HLA binding peptides and their uses

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. The immunogenic peptide compositions of the present invention comprise immunogenic peptides having an HLA binding motif, where the peptide is from a target antigen. Target antigens of the present invention include prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (e.g., MAGE-1), human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2/neu.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser. No. 09/189,702, filed Nov. 10, 1998, which is a continuation-in-part of U.S. application Ser. No. 08/205,713 filed Mar. 4, 1994. The present application is also related to U.S. Ser. No. 09/017,735, U.S. Ser. No. 08/753,622, U.S. Ser. No. 08/822,382, U.S. Ser. No. 60/013,980, U.S. Ser. No. 08/589,108, U.S. Ser. No. 08/454,033, U.S. Ser. No. 08/349,177, U.S. Ser. No. 08/073,205, and U.S. Ser. No. 08/027,146. The present application is also related to U.S. Ser. No. 09/017,524, U.S. Ser. No. 08/821,739, U.S. Ser. No. 60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No. 08/589,107, U.S. Ser. No. 08/451,913 and to U.S. Ser. No. 08/347,610, U.S. Ser. No. 08/186,266, U.S. Ser. No. 08/159,339, U.S. Ser. No. 09/116,061, U.S. Ser. No. 08/103,396, U.S. Ser. No. 08/027,746, and U.S. Ser. No. 07/926,666. The present application is also related to U.S. Ser. No. 09/017,743; U.S. Ser. No. 08/753,615; U.S. Ser. No. 08/590,298; U.S. Ser. No. 08/452,843; U.S. Ser. No. 09/115,400; U.S. Ser. No. 08/344,824; and U.S. Ser. No. 08/278,634. The present application is also related to U.S. Ser. No. 08/197,484 and U.S. Ser. No. 08/815,396. All of the above applications are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

[0002] The Sequence Listing written in file "Sequence Listing ascii.txt," 92,160 bytes, created on Aug. 2, 2007, on two identical copies of compact discs for this application, Sette et al., HLA Binding Peptides and Their Uses, is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to compositions and methods for preventing, treating or diagnosing a number of pathological 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.

[0004] MHC molecules are classified as either Class I or Class II molecules. Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc. Class II MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed. Class I MHC molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs), which then destroy the antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections.

[0005] The CTL recognizes the antigen in the form of a peptide fragment bound to the MHC class I molecules rather than the intact foreign antigen itself. The antigen must normally be 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 translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit .beta.2 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific CTLs.

[0006] Investigations of the crystal structure of the human MHC class I molecule, HLA-A2.1, indicate that a peptide binding groove is created by the folding of the .alpha.1 and .alpha.2 domains of the class I heavy chain (Bjorkman et al., Nature 329:506 (1987). In these investigations, however, the identity of peptides bound to the groove was not determined.

[0007] Buus et al., Science 242:1065 (1988) first described a method for acid elution of bound peptides from MHC. Subsequently, Rammensee and his coworkers (Falk et al., Nature 351:290 (1991) have developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules of the B type (Jardetzky, et al., Nature 353:326 (1991) and of the A2.1 type by mass spectrometry (Hunt, et al., Science 225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I has been presented by Rotzschke and Falk (Rotzschke and Falk, Immunol. Today 12:447 (1991).

[0008] Sette et al., Proc. Natl. Acad. Sci. USA 86:3296 (1989) showed that MHC allele specific motifs could be used to predict MHC binding capacity. Schaeffer et al., Proc. Natl. Acad. Sci. USA 86:4649 (1989) showed that MHC binding was related to immunogenicity. Several authors (De Bruijn et al., Eur. J. Immunol., 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. Class I motifs specific for a number of human alleles of a given class I isotype have yet to be described. It is desirable that the combined frequencies of these different alleles should be high enough to cover a large fraction or perhaps the majority of the human outbred population.

[0009] Despite the developments in the art, the prior art has yet to provide a useful human peptide-based vaccine or therapeutic agent based on this work. The present invention provides these and other advantages.

SUMMARY OF THE INVENTION

[0010] The present invention provides compositions comprising immunogenic peptides having binding motifs for HLA-A2.1 molecules. The immunogenic peptides, which bind to the appropriate MHC allele, are preferably 9 to 10 residues in length and comprise conserved residues at certain positions such as positions 2 and 9. Moreover, the peptides do not comprise negative binding residues as defined herein at other positions such as positions 1, 3, 6 and/or 7 in the case of peptides 9 amino acids in length and positions 1, 3, 4, 5, 7, 8 and/or 9 in the case of peptides 10 amino acids in length. The present invention defines positions within a motif enabling the selection of peptides which will bind efficiently to HLA A2.1.

[0011] The motifs of the inventions include peptide of 9 amino acids which have a first conserved residue at the second position from the N-terminus selected from the group consisting of I, V, A and T and a second conserved residue at the C-terminal position selected from the group consisting of V, L, I, A and M. Alternatively, the peptide may have a first conserved residue at the second position from the N-terminus selected from the group consisting of L, M, I, V, A and T; and a second conserved residue at the C-terminal position selected from the group consisting of A and M. If the peptide has 10 residues it will contain a first conserved residue at the second position from the N-terminus selected from the group consisting of L, M, I, V, A, and T; and a second conserved residue at 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.

[0012] Epitopes on a number of immunogenic target 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 core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1), Kaposi's sarcoma herpes virus (KSHV), human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT), p53 and murine p53 (mp 53), CEA, trypanosome surface antigen (TSA), members of the tyrosinas related protein (TRP) families, and Her2/neu. The peptides are thus useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

[0013] The present invention also provides compositions comprising immunogenic peptides having binding motifs for MHC Class I molecules. The immunogenic peptides are typically between about 8 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.

[0014] For instance, the motif for HLA-A3.2 comprises from the N-terminus to C-terminus 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.

[0015] The motif for HLA-A1 comprises from the N-terminus to the C-terminus 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 conserved residues are A, S or T. The first and second conserved residues are adjacent and are preferably separated from the third conserved residue by 6 to 7 residues. A second motif consists of a first conserved residue of E or D and a second conserved residue of Y where the first and second conserved residues are separated by 5 to 6 residues.

[0016] The motif for HLA-A11 comprises from the N-terminus to the C-terminus a first conserved residue of T, V, M, L, I, S, A, G, N, C D, or F at position 2 and a C-terminal conserved residue of K, R, Y or H. The first and second conserved residues are preferably separated by 6 or 7 residues.

[0017] The motif for HLA-A24.1 comprises from the N-terminus to the C-terminus a first conserved residue of Y, F or W at position 2 and a C terminal conserved residue of F, I, W, M or L. The first and second conserved residues are preferably separated by 6 to 7 residues.

[0018] Epitopes on a number of potential target proteins can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, malignant melanoma antigen (MAGE-1) Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1), papilloma virus antigens, Lassa virus, mycobacterium tuberculosis (MT), p53 and murine p53 (mp 53), CEA, and Her2/neu, and members of the tyrosinase related protein (TRP) families. The peptides are thus useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

[0019] The present invention also provides compositions comprising immunogenic peptides having binding motifs for non-A HLA alleles. The immunogenic peptides are preferably about 9 to 10 residues in length and comprise conserved residues at certain positions such as proline at position 2 and an aromatic residue (e.g., Y, W, F) or hydrophobic residue (e.g., L, I, V, M, or A) at the carboxy terminus. In particular, an advantage of the peptides of the invention is their ability to bind to two or more different HLA alleles.

[0020] Epitopes on a number of potential target proteins can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, malignant melanoma antigen (MAGE-1) Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1), papilloma virus antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, and Her2/neu. The peptides are thus useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

DEFINITIONS

[0021] The term "peptide" is used interchangeably with "oligopeptide" in the present specification to designate a series of residues, typically L-amino acids, connected one to the other typically 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 usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues.

[0022] An "immunogenic peptide" is a peptide which comprises an allele-specific motif such that the peptide will bind an MHC molecule and induce a CTL response. Immunogenic peptides of the invention are capable of binding to an appropriate HLA-A2.1 molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived.

[0023] Immunogenic peptides are conveniently identified using the algorithms of the invention. The algorithms are mathematical procedures that produce a score which enables the selection of immunogenic peptides. Typically one uses the algorithmic score with a "binding threshold" to enable selection of peptides that have a high probability of binding at a certain affinity and will in turn be immunogenic. The algorithm is based upon either the effects on MHC binding of a particular amino acid at a particular position of a peptide or the effects on binding of a particular substitution in a motif containing peptide.

[0024] A "conserved residue" is an amino acid which occurs in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. Typically a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide. At least one to three or more, preferably two, conserved residues within a peptide of defined length defines a motif for an immunogenic peptide. These residues are typically in close contact with the peptide binding groove, with their side chains buried in specific pockets of the groove itself. Typically, an immunogenic peptide will comprise up to three conserved residues, more usually two conserved residues.

[0025] As used herein, "negative binding residues" are amino acids which if present at certain positions (for example, positions 1, 3 and/or 7 of a 9-mer) will result in a peptide being a nonbinder or poor binder and in turn fail to be immunogenic i.e. induce a CTL response.

[0026] The term "motif" refers to the pattern of residues in a peptide of defined length, usually about 8 to about 11 amino acids, which is recognized by a particular MHC allele. The peptide motifs are typically different for each human MHC allele and differ in the pattern of the highly conserved residues and negative residues.

