HIV PROTEIN EPITOPES IMMUNOLOGICALLY HOMOLOGOUS TO HLA

Abstract: Immunological homologous epitopes of HLA and HIV virus proteins, particularly gp160, are described, to be used for diagnostics and for producing vaccines.

Description: HIV PROTEIN EPITOPES IMMUNOLOGICALLY HOMOLOGOUS TO HLA SPECIFICATION The invention concerns epitopes of the HIV virus gp160 protein, that are immunologically homologous to epitopes of the protein family of the human major histocompatibilty complex HLA, to be used for diagnosing and immunization. Among the mechanisms thought to be responsible of AIDS, it has been suggested that auto-immunity, namely auto- antibody induction mechanism, may play a major role (Hebeshaw J.A and Dalgleish A.G., J. Acquired Immun. Defic. Syndrome, 2, 457, 1989). The homology of portions of virus components with endogenous proteins of the human body could reduce the self- tolerance immunity mechanisms, thus causing the synthesis of antibodies, and activating cytotoxic T lymphocytes. The gp120 protein is used to produce vaccines for immunization of HIV infection, as a complex with the gp41 protein, such complex being defined as gp160 (ref.). The PCT Application No. WO89/09618 claims anti-HIV vaccines devoided of gp1 60 fragments generating HIV-1 infection enhancing antibodies. However, the cited application does not disclose any gp160 epitope able to induce auto- antibodies in HIV-infected subjects. The PCT Application No. W092/05196 concerns alloepitopes of HIV gp1 60 recognized by CD4+ T cell receptors, having a three-dimensional structure (α-helix) similar to the region of the human Major Hystocompatibiliy Complex (MHC). However, also this cited application does not disclose any gp160 epitope able to induce auto-antibodies in HIV-infected subjects. The inventor previously isolated and described two monoclonal antibodies, M38 and L31 , which react with the HIV virus gp160 protein as well as with the class I HLA protein (Beretta et al., Eur. J. Immunol., 17, 1793-1798, 1 987; Grassi et al., J. Exp. Med., 174, 53-62, 1980). The gene coding said cell protein was idendified and sequenced (Grassi et al., J. Exp. Med., 174, 53- 2562, 1980). Therefore ft results obvious the need to identify homology regions between HLA and HIV proteins, either for diagnosis purpose in order to identify crossreactive antibodies in the serum of either AIDS patients or HIV vaccinated subjects, and to produce recombinant vaccines. In the context of this invention, "epitopes" J mean fractions of an antigen molecule that are recognized and/or are able to bind a specific antibody; "immunologically homologous epitopes" mean epitopes of at least two different antigen molecules, that are recognized and/or are able to bind the same antibody; "macromolecular structures" mean either ceils, or parts thereof, or proteins, or parts, thereof. The author has analyzed sequences of both HLA and gp160 proteins, and identified immunologically homologous regions. Further tests with synthetic peptides of the HLA heavy chains have allowed to identify more precisely an immunologically homologous region of gp160 and of the heavy chain of class I HLA. This region corresponds to a specific polypeptide segment of the gp160 protein, wherein two minimum epitope sequences have been defined. The immunologically homologous regions on HLA and the minimum sequence of the epitopes have been identified too. In the gp 160 protein, said epitopes are separated by a 9 amino acid intervening sequence which is shown not to be immunogenic in the context of the natural gp160 protein, in HIV infected subjects. However, experiments published by Helseth et al. {Journ of Virol. 65, 21 19-2123, 1991 ) showed that 5 of said 9 residues are essential to the maintenance of the quaternary structure of the HIV envelope, since single amino acid substitutions within these positions result in a high dissociation rate between gp120 and gp41 . The author of the present invention has also determined that said sequence may be able to induce anti-HIV neutralizing antibodies, when used without its natural flanking sequences. Therefore an object of this invention are epitopes of the HIV gp160 protein which are immunologically homologous to at least one epitope of the HLA protein family. Preferably said HLA protein family comprises HLA Class I proteins, more preferably the HLA heavy chains, even more preferably the α-1 domain of said heavy chains. In a preferred embodiment of the invention said gp1 60 protein epitopes are comprised respectively in the following region: SELYKYKVVKIEPLGVAPTKAKRRVV. Preferably said epitopes are immunologically homologous to at least one epitope of