MXPA02008219A

MXPA02008219A - Hla binding peptides and their uses.

Info

Publication number: MXPA02008219A
Application number: MXPA02008219A
Authority: MX
Inventors: John Sidney
Original assignee: Epimmune Inc
Priority date: 2000-02-23
Filing date: 2000-02-23
Publication date: 2005-06-30
Also published as: EP1263775A4; WO2001062776A1; AU2000232427A1; CA2400215A1; CN1452634A; EP1263775A1; JP2003524016A; BR0017136A

Abstract

The present invention provides the means and methods for selecting immunogenic peptides and the immunogenic peptide compositions capable of specifically binding glycoproteins encoded by HLA alleles and inducing T cell activation in T cells restricted by the allele. The peptides are useful to elicit an immune response against a desired antigen.

Description

PEPPETS LINKING HIA AND ITS USES DESCRIPTION OF THE INVENTION The present application is a continuation in part of USSN 08/205, 713 filed on March 4, 1994. The present application also relates to USSN 09 / 017,735, USSN 08 / 753,622 , USSN 08 / 822,382, USSN 60 / 013,980, USSN 08 / 589,108, USSN 08 / 454,033, USSN 08 / 349,177, USSN 08 / 073,205, and USSN 08 / 027,146. The present application also relates to USSN 09 / 017,524, USSN 08 / 821,739, USSN 60 / 013,833, USSN 08 / 758,409, USSN 08 / 589,107, USSN 08 / 451,913 and USSN 08 / 347,610, USSN 08 / 186,266, USSN 08 / 159,339, USSN 09 / 116,061, USSN 08 / 103,396, USSN 08 / 027,746, and USSN 07 / 926,666. The present application also relates to USSN 09 / 017,743; USSN 08 / 753,615; USSN 08 / 590,298; USSN 08 / 452,843; USSN 09 / 115,400; USSN 08 / 344,824; and USSN 08 / 278,634. The present application also relates to USSN 08 / 197,484 and USSN 08 / 815,396. All of the above applications are incorporated herein by reference. The present invention relates to compositions and methods for preventing, treating or diagnosing a number of disease states such as viral diseases and cancers. In particular, it provides novel peptides capable of binding selected major histocompatibility complex (MHC) molecules and inducing an immune response.

Class I or Class II. Class II MHC molecules are expressed, mainly in cells involved in initiating and sustaining immune responses, such as T lymphocytes, lymphocytes 3, macrophages, etc. Class II MHC molecules are recognized by the attendant T lymphocytes and induce the proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide shown. MHC Class I molecules are expressed in almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs), which then destroy antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections. The CTL recognizes the antigen in the form of a peptide fragment bound to MHC Class I molecules rather than the foreign antigen itself. The antigen must be common and endogenously synthesized by the cell, and a portion of the protein antigen is degraded into small peptide fragments in the cytoplasm. Some of these small peptides are translocated within a pre-Golgi compartment and interact with heavy class I chains to facilitate proper folding and association with β2 microglobulin subunit. The peptide-MHC class I complex is then directed to the surface of the cell for expression and potential recognition by specific CTLs.

Investigations of the crystal structure of the human MHC class I molecule, HLA-A2.1, indicate that a peptide bond groove is created by the doubling of the a and a2 domains of heavy chain class I (Biorkmen et al. al., Nature 329: 506 (1987) In these investigations, however, the identity of the bound peptides to the groove was not determined, Buus et al., Science 242: 1065 (1988) first described a method for acid elution. of peptides linked from MHC Subsequently, Rammensee and his collaborators (FaLk et al., Nature 351: 290 (1991) have developed a methodology for characterizing naturally processed peptides linked to class I molecules.Other researchers have successfully achieved sequencing of direct amino acids from the most abundant peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules of type B (Jardetzky, et al., Nature 353: 326 (1991) and type A2.1 by mass spectrometry (Hunt, et al., Science 225: 1261 (1992). A review of the characterization of the naturally processed peptides in MHC Class I has been presented by Rótzschke and Falk (Rótzschke and Falk, Immunol., Today 12: 447 (1991), Sette et al., Proc. Nati. Acad. Sci. 86: 3296 (1989) showed that specific motifs of the MHC allele could be used to predict the binding capacity of MHC Schaene: et al., Proc. Nati, Acad. Sci. USA 86: 4649 (1989) of MHC was related to immunogenicity Several authors (De Bruijn et al., Eur. J. Immuno1., 21: 2963-2970 (1991); Pamer et al., 991 Nature 353: 852-955 (1991)) have provided preliminary evidence that class I binding motifs can be applied to the identification of potential immunogenic peptides in animal models. The specific Class I motifs for a number of human alleles of a given isotype of Ciase I can still be described. It is desirable that the combined frequencies of these different alleles be sufficiently high to cover a large fraction or perhaps the majority of the population produced by mixing human races. Despite development in the art, the prior art has yet to provide a vaccine based on useful human peptide or therapeutic agent based on that work. The present invention provides these and other advantages. The present invention provides compositions comprising immunogenic peptides having binding motifs for the HLA-A2.1 molecules. Immunogenic peptides, which bind to the appropriate MHC allele, are preferably 9 to 10 residues in length and comprise residues conserved er. some positions such as positions 2 and 9. In addition, the peptides do not comprise negative binding residues as defined herein in other positions such as positions 1, 3, 6 and / or 7 in the case of peptides of 9 amino acids of length and positions 1, 3, 4, 5, 7, 8 and / or 9 in the case of peptides of 10 amino acids in length. The present invention defines positions within a motif that allows the selection of peptides that will bind efficiently to HLA A2.1. The reasons for the inventions include peptides from 9 amino acids having a first residue conserved in the second position from the N term selected from the group consisting of I, V, A and T and a second residue conserved in the C-terminal position selected from the group consisting of of V, L, I, A and M. Alternatively, the peptides can have a first residue conserved in the second position from the N terminus selected from the group consisting of L, M, I, V, A and T; and a second residue conserved in the C-terminal position selected from the group consisting of A and M. If the peptide has The residue will contain a first residue conserved in the second position from the N term selected from the group consisting of L, M, I, V, A, and T; and a second residue conserved in the C-terminal position selected from the group consisting of V, I, L, A and M; wherein the first and second conserved residues are separated by 7 residues. Epitopes on a number of objective immunogenic proteins can be identified using the peptides of the invention. Examples of suitable antigens include prostate cancer specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B surface and core antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens , antigens of the immunodeficiency virus type-1 (HIV1), of the virus herpes sarcoma of Kaposi (KSHV), antigens of the human papillomavirus (HPV), Lassa virus, mycobacterium tuberculosis (MT), p53 and p53 of murine (mp53) , CEA, trypanosome surface antigen (TSA), tyrosine-related protein families (TRP), and Her2 / neu. The peptides are thus useful in pharmaceutical compositions for therapeutic and diagnostic applications in vivo and ex vivo. The present invention also provides compositions comprising immunogenic peptides having binding motifs for MHC Class I molecules. Immunogenic peptides are typically between about 3 and about 11 residues and comprise conserved residues involved in binding proteins encoded by the appropriate MHC allele. A number of allele-specific motifs have been identified. For example, the reason for HLA-A3.2 includes the term? to the term C a first conserved residue of L, M, I, V, S, A, T and F at position 2 and a second conserved residue of K, R or Y at the C-terminal end. Other first conserved residues are C, G or D and alternatively E. Other second conserved residues are H or F. The first and second conserved residues are preferably separated by 6 to 7 residues. The motif for HLA-A1 comprises from the term N up to the term C a first conserved residue of T, S or M, a second conserved residue of D or E, and a third conserved residue of Y. Other second residues conserved are A , S or T. The first and second conserved residues are adjacent and are preferably separated from the third residue conserved by 6 to 7 residues. A second reason consists of a first conserved E or D residue and a second conserved Y residue where the first and second conserved residues are separated by 5 to 6 residues. The motif for HLA-ll comprises from the N term up to the C term a first conserved residue of T, V, M, L, I, S, A, G, N, CD, or F at position 2 and a residue conserved C-terminal of K, R, Y or H. The first and second conserved residues are preferably separated by 6 or 7 residues. The motif for HLA-A24.1 comprises from the N term up to the C term a first conserved residue of Y, F or W in position 2 and a conserved terminal residue of F, I, W, M or L The first and second conserved residues are preferably separated by or to 7 residues. Epitopes on a number of potential target proteins can be identified in this form. Examples of suitable antigens include prostate specific antigen (PSA), prostate-specific membrane antigen (PSM), hepatitis 8 surface core antigens (H3Vc, HBVs) hepatitis C antigens, malignant melanoma antigen (MAGE-1) Epstein-Barr virus antigens, human immunodeficiency virus type-1 (HIV1), papilloma virus antigens, Lassa virus, mycobacterium tuberculosis (MT), p53 and murine p53 (mp53), CEA, and Her2 / neu, and members of tyrosinase-related protein families (TRP). The peptides are thus useful in pharmaceutical compositions for therapeutic and diagnostic applications in vivo and ex vivo. The present invention also provides compositions comprising immunogenic peptides having binding motifs for HLA alleles without A. Immunogenic peptides are preferably about S to 10 residues in length and comprise residues conserved in some positions such as proline in the 2-position and a aromatic residue (eg, Y, W, F) or hydrophobic residue (eg, L, I, v, M, or A) in the Q term carboxy. In particular, an advantage of the peptides of 1 = invention is their ability to bind to two or more different HLA alleles. Epitopes on a number of potential target proteins can be identified in this form. Examples of suitable antigens include prostate-specific antigen (PSA), hepatitis B surface and core antigens (HBVc, HBVs) hepatitis C antigens, malignant melanoma antigen (MAGE-1) Epstein-Barr virus antigens, immunodeficiency virus human type-1 (HIV1), papilloma virus antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, and Her2 / neu. The peptides are thus useful in pharmaceutical compositions for therapeutic and diagnostic applications in vivo and ex vivo. Definitions The term "peptide" is used interchangeably with "oligopeptide" in the present specification to designate a series of residues, typically L-amino acids, typically connected to each other by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The oligopeptides of the invention are less than about 15 residues in length and commonly consist of between about 8 and about 11 residues, preferably 9 or 10 residues. An "immunogenic peptide" is a peptide that 1 or it comprises a specific allele motif such that the peptide will bind to an MHC molecule and induce a CTL response. The immunogenic peptides of the invention are capable of binding to an appropriate ELA-A2.1 molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived. Immunogenic peptides are conveniently identified using the algorithms of the invention. Algorithms are mathematical procedures that produce a score that allows the selection of immunogenic peptides. Typically one uses the algorithmic punctuation with a "binding threshold" to allow the selection of peptides that have a high probability of binding to a certain affinity and will in turn be immunogenic. The algorithm is based on the effects on the MHC bond of a particular amino acid at a particular position of a peptide or the effects on the binding of a particular substitution on a peptide containing motif. A "conserved residue" is an amino acid that occurs at a significantly higher frequency than would be expected by the random distribution at a particular position in a peptide. Typically a conserved residue is one in which the MHC structure can provide a point of contact with the immunogenic peptide. At least one to three or more, preferably two, residues conserved within a peptide of defined length define a motif for ur. immunogenic peptide. These residues are typically er. close contact with the peptide bond groove, with its side chains enclosed, specific gaps in the groove itself. Typically, an immunogenic peptide will comprise up to three conserved residues, most commonly two conserved residues. As used herein, "negative link residues" are amino acids that if present in some positions (eg, positions 1, 3 and / or 7 of a 9-mer) will result in a peptide that is a non-binding or weak linker and in turn will fail to be immunogenic ie will induce a CTL response. The term "motif" refers to the pattern of residues in a peptide of defined length, commonly from about 8 to about 11 amino acids, which is recognized by a particular MHC allele. Peptide motifs are typically different for each human MHC allele and differ in the pattern of highly conserved residues and negative residues. The link motif for an allele can be defined with increasing degrees of precision. In one case, all conserved residues are present in the correct positions in a peptide and there are no negative residues in positions 1,3 and / or 7.