[0027] The binding motif for an allele can be defined with increasing degrees of precision. In one case, all of the conserved residues are present in the correct positions in a peptide and there are no negative residues in positions 1,3 and/or 7.

[0028] The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the peptides of this invention do not contain materials normally associated with their in situ environment, e.g., MHC I molecules on antigen presenting cells. Even where a protein has been isolated to a homogenous or dominant band, there are trace contaminants in the range of 5-10% of native protein which co-purify with the desired protein. Isolated peptides of this invention do not contain such endogenous co-purified protein.

[0029] The term "residue" refers to an amino acid or amino acid mimetic incorporated in an oligopeptide by an amide bond or amide bond mimetic.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. HLA-A2.1 Motif

[0030] The present invention relates to the determination of allele-specific peptide motifs for human Class I MHC (sometimes referred to as HLA) allele subtypes, in particular, peptide motifs recognized by HLA-A2.1 alleles. These motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases, cancers or autoiummune diseases, for which the amino acid sequence of the potential antigen or autoantigen targets is known.

[0031] Epitopes on a number of potential target proteins can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (e.g., MAGE-1), human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2/neu.

[0032] Peptides comprising the epitopes from these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in assays using, for example, purified class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules by, for instance, immunofluorescent staining and flow microfluorometry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition. 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 capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with virally infected target cells or tumor cells as potential therapeutic agents.

[0033] The MHC class I antigens are encoded by the HLA-A, B, and C loci. HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower (perhaps as much as 10-fold lower). Each of these loci have a number of alleles. The peptide binding motifs of the invention are relatively specific for each allelic subtype.

[0034] For peptide-based vaccines, the peptides of the present invention preferably comprise a motif recognized by an MHC I molecule having a wide distribution in the human population. Since the MHC alleles occur at different frequencies within different ethnic groups and races, the choice of target MHC allele may depend upon the target population. Table 1 shows the frequency of various alleles at the HLA-A locus products among different races. For instance, the majority of the Caucasoid population can be covered by peptides which bind to four HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2, and A24.1. Similarly, the majority of the Asian population is encompassed with the addition of peptides binding to a fifth allele HLA-A11.2.

TABLE-US-00001 TABLE 1 A Allele/Subtype N(69)* A(54) C(502) A1 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) A11.1 0 5.5(3) 0 A11.2 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) 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 B. DuPont, Immunobiology of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989. *N--negroid; A = Asian; C = Caucasoid. Numbers in parenthesis represent the number of individuals included in the analysis.

[0035] The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. In the formulae representing 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 physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G.

[0036] The procedures used to identify peptides of the present invention generally follow the methods disclosed in Falk et al., Nature 351:290 (1991), which is incorporated herein by reference. Briefly, the methods involve 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 isolation of the desired 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 of the above techniques.

[0037] In the typical case, immunoprecipitation is used to isolate the desired allele. A number of protocols can be used, depending upon the specificity of the antibodies used. For example, allele-specific mAb reagents can be used for the affinity purification of the HLA-A, HLA-B1, and HLA-C molecules. Several 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.

[0038] In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C mAbs, such as W6/32 and B9.12.1, and one anti-HLA-B, C mAb, B1.23.2, could be used in alternative affinity purification protocols as described in previous applications.

[0039] The peptides bound to the peptide binding groove of the isolated MHC molecules are eluted typically using acid treatment. Peptides can also be dissociated from class I molecules by a variety of standard denaturing means, such as heat, pH, detergents, salts, chaotropic agents, or a combination thereof.

[0040] Peptide fractions are further separated from the MHC molecules by reversed-phase high performance liquid chromatography (HPLC) and sequenced. 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, isoelectrofocusing, and the like.

[0041] 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 is incorporated herein by reference). Amino acid sequencing of bulk heterogenous peptides (e.g., pooled HPLC fractions) from different class 1 molecules typically reveals a characteristic sequence motif for each class I allele.

[0042] Definition of motifs specific for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class molecules is measured in a variety of different ways. One means is a Class I molecule binding assay as described in the 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 assays (Townsend, et al., Cell 62:285 (1990), and FACS based assays using mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol. 21:2963 (1991)).

[0043] Next, peptides that test positive in the MHC class I binding assay are assayed for the ability of the peptides to induce specific CTL responses in vitro. For instance, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. 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]).

[0044] Alternatively, mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-S (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 for the capacity of the peptide to induce in vitro primary CTL responses. Other eukaryotic cell lines which 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), armyworm (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line (see Schneider J. Embryol. Exp. Morphol. 27:353-365 [1927]).

[0045] Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 .mu.M of peptide in serum-free media for 4 hours under appropriate culture conditions. The peptide-loaded antigen-presenting cells are then incubated with the responder cell populations in vitro for 7 to 10 days under optimized culture conditions. Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing the endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence was derived.

[0046] Specificity and MHC restriction of the CTL is determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are referred to herein as immunogenic peptides.

[0047] The immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or from natural sources such as whole viruses or tumors. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles.

[0048] The polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.

[0049] Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. When possible, it may be desirable to optimize peptides of the invention to a length of 9 or 10 amino acid residues, commensurate in size with endogenously processed viral peptides or tumor cell peptides that are bound to MHC class I molecules on the cell surface.

[0050] Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For instance, the peptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications may be made using well known peptide synthesis procedures, as described in e.g., 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, Ill., Pierce), 2d Ed. (1984), incorporated by reference herein.

[0051] The peptides can also be modified by extending or decreasing the compound's amino acid sequence, e.g., 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 certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity. The non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-.alpha.-amino acids, or their D-isomers, but may include non-natural amino acids as well, such as .beta.-.gamma.-.delta.-amino acids, as well as many derivatives of L-.alpha.-amino acids.

[0052] Typically, a series of peptides with single amino acid substitutions are employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo-oligomers or hetero-oligomers. The number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.

[0053] Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide. Substitutional variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 2 when it is desired to finely modulate the characteristics of the peptide.

TABLE-US-00002 TABLE 2 Original Residue Exemplary Substitution Ala Ser Arg Lys, His Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Lys; Arg Ile Leu; Val Leu Ile; Val Lys Arg; His Met Leu; Ile Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val Ile; Leu Pro Gly

[0054] Substantial changes in function (e.g., affinity for MHC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in peptide properties will be those in which (a) hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a residue having an electropositive side chain, e.g., lysl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (c) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

[0055] The peptides may also comprise isosteres of two or more residues in the immunogenic peptide. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the .alpha.-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0056] Modifications of peptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11:291-302 (1986). Half life of the peptides of the present invention is conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4.degree. C.) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

[0057] The peptides of the present invention or analogs thereof which have CTL stimulating activity may be modified to provide desired attributes other than improved serum half life. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar 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 thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.

[0058] The immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

[0059] In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes CTL. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be injected directly in a micellar form, incorporated into a liposome or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0060] As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. See, Deres et al., Nature 342:561-564 (1989), incorporated herein by reference. Peptides of the invention can be coupled to P.sub.3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Further, as the induction of neutralizing antibodies can also be primed with P.sub.3CSS conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.

[0061] In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one 1.0 another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide. Modification at the C terminus in some cases may alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH.sub.2 acylation, e.g., by alkanoyl (C.sub.1-C.sub.20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

[0062] The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with 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.

[0063] Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982), which is incorporated herein by reference. Thus, fusion proteins which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.

[0064] 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. 103:3185 (1981), modification can be made 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 vectors and suitable 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 regions and usually a replication system to provide an expression vector for expression in the desired cellular 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 cell hosts may also be used, employing suitable vectors and control sequences.

[0065] The peptides of the present invention and 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 which 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.

[0066] For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual already suffering from cancer or infected with the virus of interest. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., 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 generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 .mu.g to about 5000 .mu.g of peptide for a 70 kg patient, followed by boosting dosages of from about 1.0 .mu.g to about 1000 .mu.g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening, situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

[0067] For therapeutic use, administration should begin at 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 boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.

[0068] Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.

[0069] The peptide compositions can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers. It is important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response. Thus, for treatment of chronic infection, a representative dose is in the range of about 1.0 .mu.g to about 5000 .mu.g, preferably about 5 .mu.g to 1000 .mu.g for a 70 kg patient per dose. Immunizing doses followed by boosting doses at established intervals, e.g., 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 eliminated or substantially abated and for a period thereafter.

[0070] The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0071] The concentration of CTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% 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.

[0072] The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected 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 in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of 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 guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.

[0073] For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

[0074] For solid compositions, conventional nontoxic solid carriers may 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 nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

[0075] For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-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, e.g., lecithin for intranasal delivery.

[0076] In another aspect the present invention is directed to vaccines which contain 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 up the polymer, 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, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically 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 conjugating peptides of the invention to lipids, such as P3CSS. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection.

[0077] 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 elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. Such an amount is defined to be an "immunogenically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 .mu.g to about 5000 .mu.g per 70 kilogram patient, more commonly from about 10 .mu.g to about 500 .mu.g mg per 70 kg of body weight.

[0078] In some instances it may be desirable to combine the peptide vaccines of the invention with vaccines which induce neutralizing antibody responses to the virus of interest, particularly to viral envelope antigens.

