for T cells (unpublished observation). However, the induction of anti-convolutions of any specificity does not necessarily affect the diabetes of NOD; the treatment of NOD mice with BSA, which induces high titers of anticuefos, as well as strong T cell responses (not shown) does not affect the development of diabetes (Elias and Cohén, 1994). While it was not found by the inventors herein that GAD-p35 was as strongly immunogenic to T cells as were the other peptides (Figure 3), NOD mice have been reported to manifest spontaneous T cell responses to their peptide. (Kaufman and coauthors, 1989). Finally, the association of an effective treatment with the induction of anticuefos specific for the peptide, suggests that the therapeutic effects of pl2 and p277 could be related to the activation of Th2-type T cells, which are responsible for helping the induction of IgGl anticuefos specific, antibodies regulated by the production of IL-4 (Mossman and Coffman, 1993). Such T cells could suppress Thl T cells, although it has been proposed that they are responsible for damage to beta cells (Latz and coauthors, 1995; Liblau and coauthors, 1995). In fact, it has been discovered that therapy with the peptide p277, in NOD diabetes, is associated with a discharge of T cells that produce IL-4 and IL-10 in the spleen, and a failure in the T cells that produce INF-gamma, both in the spleen and in the islets (observation not published). The appearance of specific anti-convolutions for the peptide, which carry Th2 isotypes, in response to peptide therapy, seems to be an indicator of a beneficial response.

EXAMPLE 5 ADDITIONAL PEPTIDES TO WHICH PATIENTS WITH I DM CAN MANIFEST CELLULA T ANSWERS Twenty-six patients were enrolled who had just been diagnosed with IDDM (1 to 16 weeks after diagnosis of IDDM). The age of the patients varied between 5 and 60 years. Patients were discriminated in terms of their proliferating T-cell responses in the peripheral blood, human hsp60 and synthetic human hsp60 peptides, shown in Tables 1 and 5, which are portions of the human hsp60 protein sequence. The T cells of the patients were analyzed for their proliferation responses to normal updating antigens, such as tetanus toxoid, Candida albicans and influenza and the responses were recorded as positive if the stimulus index (EI) was 2. or more (see below).

Proliferation was analyzed using the following protocol: CELL PREPARATION AND CELL PROLIFERATION PROTOCOL Patients with IDDM were taken 50 ml of peripheral blood, supplemented with 10 IU / ml of heparin, or of healthy controls. Two volumes of PBS (free of calcium and magnesium) were added. The blood-PBS preparation was mixed using a 10 ml pipette. A volume of 10 ml of Ficoll was placed in the blood mixture, followed by centrifugation at 2,000 fm for 30 minutes, at room temperature (T.A), 20-24 ° C (without brake). The peripheral blood T cells were harvested in the cotton wool coating, using a 10 ml pipette, and transferred to a new 50 ml test tube. A volume of 30 to 40 ml of PBS (free of calcium and magnesium) was added to the harvested T cells. This was then mixed and centrifuged at 1,000 fm for 20 minutes at T.A. (with the brake set). The supernatant was aspirated and the cell pellet was resuspended in AIM-V serum free culture medium (GIBC0, USA). The culture medium contains AIM-V supplemented with 1% sodium pyruvate, 1% L-glutamine (200 mM each), 1% penicillin / streptomycin (10,000 U / ml / 10,000 mg / ml and 2% Hepes (1 M, pH 7.3) Alternatively, RPMI supplemented with 10% AB serum from the blood bank was used, then the cell mixture was centrifuged at 2,000 rpm for 10 minutes at RT (brake applied). to aspirate the supernatant and resuspend the cell pellet in a smaller volume of fresh AIM-V (10-20 ml) .The cells were resuspended and counted, cell count and viability analyzes were performed using trypan blue, a hemocyte and a light microscope was used for this step, then the cell concentration was adjusted to 2 x 10 6 cells / ml in AIM-V media, and a volume of 100 μl of the cells was transferred. cells per concavity, to each concavity of a microtiter plate of 96 concavities, then 100 μl of medium containing twice the recommended antigen concentration (see list of antigens and concentrations below). The analysis was carried out in quadruplicate. Four concavities were analyzed with cells and medium without antigen, as a control. Plates were cultured at 37 ° C in a humidified incubator, with 5% CO2, for 18 hours, and then harvested. The proliferation of cells was analyzed by incorporation of 3H-thymidine into the DNA, determined using flash liquid and a beta counter reader.

