MXPA00010618A - Feline cd80, feline cd86, feline cd28, and feline ctla-4 nucleic acid and polypeptides - Google Patents

Abstract

The present invention provides isolated and purified DNA encoding feline CD80 (B7-1) ligand, feline CD86 (B7-2) ligand, feline CD28 receptor, or feline CTLA-4 (CD152) receptor, as well as vectors comprising nucleic acid encoding feline CD80, feline CD86, feline CD28, or feline CTLA-4. The present invention provides host cells transformed with CD80-encoding vectors, CD86-encoding vectors, CD28-encoding vectors, or CTLA-4-encoding vectors. The invention provides polypeptides encoded by the nucleic acid of feline CD80, feline CD86, feline CD28, or feline CTLA-4. The present invention provides a vaccine comprising an effective amount of polypeptides encoded by the nucleic acid of feline CD80, feline CD86, feline CD28, or feline CTLA-4. The present invention also provides vaccines which further comprise immunogens derived from pathogens. The invention provides for vaccines capable of enhancing an immune response. The invention also provides for vaccines capable of suppressing an immune response.

Description

NUCLEIC ACID AND FELINE CD80 POLYPEPTIDES, FELINE CD86, FELINE CD28 AND FELINE CTLA-4 Background of the Invention There are currently no effective vaccines for the prevention of feline immunodeficiency disease and feline infectious peritonitis disease in cats. Normal vaccines against feline leukemia virus are available, but their level of efficiency remains in doubt and in some cases can cause the disease. Therefore, there is a need in the art for agents and compositions that provide protection against this and other diseases, for which a vaccine does not yet exist, or which provide the efficiency of existing and commonly used vaccines. In addition, pet vaccination is difficult due to the inability to overcome maternal antibodies in such pets. Safe and effective agents are also needed to help overcome these barriers. It is considered that the stimulation of the activation and proliferation of the T-cell in response to the disease in the host, depend on two interactions: the recognition of the T-cell receptor (TCR) with immunogenic peptides within the context of the MCH molecules. class I, and_ the secondary interaction of accessory binders, such as CD80 and CD86 with their coreceptors, CD-28 and / or CTL-4 in the T-cell. The efficient interaction of these two trajectories leads to the activation and proliferation of both CD4 + and CD8 + T cells, and the increased production of Th1 and Th2 type immune regulation cytokines. In the absence of T cell co-stimulation, an anergic state can develop where the -T-cell fails to proliferate and secrete cytokines. Over time, 2 molecules have emerged as key regulators of the responses of the T-cell, CD28 and its ligands, CD80 and CD86. CD28 is the primary T-cell co-stimulatory receptor and at the time of interaction with CD80 and CD86, it increases T-cell proliferation and cytokine synthesis, preventing the death of the T-cell. CTL-4 (also called CD152), a homologue of CD-28, also plays an important role in costimulation. Although it is not completely understood because it appears to inhibit the co-stimulatory responses of the T-cell. The interaction and interplay between CD28, CTL-4 and its ligands CD80 and CD86 in co-stimulant processes, are key to the general induction and suppression of immune responses to the disease in the host. Manipulating the expression, of these 4 costimulatory molecules, it may be possible to regulate T-cell responses through augmentation, suppression or redirection, to elevate a desired immune response towards a particular pathogen or disease condition. In particular, they can be useful for vaccination against infectious diseases, treatment of infectious diseases and treatment of neoplastic, degenerative, autoimmune and immunodeficiency conditions.

The T lymphocytes of the mammalian immune system show both control and affection functions. The progenitors of the T-cell arise in the bone marrow from stem cells and migrate to the thymus. In the thymus, maturation and selection takes place to produce a naive population of immune cells, which have the capacity to recognize the antigen in the context of the main presentation of the histocompatibility of the complex (MHC), although it is not self-reactive. After thymic maturation, each T-cell has a clonally distributed T-cell receptor (TCR), which determines its antigenic specificity. In addition, CD4 + and CD8 + T cells, the two major subgroups found in most adult mammals, possess a TCR composed of subunits a and β (Allison and Lanier, 1987). The protein and gene organization of the TCR protein is similar to that observed with immunoglobulin (Ig) molecules, and shares many properties similar to the bound Ig membrane in B cells (Allison and Lanier, 1987). Like the Ig molecules, the TCR must potentially recognize a large number of potential antigen sequences. For this reason, the organization and redistribution of the TCR gene is similar in complexity to that observed in B cells (Davis and Bjorkman, 1988). As with the antibody molecules produced by B cells, the generation of ideotypic diversity in T cells comprises multiple copies of variable genes (V) in the germ line, random redistributions of subunits a and β and variability generated by the cases of union and insertion (David and Bjorkman, 1988). However, unlike B-cells, T-cells do not seem to generate diversity through somatic mutation, since the potential repertoire of the TCR seems to be as large as that of the Ig molecule (Lechler et al., 1990) . The TCR, although it is responsible for antigen recognition, does not have signal supply capabilities (Allison and Lanier, 1987). The conformational changes in the TCR, after binding to the antigen presented in the context of MHC in the cell-presenting antigen (APC), results in a signal supply, through a non-covalently associated complex, of the surface molecules including CD3, and the chains- ?, (Clevers and associates, 1988). The TCR binding results in the phosphorylation of the CD3 complex, which indirectly leads to a Ca + influx in the cell, initiating the production of IL-2 and IL-2R (Weiss and Littman, 1994). This cascade is considered an initial event in the activation of the T-cell. The TCR recognizes the antigen, only when presented in association with the MHC. There are two subgroups of HCM proteins associated with the antigen presentation of the -T-cell. The MHC class I, is found in almost all nucleated cells within the body, and functions to express on the surface the endogenously produced peptides (Matasumura et al., 1992). The peptide expressed within the context of MHC class I is recognized by T cells expressing CD8 in association with TCR (Littman, 1987). The CD8 + T cells work in immuno-surveillance to remove virally infected cells and malignancies. The recognition of non-independent molecules through the T CD8 + cell (altered independent peptides or peptides that may indicate a malignancy), results in the destruction of the cell mediated by cytotoxic -T-lymphocytes (Berke, 1994). MHC class I molecules, the second major subset of histocompatibility, are usually found only in professional antigens that present cells including, B-cells, macrophages / monocytes and dendritic cells, although induction is possible in some other cell populations in response to stimuli specific (Germain, 1993). The MHC class II molecule is responsible for the presentation of the exogenous antigen for the CD4 + T cell. The antigen that is pagocitozado, endocitozado or link Ig to the surface and absorbed by the cells that present the antigen, is processed endogenously and bound to MHC class I I (Unanue, 1987). Subsequently, the molecule is expressed on the surface and is available for recognition by T-cells that express CD4 (Littman, 1987). Antigen recognition by means of CD4 + T cells, results in the production of cytokines and growth factors necessary for the initiation and promulgation of many facets of an immunoactive response (Mosmann and Coffman, 1987). CD4 and CD8, differentiate the T-cell subgroups and define the functional properties of each group. The presentation of CD4 or CD8 in a T cell is mutually exclusive (Littman, 1987). Therefore, after thymic selection and maturation, α-T cells exhibit only CD4 or CD8. The molecules act to stabilize the interaction between TCR and the MHC-binding antigen, and determine whether the T-cell recognizes the antigen presented within the context of MHC class I or class I I (Littman, 1987). The binding field of the CD4 or CD8 molecule recognizes the respective non-polymorphic regions of the class I class I molecule (Clayberger and associates, 1994). The binding of CD4 or CD8 to these specific regions acts to stabilize the interaction of MHC-binding / TCR antigen, for the initiation of T-cell activation (Littman, 1987). Therefore, CD4 + T cells interact only functionally with the antigen that expresses APC within the context of class II, and initiate a T-helper response while CD8 + T cells only recognize the antigen presented within the context of class I, and when they are linked, they initiate a cytotoxic response (Germain, 1993). The two different phenotypes of T-cells and auxiliary CTL can be differentiated by the expression of the surface of either CD4 or CD8. The majority of T-lymphocytes containing CD4 are generally considered auxiliary cells, although there is a proposed CD4 + CTL subgroup (Yasukawa and associates, 1989). The CD4 helper T cells are important regulators of the immune response through the production of a battery of stimulating and suppressive cytokines (Mosmann and Coffman, 1987). The factors produced by these cells are important mediators in the initiation of both a response mediated by humor or by antibody and a delayed or cellular type hypersensitivity response (DTH) (Mosmann and Coffman, 1987). For T-CD4 + cells to activate and produce soluble growth factors, a complex cascade and events must occur. The antigen is detected and endocytosed by a professional APC, usually a macrophage (Unanue, 1984). The APC denatures the protein and breaks it into smaller fragments, subsequently fragments of peptides of between 15 and 18 amino acid residues are linked to the MHC in the endoplasmic reticulum and subsequently transferred to the surface for expression (Rotzschke and associates, 1994). Therefore, the antigen expressed on the surface is visible for T-lymphocytes and can be recognized by subgroups of T-cells with the appropriate TCR ideotype, and express CD4 (Germain, 1993). When the T-cell recognizes the appropriate antigen, and the appropriate accessory signals are supplied, the naive lymphocyte differentiation occurs and clonal expansion can proceed. Thus, the stimulus not yet determined results in the preferential development of a type I (cellular) response against a type I I (humoral) response (Mosmann and Coffman, 1989). It requires the help of the T-cell, for much of the activity of both a humoral and cellular response. A T-cell dependent on a B-cell response, which is required for the antibody to be elaborated for most antigens, requires the help of the T-cell for proper maturation of the B-cell. (Chesnut and associates, 1986). Once the surface expressed Ig in the B cell that has the binding antigen, the internalization, processing and expression on the MHC class I I surface of these antigens occurs (Germain, 1993). The direct contact, cell by cell, between the CD4 + T cell with the appropriate TCR ideotype and the B-cell, promotes the activation and proliferation of the -T-cell (Chesnut et al., 1986). The activated activated -T-cell may have the capacity to promote a type I I response, secreting factors necessary for the growth and differentiation of the B-cell (Mosmann and Coffman, 1989). These factors include, IL-4, IL-5 and IL-13, which induce the activation and proliferation of B-cell and are important in isotypic switching for the Ig molecule, while IL-10 acts to prevent initiation. of a type I response, which, in turn, would regulate upward humoral activity (Mosmann and Coffman, 1989). Cellular responses (type I) do not mature in the same way as humoral responses (type I I), (Sher and associates, 1992). At the time of activation and maturation of the T-cell to a type I response, factors are produced through the T cell that favor cellular immunity. IL-2, is a T-cell growth factor that also promotes CTL responses, whereas IFN? it acts to activate macrophages, CTL and neutrophils (Wang and associates, 1996). Therefore, T helper cells have the ability to mediate, in large part, two mutually exclusive responses. The pattern of cytokine secretion that leads to the initiation of a humoral response contains factors that are suppressors of a cellular response and vice versa (Mosmann and Coffman, 1989). It is not clear what determines whether a T-cell will produce a type I pattern (IL-2, IFN-α and lymphotoxins) or a type II pattern (IL-4, 5, 6, 10, and 13), although it is proposed that the type of APC that presents the antigen or soluble factors produced by the APC, can influence the type of the cytokine pattern that develops (Mosmann and Coffman, 1989). In addition to the auxiliary cells type I and type I I, there are subgroups auxiliary T type 0, in which the secretion patterns are intermediate between type I and type I I (Gajewski and associates, 1989). Although the auxiliary subgroups T have been demonstrated mainly in in vitro experiments and can be culture artefacts, they are important models for the performance of the role of the auxiliary cell T in modulating the development of specific responses in an in vivo environment. In addition to the subset of the T CD4 + helper lymphocyte, a second population of aβ T cell consists of CD8 + that has cytotoxic lymphocytes. CD8 + CTL appears as an important component of the immune surveillance system, whose main function is to destroy virally and intracellularly infected cells in a bacterial form, as well as malignancies (Berke, 1994). These cells also have the capacity to produce cytokines, but generally only those associated with induction of cellular responses (IL-2, IFNα and TNF) (Fong and Mosmann, 1990). The TCR of these cells, in association with CD8, recognize the antigen presented within the context of MHC class I (Littman, 1987). In general, all nucleated cells have expression on the surface of class I, which has endogenously synthesized peptides (Matasumara, 1992). Specific, immunoprivileged sites of the brain and testes have an expression of the lowest level protein, although it is inducible in these areas with interferon exposure (Moffett and Paden, 1994). Proteins produced in the endoplasmic reticulum through normal cell metabolism are denatured, partially degraded and bound to MHC class I for surface expression (Engelhard, 1994). Polypeptides are linearized in proteolytic form and bound in epitopes of 9 to 12 amino acids to class I, which is subsequently expressed on the surface of the cell (Engelhard, 1994). Theoretically, all endogenously produced peptides are expressed on the surface in this way, and thymic selection, ideally results in the elimination of all autoreactive T-cells, immunosurveillance can detect the presence of infected or transformed cells. virally (Berke, 1993). The recognition of external peptides expressed by class I is facilitated by antigen-specific T-cell receptors in CD8 + CTL (Lechler et al., 1990). The contact between the performing cell and the target is required for activation to proceed (Berke, 1994). When an antigen that is observed as external is detected by it, the interaction between the molecules is stabilized by CD8, binding to class I in the infected cell (Littman, 1987). Once the recognition occurs and the T-cell is activated, it forms a conjugate between the target cell and the performing T cell, and the performing cell is sent (Taylor and Cohen), 1992). Therefore, if in this way, the own proteins are altered or if the cellular machinery is picked up by a pathogen, the peptides will be available for recognition by immunosupervision and this arm of the immune system can eliminate the diseased cell (Berke, 1994). Cellular cytotoxicity seems to result from one of two main trajectories. Any of the cells is induced to undergo apoptotic death, or is lysed through the release of cytotoxic granules by CTL (Berke, 1993). Apoptosis is induced in the target cells through the release of factors, through the CTL that induces the expression of the gene, which results in cell death (Russel, 1983). One advantage of this mechanism is that cell lysis does not occur and that the potential for the release of the potentially infectious contents of the cell is reduced (Nagata and Golstein, 1995). However, cell lysis may be the most common mechanism through which target removal takes place. Perforin, which acts to perforate target cell membranes is an important constituent of CTL cytotoxic granules (Liu and associates, 1995). Although there are other types of cells involved in this form of immunosurveillance, CTL seems to be an important component of immunity. anti-viral and anti-tumor and against specific pathogens, which are considered indispensable for protection (Kupfer and Singer, 1989). Initially, cell surface proteins were used to differentiate specific cell populations. At present, functional aspects of many of these molecules have been derived, and although they are still important in the delineation of cell populations, their important role in the function of many cells becomes more evident. In T-cells and cells that present antigen, a variety of accessory and adhesion molecules that play an important role in the development of an immunoproductive response is expressed (Van Seventer and associates, 1991). Adhesion molecules are expressed at some level in most cells of the immune system. These are important in the retention of cells within an area, and in the initiation and maintenance of cell-to-cell contact (Mescher, 1992). CD-2 / LFA 3 (CD58) and LFA-1 / ICAM-1, are two molecular adhesion complexes, involved in the stabilization of T-cell / APC interactions, and in increased activity (Springer and associates, 1987) . CD2 is one of the first markers expressed in pre-T-cells, and it persists throughout the life of the cell, that LFA-1 is subsequently expressed in T-cells and is regulated in memory cells or by induction ( Springer and associates, 1987) Accessory molecular complexes also demonstrate adhesion properties, but their main function is probably the supply of an intracellular signal when binding the binder (Anderson and associates, 1998). In the establishment of an interaction between the receptor and its ligand, a conformational change takes place in the structures of the molecules, which results in the supply of a signal to the cytoplasm of one or both cells (Hutchcroft and Bierer, 1994) . The signals supplied by these molecules play a variety of roles in promoting the development of the T-cell, but in the absence of signals mediated by these molecules, the T-cells can become anergic (Leung and Linsley, 1994). The CD28 / CD80 interaction is a major component of an immune response mediated by the productive T-cell (Linsley and associates, 1993a). The interaction of the accessory molecule CD28 with its CD80 ligand is required for the complete activation and proliferation of naive T cells (Linsley and associates, 1991 a). The interaction also seems to play an important role in the proliferation of activated -T-cell and CD4 + memory and in the prevention of apoptotic cell death (Linsley et al., 1991 a). The discovery of the interaction and elucidation of these mechanisms has provided an important link in the understanding of T cell-mediated immunity.

Summary of the Invention The present invention provides an isolated and purified DNA encoding a feline CD80 (B7-1) ligand, feline CD86 (B7-2) ligand, feline CD28 receptor or feline CTLA-4 receptor (CD152), as well as vectors comprising feline CD80 encoding nucleic acid, feline CD86, feline CD28 or feline CTLA-4. The present invention provides a host cell transformed with CD80 encoding vectors, CD86 encoding vectors, CD28 encoding vectors, or CTLA-4 encoding vectors. The present invention provides polypeptides encoded by feline CD80 nucleic acid, feline CD86, feline CD28 or feline CTLA-4. The present invention provides a vaccine comprising an effective amount of polypeptides encoded by the feline CD80 nucleic acid, feline CD86, feline CD28 or feline CTLA-4. The present invention also provides vaccines that additionally comprise immunogens derived from pathogens. The present invention provides vaccines that have the ability to improve an immune response. The present invention also provides vaccines that have suppressive capacity and immune response.

Brief Description of the Figures Figure 1 A: DNA and amino acid sequence of feline CD80 (B7-1) (TAMU). (SEQ ID NO: 1 and 2) Figure 1 B Hydrophobicity trace of the amino acid sequence of feline CD80 (B7-1) (TAMU). Figure 2A: DNA and feline CD80 amino acid sequence (B7-1) (SYNTRO). (SEQ ID NO: 3 and 4). Figure 2B: Feline CD80 amino acid sequence hydrophobicity tracing (B7-1) (SYNTRO).

Figure 3A: DNA and amino acid sequence of feline CD86 (B7-2). (SEQ ID NO: 5 and 6). Figure 3B: Feline CD86 amino acid sequence hydrophobicity trace (B7-2). Figure 4A: DNA and feline CD28 amino acid sequence (SEQ ID NO: 7 and 8). Figure 4B: Feline CD28 amino acid sequence hydrophobicity trace. Figure 5A: DNA and feline CTLA-4 amino acid sequence (CD152). (SEQ ID NO: 9 and 10) Figure 5B: Feline CTLA-4 amino acid sequence hydrophobicity trace (CD1 2).

Detailed Description of the Invention.

The present invention comprises an isolated nucleic acid encoding a feline CD80 ligand or a soluble feline CD80 ligand. The present invention also comprises an isolated nucleic acid encoding a feline CD86 ligand or a soluble feline CD86 ligand. The present invention comprises an isolated nucleic acid encoding a feline CD28 receptor or a soluble feline CD28 receptor. The present invention comprises an isolated nucleic acid encoding a feline CTLA-4 receptor to a feline soluble CTLA-4 receptor. In one embodiment of the present invention, there is provided a nucleic acid encoding a feline CD80 ligand, which has the sequence shown in Figure 1 A, starting with methionine and terminating with threonine (SEQ ID NO: 1). In another embodiment, the present invention provides nucleic acid encoding feline CD86 ligand, which has the sequence shown in Figure 3A starting with methionine and terminating with glutamine (SEQ ID NO: 5). In one embodiment of the present invention, nucleic acid encoding a feline CD28 receptor shown in Figure 4A is provided, which has the sequence starting with methionine and terminating serine (SEQ ID NO: 7). In one embodiment, the present invention provides nucleic acid encoding a feline CTLA-4 receptor which has the sequence shown in Figure 5A beginning with methionine and ending with asparagine (SEQ ID NO: 9) In another embodiment of the present invention previously described, the nucleic acid is DNA or RNA. In another embodiment, the DNA is cDNA or genomic DNA. The present invention provides an oligonucleotide of at least 12 nucleotides which have a sequence complementary to the sequence present only in the nucleic acid encoding CD28, CD80, CD86 or CTLA-4 described above. Another embodiment of the present invention provides an oligonucleotide which has at least 15 or 16 nucleotides in length, which has a sequence complementary to a sequence present only in the nucleic acid encoding CD28, CD80, CD86 or CTLA-4 described previously. Another embodiment of the present invention described above, provides an oligonucleotide which is labeled in detectable form. In one embodiment, the detectable label comprises an isotope radio, a fluorophore or biotin. In another embodiment, the oligonucleotide is methylated selectively. The present invention provides a vector comprising nucleic acid encoding a feline CD80 ligand or a soluble feline CD80 ligand. Another embodiment of the present invention provides a plasmid vector designated PSI-B7-1 / 871 -35 (Accession number ATCC 209817). This plasmid was deposited on April 29, 1998 with the American Type Culture Collection (ATCC), University Boulevard, Manassas, Va 20108-0971, E.U.A. , under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The present invention provides a vector comprising a nucleic acid encoding a feline CD86 ligand or a soluble feline CD80 ligand. The present invention provides a plasmid vector designated PSI-B7-2 # 19-2 / 01 1298 (accession number ATCC 209821). This plasmid was deposited on April 29, 1998 with the American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va 20108-0971 E. U.A. under the Provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The present invention provides a vector comprising the nucleic acid encoding a feline CD28 receptor or a soluble feline CD28 receptor. The present invention provides, a plasmid vector designated PSI-CD28 # 7/100296 (Accession No. ATCC 209819). The plasmid was deposited on April 29, 1998, with the American Type Culture Collection (ATCC), 10801 University Boulevarcl, Manassas, Va 20108-0971, E.U.A. , under the Provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedures.

The present invention provides a vector comprising a nucleic acid encoding a feline CTLA-4 receptor or a soluble feline CTLA-4 receptor. The present invention provides a plasmid vector designated CTLA-4 # 1/09 1997 (ATCC Accession Number 209820). The plasmid was deposited on April 29, 1998, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va 20108-0971, E. U.A., under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures. The present invention provides a vector described above, which additionally comprises a promoter linked in operable form to the nucleic acid. In another embodiment, the present invention provides a host cell which comprises any of the vectors described above. In one embodiment, the host cell comprising one of the vectors described above is a eukaryotic or prokaryotic cell. In another embodiment, the host cell is E. coli. yeast, COS cells, PC12 cells, CHO cells or GH4C1 cells. The present invention provides a polypeptide encoded by the nucleic acid encoding a feline CD80 ligand or a soluble feline CD80 ligand. One embodiment of the present invention provides a polypeptide encoded by nucleic acid encoding a feline CD86 ligand or a soluble feline CD86 ligand. Another embodiment of the present invention provides a polypeptide encoded by a nucleic acid encoding a feline CD28 receptor or a soluble feline CD28 receptor. The present invention provides a polypeptide encoded by nucleic acid encoding a feline CTLA-4 receptor or a feline soluble CTLA-4 receptor. In another embodiment, the present invention provides a method for producing the polypeptides described above, by culturing a host cell which expresses the polypeptides, and recovering the polypeptides already produced. The present invention provides a vaccine comprising an effective amount of the polypeptides described above and a suitable transporter. In another embodiment, the present invention provides a vaccine wherein the effective amount of the polypeptide described above and of the suitable carrier is from about 0.01 mg to about 100 mg per dose. In another embodiment, the present invention provides a vaccine wherein the effective amount of the above-described polypeptide and suitable carrier is an amount of from about 0.25mgkKg of a feline body weight per day to about 25mg / kg of body weight. a feline / day. The present invention, further provides a vaccine described above which further comprises an immunogen derived from a pathogen. In another embodiment, the immunogen within the vaccine is derived from a feline pathogen, a rabies virus, chlamydia, Toxoplasmosis gondii, Dirofilaría immitis, a parasite or a bacterial pathogen. In another embodiment, the present invention provides a vaccine wherein the feline pathogen is a feline immunodeficiency virus (FIV), feline leukemia virus (FeLV), feline infectious peritonitis virus (FIP), feline panleukopenia virus. , feline calicivirus, feline reovirus type 3, feline rotavirus, feline coronavirus, feline syncytial virus, feline sarcoma virus, feline herpesvirus, feline Borna disease virus, or a feline parasite. The present invention also provides a method for inducing immunity in a feline, which comprises administering to the feline a dose of a vaccine that contains any of the immunogens described above. The present invention also provides a method for improving an immune response in a feline, which comprises an effective dose of a polypeptide, an immunogen and a suitable transporter. The present invention provides a method for administering the vaccine described above subcutaneously, intramuscularly, systemically, topically, or orally. A further embodiment of the present invention provides a method of suppressing an immunological response in a feline, which comprises administering to the feline an effective amount of suppression of the immunological response of a polypeptide encoding feline CTLA-4 nucleic acid. In another embodiment, the present invention provides a method for suppressing an immunological response in a feline, which comprises administering to the feline an effective amount of immunological response suppression of a soluble polypeptide encoding feline CD80, feline CD86 or CD28. of feline.

In another embodiment, the present invention provides a method for suppressing an immunological response in a feline, by administering from about 0.25 mg / kg body weight / day to about 25 mg / kg body weight / day of a polypeptide encoding feline CTLA-4 nucleic acid. In another embodiment, the present invention provides a method for suppressing an immunological response in a feline, administering from about 0.25 mg / kg of body weight / day to about 25 mg / kg of body weight / day of a polypeptide encoding feline CD80, Feline CD86, or feline CD28. The present invention also provides a method for suppressing an immune response in a feline suffering from an autoimmune disease, or that is a recipient of a tissue or organ transplant, by administering to the feline an effective amount of immune response suppression of a polypeptide that encodes a feline CTLA-4 nucleic acid. The present invention also provides a method for suppressing an immune response in a feline suffering from an immunological disease or that is a recipient of a tissue or organ transplant, by administering to the feline an effective amount of suppression of immune response of a polypeptide that it encodes feline CD80, feline CD86, or feline CD28. The present invention provides isolated and purified feline CD80 cDNA (B7-1) of about 941 nucleotides. The present invention also provides isolated and purified feline CD80 polypeptide of approximately 292 amino acids, the binding of the native membrane or mature form, which has a molecular mass of approximately 33.485 kDa, an isoelectric point of approximately 9.1, a net charge of pH 7.0 out of 10. The coexpression of CD80, with the co-stimulatory molecule of CD28, and a tumor antigen or an antigen from a pathogenic organism, have the ability to activate or improve the activation of T-lymphocytes, inducing production of immune stimulation cytokines, and to regulate the growth of other cell types. The coexpression of CD80, with the co-stimulatory molecule CTLA-4, has the ability to regulate the activation of T-lymphocytes. The present invention provides isolated and purified feline CD86 (B7-2) cDNA of about 1,176 nucleotides. The present invention also provides isolated and purified feline CD86 polypeptide of about 320 amino acids, the native membrane bond or mature form of which has a molecular mass of about 36.394 kDa, an isoelectric point of about 9.19, a net charge of pH 7.0 of 1 1 .27. The coexpression of CD86, with CD28 co-stimulatory molecules and a tumor antigen or an antigen from a pathogenic organism, has the ability to activate or increase the activation of T-lymphocytes, inducing the production of immune stimulation cytokines, and the regulation of the growth of other types of cells. The coexpression of CD86, with the co-stimulatory molecule CTLA-4, has the ability to regulate the activation of -T-lymphocytes. The feline CD80 or CD86 according to the present invention are obtained from native or recombinant sources. The feline CD80 or CD86 according to the present invention comprises the native and membrane binding form or a secreted form lacking a transmembrane field. The present invention provides an isolated and purified feline CD28 cDNA of approximately 689 nucleotides. The present invention also provides feline CD28 polypeptide isolated and purified from approximately 221 amino acids, the native or mature membrane binding form, which has a molecular mass of approximately 25,319 kDa, an isoelectric point of approximately 9.17, a net charge of pH 7.0 of 9.58. The present invention provides an isolated and purified feline CTLA-4 cDNA of about 749 nucleotides. The present invention, also provides isolated and purified feline CTLA-4 polypeptides of approximately 223 amino acids, the native or mature membrane binding form which has a molecular mass of approximately 24,381 kDa, an isoelectric point of approximately 6.34, a net charge of pH 7.0 -0.99. In another aspect, the present invention provides a method for improving a feline immune response to an immunogen, which is achieved by administering the immunogen before, after or substantially simultaneously with feline CD86 or feline CD86, with or without Feline CD28 or feline CTLA-4 in an effective amount to improve the immune response. In another aspect, the present invention provides a method for suppressing in an feline an immunological response to an immunogen, which is achieved by administering the immunogen before, after or substantially simultaneously with feline CD80 or feline CD86, with or without feline CD28 or feline CTLA-4, or with RNA or DNA anti-perception, in whole or in part, which encodes feline CD80 or feline CD86 or feline CD28 or feline CTLA-4, in an amount effective to suppress immune response. In another aspect, the present invention provides a vaccine for inducing an immunological response in felines to an immunogen, comprising the immunogen and an effective amount of feline CD80 to improve the immune response. The immunogen is derived, for example, from feline pathogens such as feline immunodeficiency virus, feline leukemia virus, feline parvovirus, feline coronavirus, feline leptovirus and the like. In another aspect, the present invention provides a vaccine for inducing an immunological response to an immunogen in felines, which is achieved by administering DNA or RNA from an immunogen and DNA or RNA from feline accessory molecules CD80, CD86, CD28, in any combination , which encode proteins or protein fragments in an amount effective to modulate the immune response. The feline CD80 protein has an amino acid sequence that is 59% and 46% identical to that of human and mouse proteins, respectively. The feline CD86 protein has a 68% and 64% amino acid sequence identical to the human and rabbit proteins, respectively. The feline CD28 protein has an amino acid sequence of 82% and 74% identical to the human and mouse proteins, respectively. The feline CTLA-4 protein has an amino acid sequence that is 88% and 78% identical to the human and mouse proteins, respectively. The CD80 or CD86 proteins of human or mouse can not functionally replace feline CD80 or CD86 proteins. Therefore, feline CD80, feline CD86, feline CD28 and feline CTLA-4 proteins are new reactants required for the regulation of feline immunity. The present invention comprises regulatory accessory molecules of the T-cell, CD80 (B7-1) or CD86 (B7-2) or CD28 or CTLA-4 (CD152) of feline species. The present invention provides isolated and purified nucleic acids encoding, in part or completely, feline CD80, feline CD86 or feline CD28 or feline CTLA-4, as well as polypeptides CD80, CD86, CD28 or CTLA-4. purified from any native or recombinant source. Feline CD80, CD86, CD28 or CTLA-4 produced in accordance with the present invention are used to improve the efficiency of feline vaccines against tumors and pathogenic organisms, and as a therapeutic for treating viral and bacterial diseases in cats. Feline CD80, CD86, CD28 or CTLA-4 produced in accordance with the present invention is also used to alleviate diseases due to immune, overactive, overactive or misdirected responses.