[0079] For therapeutic or immunization purposes, nucleic acids encoding one or more of the peptides of the invention can also be administered to the patient. A number of methods are conveniently used to deliver the nucleic acids to the patient. For instance, the nulceic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et. al., Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. 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 therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

[0080] A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding 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 reverse translated. A human codon usage table is used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences are directly adjoined, creating a continuous polypeptide sequence. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequence that could be reverse translated and included in the minigene sequence include: helper T lymphocyte epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal. In addition, MHC presentation of CTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.

[0081] The minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then cloned into a desired expression vector.

[0082] Standard regulatory sequences well known to those of skill in the art are included in the vector to ensure expression in the target cells. Several vector elements are required: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0083] Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences can also be considered for increasing minigene expression. 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 found to enhance immunogenicity.

[0084] In some embodiments, a bioistronic expression vector, to allow production of the minigene-encoded epitopes and a second protein included to enhance or decrease immunogenicity can be used. 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. LeIF) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may be beneficial in certain diseases.

[0085] Once an expression vector is selected, the minigene is cloned into the polylinker region downstream 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 all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

[0086] Therapeutic quantities of plasmid DNA are produced by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate fermentation medium (such as Terrific Broth), and grown to saturation in shaker flasks or a bioreactor according to well known techniques. 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 the open circular and linear forms using gel electrophoresis or other methods.

[0087] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (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 compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

[0088] Target cell sensitization can be used as a functional assay for expression and MHC class I presentation of minigene-encoded CTL epitopes. The plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas 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 fluorescence activated cell sorting (FACS). These cells are then chromium-51 labeled and used as target cells for epitope-specific CTL lines. Cytolysis, detected by 51Cr release, indicates production of MHC presentation of minigene-encoded CTL epitopes.

[0089] In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromiurn-51 labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.

[0090] Antigenic peptides may be used to elicit CTL ex vivo, as well. The resulting CTL, can be used to treat chronic infections (viral or bacterial) or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine approach of therapy. 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 appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell).

[0091] The peptides may also find use as diagnostic reagents. For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing chronic infection.

[0092] The following example is offered by way of illustration, not by way of limitation.

EXAMPLE 1

[0093] Class I antigen isolation was carried out as described in the related applications, noted above. Naturally processed peptides were then isolated and sequenced as described there. An allele-specific motif and algorithms were determined and quantitative binding assays were carried out.

[0094] Using the motifs identified above for the HLA-A2.1 allele amino acid sequences from 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.

[0095] 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-US-00003 TABLE 3 Peptide AA Sequence Source A*0201 SEQ ID NO: 17.0317 9 LQIGNIISI Flu.24 0.0130 1 38.0103 9 NLSLSCHAA CEA.432 0.0110 2 1233.11 9 YLSGANLNV CEA.605V9 0.0690 3 1295.03 9 SMPPPGTRV p53.149M2 0.0290 4 1295.04 9 SLPPPGTRV p53.149L2 0.0410 5 1317.24 9 KTCPVQLWV p53.139 0.0069 6 1323.02 9 KLLPENNVV p53.24V9 0.0130 7 1323.04 9 ALNKMFBQV p53.129B7V9 0.0260 8 1323.06 9 KLBPVQLWV p53.139L2B3 0.1100 9 1323.08 9 BLTIHYNYV p53.229B1L2V9 0.0430 10 1323.18 10 LLPPQHLIRV p53.188L2 0.0061 11 1323.29 11 YMCNSSCMGGM p53.236 0.0075 12 1323.31 11 YLCNSSCMGGV p53.236L2V11 0.2300 13 1323.34 11 KLYQGSYGFRV p53.101L2V11 0.0620 14 1324.07 9 CQLAKTCPV p53.135 0.0240 15 1325.01 9 RLPEAAPPV p53.65L2 0.0640 16 1325.02 9 GLAPPQHLV p53.187V9 0.0130 17 1325.04 9 KMAELVHFL MAGE3.112M2 0.2100 18 1325.05 9 KLAELVHFL MAGE3.112L2 0.2500 19 1326.01 9 CLLAKTCPV p53.135L2 0.0400 20 1326.02 9 KLSQHMTEV p53.164L2 0.0410 21 1326.04 9 ELAPVVAPV p53.68L2V9 0.0860 22 1326.06 10 QLAKTCPVQV p53.136 0.0320 23 1326.08 9 HLTEVVRRV p53.168L2 0.0180 24 1329.01 11 KTYQGSYGFRL 0.0028 25 1329.03 10 VVVPYEPPEV p53.216 0.0081 26 1329.14 9 BQLAKTBPV p53.135B1B7 0.0490 27 1329.15 9 BLLAKTBPV p53.135B1L2B7 0.1100 28 1330.01 9 QIIGYVIGT CEA.78 0.0160 29 1330.02 9 QLIGYVIGV CEA.78L2V9 0.5300 30 1330.05 9 YVCGIQNSV CEA.569 0.0510 31 1330.06 9 YLCGIQNSV CEA.569L2 0.1000 32 1330.07 9 ATVGIMIGV CEA.687 0.1400 33 1330.08 9 ALVGIMIGV CEA.687L2 0.5000 34 1330.09 10 VLYGPDDPTI CEA.411 0.0170 35 1330.10 10 VLYGPDDPTV CEA.411V10 0.0310 36 1331.02 9 DLMLSPDDV p53.42V9 37 1331.03 9 ALMLSPDDI p53.42A1 38 1331.04 9 ALMLSPDDV p53.42A1V9 39 1331.05 9 DLMLSPADI p53.42A7 40 1331.06 9 DLMLSPADV p53.42A7V9 41 1331.07 9 DLMLSPDAI p53.42A8 42 1331.08 9 DLMLSPDAV p53.42A8V9 43 38.0007 9 AILTFGSFV KSHV.89 0.0850 44 38.0009 9 HLRDFALAV KSHV.106 0.0183 45 38.0015 9 ALLGSIALL KSHV.155 0.0470 46 38.0018 9 ALLATILAA KSHV.161 0.0490 47 38.0019 9 LLATILAAV KSHV.162 0.1600 48 38.0022 9 RLFADELAA KSHV.14 0.0150 49 38.0024 9 YLSKCTLAV KSHV.65 0.2000 50 38.0026 9 LVYHIYSKI KSHV.153 0.0457 51 38.0029 9 SMYLCILSA KSHV.208 0.0250 52 38.0030 9 YLCILSALV KSHV.210 0.3500 53 38.0033 9 VMFSYLQSL KSHV.268 0.5000 54 38.0035 9 RLHVYAYSA KSHV.285 0.0270 55 38.0039 9 GLQTLGAFV KSHV.98 0.0110 56 38.0040 9 FVEEQMTWA KSHV.105 0.0380 57 38.0041 9 QMTWAQTVV KSHV.109 0.0110 58 38.0042 9 IILDTAIFV KSHV.130 0.6800 59 38.0043 9 AIFVCNAFV KSHV.135 0.0910 60 38.0046 9 AMGNRLVEA KSHV.172 0.0200 61 38.0047 9 RLVEACNLL KSHV.176 0.0180 62 38.0059 9 TLSIVTFSL KSHV.198 0.2200 63 38.0063 9 KLSVLLLEV KSHV.292 0.1400 64 38.0064 9 LLLEVNRSV KSHV.296 0.0270 65 38.0068 9 FVSSPTLPV KSHV.78 0.0350 66 38.0070 9 AMLYLLAEI KSHV.281 0.0820 67 38.0075 9 QMARLAWEA KSHV.1116 0.0990 68 38.0131 10 VLAIEGIFMA KSHV.10 0.0730 69 38.0132 10 YLYHPLLSPI KSHV.27 0.1400 70 38.0134 10 SLFEAMLANV KSHV.49 0.9500 71 38.0135 10 STTGINQLGL KSHV.62 0.0710 72 38.0137 10 LAILTFGSFV KSHV.88 0.0160 73 38.0139 10 ALLGSIALLA KSHV.155 0.0360 74 38.0141 10 ALLATILAAV KSHV.161 0.1100 75 38.0142 10 LLATILAAVA KSHV.162 0.0110 76 38.0143 10 RLFADELAAL KSHV.14 0.1800 77 38.0148 10 YLSKCTLAVL KSHV.65 0.0300 78 38.0150 10 LLVYHIYSKI KSHV.152 0.0130 79 38.0151 10 SMYLCILSAL KSHV.208 0.0360 80 38.0153 10 HLHRQMLSFV KSHV.68 0.0160 81 38.0163 10 LLCGKTGAFL KSHV.167 0.0100 82 38.0164 10 ETLSIVTFSL KSHV.197 0.0180 83 39.0063 9 VMCTYSPPL mp53.119 1.4000 84 39.0065 9 KLFCQLAKT mp53.129 0.0160 85 39.0067 9 ATPPAGSRV mp53.146 0.0130 86 39.0133 10 FLQSGTAKSV mp53.110 0.0180 87 39.0169 10 CMDRGLTVFV KSHV.311 0.0120 88 39.0170 10 VLLNWWRWRL KSHV.327 0.1500 89 40.0070 9 GVFTGLTHI HCV.1565 0.0110 90 40.0072 9 QMWKCLIRL HCV.1611 0.0620 91 40.0074 9 IMTCMSADL HCV.1650 0.0121 92 40.0076 9 ALAAYCLST HCV.1674 0.2500 93 40.0080 9 VLSGKPAII HCV.1692 0.0150 94 40.0082 9 FISGIQYLA HCV.1773 0.1000 95 40.0134 10 YIMTCMSADL HCV.1649 0.0300 96 40.0137 10 AIASLMAFTA HCV.1791 0.0580 97 40.0138 10 GLAGAAIGSV HCV.1838 0.0320 98 41.0058 8 MIGVLVGV CEA.692 0.0120 99 41.0061 9 VLPLAYISL TRP1 0.0110 100 41.0062 9 SLGCIFFPL TRP1 0.9700 101 41.0063 9 PLAYISLFL TRP1 0.0220 102 41.0065 9 LMLFYQVWA TRP1 0.0270 103 41.0071 9 NISIYNYFV TRP1 0.2300 104 41.0072 9 NISVYNYFV TRP1 0.0600 105 41.0075 9 FVWTHYYSV TRP1 1.5000 106 41.0077 9 FLTWHRYHL TRP1 0.5500 107 41.0078 9 LTWHRYHLL TRP1 0.1600 108 41.0082 9 MLQEPSFSL TRP1 0.6900 109 41.0083 9 SLPYWNFAT TRP1 0.0110 110 41.0088 9 RLPEPQDVA TRP1 0.0180 111 41.0090 9 VTQCLEVRV TRP1 0.0160 112 41.0096 9 LLHTFTDAV TRP1 0.2700 113 41.0100 9 NMVPFWPPV TRP1 0.6200 114 41.0104 9 AVVGALLLV TRP1 0.0210 115 41.0105 9 AVVAALLLV TRP1 0.0390 116 41.0108 9 LLVAAIFGV TRP1 1.9000 117 41.0112 9 SMDEANQPL TRP1 0.0770 118 41.0114 9 VLPLAYISV TRP1 0.1100 119 41.0115 9 SLGCIFFPV TRP1 3.2000 120 41.0116 9 PLAYISLFV TRP1 0.0310 121 41.0117 9 LLLFQQARV TRP1 0.1100 122 41.0118 9 LMLFYQVWV TRP1 2.4000 123

