MXPA97007674A - Use of a lymphosine that removes the immunodeficiency virus (isl) to inhibit the replication of viruses, in particular of the retrovi - Google Patents

Abstract

The present invention relates to an isolated polypeptide that inhibits the replication of retroviruses in peripheral blood lymphocytes and i) is encoded by the DNA sequence shown in SEQ ID No: 1 or complementary sequences, ii) is encoded by the DNA sequences which are hybridized with SEQ ID No: 1osSEQ ID No: 3 under severe conditions, iii) are encoded by the DNA sequences, which, if there is no degeneracy of the genetic code, will hybridize under severe conditions with the sequences defined in i) a ii), is useful as a therapeutic agent for the treatment of viral infections and benign and malignant proliferative diseases

Description

USE OF A LYMPHOCINE THAT SHPRTMF Fl. VIRUS DF IN UNODEFICIENCY (ISL) TO INHIBIT THE REPLICATION OF VIRUSES. IN PARTICULAR OF THE RETROVIRUS The present invention relates to the use of a "lymphokine that suppresses immunodeficiency virus" (ISL, for its acronym in English) "to inhibit the replication of viruses, therapeutic compositions containing ISL or nucleic acid molecules that encode the same ISL activity is defined by the inhibition of the rep repción of HIV in the primary hnfocitos (P BC) It is known that certain CD8 + cells, for example, those of human and animal origin which, besides being CD8 + are HLA-DR *, CD28 +, or CD11B ", show activity in the suppression of immunodeficiency virus , such as HIV and SIV This activity has been attributed to a molecule referred to as lymphokine that suppresses immunodeficiency virus or "ISL" ISL is capable of inhibiting the replication of viruses in CD4 + cells that are infected with HIV or VIS (Ennen, Findeklee, Kurth et al. (Proc Nati Acad Sci USA, Vol 91, p 7207-7211 (1994)) Without REF: 25770 However, the identity of the ISL has not been clear to date. It has been known since at least 1989 (Walker, CM, and Leighly, JA, Itnmunology, Vol. 66, p.628-630 (1989)) that there is a soluble factor secreted by downwardly regulated, stimulated human CD8 + T lymphocytes. the replication of HIV in CD4 + T cells. However, until now it has been impossible to establish if this activity results from an individual substance, and it has also been impossible to isolate and characterize the substance with this activity, although there is a group of publications in which methods to establish cell cultures are described. corresponding and methods to purify these antiviral factors are suggested. These publications are, for example, WO 94/23058 and WO 93/0883 as well as Mackewicz et al. (Lancet, 344, p.1671-1673 (1994)); Mackewicz et al (AIDS Research and Human Retroviruses, Vol.8, No. 6, p.1039-1050 (1992); Castro, Walker et al. (Cellular Immunology 132, p.246-255, (1991); Blackbourn et al. (Journal of Medical Primatology No. 23, pp. 343-354 (1994); Chen et al. (AIDS Research and Human Retroviruses Vol. 9, No. 11, p.1079-1086)); Kannagi et al. (The Journal of Immunology, Vol. 140, No. 7, pp. 2237-242 (1988)); Joag et al. (Virology 200, p.436-446 (1994)); Walker Moody et al. (Science, Vol. 234, p.1563-1566 (1986)); Walker, Erickson et al. (Journal of Virology, Vol. 65, No. 11, pp. 5921-5927 (1991)); Walker, Thomson-Honnebier et al (Cellular Immunology 137, p.420-428 (1991)); Knuchel, Bednarik et al. (Journal of Acquired Immune Deficiency Syndromes, No. 7, pp. 438-446 (1994)); Ennen, Findeklee, Kurth et al. (Proc. Nati, Acad. Sci. USA, Vol. 91, p. 7207-7211 (1994)) and Hsueh, Walker et al. (Cellular Immunology 159, p 271-279 (1994)). In spite of the intensive research mentioned above that has been carried out in activities to suppress the virus of In terms of immunodeficiency, produced from CD8 + cells for approximately seven years, the biological nature (especially the molecular structure of ISL), apart from the assumption that ISL is a protein, is completely unclear. They are also unclear: - your gene or genes if it consists of several factors; - the way in which it acts on infected and uninfected CD4 + cells. There are preliminary indications that the action of the ISL is based on a negative regulation of the transcription rate of the HIV-LTR (long terminal repeat); its mechanism of action in other infections. It can be assumed that several viruses whose transcription is regulated by transcription factors that are comparable to those of HIV will also be subject to negative regulation by the ISL; its mechanism of action in normal and malignant cell proliferation. According to the present state of knowledge, it can not be ruled out that, similar to interferons, an inhibitory effect on normal or malignant cell proliferation may be possible; the fact that if ISL could regulate the expression of CD4; Because in the case of patients infected with Does HIV occur during the time a decrease in ISL activity that is measurable in vitro ?; the fact that if ISL can also be partially responsible for the long latency period between infection and the development of the disease in HIV-infected humans, that is, it correlates positively with a positive prognosis.

Therefore, the problem has arisen from the identification of ISL and to clarify whether it represents one or several substances as well as to examine it with respect to its therapeutic action in the immunodeficiency viruses and other viruses. Cruikshank and Center (Journal of Immunology 128 (1982) 2569-2574) describe a protein called "lymphocyte chemoattractant factor" (LCF), which has a sequence completely similar to the sequence of the polypeptides of the invention. . It is expressed by human lymphocytes and is a member of the group of lymphokines. After proper purification by gel filtration, a homogeneous product with a molecular weight of about 56,200 was obtained which was cleaved by sodium dodecylsulfate in monomers with a molecular weight of about 14,400. It was assumed that this lymphokine played a role in the formation and amplification of the delayed type of immune response (delayed-type hypersensitivity reaction). The nucleic acid sequence of LCF is described by Cruikshank, W., et al., Proc. Nat. Acad. Sci. USA 91 (1994) 5109-5113. The nucleotide sequence and the sequence of proteins derived therefrom are available under accession number M90391 in the GenBank database and are shown in SEQ ID No. 3 and SEQ ID No: 4. From Cruikshank et al. ( Journal of Immunology 138, 3817-3823 (1987)) it is also shown that LCF stimulates the expression of interlucin-2 receptors (IL-2) and HLA-DR antigens in CD4 + lymphocytes. Therefore, the LCF is also referred to as a growth factor. Additionally, Cruikshank et al. Reported in Journal of Immunology 146, 2928-2934 (1991) that LCF induces CD4-dependent intracytoplasmic signals in lymphocytes and concluded in this way that these signals act as a second type of messenger. In J. Exp. Med. 173, p. 1521-1528 (1991) Rand, Cruikshank and colleagues further describe the stimulation of human eosinophils by LCF and their mass production by activated T lymphocytes. Finally in Proc. Nati Acad. Sci. USA, Vol. 91, p. 5109-5113 (1994) Cruikshank and co-workers describe a cloning of LCF by isolating the LCF cDNA from a library or expression library from mononuclear, mitogen-stimulated (PBMC: mononuclear, blood, peripheral cells) and blood cells. introduction into E. coli to produce the recombinant, biologically active LCF protein. The recombinant LCF shows an isoelectric point of 9.0 (Center, D.M., and collaborators J. Lab. Clin. Med. 125 (1995) 167-171). Cruikshank, W. et al. (Proc. Nat. Acad. Sci. USA 91 (1994) 5109-5113) describe that LCF can contribute to the reinforcement of eosinophils and CD4 + mononucleic cells, concomitantly in intracellular reactions. . Cruikshank further suggests that the activities of LCF in CD4 + cells would provide a mechanism for the accumulation of non-sensitized T cells in the tissue. Its ability to prepare CD4 + T cells for sensitivity to IL-2 could play a role in the specific expansion of this population of T cells. In WO 94/28134, the same authors suggest the use of LCF as an immunosuppressive agent or as part of the therapy of imm or nosuppression. However, an antiviral activity of LCF was neither described nor obvious from these publications. On the contrary, Center, D.N. et al. (1995), (supra) conclude that the LCF amplifies the inflammatory process.

Brief Description of the Invention The subject of the invention is the identification and molecular cloning of a lymphocyte suppression of immunodeficiency virus (ISL), for its acronym in English) and the isolation of nucleic acid molecules that code for polypeptides with ISL activity. These polypeptides have improved properties, especially higher activity than the polypeptide described in WO 94/28134. More specifically, the invention relates to those nucleic acid molecules that code for eukaryotic ISL, including human, monkeys and other species. Especially preferred are nucleic acid molecules that hybridize to SEQ ID No: 1 under severe conditions as set forth below and code for a polypeptide with ISL activity. It can be shown that the natural, synthetic and recombinantly produced ISL is able to suppress the replication of viruses, especially retroviruses, in vivo and in vitro. The nucleotide sequence according to the invention codes for a polypeptide that binds to CD4 + lymphocytes and that suppresses the replication of the virus such as, in particular, strains of HIV-1, HIV-2 and SIV.

Therefore, these polypeptides, active fragments and derivatives are also a subject of the present invention. The role of the ISL is not limited by its presentation in an MHC complex. A further subject of the invention is the use of ISL for the therapeutic treatment of viral infections, preferably retroviral infections and / or benign and malignant diseases based on viruses, and their use for the production of a therapeutic composition containing the ISL, as well as its use for the manufacture of these therapeutic agents. A further subject of the invention is a therapeutic composition containing ISL in an amount effective for the treatment of these diseases, especially viral infections. The composition or pharmaceutical agent also contains suitable pharmaceutically compatible carrier substances. A further subject of the invention is an anti-ISL, polyclonal or monoclonal antibody or an immunoactive fragment thereof, as well as methods for producing these antibodies and their use for the determination and detection by ISL of viral infections of eukaryotic cells, especially samples of mammals, preferably derived from mammalian cells Another subject of the invention is the use of ISL for the detection of mammalian cells activated by viruses, especially T cells. A further subject of the invention is a method for the determination of free or bound ISL. to the cell, soluble or insoluble This diagnostic method can be used for the detection of acute or chronic infections, to inspect the course of viral infections and / or for the inspection and detection of benign and malignant diseases based on viruses. An additional topic of the invention is the use of a nucleotide molecule that can ensure the expression of the ISL in a eucaptotic cell for the activation of ISL in human cells, for gene therapy in vivo or ex vivo Another subject of the invention is a therapeutic composition useful in the treatment of a pathological condition characterized by viral rep lication, especially retroviral replication , which comprises at least one substance that activates the activity of ISL in CD8 + cells, and a pharmaceutically acceptable carrier. Therefore, a further subject of the invention is a method for the production of a substance and a therapeutic agent for the inhibition of replication of the virus in a patient, the method comprising combining with a pharmaceutically acceptable carrier, a therapeutically effective amount of a substance that activates the expression of a protein with ISL activity, preferably a protein with the amino acid sequence shown in SEQ ID No: 2 on CD8 + T cells, in vivo and in vitro, to a degree such that Viral replication in CD4 + cells is inhibited. A further subject of the invention is a therapeutic composition useful in the treatment of a pathological condition characterized by viral replication, especially retroviral replication, comprising at least one substance that activates ISL activity in CD8 + T cells, and a carrier pharmaceutically acceptable.

Detailed description of the invention The invention is based on novel isolated polypeptides with ISL activity that inhibit the replication of HIV-1, in preferential form of HIV-1 SF2 in depleted CD8 + peripheral blood lymphocytes (PBMC) that are prepared from the surface layer buff color of blood samples, human, normal, uninfected retrovirally, in one assay (also referred to in the following as an HIV inhibition assay) a) by which CD8 + depleted PBMC are incubated with 0 1 μg of the polypeptide / 1 5 x 10s cells in 150 μl of the medium for half an hour at 37 ° C, b) the depleted PBMCs of CD8 + are subsequently infected with HIV-1, preferably with HIV-1 SF2 upon incubation 1 5 x 106 cells in 150 μl with 50 μl in concentrated solution of HIV-1 containing 50 two? 50 infectious tissue (TCIDso) for 1 hour at 37 ° C, c) infected, depleted CD8 + PBMCs are washed to remove the unbound HIV-1 and preferably the pohpeptide; d) the depleted CD8 + PBMCs are cultured at 37 ° C in a 5% CO2 atmosphere and the medium is changed and the pohpeptide is added after 3, 6, 9 and 12 days, e) the amount of HIV-1 in the culture supernatants of CD8 + depleted PBMC cells is determined on days 9 and 12 after infection by three-fold, serial dilutions of the supernatant and inoculation in wells quadrupled in 2000 cells in 150 μl of medium from a highly susceptible indicator cell line, which should be routinely inferable to a degree of 85% or greater with strains of HIV-1, preferably the MT4 cell line of lymphoma transformed by human HTLV; f) replication of the virus in each well is determined 8 days after infection by the determination of inverted transcriptase (RT) in the cell culture supernatant (Boehringer's inverted transcriptase assay is preferably applied Mannheim GmbH, Biochemica, 68298 Mannheim, Germany, Order No.: 1468 120) of each individual well following the manufacturer's instructions); g) the infectious dose50 of the tissue culture (TCID50) of the cultures of PBMC depleted of CD8 + is preferably calculated following the method published by Karber (Karber, G. 1931. Assay for statistical analysis of pharmacological experiments, Arch. Exper. Path. V. Pharmakol., 162, 148) according to the formula logTCIDSo = L-d (s-0.5), where L = log of the dilution of virus lower d = log of the dilution of virus s = sum of the positive cell cultures in virus, h) inhibition of HIV-1 referencing in cultures of CD8 + depleted PBMCs is calculated by comparing the virus content of the cell culture supernatants in an assay according to steps a) to g), and the content of virus from an assay according to steps a) to g) wherein the pohpeptide to be tested for the inhibition of HIV replication is replaced by buffer without polypeptide (untreated control), i) inhibition is found if the The amount of viral replication in the depleted CD8 + PBMCs is inhibited in such a way that the amount of the virus is about 50% less, preferably 10% less, and most preferably 1% less compared to the control not treated, and the pohpeptide i) is encoded by the DNA sequence shown in SEQ ID No. 1, ii) is encoded by the DNA sequences that hybridize with SEQ ID No. 1 or SEQ ID No: 3 under severe conditions, iii) is encoded by the DNA sequences that, if there is no degeneracy of the genetic code, will hybridize under severe conditions with the sequences defined in i) or ii),, with the proviso that the polypeptide differs from the polypeptide encoded by the DNA sequence in SEQ.