Nucleic acids, vectors, transformers In Figures 1 to 5, the cDNA sequences encoding feline CD80 (SEQ ID NO: 1), feline CD86 (SEQ ID NO: 5), feline CD28 ( SEQ ID NO: 7) or feline CTLA-4 (SEQ ID NO: 9), and Figures 1 to 5 show the anticipated amino acid sequences of feline CD80 (SEQ ID NO: 2 CD86 of feline) (SEQ ID NO: 6), feline CD28, (SEQ ID NO: 8), or CTLA-4 (SEQ ID NO: 10). The designation of these feline, CD80, CD86, CD28 or CTLA-4 polypeptides is based on the partial homology of the amino acid sequence with human or mouse or rabbit homologies of these polypeptides, and on the capacity of the CD80 or CD86 polypeptides to bind to the feline CD28 receptor (see below), or to CTLA-4 and to activate or stimulate or, otherwise regulate the activation of -T-lymphocytes. In addition, without wishing to be bound by theory, it is anticipated that feline CD80 or feline CD86 polypeptides also exhibit one or more of the following bioactivities: Activation of NK cells (general shredders), B-cell activation maturation stimulation cytotoxic T lymphocytes restricted by MHC, mast cell proliferation, interaction with cytokine receptors and induction of cytokines that regulate immunity. Due to the degeneracy of the genetic code (for example, multiple codons that encode certain amino acids), the DNA sequences other than those shown in Figures 1 through 5 also encode the amino acid sequences CD80, CD86, CD28 or Feline CTLA-4 shown in Figures 1 to 5. Said other DNAs, include those that contain "sequence-conservative" variations, in which, a change in one or more nucleotides in a given codon, gives as a result the non-alteration in the amino acid encoded in said position. In addition, a particular amino acid residue in a polypeptide can often be changed without altering the conformation and overall function of the native polypeptide. Said "function-conserving" variants, include but are not limited to the replacement of an amino acid with one having similar physical-chemical properties such as, for example, acidic, basic, hydrophobic, hydrophilic, aromatic and the like. (For example, the replacement of lysine with arginine, aspartate with glutamate, or glycine with alanine). In addition, the amino acid sequences are added or eliminated without destroying the bioactivity of the molecule. For example, additional amino acid sequences are added to either amino or carboxyl terminal ends to serve as purification tags, such as histidine tags, (eg, to allow purification in one step of the protein, after which are chemically or enzymatically removed), Alternatively, the additional sequences confer an additional cell surface binding site, or otherwise alter the specificity of the target cell of DC80, DC86, DC28 or feline CTLA-4 , such as with the addition of an antigen binding site for antibodies. CD80 feline or feline CD86 or feline CD28 or feline CTLA-4 DNAs, within the scope of the present invention, are those of Figures 1 to 5, variant DNAs that preserve the sequences, DNA sequences encoding variant polypeptides that retain function and combinations thereof. The present invention comprises fragments of feline CD80, CD86, CD28 or CTLA-4 that exhibit a useful degree of bioactivity, either alone or in combination with other sequences or components. As will be explained later, it is correct for an expert in the art to predictably manipulate the sequence of CD80, CD86, CD28 or CTLA-4 and establish whether a variant CD80, CD86, CD28 or feline CTLA-4 has a stability and adequate bioactivity for a given application, or variations that affect the binding activities of these molecules resulting in increased effectiveness. The feline CD80 and CD86, it will link each one with the CD28 co-receiver or with the CLTA-4 co-receiver. This can be achieved by expressing and purifying the polypeptide variant CD80, CD86, CD28 or CTLA-4 in a recombinant system, and by analyzing its T-cell stimulating activity and / or activity that promotes growth in cell culture and in animals. , subsequently developing tests in the application. The CD80 variant is tested for bioactivity by functional binding to CD28 or CTLA-4 receptors. The bioactivity of the CD86 variant is tested by functional binding to CD28 or CTLA-4 receptors. Similarly, the bioactivity of the variant CD28 or the variant CTLA-4 is tested. The present invention also comprises CD80, CD86, CD28 or feline CTLA-4 (and polypeptides) DNAs derived from other feline species including, but not limited to, domestic cats, lions, tigers, cheetahs, lynxes and the like. The CD80, CD86, CD28 or CTLA-4 feline homologues of the sequence, shown in Figures 1 to 5, are easily identified by selection of cDNA or genomic libraries to identify clones that hybridize the samples comprising all or part of the sequence of Figures 1 through 5. Alternatively, expression libraries are selected using antibodies that recognize CD80, CD86 CD28 or feline CTLA-4. Without wishing to be bound by theory, it is anticipated that the CD80 or CD86 genes from other feline species will share at least about 70% homology with the feline CD80, CD86, CD28 or CTLA-4 genes. Also within the scope of the present invention are the DNAs encoding CD80, CD86, CD28, CTLA-4 homologs, defined as polypeptides encoding DNA, which share at least about 25% amino acid identity with CD80, CD86, Feline CD28 or CTLA-4. Generally, nucleic acid manipulations according to the present invention utilize methods that are well known in the art, such as those described in, for example, Molecular Cloning, a Laboratory Manual (2nd Edition, Sambrook, Fritsch and Maniatis). , Cold Spring Harbor), or in Current Protocols in Molecular Biology (Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, NY, 1992). The present invention comprises cDNA and RNA sequences and perception and antiperception. The present invention also comprises CD80, CD86, CD28 or CTLA-4 genomic feline DNA sequences and flanking sequences, including, but not limited to, regulatory sequences. Nucleic acid sequences encoding feline CD80, CD86, CD28 or CTLA-4 polypeptide (s) are also associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, regions non-coding 5 'and 3' and similar. Transcriptional regulatory elements that are operably linked to the feline CD80, CD86, CD28, CTLA-4 cDNA sequence (s) include, but are not limited to those that have the ability to direct the expression of prokaryotic cell-derived genes , eukaryotic cells, prokaryotic cell viruses, eukaryotic cell viruses and any combination thereof. Other useful heterologous regulatory sequences are known to those skilled in the art. The nucleic acids of the present invention are modified by methods known to those skilled in the art to alter their stability, solubility, binding affinity and specificity. For example, the sequences are methylated selectively. The nucleic acid sequences of the present invention are also modified with a tag having the ability to provide a detectable signal, either directly or indirectly. Example labels include radioisotopes, fluorescent molecules, biotin and the like. The present invention, also provides vectors that include nucleic acids that encode in part or in whole, the CD80, CD86, CD28 or CTLA-4 polypeptide (s). Such vectors include, for example, plasmid vectors for expression in a variety of eukaryotic or prokaryotic hosts. Preferably, the vectors also include a promoter linked in operable form to the portion encoding the CD80, CD86, CD28 or feline CTLA-4 polypeptide. The feline CD80, CD86, CD28 or CTLA-4 polypeptide (s) is expressed using any suitable vectors and host cells, such as those explained in the present invention, or those otherwise known to those skilled in the art. .

Vectors suitable for use in the practice of the present invention include, without limitation, VEp352, pcDNAl (Invitrogen, Carlsbad, CA), pRc / CMV (Invitrogen), and pSFV1 (GIBCO / BRL, Gaithersburg, MD). A preferred vector for use in the present invention is pSFV1. Suitable host cells include E. coli, yeast, COS cells, PC 12 cells, CHO cells, GH4C1 cells, BHK-21 cells, and amphibian melanoforo cells. BHK-21 cells are a preferred host cell line for use in the practice of the present invention. Suitable vectors for the construction of naked DNA or genetic vaccinations, include without limitation objective (Promega, Madison, Wl), pSI (Promega, Madison, Wl) and pcDNA (Invitrogen, Carlsbad, CA). The nucleic acids encoding feline CD80, CD86, CD28 or CTLA-4 polypeptide (s) are also introduced into the cells through recombination events. For example, a sequence is microinjected into a cell, performing homologous recombination at the site of an endogenous gene encoding the polypeptide, an analog or pseudogene thereof, or a sequence with substantial identity for a CD80, CD86 polypeptide coding gene. , Feline CD28 or CTLA-4. Other methods based on recombination, such as non-homologous recombinations, and elimination of the endogenous gene by homologous recombination, especially in pluripotent cells, are also used.

The present invention provides a method for raising an immune response to an immunogen in a feline, which is achieved by administering the immunogen before, after or substantially simultaneously with feline CD80 or feline CD86 with or without feline CD28 or Feline CTLA-4, in an amount effective to increase the immune response. The present invention provides a method for improving a feline immunological response to an immunogen, which is achieved by administering an expression vector containing an immunogen derived from a feline pathogen and feline accessory CD80 feline or CD86 molecules, with or without feline CD28 or feline CTLA-4 in an effective amount to improve the immune response. The present invention provides a method for redirecting an immune response to an immunogen in feline, which is achieved by administering an expression vector which contains an immunogen derived from a feline pathogen, and feline CD80 accessory molecules or CD86 of feline with or without feline CD28 or feline CTLA-4 in an effective amount to improve the immune response. The present invention provides a method for suppressing in an feline, an immunological response to an immunogen, which is achieved by administering the immunogen before, after or substantially simultaneously with feline CD86 or feline CD86 with or without feline CD28. or feline CTLA-4, or with anti-perception RNA or DNA encoding feline CD80 or feline CD86 or feline CD28 or feline CTLA-4, in an amount effective to suppress the immune response. The present invention provides a vaccine for inducing a feline, an immunological response to an immunogen (s), comprising the immunogen and an effective amount of feline CD80 or feline CD86, with or without feline CD28 or CTLA- 4 feline to improve the immune response, or feline CD80 or feline CD86 with feline CTLA-4 for the suppression of the immune response. In another embodiment, the present invention provides a vaccine comprising an expression vector containing immunogen (s) genes, for pathogens and feline genes of CD80, CD86, with or without feline CD28 or feline CTLA-4 for the increase or suppression of the immune response.

CD80, CD86, CD28 or feline CTLA-4 polypeptides. The feline CD80 gene (the cDNA and amino acid sequence, which are shown in Figures 1 and 2) encodes a polypeptide of approximately 292 amino acids. The feline CD86 gene (the cDNA and amino acid sequence shown in Figure 3), encodes a polypeptide of approximately 320 amino acids. The feline CD28 gene (the cDNA and amino acid sequence shown in Figure 4) encodes a polypeptide of approximately 221 amino acids. The feline CTLA-4 gene (the cDNA and amino acid sequence shown in Figure 5) encodes a polypeptide of approximately 223 amino acids. The purification of feline CD80, CD86, CD28 or CTLA-4 from natural or recombinant sources is accomplished by methods well known in the art, including, but not limited to, ion exchange chromatography, reverse phase chromatography in C4 columns, gel filtration, isoelectric focusing, affinity chromatography and the like. In a preferred embodiment, large amounts of feline CD80, CD86, CD28 or CTLA-4 are obtained by constructing a recombinant DNA sequence, comprising the coding region for feline CD80, CD86, CD28 or feline CTLA-4 fused to the structure to a sequence encoding 6 C-terminal histidine residues in the pSFV1 replicate (GIBCO / BRL). The mRNA encoded by this plasmid is synthesized using techniques well known to those skilled in the art, and introduced into BHK-21 cells by electroporation. The cells synthesize and secrete mature glycolized feline CD80, CD86, CD28 or CTLA-4 polypeptides containing 6 C-terminal histidines. The modified feline CD80, CD86, CD28 or CTLA-4 polypeptides are purified from the floating cell by affinity chromatography, using a histidine binding resin (His-bind, Novagen, Madison, Wl). Cat feline CD86 or CD86 polypeptides isolated from any source are modified by methods known in the art. For example, feline CD80, CD86, CD28 or CTLA-4 are phosphorylated or dephosphorylated, glycosylated or deglycosylated and the like. Modifications that alter the solubility, stability and binding and affinity specificity of feline CD80, CD86, CD28 or CTLA-4 are especially useful.

CD80, CD86, CD28 or feline CTLA-4 Chimeric Molecules. The present invention comprises the production of chimeric molecules made from feline CD80, CD86, CD28 and feline CTLA-4 fragments in any combination. For example, the CTLA-4 binding site is introduced in place of the CD28 binding site, to increase the binding affinity of CD28, while maintaining the increase in the immune response. In one embodiment, the binding sites for CD80 or CD86 are exchanged in CTLA-4 and CD28, so that a CD28 binding region is replaced by a binding region of CTLA-4. The effect of the chimeric CD28 molecule with a CTLA-4 binding region is to increase the affinity of CD28 for CD80 or CD86 and increase the magnitude of the increase in the immune response. In an alternative embodiment, the chimeric molecules of CD80 and CD28 or CD86 and CD28, or fragments thereof, are a membrane bond and enhance the immunological augmentation capabilities of these molecules. In an alternative embodiment, the chimeric molecules of CD80 and CTLA-4 or CD86 and CTLA-4, or fragments thereof, are a membrane bond and enhance the immunological suppression capabilities of these molecules. In an alternative embodiment, the chimeric molecules of CD80 and CTLA-4 or CD86 and CTLA-4, or fragments thereof, are a membrane link and redirect the immune response to achieve the desired effect.

Anti-feline CD80, CD86, CD28 or CTLA-4 antibodies. The present invention comprises, as described above, antibodies that are specific for feline CD80, CD86, CD28 or feline CTLA-4 polypeptides. The antibodies are polyclonal or monoclonal and differentiate CD80, CD86, CD28 or feline CTLA-4 from different species, identify functional fields and the like. Said antibodies are conveniently made, using the methods and compositions described by Harlow and Lane, in Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, 1988, as well as immunological and hybridoma technologies known to those skilled in the art. When native or synthetic CD80, CD86, CD28 or CTLA-4 derived peptides are used to induce a feline CD80, CD86, CD28 or feline CTLA-4 immune response, the peptides are conveniently coupled to a suitable carrier such as KLH, and are administered in a suitable adjuvant such as Freund's. Preferably, the selected peptides are coupled, substantially in accordance with the methods of Tan (1988) Proc. Nati Acad. Sci. USA, 85: 5409-5413, to a lysine core transporter. The resulting antibodies, especially the internal image generation anti-idiotypic antibodies, are also prepared using known methods. In one embodiment, the purified feline CD80, CD86, CD28 or CTLA-4 is used to immunize mice, after which their spleens are removed, and the splenocytes are used to form cell hybrids with myeloma cell, to obtain clones of antibody secreting cells according to standard techniques in the art. The resulting monoclonal antibodies, secreted by said cells, are selected using in vitro assays for the following activities: binding to CD80, CD86, CD28 or feline CTLA-4, inhibition of the binding activity of the CD80 receptor, CD86, CD28 or CTLA -4, and inhibition of the stimulating activity of the T-cell of CD80, CD86, CD28 or CTLA-4. Anti-feline CD80, anti-feline CD86, anti-feline CD28 or anti-feline CTLA-4 antibodies are used to identify and quantitate feline CD80, CD86, CD28 or CTLA-4, using such immunoassays as ELISA, RIA, and the like. Anti-feline CD80 antibodies, anti-feline CD28 anti-feline CD86 antibodies, or anti-feline CTLA-4 antibodies are also used for feline CD80 feline CD86 or feline CD86 or feline CD28 immunoassays. CTLA-4 feline. further, these antibodies can be used to identify, isolate and purify CD80, CD86, CD28 or CTLA-4 from different sources, to perform studies of subcellular localization and histochemistry. APPLICATIONS The feline CD80 (B7-1) ligand, feline CD86 (B7-2) ligand, feline CD28 receptor or feline CTAL-4 receptor (CD152), produced in accordance with the present invention, can be used in beneficial form as a vaccine to prevent infectious diseases, or to promote growth in feline homologous or heterologous species. For example, the coexpression of CD80 or CD86 with co-stimulatory molecules CD28 or CTLA-4 in any combination, and a tumor antigen or antigens from a pathogenic organism. The co-expression of feline CD80 or CD86 with a feline CTLA-4 receptor has the ability to inhibit the activation of T-lymphocytes and suppress an immune response. A specific example would be to co-express CD80 or CD86 with FIV, FeLV, or FIP derived from immunogens in a viral vector or a DNA expression vector, which, when administered in the form of an activation vaccine, could improve or regulate the production of CD4 + and CD8 + lymphocytes and could induce immune regulatory cytokines such as IL-2, IFN- ?, IL-12, TNF-α, IL-6 and the like. Another specific example would be to express CD80, CD86, CD28 or CTLA-4 in a viral vector or in a DNA expression vector, which when administered in the form of a therapeutic, could regulate or redirect the immune response. The increase of immunity through the interaction of feline CD80 or CD86 with CD28 or CTLA-4 or, inhibition of a response, through the interaction of feline CD80 or CD86 with CTLA-4, has the advantage of the natural process of regulation instead of adding extraneous substances that could have multiple harmful effects even in general health or, in the long term. The CD80, CD86, CD28 and / or CTLA-4 molecules are administered with other recombinant molecules, such as those encoding antigens that are desirable for the induction of immunity. The feline CD80, CD86, CD28 and / or CTLA-4 gene is inserted into an expression vector and infected or transfected into a target cell and expresses the gene product within the target cell, so that it is anchored in the cell. plasma membrane of the target cell or the cell presenting the antigen, or secreted outside the target cell or cell presenting the antigen. An expression vector, such as a plasmid, Semliki Forest virus, a poxvirus or a herpesvirus, transfers the gene to the cell presenting the antigen. The CD80, CD86, CD28 and / or feline CTLA-4 gene or gene fragments in any combination, is inserted into a DNA or RNA expression vector and injected into a feline, and expresses the gene product in the feline, in the form of "naked" DNA / RNA or genetic vaccine. The coexpression of the immunogen and CD80, CD86, CD28 and / or CTLA-4 within a target or feline cell, contributes to the activation, enhanced activation or regulation of T lymphocytes, B lymphocytes and other cells. Alternatively, the expressed protein could be administered after expression in a plasmid. The feline CD80, CD86, CD28 or CTLA-4 proteins normally function anchored in the cell membrane as accessory molecules of the plasma membrane, but can be presented in other forms, particularly without membrane anchors. In one embodiment, feline CD80 and feline CD86 are soluble, lack a transmembrane field or hydrophobic region, and interact with co-stimulatory molecules CD28 or CTLA-4, either in the form of membrane binding or soluble. In an alternative embodiment, feline CD86 or feline CD86 are bound to the membrane and co-stimulatory molecules CD28 or CTLA-4 are in soluble form, lacking a transmembrane field or hydrophobic region. Soluble CD28 or CTLA-4, preferably in a dimeric form, is useful for treating diseases related to immunosuppression mediated by the T-cell in cats. The soluble CD28 or CTLA-4, avoids the rejection of transplanted tissue, and can be used to treat autoimmune diseases. Specifically, soluble CD28 or CTLA-4 is useful for preventing grafts against host diseases in a bone marrow transplant. CD28 or soluble CTLA-4 prevents the binding of a cell containing CD80 or CD86 of membrane binding feline. Sequence-preserving and function-conserving variants of feline CD80, CD86, CD28 or CTLA-4 polypeptides, or a CD80, CD86, CD28 or feline CTLA-4 fragment or subfragment are fused in structure for another sequence. , such as, a cytokine, interleukin, interferon, colony stimulation factor, antigen from a pathogenic microorganism, antibody or purification sequence, such as a his-tag or a reporter gene, such as E. coli LacZ, E. coli uidA, or green fluorescent protein.

Vaccines The present invention comprises methods and composition for improving the efficiency of an immune response in feline species. In this modality, the feline CD80, CD86, CD28 or CTLA-4 are used in conjunction with an immunogen, for which it is desired to tender a immunological response. For example, in feline vaccines containing immunogens from pathogens, such as feline immunodeficiency virus and feline leukemia virus, and other pathogens such as feline parvovirus, feline leptovirus, and feline coronavirus, it is desired to include CD80, CD86, CD28 or feline CTLA-4 in the vaccine to regulate the magnitude and quality of the immune response. For this purpose, the purified feline CD80, CD86, CD28 or CTLA-4 from native or recombinant sources, as described above, is included in the vaccine formulation at a concentration within the range of from about 0.1 to 100.0 mg per vaccine per cat. Commercial sources of feline vaccines are known to those skilled in the art (Compendium of Veterinary Pharmaceuticals, 1997) (Compendium of Veterinary Pharmaceuticals, 1997) and are used in combination with the present invention for a more effective vaccine. A vaccine for inducing and regulating in an feline an immunological response to an immunogen, is comprised in an immunogen and an effective amount of feline CD80 or feline CD86 with or without feline CD28 or feline CTLA-4 for increased feline immune response, or feline CD80 or feline CD86 with feline CTLA-4, for the suppression of the immune response. The immunogen is selected from the group comprising, but is not limited to, feline pathogens such as feline immunodeficiency virus, feline leukemia virus, feline infectious peritonitis virus, feline panleukopenia virus (parvo), feline calicivirus , feline reovirus type 3, feline rotavirus, feline coronavirus (infectious peritonitis), rabies virus, feline syncytial virus, feline sarcoma virus, feline herpesvirus (rhinotracheitis virus), Borna de feli disease virus. or, chlamydia, toxoplasmosis gondii, feline parasites, Dirofilaria immitis, parasites, bacterial pathogens, and the like. Growth regulation or regulation of the activation of a cell type, such as a T-lymphocyte, indicates that the regulatory response either stimulates or suppresses cell growth. The regulation of an immune response in a feline, indicates that the immune response is either stimulated or suppressed, to treat the disease or infectious agent in the feline.

Expression of feline CD80, CD86, CD28 or CTLA -4, alone or in any combination, in part or in whole, in an expression vector, contains a gene (s) for feline immunogens, for the purpose of administration in the form of a genetic vaccine or "naked" DNA vaccine. Vectors include but are not limited to: target (Promega, Madison, Wl), pcDNA (Invitrogen, Carlsbad, CA). (Donnelly JJ, and associates, 1997, Hassett and Whitton, 1996). The genes or fragments of the genes for feline CD80, CD86, CD28 or CTLA-4, alone or in any combination, in part or in whole, can be inserted or transfected into the chromosomes of a feline or other mammal. Said integration of these genes or fragments of these genes can be achieved with a retroviral vector or can be used as a form of gene therapy. The present invention provides methods and compositions for improving disease resistance of feline species, for medical and / or commercial purposes. In this modality, the CD80, CD86, CD28 or feline CTLA-4, expressed alone or in any combination, in part or in its entirety, and in combination with or without feline immunogens gene coding, is administered to felines, using an appropriate form of administration. For the promotion of growth or resistance to the disease, feline CD80, CD86, CD28 or CTLA-4, expressed alone or in any combination, is administered in a formulation, in a concentration within the range of from about 0.1 to 100.0 mg per vaccine per cat, in amounts preferably in one formulation with a concentration within the range of from about 0.25 mg / kg / day to about 25 mg / kg / day. It is understood that the required amount of feline CD80, CD86, CD28 or CTLA-4 can be determined by routine experimentation well known in the art, such as establishing a matrix of dosages and frequencies and comparing a group of units or subjects experimental for each point in the matrix. According to the present invention, native or recombinant feline CD80, CD86, CD28 or CTLA-4 is formulated with a physiologically acceptable carrier, such as, for example, phosphate-buffered saline or deionized water. The formulation may also contain excipients, such as lubricant (s), plasticizer (s), absorption enhancer (s), bactericide (s), and the like which are well known in the art. The feline CD80, CD86, CD28 or CTLA-4 polypeptide of the present invention is administered by any effective means, including without limitation, intravenous, subcutaneous, intramuscular, transmuscular, topical or oral routes. For example, for subcutaneous administration, the dosage form consists of CD80, CD86, CD28 or feline CTLA-4 in sterile physiological saline. For oral or respiratory administration the CD80, CD86, CD28 or feline CTLA-4, with or without excipients, is micro or macro encapsulated, for example, liposomes and microspheres. Dermal patches (or other slow-release dosage forms) can also be used.