the following α-1 domain region, allele Cw4, or an allele thereof: TQKYKRQAQADRVNLRKLRGYY. Preferably said gp160 region comprises two epitopes, more preferably having the amino acid sequences as follows: SELYKYKVVK and PTKAKRRVV. More preferably said two epitopes are immunologically homologous respectively to the following α-1 domain regions: TQKYKRQAQ and NLRKLRGYY. Another object of the present invention are epitopes of HLA protein faminly which are immunologically homologous to at least one epitope of the HIV gp160 protein, according to the invention. Preferably said HLA protein family comprises HLA Class I proteins, more preferably the HLA heavy chains, even more preferably the α-1 domain of said heavy chains, most preferably the following α-1 domain region, allele Cw4, or an allele thereof: TQKYKRQAQADRVNLRKLRGYY. According to an embodiment of this invention said epitopes are recognized by neutralizing antibodies. According to a further embodiment of this invention said epitopes are immunologically homologous to a bacterial superantigen, preferably to the SEE protein of E. coli. Always according to the invention said epitopes are of either natural, or recombinant, or synthetic origin. It is a further object of the invention the use of epitopes as disclosed in the present specification, for the preparation of compositions of immunodiagnostic and for immunization purposes. Another object of the invention are immunodiagnostic kits, comprising as specific ligand macromolecular structures comprising at least one of the epitopes according to the invention, to detect antibodies from a sample of serurrr from either AIDS affected or HIV immunized subjects, said antibodies being able to react also with at least one epitope of HLA protein family. Said immunodiagnostic diagnostic kits comprise any of the known binding-detection systems, preferably ELISA, RIA or Western Blot systems. Preferably said epitopes comprise at least one epitope of the HIV gp160 protein. In an embodiment of the invention said epitopes comprise the region having the following sequence: IEPLGVAPT. A further object of this invention are antigenic compositions for HIV vaccines which comprise a sterile carrier and the gp160 protein or parts thereof, either lacking at least one of its epitopes which are immunologically homologous to HLA , according to the invention, or being modified to make at least one of said epitopes not immunologically homologous to HLA. In a preferrred embodiment of the invention said gp160 or parts thereof do comprise the region having the following sequence: IEPLGVAPT. It is an object of the invention polyclonal or monoclonal antibodies able to recognize the gp160 region: IEPLGVAPT. The invention will be described in the following, by some explicative but not limititative examples, with reference to the annexed figures, wherein: fig. 1 shows an ELISA assay with the gp160 protein and sera obtained from mice immunized with different gp1 60 derived peptides; fig. 2 shows the binding of M38 (panel a and b) and L31 (panel c and d) antibodies to different peptides; fig. 3 is an histogram showing the M38 antibody binding to 45 one amino acid overlapping peptides, which are obtained from the HLA sequence; fig. 4 is an histogram showing the M38 antibody binding to 45 one amino acid overlapping peptides, which are obtained from the gp160 sequence; fig. 5 shows a western blot binding assay of gp1 60 with sera from AIDS patients, pre-immunoadsorbed with S: MAP HLA, MAP HLA 1 1 or MAP HLA 12 peptides and eluted; fig. 6 shows a western blot binding assay of gp160 with sera from AIDS patients, pre-immunoadsorbed with S. MAP HLA, or MAP HIV 9 peptides and eluted; fig. 7 shows a western blot binding assay of gp16O with sera from two subjects (1 and 2) vaccinated with both Vaccinia Virus and the gp160 recombinant protein; (a) "in toto", and (b) after pre-adsorption with the S.MAP HLA peptide and elution; fig. 8 shows a western blot binding assay of gp1 60 with sera of subjects treated with wild-type strain Vaccinia Virus (1 and 2), with recombinant gp160 (3 and 4) and with recombinant Vaccinia Virus expressing on its surface the HIV gp160 complex (5 and 6); (a) "in toto"; (b) after pre-adsorption with the S.MAP HIV peptide and elution; (c) after pre-adsorption with the S.MAP HLA peptide and elution; (d) pool of 5 and 6 sera pre-adsorbed with an unrelated peptide and eluted; fig. 9 shows a syncytium formation inhibition assay; fig.10 shows another syncytium formation inhibition assay; fig. 1 1 shows a binding assay of the L31 antibody with the SEE protein. Example 1 Identification of immunoαically homologous regions between