ID No: 3. Useful polypeptides with ISL activity in addition to the preferred polypeptides of SEQ ID No: 2 or SEQ ID No: 4 are for example, also preferred polypeptides of Figure 3. Figure 3 also shows the sequences of DNA that code for these polypeptides. A further preferred polypeptide is a polypeptide according to the invention wherein amino acid 26 (alanine) is deleted. This polypeptide also exists as natural allelic forms in humans and monkeys. In the examples that follow, a strain of HIV-1 known as HIV-1 SF2 is used. This is a typical North American / European strain. Its nucleotide sequence is set forth in SEQ ID No: 7, and is also accessible Via GenBank Accession No. K02007. Other strains of HIV include HIV-1SF33 (SEQ ID No: 8), as well as the strains set forth in for example, Cheng-Mayer et al., J. Virol 64 (1990) 4390-4398; Levy, J.A., et al., Science 232 (1986) 998-1001; Luciw, P.A., and collaborators Nature 312 (1984) 760-763; Sa nchez- Pescador, R., et al. Science 227 (1985) 484-492. In the test applied for the determination of ISL activity, PBMC stimulated with phytohemagglutinin (PHA) and purified by Ficoll gradient were infected with an HIV strain and cultured. Exhausted CD8 + PBMCs were used because of the improved accuracy compared to the use of PBMC. However, it is also possible to use PBMC. The depleted CD8 + cells are selected by choosing the cells using specific antibodies. These methods are widely known in the current state of the art. The culture supernatants are tested for their virus content. For this purpose, for example, the determination of the inverted transcriptase or the P24 antigen can be carried out. Another possibility is to determine the level of infection of highly susceptible indicator cell lines, referred to a virus-free cell supernatant in each case. In this test, ISL activity was found if the substance to be tested causes a reduction in the activity of the inverted transcriptase by at least about 50%, preferably 70%, more preferably 90% more. The polypeptide can be defined by its DNA sequence and by the amino acid sequence derived therefrom. The ISL polypeptide can occur in natural allelic variations that differ from individual to individual. These variations of amino acids are usually amino acid substitutions. However, there may also be deletions, insertions or additions of amino acids to the total sequence. The ISL protein according to the invention, depending both on the degree and type of the cell and the type of cell in which it is expressed, can be in the glycosylated or non-glycosylated form. Polypeptides with ISL activity can be easily identified by the HIV inhibition assay described above. Figure 3 shows a comparison of the DNA and polypeptide sequences of human ISL or different monkey. It has also been found that an allelic variant in which codon 26 (encoding Ala) is deleted exists in all of these species. As can be seen in Figure 3, ISL polypeptides and nucleic sequences coding for them are preferred, wherein amino acid 7 is Ser or Thr, amino acid 25 is Thr or Ser, amino acid 31 is Cys or Tyr , amino acid 76 is Val or lie, amino acid 86 is Gly or Ala, amino acid 112 is Me or Thr, amino acid 121 is Ser or Pro and / or amino acid 128 is Gly or Ala. Polypeptides in which the amino acid 26 is deleted are also preferred. These variations can improve the immunosuppressive and / or therapeutic (benign or malignant) activity of the ISL antiviral tumor without changing the biological properties in general. The "polypeptide with ISL or ISL activity" means any protein with minor amino acid variations but with substantially the same ISL activity. Substantially, the same means that the activities are of the same biological properties and the polypeptides preferably show at least 75% homology in the amino acid sequence. More preferably, the amino acid sequences are at least 90% identical. The "indicator cell line" means a lymphoma cell line that can be routinely infected to an extent of 85% greater with the HIV-1 strain used in the HIV inhibition assay. Preferably, this indicator cell line is MT4 which is described in Norley, S.G., and collaborators Biologicals 21 (1993) 251-258 which is incorporated herein by reference. Other useful lymphoma cell lines are described by Cheng-Mayer, C, et al., Virol, 181 (1991) 288-294 and J. Virol 65 (1991) 6931-6941 which are also incorporated herein by reference. These publications describe lymphoma cell lines that are infectious to HIV-1 to a greater or lesser degree. From the cell lines named only those cell lines that are routinely infectious to an extent of 85% higher with the HIV strain of the HIV cleavage assay are useful. "ISL activity" denotes the antiviral action of the tissue culture supernatant of lymphocytes CD8 + activated and non-activated of human origin (ISL) or animal (for example, ISL in the lymphokines of African green monkeys (ISL-agm)). The "ISL" preferably denotes the molecule whose sequence is shown in SEQ ID No: 1, 2, 3 or 4. The ISL is a polypeptide that is active in its glycosylated or non-glycosylated form. The non-glycosylated form can be produced by recombinant technology in prokaryotic cells.

ISL is produced by non-activated T lymphocytes (small amount) as well as activated. ISL binds to CD4 + lymphocytes, preferably to the CD4 receptor molecule or to a molecule associated with the CD4 molecule. ISL has suppressed the replication of all strains of HIV-1 and HIV-2 tested so far as well as all previously tested strains of SIV. This effect can be seen in CD4 + lymphocytes of the peripheral blood as well as in a number of CD4 + positive T cell lymphomas. The ISL has a specific action between species since at least the ISL of the African green monkey is able to suppress the replication of HIV in human CD4 + cells. The role of the ISL is not limited by an MHC locus incompatibility (major histocompatibility complex) and does not have a lytic action on the cells. ISL is synthesized by CD8 + lymphocytes from such symptomatic patients infected with HIV and less by cells from symptomatic patients. ISL is also produced by CD8 + cells, activated from healthy blood donors. The degree of synthesis of ISL is quantitatively correlated with the clinical status of patients infected with HIV. ISL activity in asymptomatic HIV patients is higher (with a comparable number of activated CD8 + lymphocytes) than in symptomatic patients. The antiviral action of ISL is not identical with previously known hnfocins and interferons. ISL activity has also been detected in the supernatant of the cell culture of activated CD8 + lymphocytes of chimpanzees infected and not infected with HIV, as well as African green monkeys infected and not infected with SIV, rhesus monkeys and dark gray monkeys (Ennen, J et al., Proc Nati Acad Sci USA 91 (1994 ) 7207-7211 ISL may be able to protect against super-infections with other strains of HIV / SIV (Cheng-Mayer, C, and collaborators J Virol, 64 (1990) 4390-4398) A protein with ISL activity is described in Cruikshank et al., Proc Nati Acad Sci USA 91 (1994) 5109-5113 and WO 94/28134 and is called LCF (see above) This protein is encoded by SEQ ID No. 3 and therefore has the SEQ ID No. 4 Cruikshank refers, for the sequence reported for the accession number M90391 agreed by the GenBank database While the protein sequences shown in GenBank and Figure 2 of Cruikshank are identical, the nucleic acid sequences exhibit a difference in nucleotide 1070 While in the GenBank sequence this nucleotide is T, in Figure 2 of the Cruikshank publications this nucleotide is G as TTG not it codes for Phe, but for Leu, it is clear that G is a typing error. This is confirmed by the cloning of ISL DNA derived from independent PCR amplifications. From these clones it is clear that the LCF sequence in codon 96 is truly represented by the TTT sequence. Therefore, nucleotide 1070 is clearly T. The term "nucleic acid molecule" denotes a polypeptide which can be, for example, a DNA, RNA or active RNA, of a kidney. However, DNA and / or RNA molecules are preferred. The term "hybridizes under severe conditions" means that two nucleic acid fragments are capable of hybridizing to each other under normal hybridization conditions described in Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning; A. laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61. More specifically the "severe conditions" as used herein refer to hybridization in 6.0 x SSC at about 45 ° C, followed by a 2.0 x SSC wash at 50 ° C. For severity selection, the concentration of the salt in the wash step can be selected, for example, from about 2.0 x SSC to 50 ° C, for low severity, up to about 0.2 x SSC at 50 ° C, for high severity. In addition, the temperature in the wash step can be increased from conditions of low stringency to room temperature, at about 22 ° C, to conditions of high stringency at about 65 ° C. The term "isolated" as used throughout this application refers to a nucleic acid or polypeptide having an ISL activity and is substantially free of cellular material or culture medium, when produced by recombinant DNA techniques, or chemical precursors or other chemicals, when chemically synthesized. An isolated nucleic acid is preferably free of sequences that naturally flank the nucleic acid (i.e., located at the 5 'and 3' ends of the nucleic acid) in the organism from which the nucleic acid is derived. ISL can be isolated and purified from activated T cells by affinity chromatography using a monoclonal antibody against ISL. It is also preferred to use other known protein purification techniques, including immunoprecipitation, gel filtration, ion exchange chromatography, atoconcentration, isoelectric concentration, selective precipitation, electrophoresis, and the like. The fraction isolated during purification procedures can be analyzed for the presence of ISL activity by using ISL-specific antibodies. The polypeptides according to the invention can also be produced by recombinant means, or synthetically. The non-glycosylated ISL polypeptide is obtained when it is produced recombinantly in prokaryotes. With the help of the nucleic acid sequences provided by the invention it is possible to search for the ISL gene or its variants in genomes of any of the desired cells (eg, apart from human cells, also in cells of other mammals), to identify those and isolate the desired gene that codes for the ISL protein. These processes and suitable hybridization conditions known to a person skilled in the art are described, for example, by Sambrook, J., et al., "Expression of cloned genes in E. coli" in Molecular Cloning; A. laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, and B.D. Hames, S.G. Higgins, Nucleic acid hibridisation - a practical approach (1985) IRL Pres, Oxford, England. In this case, the normal protocols described in these publications are usually used for experiments. The use of recombinant DNA technology and knowledge of the HIV inhibition assay allows the production of numerous active ISL derivatives.

These derivatives can be modified for example into individual or multiple amino acids by substitution, deletion or adhesion. Derivatization can be carried out by means of direct mutagenesis at the site. These variations can be easily carried out by a person skilled in the art (J. Sambrook, B.C. Hames, loc. Cit.). It only has to be ensured by means of the aforementioned HIV inhibition assay that the characteristic properties of the ISL (inhibition of virus replication) are maintained. Therefore, the invention is further related to an ISL polypeptide that is a product of a prokaryotic or eukaryotic expression of an exogenous DNA. The invention further relates to an isolated nucleic acid molecule encoding the polypeptide or active fragment or active derivative thereof, which inhibits replication of HIV-1 in depleted CD8 + PBMCs, PBMCs are prepared from the surface layer color of human blood samples, normal, retrovirally uninfected, in the HIV inhibition assay mentioned above, and wherein the nucleic acid molecule is selected from the group of i) DNA molecules shown in SEQ ID No 1 or the complementary sequences, n) nucleic acid molecules that hybridize with SEQ ID No. 1 or SEQ ID No. 3 under severe conditions, nor) nucleic acid molecules which, if there is no degeneracy of the genetic code, will hybridize under severe conditions with one of the sequences set forth in i) or n), with the proviso that the isolated nucleic acid molecule is not identical with SEQ ID No. 3 In a preferred embodiment of the invention, also nucleic acid molecules are unknown, which code for a popeptide of SEQ ID No 4 With the aid of These nucleic acids encoding an ISL protein, the protein according to the invention can be obtained in a reproducible manner and in large quantities. For expression in prokaryotic or eukaryotic organisms, such as human prokaryotic cells or eukaryotic host cells, the acid The nucleic acid is integrated into suitable expression vectors, according to methods familiar to a person skilled in the art. This expression vector preferably contains an adjustable / inducible promoter. These recombinant vectors are then introduced for expression in suitable host cells, such as for example E. coli as a prokaryotic host cell or Saccharomyces cerevisiae, cell line PA-1 sc 9117 of Terato carcinoma (Büttner et al., Mol. Cell Biol. 11 (1991) 3573-3583), insect cells, CHO or COS cells as eukaryotic host cells and transformed or transduced host cells are cultured under conditions that allow expression of the heterologous gene. Isolation of the protein can be carried out with known methods from the host cell or from the culture supernatant of the host cell. These methods are described for example by Ausubel I., Frederick M., Current Protocols in Mol. Biol. (1992), John Wiley and Sons, New York.

Also the in vitro reactivation of the protein may be necessary if it is not found in soluble form in the culture of the cells. The detection of transformed or transduced host cells that recombinantly reduce the ISL protein and purify the protein are preferably carried out by means of antibodies that bind to this protein. These antibodies can be obtained in a simple manner according to known methods by using the protein according to the invention as an antigen or an immunogen. The invention therefore relates in addition to the use of the protein with ISL activity according to the invention for the production of antibodies that bind to this protein. Anti-ISL antibodies are produced by immunization of appropriate vertebrate hosts with purified ISL or derivatives of ISL polypeptides, preferably with an adjuvant. These techniques are well known in the literature and are described for example by Harlow and Lane eds., Antibodies: A laboratory manual (1988), Cold Spring Harbor Laboratories Press. For this, animals that are usually used for this purpose, such as, in particular, sheep, rabbits or mice, are immunized with the protein according to the invention (preferably with the protein of Figure 3), and subsequently the antiserum it is isolated from the animals immunized according to known methods or the vessel cells of the immunized animals are fused with immortalized cells, such as, for example, myeloma cells, according to the method of Kohler and Milstein (Nature 256 (1975) 495-497). Those cells that produce a monoclonal antibody against the protein of ISLs are selected from hybridoma cells obtained in this manner and cloned. The monoclonal or polyclonal antibodies obtained in this way can be bound to a support material, such as, for example, cellulose, for a purification by immunosorption of ISL. Additionally, antibodies of this class can be used for the detection of ISL in samples, such as, for example, cut tissue or body fluids, preferably for the determination of viral infections and virally induced benign and malignant diseases, most preferably for the determination of retroviral infections in mammalian samples. In these assays, ISL binds immunologically to its antibody in the specific step. The invention therefore further relates to antibodies specific to the ISL protein, preferably the ISL proteins not described by Cruikshank, which can be obtained by immunizing an animal with the ISL protein and isolating the antibodies from the serum or cells of the vessel of immunized animals, and their use for the determination of ISL. The invention further relates to the use of a pohpeptide defined in the manner mentioned above that includes a protein of SEQ ID No. 3, for the production of an agent pharmaceutical and for the treatment of viral infections, preferably retroviral infections such as HIV infections, and for use in therapy in benign and malignant diseases, especially in tumor therapy, most preferably for the treatment of virally induced tumors. it is processed, if desired together with the auxiliary agents usually used, fillers and / or additives s, in a pharmaceutical formulation for therapeutic applications Therefore, the invention further relates to a therapeutic composition containing an ISL polypeptide according to the invention and if desired together with auxiliary agents, fillers and / or additives that are usually used When the polypeptides according to the invention are applied for therapeutic use, their doses depend on the proposed use. To find the dose and optimize the application, the properties of the polypeptide such as half-life will usually also be taken into account. and the bioavailability and the age and weight of the patient. Optimal therapeutic efficiency is achieved when the polypeptides according to the invention are applied as soon after infection as possible, preferably as soon after the first virus peak, as possible. Here it is important that a concentration of the polypeptides and substances according to the invention which effectively inhibit the replication of the virus in the blood during the early stage of the viral infection is maintained. This can be achieved, for example, by the application of 1 to 1000 μg / patient of the polypeptide according to the invention at intervals of 12 to 72 hours. The period of application can be determined, suitably, by the method of determining the virus replication or the amount of virus according to the invention or by other methods of virus determination known to those skilled in the art. The application period can be in the range from a few days to a few months. The invention further relates to the use of ISL genes or fragments thereof, preferably nucleic acid molecules that encode a polypeptide having ISL activity, or activation polynucleotides of the 5'-untranslated region, in gene therapy , and in particular for the production of drugs for gene therapy, preferably for an antiviral or immunosuppressive therapy, or a therapy of benign and malignant diseases. Gene therapy of somatic cells can be achieved by using, for example, retroviral vectors, other viral vectors, or by non-viral gene transfer (for clarity compare T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RAC DATA MANAGEMENT REPORT, June 1993). Suitable vector systems for gene therapy are, for example, retroviruses (Mulligan, RC (1991) in Nobel Symposium 8: Ethology of human disease at the DNA level (Lindsten, J. and Pattersun Editors) 143-189, Raven Press) , adeno-associated viruses (McLughlin, J. Virol., 62 (1988), 1963), vaccinia virus (Moss et al., Ann, Rev. Immunol., 5 (1987) 305), bovine papilloma virus (Rasmussen et al. Enzymol 139 (1987) 642) or viruses from the group of herpes viruses such as Epstein Barr virus (Margolskee et al., Mol.Cell Biol. 8 (1988) 2937) or herpes simplex virus.

Non-viral delivery systems are also known. For this, the "naked" nucleic acid, preferably DNA, or the nucleic acid is usually used with an auxiliary agent, such as, for example, transfer reagents (liposomes, dendromers, polylysine-transferrin conjugate (Felgner et al., Proc. Nati, Acad. Sci USA 84 (1987) 7413. Particularly preferred is an ex vivo gene therapy as described in for example Patent North American No. 5,399,346 of W.F. Anderson and collaborators. According to this method, a polypeptide according to the invention is provided to a human by introducing human cells into a human, human cells that have been treated in vitro to insert a segment of DNA encoding a polypeptide in accordance therewith. with the invention, human cells that express in vivo in human a therapeutically effective amount of the polypeptide. Since human cells are used, preferably fibroblasts or autologous, hematopoietic stem cells, which are preferably characterized by CD3 +, CD4-, CD8-. Progenitor, hematopoietic, human, primitive cells are characterized by high expression of CD34 and absence of CD38 expression, are particularly preferred. However, more differentiated hematopoietic stem cells such as CD34 + and CD38 + cells can also be used. These cells are described, for example, by Tastappen et al., Blood 77 (1991) 1218 or Huang and Terstappen, Nature 360 (1992) 745. For the transfection of fibroblasts it is preferred to use vectors based on cytomegalovirus (CMV). For the transfection of haematopoietic stem cells it is preferred to use retroviral vectors based on the murine molony leukemia vector (MMLV). These techniques are described in the state of the art, for example in the aforementioned US Patent No. 5,399,346 which is incorporated herein by reference. For the regulation of therapeutic application, the use of a suicide gene system is preferred (e.g., tk-Gen (Ganciclovir)). Another preferred method of technical therapy is based on homologous recombination. In this, you can insert either the gene that codes for the protein of ISL in one or more copies in the genome of the somatic cells and / or the ISL gene present endogenously in the cells can be modulated, preferably activated. Homologous recombination methods are described, for example in Kucherlapati, Proc. in Nucí.