EXAMPLES Example 1 A Cloning of cDNA CD80 (B7-1) -TAMU, CD86 (B7-2), CD28, and feline CTLA-4: cDNA CD80 (B7-1), CD86 (B7-2), CD28 were cloned , and feline CTLA-4, through the first RT-PCR (reverse transcriptase / polymerase chain reaction), amplifying a region between two sequences that were conserved enough to degenerate the primaries that interacted with the mRNA. The source of mRNA was peripheral blood mononuclear cells (PBMC), stimulated for at least 16 hours by Con A. This PCR product was sequenced. The sequence was used to make primaries for RACE (Rapid Amplification of End of cDNA) PCR. The 5 'end was amplified, first making the cDNA with a downstream primary complementary to the recently sequenced conserved region. An oligonucleotide was ligated to the 3 'end of the cDNA (complement with the 5' end of mRNA). This sequence served as the binding site for the ascending primary, which was PCR compatible with the primary descending PCR, which corresponded with the other region in the recently sequenced region. The degenerate primaries were used in multiple turns of nested reactions to obtain the 3 'end. This ascending primary for PCR was designated to react with a sequence in the recently sequenced region. The products were either directly sequenced or cloned into a TA cloning vector and sequenced from the plasmid. The complete open reading structure was cloned by amplifying it in its entirety by PCR with primaries constructed from the known sequences. The ORFs were cloned and sequenced three times. The B7-1 ORF was subcloned into a pSI plasmid with an SV40 promoter, and the SFV plasmid. The pSI was used to establish the functional interaction of B7-1 with feline CD28. The DNA primaries used for the RT / PCR of the feline CD80 (B7-1) cDNA were: Primary 5 ': 5'-CGCGGATCCGCACCATGGGTCACGCAGCAAAGTGGAAAAC-3'; (SEQ ID NO: 1 1) Primary 3 ': 5'-CCTAGTAGAGAAGAGCTAAAGAGGC-3'; (SEQ ID NO: 12) (See the description above to complete the list of primaries for feline CD28 cDNA). The DNA primaries used for the RT / PCR of the feline CD28 cDNA were: Primary 5 ': 5'-CGCGGATCCACCGGTAGCACAATGATCCTCAGG-3' (SEQ ID NO: 13) Primary 3 ': 5'-CGCGGATCCTCTGGATAGGGGTCCATGTCAG-3 \ (SEQ ID NO: 14) (See the description above to complete the list of primaries for feline CD28 cDNA). The DNA primaries used for the RT / PCR of the cDNA CTLA-4 feline, were: 1. Primary degenerates for the first PCR product (672 bp): Deg 5 'P: 5'-ATGGCTT (C) GCCTTGGATTT (C) CAGC (A) GG-3'; (SEQ ID NO: 15) Deg 3 'P: 5'-TCAATTG (A) ATG (A) GGAATAAAATAAGGCTG-3'; (SEQ ID NO: 16) 2. 5 'end of CTLA-4 (455 bp): specific primaries of the degenerate gene (GSP) and nested gene specific (NGSP): first round PCR: Deg 5' P: 5 ' -TGTTGGGTTTC (T) G (A) CTCTG (A) CTT (C) CCTG-3 '; (SEQ ID NO: 17) 3 'GSP: 5'-GCATAGTAGGGTGGTGGGTACATG-3'; (SEQ ID NO: 18). PCR nested with the PCR product of the first round: Deg ^ 5 'P: 5'-TGTTGGGTTTC (T) G (A) CTCTG (A) CTT (C) CCTG-3'; (SEQ ID NO: 19) 3 'NGSP: 5'-ACATGAGCTCCACCTTGCAG-3'; (SEQ I D NO: 20). End 3 'of CTLA-4: primary adapter 1 (AP1, Clonetech Lab, Inc., Palo Alto, CA); primary nested adapter (AP2, Clonetech Lab), gene-specific primary (GSP), and nested gene-specific primary (GNSP): RACE 3 'PCR: AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3'; (SEQ ID NO: 21) 5 'GSP: 5'-GTGAATATGGGTCTTCAGGCAATG-3'; (SEQ ID NO: 22) PCR RACE nested 3 'with the PCR product RACE 3': AP2: 5'-ACTCACTATAGGGCTGCGACGCGGC-3 '; (SEQ ID NO: 23) NGSP 5 ': 5'-GAAATCCGAGTGACTGTGCTGAG-3'; (SEQ ID NO: 24) 4. Primaries for the CTLA-4 Fel primary complete CTLA-4 gene 5: d'-AACCTGAACACTGCTCCCATAAAG-3 '; (SEQ ID NO: 25) Primary 3 'CTLA-4 Fel: 5'-GCCTCAGCTCTTAGAAATTGGACAG-3'; (SEQ ID NO: 26) The DNA primaries used for the RT / PCR of the feline CD86 (B7-2) cDNA were: 1. Primary degenerates for the first PCR product (423 bp): Deg 5 'P: 5'-TAGTATTTTGGCAGGACCAGG-3' (SEQ I D NO: 27) Deg 3 'P: 5'-CTGTGACATTATCTTGAGATTTC-3'; (SEQ ID NO: 28) 2. Degenerate primaries for the second PCR product (574 bp): Deg 5 'P: 5'-GA (G) CA (T) GCACT (A) ATGGGACTGAG-3; (SEQ ID NO: 29) Deg 3 'P: 5'-CTGTGACATTATCTTGAGATTTC-3'; (SEQ ID NO: ) 5 'end of CD86: AP1, AP2 (Clonetech Lab), Degenerated 3' gene specific primers (GSP) and 3 'nested gene specific (NGSP): 5' RACE PCR: AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3 '; (SEQ ID NO: 31) GSP 3 ': 5'-TGGGTAACCTTGTATAGATGAGCAGGTC-3'; (SEQ ID NO: 32) 5 'RACE PCR nested with the 5' RACE PCR product: AP2: 5'-ACTCACTATAGGGCTCGAGCGGC-3 '; (SEQ ID NO: 33) NGSP 3 ': 5'-CAGGTTGACTGAAGTTAGCAAGCAC-3'; (SEQ ID NO: 34). 3 'end of B7-2: AP1, AP2, GSP 5', and NGSP 5 ': PCR RACE 3': AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3 '; (SEQ ID NO: ) GSP 5: 5'-GGACAAGGGCACATATCACTGTTTC-3 '; (SEQ ID NO 36) PCR RACE 3 'nested with the PCR product of RACE 3': AP2: 5'-CAGTGCTTGCTAACTTCAGTCAACC-3 '; (SEQ ID NO: 37) NGSP 5 ?: 5'-CAGTGCTTGCTACTTCAGTCAACC-3 ?; (SEQ ID NO: 38) Complete CD86 gene: Primary 5 'B72 (1) Fel: 5'-CGGGAATGTCACTGAGCTTATAG-3'; (SEQ ID NO: 39) Primary 3 'B72 (1 176) Fel: 5'-GATCTTTTTCAGGTTAGCAGGGG-3'; (SEQ ID NO: 40) Example 1 B Cloning of CD80 / (B7-1) -Synthro / SPAH; Plasmid 917-19- 8/16 Feline spleen cells were taken from cats and cultured with Concanavalin A for 5 hours, the cells were pelleted, washed with PBS and used to isolate the total RNA (Qiagen RNeasy Total RNA System The total RNA was treated with DNAse I (Boehringer Mannheim) to remove the DNA contamination of the RNA preparations, then the messenger RNA was extracted from these preparations using Qiagen's Oligotex beads (Santa Clara, CA) and rapid columns. The copy DNA was generated from mRNA, in the presence of random hexamers, dNTPs, RNAsin, reverse transcriptase (Promega) and reverse transcriptase regulator (Promega), and incubated at a temperature of 42 ° C for 30 minutes. , PCR was used to generate a double-stranded full-length cDNA clone of the feline B7-1 open reading frame (ORF), using the primary of perception 5 / 97.50 (5'-ATGGGTCACGCAGC AAAGTG-3 '); (SEQ ID NO: 41) and the primary anti-perception 5 / 97.51 (5'-CTATGTAGACAGGTGAGATC-3 '); (SEQ ID NO: 42), dNTPS, cDNA B7-1 (first braid), MgSO4, Vent polymerase (BRL) and Vent polymerase regulator (BRL). The PCR conditions were as follows: 1 cycle at a temperature of 94 °, 15 seconds; 35 cycles at a temperature of 94 ° for 30 seconds, at 48 ° for 2 minutes, at 72 ° for 2 minutes; 1 cycle at a temperature of 72 ° for 10 minutes. The PCR reactions were run on a 1% low pressure agarose gel and the DNA fragments corresponding to the expected size of the B7-1 ORF were isolated, the gel was purified (Qiagen's Gel Purification Kit, Santa Clara, CA) and cloned in a plasmid vector PCR-BLUNT using the reagent kit of Invitrogen's Zero Blunt PCR Cloning Kit (San Diego, Ca). The extracted DNA from bacterial colonies resistant to kanamycin was preselected in the presence of a unique Nhel site (contained in CD 80 (B7-1) -TAMU feline). Inserts that were within the range of 800 to 900 bp in size and that contained an Nhel site were sequenced using ABI fluorescent automatic sequencing protocols, and the equipment (Perkin-Elmer-Cetus, Applied Biosystems, Inc.). The plasmid vector and the B7-1 specific primaries derived from the previously cloned B7-1 gene, which were used to generate pCR-Blunt primary DNA sequence are 1 / 97.36 (5'-CAGGAAACAGCTATGAC-3 '); (SEQ ID NO: 43) and 1 / 97.37 (5'-AATACGACTCACTATAGG-3 '); (SEQ ID NO: 44). The specific primaries of the B7-1 gene are: 12 / 96.22 (5'-AACACCATTTCATCATCCTTT-3 '): (SEQ ID NO: 45), 1 / 97.33 (5'-ATACAAGTGTATTTGCCATTGTC-3'); (SEQ ID NO: 46), 12 / 96.20 (5'-AGCTCTGACCAATAACATCA-3 '); (SEQ ID NO: 47), 12 / 96.21 (5'-ATTAGAAATCCAGTTCACTGCT-3 '); (SEQ ID NO: 48), 1 / 97.32 (5'-TCATGTCTGGCAAAGTACAAG-3); (SEQ ID NO: 49), 1 1 / 96.32 (5? TTCACTGACGTCACCGA-3 '); (SEQ ID NO: 50), 1 1 / 96.31 (5'-AAGGCTGTGGCTCTGA-3 '); (SEQ ID NO: 51). Two clones were determined to contain the full-length CD80 sequence corresponding to the original CD80 sequence, with the exception of two DNA point mutations. In said point mutation, the amino acid sequence was not made. The second mutation resulted in an amino acid change from a Leucine to an Isoleucine. The resulting feline CD80 clone was designated 917-19.8 / 16. (CD80-Syntro / SPAH). To facilitate the cloning of the feline CD80 (B7-1) gene, together with any pox promoter containing EcoRI and BamH l cloning sites, two new primaries were designated to introduce the restriction enzyme cloning sites EcoRI and BamH I in the 5 'and 3' end of CD-80 ORF, respectively. These two primaries are: primary of perception 1 /97.43 (5'- TCGAGAATTCGGGTCACGCAGCAAAGTGG-3 '): (SEQ ID NO: 52) and primary antiperception 1 / 97.6 (5'-GCTAGGATCCAATCTATGTAGACAGGTGAGAT-3'): (SEQ ID NO: 53 ). The resulting PCR fragment was digested with EcoRI and BamHl and cloned into a SPV 01 L homology vector (Accl insertion site within the M Hind M I genomic fragment of the porcine virus) for the generation of a recombinant SPV virus. This resulted in the tape 930-23. A1, a 01 L SPV homology vector containing the feline CD80 ORF together with the advanced / early synthetic px promoter, LP2EP2, and adjacent to the LacZ E. coli marker gene band, promoted by the synthetic advanced pox promoter LP2. The plasmid vector 930-23. A1, was co-transfected with SPV 001 to generate a recombinant SPV virus expressing the feline B7-1 and ß-galactocidase proteins of E. coli.

Example 1 C: Subcloning of CD-28 in the pox viral homology vector: The feline CD-28 coding region was PCR amplified with synthetic primaries containing convenient cloning sites, to facilitate cloning CD-28 along with any pox promoter for the construction of a specific homology vector of pox. The synthetic primaries were made to introduce an EcoRI and BglI cloning site at the 5 'and 3' ends of the PCR fragment, respectively. The two primary ones are: perception primary, 7 / 97.1 (5'-GATGAATTCCATGATCCTCAGGCTGGGCTTCT-3 '); (SEQ ID NO: 54) and the primary anti-perception 7 / 97.2 (5'- GATCAGATCTCAGGAACGGTATGCCGCAA-3 '); (SEQ ID NO: 55). The resulting PCR DNA fragment was digested with EcoRI and Bglol, and cloned into a SPV 01 L homology vector for the generation of a recombinant SPV virus. This resulted in the tape 930-26. A1, a homology vector 01 L SPV (Accl insertion site within genomic fragment M Hindl ll of porcine virus), containing the feline CD-28 ORF together with the advanced / early synthetic pox promoter, LP2EP2 and adjacent to Ribbon marker gene LacZ E. Coli, promoted by a synthetic advanced pox promoter LP2. The plasmid vector of homology 930-26. A1, was co-transfected with SPV 001 to generate a recombinant SPV virus expressing feline CD28 and β-galactocidose proteins.

Example 2 Characterization of cDNAs and polypeptides of CD80 (B7-1) -TAMU, CD86 (B7-2), CD28, CTLA-4 and CD80 (B7-1) -Synth / feline SPAH: CD80 cDNA (B7- 1) of isolated and purified feline of approximately 941 nucleotides, encodes an open reading frame of the feline CD80 polypeptide of approximately 292 amino acids. The native membrane bond or mature form has a molecular mass of approximately 33,485 kDa, an isoelectric point of approximately 9.1, a net charge of pH 7.0 of 10.24. The transmembrane field of the protein is approximately 241 to 271 amino acids. Feline CD80-TAMU and feline CD80-Syntro / SPAH are isolated cDNAs and polypeptides independently from two different sources, and the DNA and amino acid sequences differ slightly. The source of mRNA CD-80-TAM U were feline peripheral blood mononuclear cells stimulated by With A, and the source of mRNA CD80-Sintro / SPAH were feline spleen cells stimulated by Con A. The difference in sequence of CD80-TAMU and CD80-Syntro / SPAH is from T to C at nucleotide 351, and from C to A in nucleotide 670. In the amino acid sequence, the change in nucleotide 351 is silent and the change in nucleotide 670 results in a conservative change of neutral amino acids, leucine to isoleucine, in residue 224 of the amino acid The isolated and purified feline CD86 cDNA (B7-2) of about 1 176 nucleotides, encodes an open reading frame of feline CD86 polypeptide of about 320 amino acids, the native membrane link or mature form, has a molecular mass of about 36.394 kDa, an isoelectric point of approximately 9.19, a net charge of pH 7.0 of 11.27. The isolated and purified feline CD28 cDNA of approximately 689 nucleotides, encodes an open reading frame of feline CD28 polypeptide of approximately 221 amino acids, the native membrane binding or mature form has a molecular mass of approximately 25,319 kDa, an isoelectric point of approximately 9.17, a net charge of pH 7.0 of 9.58. Isolated and purified feline CTLA-4 cDNA of approximately 749 nucleotides, encodes an open reading structure of feline CTLA-4 polypeptide of approximately 223 amino acids, the native membrane binding or mature form has a molecular mass of approximately 24,381 kDa, a Isoelectric point of approximately 6.34, a net charge of pH 7.0 of -0.99. CD80 co-expression, with co-stimulatory molecules CD28 or CTLA-4, and a tumor antigen or an antigen from a pathogenic organism, has the ability to activate or enhance the activation of T-lymphocytes, more specifically of Th-1 lymphocytes, and to promote the growth of other cell types. The coexpression of CD80, with the co-stimulatory molecule CTLA-4, has the ability to suppress the activation of T-lymphocytes, more specifically Th-1 lymphocytes. The co-expression of CD86 with co-stimulatory molecules CD28 or CTLA-4, and a tumor antigen or an antigen from a pathogenic organism has the ability to activate or enhance the activation of T-lymphocytes, more specifically Th-1 lymphocytes and promote the growth of other cell types. The coexpression of CD86 with the co-stimulatory molecule CTLA-4, has the ability to suppress the activation of T-lymphocytes, more specifically Th-1 lymphocytes.

CD28 Feline 88 88 79 78 CTLA-4 Example 3 Use of CD80 (B7-1), CD86 (B7-2), CD28 and feline CTLA-4 in vaccines. The following experiments were carried out to evaluate the immuno enhancing activities of feline CD80, CD86, CD28 and CTLA-4 in feline vaccines. In an alternative procedure, 8-week-old cats are injected intramuscularly with 100μg of plasmid containing cDNA for feline CD80, CD86, CD28 and CTLA-4 molecules, in a mixture with a plasmid containing VIF env cDNA and gag or Felv env and gag, or alternatively, injected intramuscularly with 100μg of plasmid containing combinations in pairs expressing cDNA of CD80 and CD28, or CD80 and CTLA-4, or CD86 and CD28 or CD86 and CTLA-4, in pairs with CD28 or CTLA-4, in a mixture with a cDNA containing plasmid VI F env and gag or FeLV env and gag. The control cats do not receive CD80, CD86, CD28 and CTLA-4. Cats are stimulated with FeLV or virulent FIV and signs of disease are observed, as described above. The results of the stimulation experiments were that the cats receiving the cDNA vector containing feline CD80, CD86, CD28 and CTLA-4 and the cDNA vector containing FIV genes or FeLV genes, show 100% protection against the disease, compared to cats that receive only the cDNA vector that contains FIV genes or FeLV genes, which show a 75% protection against the disease. In an alternative procedure, 8-week-old cats are injected intramuscularly with 0.1 to 100 mg of purified protein from feline CD80, CD86, CD28 and CTLA-4 molecules, or alternatively with combinations of CD80 or CD86 pairs in pairs with CD28 or CTLA-4 proteins, from the recombinant cDNA vectors described above, and injected intramuscularly with 0.1 to 100.0 mg of a subunit vaccine containing VIF env and gag or FeLV env and gag. The control cats do not receive CD80, CD86, CD28 and CTLA-4. Cats are stimulated with a virulent VIF deformation or a FeLV strain, and the development of the disease is regularly observed. The results of the stimulus experiments, are that the cats receiving the purified protein of feline CD80, CD86, CD28 and CTLA-4 and the subunit vaccine containing FIV or FeLV, show a significantly reduced incidence of the disease, compared with cats that receive only the subunit vaccine that contains FIV or FeLV proteins.

Example 4 Use of feline CD80, CD86, CD28 and CTLA-4 to inhibit and destroy the growth of the tumor cell. Tumor cells from a cat are transfected with a feline CD80 or CD86 expressing vector in combination with CD28 or. CTLA-4. Transfected tumor cells are re-administered to the cat and the presence of CD80, CD86, CD28 and CTLA-4 on the surface of the tumor cell, rs a broad immunological response for transfected and non-transfected tumor cells, resulting in the death of localized and metastatic tumor cells. In an alternative procedure, feline CD80, CD86 expressing vectors in combination with feline CD28 and CTLA-4, which are directly injected into a tumor of a cat, result in a broad immunological response to the tumor cells, giving as a result the death of localized and metastatic tumor cells.

Example 5 Feline Cloning and Sequencing of Feline CD80 cDNA INTRODUCTION In addition to the cytokines, some surface molecules have been shown to improve or suppress a different immune response. CD80 (B7-1) is an accessory molecule that binds its CD28 receptor in T cells (Freeman and associates, 1989). This interaction works in the supply of a secondary stimulus that, together with the primary signal supplied by the T-cell recognition of the antigen presented in the context of MHC, results in the activation and proliferation of the -T-cell (Allison and Lanier, 1994). Although first described as a B-cell antigen, CD80 has subsequently been discovered to be expressed in a variety of cell types, most with antigen-presenting capabilities (Freeman and associates, 1989). In primates and rodents, the CD80 molecule is a 60 kDa polypeptide composed of approximately 290 amino acids (Freedman and associates, 1987; Freeman and associates, 1989). The confirmation of the putative amino acid sequence suggests characteristics that distinguish it as a member of the super, immunoglobulin family (IgSF) (Peach and associates, 1995). It is composed of two extracellular IgSF fields, a hydrophobic transmembrane field and a short cytoplasmic tail (Freeman and associates, 1989). The extracellular field of the mature peptide has a region similar to the variable (V) IgSF of terminal NH3 of 124 residues, followed by a field similar to the constant (C) IgSF of 100 amino acids (Freeman and associates, 1989). The human counterpart has 8 glycosylation sites, linked by N potentials, and although the mature peptide is highly glycosylated, these carbohydrate residues are not considered to be involved in the binding with CD28 or CTLA-4, since they are considered to be oriented opposite to the proposed link field (Bajorath et al., 1994). In addition, the removal of the carbohydrate residues do not seem to influence the binding capacities, rather it is proposed that their function is to increase the solubility of the extracellular portion of the molecule (Linsley and associates, 1994a). CD80 binds the two different receptors expressed at different times during the course of T-cell activation. CD28 is found in a variety of thymocytes and naive and activated T cells, and has a role that can be demonstrated in the activation of T-cell proliferation (Aruffo, 1987). The second CD80 receptor, CTLA-4, is normally found at a later time in fully activated T cells (Linsley et al., 1991b). Although a role for CTLA-4 has not been definitively established, it is hypothesized that the molecule can act to suppress an active and existing T-cell response (Hutchcroft and Bierer, 1995). The CD80 molecule by itself does not seem to have signaling capacity. The cytoplasmic region is relatively short without residues, with a signaling or enzymatic function that can be demonstrated (Hatchcock and associates, 1994). The lack of conservation in the cytoplasmic tail between murine and human mouse peptides also reflects the likelihood that CD80 lacks signaling function and acts only to cross-link CD28 or CTLA-4 (Lindsley and associates, 1994a). The interaction between CD80 and CD28 has been shown to be necessary for the maturation of naive T cells to an activated state, thus initiating a primary T-cell response (Damle and associates, 1988). Although they were first identified in activated B-cells (Freednnan and associates, 1987), they have been subsequently found in most of the subgroups of cells presenting the professional antigen, including macrophages / monocytes (Freedman and associates, 1987), Langerhan cells (Symington and associates, 1993), dendritic cells (Liu and associates, 1992), activated -T cells (Razi-Wolf and associates, 1992) and a variety of tumor lines (Chen and associates, 1992). The presence of the molecule CD80 and APC has been shown to be important in the activation of both CD4 + and CD8 + T cells (Allison and Lanier, 1994, Bellone and associates, 1994). Although the molecule is normally present only at significant levels in professional APC, some tumor cell lines have been shown to regulate the signaling molecule (Chen et al., 1992).

In response to the transformation, it appears that some oncogenic lines regulate CD80 expression. In these tumors derived from cells that do not present antigen, as well as in some immortalized cell lines, CD80 is a surface expressed at levels sufficient to promote complete activation of the T-cell (Chen et al., 1992). Although the kinetics of expression are not clear, it is possible that CD80 derived from the tumor may be a response to the "oncogenic insult", and a mechanism of evolution through which the immune system can remove transformed or tumugenic cells (Antonia and associates, 1995). The role of the CD28-B7 interaction seems important in the activation of the primary T-cell. The recognition of the antigen in the MHC context by the TCR is insufficient in itself to initiate an optimal proliferation and activation of the T-cells (Schwartz, 1992). The stimulation of TCR in the absence of accessory signals can lead to a state of anergy or hyporesponsiveness in populations of T-cells (Jenkins and associates, 1987). The binding of the CD28 molecule with the CD80 molecule in the antigen presented by the cell appears to supply the second signal required to activate the -T cell (Schwartz, 1992). When the TCR is embedded, in the absence of this second signal, the naive cells are not activated and can become anergic (Lanier et al., 1995). This important role for the CD28-CD80 interaction, has not been defined solely in the activation of naive CD4 + cells, but also in Th1 Th2 CD4 + clones, and naive and memory CD8 + -T cells derived from small peripheral blood lymphocytes at rest (Linsley and associates, 1993a). As with the CTLA-4 / CD28 family, there is also at least one additional counter-receptor related to CD80. The initial studies that try to demonstrate the importance of CD80 in a primary immune response, found problems because although the introduction of CTLA-4lg inhibited immunological responses, the addition of monoclonal antibody (mAb) to CD80, did not seem to elicit the analogous results ( Lenschow et al., 1993). The development of nonsense CD80 mice inadvertently led to the discovery of the second CTLA-4 / CD28 receptor (Freeman and associates, 1993). It was perceived that these mice would share a similar phenotype with previously developed CD28 senseless mice, which had an inadequate response of the T-cell (Freeman and associates, 1993). However, it was discovered in nonsense CD80 mice that a normal response developed and that APC had the ability to provide the second signal necessary for T cell maturation (Freeman and associates, 1993). From these results, a second receptor was hypothesized and eventually isolated. The subsequent discovery of the related CD86 receptor (B7-2 or B7-0) seemed to resolve the discrepancies found in CD80 senseless mice, and together with structural and linkage similarities, reflects the likelihood that the molecules share a function and origin common (Hatchcock and associates, 1994). CD86 (B7-2), shows some similarity with CD80, sharing a V-region and structurally similar extracellular C-region IgSF (Freeman and associates, 1993). However, the general homology between the molecules is less than 25% with scattered residues scattered on opposite sides of both extracellular fields (Bajorath et al., 1994). Although no binding region has been defined for any molecule, sequence homology has provided proposed prospect regions as potential sites of interaction (Lindsley and associates, 1994a). Despite the lack of conservation, CD80 and CD86 share similar binding capabilities for both receptors, although CD86 dissociates more rapidly from CTLA-4 (Lindsley and associates, 1995a). CD86 appears to share a similar expression pattern, with CD80 being expressed in activated B-cells, T-cells, macrophages and monocytes (Azuma et al., 1993a). However, the expression kinetics are slightly different for the two molecules (Hathcock and associates, 1994). Generally, CD86 appears earlier in an active immune response than CD80, and appears to be expressed constitutively in monocytes (Freeman and associates, 1991). Although CD80 may appear as late as 24 hours after the initial stimulation, CD86 appears earlier in the response, or is constitutively expressed at low levels of myeloid cells (Hathcock and associates, 1994). The two surface proteins evoke similar intracellular responses if they bind to their respective counter-receptors on T-cells, either CD4 + or CD8 + (Lanier and associates, 1995). There does not seem to be any difference in the ability of any molecule to initiate the activation and proliferation of T-cells or to induce CTL activity (Hathcock and associates, 1994). Therefore, the data suggest that both molecules initiate a similar signal cascade when binding to CD28 or CTLA-4 respectively (Hathcock and associates, 1994). As both molecules also seem to bind these against receptors with equal kinetics and do not elicit different effects, this is not clear for the evolutionary meaning of this system of "two binders / two receptors" (Lanier and associates, 1995). Originally, CD80 was described as a marker for B-cells, and high levels of both CD80 and CD86 were found in B cells stimulated with lipopolysaccharide (LPS), anti-lg, anti-CD40, concanavalin A (Con A), cAMP, IL-2 and IL-4 (Hathcock and associates, 1994). IFN? and IL-5 have been shown to regulate CD86 in B-cells in murine mice, although there are no data available from the human system or for CD80, with respect to these immunological regulators (Azuma et al., 1993a). The kinetics of expression are slightly different in B-cells for each molecule. CD86 is expressed early after stimulation (6 hours) whereas CD80 is not present until almost after 24 hours and does not reach peak expression until after 48 hours (Lenshow and associates, 1993). The regulation of CD80 in B-cells also seems to be regulated by signaling mediated by MHC class I I (Nabavi et al., 1992). Two other surface receptors also appear to be important in CD80 surface expression. Cross-linking of CD40 expressed in B-cell by T-cells or Ig that express the counter-receptor, resulted in increased CD80 expression (Azuma et al., 1993b), while cross-linking of the Fc receptor down-regulated the expression of both molecules (Barcy and associates, 1995). CD40 and its CD40L binder have been proposed as a pathway through which the CD80 expression in APC is regulated (Page and associates, 1994). CD40 is expressed in a variety of cell types, including B-cells, monocytes, dendritic cells, fibroblasts and human endothelial cells and can be regulated in these cells in the presence of I FN? (de Boer and associates, 1993). The CD40 ligand (CD40L) is expressed in activated CD4 + T cells. The binding of CD40 by CD40L has been shown to regulate CD80 expression in APC, although it does not appear to induce expression in other types of cells expressing the receptor, including endothelial cells (Page and associates, 1994). The CD80 and CD86 molecules, although they share less than 25% amino acid identity, have structural similarities and are considered to be distantly related (Freeman and associates, 1993). Homologous residues are concentrated in Ig-like fields, with many residues conserved in the transmembrane or cytoplasmic fields (June and associates, 1995). A family comprising the B7 genes has been proposed, which could, in addition to CD80 and CD86, include butyrophilin (BT), myelin glycoprotein / oligodendrocyte (MOG), the chicken MHC analog, B-G (Linsley and associates, 1994b). BT, MOG and B-G are encoded by the gene complex which elevates a potential evolutionary link between the MHC and the requisite co-stimulatory molecules (Linsley and associates, 1994b). Murine and human mouse T cells at rest, express low levels of CD86, while human T cells and mouse murine (and T cell clones), activated with anti-CD3, express CD80 and CD86 at appreciable levels (Hathcock and associates, 1994). Expression of both CD80 and CD28 in activated T-cells may reflect the ability of T-cells to expand through autocrine stimulation (Azuma et al., 1993b)). Surprisingly, CD80 has been shown to be regulated in CD4 + T cells infected with HIV, with down-regulation of concomitant CD28. It is proposed that this is a possible mechanism of viral transmission when non-infected CD4 + T cells initiate contact mediated by CD28 / CD80 with infected lymphocytes (Haffar and associates, 1993). Peripheral human blood monocytes express low levels of CD80 and higher levels of CD86, while exposure to GM-CSF or IFN ?, results in the up-regulation of both CD80 and CD86 surface expression (Barcy et al. associates, 1995). LPS is a strong inducer of CD80 expression in peripheral human blood monocytes (Schmittely Associates, 1994). No data are reported in peritoneal macrophages in the human system, but mouse murine mouse macrophages at rest express low levels of CD80 and CD86 ( Freeman and associates, 1991; Hathcock and associates, 1994). The stimulation of LPS and IFN? of murine mouse macrophages, increase expression on the surface, although IFN? in combination with interleukin 10, down-regulates both receptors (Ding and associates, 1993).

Splenic dendritic cells express low levels of both molecules, and Langerhan cells express low levels of CD86, although culture tends to increase expression in both cell types (Larsen and associates, 1994). In dendritic cells, CD86 appears to be upregulated more strongly after culture, appears earlier and may play the most important role in dendritic cell-mediated signaling (Hathcock and associates, 1994). Surprisingly, although I L-10 has no effect on CD80 expression in dendritic cells, it acts to down-regulate CD86 expression (Buelens et al., 1995). O'Doherty et al. (1993) reported that although the initial levels of CD80 were low, at the time of maturation, the dendritic cells show higher levels of CD80. The Langerhan cell expression of CD80 is inhibited by both I L-10 and IFNy, but the GM-CSF exposure can reverse the inhibition of I FNy, but not the inhibition of IL-10 (Ozawa et al., 1996). In humans and mice, specific cytokines have been shown to exert control over CD80 and CD86 expression. IL-4 is a strong inducer of CD86 and up to a lower point of CD80 in B-cells (Stack et al., 1994), whereas IFNy increases the expression of CD86 in a variety of cell types including monocytes and macrophages of B-cells (Hathcock et al., 1994). Although IFNy appears to upregulate CD80 expression in monocytes, it may act to down-regulate expression in macrophages. IL-10, even in the presence of I FNy, acts to down-regulate CD80 and CD86 expression in monocytes (Ding and associates, 1993). This interaction can be a potential mechanism of a switch from a Th1 (DTH) response to a Th2 (humoral) response. However, IL-10 does not influence the expression of CD80 in dendritic cells (Buelens et al., 1995). This may additionally reflect the role of these molecules in the regulation of the auxiliary subgroup T, such as dendritic cells that are considered important in the initiation of a type 2 response. IL-7, increases CD80 expression in T cells, although they are not defined effects on other cell types (Yssel et al., 1993), while CD80 expression of B-cell has been shown to be mediated through the cross-linkage of the p75 TNF receptor, expression may be increased in the presence of IL -4 (Ranheim and Kipps, 1995). Surprisingly, TNF belongs to the same family of CD40 molecules, another potent initiator of CD80 expression. GM-CSF appears to upregulate the dendritic cells and the expression of the Langerhan cell of the CD80 surface, whereas IFNy only causes upregulation of CD86 in these cells (Larsen and associates, 1994). The recognition, binding and lysis of target cells transformed and virally infected by CD8 + cytotoxic T-lymphocytes (CTL), was long but is only mediated through TCR recognition of foreign peptides expressed in the context of MHC class I (Berke, 1993) . Recently, it has been determined that a variety of surface molecules expressed by both of the CTL and target cells is required to complete the interaction that takes place (Mescher, 1992). A key participant in this interaction is CD80 and its counter receiver CD28. The accessory signal supplied by the B7-CD28 interaction is required by the resting CD8 + small lymphocytes to differentiate a lithic state (Mescher, 1992). Surprisingly, once CTL is differentiated, this secondary signal is no longer required for the expression of lithic properties (Hodge et al., 1994).

It has long been known that CTL is an important mediator of viral immunity. In human individuals infected with immunodeficiency virus (VI H), no long-term progress is associated with high CTL levels of Gag CD8 + memories, Pol and Env, and very low copy numbers of HIV DNA and RNA in peripheral blood mononuclear cells (Rinaldo, 1995). Conversely, in individuals in the advanced stages of infection, the memory CTL (mCTL) is seriously diminished (Zanussi et al., 1996). The correlation of these findings supports the idea that mCTL may be an important factor in the host control of infection, and could play an important role in the establishment of protective immunity in naive non-infected individuals (Zanussi and associates, 1996). In addition, the pathological correlations with VI H infection reflect a potential role for CD80 and CD28 in the progression of the disease. CD80 has also been proposed to play an important role in the development of the pathogenesis of an HIV infection (Haffar and associates, 1993). T cells, usually express CD80 and only at low levels, and only after activation (Schwartz, 1992). In in vitro studies of HIV infection in allo-stimulated primary T-cell lines, CD28 appears to be down-regulated, and CD80 expression appears to be improved along with MHC Cll (Haffar and associates, 1993). Although a reason for these events has not been defined, two potential damage papers can be hypothesized. The presence of CD80 on the surface together with class I I should result in increased contact between infected T-cells and uninfected CD4 + cells (Haffar et al., 1993). Although this interaction could act to improve the transmission range between T cells, another role could increase the recognition mediated by CTL, and annihilate through the supply of the secondary signal through the interaction of CD80 in the infected cell, with CD28 expressed by the CD8 + T cell (Haffar and associates, 1993). This could accelerate the decline of the CD4 + population linked to the triggering of AIDS-related diseases (Haffar and associates, 1993). In murine and human mouse systems, the expression of the B7 protein family seems to be an important factor in the immunological recognition of transformed cells (Chen et al., 1992). Although expression has been found in some transformed cells, most tumors do not normally express CD80 or CD86, making it improbable in this way that when a potentially immunogenic tumor antigen is expressed, complete recognition takes place through cells. T (Chen and associates, 1993). However, transfection experiments using the CD80 molecule to improve tumorcidal cytolysis have proved successful (Hodge et al., 1994).