41.0119 9 LLPSSGPGV TRP1 0.3700 124 41.0121 9 NLSIYNYFV TRP1 0.9700 125 41.0122 9 NLSVYNYFV TRP1 0.8700 126 41.0123 9 FLWTHYYSV TRP1 5.6000 127 41.0124 9 SLKKTFLGV TRP1 0.0224 128 41.0125 9 FLTWHRYHV TRP1 0.3800 129 41.0129 9 MLQEPSFSV TRP1 1.6000 130 41.0130 9 SLPYWNFAV TRP1 0.5700 131 41.0131 9 ALGKNVCDV TRP1 0.0160 132 41.0132 9 SLLISPNSV TRP1 0.1300 133 41.0133 9 SLFSQWRVV TRP1 0.0740 134 41.0134 9 TLGTLCNSV TRP1 0.0330 135 41.0136 9 RLPEPQDVV TRP1 0.1000 136 41.0137 9 VLQCLEVRV TRP1 0.0360 137 41.0138 9 SLNSFRNTV TRP1 0.0140 138 41.0139 9 SLDSFRNTV TRP1 0.0440 139 41.0141 9 FLNGTGGQV TRP1 0.0220 140 41.0142 9 VLLHTFTDV TRP1 0.0180 141 41.0145 9 ALVGALLLV TRP1 0.2600 142 41.0146 9 ALVAALLLV TRP1 0.5800 143 41.0147 9 LLVALIFGV TRP1 1.0000 144 41.0148 9 YLIRARRSV TRP1 0.0170 145 41.0149 9 SMDEANQPV TRP1 0.1600 146 41.0151 10 SLGCIFFPLL TRP1 0.1800 147 41.0157 10 GMCCPDLSPV TRP1 0.0950 148 41.0160 10 AACNQKILTV TRP1 0.0120 149 41.0162 10 FLTWHRYHLL TRP1 0.0830 150 41.0166 10 SLHNLAHLFL TRP1 0.3900 151 41.0174 10 LLLVAAIFGV TRP1 0.3000 152 41.0177 10 LLVAAIFGVA TRP1 0.0820 153 41.0178 10 ALIFGTASYL TRP1 0.0230 154 41.0180 10 SMDEANQPLL TRP1 0.0250 155 41.0181 10 LLTDQYQCYA TRP1 0.0320 156 41.0183 10 SLGCIFFPLV TRP1 0.3200 157 41.0186 10 FLMLFYQVWV TRP1 0.8100 158 41.0189 10 ALCDQRVLIV TRP1 0.0530 159 41.0190 10 ALCNQKILTV TRP1 0.0770 160 41.0191 10 FLTWHRYHLV TRP1 0.0510 161 41.0197 10 SLHNLAHLFV TRP1 0.5000 162 41.0198 10 NLAHLFLNGV TRP1 0.4100 163 41.0199 10 NMVPFWPPVV TRP1 0.2800 164 41.0201 10 ILVVAALLLV TRP1 0.0190 165 41.0203 10 LLVALIFGTV TRP1 0.1200 166 41.0205 10 ALIFGTASYV TRP1 0.0900 167 41.0206 10 SMDEANQPLV TRP1 0.0350 168 41.0207 10 LLTDQYQCYV TRP1 0.2100 169 41.0212 11 LLIQNIIQNDT CEA.107 0.0140 170 41.0214 11 IIQNDTGFYTL CEA.112 0.0130 171 41.0221 11 TLFNVTRNDTA CEA.201 0.0110 172 41.0235 11 LTLLSVTRNDV CEA.378 0.0150 173 41.0243 11 GLYTCQANNSA CEA.473 0.0290 174 41.0268 11 ATVGIMIGVLV CEA.687 0.0160 175 44.0075 11 GLVPPQHLIRV mp53.184.V3 0.0370 176 44.0087 11 GLAPPVHLIRV mp53.184.V6 0.0330 177 44.0092 11 GLAPPEHLIRV mp53.184.E6 0.1600 178 1227.10 9 ILIGVLVGV CEA.691.L2 0.2300 179 1234.26 10 YLIMVKCWMV Her2/neu.952.L2V10 0.3800 180 1295.06 9 LLGRDSFEV mp53.261 0.2000 181 1319.01 9 FMYSDFHFI Flu.RRP2.446 0.4400 182 1319.06 9 NMLSTVLGV Flu.RRP2.446 0.1700 183 1319.14 9 SLENFRAYV Flu.RRP2.446 0.0430 184 1325.06 KMAELVHFV Mage3.112 0.1900 185 1325.07 KLAELVHFV Mage3.112 0.3500 186 1334.01 VLIQRNPQV Her2/neu.153.V9 0.0910 187 1334.02 VLLGVVFGV Her2/neu.665.L2V9 2.1000 188 1334.03 SLISAVVGV Her2/neu.653.L2V9 0.7000 189 1334.04 YMIMVKBWMI Her2/neu.952.B7 0.2700 190 1334.05 YLIMVKBWMV Her2/neu.952.L2B7V10 0.6900 191 1334.06 KLWEELSVV Mage3.220.L2V9 0.4500 192 1334.08 AMBRWGLLV Her2/neu.5.M2B3V9 0.1400 193 1345.01 9 IJIGVLVGV CEA.691.J2 0.0570 194 1345.02 9 ATVGIJIGV CEA.687.J6 0.1595 195 1345.03 9 SJPPPGTRV p53.149.J2 0.0545 196 1345.04 10 LVFGIELJEV MAGE3.160.J8 0.7650 197 918.12 8 ILGFVFTL Flu.M1.59 0.7900 198 1095.22 9 KIFGSLAFL Her2/neu 199 1090.01 10 YLQLVFGIEV MAGE2 200 1126.01 9 MMNDQLMFL PSM 201 1126.02 10 ALVLAGGFFL PSM 202 1126.03 9 WLCAGALVL PSM 203 1126.05 9 MVFELANSI PSM 204 1126.06 10 RMMNDQLMFL PSM 205 1126.09 9 LVLAGGFFL PSM 206 1126.10 9 VLAGGFFLL PSM 207 1126.12 9 LLHETDSAV PSM 208 1126.14 9 LMYSLVHNL PSM 209 1126.16 10 QLMFLERAFI PSM 210 1126.17 9 LMFLERAFI PSM 211 1126.20 10 KLGSGNDFEV PSM 212 1129.01 10 LLQERGVAYI PSM 213 1129.04 10 GMPEGDLVYV PSM 214 1129.05 10 FLDELKAENI PSM 215 1129.08 9 ALFDIESKV PSM 216 1129.10 10 GLPSIPVHPI PSM 217

II. Non-HLA-A2 Motifs

[0096] The present invention also relates to the determination of allele-specific peptide motifs for human Class I MHC (sometimes referred to as HLA) allele subtypes. These motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases, cancers or autoimmune diseases, for which the amino acid sequence of the potential antigen or autoantigen targets is known.

[0097] Epitopes on a number of potential target proteins can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (e.g., MAGE-1), human immunodeficiency virus (HIV) antigens and human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, and Her2/neu.

[0098] Peptides comprising the epitopes from these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in assays using, for example, purified class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition. 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 capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with virally infected target cells or tumor cells as potential therapeutic agents.

[0099] The MHC class I antigens are encoded by the HLA-A, B, and C loci. HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower (perhaps as much as 10-fold lower). Each of these loci have a number of alleles. The peptide binding motifs of the invention are relatively specific for each allelic subtype.