The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free of components that commonly accompany it as it is in its native state. Thus, the peptides of this invention do not contain materials commonly associated with their in situ environment, eg, MHC I molecules on antigen presenting cells. Even where a protein has been isolated for a homogenous or dominant band, there are trace contaminants in the range of 5-10% of the native protein that co-purifies with the desired protein. The isolated peptides of this invention do not contain such endogenous co-purified protein. The term "residue" refers to an amino acid or amino acid mimetic incorporated in an oligopeptide by an amide bond or a mimetic amide linkage. I. Reason HLA-A2.1 The present invention relates to the determination of allele-specific peptide motifs for human MHC Class I allele subtypes (sometimes referred to as HLA), in particular, peptide motifs recognized by HLA-A2 alleles. 1. These motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with viral diseases, cancers or human autoimmune diseases, for which the amino acid sequence of the potential antigen or autoantigen targets is known.

Epitopes on a number of objective-potential proteins can be identified in this way. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis 3 core and surface antigen (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (e.g., MAGE-i), human immunodeficiency virus (HIV) antigen, human papilloma virus (HPV) antigen, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2 / neu. The peptides comprising the epitopes from these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in tests using, for example, purified class I molecules and radioiodinated peptides and / or expressing cells. Class I molecules empty by, for example, immunofluorescent staining and flow microfluorometry, peptide-dependent class I assembly test, and inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule that further vary by their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as by their ability to induce primary CTL responses in vivo or in vitro that can give rise to CTL populations capable of reacting with virally infected target cells or tumor cells as potential therapeutic agents. MHC class I antigens are encoded by the HLA-A, B, and C antigens. The HLA-A and B antigens are expressed on the cell surface at approximately equal densities, while the expression of HLA-C is significantly lower (maybe as much as 10 times smaller). Each of these sites has a number of alleles. The peptide linking motifs of the invention are relatively specific for each allelic subtype. For peptide-based vaccines, the peptides of the present invention preferably comprise a motif recognized by an MHC I molecule that has a wide distribution in the human population. Since MHC alleles occur at different frequencies within different ethnic groups and races, the selection of the target MHC allele may depend on the target population. Table 1 shows the frequency of several alleles in the products of the HLA-A site between different breeds. For example, the majority of Caucasoid population can be covered by peptides that bind to four subtypes of HLA-A allele, specifically HLA-A2.1, Al, A3.2, and A24.1. Likewise, the majority of the Asian population is encompassed with the addition of peptide that bind to a fifth allele HLA-A11.2.

TABLE 1 Subtype / Allele A N (69) * A (54) C (502) At 10.1 (7) 1.8 (1) 27.4 (138) A2.1 11.5 (8) 37.0 (20) 39.8 (199) A2.2 10.1 (7) 0 3.3 (17) A2.3 1 4 (1) 5.5 (3) 0.8 (4) A2.4 - - A2.5 - - A3.1 1.4 (1) 0 0.2 (0) A3.2 5.7 (4) 5.5 (3) 21.5 (108) Al l.l 0 5.5 (3) 0 Al 1.2 5.7 (4) 31.4 (17) 8.7 (44) A11.3 0 3.7 (2) 0 A23 4.3 (3) - 3.9 (20) A24 2.9 (2) 27.7 (15) 15.3 (77) A24.2 - - A24.3 - - A25 1-4 (1) - 6.9 (35) A26.1 4.3 (3) 9.2 (5) 5.9 (30) A26.2 7.2 (5) 1.0 (5) A26V - 3.7 (2) -A28.1 10.1 (7) - 1.6 (8) A28.2 1-4 (1) - 7.5 (38) A29.1 1.4 (1) - 1.4 (7) A29.2 10.1 (7) 1.8 (1) 5.3 (27) A30.1 8.6 (6) 4.9 (25) A30.2 1-4 (1) 0.2 (1) A30.3 7.2 (5) - 3.9 (20) A31 4-3 (3) 7.4 (4) 6.9 (35) A32 2.8 (2) 7.1 (36) AW33.1 8.6 (6) 2.5 (13) A 33.2 2.8 (2) 16.6 (9) 1.2 (6) A 34.1 1-4 (1) - Aw34.2 14.5 (10) - 0.8 (4) Aw36 5.9 (4) - Table compiled from B. DuPont, Iitimunobiology of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989. * N - Negroid; A = Asian; C = Caucasoid. The numbers in parentheses represent the number of individuals included in 1 O the analysis. The nomenclature used to describe the peptide compounds follows the conventional practice wherein the amino group is presented on the left (the N term) and the carboxyl group on the right (the C term) of each amino acid residue. In the formulas representing selected specific embodiments of the present invention, the terminal amino and carboxyl groups, although not specifically shown, are in the form they would assume at physiological pH values, unless otherwise specified. In amino acid structure formulas, each residue is usually represented by three letter or standard single letter designations. The L form of an amino acid residue is represented by a single uppercase letter or a first uppercase letter of a three letter symbol, and the D form for those amino acids that have D forms is represented by a single lowercase letter or a threefold symbol lowercase letters. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G. The procedures used to identify the peptides of the present invention generally follow the methods described in Falk et al., Nature 351: 290 (1991) which incorporated in the present for reference. Briefly, the methods involve the large-scale isolation of MHC class I molecules, typically by immunoprecipitation or affinity chromatography, from the appropriate cell or cell line. Examples of other methods for isolating the desired HC molecule equally well-known to the artisan include ion exchange chromatography, lectin chromatography, size exclusion, high resolution ligand chromatography, and a combination of all the above techniques. In the typical case, immunoprecipitation is used to isolate the desired allele. A number of protocols can be used, depending on the specificity of the antibodies used. For example, allele-specific mAb reagents can be used for affinity purification of the HLA-A, HLA-B1, and HLA-C molecules. Various mAb reagents for the isolation of HLA-A molecules are available. The monoclonal BB7.2 is suitable for isolating HLA-A2 molecules. Affinity columns prepared with these mAbs using standard techniques are successfully used to purify the respective HLA-A allele products. In addition to allele-specific mAbs, widely reactive anti-HLA-A-, B, C mAbs such as 6/32 and B9.12.1, and an anti-HLA-B, C mAb, Bl.23.2 could be used in protocols of alternative affinity purification as described in previous applications. The peptides linked to the peptide bond groove of the isolated MHC molecules are typically eluted after an acid treatment. Peptides can also be dissociated from class I molecules by a variety of standard denaturing media, such as heat, pH, detergents, salts, queotropic agents or a combination thereof. Peptide fractions are further separated from the MHC molecules by reverse phase high performance liquid chromatography (HPLC) and sequenced. The peptides can be separated by a variety of other standard means well known to the artisan, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, iso-electro-focusing and the like. The sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller, M.W., et al., Methods Enzymol, 91, 399

[1983]). Other methods suitable for sequencing include mass spectrometry sequencing of individual peptides as previously described (Hunt, et al., Science 225: 1261 (1992), which are incorporated herein by reference). The amino acid sequencing of mass heterogeneous peptides (e.g., pooled HPLC fractions) from different Ciase I molecules typically reveals a characteristic sequence motif for each class I allele. The definition of the specific motifs for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, the identification of potential peptide epitopes is carried out initially using a computer to examine the amino acid sequence of a desired antigen by the presence of motifs. The epitope sequences are then synthesized. The ability to bind MHC class molecules is measured in a variety of different ways. A means is a Class I molecule binding test as described in related applications, noted above. Other alternatives described in the literature include the inhibition of antigen presentation (Sette, et al., J. Immunol., 141: 3893 (1991), in vitro assembly tests (Townsend, et al., Cell 62: 285 (1990 ), and FACS-based tests using mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol., 21: 2963 (1991).) Then, the peptides that test positive in the MHC binding test. Class I are tested for the ability of the peptides to induce specific CTL responses in vitro, for example, the cells that present antigen to be incubated with a peptide can be tested for the ability to induce CTL responses in populations of responding cells. The cells that present antigen can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al., J. Exp. Mea 166: 182 (1987); Boog, Eur. J. Im unol. 18: 219

[1988]). Alternatively, the lines of mammalian mutant cells that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-5 (Karre, et al., Nature, 319: 675 ( 1986), Ljunggren, et al., Eur. J. Immunol., 21: 2963-2970 (1991)), and the human somatic T cell hybrid, T-2 (Cerundolo, et al., Nature 345: 449-452 (1990)), and which have been transfected with the appropriate human class I genes are conveniently used, when peptide is added to them, to test by the ability of the peptide to induce primary CTL responses in vitro. Other eukaryotic cell lines that could be used include various insect cell lines such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), gardama ( ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as Schneider cell lines (see Schneider J. Embryol, Exp Morphol 27: 353-365