Acids Res. And Mol. Biol. 36 (1989) 301; Thomas et al., Cell 44 (1986) 419-428; Thomas and Capecchi, Cell 51 (1987) 503-512; Doetschman et al., Proc. Nati Acad. Sci. USA 85 (1988) 8583-8587 and Doetschman et al., Nature 330 (1987) 576-578. In these methods, a portion of the DNA that is to be integrated into a specific site in the genome (ISL gene fragment) binds to target DNA. The target DNA is a DNA that is complementary (homologous) to a region (preferably in or near the ISL gene) of the genomic DNA. When two homologous portions of an individual strand DNA (e.g., target DNA and genomic DNA) are in close proximity to each other, they will hybridize and form a double stranded helix. Then, the fragment of the ISL gene and the target DNA can be integrated into the genome by means of the occurrence of recombination. This homologous recombination can be carried out in vitro and in vivo (in the patient). Preferably, a DNA encoding a protein having ISL activity, a fragment that inhibits the expression of ISL (ejection sequence) or a fragment capable of activating, after integration of the genome of a cell, is used. expression, in this cell, of a protein that has ISL activity. This fragment can be, for example, a promoter and / or enhancer region that is heterologous to the corresponding ISL region or that, after integration into the ISL gene, activates the ISL gene expressed to a truly imperceptible degree or little, transcriptionally and / or translationally. In this way, by means of this DNA, one or more ISL genes are reintroduced into the target cell, or the gene essentially imperceptible transcriptionally in the genome of a mammalian cell is activated in such a way that the mammalian cell is capable of producing the endogenous ISL protein. For this purpose, a DNA construct is inserted into the genome by homologous recombination, the DNA construct comprising the following: a DNA regulatory element capable of stimulating the expression of this gene if it is operatively linked to it; and one or more target segments of DNA that are homologous to a region in this genome, a region that is within or close to this gene. This construct is inserted into the genome of the cell in a manner such that the regulatory segment is operably linked to the gene encoding the protein having ISL activity. Preferably, the construct further comprises amplification sequences, especially if the genes encoding proteins with ISL activity are inserted into the cell. For the introduction of the ISL genes into the target cells, the construct comprises a regulatory element, one or more ISL genes and one or more target segments. The target segments are chosen in such a way that they hybridize to an appropriate region of the genome, whereby, after the homologous recombination, the exogenous, inserted ISL genes are expressed. A large number of processes are known by which homologous recombination can be initiated. Preferably, the homologous recombination takes place during the replication or mitosis of the DNA of the cells. A DNA of this kind can be used for the production of an agent for the therapeutic treatment of tumors and viral infection or for the production of homologous or heterologous protein of ISL in a host organism. A further subject of the invention is a method for the determination of ISL polypeptides, nucleic acid sequences, virus-activated cells and ISL expression, preferably in samples of the human body such as human cell preparations, cell supernatants and fluids. bodily such as blood, serum or plasma. This determination is useful for the detection of a viral infection, preferably from a population of mammalian cells, especially humans. This method is particularly useful for the determination of the activation state of these cells and for the determination of a viral infection, preferably retroviral CD4 + cells. The diagnostic method is preferably applied immediately or as soon as possible after the first peak or maximum value of the virus. A further subject of the invention is the use of an antibody that immunologically binds to a polypeptide that can be obtained by immunizing an animal with an ISL polypeptide and by isolating serum antibodies or spleen cells from immunized animals, for determination of the ratio of CD8 + and / or CD4 + cells, activated / non-activated, in body fluids, especially in blood, serum or plasma. These tests can be provided based on antibodies that are directed against part or all of the ISL polypeptides. These antibodies can be polyclonal or monoclonal antibodies, chimeric antibodies, humanized antibodies or fragments thereof such as F (ab), F (ab) 2, individual chain Fv, or the like. In this test, the antibodies are used for recognition The specific detection of ISL (with and without separation of this complex, and subsequent inspection) can be done by immunoassays that are widely known in the state of the art. For example, the antibody can be labeled by an inspection agent such as a fluorescent indicator, radioactive or enzymatic labeling Particularly preferred is a diagnostic determination of ISL concentrations in serum and other body fluids as well as the number of cells that produce ISL, for example, for the detection of Acute or chronic infections (for example, even in blood donors) or for the inspection of the course of (retroviral) viral infections (for example, in patients suffering from AIDS), wherein the antibodies that are provided with a fluorescent indicator or a radioactive or enzymatic label or with a labeled anti-antibody are reacted, contacted with ISL or cells that produce ISL, the antigen / antibody complexes are separated in a known manner and their concentration is determined via the tag or label. A suitable test method comprises the steps of incubating CD8 + T cells in vitro, with the substance to be tested and determining the ISL activity, preferably after 1 to 12 days, upon detecting the expression of ISL according to the invention or upon determining the ISL polypeptide, preferably by means of an anti-ISL antibody based test.

This test is carried out, for example, in the following manner: a) 96-well ELISA plates, commercially available, are coated with anti-ISL, monoclonal antibodies; b) the sample to be tested for the ISL content is added to a well coated with antibodies for 1 hour at room temperature and the well is washed; c) bound ISL is detected by incubation of an anti-ISL, goat, polyclonal, purified, affinity IgG preparation, followed by a specific horseradish peroxidase labeled antibody, anti-goat and subsequent visualization with OPD .

Other immunological assays based on the state of the art are also suitable.

It is also possible to provide a test based on the nucleic acid sequences of the ISL protein provided by the invention that can be used to detect the nucleic acids, preferably the ARNS, more preferably the ARNSm coding for the ISL proteins. . This test can be carried out for example in cells or cell lysates and by means of nucleic acid diagnosis. In this case, the sample to be examined is contacted with a probe that will hybridize to the nucleic acid sequence encoding the ISL protein. Hybridization between the probe and the nucleic acids in the sample indicates the presence of expressed ISL proteins. These methods are known to a person skilled in the art and are for example described in WO 89/06698, EP-A 0 200 362, USP 2915082, EP-A 0 063 879, EP-A 0 173 251, EP-A 0 128 018. In a preferred embodiment of the invention, the nucleic acid of the sample encoding an ISL protein is amplified before the test, eg, by the well-known PCR technique. A derivatized (labeled) nucleic acid probe is usually used in the field of nucleic acid diagnostics. This probe is contacted with a denatured DNA or RNA, bound to a carrier, of the sample and in this process the temperature, ionic strength, pH value and the conditions of the buffer are selected in such a way that, depending on the length of the nucleic acid sample and the resulting melting temperature of the expected hybrid, the labeled DNA or RNA can be bound to the homologous DNA or RNA (hybridization, also see Southern, EM, J. Mol. Biol. 98 (1975), 503-517; Wahl, GM et al., Proc. Nati, Acad. Sci USA 76 (1979), 3683-3687). Suitable carriers are membranes or carrier materials based on nitrocellulose (eg, Schleicher and Schüll, BA 85, Amersham Hybond, C), nitrocellulose reinforced or bound in a powder form or nylon membranes derivatized with various functional groups (e.g. , nitro group), (for example, Schleicher and Schüll, Nytran, NEN, Gene Screen, Amersham Hybond M .; Pall Biodyne). The hybridized DNA or RNA is then detected by incubating the carrier, after complete washing and saturation to prevent non-specific binding, with an antibody or antibody fragment. The antibody or antibody fragment is directed towards the substance incorporated in the nucleic acid probe during derivatization. The antibody is labeled in turn. However, it is also possible to use a directly labeled DNA After incubation with the antibodies, it is washed again in order to detect only the specifically defined antibody conjugates. The determination is then carried out via the label or labeling of the antibody or fragment of the antibody. antibodies according to well known methods Detection of ISL expression can be carried out, for example - as an in situ hybridization with immobilized whole cells using immobilized tissue smears and isolated metaphase chromosomes, such as a colony hybridization (cells ) and plate hybridization (phage and virus), - as a Northern hybridization (RNA detection), as serum analysis, (eg, cell-type analysis of serum cells by slot transfer analysis, after amplification (eg, PCR technique) The invention therefore includes a method for the detection of nucleic acids encoding an ISL protein that is characterized in that the sample to be examined is incubated with a nucleic acid probe that is selected from the group comprising: a) the DNA sequences shown in SEQ ID No: 1 and SEQ ID No: 3 or a sequence complementary to these, b) nucleic acids that hybridize under severe conditions with one of the sequences of a). the nucleic acid probe is incubated with the nucleic acid of the sample and the hybridization of the nucleic acid in the sample and the nucleic acid probe is detected, if desired, via an additional binding partner. In this way, the ISL is a valuable prognostic marker in the diagnosis of benign and malignant, viral diseases. Surprisingly it was found that according to the invention it is not necessary to use an ISL polypeptide or nucleic acid directly for the inhibition of virus replication. It is also possible to use substances that induce the production of ISL in cells. These cells are preferably human blood lymphocytes, especially CD8 + cells. For the production of ISL, these cells are incubated, in vivo or in vitro, with these activation substances. If in vitro activation is performed, the 4 cells are subsequently administered to the patient, for example, according to U.S. Patent No. 5,399,346 mentioned above. In accordance with the invention, it is easily possible to identify these substances that activate the production of ISL. It has been found that these substances are, for example, phytohemagglutinin (PHA), Concanavalin A (ConA), histamine, polypeptides or nucleic acid molecules. Nucleic acid molecules are used as vectors containing additional elements that ensure the expression of the nucleic acid molecules in the target cells. These elements are known in the state of the art (for example, regulatory sequences, promoter and / or operator regions). Target cells suitable for transfection with these nucleic acid molecules are preferably human cells, more preferably human blood cells such as lymphocytes, especially CD8 + cells. Therefore, a further subject of the invention is a method for the identification and production of a substance and a therapeutic agent for the inhibition of virus replication in a patient. This method comprises combining with a pharmaceutically acceptable carrier a therapeutically effective amount of a substance that activates the expression of a protein with ISL activity in CD8 + cells, preferably in vivo. The protein, the expression of which is activated, is preferably a protein with the amino acid sequence shown in SEQ ID No. 2 A suitable substance can be identified in an assay (substance assay) wherein a) PBMCs are isolated from healthy blood donors by gradient separation of Ficoll, b) isolates CD8 + cells by the magnetic cell concentration, c) the purity of the preparation is tested by FACS analysis, d) the preparation should have a content of approximately 95% CD8 + cells and 5% non-CD8 + contaminants to ensure adequate stimulation of CD8 + cells, e) the substance to be tested for the induction of expression of ISL activity is added a) to the cultivation of cells in a concentration range of 1 pM to 10 mM, f) IL-2 is added to the cell culture (180 U / ml of cell culture medium) or to the culture of transfected cells if the substance is a nucleic acid molecule, g) after three days, the medium is completely removed and the cells are cultured with IL-2 (180 U / ml of cell culture medium) for three days; h) the supernatant of the cell culture is centrifuged (x 1000) to remove the cells, sterilely filtered, and aliquots are formed; and is further investigated in the aforementioned HIV inhibition assay, whereby i) the depleted CD8 + PBMCs are incubated with 50 μl of the cell culture supernatants / 1.5 x 10 6 cells in 150 μl of the medium for half an hour at 37 ° C; k) CD8 + depleted PBMCs are subsequently infected with HIV-1, preferably with HIV-1SF2, by incubating 1.50 x 10 6 cells in 150 μl with 50 μl of stock or concentrate of HIV-1 containing 50 infectious tissue testes ( TCIDS0) for 1 hour at 37 ° C. I) infected CD8 + depleted PBMCs are washed to remove unbound HIV-1; m) depleted CD8 + PBMC are cultured at 37 ° C under a 5% C02 atmosphere and the medium and cell culture supernatant is replaced after 3, 6, 9 and 12 days; n) the amount of HIV-1 in the culture supernatants of CD8 + depleted PBMC cells was determined on days 9 and 12 after infection by three-fold serial dilutions of the supernatant and inoculation in quadruplicate wells in 2000 cells in 150 μl of medium from a highly susceptible indicator cell line, which should be routinely infectious to an extent of 85% higher with strains of HIV-1, eg, lymphoma cell line MT4 transformed by HTLV, human; o) virus replication is determined in each well, 8 days after infection by the determination of inverted transcriptase (RT) in the cell culture supernatant (inverted transcriptase assay, Boehringer Mannheim GmbH, Biochemica, 68298 Mannheim, Germany , Order No.: 1468-120) of each individual well following the manufacturer's instructions; p) Infectious tissue culture dose (TCID50) of PBMC cultures depleted of CD8 + is calculated following the method published by Karber (Karber, G. 1931. Assay for statistical analysis of pharmacological experiments. Arch. Exper. Path. V. Pharmakol, 162, 148 according to the formula: logTCID50 = L-d (s-0.5); where L = log of the virus dilution lower d = log of the dilution of the virus s = sum of the virus-positive cell cultures q) Inhibition of HIV-1 replication in cultures of CD8 + depleted PBMCs is calculated by comparing the virus content of the cell culture supernatants in an assay according to steps i) ap) and the content of virus from an assay according to steps i) ap) where the supernatant of the cell culture to be tested for inhibition in HIV replication is replaced by normal medium (untreated control); r) inhibition is found if the amount of viral replication in CD8 + depleted PBMCs is inhibited in such a way that the amount of the virus is only about 50%, more preferably 10%, most preferably 1% or less compared to the untreated control. A further subject of the invention is a therapeutic composition useful in the treatment of a pathological condition characterized by excessive viral replication, especially retroviral replication, comprising at least one substance that activates ISL activity in CD8 + T cells, and which characterized by the properties of the aforementioned substance assay and HIV inhibition assay, and a pharmaceutically acceptable carrier. This substance, which can be obtained and characterized by the substance test mentioned above, is useful for the induction and / or activation of the ISL in mammalian cells, for the inhibition of the replication of viruses, preferably retroviruses, especially HIV. and / or HTLV, for the therapeutic treatment of benign and malignant diseases and viral infections, especially retroviral, especially by HIV and / or HTLV. It is also particularly preferred to use the substances for the therapeutic treatment of these viral infections as soon as possible after infection, preferably as soon as possible after the first peak or maximum value of the virus. The following examples, sequence listings, and figures are provided to assist in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the disclosed procedures without departing from the spirit of the invention.

Sequence Listing SEQ ID No: 1 represents the nucleotide of ISLagm (African green monkey) and the protein sequence derived therefrom.

SEQ ID No: 2 represents the protein sequence of the ISLagm. SEQ ID NO: 3 represents the nucleotide of LCF and the protein sequence derived from the same SEQ ID No: 4 represents the sequence of LCF proteins SEQ ID No: 5 represents primer 1 for the cloning of ISL SEQ ID No: 6 represents Primer 2 for cloning of ISL SEQ ID No: 7 represents the DNA sequence of HIV-1SF2 SEQ ID No: 8 represents the DNA sequence of HIV-1SF33 Legends in Figures Figure 1 Inhibition of HIV-1SF2 replication in the H9 line of purified T cell lymphoma by recombinant ISL (B) and by a cell culture supernatant of activated human lymphocytes (ISL) (C) CD8 +. A comparable quantitative inhibition of viral replication was measured using the following strains of HIV and SIV: HIV-1 SF2, HIV-1 SP33, HIV-1SF162, HIV-2uc3 and ISagm ((A) comparison, tissue culture only) Figure 2: Inhibition of replication of HIV-1SF2 in primary CD4 + lymphocytes by recombinant ISL. LogTCID50. Logarithm of the infectious dose of the tissue culture (infectious dose of the tissue culture) quantitatively comparable inhibitions occurred with the following strains of immunodeficiency virus: HIV-1SF33, HIV-1 SFI62, HIV-2uc3 and VIS, flm.