[0100] For peptide-based vaccines, the peptides of the present invention preferably comprise a motif recognized by an MHC I molecule having a wide distribution in the human population. Since the MHC alleles occur at different frequencies within different ethnic groups and races, the choice of target MHC allele may depend upon the target population. Table 4 shows the frequency of various alleles at the HLA-A locus products among different races. For instance, the majority of the Caucasoid population can be covered by peptides which bind to four HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2, and A24.1. Similarly, the majority of the Asian population is encompassed with the addition of peptides binding to a fifth allele HLA-A11.2.

TABLE-US-00004 TABLE 4 A Allele/Subtype N(69)* A(54) C(502) A1 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) A11.1 0 5.5(3) 0 A11.2 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) 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 B. DuPont, Immunobiology of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989. *N--negroid; A = Asian; C = Caucasoid. Numbers in parenthesis represent the number of individuals included in the analysis.

[0101] The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. In the formulae representing 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 physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G.

[0102] The procedures used to identify peptides of the present invention generally follow the methods disclosed in Falk et al., Nature 351:290 (1991), which is incorporated herein by reference. Briefly, the methods involve 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 isolation of the desired 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 of the above techniques.

[0103] A large number of cells with defined MHC molecules, particularly MHC Class I molecules, are known and readily available. For example, human EBV-transformed B cell lines have been shown to be excellent sources for the preparative isolation of class I and class II MHC molecules. Well-characterized cell lines are available from private and commercial sources, such as American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," 6th edition (1988) Rockville, Md., U.S.A.); National Institute of General Medical Sciences 1990/1991 Catalog of Cell Lines (NIGMS) Human Genetic Mutant Cell Repository, Camden, N.J.; and ASHI Repository, Bingham and Women's Hospital, 75 Francis Street, Boston, Mass. 02115. Table 5 lists some B cell lines suitable for use as sources for HLA-A alleles. All of these cell lines can be grown in large batches and are therefore useful for large scale production of MHC molecules. One of skill will recognize that these are merely exemplary cell lines and that many other cell sources can be employed. 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-US-00005 TABLE 5 HUMAN CELL LINES (HLA-A SOURCES) HLA-A allele B cell line A1 MAT COX (9022) STEINLIN (9087) A2.1 JY A3.2 EHM (9080) HO301 (9055)GM3107 A24.1 T3(9107), TISI (9042) A11 BVR (GM6828A) WT100 (GM8602)WT52 (GM8603)

[0104] In the typical case, immunoprecipitation is used to isolate the desired allele. A number of protocols can be used, depending upon the specificity of the antibodies used. For example, allele-specific mAb reagents can be used for the affinity purification of the HLA-A, HLA-B, and HLA-C molecules. Several mAb reagents for the isolation of HLA-A molecules are available (Table 6). Thus, for each of the targeted HLA-A alleles, reagents are available that may be used for the direct isolation of the HLA-A molecules. Affinity columns prepared with these mabs using standard techniques are successfully used to purify the respective HLA-A allele products.

[0105] In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C mAbs, such as W6/32 and B9.12.1, and one anti-HLA-B, C mAb, B1.23.2, could be used in alternative affinity purification protocols as described in the example section below.

TABLE-US-00006 TABLE 6 ANTIBODY REAGENTS anti-HLA Name HLA-A1 12/18 HLA-A3 GAPA3 (ATCC, HB122) HLA-11, 24.1 A11.1M (ATCC, HB164) HLA-A, B, C W6/32 (ATCC, HB95) monomorphic B9.12.1 (INSERM-CNRS) HLA-B, C B.1.23.2 (INSERM-CNRS) monomorphic

[0106] The peptides bound to the peptide binding groove of the isolated MHC molecules are eluted typically using acid treatment. Peptides can also be dissociated from class I molecules by a variety of standard denaturing means, such as heat, pH, detergents, salts, chaotropic agents, or a combination thereof.

[0107] Peptide fractions are further separated from the MHC molecules by reversed-phase high performance liquid chromatography (HPLC) and sequenced. 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, isoelectrofocusing, and the like.

[0108] Sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller, M. W., et al., Methods Enzmmol. 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 is incorporated herein by reference). Amino acid sequencing of bulk heterogenous peptides (e.g., pooled HPLC fractions) from different class I molecules typically reveals a characteristic sequence motif for each class I allele.

[0109] Definition of motifs specific for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class molecules is measured in a variety of different ways. One means is a Class I molecule binding assay as described in the 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 assays (Townsend, et al., Cell 62:285 (1990), and FACS based assays using mutated ells, such as RMA.S (Melief, et al., Eur. J. Immunol. 21:2963 (1991)).

[0110] Next, peptides that test positive in the MHC class I binding assay are assayed for the ability of the peptides to induce specific CTL responses in vitro. For instance, Antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. 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]).

[0111] Alternatively, mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-S (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 for the capacity of the peptide to induce in vitro primary CTL responses. Other eukaryotic cell lines which 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), armyworm (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line (see Schneider J. Embryol. Exp. Morphol. 27:353-365 [1927]).

[0112] Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 .mu.M of peptide in serum-free media for 4 hours under appropriate culture conditions. The peptide-loaded antigen-presenting cells are then incubated with the responder cell populations in vitro for 7 to 10 days under optimized culture conditions. Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence was derived.

[0113] Specificity and MHC restriction of the CTL is determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are referred to herein as immunogenic peptides.

[0114] The immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or from natural sources such as whole viruses or tumors. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles.

[0115] The polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.

[0116] Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. When possible, it may be desirable to optimize peptides of the invention to a length of 9 or 10 amino acid residues, commensurate in size with endogenously processed viral peptides or tumor cell peptides that are bound to MHC class I molecules on the cell surface.

[0117] Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell. For instance, the peptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications may be made using well known peptide synthesis procedures, as described in e.g., 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, Ill., Pierce), 2d Ed. (1984), incorporated by reference herein.

[0118] The peptides can also be modified by extending or decreasing the compound's amino acid sequence, e.g., 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 certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity. The non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-.alpha.-amino acids, or their D-isomers, but may include non-natural amino acids as well, such as .beta.-.gamma.-.delta.-amino acids, as well as many derivatives of L-.alpha.-amino acids.

[0119] Typically, a series of peptides with single amino acid substitutions are employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo-oligomers or hetero-oligomers. The number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.

[0120] Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide. Substitutional variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 2 when it is desired to finely modulate the characteristics of the peptide.

TABLE-US-00007 TABLE 2 Original Residue Exemplary Substitution Ala Ser Arg Lys, His Asn Gln Asp Glu Cys Ser Glu Asp Gly Pro His Lys; Arg Ile Leu; Val Leu Ile; Val Lys Arg; His Met Leu; Ile Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val Ile; Leu Pro Gly

[0121] Substantial changes in function (e.g., affinity for MHC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in peptide properties will be those in which (a) hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a residue having an electropositive side chain, e.g., lysl, arginyl, or histidyl, is substitute for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (c) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

[0122] The peptides may also comprise isosteres of two or more residues in the immunogenic peptide. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the .alpha.-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0123] Modifications of peptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11:291-302 (1986). Half life of the peptides of the present invention is conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4.degree. C.) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

[0124] The peptides of the present invention or analogs thereof which have CTL stimulating activity may be modified to provide desired attributes other than improved serum half life. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar 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 thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.

[0125] The immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy Terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

[0126] In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes CTL. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be injected directly in a micellar form, incorporated into a liposome or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0127] As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to inappropriate peptide. See, Deres et al., Nature 342:561-564 (1989), incorporated herein by reference. Peptides of the invention can be coupled to P.sup.3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Further, as the induction of neutralizing antibodies can also be primed with P.sup.3CSS conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.

[0128] In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide. Modification at the C terminus in some cases may alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH.sub.2-acylation, e.g., by alkanoyl (C.sub.1-C.sub.20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

[0129] The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with 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.

[0130] Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally 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 which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.

[0131] As the coding sequence for peptide, of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981), modification can be made 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 vectors and suitable 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 regions and usually a replication system to provide an expression vector for expression in the desired cellular 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 cell hosts may also be used, employing suitable vectors and control sequences.

[0132] The peptides of the present invention and 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 which 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.

[0133] For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual already suffering from cancer or infected with the virus of interest. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, e.g., 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 generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 .mu.g to about 5000 .mu.g of peptide for a 70 kg patient, followed by boosting dosages of from about 1.0 .mu.g to about 1000 .mu.g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

[0134] For therapeutic use, administration should begin at 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 boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.

[0135] Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing (chronic infection the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.

[0136] The peptide compositions can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers. It is important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response. Thus, for treatment of chronic infection, a representative dose is in the range of about 1.0 .mu.g to about 5000 .mu.g, preferably about 5 .mu.g to 1000 .mu.g for a 70 kg patient per dose. Immunizing doses followed by boosting doses at established intervals, e.g., 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 eliminated or substantially abated and for a period thereafter.

[0137] The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0138] The concentration of CTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% 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.

[0139] The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected 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 in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of 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 guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.

[0140] For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

[0141] For solid compositions, conventional nontoxic solid carriers may 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 nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

[0142] For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-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, e.g., lecithin for intranasal delivery.

[0143] In another aspect the present invention is directed to vaccines which contain 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 up the polymer, 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, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically 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 conjugating peptides of the invention to lipids, such as P.sub.3CSS. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection.