αp160 and HLA and synthetic peptide production The homology regions between class I HLA and HIV gp160 protein are determined by analysis of the gp160 amino acid sequence and the consensus sequence of the HLA gene locus C (Grassi et al., J. Exp. Med. 174, 53-62, 1980). Among regions showing a sinificative level of sequence homology, the COOH C-5 terminal of gp160 and the α-1 heavy chain region of HLA are selected and analyzed. The amino acid alignment of the two regions, sequences of which are shown in the Table 1 herebelow, shows a sequence of three identical amino acids (K66, Y67 and K68 of HLA, according to the numeration of Grassi et al., "J. Exp. Med.", 174, 53-62, 1980 as well as K490, Y491 and K492 of gp160, according to the numeration of Wein-Obson et al., "Cell", 40, 9, 1985. TABLE 1 Peptide sequence from HIV gp160 and HLA S. MAP HIV SELYKYKVVKIEPLGVAPTKAKRRVV MAP HIV 1 1 SELYKYKVVK MAP HIV 9 IEPLGVAPT MAP HIV 12 PTKAKRRVV S. MAP HLA TQKYKRQAQADRVNLRKLRGYY MAP HLA 1 1 TQKYKRQAQ MAP HLA 12 NLRKLRGYY Synthetic peptides corresponding to HLA and gp160 sequences as above are synthesized using an automatic synthesizer equipment (ABI 431 /A from Applied Biosystems) and a FMOC chemistry procedure. All of peptides are purified to homogeneity by ion- exchange chromatography followed by a reverse phase HPLC. The peptide amino acid contents comply with the expected data. Example 2 Antiαenic homology among QP160 and HLA peptides S.MAP HIV and S. MAP HLA peptides are injected subcutaneously to BALB/c female mice together with Freund's incomplete adjuvant. After the third immunization (for a total of three immunizations at three week intervals) mice sera are tested by an ELISA assay with the recombinant gp1 60 protein (from Transgene, Strasbourg) as follows: microtiter plastic wells (NUNC) are coated with 0.2 μg of protein resuspended in phospahte buffer pH 9, for 1 .5 hours at 37 °C, followed by washing with phosphate buffer pH 7, and by further incubating for 1 hour at 37° C with bovine serum albumin (BSA, 1 mg/ml), to saturate the free sites of solid phase. After three washings, diluted sera in PBS, comprising BSA 1 % w/v, are incubated with the coated wells for 1 hour at 37 °C. After five washings in PBS, a rabbit anti-mouse Igs serum (SIGMA), 1 /1000 diluted, is added. After 45 min. incubation at 37° C, wells are washed five times and 100 μl of chromogen substrate is added to each well (SORIN BIOMEDICA, Saluggia, Italy). The adsorbance is read with an automatic ELISA reader (BIORAD). The results are shown in fig.1 . Sera from S. MAP HLA peptide immunized mice react specifically with the recombinant gp160 protein when compared with a mouse serum immunized with a control peptide (NMS). As expected, the sera of mice immunized with the S. MAP HIV peptide react at significantly higher level. The results show that the gp160 and the HLA regions cross-react immunologically and thus have common antigenic determinants. The inventor already described a murine monoclonal antibody, named M38, which selectively reacts with HLA free molecules (heavy chains not associated with β-2 microglobulin) and with the HIV gp1 60, as well as a further monoclonal antibody, L31 , which reacts with the HLA free chains, but negative when submitted to a viral gp160 western blot assay (Beretta and al., "Eur. J. Immunol.", 17, 1793-1798, 1 987; Grassi and al., "J. Exp. Med.", 174, 53-62, 1980). Therefore both antibodies could be properly used to confirm the antigenic homology of the regions already identified by means of mice polycional sera. Both antibodies are tested by ELISA assays with synthetic peptides of Table 1. The test conditions are those of Example 1 , except that the solid phase antigen (synthetic peptide) is present at a lower concentration (0.2 μg/ml). The results of fig. 2 show that both antibodies recognize both HLA peptide and gp160 peptide, thus confirming the antigen homology resulting from the previous tests. Example 3 Identification of the common epitopes of HLA and HIV gp160 As the HLA and gp160 regions, which are defined as immunologically homologous by polyclonal sera and by L31 and M38 antibodies, extend over a long portion of both HLA and gp160 proteins, the homologous epitopes are identified by a M38 monoclonal antibody assay with two sets of 45, one amino acid overlapping, synthetic peptides, which respectively extend over the HLA and gp160 region, as shown respectively in fig. 3 and 4. Purposely, a synthetic peptide commercial system (CAMBRIDGE RESEARCH BIOCHEMICALS) is used directly on the solid phase. The M38 antibody is then incubated with the solid phase peptides for 12 hours at 4°C at two