Figure 3 Comparison of DNA sequences and ISL polypeptides of human and monkey a) human; b) P. Troglodytes (chimpanzees); c) M. mulatta chin .; d) M. mulatta ind .; e) M. nemestrina; f) M. fascicularis; g) C. aethiops (AGM) Example 1 Cloning, expression and purification of the ISL 1. 1 Isolation of RNA x 10 7 PBMC (human or monkey) were cultured for 48 hours with 10 μg / ml of Concanavalin A and 180 units / ml of IL-2. In order to prepare the RNA, the cells were washed once with PBS and subsequently used with 5 ml of denaturing solution (RNA isolation kit, Stratagene). After the addition of 1 ml of Na acetate, 5 ml of phenol and 1 ml of chloroform / alcohol or methyl (24: 1), the lysate was kept on ice during minutes. The aqueous phase was subsequently mixed with 6 ml of isopropanol in order to precipitate the RNA and stored for 2 hours at -20 ° C. The precipitate was finally washed once with pure ethanol and dissolved in 150 μl of H20. The yield was determined photometrically and was 120 μg. 1. 2 cDNA synthesis The mixture for cDNA synthesis contained 10 μg of RNA, 0.2 μg oligo-dT, 13 mM DTT and 5 μl of the first strand reaction mixture, volumetric (cDNA synthesis kit of the first strand, Pharmacia) in a volume of 15 μl. The reaction was incubated for 1 hour at 37 ° C and subsequently stored at -20 ° C for the final use. 1. 3 Amplification and cloning of the ISL cDNA For the amplification of the ISL cDNA by means of PCR and for the next cloning, the following oligonucleotides were synthesized: Primer 1: GCTGCCTCTCATATGGACCTCAACTCCTCCACTGACTCT (SEQ ID No: 5) Primer 2: GATGGACAGGGATCCCTAGGAGTCTCCAGCAGCTGTGG (SEQ ID No: 6).

The primers introduced additional Ndel or BamHI cleavage sites. The PCR mixtures (100 μl reaction volumes) each contained 1 μl of cDNA (from the synthesis in section 3), 50 pmol of primer 1 and 2, 12.5 μmol of dNTPs, 10 μl of buffer 10xTAQ and 2. 5 units of Taq polymerase (Perkin-Elmer). The cycle conditions were 30 seconds, 94 ° C, 1 minute, 53 ° C and 1 minute, 72 ° C. 35 cycles are carried out. The PCR products were purified and digested for 16 hours at 37 ° C with Ndel and BamHI. For the cloning preparation, vector pET15b (Novagen) was also cleaved with Ndel and BamHI and subsequently purified on an agarose gel. The ligations were carried out for 2 hours at room temperature in 20 μl mixtures containing 100 ng of the vector, 25 ng of the PCR product (insert); 2 μl of the 10x ligase buffer, and 0.2 μl of ligase (New England Biolabs). After transformation by electroporation at 2.5 kV, 25 μ farad, 200 ohm (BIO-RAD electroporator) in E. coli DH5, the cells were plated on ampicillin resistant plates. Recombinant clones were identified by restriction analysis of the plasmid preparations (pMISLB) and transformed into strain BL21-DE3 for the proposed protein expression. The cloning of the ISL cDNA could be confirmed additionally when determining the nucleotide sequences. The sequences found were in agreement with the sequence of published LCF (Cruikshank et al. In Proc. Nati, Acad. Sci. USA, Vol. 91 (1994) 5109-5113) apart from a discrepancy in codon 96. In contrast to the published sequence, codon 96 is not composed of the TTG base sequence, but rather of the TTT sequence and thus codes for leucine and not for phenylalanine. The sequencing of the additional ISL clones that were derived from the independent PCR amplifications clearly showed that the authentic ISL sequence in codon 96 is actually represented by the TTT sequence. Proteins homologous to ISL are isolated in an analogous manner from body fluids containing CD8 + lymphocytes from animals infected with immunodeficiency virus and in particular from those that are infected without decrease, such as chimpanzees (P. troglodytes), African green monkeys (C. aethiops), dark gray monkeys, M. mulatta chin., M. mulatta ind., M. nemetrina or M. fascicularis. It is possible to use these proteins and nucleic acids in a therapeutic manner and in diagnostics in a manner similar to human ISL.

Example 2 Expression and purification of soluble recombinant ISL 2. 1 Human ISL The ISL is expressed aminoterminally in a fusion with a 6-residue histidine guide in the pET15b vector, 20 ml of the pMISLB culture was used in each case to inoculate 2 liters of the 2XTY medium / ampicillin. The cultures were shaken at 25 ° C and when they reached an OD600 of 0.4, they were induced by the addition of 1 ml of 1 M isopropyl-β-D-thiogalactoside. After an additional 4 hours, the bacteria were pelleted and frozen for 14 hours. Hours at -70 ° C.

The pellets were melted and washed once with 250 ml of PBS. The cells were used in 50 ml ice-cold PBS by adjusting the suspension to 1% P-40 N, 10 mM EDTA, 0.4 M NaCl and 50 μg / ml lysozy a. After 60 minutes of incubation on ice, the lysate was free of the insoluble components by centrifugation. The ISL with 6 histidine residues at the amino terminus (His6-ISL) was purified by means of a chromatographic step. For this, the lysate was adjusted to mM MgCl 2, 10 mM imidazole, 0.5 M NaCl and applied to a column of (Qiagen) Ni 2 + -NTA-Agarose with a flow rate of 0.1 ml / minute. 0.25 ml of Ni2 + -NTA-Agarose was used per initial culture per liter. The column was subsequently washed with 20 volumes of PBS, 25 mM imidazole and His6-ISL were finally eluted with 4 ml of PBS, 20 mM imidazole. The His6-ISL fusion protein isolated in this manner had a purity level of more than 90% after the test in the gel electrophoresis of SDS. The yields were approximately 5 mg of protein per initial culture per liter. The purified protein was finally free of lower molecular impurities such as, for example, imidazole by gel filtration on NAP-10 columns (pharmacy) and transferred to PBS. Then, the protein concentrations were 0.5-1 mg / ml (Scheme of purification see Table 1): Table 1 Flow chart of ISL purification Culture B121-DE3 of transformed E. coli using pMISL-1huB U induce with 1 mM IPTG JJ Use the cells (NP-40, EDTA, lysozyme) JJ adjust to 20 mM MgCI2, 10 mM imidazole, 0.5 M NaCl to bind Ni2 + -NTA-Agarose wash with 25 mM imidazole / PBS JJ elute with 200 mM imidazole / PBS reamor ture in PBS JJ verify the purity by SDS-PAGE i i determination of protein 2.2 LCF derived from ISL Since the LCF has an almost homologous sequence, the LCF was cloned specifically from a DNA library or library of activated CD8 + lymphocytes, human, in addition to an ISL cloning experiment, in order to examine the above for a possible antiviral efficiency. It could be shown that the LCF has an action of ISL and is capable of inhibiting the replication of HIV as well as the SIV. 2. 3 Derivative of ISL of the African green monkey (ISL-agm) A protein homologous to the human ISL (SEQ ID No: 1) can be isolated from the African green monkey (ISL-agm). The nucleotide sequences of the human ISL (ISL-hu (SEQ ID No: 2)) differ as shown in the tables below: Table 2a Comparison of the DNA sequences of the ISL-hu and the ISL-agm Table 2b Comparison of the protein sequences of the ISL-hu and the ISL-agm 2. 4 ISL derivatives of other monkeys Sequences of proteins and nucleic acids from the ISL of other monkeys can be isolated in the same way. A comparison of the sequences is shown in Figure 3. 2. 5 Recombinant expression of fusion-free ISL in E. coli The DNA sequence encoding the ISL is modified in such a way as to allow efficient expression in E. coli. For expression, an expression plasmid is transfected into a suitable E. coli strain. These strains are, in the case of the use of an expression plasmid under the control of the lac repressor such as the expression plasmid p11379, strains possessing a sufficiently high intracellular concentration of the lac repressor. These classes of strains can be prepared by transfection of a second plasmid such as pREP4 (Diagen GmbH), pUBS 500 or pUBS520 (Brinckmann et al.

Gene 85 (1989) 109-114). The E. coli strains applied should preferably have a low protease activity of the cells, as is the case, for example, with E. coli UT5600 (Earhart et al., FEMS Microbiology Letters 6 (1979) 277-280), coli BL 21 (Grodberg and Dunn, J. Bacteriol., 170 (1988) 1245-1253) or E. coli B. Then, culture of the expression is achieved in a manner according to the state of the art, as a protein aggregate, and it is continued according to the procedures described in EPO 241 022, EP 0 364 926, EP 0 219 874 and DE-A 40 37 196. In detail, for example, the following procedure is applied for this purpose: lysates containing ISL of E. coli fermentations were adjusted to 6 M guanidinium hydrochloride, 100 mM Tris-HCl at pH 8, 1 mM EDTA, subsequently adjusted to a pH of 3 to 4 and dialysed against 4 M guanidinium hydrochloride at pH 3.5. The renaturation of the solubilized protein is then carried out in 1 M argininin at pH 8, 1 mM EDTA, 5 mM GSH (reduced glutathione) and 0.5 mM GSSG (glutathione, oxidized). The ISL can be further purified by usual chromatographic techniques. 2. 6 Recombinant expression of ISL in mammalian cells.

For this, the cDNA is ligated into a vector in which it is transcribed into mammalian cells, preferably CHO or COS cells, based on a strong promoter-enhancer system. These promoters and enhancers are mainly from viruses such as SV40, hCMV, polyoma or retroviruses. As an alternative, promoter-enhancer systems that are specific to a certain cell type can also be applied by tissue type, such as, for example, WAP-, MMTV- or immune globulin promoter, or systems that are inducible such as by promoter example of metallothionein. This class of vector supplements the ISL cDNA (if the latter is used) with donor and acceptor signals for processing RNA as well as a signal for poly-A-addition. For example, pCMX-pL1 (Umesono et al., Cell 65 (1991) 1255-1266) is a suitable vector. In one and only of the EcoRI cleavage site of this vector, the cDNA provided with EcoRI linkers is ligated, where it is ensured by restriction analysis with the help of other cleavage sites in the polylinker of this vector that the cDNA is orient in the reading direction of the CMV promoter. An absolutely analogous procedure is applied when cloning into other vectors, for example, in pCDNA3 (Invitrogen, San Diego / USA) or pSG5 (Stratagene, La Jolla / USA). The DNA of the expression plasmids thus obtained is prepared from E. coli and transfected into the mammalian cells, applying techniques that are specific to the cell types in the particular case (Methods of Enzymology 185 (Goeddel, David V ., (ed.), Gene Expression Technology, Academic Press 1991, section V) After transfection, the cells are grown in MEM (Gibco) without the addition of fetal calf serum, whereby the ISL is detectable in the supernatant of the cell culture after 48 hours.

Example 3 Testing the inhibition of HIV replication by ISL in T-cell lymphoma lines; Primary lymphocytes (PBMC) and primary, purified CD4 + lymphocytes. 3. 1 Obtain concentrated solutions of HIV virus to infect the cells.

Human immunodeficiency viruses (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV) replicate in the lines of human T-cell lymphomas as well as primary CD4 + lymphocytes. Cell culture supernatants containing viruses obtained from primary lymphocyte cultures (PBMC) usually contain more infectious viruses than those obtained from the T cell lines in the case of the HIV-1 strain, HIV-1SF2, replication is only possible in PBMC since this virus does not replicate in any of the known T-cell lymphoma lines. The supernatants of the normal viruses used in all subsequent experiments were therefore produced in primary lymphocytes. For this, PBMC purified by means of a Ficoll gradient and stimulated with phytohemagglutinin (PHA) (for a detailed description of the method see below) were infected with strains of HIV-1, HIV-1SF2, HIV-1SF33, HIV-1SF162 ( C heng - M yesterday, C, et al J. Virol. 64 (1990) 4390-4398) and strain of HIV-2, HIV-2UC3 (Castro, BA, et al., Virology 178 (1990) 527-534) . These viral strains were also previously passaged exclusively in the PBMC and the supernatants of the similar culture containing the viruses were stored at -70 ° C. For the infection 120 x 106 PBMC were incubated with a multiplicity of infections (MOI) of 0.1, for 2 hours at 37 ° C, the cells were washed with RPMI medium and the cell culture medium was cultured for 12 days in 40 ml ( RPMI 1640, 20% FDCS, 2 mM glutamine, 180 U / ml IL-2) corresponding to a cell count of 3 x 106 / ml of the cell culture medium. The cell culture medium was changed on the third day in which the cell count was again adjusted to 3 x 106 / ml of the medium. On days 6, 9 and 12 the supernatant of the cell culture was collected, the cells and cell wastes were removed by centrifugation, sterilized by filtration (pore size 0.45 μm) and stored in aliquots of 0.5 ml at 70 ° C. 3. 2 Cell culture conditions for the propagation of T-cell lymphoma lines The H9 lymphoma lines of T cells, CEM, Muda 4, clone 8, MT4 and C8166 were cultured at 37 ° C and 5% C02 atmosphere in RPMI 1640 medium, supplemented with 10% FCS in 2 mM glutamine. The cells were passed every third day while the medium was changed at the same time and the cell count was adjusted to 1 x 105 / ml. 3. 3 Preparation and propagation of primary blood lymphocytes (PBMC) Peripheral blood lymphocytes of "ante-colored surface layers" that have been isolated in normal blood donors were prepared. Whole blood is used to prepare PBMC from non-human primates such as African green monkeys and rhesus monkeys. For this, the "ante-colored surface layer" of whole blood is layered on a Ficoll-Hypaque gradient and centrifuged for half an hour at 1000 xg. The supernatant of the serum is discarded, the mononuclear cells are collected and washed several times in the Hanks medium. Cells (3 x 10 6 / ml) are taken in RPMI 1640, 20% FCS, 2 mM glutamine and are stimulated for three days with 9 μg / ml phytohemagglutinin (PHA) and proliferate with 180 U / ml IL- 2. Cells are cultured at 37 ° C and 5% C02 atmosphere. Then, the medium is completely changed and the cells are further cultured without PHA at a cell count of 3 x 106 / ml culture medium or used in experiments. 3. 4 Purification of CD4 + and CD8 + lymphocytes by means of the concentration of activated cells, magnetic (MACS).

Lymphocytes isolated by means of Ficoll gradients from an "ante-colored surface layer" are redispersed in 500 μl of PBS-azide / 1 x 108 cells (phosphate-buffered saline without Ca2 + and Mg2 +, sodium azide 0.01%, 5 mM EDTA, pH 7.2). After the addition of 20 ml of microbeads CD8 / 1 x 107 cells expected (antibodies anti-human CD8, mouse, conjugated with magnetic particles, Miltenyi Biotec GmbH) are incubated for 15 minutes at 4 ° C. 2 mg of DTAF / 1 x 107 cells were added (anti-mouse IgG, conjugated FITC, Dianova Company), for an additional 5 minutes at 4 ° C. After dilution with 25 ml of PBS-azide / 1% BSA, it is again centrifuged (10 minutes, 1200 rpm, 4 ° C). The supernatant is discarded, the cells are redispersed in 2 ml of PBS / 1% BSA and the cell suspension is applied to a column that is located in a magnetic separator (Miltenyi Biotec GmbH). The CD8 + cells to which the CD8 microbeads are attached are kept in the column, the flow fraction therefore contains all the lymphocytes (approximately 80% of CD4 + cells) except the CD8 + cells. After washing, the column is taken from the support and the fraction of CD8 + cells is eluted with PBS azide / 1% BSA. The fraction of flow and the fraction of CD8 + cells are centrifuged, re-dispersed in the cell culture medium (RPMI 1640, 20% FCS, 2 mM glutamine, 180 U / ml IL-2), the cells is adjusted to 3 x 10ß / ml and the cells are stimulated with PHA (9 mg / ml). The quality of the separation of the lymphocyte subpopulation is verified by means of FACS. 3. 5 Titration of the concentrated solution of HIV virus in several host cells CD4 + lymphocytes, PBMC, and T-cell H9 lymphoma lines (Popovic, M., et al., Science 224 (1984) 497-500), Muda 4, clone 8 (Kikukawa, R., et al J. Virol 57 (1986) 1159-1162), C8166, MT4 and CEM (obtained from the American Type Culture Collection) were used as host cells. The titrations were carried out in 96-well plates. a) Titration in PBMC and CD4 + lymphocytes The concentrated solutions of HIV-1SF2 virus, HIV-1SF33, HIV-1SF162 (Cheng-Mayer, C, Quiroga M.