[1927]). Peripheral blood lymphocytes are used, conveniently after venipuncture or simple leukapheresis of normal donors or patients and are used as the cell sources that respond to CTL precursors. In one embodiment, cells that present appropriate antigen are incubated with 10-100 μ? of serum free peptide for 4 hours under appropriate culture conditions. Peptide-laden antigen-presenting cells are then incubated by copulating populations of cells that respond in vitro for 7 to 10 days under optimized culture conditions. Positive CTL activation can be determined by testing the cultures for the presence of CTLs that destroy radiolabeled target cells, pulsed specific peptide targets, as well as target cells expressing the endogenously processed form of the relevant virus or tumor antigen from which it was derived the peptide sequence. The specificity and restriction of MHC of the CTL is determined by testing against different peptides of target cells expressing appropriate or inappropriate human class I MHC. Peptides that test positive for MHC binding and give rise to specific CTL responses are referred to herein as immunogenic peptides. Immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or from natural sources such as whole virus or tumors. Although the peptide is preferably substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be conjugated synthetically in native fragments or particles. The polypeptides or peptides can be of a variety of lengths, in their neutral (uncharged) forms, or in forms that are salts and free of modifications, such as glycosylation, side chain oxidation, or phosphorylation, or containing these modifications, subject to the proviso that the modification does not destroy the biological activity of the polypeptides as described herein Desirably, the peptide will be as small as possible while still maintaining substantially all the biological activity of the large peptide. , it may be desirable to use peptides of the invention at a length of 9 or 10 amino acid residues, in proportion to size with endogenously processed viral peptides or tumor cell peptides that bind to HC class I molecules on the surface of the cell Peptides having the desired activity can be modified as necessary to provide some desired intakes, for example, improved pharmacological characteristics, simultaneously increasing or at least retaining substantially all the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For example, the peptides may undergo various changes, such as conservative or non-conservative substitutions, wherein such changes might provide some advantages in their use, such as improved MHC binding. By conservative substitutions is meant replacing one amino acid residue with another that is biologically and / or chemically similar, for example, a hydrophobic residue by another, or a polar residue by another. Substitutions include combinations such as Gly, Ala; Val, lie, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions can also be probed using D-amino acids. Such modifications can be made using well-known peptide synthesis methods, as described in, for example, Merrifield, Science 232: 341-347 (1986), Barany and Merrifield, The Peptides, Gross and Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis, (Rockford, 111., Pierce), 2d Ed. (1984), incorporated herein by reference. The peptides can also be modified by increasing or decreasing the amino acid sequence of the context, for example, by the addition or subtraction of amino acids. The peptides or analogues of the invention can also be modified by altering the order or composition of some residues, it being readily appreciated that some amino acid residues essential for biological activity, for example, those at critical contact sites or conserved residues, can generally not be altered without an adverse effect on biological activity. Critical nc amino acids do not need to be limited to those that occur naturally in proteins, such as La-amino acids or their D-isomers, but may include non-natural amino acids as well, such as ß -? - d amino acids, as well as many amino acid derivatives. amino acids. Typically, a series of peptides with simple amino acid substitutions is used to determine the effect of electrostatic charge, hydrophobicity, etc. in the link. For example, a series of amino acid substitutions positively charged (eg, Lys or Arg) or negatively charged (eg, Glu) are made along the length of the peptide revealing different patterns of sensitivity to various MHC molecules and receptors. T cell. In addition, multiple substitutions using relatively small, small portions, such as Ala, Gly, Pro, or similar residues can be employed. The substitutions can be homo-oligomers or hetero-oligomers. The number and type of residuals that are replaced or added depend on the necessary spacing between the essential points of contact and some functional attributes that are sought (for example, hydrophobicity versus hydrophilicity). The increased binding affinity for an MHC molecule or T cell receptor can also be achieved by such substitutions, compared to the affinity of the parent peptide. In any case, such substitutions should employ amino acid residues or other selected molecular fragments to avoid, for example, steric interference and charge that could disrupt the bond. Amino acid substitutions are typically single residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final peptide. Substitution variants are those in which at least one residue of a peptide has been removed and a different residue has been inserted in its place. Such substitutions are generally made according to the following Table 2 when it is desired to finely modulate the characteristics of the peptide. TABLE 2 Original Residual Example of Substitution Wing Ser Arg Lys, His Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Lys; A. g He Leu; V l Leu He; Val Lys Arg; His Met Leu; He Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val lie; Leu Pro Gly Substantial changes in function (eg, affinity for MHC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in Table 2, that is, by selecting residues that differ more significantly in their effect to maintain (a) the structure of the peptide backbone structure in the area of substitution, eg, as a leaf or helix conformation and, (b) the charge or hydrophobicity in the molecule at the target site or (c) the mass of the side chain. The substitutions that are generally expected to produce the largest changes in the properties of the peptide will be those in which (a) the hydrophilic residue, eg, seryl, is replaced by (or by) a hydrophobic residue, eg, isocyclyl, , phenylalany, valyl or alanyl; (b) a residue having an electro-positive side chain, for example, lysium, arginyl, or histidyl, are substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (c) a residue having a bulky side chain, for example phenylalanine, is substituted for (or by) one that does not have a side chain, for example, glycine. The peptides may also comprise isoesters of two or more residues in the immunogenic peptide. An "isoster" as defined herein is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence adjusts a specific binding site for the second sequence. The term specifically includes modifications of the peptide backbone structure well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, the amide carbonyl, the complete replacement of the amide bond, extensions, deletions or cross-links of the main structure. See generally, Spatola, Chemistry and Biochemistry of Amino Acids. peptides and Proteins, Voi. VII (Weinstein ed., 1983). Modifications of the peptides with various amino acid mimetics of non-natural amino acids are particularly useful for increasing the stability of the peptide in vivo. Stability can be tested in a number of ways. For example, peptidases and various biological media, such as plasma and human serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11 291-302 (1986). The half-life of the peptides of the present invention is conveniently determined using a 25"- (v / v) human serum test.The protocol is generally as follows.The assembled human serum (Type AB, not heat-inactivated) is degrease by centrifugation before use The serum is then diluted to 25% with RPMI tissue culture medium and used to test the stability of the peptide At predetermined time intervals a small amount of the reaction solution is removed and added to 6% aqueous trichloroacetic acid or ethanol.The turbid reaction sample is cooled (4 ° C) for 15 minutes and then rotated to granules in precipitated serum proteins.The presence of the peptides is then determined by phase HPLC. Inverse using stability-specific chromatography conditions The peptides of the present invention or analogs thereof having CTL-stimulating activity can be modified to provide desired attributes other than improved serum half-life. For example, the ability of the peptides to induce CTL activity can be enhanced by binding to a sequence containing at least one epitope that is capable of inducing a helper T cell response. Particularly preferred immunogenic peptides / conjugates of assistant T are linked by a spacing molecule. The spacer is typically comprised of relatively small neutral molecules, such as amino acids or mimetic amino acids, which are substantially uncharged under physiological conditions. The spacers are typically selected from, for example, Ala, Gly, or other neutral spacer of non-polar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer does not need to be comprised of the same residues and may thus be a hetero- or homo-oligomer. When present, the spade will commonly be of at least one or two residues, most commonly three to six residues. Alternatively, the CTL peptide can be linked to the assistant peptide T without a spacer. The immunogenic peptide can be linked to the assistant peptide T directly or by means of a spacer at the amino or carboxy terminus of the CTL peptide. The amino terminus of the immunogenic peptide or the assistant peptide T can be acylated. Examples of T-helper peptides include tetanus toxoid 830-843. influenza 307-319, malaria circumsporozoite 382 - 398 and 378 - 38 9. In some embodiments it may be desirable to include at least one CTL priming component in the pharmaceutical compositions of the invention. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, residues of palmitic acid can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, for example, by means of one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser , or the like, to an immunogenic peptide. The oiled peptide can then be injected directly into a micellar form, incorporated into a liposome or emulsified in an adjuvant, for example, incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid bound to alpha amino and epsilon groups of Lys, which is linked via, for example, Ser-Ser, to the amino terminus of the immunogenic peptide. As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteiniseryl-serine (P2CSS) can be used to prime virus-specific CTL when covalently bound to an appropriate peptide. See, Deres et al., Nature 342: 561-564 (1989), incorporated herein by reference. The peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Additionally, as the induction of neutralizing antibodies can also be primed with PC5S conjugated to a peptide showing an appropriate epitope, the two compositions can be combined to more effectively produce cell-mediated and humoral responses for infection. In addition, additional amino acids can be added to the terms of a peptide to provide ease of binding peptides to each other for coupling a carrier support, or larger peptides, to modify the physical or chemical properties of the peptide and oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced into the C or N terminus of the peptide or oligopeptide. The modification in the C term in some cases may alter the binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences may differ from the natural sequence when modified by NK acylation; terminal, for example, by acetylation of alkanoyl (Ci-C;-) or thioglycolyl, carboxy terminal amidation, for example, ammonia, methylamine, etc. In some cases these modifications may provide sites to bind to a support or other molecule. The peptides of the invention can be prepared in a wide variety of ways. Due to their relatively short size, the peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984), supra. Alternatively, recombinant DNA technology can be employed where a sequence of nucleotides encoding immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultured under conditions suitable for expression. These procedures are generally known in the art, as is generally described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), which is incorporated herein by reference. Thus, fusion proteins comprising one or more peptide sequences of the invention can be used to present the appropriate T cell epitope. As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Scc. 103: 3185 (1981), modifications simply by substituting the appropriate base (s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such suitable vectors and host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator sections and commonly a replication system to provide an expression vector for expression in the desired cell host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast or mammalian host cells can also be used, employing suitable vectors and control sequences. The peptides of the present invention and the pharmaceutical and vaccine compositions thereof are useful for administration to mammals, particularly humans, to treat and / or prevent viral infection and cancer. Examples of diseases that can be treated using the immunogenic peptides of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and condlyloma acuminatum. For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual who already suffers from cancer or is infected with the virus of interest. Those in the incubation phase or the acute phase of the infection can be treated with the immunogenic peptides separately or together with other treatments as appropriate. In therapeutic applications, the compositions are administered to a patient in an amount sufficient to produce an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest the symptoms and / or complications. An adequate amount to achieve this is defined as "therapeutically effective dose". The amounts effective for this use will depend on, for example, the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but it generally varies for the initial immunization (for prophylactic therapeutic administration) from about 1.0 μg to about 5000 g of peptide for a 70 kg patient, followed by thrust dosages of about 1.0 μg to about 1000 μg of peptide in tracking er. régimer of pushing for weeks to months depending on the response and condition of the patient and measuring the specific CTL activity in the patient's blood. It should be kept in mind that the peptides and compositions of the present invention can generally be employed in serious affective states, that is, life-threatening or life-threatening situations. In such cases, in view of the minimization of foreign substances and the relatively non-toxic nature of the peptides, it is possible and may be felt desirable by the physician who tries to administer substantial excess of these peptide compositions. For therapeutic use, administration should begin with the first sign of viral infection or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by pushing dose until at least the symptoms are decreased and for a period later. In chronic infection, loading doses followed by booster doses may be required. The treatment of an individual infected with the composition of the invention can precipitate the resolution of the infection in acutely infected individuals. For those susceptible or predisposed individuals) to develop chronic infection the compositions are particularly useful in methods to prevent the evolution of the infection from acute to chronic. Where susceptible individuals are identified before or during infection, for example, as described herein, the composition can be selected for them, minimizing the need for administration to a larger population. The peptide compositions can also be used for the treatment of chronic infection and for stimulating the immune system to eliminate infected cells with carrier virus. It is important to provide an amount of immunopotentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response. Thus, for the treatment of chronic infection, a representative dose is in the range of about 1.0 μg to about 5000 μg / preferably about 5 μg to 1000 g for a patient of 70 kg per dose. Immunization doses-followed by booster doses at set intervals, for example, from one to four weeks, may be required, possibly for a prolonged period of time to effectively immunize an individual. In the case of chronic infection, administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been substantially eliminated or decreased and for a later period. Pharmaceutical compositions for therapeutic treatment are intended for parenteral administration, topical, oral or local. Preferably, the pharmaceutical compositions are administered parenterally, for example, intravenously, subcutaneously, intradermally or intramuscularly. Thus, the invention provides compositions for parenteral administration comprising, a solution of the immunogenic peptides either dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, regulated water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterilized by filtration. The resulting aqueous solutions can be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting agents and regulators, tonicity adjusting agents, wetting agents and the like, eg, sodium acetate, sodium lactate, Sodium chloride, potassium chloride, calcium chloride, sorbitan monoaurate, triethanolamine oleate, etc. The concentration of the CTL stimulating peptides of the invention in the pharmaceutical formulation can vary widely, ie, less than about 0.1 '.;, commonly at or at least about 2% up to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. The peptides of the invention can also be administered by means of liposomes which will serve to select the peptides in a particular tissue, such as lymphoid tissue, or selectively selected to infect cells, as well as increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or together with a molecule that binds to, for example, a predominant receptor among the lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled or decorated with a desired peptide of the invention can be directed to the site of the lymphoid cells, where the liposomes then deliver the selected therapeutic / immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally directed by consideration of, for example, liposome size, the capacity and acid stability of the liposomes in the bloodstream. A variety of methods are available to prepare liposomes, as described in, for example, Szoka et al., Ann. Re. Biophys. Bioeng. 9: 467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference. To select immune cells, a ligand to be incorporated into the liposome may include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide can be administered intravenously, locally, topically, etc. in a dose that varies according to, inter alia, the form of administration, the peptide that is delivered, and the stage of the disease being treated. For solid compositions, conventional non-toxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any of the commonly used excipients, such as those previously listed carriers, and generally 10-95% active ingredient, that is, one or more peptides of the invention, and greater preference at a concentration of 25% -75%. For aerosol administration, the immunogenic peptides are preferably delivered in finely divided form together with a surfactant and propellant. Typical percentages of peptides are 0.01% -20% by weight, preferably 1% -10 *. The surfactant must, of course, be non-toxic, and preferably soluble in the propellant. Representative of such agents are the partial esters or esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic acid, octanoic, leuric, palmitic, stearic, linoleic, linolenic, oesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides can be employed. The surfactant may constitute 0.1¾-20% by weight of the composition, preferably 0.25-5, · -. The balance of the composition is ordinarily orogenic. A carrier can also be included, as desired, as with, for example, lecithin for intranasal delivery. In another aspect the present invention is directed to vaccines containing as an active ingredient an immunogenically effective amount of an immunogenic peptide as described herein. The peptide (s) can be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make the polymer, the additional ability to induce antibodies and / or the CTL that react with different antigenic determinants of the virus or tumor cells. Useful carriers are well known in the art, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), influenza, hepatitis B virus core protein. , recombinant hepatitis 3 virus vaccine and the like. The vaccines may also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and typically and additionally include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. And, as mentioned above, CTL responses can be primed by cgating peptides of the invention with lipids, such as P3CSS. During immunization with a peptide composition as described herein, by injection, aerosol, oral, transdermal or other route, the host's immune system responds to the vaccine by producing large amounts of the CTL specific for the desired antigen, and the host becomes at least partially immune to subsequent infection, or resistance to developing chronic infection. Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of viral infection or cancer to produce an immune response against the antigen and thereby enhance the patient's own immune response capabilities. Such an amount is defined as an "immunologically effective dose". In this use, the precise amounts again depend on the state of health and weight of the patient, the mode of administration, the nature of the formulation, etc., but it varies in general from about 1.0 g to about 5000 ug per patient of 7C kilograms , most commonly of approximately 10 μs to about 500 g per 70 kg of body weight.