different dilutions (5 and 0.5 jug/ml); the wells are washed with PBS and the M38 binding is detected by means of goat peroxidase- conjugated anti-rat Igs serum (SIGMA) at 1 /1000 dilution in PBS with 0.5 % w/v BSA. The colorimetric reaction, detected by the chromogen substrate (OPD/H2O2) is directly proportional to the antibody amount bound to the solid phase. The test results, as showed in the fig. 3 and 4, indicate that the M38 antibody recognize two different epitopes on both molecules; namely, M38 defines a first epitope, corresponding to the K66, Y67 and K68 residues and a second epitope, corresponding to L78, R79, K80, L81 and R82 residues, according to the above mentioned numeration, for HLA. For gp160, the two epitopes are respectively K490, Y491 , K492 and K505; A506, K507, R508 and R509, according to the above mentioned numeration. An amino acid motif, common to the four epitopes, is a first lysine, followed by a hydrophobic residue and a second lysine or an arginine. The two epitopes are separated on both molecules by an intervening sequence with a comparable amount of residues (12 on gp160 and 9 on HLA), thus showing a strong structural homology between both proteins. On the basis of known cristallography data (Bjorkman and al., "Nature", 329, 506, 1 987), both HLA epitopes correspond to the HLA hypervariable region, as they comprise at least one polymorphic site, that results to be oriented to the inner part of the native molecule and is involved in the binding of peptide fragments of HLA-carrying antigens. Example 4 Detection of an autoimmunitv response in AIDS affected patients An assay for the induction of auto-immunity response during the disease progression is carried out. Sera from AIDS affected subjects are immunoadsorbed on columns of either S. MAP HIV, or MAP HLA 1 1 or MAP HLA 12 peptide coupled Sepharose (CNBR activated sepharose 4B from PHARMACIA) and eluted. 1 ml of Sepharose/peptide equilibrated with 0.1 M TRIS HCI pH 8 and 0.5 M NaCI buffer is incubated for 16 hours with 100 μl of serum, washed with 0.1 M TRIS HCI ph 8 and 0.5 M NaCI. The bound antibodies are eluted with a buffer containing 0.2 M glycine pH 2 and 0.5 M NaCI. The eluted antibodies are assayed on HIV western blot commercial stripes (SORIN BIOMEDICA, Saluggia, Italy) and detected using the chemio-luminescence system from AMERSHAM. The results are shown in fig.5, showing a HIV western blot of purified antibodies by two different sera with the MAP HLA 1 2 peptide (lanes E and I), the MAP HLA 1 1 peptide (lanes F and J), both peptides mixed in equimolar amount (lanes G and K), and S. MAP HIV peptide (lanes H and L). Lane M is obtained from one of the "in toto" sera and used as control. The data show that either HLA or HIV peptide pre-adsorbed antibodies bind similarly to the viral gp160. In one case (lane E), the antibodies also bind to a 45 KD protein, which is likely corresponding to HLA heavy chains. Therefore, it can be stated that anti-gp160 antibodies cross-react with HLA. The same test is carried out using two sera of AIDS affected patients (fig.6), pre-adsorbed with the S. MAP HIV peptide (lanes A and B) or with the HIV 9 peptide (lanes C and D), and eluted. The last ones do not show any reactivity, thus showing that antibodies raised against the native gp160 protein, react with the HIV 11 and HIV 12 regions, and not with the HIV 9 regions. Example 5 Detection of an auto-immune response in gp160 vaccinated subjects The same test method is applied using sera of serum- negative vaccinated patients. Fig.7 shows a gp160 binding assay of sera of two subjects which are vaccinated with Vaccinia Virus (from Bristol- Myers), as well as with the gp160 recombinant protein (from VanSyn Microgenesis) (lanes l and 2): (a) "in toto"; (b) after pre- absorption with the S. MAP HLA peptide. Fig.8 shows a gp160 binding assay of sera of subjects vaccinated with Vaccinia Virus (lanes l and 2), recombinant gp160 (lanes 3 and 4), recombinant HIV gp160 surface expressing Vaccinia Virus, followed by a gp160 recombinant boost (HIVAC, Bristol-Myers), (lanes 5 and 6): (a) "in toto"; (b) after pre-absorption with the S.MAP HIV peptide and elution; (c) after pre-absorption with the S.MAP HLA peptide and elution; (d) pool of two pre-adsorbed sera with a un correlated peptide. These data show a positive relationship between the ability of a gp160 vaccine to induce a significant serological response and an anti-HLA response. Example 6 Neutralizing activity of anti-S. MAP HLA peptide antibodies In order to check the ability to neutralize the viral infection of sera raised against the HLA homologous regions, mice sera are prepared by