Retroviral and vaccine-based vectors, which express the functional CD80 molecule, have been used to transfect malignant cells (Li et al., 1994; Hodge et al., 1994). Subsequently, these cells expressing CD80 in addition to tumor antigens normally known to be deficient are reintroduced into the host, where it is considered that a cellular immune response is generated against tumor antigens expressed in malignant cells (Townsend and Allison, 1993). The results of these experiments with some forms of tumors have been surprisingly effective. In many cases, the host has the ability to mount a strong cellular response against the malignancy, and control or eliminate it (Hodge and associates, 1994). Subsequent re-introduction of tumor cells with or without the CD80 surface molecule in the host, results in similar levels of anti-tumor immunity (Hodge et al., 1994). Therefore, it seems that once memory has been established, the CD80 molecule is not required to sustain the response or to initiate it, if re-introduction occurs (Hodge and associates, 1994). These experiments demonstrate that the CD80 molecule is an efficient mediator of cellular immunity, and that in specific tumors, cellular responses can be induced to possibly control malignancy and prevent reestablishment (Hodge et al., 1994).

As seen in the development of some forms of autoimmunity, increased CD80 expression can have detrimental effects. It is considered that CD80 in synergy with IL-12, is important in the early development of multiple sclerosis (MS), and results in the stimulation of the T-cell, and the development of DTH (Windhagen and associates, 1995). Experimental autoimmune encephalomyelitis (EAE) can be partially inhibited by the administration of soluble CTLA-4lg to experimental subjects. The inhibition of demyelination by blocking the CD28 / CD80 interaction reflects a potential role during this interaction in the exacerbation of the disease (Arima and associates, 1996). The importance of the CDSO molecule in the progress of an effective immune response is clear. Although the cDNA encoding the protein has been isolated in rodents and primates, it has not been isolated outside of these groups. The feline is an important companion animal and potential model of retroviral disease. The cloning of immunological reagents from feline species provides tools for the investigation of diseases of veterinary importance, with potential relevance for the progress or prevention of the disease in other species.

MATERIALS AND METHODS. Isolation of an initial fragment. MRNA was extracted from peripheral blood mononuclear cells (PBMC) stimulated for 16 hours by Con A, using the RNAzolB RNA extraction reagent (Biotexc, Houston, TX). Initially, the cDNA was derived from this RNA by a reverse transcriptase (RT) reaction, which uses dT oligo as the primary 3 '. The RNA and dT oligo were briefly heated to a temperature of 75 ° C for 3 minutes, to remove the secondary structure. Subsequently RT, dNTP, regulator and distilled water were added and the mixture was incubated for 1 hour at a temperature of 42 ° C. After this incubation, the sample was heated to a temperature of 95 ° C for 5 minutes, to deactivate the RT. Subsequently, the degenerate primaries derived from the consensus regions within the sequences published by CD80 of murine human and mouse (GeneBank, Gaithersburg, MA) were used for the initial amplification of a fragment of 344 nucleotides that encodes a central region within the constant field of the gene: primary 5 'B7-2 GGC CCG AGT A (CT) A AGA ACC GGA C primary 3' B7-3 CAG (AT) TT CAG GAT C (CT) T GGG AAA (CT) TG (SEQ ID NO: 56). A heat-initiated polymerase chain reaction (PCR) protocol employing Taq polymerase was used, to amplify the product. The reaction mixture, which lacks the enzyme Taq, was initially heated, in a heat-up step, at a temperature of 95 ° C for 5 minutes to avoid the formation of primary dimers. The enzyme was added before the initiation of the temperature cycle. Subsequently, the PCR reaction was heated to a temperature of 95 ° C for 30 seconds to melt the double stranded DNA. Subsequently, the reaction was cooled to a temperature of 42 ° C for 30 seconds, to facilitate the hardening of the degenerated primaries. A low hardening temperature was used to facilitate the binding of the primaries that were not 100% analogous. Subsequently, the reaction was heated at 72 ° C for 45 seconds, the optimum temperature for the Taq polymerase to extend to the primary and copy the opposition DNA braid. The temperature cycle was repeated 30 times. After 30 cycles, a final extension step of 72 ° C was used for 7 minutes, to facilitate the extension of any products not completed. After visualization on a 1% agarose gel, during sequencing, the product was ligated overnight at a temperature of 16 ° C in the vector cloning TA (InVitrogen, San Diego, CA). Two μl of ligation reaction was used to transform competent InvaF 'cells. Transformed bacteria were stained on LB plates (50 μg / ml ampicillin), covered with 40 μl of 50 μg / ml x-gal solution. The next day, the white colonies were selected and inoculated in 5 ml of LB medium containing 100 μg / ml of ampicillin, and were grown overnight at a temperature of 37 ° C with shaking at 225 rpm. During the night, mini-crop preparations were carried out to determine clones that possessed the plasmid with the correct insert. The plasmid was extracted from the cultures, using a standard alkaline lysis procedure, the DNA being further purified by phenol: chloroform extraction (Maniatis et al., 1982). The DNA was precipitated in 2 volumes of ethanol and subsequently digested with EcoRl. The digestions were visualized on a 1% agarose gel to determine colonies with plasmid that contained the appropriate insert. Subsequently, the plasmid was purified from positive clones and sequenced, using any sequencing sequencing of radiolabeled dideoxy S35 based on Sequenase (USB, Cleveland, OH), or by sequencing fluorescent ink-killing cycle (Perkin Elmer, Norwalk CT). From the cDNA sequence, the specific 3 'and 5' primaries were constructed for use in 5 'rapid amplification of cDNA end reactions (RACE) and for the derivation of the 3' sequence together with the degenerate primaries of the 3 'untranslated region (UTR).

Isolation of the 5 'region. The Marathon cDNA amplification protocol (Clonetech, Palo Alto, CA) was used to derive the 5 'sequence of the gene. MRNA was produced, from PBMC stimulated for 12 hours by Con A, and normally for 4 hours with LPS. The mRNA was extracted using the ULTRASPEC RNA extraction reagent (Biotexc, Houston TX). The cDNA was produced with a primary dT anchor oligo with degenerate nucleotides at the 5 'end, to facilitate a link from the primary to the 5' major end of the poly A tail.

Subsequently, the cDNA was transcribed as previously described. Specific linkers were ligated to the cDNA, with T4 DNA ligase. The PCR assay was carried out in the cDNA with an internal 3 'primer specific for the previously amplified region: B7-284: TTA TAG TGA TGA GGA CAG GGA AG (SEQ ID NO: 58) B7-190: TGA TGG AGG AAA ACC TCC AG (SEQ ID NO: 59) and a primary anchor complementary to the linked linker sequence. The parameters for the PCR reaction assay using the Klentaq polymerase mixture (Clontech, Palo Alto, CA) were: 1 cycle at a temperature of 95 ° C for 5 minutes; 5 cycles at a temperature of 95 ° C for 30 seconds, at 72 ° C for 30 seconds and at 68 ° C for 45 seconds; 5 cycles at a temperature of 95 ° C for 30 seconds, at 65 ° C for 30 seconds and at 68 ° C for 45 seconds; 25 cycles at a temperature of 95 ° C for 30 seconds, at 60 ° C for 30 seconds and at 68 ° C for 45 seconds. 1 μl of this reaction was diluted in 50 μl of water, and then 5 μl of this dilution was used in a nested PCR reaction (1 cycle at a temperature of 95 ° C for 5 minutes, 30 cycles at a temperature of 95 ° C for 30 seconds, at 65 ° C for 30 seconds and at 68 ° C for 45 seconds, with the KlenTaq polymerase mixture), with the primary specific anchor of the linker and a specific 3 'primer of the gene, located 5' from the initial primary (Figure 6). B7-20: TTG TTA TCG GTG ACG TCA GTG (S EQ ID NO: 60) B7-135: CAA TAA CAT CAC CGA AGT CAG G (SEQ ID NO: 61) 20 ul of each reaction was visualized on an agarose gel at 1.5%, and the appropriate fragment was cut out of the gel. The cDNA was extracted and purified from the agarose, centrifuging the gel through a gel nebulizer and 0.22μm micropore filter (Amicon, Beverly, MA). Subsequently, the purified DNA was sequenced directly, using ink-killing cycle sequencing (Perkin Elmer, Norwalk, CN).

Isolation of the 3 'region. region 3 was derived the gene, by choosing 5 gene specific primary of from 344 nucleotide fragment and the 5 'region previously sequenced: B7-S220 GTC ATG TCT GGC AAA GTA CAA G (SEQ ID NO: 62) B7-50 CAC TGA CGT CAC CGA TAA CCA C (SEQ ID NO: 63) B7-140 CTG ACT TCG GTG ATG TTA TTG G (SEQ ID NO: 64) B7-550: GCC ATC AAC ACÁ ACÁ GTT TCC (SEQ ID NO: 65) B7-620: TAT GAC AAA CAA CCA TAG CTT C (SEQ ID NO: 66) Subsequently, the degenerated 3 'primaries were chosen from the consensus regions of the UTR 3' CD80 of human and mouse murine. B7-1281 G (A / G) A AGA (A / T) TG CCT CAT GA (G / T) CC (SEQ ID NO: 67) B7-1260 CA (C / T) (A / G) AT CCA ACÁ TAG GG (SEQ ID NO: 68) The cDNA was produced from RNA extracted with ULTRASPEC (Biotexc, Houston, TX), from stimulated PBMC by Con A and LPS as described above. The anchored dT oligo was used as the initial 3 'primer for the transcription of RNA to cDNA. PCR reactions based on Taq polymerase were carried out with this cDNA, using the degenerated primary and 3 'primary 5' primers (1 cycle at a temperature of 95 ° C for 5 minutes, 30 cycles at a temperature of 95 ° C during 30 seconds, at 42 ° C for 30 seconds and at 72 ° C for 45 seconds, at 72 ° C for 7 minutes). Two rounds of nested reactions were required before a simple fragment of the correct size was produced. This product was cut from a 1.5% agarose gel, was purified as described above and sequenced with ink-killing cycle sequencing (Perkin Elmer, Norwalk, CN). Primary primaries were constructed from the sequence data of the 5 'and 3' regions, which could amplify a region encoding the complete open reading frame of the feline CD80 gene: START B7: ATG GGT CAC GCA GCA AAG TGG ( SEQ ID NO: 69) B7-960: CCT AGT AGA GAA GAG CTA AAG AGG C (SEQ ID NO: 12) PBMC cDNA previously produced and known to contain DNA encoding the gene was used. This PCR reaction (1 cycle at a temperature of 95 ° C for 5 minutes; 30 cycles at a temperature of 95 ° C for 30 seconds, at 42 ° C for 30 seconds and at 72 ° C for 45 seconds; at 72 ° C for 7 minutes), used KlenTaq DNA polymerase, an enzyme cocktail that retains some of the 5 'exonuclease activity, in the hope of reducing random errors that are often associated with Taq polymerase. The reaction amplified a fragment of 960 base pairs (bp), which was cloned into the TA cloning vector (InVitrogen, San Diego, CA) and sequenced as described above. The final sequence of the gene included cDNA from two animals separately. Each base pair of the gene was independently verified in at least three separate sequences derived from individual PCR reactions, to reduce the possibility of error derived from errors induced by PCR.

RESULTS RNA extracted from experimental cat HK5 was used during the initial amplification attempts with CD80, however this cat was subsequently exterminated and the additional products were produced in different animals. The initial amplification of PBMC RNA stimulated by Con A HK5 resulted in a 344 bp product that shared 70% identity with the human CD80 gene (Figure 7 and 8). The primaries were specific for a region within the center of the coding sequence corresponding to the IgC similar to the field. Although additional degenerate primaries were used in these initial experiments, in order to amplify regions that encoded more of the peptide, only the combination of B7-2 (5 ') and B7-3 (3') resulted in a product of size suitable. Subsequent experiments that used these additional degenerate primaries with specific primaries were not successful. Therefore, both 5 'and 3' regions had to be derived using other methods. A battery of six new primaries specific to the gene was created, based on the sequence data obtained from the initial product. (B7-20 5 ', 135 5', 140 3 ', 50 3', 284 5 ', and 190 5'). Initially, the 3 'primers were used in the PCR RACE 5' procedure, which is somewhat similar to the procedure used to successfully amplify the 5 'region of the CD28 molecule. The product was not produced using this method.

The RACE Marathon cDNA amplification system (Clontech, Palo Alto, CA), successfully amplified a region encoding the 5 'coding sequence. The RNA derived from RNA stimulated by Con A EK6, was successfully amplified with this protocol. The initial amplification was carried out with B7-284 and B7-3 and the primary anchor AP1. This reaction did not produce a defined single band, since nested reactions using this product as a template were performed using B7-20 and B7-135 as the 3 'nested primaries and the primary anchor AP-1 as the primary 5'. A product of the appropriate length was produced, from each of these reactions (Figure 9). The sequence data derived from the direct sequencing of the RACE products, gave a total identity in the regions of 20 and 135 bp, respectively, which overlapped the 344 bp product initially sequenced. The fragments were extended from the known 5 'region through the ATG 5' start codon of the feline gene, and in the 5 'untranslated region. The identity between the 5 'coding sequence derived from these products, and the 5' region of the human CD80 gene, was lower than the identity found between the 344 bp region of the feline gene and the analogous region within the sequence of human. It was observed that a lack of similar homology is found between murine human and mouse sequences within similar regions. Comparisons of sequence data from regions outside the coding region showed a further marked decrease in conservation (data not shown). A new panel of degenerate primaries encoding the 3 'untranslated region was synthesized, from consensus regions within the 3' UTR of the human and mouse sequences. These primaries successfully amplified the transcribed cDNA from RNA isolated from PBMC stimulated by Cat A's ED A. Unlike the initial amplification procedures, a series of nested reactions had to be run to obtain the final product. Primary PCR reactions using these primary degenerate and primary anchors from within the 344 bp fragment and the 5 'region, did not initially produce identifiable and clean bands (Figure 10). However, nested reactions using the diluted primary product and additional specific 5 'primers resulted in the production of the product that encoded the remaining 3' region (Figure 11). After sequencing the 3 'product, it was found that once outside the constant region, the identity again decreased. The distant end of the coding sequence showed a very low identity which decreased further after the stop codon. From the 3 'sequence, a reverse primary begins outside the coding region at nucleotide 960 that was constructed.

This construction in combination with a primer comprising the start codon amplified a product of the anticipated size. The sequencing of the final product showed that in fact each of the previously determined regions was translocated, and this fragment provided a contiguous and complete sequence of the feline CD80 gene (Figure 12). For amplification of the final product, two samples were amplified from RNA from PMBC stimulated by Con A from animals ED3 and EK6. At least two products from each animal were completely sequenced, and each nucleic acid site was checked and confirmed with at least three different correctly read sequences. Subsequently, the product from animal EK6 was cloned into the TA cloning vector for future reference and handling. In Figure 8, the complete nucleic acid sequence is provided. The fragment of nucleotide 960 includes the start codon at position 1, the stop codon located at nucleotide 888 and an additional 72 nucleotides from the 3 'untranslated region (Figure 13). From the products produced and sequenced to comprise the entire fragment, the additional 5 'and 3' regions were sequenced (data not shown). The sequencing of the upstream region of the 5 'start codon of the RACE 5' products, demonstrated that the ATG listed as the position from 1 to 3, was the first at the site of the methionine structure and conformed a similar position in murine and human mouse sequences. The stop codon located at position 888 was also conformed to a similar location in the previously sequenced genes. The alignment of the sequence showed 77% and 62% identity, respectively, with the published sequences of human and mouse murine CD80 nucleic acids (Figure 14). The homology with the other primate and rodent sequences is comparable with the levels found in human and mice respectively. The identity with the CD86 gene of each of the species was less than 25%. Using the MacVector DNA analysis software (I BI, Rochester, NY), the nucleic acid sequence was translated into an amino acid sequence. The translation produced a peptide of 292 amino acids, similar in length, although not identical, to both murine and human mouse proteins (Figure 15). It is proposed that the signal peptide extends from position 1 to position 25. The extracellular region of the molecule is composed of the variable IgSF of base 1 15, similar to the field, which extends through residue 139 and the constant IgSF of base 100, similar to the field, which extends to approximately residue 240. The membrane that disperses the field from residue 241 to 271 is followed by a short cytoplasmic tail of 21 residues. As in the human molecule, the feline polypeptide has 8 glycosylation sites linked by N potentials, although the fields are not located in the same way. The homology between the feline, human and murine mouse peptides is significantly lower than the identity observed in the nucleic acid sequences (Table 1).

Table 1: Comparison of feline CD80 sequence homology with murine and human mice.

Species Percentage of homology with the feline sequence: Amino Acid Nucleotide Human 77% 59% Mouse 62% 46% An alignment of the proposed peptide sequence of the CD80 feline with the proposed human protein demonstrates, by far, that most of the analogy between the two molecules occurs in the constant region (residues 140 to 240). There is a small homology between the peptides in the signal sequence, and this lack of identity extends through the IgV-like field. As mentioned, conservation is strong through the constant field, however, this identity is not contiguous and very little homology was discovered between the transmembrane field, and the intracellular cytoplasmic tail of the feline peptide and the analogous regions in the human molecule (Figure 16). The alignment of the feline, human and murine mouse CD80 genes with murine and human mouse CD86 genes reveals that although only the general homology is limited between the two members of the B7 family, the residues that have been considered as indicators of the B7 family of genes are retained by the feline peptide (Figure 17). These molecules include residues that are going to be involved in the fold, and the proposed binding region. Although the homology between the human and feline CD80 sequence is not as large as the identity between the two CD28 molecules, a comparison of the proposed hydrophilicity plots demonstrates that although there are a variety of changes in the specific amino acid sequence, these changes frequently they are homologous and potentially do not alter the surface characteristics of the peptide (Figure 18).

EXPLANATION The identities of the nucleotides between the felines and humans and the feline and the murine CD80 are moderate, although the degree of nucleotide homology is not transferred to the peptide. It seems that while the genetic code is degenerate, and in some differences of molecules (eg, CD28) between the nucleotide sequences do not significantly alter the peptide, with the conservation of CD80, the overall integrity of the amino acid is not as critical, and therefore, alterations in evolution in all species throughout the molecule are more permissible. Although the general nucleic acid sequences share a relatively moderate degree of identity, there were complications in obtaining a full-length sequence. The initial CD80 product was obtained from the constant region of the molecule, an area that demonstrates the highest degree of conservation in the cDNA of the cloned species. The preparators that recognized the region and successfully amplified a product were easily produced resulting in the nucleotide fragment 344 comprising the well conserved IgC region. Unfortunately, due to the lack of homology in the signal peptide, and in the cytoplasmic field and in 3 'UTR, more complicated measures were required to isolate the sequence of these regions. However, the presence of sequence data from the central region provided a strong anchor point from which the remainder of the molecule could be elucidated. The feline CD80 is a good example of how, by obtaining a short extension of a molecule and using the RACE methods and the degeneration primers in combination with the anchor primers from the sequenced region, a full-length sequence can be easily obtained . The comparison of the putative amino acid sequence of these cloned CD80 molecules demonstrates a lack of general homology. The murine and human mouse polypeptides showed less than 50% homology at the amino acid level. This is comparable to the 59% identity between the feline and the human, and the identity of 46% between the feline and murine mouse polypeptides and reflects, perhaps, the proximity of species evolution. A comparison of the predicted hydrophilicities of the feline and human residues, which shows the amino acids that would be potentially exposed or rested due to their relative hydrophilicity, reveals that while at the amino acid level, the specific amino acids can not be retained, it seems that the Changes can be relatively preserved. This shows the potential retention of the hydrophilic / hydrophobic character of the molecule which, therefore, can reflect general polypeptides with similar structures. It appears that the surface protein has particular amino acids that can be directly involved in the binding and other structural amino acids that only need to retain a structure that will allow interaction with the binding region to take place. Although there is divergence in the identity of the amino acid residues in the CD80 molecules of the primate, rodent and feline species, there is a retention of the characteristics of the IgSF. The feline CD80 molecule consists of an amino terminal field similar to IgC and a field similar to IgV near the membrane of the associated region. As with the conservation between murine and human mouse CD80, the identities are much higher between the constant regions than between the variable regions (Freeman and associates, 1989). In general, conservation in the variable field is only above 50% while it was found that in the constant region it is 70%, with a short extension from residues 164 to 198 (the region from which they were obtained). the initial fragments of nucleotide 344) that have a much larger identity. This central region of amino acid 56 (residues from 165 to 221), within the constant region shows a homology of 87% between the human and feline sequences, with an extended region of 28 amino acids (residues 171 to 198), in which there is only one difference. The region in the feline also demonstrates significant homology to the corresponding residues in the murine mouse polypeptide. The hydrophilic nature of the amino acids within this region demonstrates a high probability of surface expression and due to the level of cross-conservation of the species, a potential involvement in the binding / receptor interaction. It has been proposed that the IgC portion of the molecule is directly involved in the presentation of the binding field for the binding / receptor interaction (Peach and associates, 1995). However, it has been determined experimentally, that both motives, the variable and the constant, are required for the effective link (Peach and associates, 1995). The concentration of homologous residues in the IgC region of the extracellular field, together with the high level of divergence within the transmembrane and cytoplasmic fields, seems to further confirm the role of CD80 as a binder, rather than having a capacity of signaling. Similar to human CD80 and mouse murine, the feline molecule is highly glycosylated. Carbohydrate residues are not considered to be directly involved in the bond, but may help increase the solubility of the extracellular portion of the molecule (Peach and Associates, 1995). Of the eight potential sites found in the human peptide, seven are located in identical positions in the feliNO protein. The site located at residue 39 of the amino acid in the feline molecule is not replicated in human CD80, although there is a site in residue 232 that has not been found in the feline protein (Freedman and associates, 1987). Seven sites were found in the mouse murine molecule, only two being in an identical location, although they were generally found in the same areas of the molecule as the feline and human peptides (Freeman and associates, 1989). The similarity in the number retained and the locations of the glycosylation sites seem to reflect the importance of the motifs in the function of the molecule. There is a wide variety of potential applications of the feline CD80 molecule. The molecule, as explained above, is critical for the correct development of a mature T cell response. Monitoring gene expression at both the RNA and protein levels will help establish the means by which the feline's immune system is handling the infection. The way in which this system handles specific pathogens, when combined with observations obtained by research in other model systems, can provide additional insight into the human immune response. In addition, a significant animal company makes sense of how the feline system can be manipulated in a beneficial way and can provide veterinary medicine with improved options.

An important future application proposed for the CD80 molecule in other species has been the induction of tumor-specific immunity by introducing the CD80 gene into transformed cells, with re-introduction into host cells to elicit tumor immunity based on the CTL (Townsend and Allison, 1993). As explained above, it is considered that as a result of the expression of the surface of the CD80 by the tumor cells, a specific CTL response is mounted against the malignancy (Hodge et al., 1994). In addition, a memory population of CD8 + T cells is established in the host (Hodge et al., 1994). Although this technology has been concentrated on tumor immunity, by analogy, it may also be applicable in the establishment of anti-viral immunity.

As explained above, the long-term non-progression in acquired immunodeficiency syndrome is considered to be mediated through the initial establishment of a strong CTL anti-VI H response (Landay and associates, 1994). It seems that those individuals who can maintain an asymptomatic condition for the longest time after infection, have the ability to mount and maintain a strong CTL response directed against the virus. Although vaccines have traditionally been directed towards the establishment of a humoral response, if a vaccine could induce the development of a VIF / anti-HIV mCTL population, this population could provide protection similar to that found in long non-progressors. term. In naive individuals, the introduction of the vaccine-based gene that combined the VI F proteins with the CD80 peptide could result in the expression of the surface of the molecule in a customary manner in combination with the presentation of the MHC Cl of the VIF epitopes . If successful, this could result in the expansion of the specific VCT mCTL population in naive individuals. In subsequent exposures to the virulent virus, the individual vaccinated would be prepared to mount a response against the cells that become infected with the virus, eliminating them before the virus has the opportunity to establish itself and begin its destruction of the components of the system. immunological EXAMPLE 6 CLONING AND ELABORATION OF SEQUENCES OF CD28 FELINE cDNA INTRODUCTION CD28 is a surface glycoprotein normally expressed as an identical disulfide compound homodimer linked to 44 kDa subunits. It is a member of the immunoglobulin supergene family, and is characterized for a simple extracellular region V, a transmembrane field and a short intracellular tail (Aruffo and Seed, 1987). Although the molecule is glycosylated, the portions do not seem to play a role in the binding, and there is a hypothesis that they increase the solubility of the extracellular field (Peach and associates, 1994). The coding peptides of human, rat, mouse and rabbit cDNA and an analogous molecule in the hen have been cloned and their sequences elaborated (Linsley and associates, 1995a). It was discovered that CD28 in most thymocytes CD4 +, CD8 + and peripheral CD4 + and CD8 + T cells with increased expression resulting from a v t i - X? 3, PHA stimulation and PMA, and the resulting deletion of the anti-CD28 binding (Linsley et al., 1993b). It was soon determined, after its discovery, that CD28 played an important role in the regulation of the activation of CD4 + and CD8 + T cells (June and associates, 1990). In addition to increasing the activation and proliferation of the T cell, it has been further demonstrated that the production of this secondary signal also acts to induce cytolytic activities in the CTL (Azuma et al., 1993c). CD28 is expressed early in the maturation of T cells. Although "immature CD3 cells are normally CD28", intermediate CD4 + CD8 + cells express low levels, and CD4 + or CD8 + thymocytes, and CD3 + express CD28 at high levels ( Turka and associates, 1991). After maturation, the receptor has been found in almost all CD4 +, and more than half of all CD8 + T cells in humans (Turka and associates, 1991), and nearly 100% in murine mouse T cells. (June and associates, 1990). The molecule is not expressed at constant levels on the cell surface (Turka and associates, 1990).

After activation of the T cell, the expression of the surface increases, while the binding of the molecule by its specific ligand or mAbs, results in little regulation of the gene at both the mRNA and the protein level, in activated cells (Linsley and associates, 1993a). Although it was found to be important in T cell lineage lymphocytes, CD28 has been reported in plasmacytomas of bone marrow biopsies (Kozber and associates, 1987), and expressed by cultures of the natural leukemic cell line, similar to the mortal one (Azuma and associates, 1992). CD28 shares a degree of structural homology with the other B7 receptor of CTLA-4, and the two are grouped as a subfamily within the IgSF group (Linsley and associates (1995a) .The two molecules have an extracellular IgV region, a single membrane which expands the field, and a short cytoplasmic signaling field (Aruffo and associates, 1987) Although the general homology between the two molecules is only about 31%, there are short regions and specific residues that are completely conserved between the two molecules , reflecting a potentially important role for these motives in the recognition of B7 and structural integrity (Leung and Linsley, 1994). The motif MYPPPY, a region of six residues, is retained in all isolated members of the CD28 / CTLA family. -4 (Peach et al., 1994) This traces a circuit region similar to CD3 within the molecules, and when altered by the mutation, results in an avidity of reduced link in both, CD28 and CTLA-4 (Peach and associates, 1994). This region has been proposed as the potential binding site of the binder in the CD28 and CTLA-4 proteins, but it has not been determined whether this region is the actual binding site for B7 or whether it provides the structural motifs indirectly required for it to have place the link (Peach and associates, 1994). Although there are conserved residues shared between CD28 and CTLA-4, CTLA-4 binds both CD80 and CD86, with a greater avidity than CD28 (Ellis and associates, 1996). Therefore, although CTLA-4 is expressed in only 2 to 3% of the level of CD28 in activated T cells, it binds with an avidity greater than 20 in vitro folds (Linsley and associates, 1995b). Although the CTLA-4 and CD28 molecules are related to evolution, and share common binders, their function and signaling capabilities seem to be unrelated (Balazano and associates, 1992). The comparison of the signaling regions of each of the molecules does not reflect a high identity and suggests that different signaling paths are initiated by each of the molecules (Hutchcroft and Bierer, 1996). Although CD28 is expressed as residing in T cells, and is up-regulated, initially in response to activation, the peak expression of CTLA-4, 48 hours after activation and returns to the levels of the baseline approximately 96 hours after activation (Linsley and associates, 1992a). The expression of CTLA-4 seems to correspond to the down-regulation of CD28 (Lindsten and associates, 1993). Additionally, the signaling pathways mediated through the ligand linkage of the CD28 molecule appear to be important in the upregulation of CTLA-4 expression (Linsley et al., 1993a). T cells that are CD28"do not express appreciable CTLA-4 in response to stimulation with PMA or calcium ionophore (Lindsten and associates, 1993)." A complete sequence of CD28-mediated signaling events remain incompletely defined, although there is a hypothesis that the cascade has been established (Hutchcroft and Bierer, 1996) It has been suggested that CD28 signaling comprises the mobilization of intracellular Ca +, the metabolism of phosphotidylinositol and the induction of tyrosine phosphorylation of the protein (Hutchcroft and Bierer, 1996).