Tung, J.W., Dina D. & Levy, J.A. (1990) HIV-2UC3 and VISagm. (Kraus, G., and collaborators Proc. Nati. Acad.

Sci .. USA 86 (1989) 2892-2896; Baler, M., and collaborators, J. Virol. 63 (1989) 5119-5123) are diluted in three steps and 50 μl of each are pipetted into four independent PBMC cultures or lymphocytes.

CD4 + (1 x 10β PBMC or CD4 + lymphocytes in each case) in the culture medium of 100 μl and incubate for one hour at 37 ° C. then viruses that do not bind to the cell are removed by washing the cells with the culture medium. The medium is changed (removal and addition of 100 μl of medium each time) 3, 6, 9 and 12 days after infection. The cell culture supernatants of each individual culture from days 6, 9 and 12 are tested for their virus content by carrying out either a) a test for the inverted transcriptase (according to the manufacturer's instructions of the test, Boehringer Mannheim GmbH, Germany), b) a p24 antigen ELISA (according to the manufacturer's instructions "Abbott") or c) highly susceptible indicator cell line infections (Ennen, J., et al., Proc. Nati. Acad. Sci. USA 91 (1994) 7207-7211). b) Titration in the T cell lymphoma H9 lines, CEM, Muda 4/8 The concentrated solutions of virus are diluted in three steps and 50 μl of each are taken with pipette in independent cell cultures (in each case 5 x 10 4 cells in 100 μl of the culture medium in 96-well cell culture plates in the form of U) and incubated for 1 hour at 37 ° C. The washing and testing for the infection is carried out according to the method described in 2a). The initial, smaller cell count compared to PBMC cultures is due to the proliferative competition of the T-cell lymphoma lines that during the course of the titration proved to grow at the critical cell density in the assigned cell culture volume of the microtiter plates of a maximum of 250 μl. c) Titration of the T cell C8166 and MT4 lymphoma lines Lines of T cell lymphoma C8166 and MT4 were titrated according to the method described in 2b). However, the virus test is not carried out using the test systems mentioned above but the cell cultures are evaluated by light microscopy. In the case of infection by viral strains HIV-1SF2, HIV-1SF33, HIV-2uc3, C8166 and MT cells were killed by the proliferation of viruses that were easily and rapidly identified using the microscope. 3. 6 Calculation of infectious tissue culture dose50 (TCID50) The TCID50 is calculated according to the method published by Karber (Karber, G. 1931. Assay for statistical analysis of pharmacological experiments.

Arch. Exper. Path. V. Pharmakol. 162, 148) according to the formula: logTCID50 = L-d (s-0.5), where L = log of the lowest virus dilution d = log of the virus dilution s - sum of virus-positive cell cultures; 3. 7 Test for inhibition of HIV replication by ISL The effectiveness of ISL in HIV replication is tested in T cell lymphoma lines as well as in primary lymphocytes. The experiments begin with the toxicity and dose-finding experiments, in the additional course of one week with a non-toxic but maximally effective ISL concentration. These tests are carried out separately in 96-well microtiter plates in quadruplicate for each T-cell line in PBMC as well as in total PBMC and also in CD4 + lymphocytes. a) Dosage and toxicity encounter experiments using ISL on T cell lines x 104 cells are incubated for 10 minutes at room temperature with various dilutions (40 μg / ml to 0.15 μg / ml) of ISL. The cells are then infected with the concentrated virus solutions mentioned under 1. They were infected in each case for one hour at 37 ° C and 5% C02 atmosphere with 50 TCID50 which was determined and calculated separately for each cell line (see above under 2). Unbound virus was removed by washing the cells with the culture medium. The cells were again dispersed in the cell culture medium and the ISL concentrations were adjusted to the initial concentration. In some experiments ISL was added again every day to ensure that the ISL concentration remained relatively constant. The medium was changed on days 3, 6, 9 and 12 after infection in the process of which the cell culture supernatants of days 3, 6, 9 and 12 were examined quantitatively for their virus content using the test described above.

Parallel to this, crop growth curves were plotted (the count of cells dissected with trypan blue by means of light microscopy) that gave an insight into the toxicity of ISL. b) Dosage and toxicity encounter experiments using ISL in primary PBMC The experiments were carried out using total PBMC as well as purified CD4 + lymphocytes. 1 x 106 cells were incubated with ISL under the conditions described supra, were infected with HIV and the cell culture supernatants were examined quantitatively for their virus content on days 6, 9 and 12 after infection. The toxicity of ISL in PBMC was determined with the help of trypan blue staining and counting the cells by light microscopy. c) Evidence of the mechanism of action of the ISL We examined whether ISL develops its inhibitory effect at the level of de novo infection or during the persistent replication of HIV. The experiments were carried out for this, in which the fully effective dose of ISL in the cell culture was present only during an infection period of one hour. Parallel to this, a constant ISL concentration was further maintained in the cell culture medium during the entire test period in another experimental mixture. This experiment allows a decision if ISL inhibits HIV infection or HIV replication or if it is effective at both levels (Figures 1 and 2).

Example 4 Qualitative and quantitative detection of ISL in body fluids and cell culture supernatants ISL was detected by means of an enzyme-linked immunosorbent assay (ELISA, for its acronym in English). For this, monoclonal antibodies against ISL were prepared. These were absorbed into the wells of an ELISA plate. The liquid to be tested for its ISL content was incubated, the monoclonal antibodies reacted specifically with the immobilized ISL. The bound ISL is detected by means of an anti-ISL antibody, polyclonal, purified by affinity (it was obtained by immunizing a goat with ISL) that by itself becomes visible by means of a color reaction using a coupled anti-goat antibody. to peroxidase Example 5 Direct detection of cells that produce ISL The monoclonal antibody against ISL described above is used for the direct detection of cells that produce ISL either for diagnostic purposes already in the case of cells of patients or for cells in tissue culture. For this, the cells are fixed with methanol while at the same time dividing the cell membrane and incubated with the monoclonal anti-ISL antibody labeled with fluorescein isothiocyanate (FITC). It is evaluated with the help of a fluorescent activated cell sorter (FACS).