immunization with either S. MAP HLA or S. MAP HIV peptides. Sera are assayed for antibody ability to inhibit syncytium cells between HIV infected cells (8E51 LAV cells, AIDS Repository National Insitute of Health, Bethesda, Mary- land USA, "Journ. Exp. Medicine, 164, 280, 1986) and not infected, CD4 + cells (MOLT3 cells, AIDS Repository National Institute of Health, Bethesda, Maryland, USA). The syncytiums are counted at the optical microscope and the inhibition percentages are evaluated with reference to syncytiums in a control antibody-free cell culture. As specifity control, sera of mice before immunization are used. The results (fig.9) show that antibodies against the HLA peptide (S.MAP HLA) comprising gp1 60 homologous regions has the same ability to neutralize the viral fusion than antibodies against the gp1 60 homologous peptide (S. MAP HIV). Accordingly, there is a positive correlation between the anti-HLA cross-reactive response and the viral infection neutralizing response. Example 7 Neutralizing activity of anti-HIV 9 peptide antibodies Experiments published by Helseth and coworkers in 1 991 (Journ of Virol. 65, 21 1 9-21 23, 1 991 ) show that 5 of the 9 residues of the peptide HIV-9 of gp1 60 (peptide IEPLGVAPT) are essential to the maintainance of the quaternary structure of the envelope since single amino acid substitutions within these positions result in a high dissociation rate between gp1 60 and gp41 . As reported in Example 4 none of sera from HIV positive subjects are reactive with the HIV-9 peptide, whereas all of them are reactive with the two flanking regions bearing the HLA homologous epitopes. In order to investigate whether the presence of the two HLA homologous epitopes flanking the IEPLGVAPT sequence influence negatively the antibody response against this region and prevent the production of antibodies against a potentially neutralizable site, BALB/c mice are immunized with the IEPLGVAPT peptide and complete Freund's agjuvant. After four antigen boosts, spleen cells of sacrified mice are fused with the myeloma cell line NS1 , to generate a number of hybridomas. All hybridomas are screened by ELISA with the IEPLGVAPT peptide. One hybridoma (C1 A) is selected for its reactivity against the IEPLGVAPT peptide and tested for inhibition of HIV induced cell fusion using the syncitia assay described in Example 6. Briefly, 8E51 LAV cells (AIDS Repository National Institute of Health, Bethesda, MD, USA, Journ. Exp. Medicine 164, 280, 1986) and CD4+ M0LT3 cells (AIDS Repository National Institute of Health, Bethesda, MD, USA) are mixed in V shaped wells, incubated for three hours and syncitia counted at the microscope. As it can be seen in fig. 9, the C1 A antibody inhibits syncitia formation in a dose dependent manner. Thus the IEPLGVAPT sequence is a neutralizable site of the HIV envelope necessary to HIV induced cell fusion which is non immunogenic in HIV infected individuals. Antibodies to this sequence may have protective effects against HIV infection. Example 8 Immunological homoloy of both HLA and QP1 60 epitopes with a bacterial superantioen Some recent experiments (fmberti et al. "Science", 254, 869, 1991 ) suggest that, in the course of the HIV infection, the T lymphocyte population of the patient suffers functional alterations, that are similar to alterations which are observed with test animals, as well with human lymphocytes in vitro when exposed to superantigenic substances. Such modifications essentially consist of a selective loss of some T receptors, which express some genes of the family of the variable jS-region. Superantigens are defined as bacterial glycoproteins being able to bind the 3-chains of the T lymphocyte receptor ando to the class 2 HLA molecules (Marrack and Kappler, Science", 248, 705, 1990), thus activating the target cell and inducing the cell death for apoctosys. Some Vβ families are eliminated by the T lymphocyte population during the HIV infection, thus suggesting an HIV superantigenic effect. In order to verify whether gp160 HLA homologous epitopes have also superantigenic features, as being able to bind the T lymphocyte receptor, an ELISA assay is carried out, under the same conditions as in Example 1 , using the L31 antibody, which showed to be specific to define the HLA/gp160 homology in the previous experiments, with a known superantigen, the SEE protein (from SERVIA BIOCHEMIA, Germany). As shown in fig.10, the test demonstrates that the HIV homologous HLA regions are able to induce anti-HLA auto- antibodies, and act as superantigens during the infection course, leading to a gradual and selective loss of certain T receptor families.