The cytoplasmic tail of the CD28 molecule has defined motifs that are considered to be involved in intracellular signaling after cross-linking by CD80 or CD86 (June and associates, 1994). The intracytoplasmic region 41 of the amino acid has no definable enzymatic activity, does not contain intracellular tyrosine activation motifs (such as TCR) or cysteine residues for the Src family linkage of cytoplasmic tyrosine kinases (June and associates, 1994). However, several potential phosphorylation sites are conserved among the isolated sequences (Hutchcroft and Bierer, 1996). Intracellular enzymatic activity and protein-protein interactions are frequently regulated through differential protein phosphorylation, although the enzymes responsible for this activity by CD28 have not been elucidated (Lu and associates, 1992). A consensus site, YMXM, located in the cytoplasmic tail, is a proposed site of the homology field Src 2 and phospho-tyrosine linker (SH2 field) dependent on the binding of phosphotidylinositol 3-kinase (PI3 kinase) (Prasad and associates, nineteen ninety five). Although this is one of the potential signaling pathways of CD28, it has been shown that PI3 kinase activity does not correlate with IL-2 activity, and as an increase in IL-2 production is a major consequence of the signaling of CD28, it is felt that other trajectories contribute to the activation resulting from intracellular signaling (June and associates, 1994). Although the role of these events is not fully understood, the co-stimulation of CD28 leads to an increased production of cytokines by the T cell. In CD28 + T cells activated with anti-CD3 or PHA, anti-CD28 increases the stable condition of RNA levels from a series of cytokines, including IL-1, IL-2, IL-3, IL-4, tumor necrosis factor (TNFa), lymphotoxin, IFNY, and factor-stimulating factor. the granulocyte-monocyte colony (GM-CSF), as well as the IL-2 receptor (Lenschow et al., 1996). The increase in the stable condition of the mRNA is due both to the stabilization of the transcripts and to the increase in transcription (Hutchcroft and Bierer, 1996). Although co-stimulation of CD28 was first documented in CD4 + T cell clones (Martin et al., 1986), it is now known that CD28 plays a role in the activation of many cell types. The co-stimulation of this trajectory has been shown to regulate the production of I FNY, a Th1-type cytokine, and the production of IL-4, a Th2-type cytokine, in subsets of CD4 + T cells in naive mice (Seder and associates, 1994). The co-stimulatory trajectory of CD28 is also important in the activation of CD8 + CTL, although it does not seem to be necessary for the conducting phase of mediated CTL (Hodge and associates, 1994). Interestingly, it also appears that CD28 plays an important role in H. pylori infection. In cultures of lymphocytes from some virus-positive individuals, the binding of CD28 with the monoclonal antibody can result in a increase in the production of HIV (Asjo and associates, 1993). In primates and rodents, the secondary signal produced by the binding of CD80 with CD28 clearly has a role that can be demonstrated in the initial activation of T cells (Aruffo and Seed, 1987). Recent data suggest, however, that an important interaction effect may also be to sustain proliferation by avoiding the presentation of apoptosis (Lenschow and associates, 1996). The rest of the T cells in the Go phase of growth can be converted into activated T cells through the formation of the TCR complex, but does not have the ability to proliferate or secrete IL-2 in the absence of cross-linking with CD28, a condition called clonal anergy (Linsley and associates, 1991 a). Mature T cells can only be activated through the binding of the TCR with the MHC in an APC, but this eventually leads to the induced activation of dead cells through apoptosis (Radvanyi and associates, 1996). Although other secondary interactions (eg, ICAM-1 costimulation) can provide proliferative helper signals, it appears that costimulation mediated by CD28 is unique in preventing the subsequent presentation of clonal anergy and apoptosis (Linsley et al. 1993a). It has been shown that CD28 can play a role in known regulatory genes that play an important role in the protection against apoptosis of T lymphocytes (Boise et al., 1995). A sustained increase in bcl-x expression is observed in T-cells co-stimulated by the cross-links of CD28 (Boise and associates, 1995). It is believed that the co-stimulation of CD28 can act to stabilize the mRNA of bcl-x.t, preventing the presentation of apoptosis, the expression of the encoded polypeptide (Radvanyi and associates, 1996). It has been shown that the CD28 binding has a role in increasing the production of a variety, both of the auxiliary cytokines T of type 1 and type 2 (Lenschow et al., 1996).

It also seems to have a role for this interaction in the development of specific subsets of T helpers. Naive mouse CD4 + T cells will normally develop a Th1 phenotype if activated in the absence of CD28 / CD80 mediated signaling (Lenschow and associates , nineteen ninety six). This may be an indirect role since the production of IL-4 could be induced by the exogenous induction of I L-2, and the role of CD28 signaling in the production of IL-2 has already been explained (Seder and associates, 1994). Additional studies comprising the CD28 of unconscious mice further support a role for the recipient in the development of Th2 cells. The mice with CD28"7" were developed by Shaninian and associates, with the intention of establishing the way in which an animal adapts to the infection, in the absence of the secondary signal derived from CD28 (Shaninian et al., 1993). The gene was interrupted in the cells of the embryonic stem, by partial replacement of the second exon with a neomycin resistance gene (Shaninian et al., 1993). The PBMC of the mouse homozigo to render it unconscious was found not to express CD28 in its T cells, whereas it found that CD28"+ heterozygotes have a reduced expression of the surface of the molecule (Shaninian et al., 1993). Mitogenic stimulation of the T cells derived from CD28"'" of the mouse had a reduced proliferation of T cells and a production of cytokines that could only be partially restored by the exogenous IL-2 (Shaninian et al., 1993). , that highly purified T cells are not activated by lectins in the absence of APC (Unanue, 1984) With this deformation of unconsciousness, it has been shown that the interaction of CD28 / CD80 is required for the mitogenicity of cell lectins T (Shaninian and associates, 1993). It was also found that the interaction is important in mediating the isotypic changes of B cells, in response to antigen (Shaninian and associates, 1993) In contrast to unconscious CD80 mice, a demonstrable role for CD28 could be determined, using the gene's unconscious technology. Still, the CTLA-4 unconscious mouse has been reported, but the overproduction of CTLA-4 Ig in a transgenic mouse has been studied (Lane and associates, 1994). As might be anticipated, this deformation has some phenotypic characteristics that are similar to the deficient deformations of CD28 (Lane and Associates, 1994). Although the isolated T cells produce normal amounts of IFNY, appreciable minor amounts of I L-4 were obtained at the stimulation (Rónchese and associates, 1994). This leads to the lack of ability of B cells to mount or maintain an appropriate humoral response (Rónchese and associates, 1994). Although there are many proposed trajectories for the differentiation of Th1 and Th2, the interaction of CD28 / B7 has a demonstrable influence on the differentiation of the subset of T cells.

The stabilization of interleukin-2 in mRNA may be a critical function of cross-linking of CD28, but it has been shown that a range of other cytokines are directly or indirectly affected by this interaction (Linsley and associates, 1991 a). Inflammatory mediators IL-1 a, I L-6 and TNFa are produced in populations of T cell memory in response to CD28 signaling, whereas in populations of naive mice only IL-1 was produced a (Cerdan and associates, 1991, van Kooten and associates, 1991). The expression of IL-4 is also regulated through the signaling path of CD28 (Seder and associates, 1994). IL-5, IL-10 and IL-13, other important mediators of the humoral response, are also up-regulated through interaction (deWaal Malefyt and associates, 1993, Minty and associates, 1993). In addition to the stimulation of the colony and the growth factors that include the GM-CSF, CSF-1 and IL-3 and the chemotactic factors that include I L-8, all are up-regulated with the signal produced by CD28 (Harán and associates, 1995). Although the potential use of CD80 in the induction of cancer immunity has been discussed above, there is a range of other clinical uses proposed for CD28 and CD80. The prevention of the interaction between CD28 and CD80 has been demonstrated in rodent model systems to facilitate the prevention or treatment of some autoimmune diseases, in the prevention of the presentation of organ rejection, or transplants against host disease, and in the prevention of the released cytokine associated with sepsis (Harán and associates, 1995; Nickoloff and associates, 1993; Thomas and associates, 1994; Zhou and associates, 1994). The addition of CTLA-4 Ig to block the CD28 / CD80 interaction in mice can avoid lupus-like symptoms in NZB / NZW mice, and can partially protect against lethal EAE and lethal nephritis in rats (Harían and associates , nineteen ninety five). Although this form of immunotherapy has not been attempted for autoimmune diseases in humans, it has been observed in humans that in psoriasis and rheumatoid arthritis, biopsies demonstrate the expression of CD80 whereas in normal biopsies, this expression is absent (Nickoloff and associates, 1993, Thomas and associates, 1994). In bone marrow, and organ transplants in mice and in human experiments in vitro, the addition of CTLA-4 Ig and the prevention of CD28 / B7 interaction may result in at least partial protection against the rejection of organs, the prevention of GVHD or the induction of specific tolerance to the antigen (Harán and associates, 1995). And finally, the release of cytokine and the presentation of sepsis that can lead to septic shock or septicemia, can be avoided in mice by the administration of CTLA-4 Ig in vivo (2.hou, et al., 1994). The manipulation of the CD28 / CD80 interaction provides a more complete understanding of T cell costimulation and provides the perception of establishing solutions for a variety of problems.

MATERIALS AND METHODS Isolation of an initial fragment of CD28 The mRNA was extracted from the HK5 peripheral blood lymphocytes stimulated for 16 hours with a Con A, using the ARNzolB RNA extraction reagent (Biotexc, Houston, TX). Initially, the cDNA was derived from this RNA by reverse transcriptase (RT), a reaction that uses the oligo dT as the 3 'preparer. Briefly, RNA and oligo dT were heated to a temperature of 75 ° C for 3 minutes to remove the secondary structure. Subsequently, RT, dNTP, regulator and distilled water were added, and the mixture was incubated for 1 hour at a temperature of 42 ° C. After this incubation, the sample was heated to a temperature of 95 ° C for 5 minutes to deactivate the RT. Preparers of the degenerate derived from consensus regions found within CD28 of human, murine mice and rabbits, published nucleic acid sequences (GeneBank, Bethesda, MD), were then employed for the initial amplification of a 673 nucleotide fragment encoding most of the open reading structure.

CD28-1 13: CAA CCT TAG CTG CAA GTA CAC (NO.I D. SEC: 70) CD28-768: GGC TTC TGG ATA GGG ATA GG (SEQ ID NO: 71) To amplify the product, a warm start PCR protocol using Taq polymerase (95 ° C for 5 minutes, 1 cycle, 95 ° C for 30 seconds, 48 ° C for 30 seconds and 72 ° C for 45 seconds, 30 cycles, 72 ° C for 7 minutes , 1 cycle). Subsequently, the fragment was visualized on a 1% agarose gel, and ligated into the TA cloning vector (In Vitrogen, San Diego, CA), and sequenced as described above. From the frequency of the cDNA, specific 3 'primers were derived and synthesized for use in the 5' RACE reactions.

CD28190: CGG AGG TAG AAT TGC ACT GTC C (SEQ ID NO: 72) CD28239: ATT TTG CAG AAG TAA ATA TCC (SEQ ID NO: 73) Isolation of the 5 'region A modified RACE 5' GI BCO protocol (Gibco BRL, Gaithersburg, MD) was employed to obtain the remaining 5 'sequence of the CD28 molecule from feliNO. The RNA was extracted from a PBMC stimulated With A for 16 hours. A specific preparer of the 3 'gene was used for the first row of cDNA synthesis. The RNA and the preparator were heated to a temperature of 75 ° C for 5 minutes before the addition of the other RT reagents. After denaturation, the mixture was cooled to a temperature of 4 ° C and the reaction regulator, magnesium chloride, dNTP, DTT and SuperScript RT (Gibco BRL, Gaithersburg, MD) were added. The RT mixture was incubated at a temperature of 42 ° C for 30 minutes and then heated to a temperature of 70 ° C for 15 minutes to denature the RT. The RNase cocktail was then added to the incubated reaction at a temperature of 55 ° C for 10 minutes for the removal of the residual RNA and to prevent the extension of the wrong terminal transferase (TdT). The cDNA was then purified on a GlassMax agitator column (Gibco BRL, Gaithersburg, MD) to remove the unincorporated dNTP and preparer. The purified cDNA eluted from the column was then tested with TdT. The TdT was used to add a tail dC from nucleotide 20 to 30 to the cDNA. The enzyme was added to a mixture of purified cDNA, magnesium chloride, reaction regulator, and dCTP after denaturation of the cDNA at a temperature of 95 ° C for 3 minutes. The reaction was incubated at a temperature of 37 ° C for 10 minutes, and then the enzyme turned off, was heated to a temperature of 70 ° C for an additional 10 minutes. The added cDNA was amplified in a Taq polymerase based on the warm start PCR reaction (95 ° C for 5 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 45 seconds, 35 cycles; ° C for 7 minutes). The primers for this reaction included 3 'preparer located in the 5' synthesis preparer of the cDNA, and an anchor preparer specific for the dC linker and largely composed of dG with few di. One μl of this reaction was diluted in 50 μl of water and 5 μl of this solution were then used in a nested PCR reaction (95 ° C for 5 minutes, 1 cycle; 95 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 45 seconds, 30 cycles with a KlenTaq polymerase mixture), with a 5 'dG / dl anchor preparer and an additional 3' specific gene preparer upward. Then thirty μl of the nested reaction was visualized on a 1.5% agarose gel, and the appropriate fragment was extracted from the gel (Figure 19). The cDNA was purified as described above with the Amicon gel nebulizer, and the micropuric filter (Amicon, Beverly, MA). The purified cDNA sample was sequenced through a dye finisher cycle, from the dye finalizer cycle sequence elaboration (Perkin Elmer, Norwalk, CN). From the completed fragments, a consensus sequence was derived. From the sequence, a pair of primers comprising the full open reading frame of the feline CD28 gene was synthesized: feCD28 5 ': CGC GGA TCC ACC GGT AGC ACÁ ATG ATC CTC AGG (NO.I D. SEC: 13) feCD28 3 ': CGC GGA TCC TCT GGA TAG GGG TCC ATG TCA G (SEQ ID NO: 14) Using these primers, a cDNA molecule that includes the entire coding region was amplified from the stimulated EK6 and ED3 by means of Con A from the cDNA derived from the PBMC. The cDNA of PBMC was produced previously and has been shown to contain the RNA encoding the gene. This PCR reaction (95 ° C for 5 minutes, 1 cycle, 95 ° C for 30 seconds, 42 ° C for 30 seconds and 72 ° C for 45 seconds, 30 cycles, 72 ° C for 7 minutes), using DNA polymerase KlenTaq in the hope of reducing random errors, frequently associated with Taq polymerase, a 754 bp fragment was produced, which was cloned into a cloning TA vector and sequenced as described above. As with the CD80 molecule, each of the nucleotide sites was confirmed by at least three independently derived sequences.

RESULTS The degeneration of selected primers from the consensus regions within murine, human and rabbit mouse CD28 cDNA sequences were used in the PCR reaction and successfully produced a product comprising almost all the coding sequences of feliNO. Due to the higher degree of conservation found in the CD28 molecule, the initial amplification using degenerate primers produced virtually the entire molecule. In contrast to the feline CD80 molecule, in which only a small central fragment was initially produced, only the majority 5 'of nucleotides 1 13 of the open reading frame of the CD28 cDNA were missing (Figure 20). This sequence of the initial fragment shared a homology of 86% with the analogous region within the human sequence, 86% identity with the rabbit cDNA and a homology of 79% with the murine mouse coding sequence. The 5 'ATG, and the additional 1 10 nucleotides, as well as some 5' ascending sequences, were isolated using a 5 'RACE PCR (Gibco, Gaithersburg, MA). The cDNA transcribed from the EK6 stimulated by Con A of the PBMC was used in the reactions of the tails. With this material, the amplification with preparer CD28-786 and the anchor preparer dG produced a poorly definable material (Figure 21). Although unidentifiable bands were amplified with the dG / CD-786 preparator combination, the diluted cDNA of this reaction was amplified using nested CD28 primers, CD28-182 and CD28-239. A visible band was present at approximately 600 bp. This product, when isolated from an agarose gel and sequenced, contained a 5 'upstream sequence that included and continued through the initial codon (Figure 22). From the sequence of these products, a 5 'preparer was derived which included the initial codon. This preparer, in combination with the 3 'construct were used to amplify the cDNA of the RNA extracted from EK6 and ED3, the PBMC stimulated with Con A producing a 754 bp product (Figure 23). At least two of the products of each animal were completely sequenced, and each nucleic acid site was checked and confirmed with at least three correctly independent reading sequences. After processing of the full-length product sequence, the sequence of the TA cloning vector to ensure a consistent and reproducible product. Within the final 685 bp fragment encoding the complete open reading frame, the ATG 5 'was located at position 1, the stop codon was found at position 664-666, with the additional 19 nucleotides in the 3' UTR (Figure 24). In a similar manner as with the CD80 faith molecule, the 5 'position of the ATG codon was confirmed through the sequencing of the 5' RACE PCR products (data not shown). Once sequenced, the feline CD28 gene demonstrated a general identity that was closest to the rabbit and human sequences (Figure 25). The murine mouse cDNA homology was still strong, although the identity with the hen sequence was more divergent, so that it is comparable to that seen between the hen sequence and other mammalian genes (Table 2).

Table 2: Comparison of the sequence homology of the feline CD28 with murine, human, chicken and rabbit mice.

Species Percentage of homology with the feline sequence: Amino Acid Nucleotide Human 85% 82% Mouse 77% 74% Rabbit 84% 84% Chicken 59% 50% An amino acid peptide sequence was derived from the nucleic acid sequence, as described above. The identity with the peptide sequences derived from the other published genes was comparable with the identity at the nucleotide level. The signal sequence of the peptide extends from the 5 'methionine through residue 19. It appears that the feline molecule, as well as in other cloned CD28 polypeptides, the single extracellular IgSF field that appears variable extends from residue 19 to 153. The hydrophobic membrane that traverses the field extends for the next 27 residues, and is followed by the cytoplasmic tail of amino acid 41. As with the human CD28 molecule, the feline polypeptide has 5 potential N-linked glycosylation sites (Figure 26). Comparison of the anticipated amino acid sequences of feline and human CD28 proteins demonstrated regions of homology with some differences (Figure 27). Most changes can be found in the transmembrane field and in the signal sequence and in the NH3 terminal field. The highest degree of homology is found in the central IgSF field similar to V, and in the cytoplasmic tail. Comparisons of the feline CD28 molecule with the anticipated human amino acid sequences and the murine members of the CD28 / CTLA-4 family demonstrate that, although there is only a general 25% homology among the members of this group, those specific regions and the waste is maintained. The MYPPPY motive is retained by all members of this group. Additional residues retained in the feline molecule appear that are anticipated to be important for structural integrity, including a number of conserved cysteine residues (Figure 28). The cytoplasmic field of the feline CD28 molecule is conserved moderately with other published sequences, especially mammalian sequences (Figure 29). It is proposed that a variety of intracellular signaling pathways are mediated as a result of cross-linking of the extracellular portion of the receptor (Hutchcroft and Bierer, 1995). The hydrophilicity plots of the anticipated amino acid sequence of the feline CD28 when compared to similar plots of the human polypeptide, further demonstrates the probability that each of the proteins retains a similar structural integrity. However, when changes occur in the amino acid sequence, there appear to be no significant changes in the hydrophilicity of the molecule, which reflect that changes in the amino acid sequence are homologous in an important way. It is important to know that the hydrophobic membrane that crosses the field, in which the feline and human peptides only share a 75% homology, although they have very similar hydrophilicity profiles (Figure 30).

EXPLANATION The sequences of the cloned CD28 molecules showed all, a moderate level of conservation of evolution. It can be hypothesized that the role of the molecule in the activation and mediation of T cell-mediated immunity is retained in a higher vertebrate variety, from poultry, to rodents and carnivores, and that includes the older primates. The comparison of the putative amino acid sequences of each of the molecules shows a moderate homology in the extracellular field portions, which are proposed to be involved with the binding of the ligand and in the intracellular regions, it is proposed that they promote intracellular signaling . In general, the highest degree of homology is found in the vicinity of the proposed site region of the binder bond, MYPPPY, located in the IgV field of residues 1 18 to 123 in the feliNO polypeptide. The proposed signal sequence of the peptide extends from 5 'methionine to residue 19 (Aruffo et al., 1987). Monomeric CD28 is composed of a single extracellular IgSF field similar to the variable, which extends from residue 19 to 153 (Aruffo et al., 1987). The hydrophobic membrane that traverses the field extends for the next 27 residues, and is followed by a cytoplasmic tail of 41 amino acids (Aruffo and associates, 1987). The feline protein has 5 potential glycosylation sites linked in positions identical to those found in the human protein. Interestingly, the glycosylation site located at residue 105 in feline protein is NQS, whereas the sequence in human is NQT. This divergence of the amino acid further reflects that, although there are frequency changes between the molecules, the general characteristics of the structure are retained. As might be expected, due to the level of homology shared by the protein, the comparison of the feline and human CD28 hydrophilicity plots demonstrates that the molecules share potentially similar conformational patterns. However, it also reveals that when there is a residue that is altered, the change is generally homologous. Although the transmembrane field is the area of the molecule with the lowest degree of conservation, it simply retains its required hydrophobic character, the cytoplasmic field of the feline CD28 molecule is moderately conserved with other published sequences. It is proposed that a variety of intracellular signaling and pathways are mediated through cross-linking of the extracellular portion of the receptor and although the intracellular portion of the CD28 polypeptide has no intrinsic enzymatic activity, but rather a binder bond, results in activation of intracellular molecules (Aruffo and associates, 1987). There are four conserved tyrosine residues (? I73j? I8ßj? O? And Y200J which has been proposed as potential sites for tyrosine phosphorylation (Lu and associates, 1992) .In addition, the MNM sequence that starts at the residue 193 of the feline molecule, is proposed as a site of a SH2 field in murine and human mouse proteins (Prasad and associates, 1995) A potential site of phosphorylation by protein kinase C is retained in S185, whereas T202 may be a site of the proline Erk1 or Erk2 of the serine / threonine-directed kinase activity (Hutchcroft and Bierer, 1996) .As explained above, the signaling function of the CD28 receptor is multifaceted, so It is not surprising that the cytoplasmic tail of the peptide has multiple potential signaling mediators.Future applications of the feline CD28 molecule should include the development of tools to detect the expression of the receptor surface and monitor the expression of CD28 after viral infections, such as FIV. If the tools for detecting proteins can be combined by existing methods of message detection, valuable information about expression levels can be determined during the course of infection. An additional correlation of expression patterns of CD28 expression during the course of chronic FIV infection will act to exemplify the feline system, as the appropriate model of HIV infection in humans, and may also lead to more definitive responses with respect to the course of the infection in both systems.

EXAMPLE 7 EXPRESSION OF PROTEIN CD28 / D80 INTRODUCTION Although the communication of the immune system is mediated largely through soluble factors, the initiation of a response of primary T cells in primates and rodents has been determined to require direct cell-to-cell contact (Mescher, 1992). Originally, we had the idea that this interaction comprised only the interaction between the TCR in the T cells and the MHC in the antigen presentation cells, but it has been clarified, that for the complete activation of the T cells, the link between the accessory molecules (Schwartz, 1992). As explained, the evidence supports the interaction between CD28 and CD80 as the mediator of this accessory signal (Linsley and associates, 1991 a). Many of the receptors and binders in vertebrates are members of the IgSF superfamily (Springer, 1990). The molecules are characterized by the presence of a region similar to immunoglobulin, usually in the extracellular portion of the molecule (Buck, 1992). Although conservation varies, it is often limited to those residues required to generate the fold of Ig (Beale, 1985). The characteristics of the Ig field include two parallel, closely associated ß threads connected by circuits that follow the conserved topology (Williams and Barclay, 1988). Although there is a participation of structural properties among the members of this family, there is a diverse distribution of linkage interactions, and the signaling properties particular to this family (Anderson and associates, 1988). As members of the IgSF family, both CD28 and CD80 share a degree of structural similarity in their extracellular fields. CD28 has a simple extracellular V region, although it is expressed as a disulfide-linked heterodimer (Aruffo and associates, 1987). The extracellular region of the CD80 molecule, however, has C field types and similar to V, and is expressed as a monomer (Freedman et al., 1989). Because the members of the IgSF family share structural characteristics, templates can be used, in a limited way, in order to establish an idea of the three-dimensional structure of related molecules that have not been crystallized (Bajorath et al., 1993). . Although neither the CD28 nor the CD80 polypeptide have been crystallized, the CD2 molecules (Driscoll and associates, 1991) and CDd (Leahy and associates, 1992), with analogous extracellular fields, have been examined by X-ray crystallography. to give us some idea with respect to the structure of the related members of the IgSF group (Linsley and associates, 1995a).

As explained above, CD80 and CD66 share a similar link avidity with CD2d and CTLA-4. However, CD2d is a low affinity receptor for ligands while CTLA-4 has high affinity for both molecules (Linsley and associates, 1994a). Although a potential mechanism has been proposed, it is unclear how the low affinity receptor, with a rapid dissociation range, as possessed by CD2d, can produce the costimulatory signal necessary for the development of T cells (Linsley and Associates, 1995a). It is hypothesized that the binding of CDdO to CD2d on the surface of T cells can promote oligomerization of the receptor which would facilitate productive cross-linking, and signal production (Linsley and associates, 1995a). It has been discovered that CD2d is evenly distributed in activated T cells and therefore, it is proposed that the molecule migrates in the membrane after it fits into the T cell (Damle and associates, 1994). The high concentrations of the oligomerized CD2d would promote the new association of free CD2d, in the cell-to-cell contact region, and would promote signal production despite the rapid dissociation range (Linsley and associates, 1995a). This process, which is called mutual capping, although not directly observed in the interaction of CD2d / CDdO, has been demonstrated for other receptors in which a similar initiation of cell-to-cell contact is required (Singer, 1992). Although it has been demonstrated that the interaction of CD2d / CD80 is critical for the promulgation of the immune response mediated by the T cell, a large part remains unclear about the exact mechanisms of this signaling path (Linsley et al. , 1993a). The existence of two receptors and two binders in this interaction raises questions regarding the role each plays in the activity of the T cell (Linsley and associates, 1992b). Although CTLA-4 binds in a stronger manner, it is expressed much later after activation, and although signaling pathways for CD2d have been proposed, it has not been determined whether a signal is produced at the binding of the binder with the CTLA-4 (Linsley and associates, 1995a).

MATERIALS AND METHODS Preparation of Inserts. The following primers were used to amplify the complete open reading frame of the CD2d and feline CDdO genes for insertion into the expression vectors: feCDdO 5 ': CGC GGA TCC GCA CCA TGG GTC ACG CAG CAA AGT GGA AAA C (SEC ID SECTION 11) faith CDdO-960: CCT AGT AGA GAA GAG CTA AG AG C (NO SEC ID: 12) feCD2d 5 ': CGC GGA TCC ACC GGT AGC ACA ATG ATC CTC AGG (NO SEC ID: 13) feCD2d 3 ': CGC GGA TCC TCT GGA TAG GGG TCC ATG TCA G (SEQ ID NO: 14) The CDdO 5 'preparer and both CD28 primers were designed with the appropriate BamH1 sites and linkers to facilitate insertion into multiple cloning sites. The 3 'BamHl site was designed on the CDdO sequence by means of the exit digestion of the TA cloning vector. The 5 'primers also contain a Kosak box, and the 5' ATG for both genes. In each case, the primers were used to amplify them from the template, coding the complete sequence of each gene that had previously been cloned, into the TA cloning vector, described above. Approximately ten nanograms of each plasmid were used in a PCR amplification based on Taq polymerase (95 ° C for 5 minutes, one cycle, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 6d ° C for 45 seconds, 30 cycles 68 ° C for 7 minutes 1 cycle). The amplified products were visualized by electrophoresis in an agarose gel and subsequently, they were ligated into the TA cloning vector (In Vitrogen, San Diego, CA), as described above. The cross-linking reaction to transform the InvaF 'component cells, and the positive clones were classified and selected, as described above.

Cloning within the pSI. For cloning within the pSI vector to be used in the transformation of COS-7 cells, the plasmid was digested with EcoRI, then the enzyme was removed with the spinning column Micropure EZ (Amicon, Beverly, MA). After removal of the enzyme, the plasmid was treated with phenolxloroform to remove any residual protein and precipitated in alcohol. The inserts were digested from 50 μg of purified QIAGEN plasmid DNA (Qiagen, Chatsworth, CA) of the clones contained in the TA cloning vector with the appropriate inserts, using the EcoRI sites found in the vector flanking the insert. The digestion of 100 μl was subjected to electrophoresis, in a 1.5% agarose gel, and the digested fragment was cut. Subsequently, the insert was purified from the agarose with a gel nebulizer and a microcon filter unit (Amicon, Beverly, MA). The treatment with alkaline phosphatase of the pSI digested with EcoRI, reduced the chances of self-ligating the vector. One hour of treatment at a temperature of 37 ° C with 0.1 U / μg of calf intestinal alkaline phosphatase (CIP) dephosphorylated the digested ends of the vector. The CIP was removed by heat denaturation at a temperature of 65 ° C for 35 minutes followed by purification by shaking with a micropure EZ agitation column (Amicon, Beverly, MA). The inserts were ligated directly into the cut and the dephosphorylated pSI vector, overnight at a temperature of 16 ° C using a ligand of T4 DNA. The molar ratio of the binder to the vector was approximately three to one, with an insert of CD28 or CDdO of 0.05 μg to 0.1 μg of pSI. Then one μl of the ligation reaction was used to transform the competent cell and InvaF '. The cells were passed on LB plates with a content of 50 μg / ml of ampicillin. The plates were incubated overnight at a temperature of 37 ° C and the next day the colonies were inoculated into 5 ml of LB medium containing 100 μg / ml of ampicillin. After incubation overnight at a temperature of 35 ° C with shaking at 220 rpm, the plasmid DNA was extracted, with alkaline lysis, the DNA was purified by extraction with phenolxloroform, and precipitated with two volumes of 95% ethanol. The DNA was treated with RNase and then digested with 10 U of EcoRl. Digests were visualized on a 1% agarose gel to identify positive clones. The plasmid DNA was then extracted from 5 ml of the overnight culture of the positive clones, using the QISprep shaking columns (QIAGEN, Chatsworth, Ca) (Figure 31). Then, the purified DNA was sequenced by elaborating the terminator dye cycle sequence with an internal 3 'primer to determine the orientation of the insert in the plasmid. The location of the preparer was such that the sequence preparation had proceeded throughout the union between the vector and the insert to ensure that the orientation was complete. A clone of each of the genes with the plasmid in the appropriate orientation was then cultured in a 100 ml culture, and the plasmid extracted with a QIAGEN maxi-prep column.

(Chatsworth, CA).

Cloning within the SFV. For insertion into the SFV vector, the vectors and the plasmid were treated in a very similar way. One hundred μg of the SFV sector were digested with 120 U of BamH l for one hour at a temperature of 37 ° C (Figure 32). The enzyme was removed from the digest by centrifugation through a micropure EZ filter (Amicon, Beverly, MA). Then the plasmid was treated with CIP. The CIP was heated and deactivated and then the plasmid was again purified with a micropure EZ filter. The inserts were extracted from the DNA of the TA cloning vector purified by the digestion of BamH I. The inserts were purified and ligated into the vector, as described above. After transformation of the competent InvaaF 'cells, the plasmid insert and orientation were confirmed with the elaboration of terminator dye cycle sequence, as described above. A large scale of the plasmid preparation was performed in a positive clone of each gene.