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USA 76 (1979) 3683-3687 Walker, C.M. and Leighly, J.A., Immunology, Vol. 66 (1989) 628-630 Walker, Erickson, et al, Journal of Virology, Vol. 65 (1991) No. 11, 5921-5927 Walker, Moody, et al, Science, Vol. 234 (1986) 1563-1566 Walker Thomson-Honnebier, et al Cellular Immunology 137 (1991) 420-428 WO 89/06698 WO 93/0883 WO 94/23058 WO 94/28134 LIST OF SEQUENCE (1) GENERAL INFORMATION (i) APPLICANT (A) NAME Bundesrepubhk Deutschland, vertreten durch den Bundesminister f Gesundheit, (B) STREET (C) CITY Bonn (E) COUNTRY Germany (F) POSTAL CODE (ZIP) D-53108 00 TITLE OF THE INVENTION Use of a "lymphokine that suppresses immunodeficiency virus (ISL)" to inhibit virus rephcation , in particular of retroviruses (ui) NUMBER OF SEQUENCES 8 (iv) COMPUTER LEGIBLE FORM (A) TYPE OF MEDIUM Flexible disk (B) COMPUTER compatible with IBM PC (C) OPERATING SYSTEM PC-DOS / MS-DOS (D) ) Patentln Reléase Program # 1 0, Version # 1 30B (EPO) (vi) DATA FROM THE PREVIOUS APPLICATION (A) NUMBER OF APPLICATION FOR 195 13 152 5 (B) DATE OF SUBMISSION 07 April 1995 (vi) DATA OF THE PREVIOUS APPLICATION (A) APPLICATION NUMBER EP 95113013 2 (B) DATE OF SUBMISSION August 18, 1995 (2) INFORMATION FOR SEQ ID NO 1 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 393 base pairs (B) TYPE of nucleic acid (C) TYPE OF DOUBLE D) TOPOLOGY linear (p) TYPE OF MOLECULE DNA (ix) CHARACTERISTIC (A) NAME / KEY CDS (B) LOCATION 1 393 (xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO 1 ATG CCC GAC CTC AAC TCC ACC ACT GAC TCT GCA GCC TCA GCC TCT GCA 48 Leu Asn Ser Thr Thr Asp Ser Wing Wing Wing Wing Wing 5 10 15 GCC AOT GAT GTT TC GTA GAA TCC TCA GCA aAß GCC ACA GTC TAC ACG 96 Ala Ser Asp val Sar Val ßlu Ser Sar? Glu? Thr Val Tyr Thr 20 25 30 = GT? ACA CTG GAO AAG ATG TCT GCA GG? TCT «C 'TTC A? C CT? GA? GGA. i val Thr Lau Glu Lya Mat Be Wing Gly Leu Gly Phß Sar Lau Glu Gly 35 40 * • »aaa AAG GQC TCC CTG CAT GGA ene ?? ß- CCT ero ACC ATT AAC AO» ATT 192, -aly Lys Gly Sar Leu His Gly? Ap Lys Pr? Lau Thr Ha? Sn Arg? La 30 55 l? TTC AAA GGA GCA GCC TCA GA? CAÁ AßT 0 »AOk A? © CA? CCT GG? GATf 21, Phe Lys Gly? The? The Ser Glu Gn Sar Glu Thr the Gln Pro Gly Asp «3 70 7S" Q0 G? A ATC TTG CAG CTG GCT GGC ACT GCC AJfß CAO GGC CTC ACÁ COL TTT 288 Glu lia Leu Gn Lau Ala Gly Thr Ala Het Gln Gly Lau Thr Arg Phe 85 99 9 »G ?? GCC TGG AAC ATC ASC AAG GCC CTG CCT 'ß * T Gß? CCT GS ACB A3V 334 Glu? La Trp? Sn Ha lia Lys Ala Leu Pro? ßp Gly Pro val Thr lía 100 105 110 OTA ATT AGG AGG A ?? AGC CTC CAA CCC AAG GAA AOC ACA GßT, GCT GCA 344 to He? Rg Arg Lys Smx Lau Gln Pro Lys Glu Thr? La? La? 115? 120A? 121 GAC TCC TAG_393_Asp Sar * 130 (2) INFORMATION FOR LASEQ IDNO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 131 amino acids (B) TYPE: amino acid D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Pro Asp Leu Asn Ser Thr Thr? sp Ser? la? la Sar? la Ala Ala 5 ': • "10 •• -. - • A < .- • ..- 15 - .. "? the Ser Asp Val Ser Val 'Glu Sar Sar Ala Glu? the Uve-Val, Tyr Th» 20 25 30 Val Thr Lau Glu Lys Met Ser? la Gly Leu Gly Phe Sar Leu Glu Gly 35 '40 *., Gly Lys Gly Ser Lau His Gly Asp Lys Pro Lau Thr.? Asn Arg Ha- 50 35 80 Phß Lys Gly? The Wing Ser Glu ain Ser Glu Thr líe Gln Pro Gly Asp 65 70 75 80 Glu Ha Lau Gln Lau Wing Gly Thr Wing Met G --- to Gly Leu Thr Arg Phe.- 85 90 95 Glu Wing Trp? Sn Ha Ha Lys? The Lau Pro? Sp Gly Pro val Thr Zla 100 105. 11Q Val Ha Arg Arg Lys Sar Lau ßln Pro Lys Glu Th • -Thr? Ala Ala- 115 120 125 Asp Sar * 130 C (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2151 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (i) TYPE OF MOLECULE: (genomics) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 783 .. 1 175 (ix) FEATURE: (A) NAME / KEY: peptide-mat 4 (B) LOCATION: 783 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TTCCTCGAG? GCTGTC? C? CAGGCTGAGG AATCTCAAGG CCCAGTGCTC AAGATGCCT? 60 GCCAGCGAGC ACGGAGCTTC CCCCTGACC? GGTCCCAGTC CTGTGAGACG A? GCT? CTTG 120 ACGAAAAGAC CAGCAAACTC TATTCZATCA CC? GCCAGTS TCATCGGCTQ TCATGAAATC 180 CTTGCTGTGC CTTCCATCTT CTATCTCCTG TGCCCAGACT CCCTGCATCC CC ?? GG ?? GG 240 GGC? -rCTCCA ACATCATCAT CCAACGAAGA CTCAGCTGC? AATGGTTCTG CTG ??? CATC 300 TGCCTTGGAC ACGGGTTCT CGCTC ?? CCT TTCAGAGCTG AG? SAAT? T? CAGACGCTCT 360 CACGGAAGCC AAGGAAGACG AXGATGGGG? CC? CAGTTCC TTCAGTCTGG TCAGTCCGTT 420 A-rsrcCCTGC TGAGCTCAG? AGAATTAA ?? A ?? CTCATCG AGG? GGTG ?? GGGTCTGGAI 480 GAAGCAACAX T? A? ßC ?? TT AGACGGC? TC CAIGTCACC? TCTTACACAA GGAGGAAGGT 540 WIWGC? Í'U GGTTCAGCTT GGCAGGAGG ^ GCAGACTAG A ??? C ?? GGT ATTACGGTT 600 CACAGAGTGT TTCC ?? AJGG GCTGGCCTCC C? GGAAGGGA CTASTCAGAA GGGCA? TßAO 660 GTTCTTTCC? TCAACGßCA? GTCTCTC ?? G GpGADSACqC ACCAIGAIGC CTTGGCCATC 720 CTCCGCGAJU5 CTCGGAGCC CAfiGC ?? GC? GTGAST? -TCA CAAOGAAGCT GACTCC? SAG 780 CC ATG CCC GAC CTC AAC TCC TCC? CT GAC TCT GC? GCC TCA GCC TCT 827 Mat Pro? Sp Leu? Sn be ser Thr? Ap Ser Allah Alaâ € ™ Sar Ala Ser 1 5. ltt 15 GC? GCC AGT GAT GT TCT GT ?: GA? -TCT AC? GC? GAG GC? ACA GTC TGC 875 Ala Ala Ser? Ap Val Ser Val Glu Sar Thr? Glu Ala'Thr Vaí Cyr Z9 2 $ 30. ACG GTG AC? CTG GAG AAG ATG TCG GC? Gßß CTß 5GC TTC AflC CTG GAA 923 Thr Val Thr Leu Glu Lys Mat Sar? The ßly Dtii Gly 'Phß be Leu Glu' 35 4Q 45 GGA Gßß AAG GGC TCC, CTA CAC GGA G? C AM OCT CTC ACC ATT AAC AGG 971 Gly Gly Lys Gly Ser Lau His Gly? Sp Lys Pro Leu Thr XI *? Sn Arg 50 51 «0 ATT TTC AAA Gß? GC? GCC TCA G ?? CAA AGT GAG ACA GTC CAG CCT GG? 09 Ha Pha Lys Gly? The? Ser Glu Gln Sa 'G1U Thr Váí' «Without Pro Gly 65 70 7 GAS GAA- ATC TTG CAG CTG GGT GGC ACT GCC AXG C? GGGC CTC AC? CGß 1067 Aap Glu He Leu Gln Leu Gly aly Thr 'Allah Met Glh Gly Lau Thr Arg 80 85 90 95 TTT G ??. GCC TGG. AAC ATC AS? C AA? GCA CTG CCT GAI GGA CCT GTC ACG 1HS Phe Glu? Txp? Sn He He Lys? Eu fro Asp aly Pro Val T-ur .100 .. 105 110? STGTCATC? GG? G? AA ?? GCCTCCAß TCC. AAG. G ?? ACC AC? GCT GCT 1163 Xlß Val lié Arg? Rg Lya Sar Lau Gln Ser Lys Glu Thr-Thr? Wing 11 * 120 125 GGA GAC TCC T? G- GCAGGACATG CTGA? GCCA? AGCC? TAAC ACACAGCTAA 1215 Gly? Sp Ser * '' 130 CACACAGCTC CCAT? CCGC TGAITCTC? GGTCTCTGCT SCCGCCCCAC CCAGATG? G? 1273 GAAAGCACAG GTGGGCTTCC CAGTGGCTGC TGCCCAGGCC CAGACCTTCT AGGACGCCAC 1335 CCAGCAAAAG GTTGTTCCT? A ?? TAAGGGC AGAGTCACAC TGGGGCAGCT G? TAC ??? TT 1395 GCAGACTGTG TA ??? AGAG? GCTTAATG? T AAT? TTGTGG TGCCACA ?? T A ??? TGGATT 1455 T? TTAG ?? TT TCATATGAC? TTCATßCCT GCTTCGCA ?? A-KT TCAAG TACTGTAACT 1515 GTGTCATGAT TCACCCCC ?? C GTGACAT TT? TTTTTCT C-VTGAA-CCTG CAATßTßGGC 1573 AGAG? -TTGGA? TGGGCAGCT C TCTCTGTC CC &CTTGGCA TC3U3CTGGCS TCATGC ??? G 1635 TCATGC ?? AG GCTGGGACC? CGTG? GATC? TTCACTCAI? CATCTGßCCG TTGATGTTGG 1695 CTGGGAACTC ACCTGGGGCT GCTGßCCTG? ATOCTTAIAG GTGGCCTCTC CTTGTGGCCT 1755 GGGCTCCTC? C? ACATGGTß TCTßßATTCC CAGGATGAßC ATCCCCGAT CGCAAGA3CC 1815 ACGTAG? AGC TGCATCTTßT TTATACCTTT GCCTTOGAAß- TTGC-STGßC? TCACCTCCAC 1875 CATACTCCAT CAßTTAG? OC TGACACAAAC CTGCCTßGßr TTAAGGGGAG AGGAAATATT "1935 GCTTGßßTC? TTTATG ???? AXACAßVTTß TCACATC-DIAA C? TTTßC ??? ASTGTTTTTG '1995 r GTTGGATTGG AGAAOTA? TC CTACGOAAGO GTßßTßGAG CAGTAAATAG- AGGAGT? C? G 2055 GTG ?? ßCACC AAGCrCAAAG CGTW? CA? T TGTGCCOAC? GAAGGAACCA sCGTGTAIAT '2115 GAGGGTATC? ?? T? TT? TACTACT? I CCTACC' '2131 (2) INFORMATION FOR SEQ ID NO 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 131 amino acids (B) TYPE-amino acid D) TOPOLOGY, linear (p) TIPODE MOLECULE protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Met Pro? S Lau Asn Sar Ser Thr Aap Sar? The? Sar? Ser? La. 1 5 - - '- Iß - "' - ÍS Ala Sar? Ap Val Ser Val ßlu Ser Thr Al * Glü? The Thr Val Cys Thr 20 25 30 Val Thr Leu Glu Lys Met Ser? The Gly Lau Gly Phe Ser Lau Glu Gly 35 '40 45 Gly Ly »Gly ßer Leu His ßly ißfe Lys Pro Leu Thr Ha? Angl ia 30 55 60. Phe Lya Gly? The? The Ser Glu Gln Ser Glu Thr Val GI? Pro Gly? Sp 65 70 • '"'" 73"80 Glu He Leu GI? Leu Gly ßly Thr? The Met Gln Gly Leu Thr? Rg Ph? 85 90 95 Glu? The Trp? Sn lie He Ly?? The Lau Pro? Sp ßly Pro Val Thr Ha 100 105.; HO val Zle Arg Arg Lys Ser Leu Gln Ser Lys Glu Thr Thr? La? Le Gly 115 120"'* 125- Asp Ser * 130 (2) INFORMATION FOR SEQ ID NO: 5: (0 CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GCTGCCTCTC ATATGGACCT CAACTCCTCC ACTGACTCT 39 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: 38 GATGGAACAGG GATCCCTAGG AGTCTCCAGC AGCTGTGG (2) INFORMATION FOR SEQ ID NO: 7: (0 CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9737 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: (genomics) (xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO 7 CTGGAAGGGC TAATTTGGTC CCAAAGAAG? CAAGAGATCC TTGATC? OTS OATCTACCAC 60 ACACAAGGCT ACTTCCCTG? pGGCAGAAT T? CACACCAß GGCCAGßß? T CAGATATCCA 120 CTGACCTTTG GATGGTGCTT CAAGCTAGT? CCAßTTGAßC CAGAGAAOOT AßAAGAQGCC 180 AATGAAGGAG AGAACAACAG CTTGTTACAC CCTATGAGCC TGCA-TGGGAT GG? ßß? CGCG 240 GAGAAAGAAG TGTT? GTGTG G GGTTTßAC AGC? AACTAß C-A-TTTCATCA C? TGGCCCGA 300 GAGCTGCATC CGG? GTACT? C AGACTGC TGACATCGAG CTTTCTACAA GGGACTTTCC 360 GCTGGGGACT TTCCAGGßAG GCGTGGCCTG GGCGGGACTß GGGAUTOGC8 TCCCTCA-3AT 42CT GCTGCAT? T? AGCAGCTGCT TTTTGCCTGT ACTGßßTCTC TCTßSTSAß * CCA ---? TCTSA'- '489 GCCTßßßAGC TCTCTGGCT? ACTAGßG ?? C, CCACTGCTT? AßCCTCAA-EK ?? ß RGCCT 540 TGAGTGCTTC A? GT? ßTGTG TGCCCGTCTß TTGTßTGACT CTGGTAACT? GA --- A-TCCCTC '600 AG? CCCTTTT AGTCAGTGTß GAAAAATCTC TAGCAGTßGC. ßCCOGAACAfl. GCAC0O9AAA 660 GCGAAAGTAG AACC? GAGG? GCTCTCTCG? CGC? GGACTC GGCTTßCTß? A-8C0O6Cte? 720 GCAAGAGGCG AGOGGCGGCG ACTGGTGAGT ACGCCAATTE TTG? CTAGCG GAGGCTÁGAA 780 GG? GAGAGAG ATGGGTGCG? GAfiCGTCGGT ATTAAGCGGß GOAGAATTAG AZAA? TGGG? 840 AAA ?? TTCGß TTAAßßCCAG GGGGAAAG ?? ???? TAT ?? ß TTAAAACATA TAOT? TOGGC 900 AAGCAGGGAG CT? GAACGAT TCGCAGTCAA .TCCTGGCCTG TTAG ??? CAT CAGAAGGCTG 960 CAGACAA? T? TTGGGACAGC TACAGCCASC CCTTCAGAC? RGGATC? S? Aß AACTTAGATC 1020 ATTATATAAT ACAGTAGC ?? CCCTCT ^ TTG TGTACATC ?? AGGATACATß TAAAAOACAC 1000 CAAGGAAGCT TTAGAGAAG? TAGAGGAAG? GCAAAACA ?? AGT ?? G ???? AGGC? CKX-A 1140 AGCAGCAGCT GCAGCTGGC? CAGGA ?? CAG CAGGCAGGTC AGCCAAAATT ACCCTATAßr 1200 GC? GAACCTA CAGGTGC ??? TGGTAC? TC? GGCCATATC? CCTAG ?? CTT TAAATGCATs' 1269 GGTAA? AGT? GTAG? AGAA? AGGCTTTCAß CCCAß ?? GTA ATACCCATGT- rTTCAGCA-TT 1320 ATCAGAAGG? GCCACCCCAC A? G? TTT ??? C? CCATGCT? A? C? C? ßTGG GGG? CA-R? 1380 AGCAGCC? TG CAAATGTTAA AAGAGACTAT C ?? XGAGGA? GCTGC? Fi? Kr GCßA-TAGAG 1440 GCATCCAGTG CA-TGCAGGGC CTATTGCACC AGGCCAAATß AGAGAACC ?? GGGGAAGTG? 1500 CATAGCAGGA ACTACTAGT? CCCTTCAGG? ACAAATAGG? TGGATG? C ?? AT ?? TCCACC 1560 TATCCCAGTA GGAGAAATCT? T? AAAGATG GATAATCCTG GG? TTA ?? T? AA? TAGTA? G 1620 AATGTATAGC CCTACCAGC? TTCTGG? C? T AAGACAAGG? CCAAAGGAAC CCTTT? GAG? 1680 TTATGTAGAC CGGTTCTAT? AAACTCT ?? G AGCCGAAC ?? GCTTCACAGG ATGT ?????? 1740 TTGGATGAC? G ??? CCTTGT TGGTCC ???? TGC ??? CCC? GATTGTAAGA CTATTTTA ?? 1800 AGCATTGOG? CCAGCAflCT? CACTAGAAG? AATGATGAC? GCATßTCAßß GAGTGGGGGG 1860 ACCCGGCCAT A ?? GCAAGAG TTTTGGCTG? AGCCATGAGC CA? GT? ACA? ATCCAGCTA? 1920 CATAAXGATG CAfiAGAGGC? ATTTTAGGA? CCAAAGAAAG ACTßTT ?? GT GTTTCA? TTG 1980 TGGC? A GA? GßßCACAZAG CC? A ??? TTG CAG6CCCCCT AGG ????? GG GCTßTTOGAG 2040 - • .TGTGGAAGG GAAGGACACC AA? TG? AAG? TTGCACTGAG AG? CAGGCT? ATTTTTTAGG 2100 GAAGATCTGG CCTTCCT? C? ? 3GGAAGGCC AGGGAATTTT CTTC? ß? GC? GACCAflAGCC 2160 A? C? SCCCC? CC? ß ?? ßAG? GCTTGAGGTT TGGGGAGGA0 A ??? C ?? CTC CCTCTCAG ?? 2220 GC? GG? GCCG AT? ß? C? GG AACTGTATCC TTT? CTTC ^ C CTCAGATCAC TCTTTGGC ?? 2280 CGACCCCTCG TCAC ?? X? AG GAT? ßßßßßß? CA? CTA? AGG AAGCTCTATT AGA AGAGG? 2340 GCAG? TGAX? CAGTATTAG? AG ??? TG ?? T TTGCC? ßG? A? TGG ??? CC A ???? TGAZ? 2400 GCGGCAATTG GAGGTTT? AT CAA? GS ?? ß? CAGTACG $ CC Afi ??? TCTßT 2460 GGAC? Z ??? G CTAT? ßGTAC AGIA-TTASTA GGACCTAC? C CTGTC ?? CAT AATTGGAAGA 2520 AATCTGTTG? CTC? G? TTßG TTGTACTTT? AATTTCCCCA TTAGTCCTAT TGAAACTGT? 2580 CCAGTAAAAT T ??? GCCAGG AATGG? TGGC CCAA? AßT? AOCAATGGCC ATTß? CAG ?? 2640 GA ???????? AACCATTAGT AGAGATATGT AC? G ??? TGG AAAACGA? GG G? AA? XTTC? 2700 A ??? TTGGGC CTGA ?? A-rCC AZAC ?? T? C. CC? ßT? TTTß CTAX ??? G ?? AAAAGACAGT 2760 ACTAAATGGA G ?? A? CTAG-. AG? TTTC? G? GAACTT ?? T? AAAG ?? CTC? AGACTTCTGß 2820 GAAGTTCAGT T? GGA? TACC ACACCCCGC? GGGTTAAAA? ? G? A ?? AATC AGTAACAGTA 2880 TTGGAZGTGG GTG? TGCAT? CTTTTCAGTT CCCTTAGAT? A? GACTTTAß AAAGTAT? CT 2940 GC? TTTACC? TACCT? GTAI A ?? G ?? TG? ß AC? CCAGGG? TTAGATATCA GTAC ?? TßTß 3000 CTGCCACAGß GATGGA ?? ßß ATC? CCAGC? ? XA-TTCC? A? ßTAGCATGAC A ??? ATCTT? 3060 GAGCCTTTT? G ??? ACAG ?? TCCAGACAI? GTTATCT? TC AAS? CATßß? TG? TTTsT? T 3120 GTAGGATCTG ACTTAGAAA? AGGGCAGCAI? G ?? CA ???? TAGAGGA? CT GAGACAGCAT 3180 CTGTTGAGGT GGGGATTTAC CACACCAGAC AAA? AACATC AG ??? ßAACC TCC? TTCCTT 3240 TGGATGGGTT ATG ?? CTCCA TCCTG? TA ?? TGGAC? ßTAC AGCCTAX ?? Z GCTGCC GA? 3300 AAAGACAGCT GGACTGTCAA TGACATAC? ß AAßTTAOTßß GAA ?? TTG ?? TTGGGCAAßT 3360 -AGATTTAXG CAGGGATTAA AGT? AAGCAG TT? TGT ??? C TCCTTAGAGQ A? CC? AAGC? 3420 CT? ACAGAAG TAATACCACT AACAG ?? G ?? GCAGAGCTAG AACTGGCAG? AA? CAGGGAß 3480 A-rrCTAAAAß AACCAGTAC? TG? ßTAT? T TATGACCCAT CAAAAG? CTT AGTAGCAGA? 3540 ATAC? G ?? ßC AGGGGC ?? Gß CC ?? TGG? C? T? TC ??? TTT ATCAAG? GCC? TTT ????? 3600 CTGAA ?? CAG G ??? GT? TGC AAGGATGAG6 ßGTGCCCAC? CTAATGATGT A? AAC? GTT? 3660 ACAGAGGC? ß TGCAAA? GT? TCC? CAG ?? AGCATAGTAA TATGßGß ??? G? LTCCT ??? 