Tested antigens Tetanus toxoid concentration (Connaught Lab., 5 g / ml US Pen, Candida albicans 20 μg / ml human recombinant hsp60 (StressGen, 2-5 μg / ml Canada) synthetic hsp60 human peptides 20 μg / ml The proliferation responses were measured according to the 3H-thymidine incubated by the DNA of the T cells (Elias and coauthors, 1991) The radioactive counts per minute (Cpm) were compared between the cultured cells with the tested antigens or without antigen (only medium) as control Proliferation values were represented as a stimulus index (IE) value: Average CPM, with the antigen divided between the average CPM without the antigen. The values of I.E. of more than 2, or equal to 2, were considered positive. The results showed that the incidence of individuals reactive to human hsp60 or the human hsp60 peptide was higher among IDDM patients (84) than among healthy people (30%). The p-value of Fisher's exact test, of the difference, is 0.0044, which is highly significant. The proliferation responses of two representative patients with IDDM and a healthy individual, the human hsp60 protein, the synthetic hsp60 peptides and various reinforcing antigens, are shown in Figures 7, 8 and 9. Table 5 shows two examples Individuals of the synthetic hsp60 peptides (pl9, p21) to which the patients with IDDM did not respond (see also Figures 10A and 10B). It can be seen that each of the patients responded to the reinforcement antigens (Candida, tetanus or influenza) and to the human protein hsp60 and to various peptides of human hsp60. The control person responded only to the control booster antigens. The sequences of the synthetic hsp60 peptides to which at least one of the patients with IDDM responded are shown in Table 1. Each of these peptides has therapeutic potential to treat IDDM.

TABLE 5 SYNTHETIC PEPTIDES OF HSP60 AND ITS SEQUENCE. TO WHOM PATIENTS WITH IDDM DID NOT RESPOND Residues No. Amino acid sequence Peptides of SEQ ID N0: 1 (single letter code) pl9 271-290 LVIIAEDVDGEALSTLVLNR p21 301-320 KAPGFGDNRKNQLKDMAIAT The foregoing description of the specific embodiments will so completely reveal the general nature of the invention that, when applying the knowledge within the experience of the art (including the contents of the references cited herein), others may easily modify and / or adapt for various applications said specific modalities, without undue experimentation, without departing from the general concept of the present invention. Therefore, it is intended that these adaptations and modifications are within the meaning and scale of equivalences of the described modalities, based on the teaching and guidance presented here. It should be understood that the phraseology and terminology of the present is intended to be descriptive and not a limitation, so that the terminology or phraseology of the present specification should be understood by those skilled in the art in light of the teachings. and the guide presented here, in combination with the knowledge of who is an expert in the field.

REFERENCES Bach JF. (1994) Insulin-Dependent Diabetes Mellitus as an Autoimmune Disease. Endocrine Reviews 15: 516-542. Bendelac A, Carnaud C, Boitard C, Bach JF. (1987) Syngeneic transfer of autoimmune diabetes from diabetic NOD ice to healthy nonate. Requirement for both L3T4 + and Ly + -2 + T cells. J Exp Med. 166: 823-32. Birk OS, Elias D, Weiss AS, Rosen A, Rosen A, Van-der Zee R, Walter MD, Cohen IR. (1996) NOD mouse diabetes: The ubiquitous mouse hsp60 is a beta-cell target antigen of autoimmune T cells. J. Autoimmun. 9: 159-166.

Bowman MA, Leiter EH and Atkinson MA. (1994) Prevention of diabetes in the NOD mouse: implications for therapeutic intervention in human disease. Immunology Today. 15: 115-20. Chestnut L, Eieenbarth GS. (1990) Type-I diabetes: a chronic autoimmune disease of human, mouse, and rat. Annu Rev Im inol. 8: 647-79. Cohen GO. (1991) Autoimmunity to chaperonins in the pathogenesis of arthritis in diabetes. Annu Rev Immunol 9: 567-589. Elias, Dana. (1994) The NOD mouse: A model for autoimmune insulin-dependent diabetes. Autoimmune Disease Models, A Guidebook, pp 147-61. Elias D, Cohen IR. (1995) Treatment of autoimmune diabetes and isulitis in NOD mice with heat shock protein 60 peptide p277. Diabetes 44: 1132-1138. Elias D, Reshef T, Birk OS, van der Zee R, Walker MD, Cohen IR. (1991) Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65 kDa heat shock protein. Proc. Nati Acad Sci USA. 88: 3088-91. Elias D. and Cohen IR. (1994) Peptide therapy for deabetes in NOd mice. The Lancet. 343: 704-706. Elias D, Cohen IR. (1996) The hsp60 peptide p277 arrests the autoimmune diabetes induced by the toxin streptozocin. Diabetes (in press). Katz Jd, Benoist C, Mathis D. (1995) T helper cell subsets in insulin-dependent diabetes. Science 268: 1185-1188.

Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GSP, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. (1993) Spontaneous loss of T-cell tolerance to glutamic acid decaFoxilase in murine insulin-dependent diabetes. Nature 366: 69-72. Lablau RS, Singer SM, McDevitt HO. (1995) Thl and Th2 CD4 + T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 16: 34-38. Mossman TR, Coffman RL. (1989) THl and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 9: 145-173. Tisch R, Yang XD, Singer SM, Liblay RS, Fuggar L, McDevitt HO. (1993) Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366: 72-75.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: YEDA RESEARCH &; DEVELOPMENT CO. LTD., At the Weizmann Institute of Sciences. (B) ADDRESS: P. 0. Box 95 (C) CITY: Rehovot.- (E) COUNTRY: Israel (F) POSTAL CODE: (CP) 76100 (G) TELEPHONE: 972-8-9470617 (H) TELEFAX: 972-8-9470739 (A NAME: I run R. COHÉN (B ADDRESS: 11 Hankin Street (C CITY: Rehovot.- (E) COUNTRY: Israel CF. POSTAL CODE: (ZIP) 76354 (TO NAME: Dana ELIAS ( B ADDRESS: 57 Derech Yavne (C CITY: Rehovot.- (E) COUNTRY: Israel (F ZIP CODE: (ZIP) 78344 (A NAME: Rivka BULAFIA (B ADDRESS: 31/11 Weizmann Street (C CITY: Yahud.- (E) COUNTRY: Israel (F) ZIP CODE: (ZIP) 56000 (A NAME: Jana BOCKOVA a / c Ms Joan Fraser NYS / Pediatrics, SUNY Health Sciences Center (B ADDRESS: 11 Floor, Room 040 (C CITY: Stony Brook (D STATE: NY (E COUNTRY: USA (F POSTAL CODE: (CP) 11794 (ii) TITLE OF THE INVENTION: NOVEDOUS PEPTIDES DERIVED FROM PROTEIN 60 THERMAL HUMAN SHOCK, FOR THE TREATMENT OF DIABETES; COMPOSITIONS, METHODS AND EQUIPMENT (iii) NUMBER OF SEQUENCES: 6 (iv) FORM THAT MAY BE LE COMPUTER INDICATION: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) APPLICATION PROGRAM: Patentln Relay # 1.0, Version # 1.30 (EPO). (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: IL 114407 (B) SUBMISSION DATE: June 30, 1995 5 (2) INFORMATION FOR SEQ. ID. NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 573 amino acids (B) TYPE: Amino acid (C) No. OF FILAMENTS: one only 10 (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Leu Arg Leu Pro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arq 1 5 10 15 Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 1 - . fifteen? '25 30 Gly Wing Asp Wing Arg Wing Leu Met Leu Gln Gly Val Asp Leu Leu Wing 35 40 45 Asp Wing Val Wing Val Thr Met Gly Pro Lys Gly Arg Thr Val lie n 50 50 60 Glu Gln Gly Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 75 80 Wing Lys Ser He Asp Leu Lys Asp Lys Tyr Lys Asn He Gly Wing Lys 0 T5 90 95 Leu Val Gln Asp Val Wing Asn Asn Thr Asn Glu 100 Glu Wing Gly Asp Gly 105 110 Thr Thr Thr Wing Thr Val Leu Wing Arg Ser He Wing Lys Glu Gly Phe 115 120 125 Glu Lys He Ser Lys Gly Wing Asn Pro Val Glu 11 135 e Arg Arg Gly Val 140 Met Leu Wing Val Asp Wing Val He Wing Glu Leu Lys Lys Gln Ser Lys 5 145 150 155 160 Pro Val Thr Thr Pro Glu Glu He Wing Gln Val Wing Thr He Be Wing 165 170 175 Asn Gly Asp Lys Glu He Gly Asn He He Ser Asp Wing Met Lys Lys 180 185 190 Val Gly Arg Lys Gly Val He Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205 Aßp Glu Leu Glu He He Glu Gly Met Lys Phe Asp Arg Gly Tyr He 210 215 220 Ser Pro Tyr Phe He Asn Thr Ser Lys Gly Gln Lys Cys Glu Phe Gln 225 230 235 240 Aso Ala Tyr Val Leu Leu Ser Glu Lys Lys He Ser Ser He Gln Ser 245 250 255 He Val Pro Ala Leu Glu He Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270 He He Wing Glu Asp Val Asp Gly Glu Wing Leu Ser Thr Leu Val Leu 275 280 285 Asn Arg Leu Lys Val Gly Leu Gln Val Val Wing Val Lys Wing Pro Gly 290 295 300 Phe Gly Asp Asn Arg Lys Asn Gln Leu Lys Asp Met Ala He Ala Thr 305 310 315 320 Gly Gly Wing Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335 Val Gln Pro His Asp Leu Gly Lys Val Gly Glu Val He Val Thr Lys 340 345 350 Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln He Glu 355 360 365 Lys Arg He Gln Glu He He Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380 Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 85 390 395 400 Val Val Val Leu Lys Val Gly Gly Thr Ser Aso Val Glu Val Asn Glu 405? O 4? S Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 25 430 Glu Glu Gly He Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys He 435 4 or 44S Pro Wing Leu Asp Ser Leu Thr Pro Wing Asn Glu Asp Gln Lys He Gly 450 455 460 He Glu He He Lys Arg Thr Leu Lys He Pro Ala Met Thr He Ala 465 470 475 480 Lys Asn Wing Gly Val Glu Gly Ser Leu He Val Glu Lys He Met Gln 485 490 495 Being Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510 Met Val Glu Lys Gly He He Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525 Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala 530 Glu Val 535 540 Val Val Thr Glu He Pro Lys Glu Glu Lys Aso Pro Gly 545 Met Gly Ala 550 555 560 Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 570 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) No FILAMENTS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Val Leu Gly Gly Val Val Leu Leu Arg Val He Pro Ala Leu Asp 1 5 10 15 Ser Leu Thr Pro Wing Asn Glu Asp 20 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) No. DE FILAMENTS: one only (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ. ID. DO NOT. 3: Gl u Glu He Wing Gl n Val Wing Th r He Se r Wing Asn Gly Asp Lys 1 5 10 15 Asp He Gly Asn He 20 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one (D) ) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ. ID. DO NOT. 4: Glu Gly Asp Glu Wing Thr Gly Wing Asn He Val Val Lys Ala Ala 1 5 10 15 Glu Ala (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear . (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ. ID. DO NOT. 5: Ser Arg Leu Ser Lys Val Wing Pro Val He Lys Wing Arg Met Met 1 5 10 15 Glu Tyr Gly Thr Thr 20 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ. ID. DO NOT. 6: Pro Gly Met Gly Wing Met Gly Gly Met Gly Gly Gly Met Gly Gly 1 5 10 15 Gly Met Phe