[0144] 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 elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. Such an amount is defined to be an "immunogenically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 .mu.g to about 5000 .mu.g per 70 kilogram patient, more commonly from about 10 .mu.g to about 500 .mu.g mg per 70 kg of body weight.

[0145] In some instances it may be desirable to combine the peptide vaccines of the invention with vaccines which induce neutralizing antibody responses to the virus of interest, particularly to viral envelope antigens.

[0146] For therapeutic or immunization purposes, nucleic acids encoding one or more of the peptides of the invention can also be administered to the patient. A number of methods are conveniently used to deliver the nucleic acids to the patient. For instance, the nulceic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et. al., Science 247:1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7):682-691; Rose U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. 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 therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

[0147] A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding 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 reverse translated. A human codon usage table is used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences are directly adjoined, creating a continuous polypeptide sequence. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequence that could be reverse translated and included in the minigene sequence include: helper T lymphocyte epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal. In addition, MHC presentation of CTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.

[0148] The minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then cloned into a desired expression vector.

[0149] Standard regulatory sequences well known to those of skill in the art are included in the vector to ensure expression in the target cells. Several vector elements are required: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0150] Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences can also be considered for increasing minigene expression. 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 found to enhance immunogenicity.

[0151] In some embodiments, a bioistronic expression vector, to allow production of the minigene-encoded epitopes and a second protein included to enhance or decrease immunogenicity can be used. 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. LeIF) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may be beneficial in certain diseases.

[0152] Once an expression vector is selected, the minigene is cloned into the polylinker region downstream 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 all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

[0153] Therapeutic quantities of plasmid DNA are produced by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate fermentation medium (such as Terrific Broth), and grown to saturation in shaker flasks or a bioreactor according to well known techniques. 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 the open circular and linear forms using gel electrophoresis or other methods.

[0154] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (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 compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

[0155] Target cell sensitization can be uses as a functional assay for expression and MHC class I presentation of minigene-encoded CTL epitopes. The plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas 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 fluorescence activated cell sorting (FACS). These cells are then chromium-51 labeled and used as target cells for epitope-specific CTL lines. Cytolysis, detected by 51 Cr release, indicates production of MHC presentation of minigene-encoded CTL epitopes.

[0156] In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.

[0157] Antigenic peptides may be used to elicit CTL ex vivo, as well. The resulting CTL, can be used to treat chronic infections (viral or bacterial) or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine approach of therapy. 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 appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell). In order to optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is maintained in an appropriate serum-free medium.

[0158] Prior to incubation of the stimulator cells with the cells to be activated, e.g., precursor CD8+ cells, an amount of antigenic peptide is added to the stimulator cell culture, of sufficient quantity to become 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 about 200, and preferably 200 or more, human Class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell. Preferably, the stimulator cells are incubated with >20 .mu.g/ml peptide.

[0159] Resting or precursor CD8+ cells are then incubated in culture with the appropriate stimulator cells for a time period sufficient to activate the CD8+ cells. Preferably, the CD8+ cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8+ (effector) cells to stimulator cells may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the within-described treatment modality is used. Preferably, however, the lymphocyte:stimulator cell ratio is in the range of about 30:1 to 300:1. The effector/stimulator culture may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CD8+ cells.

[0160] The induction of CTL in vitro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC. The number of specific MHC/peptide complexes per APC is crucial for the stimulation of CTL, particularly in primary immune responses. While small amounts of peptide/MHC complexes per cell are 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 primary response requires a significantly higher number of MHC/peptide complexes. Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses. Peptide loading of empty major histocompatibility complex molecules on cells enables the induction of primary cytotoxic T lymphocyte responses.

[0161] Since mutant cell lines do not exist for every human MHC allele, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, 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 cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies. This application discloses methods for stripping the endogenous MHC-associated peptides from the surface of APC followed by the loading of desired peptides.

[0162] A stable MHC class I molecule is a trimeric complex formed of the following elements: 1) a peptide usually of 8-10 residues, 2) a transmembrane heavy polymorphic protein chain which bears the peptide-binding site in its .alpha.1 and .alpha.2 domains, and 3) a non-covalently associated non-polymorphic light chain, .beta..sub.2 microglobulin. Removing the bound peptides and/or dissociating the .beta..sub.2 microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to MHC class I molecules on the APC without causing their degradation before exogenous peptides can be added to them.

[0163] Two possible ways to free up MHC class I molecules of bound peptides include lowering the culture temperature from 37.degree. C. to 26.degree. C. overnight to destabilize .beta..sub.2 microglobulin and stripping the endogenous peptides from the cell using a mild acid treatment. The methods release previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to the empty class I molecules. The cold-temperature incubation method enables exogenous peptides to bind efficiently to the MHC complex, but requires an overnight incubation at 26.degree. C. which may slow the cell's metabolic rate. It is also likely that cells not actively synthesizing MHC molecules (e.g., resting PBMC) would not produce high amounts of empty surface MHC molecules by the cold temperature procedure.

[0164] Harsh acid stripping involves extraction of the peptides with trifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinity purified class I-peptide complexes. These methods are not feasible for CTL induction, since it is important to remove the endogenous peptides while preserving APC viability and an optimal metabolic state which is critical for antigen presentation. Mild acid solutions of pH 3 such as glycine or citrate-phosphate buffers have been used to identify endogenous peptides and to identify tumor associated T cell epitopes. The treatment is especially effective, in that only the MHC class I molecules are destabilized (and associated peptides released), while other surface antigens remain intact, including MHC class II molecules. Most importantly, treatment of cells with the mild acid solutions do not affect the cell's viability or metabolic state. The mild acid treatment is rapid since the stripping of the endogenous peptides occurs in two minutes at 4.degree. C. and the APC is ready to perform its function after the appropriate peptides are loaded. The technique is utilized herein to make peptide-specific APCs for the generation of primary antigen-specific CTL. The resulting APC are efficient in inducing peptide-specific CD8+ CTL.

[0165] Activated CD8+ cells may be effectively separated from the stimulator cells using one of a variety of known methods. For example, monoclonal antibodies specific for the stimulator cells, for the peptides loaded onto the stimulator cells, or for the CD8+ cells (or a segment thereof) may be utilized to bind their appropriate complementary ligand. Antibody-tagged molecules may then be extracted from the stimulator-effector cell admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.

[0166] Effective, cytotoxic amounts of the activated CD8+ cells can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1.times.10.sup.6 to about 1.times.10.sup.12, more preferably about 1.times.10.sup.8 to about 1.times.10.sup.11, and even more preferably, about 1.times.10.sup.9 to about 1.times.10.sup.10 activated CD8+ cells are utilized for adult humans, compared to about 5.times.10.sup.6-5.times.10.sup.7 cells used in mice.

[0167] Preferably, as discussed above, the activated CD8+ cells are harvested from the cell culture prior to administration of the CD8+ cells to the individual being treated. 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 complete separation of stimulator cells and activated CD8+ cells is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulator cells, whereas administration of mammalian tumor-promoting cells may be extremely hazardous.

[0168] Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example, administration of activated CD8+ cells via intravenous infusion is appropriate.

[0169] The immunogenic peptides of this invention may also be used to make monoclonal antibodies. Such antibodies may be useful as potential diagnostic or therapeutic agents.

[0170] The peptides may also find use as diagnostic reagents. For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing chronic infection.

[0171] To identify peptides of the invention, class I antigen isolation, and isolation and sequencing of 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, A1, A11, and A24.1. These motifs are described on page 3, above. The motifs described in Tables 8-11, below, are defined from pool sequencing data of naturally processed peptides as described in the related applications.

TABLE-US-00008 TABLE 8 Summary HLA-A3.2 Allele-Specific Motif (SEQ ID NO: 378) Position Conserved Residues 1 -- 2 V, L, M 3 Y, D 4 -- 5 -- 6 -- 7 I 8 Q, N 9 K 10 K

TABLE-US-00009 TABLE 9 Summary HLA-A1 Allele-Specific Motif SEQ ID NO: 218 Position Conserved Residues 1 -- 2 S, T 3 D, E 4 P 5 -- 6 -- 7 L 8 -- 9 Y 10 K

TABLE-US-00010 TABLE 10 Summary HLA-A11 Allele-Specific Motif (SEQ ID NO: 379) Position Conserved Residues 1 -- 2 T, V 3 M, F 4 -- 5 -- 6 -- 7 -- 8 Q 9 K 10 K

TABLE-US-00011 TABLE 11 Summary HLA-A24.1 Allele-Specific Motif (SEQ ID NO: 380) Position Conserved Residues 1 -- 2 Y 3 I, M 4 D, E, G, K, P 5 L, M, N 6 V 7 N, V 8 A, E, K, Q, S 9 F, L 10 F, A

Example 2

Identification of Immunogenic Peptides

[0172] Using the motifs identified above for various MHC class I allele amino acid sequences from various pathogens and tumor-related proteins were analyzed for the presence of these motifs. Screening was carried out described in the related applications. Table 12 provides the results of searches of the antigens.