In some cases it may be desirable to combine the peptide vaccines of the invention with vaccines that induce neutralizing antibody responses to the virus of interest, particularly viral envelope antigens. For therapeutic or immunization purposes, the nucleic acids encoding one or more of the peptides of the invention may also be administered to the patient. A number of methods are conveniently used to deliver the nucleic acids to the patient. For example, the nucleic acid can be delivered directly, such as "naked DNA". This proposal is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Patent Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using a ballistic delivery as described, for example, in U.S. Patent No. 5,204,253. Particles comprised only of DNA can be administered. Alternatively, the DNA can adhere to particles, such as gold particles. The nucleic acids can also be supplied in complex to cationic compounds, such as cationic lipids. Methods of supplying genes mediated by lipid are described, for example, in WO 96/18372; WC 93/24640; annino and Gould-Fogerite (1988) BioTechniquas 6 (7): 682-691; Rose U.S. Pat No. 5, 279,833; WO 91/06309; and Felgner et al. (1987) Proc. Nati Acad. Sci. USA 84: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as poultry vaccination or pustulation. This proposal involves the use of the vaccine virus for a vector to express nucleotide sequences encoding the peptides of the invention. During introduction into an acutely or chronically infected host or within an uninfected host, the recombinant vaccine virus expresses the immunogenic peptide, and thereby produces a host CTL response. Vaccine vectors and methods useful in immunization protocols are described in, for example, U.S. Patent No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)) which is incorporated herein by reference. A wide variety of other vectors useful for administration or therapeutic immunization of the peptides of the invention, for example the Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein. A preferred means for administering nucleic acids encoding the peptides of the invention uses minigene constructs that encode multiple epitopes of the invention. To create a DNA sequence encoding the selected CTL epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes are translated in reverse. A table of use of human codons is used to drive codon selection for each amino acid. These DNA sequences that encode, epitope are attached directly, creating a continuous polypeptide sequence. To optimize expression and / or immunogenicity, additional elements can be incorporated into the design of the minigene. Examples of amino acid sequences that could be translated in reverse and included in the sequence of the minigene include: helper T lymphocyte epitopes, a leader sequence (signal), and an endoplasmic reticulum retention signal. In addition, the MHC presentation of the CTL epitopes can be improved by including synthetic flanking sequences (eg poly-aianine) or occurring naturally adjacent to the CTL epitopes. The minigene sequence is converted into DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Oligonucleotides (30-100 bases long) are synthesized which overlap, phosphorylate, purify and temper under appropriate conditions using bier techniques. known. The ends of the oligonucleotides are ligated using T4 of DNA ligase. This synthetic minigen, which encodes the CTL epitope polypeptide, can then be cloned into a desired expression vector. Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in the target cells. Several vector elements were required: a promoter with a cloning site towards the 3 'end for the insertion of the minigen; a polyadenylation signal for efficient transcription termination; an origin of repiication of E. coli; and a selectable E. coli marker (e.g., resistance to ampicillin or kanamycin). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus promoter (hC V). See, US Pat. Nos. 5,580,359 and 5,589,466 for other suitable promoter sequences. Additional vector modifications may be desired to optimize the expression and immunogenicity of the minigene. In some cases introns are required for efficient gene expression and one or more naturally occurring synthetic introns could be incorporated within the transcribed minigene region. The inclusion of mRNA stabilization sequences can also be considered to increase the expression of the minigene. It has recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a role in the immunogenicity of DNA vaccines. These sequences could be included in the vector, outside the minigene coding sequence if they are found to improve immunogenicity.

In some embodiments, a bioistronic expression vector can be used, to allow the production of the epitopes encoded by the minigend and a second included protein to improve or decrease immunogenicity. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (eg, IL2, IL12, GM-CSF), cytokine-inducing molecules (eg LelF) or costimulatory molecules. The attendant epitopes (HTL) could bind to intracellular selection signals and be expressed separately from the CTL epitopes. This would allow the targeting of the HTL epitopes to a cell compartment different from the CTL epitopes. If required, this could more efficiently facilitate the entry of HTL epitopes into the MHC class II route, thereby enhancing the CTL induction. In contrast to the induction of CTL, specifically decreasing the immune response by co-expressing immunosuppressive molecules (eg TGF-β) may be of benefit in some diseases. Once an expression vector is selected, the minigene is cloned into the polylinker region towards the 3 'end of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as the other elements included in the vector, are confirmed using restriction mapping of DNA sequence analysis. The bacterial cells that harbor the correct plasmid can be stored as a bank of master cells, and a bank of working cells. Therapeutic amounts of plasmid DNA are produced by fermentation in E. coli, followed by purification. The aliquots from the working cell bank are used to inoculate the fermentation medium (such as a Fantastic Broth), and are grown to saturation in shake flasks or a bioreactor according to well-known techniques. The plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion exchange resins supplied by Quiagen. If required, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other methods. Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in saline regulated with sterile phosphate (P3S). A variety of methods have been described, and new techniques may become available. As noted above, the nucleic acids are conveniently formulated with cationic ioids. In addition, glycolipids, fuscogenic liposomes, peptides and collectively preferred, protective, interactive, non-condensing (PINC) compounds could also complex with purified plasmid DNA for varying influences such as stability, intramuscular dispersion, or trafficking to specific organs or cell types. . Sensitization of the target cell can be used as a functional test for expression and presentation of MHC Class I CTL epitopes encoded by minigens. Plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release tests. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be cotranslated to allow enrichment of transfected cells using activated fluorescence cell distribution (SACF). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. Cytolysis, detected by the release of CR51, indicates the production of the MHC presentation of CTL epitopes encoded by minigens. Immunogenicity in vivo is a second proposal for the functional test of minigene DNA formulations.

Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are dependent on the formulation (for example IN for DNA in PBS, IP for DNA in lipid complex). Twenty-one days after immunization, the splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. These effector cells (the CTLs) are tested by cytolysis of target cells labeled with peptide-loaded chromium-5I using standard techniques. Lysis of target cells sensitized to peptide MHC loading corresponding to epitopes encoded by minigens demonstrate the role of the DNA vaccine for in vivo induction of CTL. The antigenic peptides can be used to produce CTL ex vivo as well. The resulting CTL can be used to treat chronic infections (viral or bacterial) or tumors in patients who do not respond to other conventional forms of therapy, or who will not respond to a proposed peptide therapy vaccine. Ex vivo CTL responses to a particular pathogen (infectious agent or tumor antigen) are induced by incubating in tissue culture the patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the immunogenic peptide. appropriate. After an appropriate incubation time (typically 1-4 weeks) in which the CTLp is activated and matures and expands within the effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (one cell infected or a tumor cell). The peptides may also find use as diagnostic reagents. For example, a peptide of the invention can be used to determine the susceptibility of a particular individual to a treatment regimen employing the peptide or related peptides, and thus it can be helpful to modify an existing treatment protocol or to determine a prognosis for an affected individual. In addition, peptides can also be used to predict which individuals will be at substantial risk of developing chronic infection. The following example is presented by way of illustration, not by way of limitation. EXAMPLE 1 The isolation of the Class I antigen was carried out as described in the related applications, noted above. The naturally processed peptides were then isolated and sequenced as described therein. A specific allele and algorithm motive was determined and the quantitative binding tests were carried out. Using the motifs identified above for the amino acid sequences of the HLA-2 allele, a number of antigens were analyzed for the presence of these motifs. Table 3 provides the results of these searches. The letter "J" represents norleucine. The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference.

Table 3 PeDtid AA Sequence Source A * 0201 17. 0317 9 LQIGKQSI Flu.24 0.0130 38. 0103 9 NLSLSCHAA CEA..432 0.01 10 1233. 1 1 9 YLSGANLNV CEA..605V9 0.0690 1295. 03 9 SMPPPGTRV P53.149M2 0.0290 1295. 04 9 SLPPPGTRV P53.149L2 0.0410 1317. 24 9 KTCPVQLWV P53.139 0.0069 1323. 02 9 LLPENNW p53.2 V9 0.0130 1323. 04 9 ALNKMFBQV P53.129B7V9 0.0260 1323. OIS 9 KLBPVQLWV P53.139L2B3 0.1 100 1323. 08 9 BLTIHY YV P53.229B1L2V9 0.0430 1323. 18 10 LLPPQHL1RV P53.188L2 0.0061 1323. 29 11 YMCNSSCMGGM P53.236 0.0075 1323. 31 11 YLCNSSCMGGV P53.236L2V1 1 0.2300 1323. 34 1 1 KLYQGSYGFRV P53.101L2V1 1 0.0620 1324. 07 9 CQLAKTCPV P53.135 0.0240 1325. 01 9 RLPEAAPPV P53.65L2 0.0640 1325. 02 9 GLAPPQHLV p53.187V9 0.0130 1325. 04 9 KMAELVHFL MAGE3.1 12M2 0.2100 Peptide A Sequence Source A * 0201 1325. 05 9 KLAELVHFL MAGE3.1 12L2 0.2500 1326. 01 9 CLLA TCPV p53. 5L2 0.0400 1326. 02 9 LSQHMTEV p53.l64L2 0.0410 1326. 04 9 ELAPWAPV p53.68L2V9 0.0860 1326. 06 10 QLAKTCPVQV p53.136 0.0320 1326. 08 9 HLTEWRRV p53.168L2 0.0180 1329. 01 11 KTYQGSYGFRL 0.0028 1329. 03 10 VWPYEPPEV p53.216 0.0081 1329. 14 9 BQLA TBPV P53.135B1B7 0.0490 1329. 15 9 BLLAKTBPV p53.135BlL2B7 0.1100 1330. 01 9 QHGYVIGT CEA.78 0.0160 1330. 02 9 QLIGYVIGV CEA.78L2V9 0.5300 1330.05 9 YVCGIQNSV CEA.569. 0.0510 1330. 06 9 YLCGIQNSV CEA.569L2 0.1000 1330. 07 9 ATVGIMIGV CEA.687 0.1400 1330. 08 9 ALVGIMIGV CEA.687L2 0.5000 1330. 09 10 VLYGPDDPTI CEA. 11 0.0170 1330. 10 10 VLYGPDDPTV CEA.411V10 0.0310 1331. 02 9 DLMLSPDDV p53.42V9 1331.03 9 ALMLSPDDI p53.42Al 1331.04 9 ALMLSPDDV P53.42A1V9 1331.05 9 DLMLSPA I p53.42A7 1331.06 9 DLMLSPADV p53.42A7V9 1331.07 9 DLMLSPDAI p53.42A8 1331.08 9 DL LSPDAV p53.42A8V9 38.0007 9 AILTFGSFV KSHV.89 0.0850 38. 0009 9 HL DFALAV KSHV.106 0.0183 38. 0015 9 ALLGSIALL KSHV.155 0.0470 38. 0018 9 ALLATHAA KSHV.161 0.0490 38. 0019 9 LLATELAAV KSHV.162 0.1600 38. 0022 9 RLFADELAA KSHV.14 0.0150 Peptide AA Sequence Source? * 020? 8.0024 9 YLSKCTLAV KSHV.65 0.2000 8.0026 9 LVYHIYSKI KSHV.153 0.0457 8.0029 9 S YLCILSA SHV.208 0.0250 8.0030 9 YLCILSALV KSHV.210 0.3500 38. 0033 9 VMFSYLQSL KSHV.268 0.5000 38. 0035 9 RLHVYAYSA SHV.285 0.0270 38. 0039 9 GLQTLGAFV SHV.98 0.0110 38. 0040 9 FVEEQMTWA KSHV.105 0.0380 38. 0041 9 QMTWAQTW KSHV.109 0.0110 38. 0042 9 IILDTAIFV KSHV.130 0.6800 38. 0043 9 AIFVCNAFV KSHV.135 0.0910 38. 0046 9 A GNRLVEA KSHV.172 0.0200 38. 0047 9 LVEAC LL KSHV.176 0.0180 38. 0059 9 TLSIVTFSL SHY.198 0.2200 38. 0063 9 LSVLLLEV KSHV.292 0.1400 38. 0064 9 LLLEV RSV SHV.296 0.0270 38. 0068 9 FVSSPTLPV KSHV.78 0.0350 38. 0070 9 AMLVLLAEI KSHV.281 0.0820 38. 0075 9 QMARLAWEA KSHV.1116 0.0990 38. 0131 10 VLAECHFMA KSHV.10 0.0730 3 41. 0118 9 LMLFYQVWV TRP1 2.4000 41. 0119 9 LLPSSGPGV T P1 0.3700 41. 0121 9 NLSIYNYFV TRP1 0.9700 41. 0122 9 NLSVYNYFV TRP1 0.8700 41. 0123 9 FLWTHYYSV TRP1 5.6000 41. 0124 9 SL KTFLGV TRPl 0.0224 41. 0125 9 FLTWHRYHV TRP1 0.3800 41. 0129 9 MLQEPSFSV TRP1 1.6000 41. 0130 9 SLPYWNFAV TRPl 0.5700 41. 0131 9 ALGKNVCDV TRP1 0.0160 41. 0132 9 SLLISPNSV TRPl 0.1300 41. 0133 9 SLFSQWRW TRPl 0.0740 41. 0134 9 TLGTLCNSV TRPl 0.0330 41. 0136 9. RLPEPQDW TRPl 0.1000 41. 0137 9 VLQCLEVRV TRPl 0.0360 41. 0138 9 SLNSFRNTV TRPl 0.0140 41. 0139 9 SLDSFRNTV TRPl 0.0440 41. 0141 9 FLNGTGGQV TRPl 0.0220 41. 0142 9 VLLHTKl'DV TRPl 0.0180 41. 0145 9 ALVGALLLV TRPl 0.2600 Peptide ?? Sequence Source A * 0201 41. 0146 9 ALVAALLLV TRJP 1 0.5800 41. #, hepatitis B nuclei and surface antigens (HBVc, HBVs), hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (eg, MAGE-1), human immunodeficiency virus (HIV) antigens, and antigens of the human papilloma virus (HPV), Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, and Her2 / neu. The peptides comprising the epitopes of these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in tests using, for example, purified class I molecules and radioiodinated peptides and / or cells expressing class molecules. I vacuums by, for example, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly tests, and inhibition of CTL recognition by peptide competence. Those peptides that bind to the class I molecule are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their ability to induce CTL responses in vitro or in vivo that can give erect CTL populations capable of reacting with virally infected target cells or tumor cells as potential therapeutic agents. The MHC class I antigens are encoded by the HLA-A, B, and C sites. The HLA-A and B antigens are expressed on the cell surface at approximately equal densities, while the expression of HLA-C is significantly lower ( maybe as much as 10 times smaller). Each of these sites has a number of alleles. The peptide bonding motifs of the invention are relatively specific for each allelic subtype. For peptide-based vaccines, the peptides of the present invention preferably comprise a motif recognized by an MHC I molecule that has a wide distribution in the human population. Since MHC alleles occur at different frequencies within different ethnic groups and rats, the selection of the target MHC allele may depend on the target population. Table 4 shows the frequency of various alleles in the products of the HLA-A site between different breeds. For example, the majority of Caucasoid population can be covered by peptides that bind to four subtypes of HLA-A allele, specifically HLA -A2.1, Al, A3.2, and A24.1. Likewise, the majority of the Asian population is covered by the addition of peptides that bind to a fifth allele HLA-All.2.