Claims: CLAIMS I . Epitopes of the HIV gp160 protein which are immunologically homologous to at least one epitope of the HLA protein family. 2. Epitopes according to claim 1 , wherein said HLA protein family comprises the HLA class Iproteins. 3. Epitopes according to claim 2, wherein said HLA class Iproteins comprise the HLA heavy chains. 4. Epitopes according to claim 3, wherein said HLA heavy chains comprise the α-1 domain. 5. Epitopes according to claim 4 comprised in the region having the following amino acid sequence: SELYKYKVVKIEPLGVAPTKAKRRVV. 6. Epitopes according to claim 5 immunologically homologous to at least one epitope of the following α-1 domain region, allele Cw4, or an allele thereof: TQKYKRQAQADRVNLRKLRGYY. 7. Epitopes according to claims 5 or 6 wherein said region comprises two epitopes . 8. Epitopes according to claim 7 wherein said two epitopes are comprised respectively in the regions having amino acid sequences as follows: SELYKYKVVK and PTKAKRRVV. 9. Epitopes according to claim 8 wherein said two epitopes are immunologically homologous respectively to the following α-1 domain regions: TQKYKRQAQ and NLRKLRGYY. 10. Epitopes of HLA which are immunologically homologous to at least one epitope of the HIV gp160 protein according to any of previous claims. I I . Epitopes of HLA according to Claim 10 comprised in the HLA Class I proteins. 12. Epitopes of HLA according to Claim 11 comprised in the HLA heavy chains. 13. Epitopes of HLA according to Claim 12 comprised in the α-1 domain of said heavy chains. 14. Epitopes of HLA according to Claim 13 comprised in the following α-1 domain region, allele Cw4, or in an allele thereof: TQKYKRQAQADRVNLRKLRGYY. 15. Epitopes according to any of previous claims characterised in being recognized by neutralizing antibodies. 16. Epitopes according to any of previous claims characterized in having a superantigen ability. 17. Epitopes according to any of previous claims characterized in being of natural, recombinant or synthetic origin. 18. Use of epitopes according to any of previous claims for the preparation of compositions of immunodiagnostic and for immunization purposes. 19. An immunodiagnostic kit comprising as specifc ligand macromolecular structures comprising at least one of the epitopes according to claim 17, to detect antibodies from a sample of serum from either AIDS affected or HIV immunized subjects, said antibodies being able to react also with at least one epitope of HLA protein family. 20. An immunodiagnostic kit according to claim 19 comprising any of the known binding-detection systems, preferably ELISA, RIA or Western Blot systems. 21 . An immunodiagnostic kit according to claim 1 9 or 20 wherein said epitopes comprise at least one epitope of the gp1 60 protein. 22. An immunodiagnostic kit according to claim 21 wherein said epitopes comprise the region having the following sequence: IEPLGVAPT. 23. An antigenic composition for HIV vaccines comprising a sterile carrier and the gp160 protein or parts thereof, either lacking at least one of its epitopes which are immunologically homologous to HLA, according to the invention, or being modified to make at least one of said epitopes not immunologically homologous to HLA. 24. An antigenic composition for HIV vaccines according to claim 23 wherein said gp! 60 or parts thereof does comprise the region having the following sequence: lEPLGVAPT. 25. Polyclonal or monoclonal antibodies able to recognize the gp! 60 region: lEPLGVAPT.

Inventors: BERETTA ALBERTO, BERETTA, ALBERTO
Applicants: BERETTA ALBERTO, IST SUPERIORE SANITA, SAN ROMANELLO CENTRO FOND, FONDAZIONE CENTRO SAN ROMANELLO DEL MONTE TABOR, ISTITUTO SUPERIORE DI SANITA'
Application year: 1993
Main classification: C07K7
IPC codes: A61K39, A61P31, C07K14, C12P21