37.20 TTT ??? CTAC CCATACAAAA GG ??? C? -rsß GAAGCATGGT GGATGGAGTA TTGGC ?? GCT 3780 ACCTGGATTC CTGAGTGGG? GTTTGTC ?? T ACCCCTCCCT TAGTGA ?? TT ATGGTACCAG 3840 TTAGAGAAAG A? CCCAT? GT AGGAGCAG ?? A TTTCTAZG TAGATGGGGC AGCT ?? T? ßß 3900 GAGACTA ?? T TAGG ???? GC AGßAZA-TßTT AC? GACAGAß GAAGAC ???? GTTGTCTCC 3960 ATAGCTß? C? CAACAAATC? G ?? ßACTG ?? TTACAAGCAA '1TCATCTAGC TTTGC? Sß? T 4020 TCGGG? TTAG AAGT ??? CAT AβTAAC? BAC TCACAATATG CATTAGGAAT C? TTCA? ßC? 4080 CAACCAGATA AGAGTGAATC AGAGTTAGTC AGTC ??? T ?? TAGAGC? ßTT AATA? AA ?? G 4140 GAAAAGGTCT ACCTGGCAXO GGTACCAßC? C? CAAAGGAA TTGG ßG ??? TGAACAAGT? 4200 G? TAAATTAG TCAGTGCTGG AATCAGG ??? GTACTATTTT TG? ATGG ?? T AGATAAGGCC 4260 CAAGAAG? AC ATGAGAAATA TCACAßt? T TG? GAAGCAA TGGCTA-TTGA TTTTAACCTG '4320 CCACCTsTAß TAGC ??? AG? ? ATAGTAQCC AGCTGTGAT? A? TGTCAGCT A ?? AGGAGA? 4380 GCCATGCATS GAC ?? GTAGX CTGTAGTCCA GGAAIA-TGGC AACT? GATTG TACACATCTA 4440 G ?? GGAAAA? TTATCCTGGT AGC? STTCAT GTAOCCAGTG GATATAT? G? AGCAG ?? GTT 4500 ATTCCAGCAG AGACAGGGC? GG ?? AC? GC? TATTTTC? CT T ???? TTAGC? ßß ?? GATGß 4560 CCAGTAA ??? CAATACAZ? C AGACAATGGC AOCA? TTTC? CCAGTACTAC GGTTAAGGCC 4620 GCCTGTTGGT GGGCAGGGAT C ?? GCAGGA? ITGGCATTC CCTACAATCC CC ??? GTCA? 4680 GGAGTAGTAG AATCT? TG? TAATG ?? TT? AAGAAA? TT? TAGGAC? ßßT AAGAGATCAG 4740 GCTGAACACC TTAAGACAGC AGTAC ??? Tß GCAGTAITC? TCCACAATTT TAAAAG ???? 4800 GGGGGG? TTG GGGGATACAG TGCAGGGG ?? AG ?? TAGTAG ACAT? ATAGC AACAGACATA 4860 CAAACTAAAG AACTACAAAA GCAAATTAC? AA ?? TTCAAA ATTTTCGGGT TTATTACAGG 4920 GACA? CAAAG? TCCCCTTT? GAA? GGACC? GC ??? GCTTC TCTGG ??? GG TGAAGGGGC? 4980 GTAGT ?? TAC AAGATAATAG TGACAT ???? GTAGTGCCAA G ?? G ???? GC ???? ATC? TT 5040 AGGGATTATG GA? AACAGAT GGCAGGTß T GATTGTGTGG CAAGT-? G? C? GG? TGAGGAT 5100 TAG ?? CATGG AAAAGTTTAG TAAAACACC? TATGTAZATT TC ??? G ??? G CTA? AGGATG 5160 GTTTT? TAG? C? XCACTA? G AAAGT? CTC? TCC ?? ßAQT? AGTTCAG? AG TACACATCCC 5220 CCTAGGGGAT GCTAAATTGG T ?? T ?? CAAC A-CATTGGGGT CTGCAT? C? G O? GAAAGAG? 5280 ATGGCA-rrrß OGCCAGGGAG TCGCCAXAGA AXGGAGGAAA AAGAAAGATA GCACACAAGT 5340 AGACCCTßßC CTAGCAGACC AACTAATTCA TCTßC? TTAT TTTßASTGTT TTTCAG ?? TC 5400 TGCZATA ??? A? TTCCAT? T TAGGATAX? G AGTTAGTCCT AGGTGTGAAT ATCAAGCAGG 5460 ACATA? CAAG GTAGGATCTC TACAATACTT GGCACT- | -SC? GCAST ?? T ?? CACC ???? AA 3520 GAC-f-AAGCC? CCTTTßCCT? GTGTT ?? G ?? ACTGACAGAG GATAGAXGGA AC ?? ßCCCC? 5580 GAAGACCAAG GGCCACAGAG GGACCCATAC AATGAATGGA CACTAGAGCT TTT? GAGGAG 5640 CTTA GAGAG ?? GCTGTTAG ACAITTTCCT AG? CCAZGGC TCCAXAGCTT AßßAC ?? T? T 5700 ATCTATß? AA CTTATGGGG? TACTTßßGC? GGAGTGGAAG CCAX ?? T ?? G AATTCTGCAA 5760 CAACTGCTGT TTAITCATTT CAGA? TTGGG TGXC ?? C? T? ßCAG ?? XAGß CATTATTC ?? 5820 CAGAGGAGAG C ?? G ?? ßAAA TGGAGCCAOT AGATCCTAAI CTAGAGCCCT GGAAGC? TCC 5880 AGGAAGTCAG CCTAGGACTG CTTßTAAC ?? TTßCTATTGT AAAAAGTGTT GCTTTCATTG 5940 CTACGCGTGT TTCACAAG ?? A? GGCTTAGG CATCTCCTAT GGCAGGAAG? AGCGGAG? C? 6000 GCGACGAAG? GCTCCTCAßG AC? TFTCASAC TC? XC ?? GCT TCTCTATCAA AGCAGT ?? GT 6060 AGTAAATGTA ATGC ?? TCTT TACAAATATT AßCAAT? GT? TCArTAGTAG TAGTAGCAAT 6120 A? ZAGCA? T? GTTGTßTGG? CCATAGTACT C? TAG ?? T? T AOGA ??? T? T T ?? GAC ??? ß 6180 A? A? T? GAC? G? TT ?? TTG? TAß ?? T ?? G? G ????? GCAß AAGACAGTGß C ?? TG ??? ßT 6240 GAAGGGGACC AßaAGG? ATT ATC? ßCACTT GTGQAG? TGG GGCACCTTGC TCCTTGGGAT 6300 GTTGATGATC TGTAGTGCT? CAGAAAAATT GTGGGTCACA GTTT? TTATG GAGTACCTGT 6360 GTGGAAAGAA GCAACTACCA. CTCSATTTTG TGCAICAGAI GCTAGAGCAT? TGATACAG? 6420 GGTACATAAT GTTTGGGCCA CACA? GCCTG TGTACCCAC? GACCCCA? CC CACAAGAAGT 6480 AGTATTGGG? AATGTG? CAG A ?? ATTTT ?? CATGTGG ??? A? TAACATGG TAGAACAGAT 6540 GCAGGAGGAT ATAATCAGTT TATTTG? TC? AAGCCTAAAG CCATGTGTAA ?? TTA? CCCC 6600 ACTCTGTGTT ACTTTAAATT GCACTGAXTT GGGGAA6TCT ACT ?? T? CC? ATAGTAGT ?? 6660 TTGG ??? G ?? G? AATAAAAG GAG ??? TA ?? A ?? CTGCTCT TTCAATATCA CC? C ?? ßG? T 6720 AAGAGATAAG AZTCAGAAAG A ?? ATGC? CT TTTTCGTAAC CTTGATGTAß TACCAATAGA 6780 T ?? TGCTAGT ACTACTACC? ACTATACC ?? CTATAGGTTG ATACATTOTA ACAGATCAGT 6840 CAITACACAG GCCTGTCC ?? AGGTATCATT TG? GCC? TT CCCATACATT ATTGT? CCCC 6900 GGCTGGTTTT GCG? TTCT ?? AGTGTAAT ?? TA ??? CGTTC A? TGG ??? AG GACCATCTAC 6960 ??? TGTCATC ACAGTACAAT GTACACAXGG A? TTAGGCC? AT? GTßTC ?? CTC ?? CTGCT 7020 GTT ??? TßGC? STCTAGCAG ?? ß? AßAßGT? ST ?? TTAG? TCTGACA8T TC? CGAAC ?? 7080 TGCTAA? CC ATA? T? GTAC AGCTG ?? TG? ATCTGTAßC? ATTAACTGTA C ?? ß? CCC ?? 7140 CAAC ?? T? C? ? G ????? GT? TCTATATAGG ACCAGßG? G? GC? ZTTC? LA CAACAOGAAG 7200 AAT ?? TAGG? G? -CAT ?? ßA? AAGCACATTG TAACATTAGT AGAOCAC? AT Gß ?? lACAC 7260 TTTAG? AC? G ATAGTT ???? - ??? T ?? G? ß? AC? G pßßß AAT ?? T ???? CAATAGTCTT 7320 TAATCAATCC TCAGGAGGGß ACCCAGAAAT TGTAATGCAC AGTTTTAATT GTAG-M-ßGG? 7380 ATTTTTTCTAC TßT ?? TACA? CACAACTGTT T ?? - T ?? TAC? TGG? GGTTA? ASC? CACTß? 7440 AGGAACT ??? GG ??? TGAC? C? TCAT? CT CCCATCTTAGA ATA ??? C ??? TTAXA ?? C? T 7500 GTGGC? GG ?? GTAGG ???? ß C ?? TGTATGC CCCTCCCATT GC? CG? C ??? TTAOTTßTTC 7560TATT ACAGGGCTGC TATTAACAAG A? TGGTGGT? C ??? TTTA? CTAATGACAC 7620 CGAGGTCTTC AGACCTGGAG GAGGAGATAT GAOGOACAAT TGGAGAAGTG AATTATATAA 7680 AT? T ??? GT? ATAA ??? TTG A? CCAXTAG? AAT? GC? CCC ACCAAßßC ?? Aß? G ?? GAGT 7740 GGTGCAGAG? GA ????? GAG CAGTGOGAAT AßTAGGAGCT? TTTTCCTTG GGTTCTTGGG 7800 AGCAGCAGG? AGCACTATGß GCGCAGTGTC ATTß? CGCTG ACGGT? C? ßß CCAGACAATT 7860 ATTGTCTGGT ATAGTOCAAC AGCAG ?? CAA TTTGCTGAGß GCTATTGAGG CGC ?? C ?? C? 7920 TCTGTTGC ?? CTCAC? GTCT GGTGC? TC ?? GC? FlCTCC? G GCAAGAGTCC TGGCTGTG? 7980 ?? GAT? CCT? AGGGATCAAC AGCTCCTAGG GATTTGGGGT TGCTCTßß ?? A? CTC? TTTG 8040 CACCACTGCT GTGCCTTGß? ? XGCTASTTT. G? GT ?? T ?? A TCTCTGGAA? ACATTTGGG? 8100 T? AC? TGACC TGG? TGCAGT GGGA ?? G? G? A? TTGAC ?? T TACACA ?? C? CAATATAC? C 8160 CTTACTTG ?? GAATCGCAG? ACCAAC? Aß? AAAGAATGAA CA? GAATTAT TAfi ?? TTGG? 8220 T ?? GTGGGC? AGTTTGTGG? AZTGßTTTAG CATA? CAAAC TGGCTGTGGT ATATAAAGAT 8280 AITC? TA? TG? TAGTAGGAG GCTTGGT? G? TTTAAGAATA GTTTTT? TG? TGCTTTCTAT 8340 AGTGAATAGA GTT? ßßCAGß G? TACTCACC ATTGTCATTT CAGACCCGCC TCCCAGTOCC 8400 G-AGßGGACCC GACAGGCCCG ACGGAATCG? AGAAGAAGGT GG? G? GAG? G ACAGAGACAG 8460 ATCCGTTCG? TTAßT -pATG G? TTCTTAßC ACTTATCTGß GAAGATCTGC GG? GCCTCTß 8520 CCTCTTCAGC T? CCGCCGCT TGAG? SACTT ACTCTTG? TT GCAGCGAGGA CTOTG? AT 8580 TCTGGGGCAC AGßGGGTGGG A? GCCCTCA? AT? TTGGTGG AGTCTCCTGC AGT? TTGG? T 8640 TCAGG ?? CT? A? G ?? T? ßT? CTGTTAGCTG GCTCAACGCC ACACCTATAG C? GT ?? CTß? 8700 GßßG? C? G? T AGGGTT? TAG AAGTAGC? C? A? G? ßCTTAT AG? ßCT? TTC TCCACATACA 8760 TAfi ?? G ?? TT AGAC? ßßßCT TGG ?? GGCT TTTGCT? X ?? G? TGßßTGGC AAGTGOTCAA 1 820 ?? CGTAGTAT GßßTßßATßß TCTGCTATAA GGG ??? G ?? T G? G? CGAGCT G? GCCACGAG 8880 CTGAGCCAGC AGCAGATßßß GTGGG? GCAG TATCTCTAG? CCTGß? A ??? CATGGAGCAA 8940 TC? CAAGT? G C ?? TAC? GC? GCTACT ?? Tß CTG? TTGTGC CTGGCT? ß ?? GC? C ?? ß? ßß 9000 AGGAAGAGOT GGGTT TCC? ßTCAGACCTC AGGTACCTTT AAGACCAATß ACTTACA? ßß 9060 CAGCTTTAG? TATTAGCCAC TTTTT ????? AA ?? GGGGG? ACTGG ?? Gßß CT ?? TTTßßT 9120 CCC? AAG ?? ß ACAAGAGATC CTTGATCTGT GGATCTACCA CACACA? ßßC TACTTCCCTO 9180 ATTGGCAßA? TTACAC? CC? GGGQC? ßßß? TCAG? T? TCC ACTGACCTTT GGATGGTGCT 9240 TCAAGCTAGT ACCAGTTGAG CCAGAG ?? GG T? ß ?? G? GGC CAATGAAGG? GAG ?? C ?? C? 9300 GCTTGTTAC? CCCT? TGAGC CTGC? TGGG? TGGAGGACGC GG? ß ??? G ?? GTGZTAGTGT 9360 GGAGGTTTG? CAGCAA? CT? GC? XTTC? TC ACATGGCCCG AGAGCTGCAT CCGGAGTACT 9420 ACAAAGACTG CTGAC? TCG? TCTTTCTAC? ? GG? CTTTC CGCTGGGG? C TTTCCAGGG? 9480 GGCGTGGCCT GGGCGGG? CT ßßGGAGTGGC GTCCCTCAG? TGCTGCAT? X A? GC? ßCTGC 9540 TTTTTGCCTG TACTGGGTCT CTCTß TTAG ACC? G? TCTß AGCCTßßG? ß CTCTCTGGCT 9600 AACTAGGG ?? CCCACTGCTT A? ßCCTC ?? T ??? ßCTTGCC TTßAGTGCTT CAAGTAGTGT 9660 GTGCCCGTCT GTTGTßTGAP TCTGGTAACT AGAGAZCCCT CAGACCCTTT TAGTC? GTGT. 9720 GGAAAAATCT CTAGCAG 9737 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4527 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA, de D) TOPOLOGY, linear (0) TYPE OF MOLÉC ULA: (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:. . GAATTCTGC? AC ?? CTGCT? TTTAXTCATT TCAfi ?? TTGG GTCCCAACAT AGCAGAATAG_60_GC? TTACTCG AC? - '? GA GC ?? G ??? TG G? GCCAGTAÂ AT ATCCTAACCT A-3A0CCCTG6 120 AAGCATCCAG GAAOTCAGCC TAGG? CTGCT TÂCACACACT GCTATTCTAA AA? GTGTTGC 180 C TTTCATTGCC AAGTTTGCT CAT ?? C ?? A? GOCTTAGCCA TATCCTATGß CAGGA? OAAG 240 CGGAGGCAGC G? CAG? ßAGC TCCTßACAGC AGTCAG ?? TC ATCAAGATTC TCTATCAAAG 300 CAOTAAGT? ß TACATOT ?? T GT? ASCTTT? ACAATATTAG C? AX? ßTAGC ?? TAGTA --- T? 60 GZ? ACA? TA? TAGCAATAGT TATATGGACC AZAGTAZT ?? T ???? TATAG G? A ?? TATT? 420 AG? C ?? AG ?? A ?? TAG? CAa ATTAATTGAT AG ?? TA? G? ß A ?? ß? ßCJ S? AGACAGTQGC 480 AATGAG? GCß AGGGAGACC? GG ?? GAATT? TCAGTGCTTG TGßAG? TGGG GCACßATGCT 540 CCTTGGß? TG TT? ATGATCT sr? STGCTGC AGAAA? TTT? TGGGTCAC? S TTT? TT? TTG 600 GGTACCTGT? TGG ??? G? TG C ?? CC? CTAC TCT? TTTTGT GCATCAGATG CTA ?? ßC? T? 660 TG? ZACAG? GTACAT ?? TG rrTTTGCC? C ACATGCTTCT GTACCCACAG ACCCCAACCC '720 C: CCAAßAAGT? GTATTGGGAX ATGTß? C? G? AAATTTT? AC ATGTGGAAAA AT ?? CATGGT 780 AGACCAGATG CATGAGGAT? TAGTCAGTTT ATGGGATCAA AGCCT? AAGC CATGTGTAAA 840 ATT ?? CCCC? CTCTGTGTT? CTTT ??? TTG CACTGAÍTAT TTßßßG ?? ZG CTACTAATAC 900 CAACAATAGT AGTGGGGG ?? CGGTGGAG ?? AGAAG ??? TA AA ??? CTTCT CTTTCAATAT 960 CACCACAGGC? TAAGAGAT? AGßTACAG ?? GGC? X? TGC? T? TTTTT? T? AACTTß? TGT 1020 AGTACC ?? T? GATGATGATA ATACT ?? TAC CAGCTATAGG TTGATACATT CTAATTCCTC 1080 AGTCATTAC? CAGACCTGTC CA ?? GGT? TC CTTTGAGCC ATTCCTATAC A-TTATTGTGC 1140 CCCGGCTGGT TTTGCGATTC TAA? GTGT ?? T ?? TA? G ?? ß TTCAOTGG ?? AAGGTCAATG 1200 TACAAATGTC AGCACAGTAC A? TGTACAC? TGG ?? TTAAG CCAGTAGTGT C ?? CTC ?? CT 1260 - 6 GCTGTTA? AT GGCAGTCTAG CAG ?? GAAG? GGTAGTAATT AGATCTß? C? ? TTTCACG ?? 1320 C ?? TGCT ??? ACCATATTAG T? CAGCTGAA TGTATCTGTA GAAATTAATT GTACAAGACC 1380 CA? CAACAAT AGAAGAAGAA GGATAACTAG TGGACCAGGG AAAGTACTTT ATACAACAGG 1440 AGAAATAATA GGAGATATAA GAAAAGCAZ? TTGTAACATT AGTAGAGC ?? A? TGGAATA? 1500 AACTTTAC- ?? CAGGTAGCT? C ???? TTAAG AGAAC ?? TTT GGGAATA ??? C? TAOTATT 1560 T ??? C? TCC TCAGG? GGAG ACCC? G ??? T TGTA? TGCAC AGTTTT ?? TT GTAGAGGGG? 1620 ATTTTTTCTAC TGTAATACAA CA ?? CTGTT T ?? TAGTACT TGGAATG ??? ATAGTACTTG 1680 GAATGCTACT GGAAATGAC? CTATCACACT CCCA G? AG? AT ???? C ??? TTATAAACAT 1740 GTßßCAGG ?? GTAGG? AAAß CAATGTATGC CCC «3C? TC GAAGG? C? A? TTAGATßTTC 1800 ATCAAATATT ACAGGGCTGC TATT ?? C ?? ß AGATGGTGGT GGTGAC ?? G? ACAOTACC? C 1860 CGAGATCTTT AGACCTGCAG GAGGAAATAT GAAGGACAAT Tßß? ß ?? GTG AATTAXATAA 1920 AT? ZAAAGT? GT ????? TTG A? CCATT? GT AßTAGCACCC ACCAAGGC ?? AGAG ?? ßAGT 1980 GGTGC? AAG? G ?????? GAG CAGTGGGAGT GATAGOAGCT ATGTTCCTTß GGTTCTTGGß 2040 AGC? GCAGG? AGCACT • TGG GCGC • ßCßTC AA • AACGCTG • CßßTACAGß CCAGAAAACT 2100 ATTGTCTGßT ATAGTGC ?? C AGC? ßAAC ?? TCTGCTGAß? GCTATTGAGG CGCAACAGC? 2160 TCTGTTGCAA CTCACAGTCT GGGGCATCAA GC? GCTCC? ß GCAAGAGTCC TGßCTßTGGA 2220 AAGATACCTA AGAGASCAAC AGCTCCTAGG GATTTGGGGT TGCTCTGGAA AACTCATTTG 2280 CACC? CTACT GTGCCTTGG? ATACTAGTTß GAGT ?? T ??? TCTCT GATA AGATTTGß ?? 2340 TAAC? TGACT TGGATGGAGT GGG ??? GAG? A? TTGAC ?? T TACACAAGCT TA? T? TACAC 2400 CTTACTTG ?? GAATCGC ??? ? CCAAC ?? G? AAAßAXTßA? C? G? GTT? TGGAATTG? 2460 TAAGTGGGC? AGTTTGTGG? A? TGGTTTA? CAZ ?? C ??? C TGGCTGTGGT ATATAAGAAT 2520 ATTCATAATG AT? GTAGGAG GCTTGAZAGG TTTAAG ?? T? ? TTTTTTGCTß TGCTTTCTAT 2580 AGTAAATAG? GTTAGGCAGß GATACTCACC ATTATCATTT C? G? CCCTC? TCCCAGCCCA 2840 GAGGGGACACG G? CAGGCCCG AAGGAATCGA AG ?? GGAGGT GGAGAGAGAG ACAGAGACAß 2700 ATCCACTCG? TTAGTGAACß G? TTCTTAGC ACTGTTCTGG GACGATCTTC GGAGCCTGT6 2760 CCTCTTCAGC TACCACCGCT TGACAGACTT ACTCTTGATT GTAGCG? ßß? TTGTGG ?? CT 2820 TCTGGGACGC AGGGßßTGGG ?? GTCCTC ?? ATATTGGTGG A? TCTCCTGC TGTATTGGAG 2880 TC? GG? ACT? A? GAAT? ßTG CTGTTAGCTT GCTCAACGCC ACAGCTATAG- CAGTAGCTGA 2940 AGGG? CAGAT AGGGTTAT? G ?? ßT? ßT? C? AAGAGTGGGT AGAGCTAITC TCCAC? T? CC 3000 TACAAGAATA AGACAGGGCT TTOA ?? GGGC TTTGCTAT ?? G? XGGGTGGC A? GTGGTCA? 3060 AAAGTAAAAT GGGATGGCCT GCTGTAAGGG A? AGAATG ?? GCGAGCTßAG CCAGCAGC? S 3120 ATGGGGTGGG AGCAGCATCT AGAGACCTGG AAA ?? C? TTT AGCACTCACA AGTAGCAATA 3180 CAGCAGCTAC T ?? TGCTG? T TGTGCCTGTC TAG ?? GCAC? A-5AGGATGAG G? AaTGßßTT 3240 TTCCAGTCAA ACCTCAGGT? CCTTTA? GAC CAAZßACTT? CAA? GCACCT TTAG? TCTT? 3300 ßCCACTTTTT A ??? GA ?? AG GGGGGACTGG AAGGGCTAGT TT? CTCCC ?? AA ?? GAC? AG 3360 ATATCCTTG? TCTGTGG? TC T? CC? CAC? C AAG6CTACTT CCCT? TTGG CAGA? CTAC? 3420 CACCAGGGCC AGGGGTCAG? TTTCCACTG? CCTTTTTAT6 GTGCTTCAAß TTAOTACCAß 3480 TAGAGCCAG? G ??? GTAGA? GAGGCCAATG AAGGAGAG ?? CAACAGCTTG TTACACCCTA 3540 TGAGCCTGC? TGGGATTGAG GACCCGGAGA AAGAAGTGTT AßTGTGGAAG TTTGACATCC 3600 ACCTAGCATT TCGTCACATG GCCCGAGAGC TGCATCCGG? GTACTACAAA ß? CTGCTß? C 3660 ATCGAGTTTT CTACAAGGG? CTTTCCGCTG GGGACTTTCC AGGGßAGGCG TßGCCTGGGC 3720 GGGACTGGGG AGTGGCGAGC CCTCAG? TSSC TGCAX? TAAG CAGCTGCTTT TTGCCTGTAC 3780 GGGGTCTCTC TGGTTAGACC AGASCTGAGC CTTGGAGCTC TCTßGCTAAC T? GGß ?? CCC 3840 ACTGCTTAAG CCTCAATAAA ÜÜinX-? L? 'TU AGTGCTTCA? GTAGTGTGTG CCCßTCTGTT 3900 GTGTGACTCT GCTATCTAGA GATCCCTCAG ACCCTTTTAG TCAGTGTGG? AAATCTCT? ß 3960 CAATAT? T ?? AT? T? TCTTT GACCTTTAC? GCAT? TßßT? AT ?? CTTA ?? AATTATATGC: 4020 CTAATTGTG? AAAAAAAAAA AGA ????? G? ACTCTTCTTG CCAG? CBT? AßTCCCATß? 4080 AAGTAGCCAA TGCTGTCTC? TT? GTTAGT? AGCTAATGG? AATGTTTCC? GC? TTTCTTT 4140 CAGTGTCTAG AAAACAG? GT GTGCAATGTG CCA? GTCTTC ACTGATTTAT TTTTGTAAGC 4200 AGCAGTGTAA TAAACCC ??? GAAGCCAAA? AAGCAAATTT TT ?? AA ?? X? AAT? TTC? TT 4260 TGCTATC ?? G ATGGGTATG? CCTTTTTTACC CAAGCCTATT ACTGACA? TT CAGAAAG? CT 4320 ATGTGAAAT? GTCACTCATT TATCTTAATT GCATTTGCA6 GTACTACCAC CACTCAAGTT 4380 TTAAAATGTT TTTAAACACT CAAGTTTGC? TTCCTTTAGC TTTTAXAC ?? G ??? CC? CAT 4440 TATTTTACAT ACATATTAAT TA-rTTTCTGA CX.l "nt.AGGA AAACCCA? X? AXATAAATCT 4500 ACAAAATGAA? TAATACTCA AGA? TTC 4527 • It is noted that in relation to this date, the best method known to the applicant to carry out the present invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (18)