TABLE 4 Subtype / Allele A N (69 ^ »A (54 CÍ502) Al io.i (7) 1.8 (1) 27.4 (138) A2.1 11.5 (8) 37.0 (20) 39.8 (199) A2.2 10.1 (7) 0 3.3 (17) A2.3 1 4 (1) 5-5 (3) 0.8 (4). A2.4 A2.5 A3.1 1 4 (1) O 0.2 (0) A3.2 5.7C4) 3 5 (3) 21.5 (108) AU. l O 5 5 (3) O Al 1.2 5.7 (4) 31.4 (17) 8.7 (44) A1 I3 O 3.7 (2) O A23 4.3 (3) 3.9 (20) A24 2.9 (2) 27.7 (15) 15.3 (77) A24.2 A24.3 A25 1 4 (1) 6.9 (35) A26.1 4.3 (3) 9.2 (5) 5.9 (30) A26.2 7.2 (5) 1 -0 (5) A26V 3.7 (2) A28.1 10.1 (7) 1.6 (8) A28.2 1.4 (1) 7.5 (38) A29.1 1.4 (1) 1.4 (7) A29.2 10.1 (7) 1 8 (1) 5.3 ( 27) A30. 1 8.6 (6) 4.9 (25) A30.2 1 4 (1) 0.2 (1) A30.3 7-2 (5) 3.9 (20) A31 4.3 (3) 7.4 (4) 6.9 (35) A32 2.8 ( 2) 7.1 (36) Aw33. 1 8.6 (6) 2.5 (13) Aw33.2 2.8 (2) 16.6 (9) 1.2 (6) Aw34. 1 1 4 (1) Aw34.2 14.5 (10) 0.8 (4) Aw36 5.9 (4) Table compiled from 3. DuPont, Immunobiology of HLA, Vol. I, Hiscocompatibility Testinc 1987, Springer-Verlag, New York 1989 . * N - Negroid; A = Asian; C = Caucasoid. The numbers in parentheses represent the number of individuals included in the analyzes. The nomenclature used to describe the peptide compounds follows the conventional practice wherein the amino group is presented on the left (the N term) and the carboxyl group on the right (the C term) of each amino acid residue. In the formulas representing the selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiological pH values, unless otherwise specified. In amino acid structure formulas, each residue is usually represented by standard three letter or single letter designations. The L form of an amino acid residue is represented by a single upper case letter or a first uppercase letter of a three letter symbol, and the D form for those amino acids is represented by a lowercase single letter or a lowercase three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G. The procedures used to identify the peptides of the present invention generally follow the methods described in Falk et al., Nature 351: 290 (1991). which is incorporated herein by reference. Briefly, the methods involve the large-scale isolation of MHC class I molecules, typically by immunoprecipitation or affinity chromatography, from the appropriate cell or cell line. Examples of other methods for isolating the MHC molecule equally well known to the artisan include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and a combination of all the above techniques. A large number of cells with defined MKC molecules, particularly MHC Class I molecules, are known and readily available. For example, transformed human EBV B cell lines have been shown to be excellent sources for the preparative isolation of MHC class I and class II molecules. Well characterized cell lines are available from private and commercial sources, such as American Type Culture Collection ("Catalog of Cell Lines and Hybridomas", 6th edition (1988) Rockville, Maryland, U.S.A.); National Institute of General Medical Sciences 1990/1991 Catalog of Cell Lines (NIGMS) Human Genetic Mutant Cell Repository, Camden, NJ; and ASHI Repository, Bingham and Women's Hospital, 75 Francis Street, Boston, MA 02115. Table 5 lists some B cell lines suitable for use as sources of HLA-A alloys. All these cell lines can be grown in large batches and are therefore useful for the large-scale production of MHC molecules. Someone with experience will recognize that these are only examples of cell lines and that they can be used in many other cell sources. Similar EBV B cell lines homozygous for HLA-B and HLA-C could serve as sources for HLA-B and HLA-C alleles, respectively. TABLE 5 HUMAN CELL LINES (SOURCES HLA-A) HLA-A allele B cell line To MAT COX (9022) STEINLIN (9087) A2.1 JY A3.2- ?? (9080) HO301 (9055) GM3107 A24.1 T3 (9107), TISI (9042) All BVR (GM6828A WT100 (GM8602) WT52 (GM8603) In the typical case, immunoprecipitation is used to isolate the desired allele. A number of the protocols, depending on the specificity of the antibodies used, for example, reagents, specific for mAb allele can be used for the affinity purification of the HLA-A, HLA-B, and HLA-C molecules. various mAb reagents for the isolation of HLA-A molecules (Table 6) Thus, for each of the selectable HLA-A alleles, reagents are available that can be used for the direct isolation of HLA-A molecules. affinities prepared with these mAbs using standard techniques are successfully used to purify the respective HLA-A allele products In addition to the allele-specific mAbs, the widely reactive anti-HLA-A, B, C mAbs, such as W6, could be used. / 32 and B9.12.1, and a mAb Bl.23. 2 of anti-HLA-3, C, in alternative affinity purification protocols as described in the example section below. TABLE 6 REAGENTS OF ANTI-HLA ANTIBODIES Name HLA-A1 12/18 HLA-A3 GAPA3 (ATCC, H3122) HLA-11.2.1 A11.1M (ATCC, HB164) 6 / HLA-A, B, C W6 / 32 (ATCC, HB9 monomorphic B9.12.1 (INSERM-CNRS HLA-B, C B .1.23.2 (INSERM-CNRS) monomorphic The peptides linked to the peptide bond groove of the isolated MHC molecules are typically eluted using acid treatment. The peptides can also be dissociated from Class I molecules by a variety of standard denaturing media, such as heat, pH, detergents, salts, chaotropic agents, or a combination thereof. Peptide fractions are further separated from the MHC molecules by reverse phase high performance liquid chromatography (HPLC) and sequenced. The peptides can be well separated by a variety of other standard means well known to the artisan, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, and the like. The sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller, M.W., et al., Merhods Enzymol, 91, 399

[1983]). Other methods suitable for sequencing include sequencing of mass spectrometry of individual peptides as previously described (Hunt, et al., Science 225: 1261 (1992), which are incorporated herein by reference). The amino acid sequencing of mass heterogeneous peptides (eg, assembled HPLC fractions) from different class I molecules typically reveals a characteristic sequence motif for each class I allele. The definition of specific motifs for different class alleles I allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, the identification of potential peptide epitopes is carried out initially using a computer to examine the amino acid sequence of a desired antigen by the presence of motifs. The epitope sequences are then synthesized. The ability to bind MHC class molecules is measured in a variety of different ways. A means is a Class I molecule binding test as described in related applications, noted above. Other alternatives described in the literature include inhibition of antigen presentation (Sette, et al., J. Immunol., 141: 3893 (1991), in vitro assembly tests (Townsend, et al., Cell 62: 285 (1990 ), and FACS-based tests using mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol., 21: 2963 (1991).) Then, the peptides that test positive in the MHC binding test. Class I are tested for the ability of peptides to induce specific CTL responses in vitro.For example, antigen-presenting cells that have been incubated with a peptide can be tested for the ability to induce CTL responses in populations of responding cells. antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al., J. Exp. Med. 166: 182 (1987); Boog, Eur. J. Immunol., 18: 219

[1988]) Alternatively, the lines of mammalian mutant cells that are capable of loading class I molecules with internally processed peptides, such as RMA-S mouse cell lines (Karre, et al., ature, 319: 675 (1986); Ljunggren, et al., Eur. J. Immunol. 21: 2963-2970 (1991)), and the human somatic T cell hybrid, T-2 (Cerundolo, et al., Nature 345: 449-452 (1990)), and which have been transfected with the class genes. Appropriate humans are conveniently used, when peptide is added thereto, to test for the ability of the peptide to induce primary CTL responses in vitro. Other eukaryotic cell lines that could be used include various insect cell lines such as mosquito larvae (ATCC CCL 125 cell lines)., 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), gardama (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as Schneider cell lines (see Schneider J. Embryol, Exp Morphol 27: 353-365