Claims (32)

NOVELTY OF THE INVENTION CLAIMS

1. - A peptide, characterized in that it is selected from the peptides identified in Table 1, and its salts and functional derivatives thereof.

2. The peptide according to claim 1, further characterized in that it is designated here p2.

3. The peptide according to the claim 1, further characterized in that p32 is designated here.

4. The use of a peptide according to claim 1 for the diagnosis of diabetes mellitus, dependent on insulin (hereinafter referred to as IDDM).

5. A method for diagnosing the presence or incipience of IDDM in a patient, characterized in that it comprises testing the patient's blood or urine with a peptide according to claim 1, as an antigen for the presence of antibodies or T cells, which are immunologically reactive with human hsp60.

6. A method according to claim 5, further characterized in that it comprises testing patients for the presence of anti-hsp60 antibodies or a T cell that immunoreacts with hsp60, thereby a result indicating the presence Positive anti-hsp60 antibody or a T cell, which immunoreacts with hsp60, indicates a high probability of the presence or the incipience of IDDM.

7. A method according to claim 5 or 6, further characterized in that the patient is tested for the presence of anti-hsp60 antibodies.

8. A method according to claim 7, further characterized in that the test method comprises a radioimmunoassay.

9. A method according to claim 7, further characterized in that the test method comprises an ELISA analysis.

10. A device for diagnosing the presence of IDDM by analyzing the presence of anti-hsp60 antibodies according to the method of any of claims 5 to 9, characterized in that it comprises: (i) an antigen that is a peptide of the sequences of claim 1; and (ii) a labeled antibody, capable of recognizing the non-variable region of said anti-hsp60 antibodies that are to be detected.

11. Equipment according to claim 10, further characterized in that the antigen is the peptide according to claim 2.

12. Equipment according to claim 10, further characterized in that the antigen is the peptide in accordance with claim 3.

13. Equipment according to any of claims 10 to 12, further characterized in that the antigen is immobilized on a solid phase.