TABLE-US-00012 TABLE 12 SEQ Peptide AA Sequence Source A*0301 A*1101 ID NO: 28.0719 10 ILEQWVAGRK HDV.nuc.16 0.0170 0.0012 219 28.0727 10 LSAGGKNLSK HDV.nuc.115 0.0097 0.0150 220 1259.02 11 STDTVDTVLEK Flu.HA.29 0.0001 0.0670 221 1259.04 9 GIAPLQLGK Flu.HA.63 0.6100 0.2000 222 1259.06 10 VTAACSHAGK Flu.HA.149 0.0380 0.0490 223 1259.08 9 GIHHPSNSK Flu.HA.195 0.1300 0.0140 224 1259.10 10 RMNYYWTLLK Flu.HA.243 2.5000 2.3000 225 1259.12 11 ITNKVNSVIEK Flu.HA.392 0.0200 0.0670 226 1259.13 11 KMNIQFTAVGK Flu.HA.402 0.0280 0.0092 227 1259.14 9 NIQFTAVGK Flu.HA.404 0.0017 0.0330 228 1259.16 11 AVGKEFNKLEK Flu.HA.409 0.0210 0.0460 229 1259.19 11 KVKSQLKNNAK Flu.HA.465 0.0470 0.0031 230 1259.20 11 SVRNGTYDYPK Flu.HA.495 0.0410 0.1400 231 1259.21 9 SIIPSGPLK Flu.VMT1.13 0.7800 8.8000 232 1259.25 10 RMVLASTTAK Flu.VMT1.178 0.5500 0.0350 233 1259.26 9 MVLASTTAK Flu.VMT1.179 1.7000 1.4000 234 1259.28 10 RMGVQMQRFK Flu.VMT1.243 0.1000 0.0059 235 1259.33 10 ATEIRASVGK Flu.VNUC.22 0.1400 0.3000 236 1259.37 11 TMVMELVRMIK Flu.VNUC.188 0.0890 0.0310 237 1259.43 10 RVLSFIKGTK Flu.VNUC.342 0.8000 0.0830 238 F119.01 9 MSLQRQFLR ORF3P 0.2000 0.7200 239 F119.02 9 LLGPGRPYR TRP.197 0.0190 0.0091 240 F119.03 9 LLGPGRPYK TRP.197K9 2.2000 0.6800 241 34.0019 8 RVYPELPK CEA.139 0.0130 0.0440 242 34.0020 8 TVSAELPK CEA.495 0.0037 0.0320 243 34.0021 8 TVYAEPPK CEA.317 0.0160 0.0220 244 34.0029 8 TINYTLWR MAGE2.74 0.0140 0.0550 245 34.0030 8 LVHFLLLK MAGE2.116 0.0290 0.1500 246 34.0031 8 SVFAHPRK MAGE2.237 0.1410 0.0810 247 34.0043 8 KVLHHMVK MAGE3.285 0.0580 0.0190 248 34.0050 8 RVCACPGR p53.273 0.3500 0.0490 249 34.0051 8 KMFCQLAK p53.132 0.3800 0.3600 250 34.0062 8 RAHSSHLK p53.363 0.5500 0.0071 251 34.0148 9 FVSNLATGR CEA.656 0.0019 0.0490 252 34.0152 9 RLQLSNGNK CEA.546 0.0250 0.0110 253 34.0153 9 RINGIPQQK CEA.628 0.0400 0.0780 254 34.0154 9 KIRKYTMIRK HER2/neu.681 0.0620 0.0055 255 34.0155 9 LVHFLLLKK MAGE2.116 0.5220 1.4000 256 34.0156 9 SMLEVFEGK MAGE2.226 0.0950 1.6000 257 34.0157 9 SSFSTTINK MAGE2.69 0.1600 2.0000 258 34.0158 9 TSYVKVLHK MAGE2.281 0.5300 0.1500 259 34.0159 9 VIFSKASEK MAGE2.149 0.4900 0.0530 260 34.0160 9 GSVVGNWQK MAGE3.130 0.0040 0.2060 261 34.0161 9 SSLPTTMNK MAGE3.69 0.6180 0.7100 262 34.0162 9 SVLEVFEGK MAGE3.226 0.1330 0.9000 263 34.0171 9 SSBMGGMNK p53.240 0.5440 1.1000 264 34.0172 9 SSCMGGMNK p53.240 0.0090 0.0490 265 34.0211 10 RTLTLFNVTK CEA.554 0.2200 1.3000 266 34.0212 10 TISPLNTSYK CEA.241 0.1800 0.0330 267 34.0214 10 STTINYTLWK MAGE2.72 0.0870 0.6500 268 34.0215 10 ASSLPTTMNK MAGE3.68 0.0420 0.0270 269 34.0225 10 KTYQGSYGFK p53.101 0.4900 0.4200 270 34.0226 10 VVRRBPHHEK p53.172 0.1800 0.2100 271 34.0228 10 GLAPPQHLIK p53.187 0.0570 0.0160 272 34.0229 10 NSSCMGGMNK p53.239 0.0071 0.0290 273 34.0230 10 SSBMGGMNRK p53.240 0.0420 0.1600 274 34.0232 10 RVCACPGRDK p53.273 0.0190 0.0250 275 34.0295 11 KTITVSAELPK CEA.492 0.3600 0.1600 276 34.0296 11 TTITVYAEPPK CEA.314 0.0200 0.0280 277 34.0298 11 PTISPSYTYYR CEA.418 (0.0002) 0.1300 278 34.0301 11 GLLGDNQVMPK MAGE2.188 0.0780 0.0047 279 34.0306 11 MVELVHFLLLK MAGE2.113 0.0200 0.0120 280 34.0308 11 FSTTINYTLWR MAGE2.71 0.0110 0.0170 281 34.0311 11 GLLGDNQIMPK MAGE3.188 0.1300 0.0570 282 34.0317 11 RLGFLHSGTAK p53.110 0.0430 0.0001 283 34.0318 11 ALNKMFCQLAK p53.129 0.4400 0.0420 284 34.0323 11 RVCACPGRDRR p53.273 0.0290 0.0290 285 34.0324 11 LSQETFSDLWK p53.14 (0.0009) 0.0470 286 34.0328 11 RAHSSHLKSKK p53.363 0.0270 0.0038 287 34.0329 11 VTCTYSPALNK p53.122 0.0700 0.1200 288 34.0330 11 GTRVRAMAIYK p53.154 1.1000 0.3300 289 34.0332 11 STSRHKKLMFK p53.376 0.3100 0.1300 290 40.0107 9 LAARNVLVK Her2/neu.846 0.0580 0.0285 291 40.0109 9 MALESILRR Her2/neu.889 0.0034 0.0237 292 40.0145 10 ISWLGLRSLR Her2/neu.450 0.0410 0.0027 293 40.0147 10 GSGAFGTVYK Her2/neu.727 0.0660 0.1300 294 40.0153 10 ASPLDSTFYR Her2/neu.997 0.0003 0.0670 295

Example 3

Identification of Immunogenic Peptides

[0173] Using the B7-like supermotifs identified in the related applications described above, sequences from various pathogens and tumor-related proteins were analyzed for the presence of these motifs. Screening was carried out described in the related applications. Table 13 provides the results of searches of the antigens.

TABLE-US-00013 TABLE 13 SEQ Peptide Sequence Source ID NO: 40.0013 SPGLSAGI CEA.680I8 296 40.0022 KPYDGIPA Her2/neu.921 297 40.0023 KPYDGIPI Her2/neu.921I8 298 40.0050 APRMPEAA p53.63 299 40.0051 APRMPEAI p53.63I8 300 40.0055 APAAPTPI p53.76I8 301 40.0057 APTPAAPI p53.79I8 302 40.0059 TPAAPAPI p53.81I8 303 40.0061 APAPAPSI p53.84I8 304 40.0062 SPALNKMF p53.127 305 40.0063 SPALNKMI p53.127I8 306 40.0117 SPSAPPHRI CEA.3I9 307 40.0119 PPHRWCIPI CEA.7I9 308 40.0120 GPAYSGREI CEA.92 309 40.0156 MPNQAQMRILI Her2/neu.706I10 310 40.0157 MPYGCLLDHVI Her2/neu.801I10 311 40.0161 APPHRWCIPW CEA.6 312 40.0162 APPHRWCIPI CEA.6I10 313 40.0163 IPWQRLLLTA CEA.13 314 40.0164 IPWQRLLLTI CEA.13I10 315 40.0166 LPQHLFGYSI CEA.58I10 316 40.0201 RPRFRELVSEF Her2/neu.966 317 40.0202 RPRFRELVSEI Her2/neu.966I11 318 40.0205 PPSPREGPLPA Her2/neu.1149 319 40.0206 PPSPREGPLPI Her2/neu.1149I11 320 40.0207 GPLPAARPAGA Her2/neu.1155 321 40.0208 GPLPAARPAGI Her2/neu.1155I11 322 40.0231 APAPAAPTPAA p53.74 323 40.0232 APAPAAPTPAI p53.74I11 324 40.0233 APAAPTPAAPA p53.76 325 40.0234 APAAPTPAAPI p53.76I11 326 45.0003 IPWQRLLI CEA.13.I8 327 45.0004 LPQHLFGI CEA.58.I8 328 45.0007 RPGVNLSI CEA.428.I8 329 45.0010 IPQQHTQI CEA.632.I8 330 45.0011 TPNNNGTI CEA.646.I8 331 45.0016 CPLHNQEI Her2/neu.315.I8 332 45.0017 KPCARVCI Her2/neu.336.I8 333 45.0019 WPDSLPDI Her2/neu.415.I8 334 45.0023 SPYVSRLI Her2/neu.779.I8 335 45.0024 VPIKWMAI Her2/neu.884.I8 336 45.0026 RPRFRELI Her2/neu.966.I8 337 45.0028 APGAGGMI Her2/neu.1036.I8 338 45.0031 SPGKNGVI Her2/neu.1174.I8 339 45.0037 SPQGASSI MAGE3.64.I8 340 45.0038 YPLWSQSI MAGE3.77.I8 341 45.0044 SPLPSQAI p53.33.I8 342 45.0046 MPEAAPPI p53.66.I8 343 45.0047 APAPSWPI p53.86.I8 344 45.0051 KPVEDKDAI CEA.155.I9 345 45.0054 IPQQHTQVI CEA.632.I9 346 45.0060 APPVAPAPI p53.70.I9 347 45.0062 APAAPTPAI p53.76.I9 348 45.0064 PPGTRVRAI p53.152.I9 349 45.0065 APPQHLIRI p53.189.I9 350 45.0071 IPQQHTQVLI CEA.632.I10 351 45.0072 SPGLSAGATI CEA.680.I10 352 45.0073 SPMCKGSRCI Her2/neu.196.I10 353 45.0074 MPNPEGRYTI Her2/neu.282.I10 354 45.0076 CPLHNQEVTI Her2/neu.315.I10 355 45.0079 KPDLSYMPII Her2/neu.605.I10 356 45.0080 TPSGAMPNQI Her2/neu.701.I10 357 45.0084 GPASPLDSTI Her2/neu.995.I10 358 45.0091 APPVAPAPAI p53.70.I10 359 45.0092 APAPAAPTPI p53.74.I10 360 45.0093 APTPAAPAPI p53.79.I10 361 45.0094 APSWPLSSSI p53.88.I10 362 45.0103 APTISPLNTSI CEA.239.I11 363 45.0108 SPSYTYYRPGI CEA.421.I11 364 45.0117 CPSGVKPDLSI Her2/neu.600.I11 365 45.0118 SPLTSIISAVI Her2/neu.649.I11 366 45.0119 IPDGENVKIPI Her2/neu.740.I11 367 45.0124 SPLDSTFYRSI Her2/neu.998.I11 368 45.0128 LPAARPAGATI Her2/neu.1157.I11 369 45.0134 HPRKLLMQDLI MAGE2.241.I11 370 45.0135 GPRALIETSYI MAGE2.274.I11 371 45.0139 GPRALVETSYI MAGE3.274.I11 372 45.0140 APRMPEAAPPI p53.63.I11 373 45.0141 VPSQKTYQGSI p53.97.I11 374 1145.10 FPHCLAFAY HBV POL 541 analog 375 1145.09 FPVCLAFSY HBV POL 541 analog 376 26.0570 YPALMPLYACI HBV.pol.645 377