[1927]). Peripheral blood lymphocytes are conveniently used following venipuncture or simple leukapheresis from normal donors or patients and are used as sources of cells that respond to CTL precursors. In one embodiment, the cells that present appropriate antigen are incubated with 10-100 μ of serum free peptide for 4 hours under appropriate culture conditions. The antigen-loaded peptide cells are then incubated with cell populations that respond in vitro for 7 to 10 days under optimized culture conditions. Positive CTL activation can be determined by testing the cultures for the presence of the CTL that destroy the radiolabeled target cells, pulsed specific peptide targets, as well as target cells that express the endogenously antigen form of the relevant virus or tumor from which it was derived. the peptide sequence. The specificity and restriction of MHC of the CTL is determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC I. Peptides that test positive in MHC binding assays give rise to specific CTL responses are referred to herein as immunogenic peptides. Immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or from natural sources such as viruses or whole tumors. Although the peptide will be preferable and substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be conjugated synthetically with native fragments or particles. The polypeptides or peptides can be a variety of lengths, in their neutral uncharged forms) or in forms that are salts and free of modifications, such as glycosylation, side chain oxidation, or phosphorylation, or containing these modifications, the condition is accepted that the modification does not destroy the biological activity of the polypeptides as described herein. Desirably, the peptide will be as small as possible while maintaining substantially all the biological activity of the large peptide. Where possible, it may be desirable to optimize peptides of the invention to a length of 9 or 10 amino acid residues, provide the size with endogenously processed viral peptides or tumor cell peptides that bind to KC class I molecules on the cell surface. Peptides having the desired activity can be modified as necessary to provide the desired attributes, for example, improved pharmacological characteristics, while simultaneously increasing or retaining at least substantially all the biological activity of the unmodified peptide to bind the desired MHC molecule. and activate the appropriate T cell. For example, the peptides may be subjected to various changes, such as conservative or non-conservative substitutions, wherein such changes have had advantageous advantages in their use, such as improved MHC linkage. By conservative substitutions is meant replacing an amino acid residue with another that is biologically and / or chemically similar, for example, a hydrophobic residue for another, or a polar residue for another. Substitutions include combinations such as Gly, Ala; Val, lie, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions can be probed using D-amino acid. Such modifications can be made in well known peptide synthesis methods, as described in, for example, Merrifield, Science 232: 341-347 (1986), Barany and Merrifield, The Peptides, Gross and Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis, (Rockford, 111., Pierce), 2d Ed. (1934), incorporated for reference herein. The peptides can also be modified by increasing or decreasing the amino acid sequence of the context, for example, by the addition or deletion of amino acids. The peptides or analogs of the invention can also be modified by altering the order or composition of some residues, it being readily appreciated that some amino acid residues essential for biological activity, for example those at critical contact sites or conserved residues, can not be altered generally. without an adverse effect on biological activity. Non-critical amino acids do not need to be limited to those that occur naturally in proteins, such as 1-a-amino acids or their D-isomers, but may include non-natural amino acids as well, such as ß -? - d amino acids, as well as many derivatives of The amino acids. Typically, a series of peptides with simple amino acid substitutions is used to determine the effect of electrostatic charge, hydro-obesity, etc. in the link. For example, a series of amino acid substitutions positively charged (eg, Lys or Arg) or negatively charged (eg, Glu) are made along the length of the peptide revealing different patterns of sensitivity to various MHC molecules and receptors. T cell. In addition, multiple substitutions using relatively small, small portions, such as Ala, Gly, Pro, or similar residues can be employed. The substitutions can be homo-oligomers or hetero-oligomers. The number and type of residuals that are replaced or added depend on the necessary spacing between the essential points of contact and some functional attributes that are sought (for example, hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor can also be achieved by such substitutions, compared to the affinity of the parent peptide. In any case, such substitutions must employ amino acid residues or other selected molecular fragments to avoid, for example, steric interference and charge that could disrupt the bond. Amino acid substitutions are typically single residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final peptide. Substitution variants are those in which at least one residue of a peptide has been removed and a different residue has been inserted in its place. Such substitutions are generally made according to the following Table 2 when it is desired to finely modulate the characteristics of the peptide. TABLE 2 Original Residue Example of Substitution Wing Ser Arg Lys, His Asn Gln Asp Glu Cys Ser Glu Asp Gly Pro His Lys; Arg Ile Leu; Val Leu lie; Val Lys Arg; His Met Leu; lie Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val lie; Leu Pro Gly Substantial changes in function (eg, affinity for HC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in Table 2, that is, by selecting residues that differ more significantly in their effect to maintain (a) the structure of the main peptide structure in the area of substitution, for example, as a sheet or helix conformation, (b) the charge or hydrophobicidate in the molecule at the target site or (c) the mass of the side chain. The substitutions that are generally expected to produce the largest changes in the properties of the peptide will be those in which (a) the hydrophilic residue, for example seryl, is substituted for (or by) a hydrophobic residue, for example leucyl, isoleucyl , phenylalany, valyl or alanyl; (b) a residue having an electro-positive side chain, for example, lysium, arginyl, or histidyl, are substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (c) a residue having a bulky side chain, for example phenylalanine, is substituted for (or by) one that does not have a side chain, for example, glycine. The peptides may also comprise isoesters of two or more residues in the immunogenic peptide. An "isoster" as defined herein is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence adjusts a specific binding site for the second sequence. The term specifically includes modifications of the peptide backbone structure well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or crosslinks of the main structure. See generally, Spatola, Chemistry and Biochemistry of Amino Acids. peptides and Proteins, Voi. VII (Weinstein ed., 1983). Modifications of the peptides with various amino acid mimetics of non-natural amino acids are particularly useful for increasing the stability of the peptide in vivo. Stability can be tested in a number of ways. For example, peptidases and various biological media, such as plasma and human serum, have been used to test stability. See, for example, Verhoef et al-, Eur. J. Drug Metab. Pharmacokin. 11: 291-302 (1986). The half-life of the peptides of the present invention is conveniently determined using a 25% human serum test (v / v). The protocol is generally as follows. The pooled human serum (Type AB, not heat-inactivated) is degreased by centrifugation before use. The serum is then diluted to 25% with an RPMI tissue culture medium and used to test the stability of the peptide. At predetermined time intervals a small amount of the reaction solution is removed and added to 6% aqueous trichloroacetic acid or ethanol. The turbid reaction sample is cooled (4 ° C) for 15 minutes and then rotated to granules in precipitated whey proteins. The presence of the peptides is then determined by reverse phase HPLC using stability-specific chromatography conditions.

The peptides of the present invention or analogs thereof having CTL-stimulating activity can. modified to provide desired attributes other than the improved half-life of the serum. For example, the ability of the peptides to induce CTL activity can be enhanced by binding to a sequence containing at least one epitope that is capable of inducing a helper T cell response. Particularly preferred immunogenic peptides / conjugates of assistant T are linked by a spacer molecule. The spacer is typically comprised of relatively small neutral molecules, such as amino acids or mimetic amino acids, which are substantially uncharged under physiological conditions. The spacers are typically selected from, for example, Ala, Gly, or other neutral spacer of non-polar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and may thus be a hetero- or oligomer. When present, the spacer will commonly be at least one or two residues, most commonly three to six residues. Alternatively, the CTL peptide can be linked to the assistant peptide T without a spacer. The immunogenic peptide can be linked to the assistant peptide T directly or via a spacer at the amino or carboxy terminus of the CTL peptide. The amino terminus of the immunogenic peptide or the assistant peptide 7 can. acilarse Examples of T-helper peptides include tetanus toxoid 830-843. influenza 307-319, alaria circunesporozoita 382-398 and 378-389. In some embodiments it may be desirable to include at least one CTL priming component in the pharmaceutical compositions of the invention. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, residues of palmitic acid can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, for example, by means of one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser , or the like, to an immunogenic peptide. The oiled peptide can then be injected directly into a micellar form, incorporated into a liposome or emulsified in an adjuvant, for example, incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid bound to alpha amino and epsilon groups of Lys, which is linked via, for example, Ser-Ser, to the amino terminus of the immunogenic peptide. As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinyl serine (P3CSS) can be used to prime virus-specific CTL when covalently bound to an appropriate peptide. See, Deres et al., Nature 342: 561-564 (1989), incorporated herein by reference. The peptides of the invention can be coupled to PJCSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Additionally, since the induction of neutralizing antibodies can also be primed with P'CSS conjugated to a peptide displaying an appropriate epitope, the two compositions can be combined to more effectively produce cell-mediated and humoral responses for infection. In addition, additional amino acids can be added to the terms of a peptide to provide ease of binding peptides to one another, to couple a carrier support, or larger peptides, to modify the physical or chemical properties of the peptide and oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced into the C or N terminus of the peptide or oligopeptide. The modification in the C term in some cases may alter the binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences may differ from the natural sequence when modified by NH-terminal acylation, for example, by acetylation of alkanoyl (C.-Cr-) or thioglycolyl, carboxyl-terminal amidation, eg, ammonia. , methylamine, etc. In some cases these modifications may provide sites to link to ur. support or another molecule. The peptides of the invention can be prepared in a wide variety of ways. Due to their relatively short size, the peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. See, for example Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984), supra. Alternatively, recombinant DNA technology can be employed where a sequence of nucleotides encoding immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultured under conditions suitable for expression. These procedures are generally known in the art, as is generally described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), which is incorporated herein by reference. Thus, fusion proteins comprising one or more peptide sequences of the invention can be used to present the appropriate T cell epitope. 32 As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103: 3135 (1981), the modification it can be done simply by replacing the appropriate base (s) with those that code for the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such suitable vectors and host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator sections and commonly a replication system to provide an expression vector for expression in the desired cell host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast or mammalian host cells can also be used, employing suitable vectors and control sequences. The peptides of the present invention and the pharmaceutical and vaccine compositions thereof are useful for administration to mammals, particularly humans, to treat and / or prevent viral infection and cancer. Examples of diseases that can be treated using the immunogenic peptides of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and condlyloma acuminatum. For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual who already suffers from cancer or is infected with the virus of interest. Those in the incubation phase or the acute phase of the infection can be treated with the immunogenic peptides separately or together with other treatments as appropriate. In therapeutic applications, the compositions are administered to a patient in an amount sufficient to produce an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest the symptoms and / or complications. An adequate amount to achieve this is defined as "therapeutically effective dose". The amounts effective for this use will depend on, for example, the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but it generally varies for the initial immunization (for prophylactic therapeutic administration) from about 1.0 g to about 5000 g of peptide for a 70 kg patient, followed by thrust dosages of about 1.0 ug up to about 1000 μg of peptide being monitored at regimen - Push for weeks to months depending on the response and condition of the patient and measuring specific CTL activity in the patient's blood. It should be borne in mind that the peptides and compositions of the present invention can generally be employed in serious affective states, that is, life-threatening or life-threatening situations. In such cases, in view of the minimization of foreign substances and the relatively non-toxic nature of the peptides, it is possible and may be felt desirable by the physician who tries to administer substantial excess of these peptide compositions. For therapeutic use, administration should begin with the first sign of viral infection or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by pushing dose until at least the symptoms are decreased and for a period later. In chronic infection, loading doses followed by booster doses may be required.

The treatment of an individual infected with the composition of the invention can precipitate resolution of the infection in acutely infected individuals. For those susceptible (or predisposed) individuals to develop chronic infection, the compositions are particularly useful in methods to prevent the evolution of the infection from acute to chronic. Where susceptible individuals are identified before or during infection, for example, as described herein, the composition can be selected for them, minimizing the need for administration to a larger population. The peptide compositions can also be used for the treatment of chronic infection and for stimulating the immune system to eliminate virus-infected cells in carriers. It is important to provide an amount of immunopotentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response. Thus, for the treatment of chronic infection, a representative dose is in the range of about 1.0 g to about 5000 μg, preferably about 5 g to 1000 μg for a patient of 70 kg per dose. Immunization doses followed by booster doses at set intervals, for example, from one to four weeks, may be required, possibly over a prolonged period of time to effectively immunize an individual. In the case of chronic infection, administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been substantially eliminated or decreased and for a later period. Pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, for example, intravenously, subcutaneously, intradermally or intramuscularly. Thus, the invention provides compositions for parenteral administration comprising a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, regulated water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterilized by filtration. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required for approaching physiological conditions, such as pH adjusting agents and regulators, agents that would adjust tonicity, wetting agents and the like, eg, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride , sorbitan monolaurate, thanolamine oleate, etc. The concentration of the CTL stimulating peptides of the invention in the pharmaceutical formulation can vary widely, ie, from less than about 0.1¾, commonly to or at least about 2% up to as much as 20% to 50% or more by weight, and will be selected mainly by fluid volumes, viscosities, etc., according to the particular mode of selected administration. The peptides of the invention can also be administered by means of liposomes which will serve to select the peptides in a particular tissue, such as lymphoid tissue, or selectively selected to infect cells, as well as increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or together with a molecule that binds to, for example, a predominant receptor among the lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or to other therapeutic or in-oenogenic compositions. Thus, liposomes filled or decorated with a desired peptide of the invention can be directed to the site of the lymphoid cells, where the liposomes then deliver the selected therapeutic / immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally directed by consideration of, for example, liposome size, the capacity and acid stability of the liposomes in the blood stream. A variety of methods are available to prepare liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Patent Nos. 4,235,871, 4, 501,728, 4, 837, 028, and 5, 019, 369, incorporated herein by reference. To select immune cells, a ligand to be incorporated into the liposome may include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide can be administered intravenously, locally, topically, etc. in a dose that varies according to, inter alia, the form of administration, the peptide that is delivered, and the stage of the disease being treated. For solid compositions, conventional non-toxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any of the commonly used excipients, such as those previously listed carriers, and generally 10-95% active ingredient, that is, one or more peptides of the invention, and greater preference at a concentration of 25% -75%. For aerosol administration, the immunogenic peptides are preferably delivered in finely divided form together with a surfactant and propellant. Typical percentages of peptides are 0.01-20% by weight, preferably 1-10. The surfactant must, of course, be non-toxic, and preferably soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic acid, octanoic acid, leuric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, oleic acid and oleic acid with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides can be employed. The surfactant may constitute O.li-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, for example, lecithin for intranasal delivery. In another aspect the present invention is directed to vaccines containing as an active ingredient an immunogenically effective amount of an immunogenic peptide as described herein. The peptide (s) may be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make the polyr.erc, the additional ability to induce antibodies and / or CTLs that react with different antigenic determinants of the virus or tumor cells. Useful carriers are well known in the art, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine glutamic acid), influenza, hepatitis B virus core protein, recombinant hepatitis B virus vaccine and the like.