1. An isolated polypeptide characterized in that it inhibits replication of HIV-1 in CD8 + depleted peripheral blood lymphocytes (PBMC) which are prepared from a colored surface layer in front of normal, human, non-retrovirally infected blood samples in the inhibition assay of HIV (ISL activity) according to the sequence ID No: 2.

2. An isolated nucleic acid molecule, characterized in that it encodes a polypeptide or active fragment or derivative thereof, which inhibits the replication of HIV-1 in PBMC depleted of CD8 +, PBMC which are prepared from the colored surface layer in front of normal, non-retrovirally infected human blood samples in the HIV inhibition assay (ISL activity), and wherein the nucleic acid molecule is selected from the group of i) DNA molecules shown in SEQ ID No: 1 or the complementary sequences, ii) nucleic acid molecules that hybridize with SEQ ID No: 1 under severe conditions, iii) nucleic acid molecules that, if there is no degeneracy of the genetic code, they will be hybridized under severe conditions with one of the sequences listed in i) or ii).

3. The isolated nucleic acid molecule according to claim 2, characterized in that it is with a sequence shown in SEQ ID No: 1 or with a sequence in which codon 7 codes for threonine codon 25 codes for serine, codon 31 codes for thyronine, codon 76 codes for isoleucine codon 86 codes for alanine, codon 112 codes for threonine, codon 121 codes for proline and / or codon 128 codes for alanine and / or codon 26 is deleted.

4. The use of a nucleic acid molecule to ensure expression in a prokaryotic or eukaryotic host cell of a polypeptide according to claim 1, wherein the sequence of the nucleic acid molecule is shown in SEQ ID No: 1 or the sequence complementary

5. A recombinant expression vector characterized in that it contains a nucleic acid according to claim 2, and that it expresses the nucleic acid in a transformed microorganism or a transformed eukaryotic cell.

6. A prokaryotic or eukaryotic host cell, characterized in that it is transfected with a DNA according to claim 2.

7. Process for the recombinant production of a polypeptide that inhibits replication of HIV-1 in peripheral blood lymphocytes, characterized by the expression of a nucleic acid as claimed in claim 2 in a suitable host cell and the isolation of the protein from the guest card or the supernatant of the host card culture.

8. Process for the production of a polypeptide according to claims 1 or 7, characterized in that it comprises the steps of expressing in a mammalian cell the endogenous gene for this protein by inserting a DNA construct into the genome of the card by homologous recombination, the DNA construct comprising a DNA regulatory element capable of stimulating gene expression if it is operably linked thereto, and comprising one or more target DNA segments homologous to a region in this genome, region that is within or close to this gene; Cultivate the cells and recover the protein from the cells or the supernatant of the cell culture.

9. The use of a polypeptide according to claims 1 or 7, as an antigen or immunogen for the production of antibodies that bind to the polypeptide or for the production of a therapeutic agent for the treatment of infections or proliferative diseases, benign and malignant .

10. The use according to claim 8, for the production of a therapeutic agent for the treatment of HIV infections.

11. Therapeutic composition characterized in that it contains a polypeptide according to claim 1 or 7, if desired, together with pharmaceutical substances, auxiliaries, compatible carrier substances, fillers and / or additives, wherein the polypeptide inhibits the replication of HIV-1. in CD8 + depleted peripheral blood lymphocytes (PBMC) which are prepared from the colored surface layer in front of normal, non-retrovirally infected human blood samples in the inhibition test HIV

12. The use of a nucleic acid molecule encoding a polypeptide having ISL activity or activating the polynucleotides of the 5 'untranslated region of the ISL gene to prepare a therapeutic agent for gene therapy.

13. The use of a DNA construct that can be introduced into a genome of mammalian cells by homologous recombination, the DNA construct, characterized in that a DNA regulatory element capable of modulating the expression of a gene is operably linked to it , the gene encoding a polypeptide according to claim 1 or 7, and one or more target DNA segments homologous to a region in this genome, region that is within or close to the gene, and wherein the construct can be inserted in the genome of the mammalian cell in such a way that the regulatory element is operably linked to the gene encoding the protein, to prepare an antiviral agent or a therapeutic agent for the treatment of benign and malignant diseases.

14. Method for identifying a compound capable of activating the production of ISL in human blood lymphocytes, the method is characterized in that it comprises a) isolating PBMC from healthy blood donors by Ficoll gradient separation, isolating CD8 + cells by cell magnetic concentration , test the purity of the preparation by FACS analysis, where the preparation should have a content of approximately 95% of CD8 + cells and 5% of non-CD8 + contaminants to ensure adequate stimulation of CD8 + cells, add the substance that is to be tested for the induction of the expression of ISL activity to the cell culture in a concentration range from 1 pM to 10 mM, add IL-2 to the cell culture (180 U / ml of cell culture medium), remove the medium completely after three days and culture the cells with IL-2 (180 U / ml of cell culture medium) for three days, centrifuge the supernatant from cell culture (x 1000) to remove the cells, and filter sterilely and aliquot; b) further investigating the ISL activation activity of the compound in the HIV inhibition assay; c) find inhibition indicating that this compound is capable of activating the production of ISL in human blood lymphocytes.

15. A method for identifying a nucleic acid molecule capable of activating the production of ISL in human blood lymphocytes, the method is characterized in that it comprises a) isolating PBMCs from healthy blood donors by gradient separation. Ficoll, isolate the CD8 + cells by cellular magnetic concentration, test the purity of the preparation by FACS analysis, where the preparation should have a content of approximately 95% of CD8 + cells and 5% of non-CD8 + contaminants to ensure stimulation adequate CD8 + cells, transfect the CD8 + cells with the nucleic acid molecule that is capable of being expressed in the CD8 + cells, add IL-2 to the culture of the transfected cells (180 U / ml of the cell culture medium), remove the medium completely after three days and culture the cells with IL-2 (180 U / ml of cell culture medium) for three days, centrifuge the supernatant of the cell culture (x 1000) to remove the cells, and filter thoroughly. sterile and put in aliquots; b) further investigate the activation activity of ISL nucleic acid in the HIV inhibition assay; c) find the inhibition that indicates that nucleic acid inhibition is capable of activating the production of ISL in human blood lymphocytes after transfection of lymphocytes.

16. A nucleic acid molecule characterized in that it is capable of activating the production of ISL in human blood lymphocytes.

17. Diagnostic determination of ISL concentrations in cell preparations, in serum and other bodily fluids as well as the number of CD8 + lymphocytes that produce ISL, for example, for the detection of acute or chronic infections (for example, in blood donors) or for the inspection of the course of (retro) viral infections ) (for example, with AIDS), characterized in that antibodies that are provided with a tag or label or with a labeled anti-antibody are reacted, contacted with ISL or cells that produce ISL, antigen complexes are separated / antibody in a known manner and its concentration is determined via the label or tag.

18. The use of an antibody, which binds immunologically to a polypeptide according to claim 1, which can be obtained by immunizing an animal with a polypeptide according to one of claims 1 or 7, and by isolating the antibodies from the serum or cells of the vessel of immunized animals, for the determination of the ratio of CD8 + and / or CD4 + cells activated / not activated in body fluids, especially blood, serum or plasma.