14. - A kit according to any of claims 10 to 13, further characterized in that it additionally includes instructions for the use of the equipment in the diagnosis of IDDM.

15. A kit according to any of claims 10 to 14, further characterized in that a marker of the group consisting of radioisotopes, enzymes, chromophores and fluorophores is selected.

16. A method according to claim 5 or 6, further characterized in that the patient is tested for the presence of a T cell that immunoreacts with hsp60.

17. A method according to claim 16, further characterized in that the test method comprises a T-cell proliferation test, comprising the following steps: (i) preparing a mononuclear cell fraction, containing T cells, a from a sample of blood obtained from the patient; (ii) adding to the mononuclear cell fraction an antigen selected from the peptide according to claim 1; (iii) incubating the cell fraction in the presence of the antigen, for an adequate period of time and under suitable culture conditions; (iv) adding a labeled nucleotide to the incubated cell culture of (iii) at an appropriate time, before the end of the incubation period, to provide for the incorporation of the labeled nucleotide into the DNA of the proliferating T cells; and (v) determining the number of proliferating T cells, by analyzing the amount of labeled nucleotide incorporated in the T cells. 18.- A device to diagnose the presence of IDDM by proving the presence of a T cell that immunoreacts with hsp60 according to the method of any of claims 5, 6 and 17, characterized in that it comprises: (i) an antigen selected from the peptide according to claim 1; (ii) a labeled nucleotide; and (iii) a suitable medium for the culture of lymphocytes. 19. Equipment according to claim 18, further characterized in that it includes instructions for the use of the kit in the diagnosis of IDDM. 20. A method according to claim 16, further characterized in that the test method comprises a T-cell cytokine response test, comprising the following steps: (i) preparing a mononuclear cell fraction containing T cells from of a blood sample obtained from the patient; (ii) adding to said mononuclear cell fraction an antigen selected from the peptide according to claim 1; (iii) incubating the cell fraction in the presence of the antigen, for an adequate period of time and under suitable culture conditions; and (iv) measuring the presence of a cytokine secreted by the responding lymphocytes, in the medium. 21. A method according to claim 20, further characterized in that the cytokine is IFN-gamma, IL-2, IL-4, IL-6, IL-10, TNFalpha or TGFbeta. 22. A device for diagnosing the presence of IDDM by testing the presence of a T cell that immunoreacts with hsp60 according to the method of any of claims 5, 6, 20 and 21, characterized in that it comprises: (i) a antigen, selected from the peptide according to claim 1; (ii) a suitable medium for the culture of lymphocytes; and (ii) an analysis equipment to measure the presence of the cytokine secreted by the lymphocytes that respond, in the medium. 23. A device according to claim 22, further characterized in that it additionally includes instructions for the use of the equipment in the diagnosis of IDDM. 24.- A method in accordance with the claim 16, further characterized by injecting an antigen selected from a peptide according to claim 1, in a patient, subcutaneously; and the occurrence of a detectable reaction on the skin is observed. 25. A preparation for preventing or treating IDDM, characterized in that it comprises: (a) T cells having specificity developed for a protein or a peptide, which is immunologically reactive in a mutual manner with a peptide according to claim 1; said cells have been activated by incubating them in the presence of the peptide; (b) T cells that have been irradiated or otherwise attenuated; (c9 T cells that have been subjected to a pressure treatment, by means of hydrostatic pressure, treatment with chemical interlacing agent and / or treatment with a cytoskeletal interlacing agent, (d) fragments of surface proteins, detached from (a) ), (b) or (c), or (e) a peptide consisting essentially of the variable region of the receptor of (a) specific for said protein or a salt, a functional derivative, a precursor or an active fraction thereof. 26. A preparation according to claim 25, further characterized in that the T cells of (a) are human T cells, and the specificity of the T cells has been developed by in vitro contact with the peptide. A preparation according to claim 2 or 26, further characterized in that the T cells have developed in vitro specificity for a peptide according to claim 1. 28.- A pharmaceutical composition, characterized in that it comprises a peptide according to claim 1 and a pharmaceutically acceptable carrier. 29. A pharmaceutical composition according to claim 28, for the prevention or treatment of IDDM. 30. A pharmaceutical composition according to claim 28 or 29, further characterized in that the peptide is the peptide according to claim 2. 31.- A pharmaceutical composition according to claim 28 or 29, further characterized by the peptide is the peptide according to claim 3. 32.- The use of a peptide according to any of claims 3, for the preparation of a pharmaceutical composition for the treatment of IDDM.