The vaccines may also contain a physiologically tolerable (tai) digestion such as water, phosphate buffered saline, or saline, and typically include an adjuvant additionally. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. And, as mentioned above, CTL responses can be primed by conjugating peptides of the invention with lipids, such as PiCSS. During immunization with a peptide composition as described herein, by injection, aerosol, oral, transdermal or other route, the in vitro system of the host responds to the vaccine by producing large quantities of the CTL specific for the desired antigen, and the host becomes at least partially immune to subsequent infection, or resistance to developing chronic infection. The vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of viral infection or cancer to produce an immune response against the antigen and thus improve the patient's own immune response capabilities. Such an amount is defined as an "immunogenically effective dose". In this use, the precise amounts again depend on the state of health and weight of the patient, the mode of administration, the nature of the formulation, etc., but vary in general from about 1.0 μg to about 5000 μa per 70 kg patient. , more commonly from about 10 μg to about 500 μg per 70 kg of body weight. In some cases it may be desirable to combine the peptide vaccines of the invention with vaccines that induce neutralizing antibody responses to the virus of interest, particularly viral envelope antigens. For therapeutic or immunization purposes, the nucleic acids encoding one or more of the peptides of the invention may also be administered to the patient. A number of methods are conveniently used to deliver the nucleic acids to the patient. For example, the nucleic acid can be delivered directly, such as "naked DNA". This proposal is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Patent Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using a ballistic delivery as described, for example, in U.S. Patent No. 5,204,253. Particles comprised only of DNA can be administered. Alternatively, the DNA can adhere to particles, such as gold particles. The nucleic acids can also be supplied in complex to cationic compounds, such as cationic lipids. Lipid mediated gene delivery methods are described, for example, in O 96/18372; WC 93/24640; Mannino and Gould-Fogerite (1988) 3ioTec niques 6 (7): 682-691; Rose U.S. Pat No. 5,279,833; WO 91/06309; and Felgner et al. (1987) Proc. Nati Acad. Sci. USA 34: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as poultry vaccination or pustulation. This proposal involves the use of the vaccine virus for a vector to express nucleotide sequences encoding the peptides of the invention. During introduction into an acutely or chronically infected host or within an uninfected host, the recombinant vaccine virus expresses the immunogenic peptide, and thereby produces a host CTL response. Vaccine vectors and methods useful in immunization protocols are described in, for example, U.S. Patent No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)) which is incorporated herein by reference. A wide variety of other vectors useful for administration or therapeutic immunization of the peptides of the invention, for example the Salmonelia typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

A preferred means for administering nucleic acids encoding the peptides of the invention uses minigene constructs that encode multiple epitopes of the invention. To create a DNA sequence encoding the selected CTL epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes are translated in reverse. A table of use of human codons is used to drive codon selection for each amino acid. These DNA sequences encoding epitope are attached directly, creating a continuous polypeptide sequence. To optimize expression and / or immunogenicity, additional elements can be incorporated into the design of the minigene. Examples of amino acid sequences that could be translated in reverse and included in the minigene sequence include: helper T lymphocyte epitopes, a leader sequence (signal), and an endoplasmic reticulum retention signal. In addition, the MHC presentation of the CTL epitopes can be enhanced by including synthetic flanking sequences (e.g. poly-alanine) or occurring naturally adjacent to the CTL epitopes. The minigene sequence is converted into DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Oligonucleotides (30-100 bases long) are synthesized which overlap, phosphorylate, purify and anneal under appropriate conditions using well-known techniques. The ends of the oligonucleotides are joined. using T4 DNA ligase. This synthetic minigene, which encodes the CTL epitope polypeptide, can then be cloned into a desired expression vector. Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in the target cells. Various vector elements were required: a promoter with a cloning site towards the 3 'end for insertion of the minigene; a polyadenylation signal for efficient transcription termination; an origin of replication of E. coli; and a selectable marker of E. coli (for example resistance to ampicillin or kanamycin). Numerous promoters can be used for this purpose, for example, the human cytomegalovirus promoter (hCMV). See, US Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. Additional vector modifications may be desired to optimize the expression and immunogenicity of the minigene. In some cases introns are required for efficient gene expression and one or more naturally occurring synthetic introns could be incorporated within the transcribed minigene region. The inclusion of mRNA stabilization sequences can also be considered to increase the expression of the minigene. It has recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a role in the immunogenicity of DNA vaccines. These sequences could be included in the vector, outside the minigene coding sequence if they are found to improve immunogenicity. In some embodiments, a bioistronic expression vector may be used to allow production of the epitopes encoded by the minigend and a second included protein to improve or decrease immunogenicity. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LelF) or costimulatory molecules. The attendant epitopes (HTL) could bind to intracellular selection signals and be expressed separately from the CTL epitopes. This would allow the targeting of the HTL epitopes to a cell compartment different from the CTL epitopes. If required, this could more efficiently facilitate the entry of HTL epitopes into the MHC class II route, thereby improving the CTL induction. In contrast to the induction of CTL, specifically decreasing the immune response by co-expressing immunosuppressive molecules (eg TGF-β) may be of benefit in some diseases.

Once an expression vector is selected, the minigene is cloned into the polylinker region towards the 3 'end of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as the other elements included in the vector, are confirmed using restriction mapping of DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a bank of master cells, and a bank of working cells. Therapeutic amounts of plasmid DNA are produced by fermentation in E. coli, followed by purification. The aliquots from the working cell bank are used to inoculate the fermentation medium (such as a Fantastic Broth), and are grown to saturation in stirred flasks or a bioreactor according to well-known techniques. The plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion exchange resins supplied by Quiagen. If required, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other methods. Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in saline regulated with sterile phosphate (PBS). A variety of methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and collectively preferred, protective, interactive, non-condensing (PINC) compounds could also complex with purified plasmid DNA for varying influences such as stability, intramuscular dispersion, or trafficking to specific organs or cell types. . Sensitization of the target cell can be used as a functional test for expression and presentation of MHC Class I CTL epitopes encoded by minigens. Plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release tests. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using activated fluorescence cell distribution (SACF). These cells are then arced with chromium-51 and used as target cells for epitope-specific CTL lines. Cytolysis, detected by the release of CR51, indicates the production of the MHC presentation of CTL epitopes encoded by minigens. Immunogenicity in vivo is a second proposal for the functional test of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are dependent on the formulation (for example IN for DNA in PBS, IP for DNA in lipid complex). Twenty-one days after immunization, the splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. These effector cells (the CTLs) are tested by cytolysis of chrome-5i-labeled target cells loaded with peptide using standard techniques. Lysis of target cells sensitized to peptide MHC loading corresponding to epitopes encoded by minigens demonstrate the role of the DNA vaccine for in vivo induction of CTL. The antigenic peptides can be used to produce CTL ex vivo as well. The resulting CTL can be used to treat chronic infections (viral or bacterial) or tumors in patients who do not respond to other conventional forms of therapy., or that they will not respond to a proposed peptide therapy vaccine. Ex vivo CTL responses to a particular pathogen (infectious agent or tumor antigen) are induced by incubating the patient's CTL precursor cells (CTLp) in culture with a source of antigen-presenting cells (APC) and the peptide. immunogenic. After an appropriate incubation time (typically 1-4 weeks) in which the CTLp is activated and matures and expands within the effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (one cell infected or a tumor cell). The peptides may also find use as diagnostic reagents. To optimize the in vi tro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is maintained in an appropriate serum-free medium. Before incubation of the stimulator cells with the cells to be activated, for example, precursor CD8 cells, an amount of antigenic peptide is added to the stimulator cell culture, in sufficient quantity to be loaded onto the human Class I molecules to be expressed on the surface of the stimulator cells. In the present invention, a sufficient amount of peptide is an amount that will allow approximately 200, and preferably 200 or more, MHC Class? loaded with peptide to be expressed on the surface of each stimulator cell. Preferably, the stimulator cells are incubated with >20uc / ml of peptide. The precursor or inactive CD8 + cells are then incubated in culture with the appropriate stimulator cells for a sufficient period of time to activate the CD8 + cells. Preferably, the CD8 + cells are activated in a specific form of antigen. The proportion of precursor or inactive CD8 + (effector) cells to stimulator cells can vary from individual to individual and may additionally depend on such values as the function of lymphocytes of an individual to culture conditions and the nature and severity of the affective condition and another condition for which the treatment modality described in the above is used. Preferably, however, the ratio of lymphocyte: stimulator cell is in a range of about 30: 1 to 300: 1. The effector / stimulator culture can be maintained for as long as necessary to stimulate a therapeutically useful or effective CD8 número cell number. The induction of CTL in vi tro requires the specific recognition of peptides that bind to MHC class I molecules specific for allele on A? C. The number of specific MHC / peptide complexes per APC is crucial for the stimulation of CTL, particularly er. primary immune responses. While small amounts of complex peptides / MHC per cell sor. sufficient to render a cell susceptible to lysis by CTL, or to stimulate a secondary CTL response, the successful activation of a CTL precursor (pCTL) during the primary response requires a significantly higher number of MHC / peptide complexes. The peptide loading of the major empty histocompatability complex molecules on the cells allows the induction of primary cytotoxic T lymphocyte responses. The peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses. Since mutant cell lines do not exist for each human MHC allele, it is advantageous to use a technique to remove the endogenous MHC-associated peptides from the APC surface, followed by loading the resulting empty MHC molecules with the immunogenic peptides. of interest. The use of non-transformed (non-tumorigenic), non-infected cells, and preferably, autologous patient cells such as APC is desirable for the design of CTL induction protocols directed towards the development of ex vivo CTL therapy. This application describes methods for removing the peptides associated with endogenous MHC from the surface of APC followed by loading the desired peptides.