Protein expression pSI. For the transformation of the pSI protein of eukaryotic cells, COS-7 cells were obtained from an American culture-type culture (ATCC) collection. The frozen material was resuspended in 15 ml of DMEM plus 10% fetal calf serum (FCS). Then, the cultures were grown as a monolayer in T-75 flasks. One night before the transfection, the cells were removed from the flasks after their treatment with a 0.25% solution of EDTA trypsin, washing them with PBS. The cells were then seeded at a confluence of -20% into 100 mm tissue culture flasks and allowed to grow to a confluence of -50% during the next day. For each of the dishes to be transfected, 5 ml of DMEM-NuSuero (Collaborative Biomedical Products, Bedford, MA) was mixed with 0.2 ml of a DEAE-dextran / chloroquine solution. Ten μg / ml of the purified pSI plasmid was then added to the mixture. The medium was aspirated from the COS cells and the DMEM-NuSuero / DEAE-dextran / chloroquine / DNA solution was added to the cells. The culture was incubated for 3.5 hours in a 5% C02 incubator followed by medium removal and replacement with 5 ml of 10% DMSO in PBS. After 2 minutes this solution was aspirated, and the cells cultured overnight in 5 ml of DMEM / 10% FBS. The next day, the cells were divided into two 100mm culture dishes. After 3 days, the medium was aspirated and the transformed cells removed with PBS / 0.5 μM EDTA. The PBS / EDTA mixture was added to the cells and then incubated for 15 minutes at a temperature of 37 ° C. The float was removed, and combined with subsequent PBS washes. The float and the washes were subsequently centrifuged. The resulting pill was resuspended in DMEM / FBS, and COS cells were counted.

Expression of the SFV Protein. Transfection with the SFV vector was performed in baby hamster kidney (BHK) cells. Thirty μg of purified plasmid were digested with Spel for 1 hour at a temperature of 37 ° C. The enzyme was then removed with a micropure EZ filter (Amico, Beverly, MA) and the DNA precipitated with 2.5 volumes of 95% EtOH. Then one and a half μg of the plasmid was used as a template for in vitro transcription mediated by Sp6. Briefly, the DNA was incubated for one hour at a temperature of 37 ° C with: transcription buffer, 100 mM DTT, 10 mM mixture of G (5 ') ppp (5') G, rNTP, water, RNasin, and 60 U of Sp6 RNA polymerase. After transcription, the reaction was divided into aliquots and a sample was visualized on a 1% agarose gel. Forty-five μl of the transcription reaction was used to transfect BHK cells at a confluence of -80% in T-75 flasks. The GMEM medium plus the FCS medium 10% was aspirated from the cells and replaced by an Opti-MEM medium. After a 2 minute incubation, this medium was replaced with Opti-MEM medium / 9 μg / ml of lipofectin / transcript RNA. The cultures were incubated for two hours at a temperature of 37 ° C in a C02 at 5% with frequent manual agitation. After 2 hours, the medium was removed, and replaced with 10% GMEM-FCS. The cultures were incubated for a period of 7 to 9 hours, and the cells were subsequently removed by trypsinization.

Cloning within the pQE. A pQE bacterial expression vector was also constructed with the feline CD28 and CDdO gene. The cut, and the pQE plasmid treated with CIP prepared and purified as described above, was ligated with DNA ligase T4 in a molar proportion of insert plasmids from four to one with 50 ng of CD2d or gel-purified CDdO. The ligation was incubated for 16 hours at a temperature of 16 ° C, then two μl of the ligation reaction was used to transform the competent INVaF 'cells. The positive colonies were selected and the orientation of the insert confirmed by means of the elaboration of the sequence. The large scale preparation of the purified plasmids was performed and was used to transform the competent M pREP4 M cells by means of a treatment with rubidium chloride. The transformed cells were grown on LB plates with 50 μg / ml of Canamycin and ampicillin to ensure that both the pQE and the pREP4 helper plasmids were retained in these colonies. Then the positive colonies were selected by means of mini-preparations of alkaline lysis, and restriction digestion of BamH l. Colonies with confirmed inserts were frozen in a solution of 50% glycerol material for future use.

Link Test.

The binding assays for transfected cells expressing feline CDdO and feline CD2d were performed after the protocol described in Linsley et al., 1994a. One day after transfection, COS-7 cells expressing CD2d were removed from the T-75 flasks with a trypsin-EDTA treatment. The cells were allowed to adhere to the plates of 24 vessels at a cell concentration of 1x105 per ml. Two days later, the feCDdO / pSI cells transfected with COS-7 were removed from the T-75 flasks with PBS / 0.5 μM EDTA. These cells were then fluorescently labeled with a 5 μM solution of Calcein AM (Molecular Probes, Eugene, OR) in sterile PBS / 1% BSA for 30 minutes at a temperature of 37 ° C (Akeson and Woods, 1993) . Transfected infected COS-7 cells were labeled in the same way. The labeled cells were then washed three times with DMEM plus 10% FCS to remove the unincorporated label, counted and added directly to the monolayer. The two cell populations were allowed to interact for 1 hour at a temperature of 37 ° C. Non-adherent cells were removed by gentle washing of the monolayer 3 times with DMEM + 10% FCS. After washing, the fluorescence of each of the containers was quantified in a microplate fluorometer. Fluorescence in the containers containing infected populations was compared to the vessels, in which the CDdO expression cells were added to the COS-7 cells transfected with only the pSI plasmid. The competitive binding assays, using CTLA-4 Ig and CDdO Ig fusion proteins (kindly provided by P. Linsley, Bristol-Meyers Squibb) to inhibit cell / cell interactions, were designed to demonstrate the specificity of the interaction. After labeling with calcein, but before addition of the CDdO expression cells to the monolayer, CTLA-4 Ig cells were incubated in DMEM / FCS at a concentration of 1 μg / ml with the labeled transfected cells for 30 minutes. The cells were washed twice in DMEM / FCS and added to the monolayer. Alternatively, the CD28 expression cells in the monolayer were incubated for 30 minutes, with a CDdO Ig concentration of 1 μg / ml in DMEM / FCS and followed by washing, and fluorescently labeled CDdO expression cells were added thereto. The inhibition of binding by the fusion proteins was calibrated by comparing the fluorescence in these vessels with the bond observed in the vessels without the competitors.

RT-PCR. The transfected COS cells were also assayed by mRNA transcription by RT-PCR. After three days, the RNA was extracted from the cells transfected with pSI by means of feCD28, feCD80 or without insert. The RNA was treated with RNase-free DNase to remove the DNA contamination potential. One half of μg of RNA was then subjected to reverse transcription to cDNA using an oligo dT preparer, and MuMLV reverse transcriptase. Each cDNA sample was then amplified with specific preparation sets for CD2d, CDdO and G3PDH with the following temperature cycle; 95 ° C 5 minutes 1 cycle; 95 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds, 30 cycles; 72 ° C 5 minutes, 1 cycle. Then 20 μg of each of the reactions were visualized on a 1% agarose gel. The feline CD2d and CDdO genes were successfully inserted into three protein expression vectors (pSI, SFV and pQE). After ligation of the respective vectors, the genes were used to transform the competent INVaF 'cells. Figures 33 through 33 show each of the vectors with the appropriate cDNA inserts. Binding assays were performed to demonstrate that the functional protein could be expressed. Initial tests were performed to determine the relationship between the binding of COS-7 transfected by CD2d and CDdO, and transfected CD2d and transfected COS-7 imitation cells. Fluorescence in the containers in which the non-adherent transfected CDdO cells were fluorescently labeled were added where they were larger than the control vessels in the two initial dilutions. The interaction was in response to the dose, and after two initial dilutions, the retention of fluorescence was the same in the vessels in which the surface protein expressed from the adherent cells as in the transfected imitation controls (Figure 39). ). To illustrate that this interaction could be inhibited, the transfected cell lines were incubated with a soluble counter receptor before mixing. At concentrations of 5x105 and 1 x105 cells, the fluorescence in the vessels containing the COS cells expressing CD23 and CDdO were similar to those observed in the previous experiments. In containers in which the adherent cells were incubated with the CDdOlg receptor-counter prior to mixing, the retention of fluorescence was comparable to that found in the transfected imitation cell vessels used for the control. When COS cells transfected with CDDO pSI were incubated with soluble CTLA-4, prior to their exposure to CD2d cells transfected by pSI, however, the fluorescence was not so completely inhibited. Although the levels are not as significant as those seen with the non-inhibited groups, this was clearly greater than with any of the control groups or other experimental groups (Figure 40). RT-PCR was carried out in RNA derived from COS-7 cells transformed with pSI-CD2d, pSI-CDdO and transfected imitation cells to demonstrate the specific presence of mRNA for each gene in the cell line. Figure 32 illustrates the 1% agarose gel for each cell line. The gene for G3PDH was amplified in each set to show RNA integrity, and as a positive control the transfected mimic set. COS-7 cells transfected by CDDO pSI expressed a mRAN CDdO, and a G3PDH message, while COS-7 cells transfected with CD23 pSI expressed the genes for CD2d and G3PDH. Transfected imitation cells expressed only G3PDH (Figure 41).

EXPLANATION. The lack of adequate antibodies indicated that a direct assay could not be performed to demonstrate the expression of the peptide. The commercially available anti-Hu CD2d and CDdO monoclonal antibodies were tested on isolated lymphocytes to determine if cross-reactivity existed. The FACS analysis using the antibodies in the cells confirmed by the PCR as cells expressing messages for both surface proteins was unsuccessful. This in combination with the fact that antibodies do not recognize surface expression in cells transfected by pSI, led us to the conclusion that these antibodies were not cross-reactive. The CDdO antibody was not expected to be cross-reactivated. The limited homology between the human and feline CDdO molecule would limit the potential for cross-reactivity in a monoclonally derived antibody. This was somewhat surprising, however, that the anti-hu CD2d was not cross-reactive. Although there is a higher degree of conservation among the cloned CD28 molecules, a commercially available anti-hu CD2d antibody that cross-reacted with the mouse protein was not found. As with the anti-hu CDdO antibody, the monoclonal CD2d hu test was also unsuccessful. So we had to devise an essay that could demonstrate, not only the probability that the peptides had been expressed, but also show that they were functional and capable of interaction. The CDDO and CD2d feline cDNAs were successfully inserted into a series of expression vectors. Although the pSI vector met the requirements necessary to perform the binding assay, additional vectors can facilitate future protein expressions. After transfection, expression of the CD2d mRNA and CDdO by the transformed COS-7 cell lines was confirmed by RT-PCR. The DNase treatment of the RNA before the PCR reaction must have significantly reduced any possibility of genomic or plasmid DNA contamination. In addition, it was not perceived that the COS-7 cell was expressed naturally, either of ligand which was further confirmed by the lack of message of either of the surface proteins and the control and imitation cells transfected used for control. The amplification of the correct RNA message from the transfected cells seems to reflect that the pSI template was present inside the cells, and that the message encoded by the plasmid was being transcribed. The linkage assays performed were designed after those performed by Peter Linsley to demonstrate the similar binding avidity of CDdO and human CDd6 (Linsley and associates, 1994 a). A modified format was used to demonstrate that feline CD2d expressed on the surface bound to feline CDdO expressed on the surface, and that the interaction could be inhibited with the soluble receptor. The level of binding could be deduced from the fluorescent retention of the cells in the specific containers. Because a fluorescent plate reader was used, the cells were not required to be used before the fluorescence measurement. The initial assay showed that the retention of COS cells transfected with fluorescently labeled CDdO-pSI was higher in the vessels in which the adherent cells were transfected with CD2d-pSI, than in the vessels in which the adherent cells were transfected as imitation. The control cells, and the COS imitation cells transfected with the pSI lacking an insert, ensured that neither the presence of the vector, nor the transfection process itself, nor the adhesive properties of the cells, result in adhesion between the cells. cells mediated by the interaction between CDdO and CD28 expressed on the surface. At the initial dilution of 1 x 106 cells, the fluorescence in the vessels in which the cells were expressing CD23 was approximately 5 times greater than that of the vessels, in which the cells transfected with CDdO fluorescently labeled were introduced into the cells. of the containers containing the transfected cells by imitation. In the 5x105 cells, the fluorescence drops were due, significantly, to the reduction in the number of cells, but the level is still significantly higher than the fluorescence in the control cells. By means of a cell concentration of 1 x 105, it is not possible to distinguish in a statistical way, the difference between the experimental cells and those of control and those of 1 x104 they are closely identical. This assay indicates that an interaction between COS cells transfected with CDdO and CD2d is occurring, which results in the retention of transduced CDdO cells fluorescently labeled in the containers. When the adherent cells were not surface expression proteins, however, the cells transfected with CDdO were removed by means of a gentle wash, this effect could be titrated, and by means of the fluorescence of the cells 1 x 10 4 in the recipients They were virtually identical. In order to confirm that an interaction was occurring, soluble receptors were introduced to inhibit the interaction of CD28 / CD80. The second trial comprised the introduction of soluble forms of receptor-counters for each of the peptides, in an attempt to inhibit the interaction between the adhesion partners. A soluble receptor for CDdO, huCTLA-4lg and for CD2d, huCDdO-Ig were incubated with the respective transfected COS cells expressing the receptor-counter of each molecule before mixing the two cell types. Although the soluble proteins were not of feline origin, it was felt that due to the level of conservation found between the proposed binding regions of the human molecules and the analogous regions of the feline molecules, there would be sufficient cross-reactivity. In addition, fusion partners of human molecules bind to their receptor-counters of murine mouse molecules (P. Linsley, personal communication). Due to the number of cells required in the assay, it was not possible to perform the 1 x 106 cells assay. The first concentration was from 5x105 cells demonstrating the transfected CD2d / CDdO cells alone, an average of similar fluorescence found in the previous experiment. It was not clear why the adherent cells incubated with the soluble CDdO receptor had a fluorescence close to that of the transfected imitation control, whereas the transfected CDdO cells non-adherently fluorescently labeled, incubated with the soluble CTLA-4 had approximately one fold from two to three higher fluorescence of two to three folds. Regardless of the differences mediated by the type of soluble receptor, there was a marked reduction in the amount of fluorescence in the containers, in which the soluble receptor was introduced onto the containers in which the receptor was not present. The interaction demonstrated by the above assay can be inhibited by introducing the appropriate soluble receptor-counter prior to mixing the cells. Although monoclonal antibodies specific for surface proteins would, in general, appear preferable for this type of assay, in the absence of appropriate reagents it seems a viable format through which the expression of functional receptor-counters can be demonstrated. The results of the initial binding assays in combination with the competitive binding assays confirm that the cDNA of the CDdO and the functional feline CD2d have been isolated, and also that the proteins expressed by the message functionality interact. Although the applications of this type of binding assay are limited, it remains an efficient system to demonstrate the probability of functional surface expression and interaction.

Example 8 INFECTION. INTRODUCTION The Lwoff viruses defined as "strictly intracellular and potentially pathogenic entities with an infectious phase and 1) that possess only one type of nucleic acid, 2) that multiply in the form of their genetic material, 3) that do not have the capacity to grow and pass through binary fission, and 4) are devoid of a Lippmann system "(Lwoff, 1957). Viruses are not cellular in nature, whose genome, either RNA or DNA, directs the synthesis of additional viral particles through an infected host cell (Luria and Darnell, 196d). Viral diseases represent an interesting system, in which the practical applications of the B7 / CD2d signaling complex can be demonstrated. Among retroviruses, infection with VI H in humans, and feline immunodeficiency virus (VI F) in cats, results in the destruction of normal immune function, which is hypothesized to occur through the elimination of CD4 + T cells (Fauci and associates, 19d4, Pedersen and associates, 1967). It is considered that the CD2d / CDdO signaling complex plays a role in the disease, and that the manipulation of the expression of the receptor can exacerbate the infection (Harán and associates, 1965). FIV is a very real clinical problem in domestic cats, which causes a series of clinical and subclinical manifestations that closely resemble those of HIV infection in humans (Pedersen and associates, 19d7). As more information is accumulated about FIV, the appropriateness of this information to an animal model for human AIDS is becoming increasingly evident and that it is not the primate model that more closely mimicked the progress of the disease in humans (Pedersen and associates, 19d7). The molecular, biological and pathogenic similarities also suggest that much of the information obtained in VI H studies may accelerate the understanding of FIV infection in cats. Initially, VI H infection is manifested by a transient lymphopenia with development similar to the mononucleosis syndrome, during the time of seroconversion (Clark et al., 1991). There is a decrease in the population of CD4 + T cells and CDd + T cells in the short term, which results in an initial decrease in the CD4: CD8 proportions that may contribute to an additional decrease during the asymptomatic phase of the disease (Cooper et al. , 1964). Through the presentation of symptoms related to SI DA, the population of CD4 + cells decreases in a serious way, and as the disease progresses to its terminal phase, the entire population of lymphocytes is drastically reduced (Fauci et al. associated, 19d4). Although the initial lymphocytopenia is probably caused by the cortico-steroid-induced changes in the immune cell population, as seen in other viral diseases, the additional loss of CD4 + T cells and the expansion of T cells are considered. CDd is related to viral production and to pathogenesis (Fauci and Dale, 1975; Fauci and associates, 19d4). The development of appropriate model systems is a critical step in the additional mechanisms of elucidation of infection and virally induced disease.

FIV, a T lymphotropic retrovirus, was originally described in a colony of cats in California in which multiple chronic infections occurred frequently (Pedersen and associates, 19d7). Although the disease manifests itself in a manner similar to VI H in humans, and is taxonomically distant, it is antigenically distinct from the causative agent of SI DA in humans (Siebelink and associates, 1990). The transmission of infection occurs through the exchange of infected body fluids as with VI H, but unlike VI H in which sexual transmission is the main route of infection, it seems that with FIV most infections occur through transmission through saliva, through bites (Yamamoto and associates, 1969). Despite the differences in transmission, the resulting immunodeficiency syndrome is one of the best models of related diseases in humans (Siebelink and associates, 1990). The clinical progress of FIV is similar to that of VI H, with the disease subdivided into five clinical stages. The initial stage is characterized by fever, malaise and lymphodenopathy, and follows a long phase asymptomatic to the infection and precedes the presentation of the three final stages in which weight loss occurs, and multiple opportunistic and secondary infections (English and associates, 1994). Although it is not clear whether the route of cellular infection is the same, LF is tropic for CD4 + T cells as well as CDd + T cells (Brown and associates, 1991). Virally infected animals experience a decrease in the activity of the CD4 + T cell - perhaps due to the formation of sincia lysis of the cells (Siebelink and associates, 1990). The presentation of the final stage of the infection coincides with a significant loss of CD4 + T cells and a decrease in the proportion of CD4: CDd (Novotney and associates, 1990). Although HIV and HIV-induced disease and IVF can not mediate the loss of CD4 + T cells in the same way, the resulting phenotype and malfunction of the immune system, it seems to be manifested in a fairly similar way. Although it is clear that the infection of CD4 + T cells with VI H adversely affects the development of the normal immune response, the exact mechanism of the interaction that results in immunodeficiency has not been conclusively defined. In the later stages of infection, the events resulting in the reduction of CD4 + T cells is not defined (Connor and associates, 1993). While the development of syncytia, the induction of apoptosis, and elimination by means of CTL, all have been shown to reduce T cell populations in HIV infections (Schattner and Laurence, 1994; Fouchier and associates, 1996), a mechanism mediated by CD2d, has also been proposed (Haffar and associates, 1995). The infected T cell lines have been shown to downregulate the expression of CD2d in both proteins, and the level of mRNA in the allo-antigen stimulation (Haffar and associates, 1995). As explained above, CD2d cross-links are a critical signal of the maturation of a T cell response (Linsley et al., 1991 a). If HIV infection results in down-regulation of CD2d surface expression, then infected T cells that recognize the presented antigen can become apoptotic, rather than being fully activated (Schattner and Laurence, 1994). While apoptosis is a normal mechanism of death of HIV-infected cells, this trajectory may be an additional contributor to the concomitant elimination of T cells (Brinchmann and associates, 1994). The CTL of CDd + has been related to the development of long-term survival of HIV infection, with high levels of CTL associated with the long-term non-progression of individuals infected with AIDS (Landay and associates, 1994) . In contrast, humoral immunity has been generally ineffective only in the control of associated lenti-viral disease, has shown that antibodies can actually increase the disease (Lombardi and associates, 1994, Siebelink and associates, 1995). The presentation of the final clinical stage of HIV infection and concomitant immunodeficiency are correlated with the change from a type 1 cellular response to a type 2 humoral response in many patients (Schattner and Laurence, 1994). This coincides with the observations that the progress of a healthy condition to the development of AIDS is related to a decrease in the antiviral activity of CD8 + mediated by the CTL (Lewis and associates, 1994). The expression of CD28 on the CDL of CDd + also seems to be related to a strong antiviral activity mediated by CTL associated with the expression of CD2d populations in CDd within infected individuals (Landay and associates, 1993). Surface expression of CD2d, although it has been proposed as a mediator that requires resistance to promote HIV, is adversely affected by the presence of HIV in both infected and uninfected T cells (Caruso and associates , 1994). In the beginning of the asymptomatic stages of a VI H infection, a reduction in the percentage of CD2d T cells carrying CD4 + and CDd + is detected (Lewis et al., 1994). It is proposed that this may be responsible for the abnormalities in the cytokine secretion patterns seen at the beginning of the infection (Caruso and associates, 1994) as well as the response of altered CDd + T cells in the final stages (Zanussi and associates, nineteen ninety six). In people infected with VI H, it is proposed that the reduction in the proliferation of CDd + T cells in the initial stage of infection is related to the down regulation of CD2d since only CD4 + T cells expressing CD2d proliferate in response to I L-2 (Brinchmann and associates, 1994). Unfortunately, in infected persons, CD2d T cells, CDd + cells can constitute as much as 75% of the CDd + population while in normal individuals, they form only 25% of the population (Saukkonen and associates, 1993)..

Therefore, while CD8 + populations may remain normal in infected persons, the effectiveness of this population in its ability to mount an effective antiviral immune response may be adversely affected, even in the initial phase of infection ( Caruso and associates, 1994). Studies have also shown that signal transduction of CD2d may be involved in the activity of the virus (Asjo and associates, 1993, Smithgall and associates, 1995). The co-stimulation of peripheral blood CD4 + T cells infected with HIV with anti-CD3 and an1 i-CD2d results in a higher viral replication than with stimulation with anti-CD3 alone (Smithgall and associates, 1995). This response can be ablated by the addition of CTLA-4 Ig as a soluble form of the CDdO receptor, and to a lesser extent by anti-IL-2 (Smithgall et al., 1995). In other studies with infected CD4 + T lymphocytes, in 40% of patients it was demonstrated that the binding of CD28 alone, resulted in an up-regulation in the production of the virus, without requiring an additional stimulus (Asjo and associate, 1993). The pretreatment of the lymphocyte population with the HIV gp120 surface glycoprotein results in the down-regulation of CDdO on the surface of the APC (Chirmule, 1995). Although the expression of CD28 in T cells seems to be downregulated by HIV infection, CDdO expression is upregulated in these cells (Haffer and associates, 1993). This is a proposed mechanism by which the infection can be transferred to uninfected T cells according to the interaction between CD2d in uninfected T cells and CDdO in infected T cells can facilitate cell-to-cell contact allows the transfer of the virus (Haffar and associates, 1993). Although the effects of CD2d on HIV have been explored, the role of the surface protein in VI F has not been distinguished. If similar results can be demonstrated in cats, as they are observed in the human system, this would further confirm the utility of the feline as a retroviral model.

MATERIALS AND METHODS. In vivo infection. Three specific adult female pathogen free (SPF) cats were intravenously infected with 1 x105 TCID50 from the Maryland strain of the VI F virus. Two similar females were simulated infected with a serum that does not contain the virus that serves as a control. The blood was drawn before the infection and once every week for seven weeks. In the initial week of infection, the cats were monitored twice a day to ensure that there was no initial reaction from the injection. As the infection progressed, the animals were monitored on a daily basis. Every week during the acute stage of the clinical disease, blood samples of 5 to 10 ml were collected for the determination of the CBC and the isolation of the PBMC. The CBC was determined by counting the cell types in a spotted blood sample by rapid immersion (Jorgensen Lab., Loveland, CO). The PBMC was extracted from the blood, by separation by a histopaque gradient (Sigma, St. Louis, MO). After the initial wash with an Alsever solution, ~ 5x105 cells were removed and divided into 5 recipients of a dish with 4d containers. The cells were resuspended in 50 μl of complete RPMI, and then labeled with antibodies directly against either CD4 or CDd. After one hour of incubation at room temperature, with gentle rolling, the cells were washed twice with PBS. After washing, the secondary antibody, and the goat anti-mouse IgG (H + L) labeled FITC (KP &L, Gaithersburg, MD), at a concentration of 1: 500 was added and incubated at room temperature. 1 hour with gentle agitation. The cells were then washed three times with PBS and fixed with 3.7% formaldehyde. The populations fluorescently labeled were quantified in a FACSCalibur flow cytometer. The remaining PBMC was washed for an additional time with 10 mL of Alsever solution. After centrifugation, the float was removed, and 1 mL of ULTFtASPEC (Biotexc, Houston, TX) was added for RNA extraction. The RNA was purified and precipitated as described above. Then, the concentration was quantified by measuring the absorbance at 260 nm in a spectrophotometer. The RNA was subsequently resuspended in 50 μl of DEPC treated with water and frozen at a temperature of -70 ° C for later use.

Semi-quantitative RT-PCR Prior to PCR amplification of the RNA sample, 60 mL of blood from a terminally bled cat was collected. The PBMC was isolated as described above. Cells were counted in a hemacytometer and divided into 4 flasks at a concentration of 5x105 cells per mL. The cells were stimulated with Con A for 0, 8, 16 and 24 hours before centrifugation and extraction of the RNA from the cellular pill with ULTRASPEC as described above. The RT-PCR was reviewed using 1.5 μg of RNA transcribed to the cDNA with MMLV reverse transcriptase and a 3 'oligo dT preparer. The RNA, the dT and dH20 preparator treated with DEPC were incubated for 5 minutes at a temperature of 70 ° C to remove the secondary structure of the RNA and allow cooking of the preparator. The transcription buffer, MgC, dNTP, and DTT were then added and the mixture was incubated for 2 minutes at a temperature of 42 ° C. Then one μl of reverse transcriptase was added and the reaction was allowed to proceed for 30 minutes. Then, four μl of the reaction of the 25 μl of RT were added to each one of the three tubes for the amplification of the CDdO and three tubes for the amplification of the CD2d. Then the 10X PCR regulator, dNTP and the CD2d or CDdO specific primers were added to the mix. The CDdO preparers were: 5 'B7-S220 Preparer: CAT GTC TGG CAA AGT ACÁ AG (No. SEC ID: 74) Preparer 3' B7-264: TTA TAC TAG TGA GGA CAG GGA AG (SEQ ID NO: 75) while the primers used for the CD28 were: Start 5 'CD2d: CGC GGA TCC ACC GGT AGC ACÁ ATG ATC CTC AGG (SEQ ID NO: 13) 3 'CD28-239: ATT TTG CAG AAG TAA ATA TCC (SEQ ID NO: 73) The three tubes of each product were subsequently incubated at a temperature of 95 ° C for 5 minutes and then 0.25 μl of Taq polymerase in 10 μl of water was added to each tube. The reactions were subjected to the cycles of the following temperature profiles: 95 ° C 30 seconds, 55 ° C 30 seconds, and 72 ° C 30 seconds. One tube was removed in 20, 25 and 30 cycles respectively. Twenty μl of each reaction was visualized on a 1% agarose gel. The agarose gel was photographed and the number of cycles in which the product appeared was determined. After these preliminary experiments, the RNA previously extracted from the infected animal and the control animal were amplified in a similar way.

In vitro infection The T cell lines infected with FIV in vitro were stimulated during Con A for 0, and 16 hours, and the expression of CD28 and CDdO were assayed by the semi-quantitative RT-PCR method. The FETJ cell line is a population of mixed T lymphocytes that grows in the absence of IL-2 in the culture medium. The independent subpopulations of these cells have been exposed to Maryland deformation and Petaluma deformation of the FIV virus. Approximately twenty million cells infected with Petaluma and infected with FETJ from Maryland were stimulated during 0 and 16 hours with d μg / mL of Con A. The RNA was extracted from these cells after incubation with the reagent for extraction of RNA ULTRASPEC ( Biotexc, Houston, TX) and were purified as described above. MCH 5.4 is a T cell line derived from a cat in a colony that had multiple FIV infections, although this line is not chronically infected with FIV. Pills of approximately twenty million 5.4 MCH cells were made by centrifugation, and resuspended in 5 mL of a concentrated float of infected FIV cells. The cells were incubated at this concentration for 30 minutes in a 5% CO2 incubator at a temperature of 37 ° C before adjusting to a concentration of -5x10 and cultured for 24 hours. After normal cells and infected MCH 5.4 cells were stimulated for 0 and 16 hours with 3 μg / mL of Con A. The RNA was extracted with the ULTRASPEC RNA extraction reagent (Biotexc, Houston, TX) and purified as described above.

Northern Spot Analysis For analysis of northern spots, RNA concentrations were determined by means of a spectrophotometric analysis at 260 nm. Fifteen μg of each RNA sample was concentrated at 3 μg / μl and resuspended in 3 volumes of the charge regulator of the sample. The samples were then heated to a temperature of 70 ° C for 15 minutes to denature the RNA, and remove the secondary structure. The samples, in a volume of 20 μl, were loaded on gel from 1% denatured agarose and then subjected to electrophoresis at 70 volts for 2.5 hours until the bromophenol blue dye front was reached within 2 centimeters of the gel bottom. . The RNA was then transferred from the gel to a nylon Genescreen membrane (Dupont, NEN, Boston, MA) by descending capillary action. The RNA was crosslinked with UV rays on the membrane by exposure to low intensity UV light for 3 minutes and then the bands were visualized to see the integrity of the ribosome bands of the UV shadows.

A specific feline sample of CD2d was constructed using randomly prepared cDNA labeling. A full-length CD2d molecule was cut from the TA cloning vector using the EcoRI flanking sites. The fragment was purified by gel electrophoresis and extracted from the agarose with an Amicon gel nebulizer (Amicon, Beverly, MA). 25 ng of the purified product was incubated with random decamers for 5 minutes at a temperature of 95 ° C to remove the secondary structure (Ambion, Austin, TX). Then the reaction was rapidly quenched in liquid nitrogen, and the dNTP lacking dATP was added to the P32adATP mixture. After 1 minute of incubation at a temperature of 37 ° C, 1 μl of Klenow DNA polymerase was added, and the mixture was incubated for 30 minutes. The reaction was stopped with the addition of 1 μl of 0.5 M EDTA, and subsequently purified on a spin column of Sephadex G-50 (Sigma, St. Louis, MO) to remove the unincorporated radiolabelled nucleotide. 1 μl of the reaction was diluted in 1 mL of scintillation fluid and the activity of the sample was determined in a scintillation counter. The spots were prehybridized for 15 minutes at a temperature of 65 ° C in 5 mL of a Rapid Hyb hybridization fluid (Amersham Life Science, Cleveland, OH). Five μl of the sample, at a concentration of 3-5x106 cpm / μl, was added to each spot, and was incubated with rotation for 1.5 hours at a temperature of 65 ° C. The sample was removed, and the stains washed twice in a solution of 1% SSC / 0.1% SDS at room temperature for 15 minutes. The samples were then scanned with a Geiger counter and rewashed if necessary at a temperature of 65 ° C. The labeling was quantified in a Be agen scanner. A final wash was carried out at a temperature of 65 ° C for 15 minutes, and the stain placed on the film for 16-24 hours at a temperature of -70 ° C with an intensifying strainer. Autoradiography was developed, and the bands were quantified by means of densitometry. RNA integrity and concentration were confirmed with a specific G3PDH sample (kindly provided by Prof. J. Piedrahita, Texas A &M University), labeled and hybridized in a similar manner.

Semi-quantitative RT-PCR from infected cells in vitro The presence of CD23 was further measured by semi-quantitative PCR. As described above, the concentration of extracted RNA was estimated by means of spectrophotometric readings at 260 nm. Two μg of RNA (25 μl final volume) was transcribed into the cDNA using an oligo dT preparator and MMLV reverse transcriptase as described above. Three and a half μl of the RT reaction were then transferred to seven PCR tubes. Three tubes were amplified using CDdO-specific primers, three tubes were amplified using specific CD2d primers and the remaining tube was amplified with specific G3PDH primers: G3PDH 5 ': CCT TCA TTG ACC TCA ACT AC T (NO SEC ID: 76) G3PDH 3': CCA AAG TTG TCA TGG ATG ACC (SEC ID SEC: 77) As described above, the tubes were removed in 20, 25 and 30 cycles. Twenty μl of each sample was visualized on a 1% agarose gel. In addition, the presence of certain cytokines derived from T cells was assayed in a similar way, from the RNA of FETJ and MCH 5.4 cells. The specific preparers for: IL-2 5 ': CAA CCC CAA ACT CTC CAG GAT G (No. SEC ID: 7d) IL-2 3': GGT CAG CGT TGA GAA GAT GCT TTG (NO.ID.ID: 79) IL-4 5 ': TAT TAA TGG GTC TCA CCT ACC (NO SEC ID: dO) IL-4 3 ': TTG GCT TCA TTC ACÁ GAA CAG (NO SEC ID: 81) IFNY 5': GGG TCG CTT TTC GTA GAC ATT TTG (NO. SEC ID: 82) I FNY 3 ': CAG GCA GGA CAA CCA TTA TTT C (SEQ ID NO: 83) were used to amplify the transcribed cDNA of 1.25 μg of RNA. Twenty percent of the transcription reaction was amplified by each cytokine. The remaining cDNA was amplified using specific G3PDH primers. The cDNA was amplified for 30 cycles using the following parameters: 95 ° C 5 minutes 1 cycle; 95 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds 30 cycles; 72 ° C 5 minutes 1 cycle. Then twenty μl of the reaction was visualized on a 1% agarose gel.

RT-PCR to determine the infection The infection of the FETJ and MCH 5.4 cells was confirmed through the RT-PCR amplification of the specific gag sequence. The RNA at a concentration of 1.25 μg was transcribed to the cDNA using the parameters described above and using MMLV RT and a specific gag 3 'preparer. Ten μl of the RT reaction was amplified by hot start PCR with the following parameters: 95 ° C 5 minutes; 95 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds, 30 cycles; 72 ° C 5 minutes. After amplification, 20 μl of each sample was visualized on a 1% agarose gel.

RESULTS In order to determine the effects of acute infection in vivo on CD28 expression, and cat AU04, AUU3 and OAC2 were infected with the virus by intravenous injection, while cat AWG3 and OAE6 were injected only with medium. The FACS analysis showed some differences between those in an almost constant proportion of around two to one, there were some fluctuations in the experimental group with the immersion of the proportion as low as one to one in an animal (Table 3).

Table 3: Proportions of CD4 T lymphocyte: CD8 from PBMC extracted from acutely infected and uninfected cats.

The time course test of the expression of CD80 and CD28 RNA to demonstrate that the message for each molecule was present and that it could be amplified by means of the PCR at the time points of 0, 8, 16 and 24 hours after stimulation with Con A. This PCR procedure Semi-quantitative, observed to detect a visible band in the lower number of amplification cycles which could be deduced as a relative measure of message abundance. A definite specific CD80 was not observed in a range of 20 to 25 cycles at any time after infection, so the CD80 message was visible in the gel in the 30 cycles in each of the experimental groups (Figure 42) . The CD28 message was also visible at each time point in the 30 cycles, so the 16-hour time point had a slightly visible band in 25 cycles (Figure 43). A similar protocol was used with the RNA extracted from the PBMC of cats infected with FIV and those not infected. The RT-PCR amplification of the specific RNA of CD28 and CDdO was used to demonstrate a relative idea of the amount of the message transcribed by each peptide. As was done previously in the time course experiments, the samples were removed in 20, 25 and 30 cycles. No message could be detected after 20 cycles, although both CD80 and CD28 products were visible in 25 cycles (Table 4). There were no demonstrable differences in the expression of any of the messages between the experimental and control groups. Both subsets had fluctuations in the point of the amplification cycle in which that product was visible. Table 4: Semi-quantitative determination of RT-PCR products of CD80 and CD28 amplified from infected and uninfected PBMC RNA at intervals during the acute stage of a FIV infection. BEFORE CYCLE NO. WEEK 1 WEEK 3 WEEK 4 WEEK 6 WEEK INFECTION B / C INFECTED 30/30 30/30 30/30 - / 25 30/25 25/25 AU04 30/30 30/30 25/25 - / 25 30/25 - / 30 AUU3 30/30 30/30 30/30 - / 30 25/30 25/25 OAC2 NOT INFECTED AWG3 25/30 30/30 30/25 - / 30 30/25 25/25 OAE6 30/30 - / 30 - / 30 - / 25 30/30 25/25 PCR amplification of the mRNA of FETJ and MCH 5.4 cells using specific gag VIF primers showed that the MCH 5.4 cell line could be infected , and transported an active infection while the FETJ cell lines did not produce specific amplified gag RNA. The specific VIF product was easily amplifiable from the RNA extracted from the infected MCH 5.4 samples but not from the RNA extracted in a similar manner from the uninfected controls (Figure 44). Similar reactions were carried out on the RNA extracted from the lines of FETJ cells exposed to the Petaluma and Maryland VI VI deformations that had no visible product under similar conditions (data not shown). The Northern spot and semi-quantitative PCR assays were carried out to detect CD28 messages on normal FETJ and MCH 5.4 cell lines. The MCH 5.4 cell line had an infected experimental group and an uninfected control group, while the FETJ cell line was used as a non-permissible T cell control. The semi-quantitative PCR demonstrated that each cell line had the ability to produce a message from CD28. Amplification in uninfected control FETJ cells demonstrated an expression pattern similar to that seen in the time course test for CD28 explained above. Although in a period of 0 hours after the stimulation, a band was not visible until after 30 cycles, at 16 hours after amplification in the uninfected control FETJ cells it showed an expression pattern similar to that seen in the time course test for CD28 explained above. Although in a period of 0 hours after the stimulation, a band was not visible until after 30 cycles, at 16 hours after the stimulation, the band was visible after 25 cycles (Figure 45). A similar pattern was observed in the uninfected MCH 5.4 cell line with no visible bands before 30 cycles in 0 hours and visible bands after 25 cycles after 16 hours of incubation. Interestingly, the MCH 5.4 Infected cell lines showed a different pattern from each of the controls. In the experimental group, the message was not visible in 0 or in 16 hours until the 30 cycles. The G3PDH message was amplified as a control to ensure RNA integrity and concentration (Figure 46). The infection seemed to influence the expression of the CD28 RNA message. A Northern blot analysis was used to confirm the data discovered using the semi-quantitative RT-PCR techniques. RNA from the 16 hours of MCH 5.4 cells stimulated by Con A that were not infected demonstrated hybridization of the stronger sample. The unstimulated samples from the uninfected line had a greater range of hybridization than the RNA from any of the stimulated or unstimulated MCH 5.4 cell lines (Figure 47).

In addition to the autoradiographies, radioactivity was measured in BetaGen. Simple counts of CD28 hybridization were standardized with counts obtained from a subsequent GAPDH sample from the blot (Figure 48) (Table 5).

Table 5: Normalized counts for spots of the North of CD28 of BetaGen.

The RT-PCR amplification of the cytokine RNA of the MCH 5.4 cell lines showed a message that could be amplified only for IL-2. Neither IL-4, IL-6 or IFNY could be amplified with 30 cycles, so the message I L-2 was easily detectable (Figure 49).

EXPLANATION The message of CD28 was measured in infection in vivo and in vitro to determine that the expression of CD28 could be assayed, and if the infection was with the retrovirus they altered the expressions of the message. When sufficient RNA could be recovered, the CD28 message was measured by Northern blots and by a semi-quantitative RT-PCR assay when recovery was limited. After the in vivo experiments, the infection of three animals with Maryland deformations of VI F, as explained above, were subjected to the amplification of the CD28 message and specific CDdO of the RNA extracted from the PBMC cells of blood isolates of these infected animals and uninfected controls. Although determination by Northern spotting would have been preferable, semi-quantitative RT-PCR was used due to limitations in the number of cells and the amount of RNA available. The cats were bled at weekly intervals, so a maximum of 10mL of blood was available for each experiment. The FACS analysis of the CD4 / CDd ratios of PBMC from infected animals decreased over a period of d weeks as compared to uninfected animals, in which the proportions remained relatively constant. There are differences between the two experimental groups. Although the CD4 / CDd ratios appear to differ in the infected animals against non-infected animals, no real differences in the expression of CBC or CDdO and CD2d were detected (data not shown).

In order to optimally determine the expression of the CD2d, pure T cells were required. Of the isolated PBMC, on average, perhaps 40% of the cells were in fact T cells. Of these cells, at some times up to half, they would be CDd + T cells which do not express CD2d at the same concentration as the cells T CD4 +. This, in conjunction with the fact that in other species, CD2d not expressed at high levels by resting the T cells (which constitute the majority of the T cells in circulation) led to the decision to use the PCR determination instead of the northern spots. Preliminary experiments attempting to detect the CD2d message of 20 μg of RNA extracted from PBMC were unsuccessful. The semi-quantitative RT-PCR reaction detects the presence of the RNA encoding CD2d. This technique was also used to amplify the specific message for the CDdO. In vitro cell lines provided RNA from which the CD2d message was detected by the Northern blot test. MCH 5.4 was selected because, as a T cell line, all cells potentially express the CD2d message, and a derived line had previously been used as a bridge for the next viral infection. A final benefit was that as a cell line, there was an available RNA container larger than that of blood lymphocytes extracted from a single animal. Attempts were also made to detect the message of the CDdO by means of spots of the North. Although CDdO is present in a lower concentration in resting B cells, and monocytes, higher levels of expression were found in stimulated monocytes and macrophages. A feline antigen presenting cell lines was not available, and normally T cells only express the peptide at low levels. Experiments to detect the specific message of CDdO from RNA extracted from PBMC were probably unsuccessful for reasons similar to those explained with CD2d. Alternatively, semi-quantitative PCR was used with cells derived from infected animals to demonstrate the message of CDdO and CD2d. Although no definitive answer was obtained regarding the amount of message, this essay does not show that there is a present message and a relative display of abundance. Northern blot analysis of the CD28 message from the feline T cell line was successful. When the message for CD2d was compared in the infected and uninfected cells, there were differences in expression patterns. The CD2d message was more abundant in uninfected cells exposed to Con A for 16 hours. The message was also detected in the unstimulated and uninfected cells. While the message could be detected in the RNA of the cells stimulated and not stimulated FIV, the levels were markedly lower than those of the non-infected cells. These data correlate well with similar findings found using the RT-PCR detection technique. The ability to detect the CD2d message is not hampered by the same limitations found with the CDdO molecule. However, if a large population of cells can be isolated with CDdO expression, it would certainly be possible to detect the message through Northern blots. When peptide-specific monoclonal antibodies are developed for these surface proteins, it would be interesting to correlate message levels with the amount of surface expression for each peptide. The cytokine cDNA was amplified to ensure there was no difference in the infected and uninfected lines. The IL-2 of each group was amplified regardless of the infection condition. No other cytokine messages were amplified. The northern spot indicates that the CD28 expression at the mRNA level is downregulated in vitro by the presence of FIV. Although this discovery must be confirmed by measurement of surface expression of the protein, it appears that VI F infection in vitro can influence the expression of CD28 as demonstrated in human T cells infected with VI H ( Brinchmann and associates, 1994).

CONCLUSION The cloning and elaboration of cDNA sequences coding for the CD28 and CD80 signaling complex of the feline system yielded products analogous to that of molecules isolated by other systems. Although the putative amino acid sequence of the feline protein demonstrated a relatively low identity with murine human and mouse polypeptides, comparisons of homology between previously cloned molecules, retention of the residues of the characteristics, and the fact that they did not it is considered that the surface binder has a signaling function, led us to the conclusion that, in effect, the isolated product was a feline analog of CD80. In contrast, the feline molecule CD28 retained a moderate identity, both in the nucleic acid and in the putative levels of amino acids and was analogous to the molecules cloned in other species. The nature of the molecules was further identified by means of the interaction shown by the binding assays. The monoclonal antibodies directed against the analogous proteins in another species, could not react with the feline proteins expressed. Therefore, a set of binding assays was designed to demonstrate that the interaction occurred, and that this interaction could be inhibited by the soluble receptor. In these assays, the binding was demonstrated by means of the retention of the fluorescently labeled cells, which could be inhibited by the introduction of soluble counter-receptors. This assay demonstrated, not only that the protein for feline CD80 and CD28 could be expressed, but also that the surface molecules could interact. The expression of the molecules in an active infection was also characterized. The expression of CD28 and CDdO were tested in in vivo and in vitro systems exposed to FIV virus. The expression of CD28, which has been shown to be altered in human cells infected with the HIV virus (Asjo and associates, 1993) was also adversely affected by VI F infection of feline T cells. Additional information regarding the expression of each of these molecules in the progression of the disease should continue to establish the feline system as an important model of retroviral infection. The long-term applications of these molecules are potentially vast. An understanding of the evolutionary divergent immune systems of human systems can only lead to a broader understanding of how systems work in man. In addition, the importance of the feline species as a model in retroviral infection is established in a clear manner (Siebelink and associates, 1990). CDdO has been proposed as a potential adjuvant to induce memory CTL in anti-retroviral vaccines. The feline system would be an exceptional model in which to test the effectiveness of this system.