A stable MHC class I molecule is a trimeric complex formed of the following elements: 1) a. peptide of commonly 8-10 residues, 2) a transmembrane heavy polymorphic protein chain carrying the peptide binding site in its a and a2, 3 domains) a non-covalently associated non-polymorphic light chain, β-microglobulin. Remove the bound peptides and / or dissociate the β-microglobulin; from the complex MHC class I molecules become non-functional or injectable, resulting in rapid degradation. All MHC class I molecules isolated from PBMC have endogenous peptides linked to them. Therefore, the first step is to remove all endogenous peptides linked to MHC class I molecules on the APC without causing their degradation before the exogenous peptides can be added to them. Two possible ways to release MHC class I molecules from bound peptides include lowering the culture temperature from 37 ° C to 26 ° C overnight to destabilize the β2 microglobulin and remove the endogenous peptides from the cell using an oderable acid treatment . The methods release previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to empty class I molecules. The cold temperature incubation method allows the exogenous peptides to bind efficiently to the MKC complex, but requires an overnight incubation at 26 ° C which can decrease the metabolic rate of the cell. It is also likely that cells that do not actively synthesize MHC molecules (eg, inactive PBMC) would not produce high quantities of MHC molecules of empty surface by the cold temperature procedure. Stripping with stringent acid involves extraction of the peptides with trifluoroacetic acid, pH 2, or acid denaturation of the class I peptides purified by immunoaffinity. These methods are not feasible for CTL induction, since it is important to eliminate the endogenous peptides while preserving the viability of APC and an optimal metabolic state that is critical for the presentation of the antigen. Moderate pH 3 acid solutions such as glycine or citrate-phosphate regulators have been used to identify endogenous peptides and to identify tumor-associated T cell epitopes. The treatment is especially effective, because only MHC class I molecules (and associated peptides released) are destabilized, while other surface antigens remain intact, including MHC class II molecules. More importantly, treating the cells with moderate acid solutions does not affect the cell's viability or the metabolic state. The treatment of moderate acid is rapid since the stripping of the endogenous peptides occurs in two minutes at 4 ° C and the APC is ready to perform its function after the appropriate peptides are loaded. The technique is used in the present to make the peptide-specific APCs for the primary antigen-specific CTL gelation.The resulting A? C is efficient to induce CD8 + peptide-specific CTL.The activated CD8 + cells can be separated effectively from the stimulator cells using a variety of known methods. For example, monoclonal antibodies specific for the stimulator cells, for the peptides loaded on the stimulator cells, or for the CD8 + cells (or a segment thereof) can be used to bind their complementary ligand The labeled antibody molecules can then be extracted from the stimulator-effector cell mixture by means of appropriate means, for example, by well-known immunoprecipitation or immunoassay methods.The effective cytotoxic amounts of the activated CD8 + cells may vary between uses in vi tro and in vivo, as well as with the amount and type of cells that are the ultimate goal of these destructive cells. The amount will also vary depending on the patient's condition and should be determined by considering all the factors appropriate to the practitioner. Preferably, however, about 1 X 10 ° to about i Y. 10'-, more preferably about 1 X 10r to about 1 X 11, and even more preferably about 1 X 10 to about 1 X 10"activated CD8 + cells are used for adult humans, compared to approximately 5 X 10 ° - 5 X 10 cells used in mice Preferably, as discussed above, activated CD8 + cells are harvested from the cell culture prior to administration of the CD8 + cells to the individual It is important to note, however, that unlike other present and proposed treatment modalities, the present method uses a cell culture system that is not tumorigenic.Therefore, if the separation-complete of the stimulator cells and activated CD3 células cells is not achieved, there is no inherent danger rc idc associated with the administration of a small number of stimulatory cells, while The administration of cells that promote mammalian tumor can be extremely dangerous. Methods for re-introducing cellular components into the art are known and include such procedures as those exemplified in U.S. Patent No. 4,844,893 to Honsik, et al., And U.S. Patent No. 4,690,915 to Rosenberg. For example, the administrator. of activated CD8 + cells by intravenous infusion is appropriate. The immunogenic peptides of this invention can also be used to make monoclonal antibodies. Such antibodies may be useful as potential diagnostic or therapeutic agents. The peptides may also find use as diagnostic reagents. For example, a peptide of the invention can be used to determine the susceptibility of a particular individual to a treatment regimen that employs the related peptide or peptides, and thus may be of assistance in modifying an existing treatment protocol or in determining a prognosis of an affected individual. In addition, peptides can also be used to predict which individuals will be at substantial risk of developing chronic infection. To identify the peptides of the invention, the isolation of the class I antigen was carried out, and the isolation and sequencing of the naturally processed peptides was carried out as described in the related applications. These peptides were then used to define specific binding motifs for each of the following alleles A3.2, Al, All, and A24.1. These reasons are described on page 3, above. The motifs described in Tables 8-11, below, are defined from sequencing data. 103 assembled of naturally processed peptides as described in the related applications. TABLE 8 Summary Specific Reason for Allele HLA-A3.2 Position Residues Conserved 1 2 V, L, M 3 Y, D 4 5 6 7 I 8 Q, N 9 K 10 K TABLE 9 Summary Specific Reason for Allele HLA-Al Position Residues Conserved 1 2 S, T 3 D, E 4 P 10 K TABLE 10 Summary Specific Reason for Allele HLA-Al 1 Position Residues Conserved 1 2 T, V 3 M, F 4 5 6 7 8 Q 9 K 10 K TABLE 11 Summary Specific Reason for Allele HLA-A2 .1 Position Residual Conserved 3?,? 4 D, E, G, K, P 5 L,?,? 6 V 7 N, V 8 A, E, K, Q, S 9 F, L 10 F, A Example 2 Identification of immunogenic peptides Using the motifs identified above for various amino acid sequences of the MHC class I allele from various proteins Related to tumor and pathogen were analyzed for the presence of these motifs. The selection described in the related applications was carried out. Table 12 provides the results of the antigen searches.

? ? : Table 12 Peptide AA Sequence Source A'0301 A * 110! 28. 0719 10 ELEQWVAGR HDV.nuc.16 0.0170 0.0012 28. 0727 10 LSAGGKNLS HDV.nuc.115 0.0097 0.0150 1259. 02 1 1 STDTVDTVLEK Flu.HA.29 0.0001 0.0670 1259. 04 9 G1APLQLGK Hu.HA.63 0.6100 0.2000 1259. 06 10 VTAACSHAG Flu.HA.149 0.0380 0.0490 1259. 08 9 GIHHPSNSK Flu.HA.195 0.1300 0.0140 1259. 10 10 RMNYYWTLL FKLHA.243 2.5000 2.3000 1259. 12 11 rTNKVNSVDE FlulHA.392 0.0200 0.0670 1259. 13 11 MNIQFTAVGK Flu.HA.402 0.0280 0.0092 1259. 14 9 NIQFTAVG Flu.HA.404 0.0017 0.0330 1259. 16 11 AVGKEFNKLEK Flu-HA.409 0.0210 0.0460 1259. 19 11 KVKSQLKNNAK Flu-HA.465 0.0470 0.0031 1259. 20 11 SVKNGTYDYPK F1U.HA495 0.0410 0.1400 1259. 21 9 SHPSGPLK Flu.VMTl.13 0.7800 8.8000 1259. 25 10 RMVLASTTAK Flu.VMTl.178 0.5500 0.0350 1259. 26 9 MVLASTTAK Flu.VMTl.179 1.7000 1.4000 1259. 28 10 RMGVQMQRFK Flu.VMTl.243 0.1000 0.0059 1259. 33 10 ATEI ASVGK Flu.VNUC.22 0.1400 0.3000 1259. 37 11 TMVMELVRMIK Flu.VNUC.188 0.0890 0.0310 1259. 43 10 RVLSFKGTK Flu.VNUC.342 0.8000 0.0830 Fl 19.01 9 MSLQRQFLR ORF3P 0.2000 0.7200 Fl 19.02 9 LLGPGRPYR TRP.197 0.0190 0.0091 Fl 19.03 9 LLGPGRPYK TRP.197K9 2.2000 0.6800 34. 0019 8 RVYPELPK CEA.139 0.0130 0.0440 34. 0020 8 TVSAELPK 34. 0148 9 FVSNLATGR CEA.656 0.0019 0.0490 34. 0152 9 RLQLSNG K CEA.546 0.0250 0.0110 34. 0153 9 JNGIPQQK CEA.628 0.0400 0.0780 34. 0154 9 KffiJCYTMRK HER2 / neu.681 0.0620 0.0055 34. 0155 9 LVHFLLL K MAGE2.116 0.5220 1.4000 34. 0156 9 SMLEVFEG MAGE2.226 0.0950 1.6000 34. 0157 9 SSFSTTIN MAGE2.69 0.1600 2.0000 34. 0158 9 TSYV VLHK MAGE2.281 0.5300 0.1500 34. 0159 9 VIFSKASEK MAGE2.149 0.4900 0.0530 34. 0160 9 GSWGNWQK MAGE3.130 0.0040 0.2060 34. 0161 9 SSLPTTMNK MAGE3.69 0.6180 0.7100 34. 0162 9 SVLEVFEG MAGE3.226 0.1330 0.9000 34. 0171 9 SSBMGGMNK p53.240 0.5440 1.1000 34. 0172 9 SSC GG p53.240 0.0090 0.0490 34. 0211 10 RTLTLFNVT CEA.554 02200 1.3000 34. 0212 10 TISPL TSYK CEA.241 0.1800 0.0330 34. 0214 10 STTINYTLW MAGE2.72 0.0870 0.6500 34. 0215 10 ASSLFTTMNK MAGE3.68 0.0420 0.0270 34. 0225 10 TYQGSYGF p53.101 0.4900 0.4200 34. 0226 10 WRRBPHHEK p53.172 0.1800 0.2100 34. 0228 10 GLAPPQHLK P53.187 0.0570 0.0160 34. 0229 10 NSSCMGGMNK P53.239 0.0071 0.0290 34. 0230 10 SSBMGGMNRK p53-240 0.0420 0.1600 34. 0232 10 RVCACPGRDK p53.273 0.0190 0.0250 34. 0295 11 KTTTVSAELPK CEA.492 0.3600 0.1600 34. 45. 0047 APAPSWPI p53.86J8 45. 0051 KPVED DAI CEA.155.19 45. 0054 IPQQHTQVI CEA.632.I9 45. 0060 APPVAPAPI p53.70J9 45. 0062 APAAPTPAI p53.76.I9 45. 0064 PPGTRVRAI p53.152.I9 45. 0065 APPQHLIRI p53.189.I9 45. 0071 IPQQHTQVLI CEA.632J10 45. 0072 SPGLSAGATI CEA.680J10 45. 0073 SPMCKGSRCI Hcr2 / ncu.l96.I10 45. 0074 MP PEGRYTI Her2 / ne L282.I10 45. 0076 CPLHNQEVTI Her2 neu.315.I10 45. 0079 KPDLSYMPn Her2 nea.605.I10 45. 0080 TPSGAMPNQI Her2Aieu.701.I10 45. 0084 GPASPLDSTC Her2 / neu.995.I10 45. 0091 APPVAPAPAI p53.70.I10 45. 0092 APAPAAPTPI p53.74.I10 45. 0093 ?????????? p53.79J10 45. 0094 APSWPLSSSI p53.88.I10 45. 0103 APTISPL TSI CEA.239.il 1 45. 0108 SPSYTYYRPGI CEA.421J11 45. 0117 CPSGVKPDLSI Her2yneu.600.Ill 45. 0118 SPLTSHSAVI Her2 / neu.649.Ill 45. 0119 IPDGENVKIPI Hcr2 ncu.740.Il l SPLDSTFYRSI Her2 neu.998.Il l LPAARPAGATI Hei2 / neu.1157,111 HPRKLLMQDLI MAGE2.24l.Ill GPRALIETSYI MAGE2.274.il 1 GPRALVETSYI MAGE3.274.il 1 APR PEAAPPI p53.63 .Ill VPSQKTYQGSI p53.97.Il l FPHCLAFAY HBV POL 541 analog FPVCLAFSY HBV POL 541 analog YPALMPLYAC1 HBV.pol.645 The above description is provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference.

Claims

ES! RE IVIND CATIONE S 1 A composition characterized in that it comprises an immunogenic peptide having a binding motif whose immunogenic peptide is selected from a group consisting of AMG RLVEAC ISVY SLPYW SMDEA LSIY SLK VLQCLEV FLNGTGGQV VLLHTFTDV ALVGALLLV YLIRA SMDEA AACNQKILTV SLH LAHLFL LLLVAAIFGV ALCNQ TLFNVTR DTA GLAPPVHLI N LSTVLGV SLENF KLAELVHFV AND YLIMV L FLE LLQE and GLPSIPVHPL 2 A method for inducing a cytotoxic T cell response against a preselected antigen in a patient expressing a product characterized in that the method comprises contacting T cells cytotoxic patient with a composition comprising an immunogenic peptide selected from the group consisting AIFVC AMG RLVEACNLL QMA YLS LLVYHTYS KLFCQLA SLK SLPY VLQCLEV FLNGTGGQV VLLHTFTDV ALVGALLLV YLIRA SLHNLAHLFL LLLVAAIFGV S LTLLSVTR GLYTCQA GLAPPVHLI YLIMV NMLSTVLGV KLAELVHFV B YLIMV B LMYSLVH and 3 a composition characterized because a p immunogenic peptide selected from a group consists of TVSAELP TI LVHFLLL SSFSTTU VEFS SSLPTTMN SSCMGGM ASSLPTTMN MFCQLA MALES and A method for inducing a cytotoxic T cell response against a preselected antigen in a characterized in that the method comprises contacting cytotoxic T cells patient with a composition comprising a peptide selected from the group consisting of TVSAELP TVYAEPP FVS SSBMGGM RLGFLHSGTA IS A composition characterized in that it comprises an immunogenic peptide selected from a group consisting of the peptides listed in Table 6 A method for inducing a cytotoxic T cell response against a preselected antigen in a characterized in that the method comprises contacting the patient's cytotoxic T cells with a composition comprising an immunogenic peptide selected from the group consisting of the peptides listed in the Table SUMMARY The present invention provides methods and methods for selecting peptides and immunogenic peptide compositions capable of specifically binding glycoproteins encoded by KLA alleles and activating induction T cells in cells restricted by the peptides. The peptides are useful for producing an immune response against an insufficient OCRQuality antigen.