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LIST OF SEQUENCES < 110 > Collisson, Ellen Hash, Stephen M. Insou, Cho. < 120 > Nucleotide and Polypeptide Acid CD80, Feline CD86, Feline, CTLA-4 < 130 > 54954-A-PCT < 140 > Still not known < 141 > 1999-04-30 < 150 > 09 / 071,699 < 151 > 1998-05-01 < 160 > 55 < 170 > Patent in Ver. 2.0 < 210 > 1 < 211 > 941 < 212 > DNA < 213 > CD80 Felino < 220 > < 221 > CDS < 222 > (1) .. (876) < 400 > 1 acg ggt cac gca gca aag tgg aaa ac cca cta cg aag cac cca tat 48 Met Gly His Wing Ala Lys Trp Lys Thr Pro Leu Leu Lys His Pro Tyr 1 5 10 15 ccc aag etc ttt ccg etc ttg atg cta gct agt ctt ttt tac ttc tgt 96 Pro Lys Leu Phe Pro Leu Leu Met Leu Ala Ser Leu Phe Tyr Phe Cys 20 25 30 tea ggt ate ate cag gtg aac aa g aa gtg gaa gaa gta gca gta cta 144 Ser Gly lie lie Gln Val Asn Lys Thr Val Glu Glu Val Ala Val Leu 35 40 45 tec tgt gat tac aac att tec acc aaa gaa ctg acg gaa att cga at? 192 Ser Cys Asp Tyr Asn lie Thr Lys Glu Leu Thr Glu lie Arg lie 50 55 60 tat tgg caa aag gat gat gaa atg gtg ttg gct gtc atg tet ggc aaa 240 Tyr Trp Gln Lys Asp Asp Glu Met Val Leu Ala Val Met Ser Gly Lys 65 70 75 80 gta ca gtg tgg ccc aag tac aag aac a cc ac tc ac act gac gtc acc 288 Val Gln Val Trp Pro Lys Tyr Lys Asn Arg Thr Phe Thr Asp Val Thr 85 90 95 gat aac cac tec att gtg ate atg gct ctg cgc ctg tea gac aat ggc 336 Asp Asn Hxs Ser He Val He Met Ala Leu Arg Leu Ser Asp Asn Gly 100 105 110 aaa tac act tgt att att caa aag att gaa aaa ggg tet tac aaa gtg 384 Lys Tyr Thr Cys He He Gln Lys He Glu Lys Gly Ser Tyr Lys Val 115 120 125 aaa cac ctg act tcg gtg atg tta ttg gtc aga gct gac tct cct gtc 432 Lys His Leu Thr Ser Val Met Leu Leu Val Arg Ala Asp Phe Pro Val 130 135 140 cct agt ata act gat ctt gga aat cca tet cat aac ate aaa agg ata 480 Pro Ser He Thr Asp Leu Gly Asn Pro Ser His Asn He Lys Arg He 145 150 155 160 atg tgc tta act tet gga ggt ttt cca aag cct cac etc tec tgg ctg 528 Met Cys Leu Thr Ser Gly Gly Phe Pro Lys Pro His Leu Ser Trp Leu 165 170 175 gaa aat gaa gaa gata tta aat gcc ate aac here gtt tec caa gat 576 Glu Asn Glu Glu Glu Leu Asn Ala He Asn Thr Thr Val Ser Gln Asp 180 185 190 cct gaa act gag etc tac act att age agt gaa ctg gat ttc aat atg 624 Pro Glu Thr Glu Leu Tyr Thr Be Ser Glu Leu Asp Phe Asn Met 195 200 205 aac aac aac cat age ttc ctg tgt ctt gtc aag tat gga aac tta cta 672 Thr Asn Asn Hxs Ser Phe Leu Cys Leu Val Lys Tyr Gly Asn Leu Leu 210 215 220 gta tea cag ate ttc aac tgg aaa tea gag cca cag cct tet aat 720 Val Ser Gln He Phe Asn Trp Gln Lys Ser Glu Pro Gln Pro Ser Asn 225 230 235 240 aat cag etc tgg ate att ate ctg age tea gta gta agt ggg att gtt 768 Asn Gln Leu Trp He He He Le Le Ser Ser Val Val Ser Gly He Val 245 250 255 gtg ate act gca ctt acc tta aga tgc cta gtc cac aga cct gct gca 816 Val He Thr Ala Leu Thr Leu Arg Cys Leu Val His Arg PJGO Ala Ala 260 265 270 agg tgg aga caga aga gaa atg ggg aga gcg cgg aaa tgg a.ia aga tet 864 Arg Trp Arg Gln Arg Glu Met Gly Arg Wing Arg Lys Trp Lys Arg Ser 275 280 285 cac ctg tet here tagattctgc agaaccactg tatgeagage atctggaggt 916 His Leu Ser Thr 290 agectettta gctcttctct actag 941 < 210 > 2 < 211 > 292 < 212 > PRT < 213 > CD80 Feline < 400 > 2 Met Gly His Ala Ala Lys Trp Lys Thr Pro Leu Leu Lys His Pro Tyr 1 5 10 15 Pro Lys Leu Phe Pro Leu Leu Met Leu Wing Ser Leu Phe Tyr Phe Cys 20 25 30 Ser Gly He He Gln Val Asn Lys Thr Val Glu Val Val Ala Val Val Leu 35 40 45 Being Cys Asp Tyr Asn He Being Thr Lys Glu Leu Thr Glu He Arg He 50 55 60 Tyr Trp Gln Lys Asp Asp Glu Met Val Leu Wing Val Met Ser Gly Lys 65 70 75 80 Val Gln Val Trp Pro Lys Tyr Lys Asn Arg Thr Phe Thr Asp Val Thr 85 90 95 Asp Asn Hxs Ser He Val He Met Ala Leu Arg Leu Ser Asp Asn Gly 100 105 110 Lys Tyr Thr Cys He He Gln Lys He Glu Lys Gly Ser Tyr Lys Val 115 120 125 Lys His Leu Thr Ser Val Met Leu Leu Val Arg Ala Asp Phe Pro Val 130 135 140 Pro Ser He Thr Asp Leu Gly Asn Pro Ser His Asn He Lys Arg He 145 150 155 160 Met Cys Leu Thr Ser Gly Gly Phe Pro Lys Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Glu Glu Glu Leu Asn Wing He Asn Thr Thr Val Ser Gln Asp _ 180 185 L90 O Pro Glu Thr Glu Leu Tyr Thr He Ser Ser Glu Leu Asp Phe Asn Met 195 200 205 Thr Asn Asn His Ser Phe Leu Cys Leu Val Lys Tyr Gly Asn Leu Leu 210 215 220 Val Ser Gln He Phe Asn Trp Gln Lys Ser Glu Pro Gln Pro be Asn 225 230 235 240 0 Asn Gln Leu Trp He He He He Leu Ser Val Val Ser Gly He Val 245 250 255 Val He Thr Ala Leu Thr Leu Arg Cys Leu Val His Arg Pro Ala Wing 260 265 270 Arg Trp Arg Gln Arg Glu Met Gly Arg Ala Arg Lys Trp Lys Arg Ser 275 280 285 His Leu Ser Thr 5 290 < 210 > 3 < 211 > 879 < 212 > DNA < 213 > CDdO Felino < 220 > < 221 > CDS < 222 > (1) .. (876) 0 < 400 > 3 atg ggt cac gca gca aag tgg aaa here cca cta ctg aag cac cca tat 48 Met Gly His Ala Ala Lys Trp Lys Thr Pro Leu Leu Ly. »His Pro Tyr 1 5 10 15 ccc aag etc ttt ccg etc ttg atg cta gct agt ctt tt tac ttc tgt 96 Pro Lys Leu Phe Pro Leu Leu Met Leu Wing Ser Leu Phe Tyr Phe Cys 20 25 30 tea ggt ate ate cag gtg aac aag gtg gaa gaa gta gta gta gta cta 144 Ser Gly He He Gln Val Asn Lys Thr Val Glu Val Glu Val Val Leu 35 40 45 tec tgt gat tac aac att tec acc aaa gaa ctg acg gaa att cga ate 192 Ser Cys Asp Tyr Asn I have been Thr Lys Glu Leu Thr Glu He Arg He 50 55 60 tat tgg caa aag gat gat gaa atg gtg ttg gct gtc atg tet ggc aaa 240 Tyr Trp Gln Lys Asp Asp Glu Met Val Leu Wing Val Met be Gly Lys 65 70 75 80 gta caa gtg tgg ccc aag taac aag aac cgc ac ttac act gac gtc acc 288 Val Gln Val Trp Pro Lys Tyr Lys Asn Arg Thr Phe Thr Asp Val Thr 85 90 95 gat aaccac tec att gtg ate atg gct ctg cgc ctg tea gac aat ggc 336 Asp Asn His Ser He Val He Met Ala Leu Arg Leu Ser Asp Asn Gly 100 105 110 aaa tac act tgt ate att caa aag att caa aaa ggg tet tac aaa gtg 384 Lys Tyr Thr Cys He He Gln Lys He Gln Lys Gly Ser Tyr Lys Val 115 120 125 aaa cac ctg act tcg gtg atg tta ttg gtc aga gct gac t .tc cct gtc 432 Lys Hxs Leu Thr Ser Val Met Leu Leu Val Arg Wing Asp Phe Pro Val 130 135 140 cct agt ata act gat ctt gga aat cca tet cat aac ate aaa agg ata 480 Pro Ser He Thr Asp Leu Gly Asn Pro Ser His Asn He Lys Arg He 145 150 155 160 atg tgc tta act tet gga ggt ttt cca aag cct cac etc tec tgg ctg 528 Met Cys Leu Thr Ser Gly Gly Phe Pro Lys Pro His Leu Ser Trp Leu 165 170 175 gaa aat gaa gaa gata tta aat gcc ate aac here gtt tec caa gat 576 Glu A = n Glu Glu Glu Leu Asn Ala He Asn Thr Thr Val Ser Gln Asp 180 185 190 cct gaa act gag etc tac act att age agt gaa ctg gat ttc aat atg 624 Pro Glu Thr Glu Leu Tyr Thr He Ser Ser Glu Leu Asp Phe Asn Met 195 200 205 here aac aac cat age ttc ctg tgt ctt gtc aag tat gga aac tta ata 672 Thr Asn Asn His Ser Phe Leu Cys Leu Val Lys Tyr Gly Asn Leu He 210 215 220 gta tea cag ate ttc aac tgg aa tea gag cca cag cct tet aat 720 Val Ser Gln He Phe Asn Trp Gln Lys Ser Glu Pro Gln Pro Ser Asn 225 230 235 240 aat cag etc tgg ate att ate ctg age tea gta gta agt ggg att gtt 768 Asn Gln Leu Trp He He He Leu Ser Ser Val Val Ser Gly He Val 245 250 255 gtg ate act gca ctt acc tta aga tgc cta gtc cac aga cct gct gca 816 Val He Thr Wing Leu Thr Leu Arg Cys Leu Val His Arg Pro Wing Wing 260 265 270 agg tgg aga aga gaa atg ggg aga gcg cgg aaa tgg aaa aga tet 864 Arg Trp Arg Gln Arg Glu Met Gly Arg Ala Arg Lys Trp Lys Arg Ser 275 280 285 cac ctg tet here tag 879 His Leu Ser Thr 290 < 210 > 4 < 211 > 292 < 212 > PRT < 213 > CD80 Felino < 400 > 4 Met Gly His Wing Wing Lys Trp Lys Thr Pro Leu Leu Lys His Pro Tyr 1 5 10 15 Pro Lys Leu Phe Pro Leu Leu Met Leu Wing Ser Leu Phe Tyr Phe Cys 20 25 30 Ser Gly He He Gln Val Asn Lys Thr Val Glu Glu Val Wing Val Leu 35 40 45 Being Cys Asp Tyr Asn He Being Thr Lys Glu Leu Thr Glu He Arg He 50 55 60 Tyr Trp Gln Lys Asp Asp Glu Met Val Leu Wing Val Met Ser Gly Lys 65 70 75 80 Val Gln Val Trp Pro Lys Tyr Lys Asn Arg Thr Phe Thr Asp Val Thr 85 90 95 Asp Asn His Ser He Val He Met Ala Leu Arg Leu Ser Asp Asn Gly 100 105 110 Lys Tyr Thr Cys He He Gln Lys He Gln Lys Gly Ser Tyr Lys Val 115 120 125 Lys His Leu Thr Ser Val Val Leu Leu Val Arg Ala Asp Phe Pro Val 130 135 140 Pro Ser He Thr Asp Leu Gly Asn Pro Ser His Asn He Lys Arg He 145 150 155 160 Met Cys Leu Thr Ser Gly Gly Phe Pro Lys Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Glu Glu Glu Leu Asn Wing He Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Thr Be Ser Glu Leu Asp Phe Asn Met 195 200 205 Thr Asn Asn His Ser Phe Leu Cys Leu Val Lys Tyr Gly Asn Leu He 210 215 220 Val Ser Gln He Phe Asn Trp Gln Lys Ser Glu Pro Gln Pro Ser Asn 225 230 235 240 Asn Gln Leu Trp He He He Leu Ser Ser Val Val Ser Gly He Val 245 250 255 Val He Thr Ala Leu Thr Leu Arg Cys Leu Val His Arg Pro Ala Ala 260 265 270 Arg Trp Arg Gln Arg Glu Met Gly Arg Ala Arg Lys Trp Lys Arg Ser 275 280 285 His Leu Ser Thr 290 < 210 > 5 < 211 > 1080 < 212 > DNA < 213 > CD86 Feline < 220 > < 221 > CDS < 222 > (63) .. (1052) < 400 > 5 gtttctgtgt tcctcgggaa tgtcactgag cttatacatc tggtctctgg gagctgcagt 60 gg atg ggc att tgt gac age act atg gga ctg agt cac act etc ctt 107 Met Gly He Cys Asp Being Thr Met Gly Leu Being His Thr Leu Leu 1 5 10 15 gtg atg gcc etc ctg etc tet ggt gtt tet tec atg aag agt caa gca 155 Val Met Wing Leu Leu Leu Being Gly Val Ser Being Met Lys Ser Gln Wing 20 25 30 tat ttc aac aac aga gga act gga ctg cca tgc cat ttt here aac tet caa 203 Tyr Phe Asn Lys Thr Gly Glu Leu Pro Cys His Phe Thr Asn Ser Gln 35 40 45 aac ata age ctg gat gag ctg gta gta ttt tgg cag gac cag gat aag 251 Asn He Ser Leu Asp Glu Leu Val Val Phe Trp Gln Asp Gln Asp Lys 50 55 60 ctg gtt ctg tat gag ata ttc aga ggc aaa gag aac cct caa aat gtt 299 Leu Val Leu Tyr Glu He Phe Arg Gly Lys Glu Asn Pro Gln Asn Val 65 70 75 cat etc aaa tat aag ggc cgt here age ttt gac aag gac aac tgg acc 347 His Leu Lys Tyr Lys Gly Arg Thr Ser Phe Asp Lys Asp Asn Trp Thr 80 85 90 95 ctg aga etc cac aat gtt cag ate aag gac aag ggc here tat cac tgt 395 Leu Arg Leu His Asn Val Gln He Lys Asp Lys Gly Thr Tyr His Cys 100 105 110 ttc att cat tat aaa ggg ccc aaa gga cta gtt ccc atg cac caa atg 443 Phe He His Tyr Lys Gly Pro Lys Gly Leu Val Pro Met His Gln Met 115 120 125 agt tet gac cta tea gtg ctt gct aac ttc agt caa cct gaa tie here 491 Ser Ser Asp Leu Ser Val Leu Wing Asn Phe Ser Gln Pro Glu He Thr 130 135 140 gta act tet aat aga here gaa aat tet ggc ate ata aat ttg acc tgc 539 Val Thr Ser Asn Arg Thr Glu Asn Ser Gly He He Asn Leu Thr Cys 145 150 155 tea tet ata caa ggt tac cca gaa cct aag gag atg tat ttt cag cta 587 Ser Ser He Gln Gly Tyr Pro Glu Pro Lys Glu Met Tyr Phe Gln Leu 160 165 170 175 aac act gag aat tea act act aag tat gat gtc atg aag aaa tet 635 Asn Thr Glu Asn Ser Thr Thr Lys Tyr Asp Thr Val Met Lys Lys Ser 180 185 190 caa aat aat aat gtg here gaa ctg tac aac gtt tet ate age ttg cct ttt 683 Gln Asn Asn Val Thr Glu Leu Tyr Asn Val Ser He Ser Leu Pro Phe 195 200 205 tea gtc cct gaa gca cac aat gtg age gtc ttt tgt gcc ctg aaa ctg 731 Ser Val Pro Glu Ala His Asn Val Ser Val Phe Cys Ala Leu Lys Leu 210 215 220 gag here ctg gag atg ctg etc tec cta cct ttc aat ata gat gca caa 779 Glu Thr Leu Glu Met Leu Leu Ser Leu Pro Phe Asn He Asp Wing Gln 225 230 235 cct aag gat aaa gac cct gaa cag gcc cac ttc etc tgg att gcg gct 827 Pro Lys Asp Lys Asp Pro Glu Gln Gly His Phe Leu Trp He Ala Al a 240 245 250 255 gta ctt gta atg ttt gtt gtt ttt tgt ggg atg gtg tec ttt aaa here 875 Val Leu Val Met Phe Val Val Phe Cys Gly Met Val Ser Phe Lys Thr 260 265 270 cta agg aaa agg aag aag aag cag cct ggc ccc tet cat gaa tgt gaa 923 Leu Arg Lys Arg Lys Lys Lys Gln Pro Gly Pro Ser Hxs Glu Cys Glu 275 280 2E5 acc ate aaa agg gag aga aga gag age aaa cag acc aac gsia aga gta 971 Thr He Lys Arg Glu Arg Lys Glu Ser Lys Gln Thr Asn G.u Arg Val 290 295 300 cca tac cac gta cct gag aga tet gat gaa gcc cag tgt gt.t aac att 1019 Pro Tyr Hxs Val Pro Glu Arg Ser Asp Glu Wing Gln Cys Val Asn He 305 310 315 ttg aag ac gcc te ggg gac aaa aat cag tag gaaaatggtg gcttggcgtg 1072 Leu Lys Thr Wing Ser Gly Asp Lys Asn Gln 320 325 330 ctgacaat 1080 < 210 > 6 < 211 > 329 < 212 > PRT < 213 > CD86 Feline < 400 > 6 Met Gly He Cys Asp Being Thr Met Gly Leu Being Hxs Thr Leu Leu Val 1 5 10 15 Met Ala Leu Leu Leu Ser Gly Val Ser Ser Met Lys Ser Gln Ala Tyr 20 25 30 Phe Asn Lys Thr Gly Glu Leu Pro Cys His Phe Thr Asn Ser Gln Asn 35 40 45 He Ser Leu Asp Glu Leu Val Val Phe Trp Gln Asp Gln Asp Lys Leu 50 55 60 Val Leu Tyr Glu He Phe Arg Gly Lys Glu Asn Pro Gln Asn Val His 65 70 75 80 Leu Lys Tyr Lys Gly Arg Thr Ser Phe Asp Lys Asp Asn Trp Thr Leu 85 90 95 Arg Leu Hxs Asn Val Gln He Lys Asp Lys Gly Thr Tyr His Cys Phe 100 105 • 110 He His Tyr Lys Gly Pro Lys Gly Leu Val Pro Met Hxs Gln Met Ser 115 120 125 Being Asp Leu Being Val Leu Asn Phe Being Gln Pro Glu He Thr Val 130 135 140 Thr Ser Asn Arg Thr Glu Asn Ser Gly He He Asn Leu Thr Cys Ser 145 150 155 160 Ser He Gln Gly Tyr Pro Glu Pro Lys Glu Met Tyr Phe Gln Leu Asn 165 170 175 Thr Glu Asn Ser Thr Thr Lys Tyr Asp Thr Val Met Lys Lys Ser Gln 180 185 190 Asn Asn Val Thr Glu Leu Tyr Asn Val Ser Be Ser Leu Pro Phe Ser 195 200 205 Val Pro Glu Wing Hxs Asn Val Ser Val Phe Cys Wing Leu Lys Leu Glu 210 215 220 Thr Leu Glu Met Leu Leu Ser Leu Pro Phe Asn He Asp Wing Gln Pro 225 230 235 240 Lys Asp Lys Asp Pro Glu Gln Gly His Phe Leu Trp He Ala Wing Val 245 250 255 Leu Val Met Phe Val Val Phe Cys Gly Met Val Ser Phe Lys Thr Leu 260 265 270 Arg Lys Arg Lys Lys Lys Gln Pro Gly Pro Ser His Glu Cys Glu Thr 275 280 285 He Lys Arg Glu Arg Lys Glu Ser Lys Gln Thr Asn Glu Arg Val Pro 290 295 300 Tyr His Val Pro Glu Arg Ser Asp Glu Wing Gln Cys Val? Sn He Leu 305 310 315 320 Lys Thr Ala Ser Gly Asp Lys Asn Gln 325 < 210 > 7 < 211 > 688 < 212 > DNA < 213 > CD28 Felino < 220 > < 221 > CDS < 222 > (1) . (663) < 400 > 7 atg ate etc agg ctg ctt ctg gct etc aac ttc ttc ccc tea att caa 48 Met He Leu Arg Leu Leu Leu Ala Leu Asn Phe Phe Pro Ser He Gln 1 5 10 15 gta here gaa aac aag att ttg gtg aag cag ttg ccc agg ctt gtg gtg 96 Val Thr Glu Asn Lys He Leu Val Lys Gln Leu Pro Arg Leu Val Val 20 25 30 tac aac aat gag gtc aac ctt age tgc aag tac act cac aac ttc ttc 144 Tyr Asn Asn Glu Val Asn Leu Ser Cys Lys Tyr Thr Hxs Asn Phe Phe 35 40 45 tea aag gag ttc cgg gca tec ctt tat aag gga gta gta gat gtg gtg 192 Ser Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asp Ser Ala Val 50 55 60 gaa gtc tgc gtt gtg aat gga aat tac tec cat cag cct cag ttc tac 240 Glu Val Cys Val Val Asn Gly Asn Tyr Ser Hxs Gln Pro Gln Phe Tyr 65 70 75 80 tea agt here gga ttc gac tgt gat ggg aaa ttg ggc aat gaa here gtg 288 Being Ser Thr Gly Phe Asp Cys Asp Gly Lys Leu Gly Asn Glu Thr Val 85 90 95 here ttc tac etc cga aat ttg ttt gtt aac caa acg gat att tac ttc 336 Thr Phe Tyr Leu Arg Asn Leu Phe Val Asn Gln Thr Asp He Tyr Phe 100 105 110 tgc aaa att gaa gtc atg tat cca cct cct tac ata gac aat gag aag 384 Cys Lys He Glu Val Met Tyr Pro Pro Pro Tyr He Asp Asn Glu Lys H5 120 125 age aat ggg acc att ate cac gtg aaa gag aaa cat ctt tgt cca gct 432 Being Asn Gly Thr He He His Val Lys Glu Lys His Leu Cys Pro Wing 130 «5 140 cag ctg tet cct gaa tet tec aag cca ttt tgg gca ctg gtg gtg gtt 480 Gln Leu ser Pro Glu Ser Ser Lys Pro Phe Trp Ala Leu Val Val Val 145 150 155 160 ggt gga ate cta ggt ttc tac age ttg cta gca here gtg gct ctt ggt 528 Gly Gly He Leu Gly Phe Tyr Ser Leu Leu Ala Thr Val Ala Leu Gly 165 170 175 gct tgc tgg atg aag acc aag agg agt agg ate ctt cag agt gac tat 576 Wing Cys Trp Met Lys Thr Lys Arg Ser Arg He Leu Gln Ser Asp Tyr 180 185 190 atg aac acc ccc cgg agg cca ggg ccc acc cga agg cac tac caa 624 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Arg Hxs Tyr Gln 195 200 205 cct tac gcc cca gca cgc gac ttt gcg gca tac cgt tec tgacatggac 673 Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 ccctatccag aagee 688 < 210 > 8 < 211 > 221 < 212 > PRT < 213 > CD28 Felino < 400 > 8 Met He Leu Arg Leu Leu Leu Ala Leu Asn Phe Phe Pro Ser He Gln 1 5 10 15 Val Thr Glu Asn Lys He Leu Val Lys Gln Leu Pro Arg Leu Val Val 20 25 30 Tyr Asn Asn Glu Val Asn Leu Ser Cys Lys Tyr Thr Hxs Asn Phe Phe 35 40 45 Ser Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asp Ser Wing Val 50 55 60 Glu Val Cys Val Val Asn Gly Asn Tyr Ser Hxs Gln Pro Gln Phe Tyr 65 70 75 80 Be Ser Thr Gly Phe Asp Cys Asp Gly Lys Leu Gly Asn Glu Thr Val 85 90 95 Thr Phe Tyr Leu Arg Asn Leu Phe Val Asn Gln Thr Asp He Tyr Phe 100 105 110 Cys Lys He Glu Val Met Tyr Pro Pro Pro Tyr He Asp Asn Glu Lys 115 120 125 be Asn Gly Thr He He Hxs Val Lys Glu Lys His Leu Cys Pro Wing 130 135 140 Gln Leu Ser Pro Glu Ser Ser Lys Pro Phe Trp Wing Leu Val Val Val 145 150 155 160 Gly Gly He Leu Gly Phe Tyr Ser Leu Leu Wing Thr Val Wing Leu Gly 165 170 175 Wing Cys Trp Met Lys Thr Lys Arg Ser Arg He Leu Gln Ser Asp Tyr 180 185 190 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Arg Hxs Tyr Gln 195 200 205 Pro Tyr Wing Pro Wing Arg Asp Phe Wing Wing Tyr Arg Ser 210 215 220 < 210 > 9 < 211 > 749 < 212 > DNA < 213 > CTLA-4 Felino < 220 > < 221 > CDS < 222 > (27) .. (698) < 400 > 9 aacctgaaca ctgctcccat aaagcc atg gct tgc ttt gga ttc cgg agg cat 53 Met Ala Cys Phe Gly Phe Arg Arg His ggg gct cag ctg gac ctg gct tet agg acc tgg ccc tgc act gct ctg 101 Gly Ala Gln Leu Asp Leu Ala Ser Arg Thr Trp Pro Cys Thr Ala Leu 15 20 25 ttt tet ctt etc ttt ate ccc gtc ttc tec aaa ggg atg cat gtg gcc 149 Phe Ser Leu Leu Phe He Pro Val Phe Ser Lys Gly Met Hxs Val Wing 30 35 40 cac cct gca gtg gtg ctg gcc age age cga ggt gtc gcc age ttc gtg 197 Hxs Pro Ala Val Val Leu Ala Ser Ser Arg Gly Val Ala Ser Phe Val 45 50 55 tgt gaa tat ggg tet tea ggc aat gcc aaa ttc cga gtg act gtg 245 Cys Glu Tyr Gly Ser Ser Gly Asn / Via Wing Lys Phe Arg Val Thr Val 60 65 70 ctg agg caa act ggc age caa atg act gaa gtc tgt gct gcg here tac 293 Leu Arg Gln Thr Gly Ser Gln Met Thr Glu Val Cys Ala Wing Thr Tyr 75 80 85 here gtg gag aat gag ttg gcc ttc cta aat gat tec ac tgc act ggc 341 Thr Val Glu Asn Glu Leu Wing Phe Leu Asn Asp Ser Thr Cys Thr Gly 90 95 100 105 ate tec age gga aac aaa gtg aac etc acc ate ca ggg ttg agg gcc 389 Be Ser Gly Asn Lys Val Asn Leu Thr He Gln Gly Leu Arg Wing 110 115 120 atg gac acg gga etc tac ate tgc aag gtg gag etc. atg tac cca cca cca 437 Met Asp Thr Gly Leu Tyr He Cys Lys Val Glu Leu Met Tyr Pro Pro 125 130 135 ccc tac tat gca ggc atg ggc aat gga acc cag att tat gtc ate gat 485 Pro Tyr Tyr Ala Gly Met Gly Asn Gly Thr Gln He Tyr Val He Asp 140 145 150 cct gaa cct tgc cca gat tet gac ttc etc etc tgg tetc etc gca gca 533 Pro Glu Pro Cys Pro Asp Ser Asp Phe Leu Leu Trp He Leu Wing Ala 155 160 165 gtc gtc gtc ttg gtc ttt ttt tat age ttc ctt tetc here gct gtt tet 581 Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu He Thr Wing Val Ser 170 175 180 185 ttg age aaa atg cta aag aaa aga age cct ctt act here ggg gtc tat 629 Leμ Ser Lys Met Leu Lys Arg Ser Pro Leu Thr Thr Gly Val Tyr 190 195 200 gtg aaa atg ccc cca here gag cca gaa tgt gaa aag caa ttt cag cct 677 Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu Lys Gln Phe Gln Pro 205 210 215 tat ttt att ccc ate aat tga cacaccgtta tgaagaagga agaacactgt 728 Tyr Phe He Pro He Asn 220 ecaattteta agagctgagg c 749 < 210 > 10 < 211 > 223 < 212 > PRT < 213 > CTLA-4 Feline < 400 > 10 Met Wing Cys Phe Gly Phe Arg Arg Hxs Gly Wing Gln Leu Asp Leu Wing 1 5 10 15 Ser Arg Thr Trp Pro Cys Thr Ala Leu Phe Ser Leu Leu Phe He Pro 20 25 30 Val Phe Ser Lys Gly Met Hxs Val Wing Hxs Pro Wing Val Val Leu Wing 35 40 45 Ser Ser Arg Gly Val Wing Ser Phe Val Cys Glu Tyr Gly Ser Ser Gly 50 55 60 Asn Wing Wing Lys Phe Arg Val Thr Val Leu Arg Gln Thr Gly Ser Gln 65 70 75 80 Met Thr Glu Val Cys Ala Wing Thr Tyr Thr Val Glu Asn Glu Leu Wing 85 90 95 Phe Leu Asn Asp Ser Thr Cys Thr Gly He Ser Ser Gly Asn Lys Val 100 105 110 Asn Leu Thr He Gln Gly Leu Arg Wing Met Asp Thr Gly Leu Tyr He 115 120 125 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Wing Gly Met Gly 130 135 140 Asn Gly Thr Gln He Tyr Val He Asp Pro Glu Pro Cys Pro Asp Ser 145 150 155 160 sp Phe Leu Leu Trp Le Le Wing Wing Val Ser Ser Gly Leu Phe Phß 165 170 175 Tyr Ser Phe Leu He Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys 180 185 190 Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu 95 200 205 Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe He Pro He Asn 210 215 220 < 210 > 11 < 211 > 40 < 212 > DNA < 213 > Catcher CD80 Preparer < 400 > 11 cgcggatccg caccatgggt cacgcagcaa agtggaaaac 40 < 210 > 12 < 211 > 25 < 212 > DNA < 213 > Catcher CD80 Preparer < 400 > 12 cctagtagag aagagctaaa gaggc 25 < 210 > 13 < 211 > 33 < 212 > DNA < 213 > CD28 Preparer Felino < 400 > 13 cgcggatcca ccggtagcac aatgatcctc agg 33 < 210 > 14 < 211 > 31 < 212 > DNA < 213 > CD28 Preparer Felino < 400 > 14 cgcggatcct ctggataggg gtccatgtca g 31 < 210 > 15 < 211 > 27 < 212 > DNA < 213 > Feline CTLA-4 Preparator < 400 > 15 atggcttcgc cttggatttc cagcagg 27 < 210 > 16 < 211 > 29 < 212 > DNA < 213 > Feline CTLA-4 Trainer < 400 > 16 tcaattgaat gaggaataaa ataaggetg 29 < 210 > 17 < 211 > 28 < 212 > DNA < 213 > Feline CTLA-4 trainer < 400 > 17 tgttgggttt ctgactctga cttccctg 28 < 210 > 18 < 211 > 24 < 212 > DNA < 213 > Feline CTLA-4 Preparator < 400 > 18 gcatagtagg gtggtgggta catg 24 < 210 > 19 < 211 > 28 < 212 > DNA < 213 > Feline CTLA-4 Preparator < 400 > 19 tgttgggttt ctgactctga cttccctg 28 < 210 > 20 < 211 > 20 < 212 > DNA < 213 > Felxno CTLA-4 Preparator < 400 > 20 acatgagctc caccttgcag 20 < 210 > 21 < 211 > 27 < 212 > DNA < 213 > Feline CTLA-4 trainer < 400 > 21 ccatcctaat acgactcact atagggc 27 < 210 > 22 < 211 > 24 < 212 > DNA < 213 > Feline CTLA-4 trainer < 400 > 22 gtgaatatgg gtcttcaggc aatg 24 < 210 > 23 < 211 > 23 < 212 > DNA < 213 > Feline CTLA-4 trainer < 400 > 23 actcactata gggctcgagc ggc 23 < 210 > 24 < 211 > 23 < 212 > DNA < 213 > Feline CTLA-4 trainer < 400 > 24 gaaatccgag tgactgtgct gag 23 < 210 > 25 < 211 > 24 < 212 > DNA < 213 > Feline CTLA-4 Preparator < 400 > 25 aacctgaaca ctgctcccat aaag 24 < 210 > 26 < 211 > 25 < 212 > DNA < 213 > Feline CTLA-4 Preparator < 400 > 26 gcctcagctc ttagaaattg gacag 25 < 210 > 27 < 211 > 21 < 212 > DNA < 213 > Feld CDd6 Trainer < 400 > 27 tagtattttg gcaggaccag g 21 < 210 > 28 < 211 > 23 < 212 > DNA < 213 > CD86 Preparer Felino < 400 > 28 ctgtgacatt atcttgagat ttc 23 < 210 > 29 < 211 > 23 < 212 > DNA < 213 > CD86 Preparer Felino < 400 > 29 gagcatgcac taatgggact gag 23 < 210 > 30 < 211 > 23 < 212 > DNA < 213 > CD86 Preparer Felino < 400 > 30 ctgtgacatt atcttgagat ttc 23 < 210 > 31 < 211 > 27 < 212 > DNA < 213 > CD86 Preparer Felino < 400 > 31 ccatcctaat acgaetcact atagggc 27 < 210 > 32 < 211 > 28 < 212 > DNA < 213 > Preparer CD66 Felino < 400 > 32 tgggtaacct tgtatagatg agcaggtc 28 < 210 > 33 < 211 > 23 < 212 > DNA < 213 > Preparer CD66 Felino < 400 > 33 actcactata gggctcgagc ggc 23 ^ 210 > 34 < 211 > 25 < -212 > DNA < 213 > CD86 Preparer Felino < 400 > 34 caggttgact gaagttagca agcac 25 < 210 > 35 < 211 > 27 < 212 > DNA < 213 > Feline CD86 Preparer .400 > 35 ccatcctaat acgactcact atagggc 27 < 210 > 36 < 211 > 25 < 212 > DNA < 213 > Preparer CDdß Felino < 400 > 36 ggacaagggc acatatcact gtttc 25 < 210 > 37 < 211 > 23 < 212 > DNA < 213 > Preparer CDßß Felino < 400 > 37 actcactata gggctcgagc ggc 23 < 210 > 38 < 211 > 25 < 212 > DNA < 213 > Preparer CDdß Felino < 400 > 38 cagtgcttgc taacttcagt caacc 25 < 210 > 39 < 211 > 23 < 212 > DNA < 213 > CD86 Preparer Felino < 400 > 39 cgggaatgtc actgagctta tag 23 < 210 > 40 < 211 > 23 < 212 > DNA < 213 > Preparer CDdß Felino < 400 > 40 gatctttttc aggttagcag ggg 23 < 210 > 41 < 211 > 20 < 212 > DNA < 213 > Preparer CDßß Felino < 400 > 41 atgggtcacg cagcaaagtg 20 < 210 > 42 < 211 > 20 < 212 > DNA < 213 > Catcher CD80 Preparer < 400 > 42 ctatgtagac aggtgagatc 20 < 210 > 43 < 211 > 17 < 212 > DNA < 213 > Catcher CD80 Preparer < 400 > 43 caggaaacag ctatgac 17 < 210 > 44 < 211 > 18 < 212 > DNA < 213 > Catcher CD80 Preparer < 400 > 44 aatacgactc actatagg lß < 210 > 45 < 211 > 21 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 45 aacaccattt catcatcctt t 21 < 210 > 46 < 211 > 23 < 212 > AD < 213 > Felino CDßO Trainer < 400 > 46 atacaagtgt atttgccatt gtc 23 < 210 > 47 < 211 > 20 < 212 > DNA < 213 > CDdO Felino Trainer < 400 > 47 agctctgacc aataacatea 20 < 210 > 48 < 211 > 22 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 48 attagaaatc cagttcactg ct 22 < 210 > 49 < 211 > 21 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 49 tcatgtctgg caaagtacaa g 21 < 210 > 50 < 211 > 18 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 50 attcactgac gtcaccga 18 < 210 > 51 < 211 > 16 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 51 aaggctgtgg ctctga 16 < 210 > 52 < 211 > 29 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 52 tcgagaattc gggtcacgca gcaaagtgg 29 < 210 > 53 < 211 > 32 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 53 gctaggatcc aatctatgta gacaggtgag at 32 < 210 > 54 < 211 > 32 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 54 gatgaattec atgatcctca ggctgggctt ct 32 < 210 > 55 < 211 > 29 < 212 > DNA < 213 > Felino CDßO Trainer < 400 > 55 gatcagatct caggaaeggt atgccgcaa 29

Claims (30)

1. R E I V I N D I C A C I O N S Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1 . An isolated nucleic acid encoding the feline CD80 ligand or a soluble CDdO ligand.
2. 2. An isolated nucleic acid encoding a feline CDdO binder or a soluble feline CDdβ binder.
3. 3. An isolated nucleic acid encoding a feline CD23 receptor or a soluble feline CD2d receptor.
4. An isolated nucleic acid encoding a feline CTLA-4 receptor or a soluble feline CTLA-4 receptor.
5. The nucleic acid as described in Claim 1, further characterized in that the feline CDdO binder has the sequence shown in Figure 1 A which starts with meteonin and ends with threonine (Sequence Identification No.: 1).
6. The nucleic acid as described in Claim 2, further characterized in that the feline CDdβ ligand has the sequence shown in Figure 3A which starts with meteonin and ends with isoleucine (Sequence Identification No.: 5).
7. 7. The nucleic acid as described in Claim 3, further characterized in that the feline CD86 receptor shown in Figure 4A has the sequence beginning with meteonin and ending with serine (Sequence Identification No.: 7). d.
8. The nucleic acid as described in Claim 4, further characterized in that the feline CTLA-4 receptor has the sequence shown in Figure 5A starting with meteonin and ending with asparagine (Sequence Identification No.: 9).
9. 9. The nucleic acid as described in any one of Claims 1 to 4, further characterized in that the nucleic acid is DNA or RNA.
10. 10. The nucleic acid as described in Claim 9, further characterized in that the DNA is a cDNA or a genomic DNA. eleven .
11. An oligonucleotide of at least 12 nucleotides which has a sequence complementary to a sequence present only in the nucleic acid as described in any of Claims 1 to 4.
12. 12. The oligonucleotide as described in Claim 1 1, further characterized by having a length of at least 15 or 16 nucleotides.
13. 13. The oligonucleotide as described in Claims 1 1 or 12, further characterized in that the oligonucleotide can be detected by labeling.
14. 14. The oligonucleotide as described in Claim 13, further characterized in that the detectable label comprises a radioisotope, a fluoropor or biotin.
15. 15. The oligonucleotide as described in Claims 1 1 or 12, further characterized in that the oligonucleotide is selectively methylated.
16. 6. A vector comprising the nucleic acid as described in Claim 1.
17. 17. The plasmid vector as described in Claim 16, further characterized in that it is designated by PSI-B7-1 / 871 -35 (Access ATCC No. 209617).
18. 18. A vector comprising the nucleic acid as described in Claim 2.
19. 19. The plasmid vector as described in Claim 18, further characterized in that it is designated by B7-2 # 19- 2/01 129d (ATCC Access No. 209621).
20. 20. A vector comprising the nucleic acid as described in Claim 3. twenty-one .
21. The plasmid vector as described in Claim 20, further characterized in that it is designated by PSI-CD2d # 7/100298 (Access ATCC No. 209819).
22. 22. A vector comprising the nucleic acid as described in Claim 4.
23. 23. The plasmid vector as described in Claim 22, further characterized in that it is designated by CTLA-4 # 1/091997 (Access ATCC No. 209620).
24. 24. The vector as described in any of Claims 16 to 23, further characterized in that it comprises an operator operably linked to the nucleic acid.
25. 25. A host cell which comprises a vector as described in any of Claims 16 to 24.
26. 26. The host cell as described in Claim 25, further characterized in that the host cell is a eukaryotic or prokaryotic cell.
27. 27. The host cell as described in Claim 26, further characterized in that the host cell is selected from the group consisting of: E. coli, yeast, COS cells, PC12 cells, CHO cells and GH4C1 cells.
28. 28. A polypeptide encoded by the nucleic acid as described in Claim 1.
29. 29. A polypeptide encoded by the nucleic acid as described in Claim 2. A polypeptide encoded by the nucleic acid as described in Claim 3. A polypeptide encoded by the nucleic acid as described in Claim 4. A method of producing the polypeptide as described in any of Claims 28 to 31, which comprises culturing a host cell which expresses the polypeptide and recovering the polypeptide produced in this manner. A vaccine which comprises an effective amount of a polypeptide as described in any of Claims 28 through 30, and a suitable vehicle. A vaccine as described in Claim 23, further characterized in that the effective amount is an amount from about 0.01 mg. to approximately 100 mg. per dose. A vaccine as described in Claim 33, further characterized in that the effective amount is an amount from about 0.25 mg / kg body weight of a feline / day to about 25 mg / kg per weight of a feline / day. A vaccine as described in any of Claims 33 to 35, further characterized in that it additionally comprises an immunogen derived from a pathogen. A vaccine as described in Claim 36, further characterized in that the pathogen is a feline pathogen of rabies virus, chlamydia, Toxoplasmosis gondii, Dirofilaria immitis, a flea or a bacterial pathogen. A vaccine as described in Claim 37, further characterized in that the feline pathogen is feline immunodeficiency virus (VI F), feline leukemia virus (FeLV), feline infectious peritonitis virus (FIP), feline depanleukopenia virus, feline calicivirus, feline reovirus type 3, feline rotavirus, feline coronavirus, feline syncytial virus, feline sarcoma virus, feline herpes virus, feline Borna disease virus, or a feline parasite. A method for the induction of immunity in a feline which comprises administering to the feline a dose of a vaccine as described in any of the Claims 36 to 3d. A method for improving the immune response in a feline which comprises administering to the feline a dose of a vaccine as described in any of the Claims 33 to 3d. The method as described in Claims 39 or 40, further characterized in that the vaccine is administered subcutaneously, intramuscularly, systematically, topically or orally. A method for suppressing the immune response in a feline, which comprises administering to the feline an effective amount of the immune response suppressor of a polypeptide as described in Claim 31. A method for suppressing the immune response in a feline, which comprises administering to the feline an effective amount of the immune response suppressor of a soluble polypeptide as described in Claims from 2d to
30. 30. A method as described in claims 42 or 43, further characterized in that the amount is from about 0.25 mg / kg per body weight / day to about 25 mg // kb per weight / day. A method as described in claims 42 or 43, further characterized in that the feline is suffering from a disease of the autoimmune system, or is the recipient of a tissue or organ transplant. SUMMARY The present invention provides isolated and purified DNA encoding the feline binder CDdO (B7-1), the feline binder CDd6 (B7-2), the feline receptor CD28, or the feline receptor CTILA-4 (CD152), as well as vectors comprising feline CDdO encoding nucleic acid, feline CD66, feline CD2d or feline CTLA-4. The present invention provides transformed host cells with CDdO coding vectors, CDd6 coding vectors, CD28 coding vectors or CTLA-4 coding vectors. The present invention provides polypeptides encoded by the nucleic acid, feline CD80, feline CDd6, feline CD28 or feline CTLA-4. The present invention provides a vaccine which comprises an effective amount of polypeptides encoded by the feline CDdO, feline CDd6, feline CD28 or feline CTLA-4 nucleic acid. The present invention also provides vaccines which additionally comprise immunogens derived from the pathogens. The present invention provides vaccines with the ability to improve an immune response. The present invention also provides vaccines with the ability to suppress an immune response.