[0174] 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.

Sequence CWU 1

1

38019PRTArtificial SequenceFlu.24 peptide 17.0317 1Leu Gln Ile Gly Asn Ile Ile Ser Ile1 529PRTArtificial SequenceCEA.432 peptide 38.0103 2Asn Leu Ser Leu Ser Cys His Ala Ala1 539PRTArtificial SequenceCEA.605V9 peptide 1233.11 3Tyr Leu Ser Gly Ala Asn Leu Asn Val1 549PRTArtificial Sequencep53.149M2 peptide 1295.03 4Ser Met Pro Pro Pro Gly Thr Arg Val1 559PRTArtificial Sequencep53.149L2 peptide 1295.04 5Ser Leu Pro Pro Pro Gly Thr Arg Val1 569PRTArtificial Sequencep53.139 peptide 1317.24 6Lys Thr Cys Pro Val Gln Leu Trp Val1 579PRTArtificial Sequencep53.24V9 peptide 1323.02 7Lys Leu Leu Pro Glu Asn Asn Val Val1 589PRTArtificial Sequencep53.129B7V9 peptide 1323.04 8Ala Leu Asn Lys Met Phe Asx Gln Val1 599PRTArtificial Sequencep53.139L2B3 peptide 1323.06 9Lys Leu Asx Pro Val Gln Leu Trp Val1 5109PRTArtificial Sequencep53.229B1L2V9 peptide 1323.08 10Asx Leu Thr Ile His Tyr Asn Tyr Val1 51110PRTArtificial Sequencep53.188L2 peptide 1323.18 11Leu Leu Pro Pro Gln His Leu Ile Arg Val1 5 101211PRTArtificial Sequencep53.236 peptide 1323.29 12Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met1 5 101311PRTArtificial Sequencep53.236L2V11 peptide 1323.31 13Tyr Leu Cys Asn Ser Ser Cys Met Gly Gly Val1 5 101411PRTArtificial Sequencep53.101L2V11 peptide 1323.34 14Lys Leu Tyr Gln Gly Ser Tyr Gly Phe Arg Val1 5 10159PRTArtificial Sequencep53.135 peptide 1324.07 15Cys Gln Leu Ala Lys Thr Cys Pro Val1 5169PRTArtificial Sequencep53.65L2 peptide 1325.01 16Arg Leu Pro Glu Ala Ala Pro Pro Val1 5179PRTArtificial Sequencep53.187V9 peptide 1325.02 17Gly Leu Ala Pro Pro Gln His Leu Val1 5189PRTArtificial SequenceMAGE3.112M2 peptide 1325.04 18Lys Met Ala Glu Leu Val His Phe Leu1 5199PRTArtificial SequenceMAGE3.112L2 peptide 1325.05 19Lys Leu Ala Glu Leu Val His Phe Leu1 5209PRTArtificial Sequencep53.135L2 peptide 1326.01 20Cys Leu Leu Ala Lys Thr Cys Pro Val1 5219PRTArtificial Sequencep53.164L2 peptide 1326.02 21Lys Leu Ser Gln His Met Thr Glu Val1 5229PRTArtificial Sequencep53.68L2V9 peptide 1326.04 22Glu Leu Ala Pro Val Val Ala Pro Val1 52310PRTArtificial Sequencep53.136 peptide 1326.06 23Gln Leu Ala Lys Thr Cys Pro Val Gln Val1 5 10249PRTArtificial Sequencep53.168L2 peptide 1326.08 24His Leu Thr Glu Val Val Arg Arg Val1 52511PRTArtificial Sequencepeptide 1329.01 25Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu1 5 102610PRTArtificial Sequencep53.216 peptide 1329.03 26Val Val Val Pro Tyr Glu Pro Pro Glu Val1 5 10279PRTArtificial Sequencep53.135B1B7 peptide 1329.14 27Asx Gln Leu Ala Lys Thr Asx Pro Val1 5289PRTArtificial Sequencep53.135B1L2B7 peptide 1329.15 28Asx Leu Leu Ala Lys Thr Asx Pro Val1 5299PRTArtificial SequenceCEA.78 peptide 1330.01 29Gln Ile Ile Gly Tyr Val Ile Gly Thr1 5309PRTArtificial SequenceCEA.78L2V9 peptide 1330.02 30Gln Leu Ile Gly Tyr Val Ile Gly Val1 5319PRTArtificial SequenceCEA.569 peptide 1330.05 31Tyr Val Cys Gly Ile Gln Asn Ser Val1 5329PRTArtificial SequenceCEA.569L2 peptide 1330.06 32Tyr Leu Cys Gly Ile Gln Asn Ser Val1 5339PRTArtificial SequenceCEA.687 peptide 1330.07 33Ala Thr Val Gly Ile Met Ile Gly Val1 5349PRTArtificial SequenceCEA.687L2 peptide 1330.08 34Ala Leu Val Gly Ile Met Ile Gly Val1 53510PRTArtificial SequenceCEA.411 peptide 1330.09 35Val Leu Tyr Gly Pro Asp Asp Pro Thr Ile1 5 103610PRTArtificial SequenceCEA.411V10 peptide 1330.10 36Val Leu Tyr Gly Pro Asp Asp Pro Thr Val1 5 10379PRTArtificial Sequencep53.42V9 peptide 1331.02 37Asp Leu Met Leu Ser Pro Asp Asp Val1 5389PRTArtificial Sequencep53.42A1 peptide 1331.03 38Ala Leu Met Leu Ser Pro Asp Asp Ile1 5399PRTArtificial Sequencep53.42A1V9 peptide 1331.04 39Ala Leu Met Leu Ser Pro Asp Asp Val1 5409PRTArtificial Sequencep53.42A7 peptide 1331.05 40Asp Leu Met Leu Ser Pro Ala Asp Ile1 5419PRTArtificial Sequencep53.42A7V9 peptide 1331.06 41Asp Leu Met Leu Ser Pro Ala Asp Val1 5429PRTArtificial Sequencep53.42A8 peptide 1331.07 42Asp Leu Met Leu Ser Pro Asp Ala Ile1 5439PRTArtificial Sequencep53.42A8V9 peptide 1331.08 43Asp Leu Met Leu Ser Pro Asp Ala Val1 5449PRTArtificial SequenceKSHV.89 peptide 38.0007 44Ala Ile Leu Thr Phe Gly Ser Phe Val1 5459PRTArtificial SequenceKSHV.106 peptide 38.0009 45His Leu Arg Asp Phe Ala Leu Ala Val1 5469PRTArtificial SequenceKSHV.155 peptide 38.0015 46Ala Leu Leu Gly Ser Ile Ala Leu Leu1 5479PRTArtificial SequenceKSHV.161 peptide 38.0018 47Ala Leu Leu Ala Thr Ile Leu Ala Ala1 5489PRTArtificial SequenceKSHV.162 peptide 38.0019 48Leu Leu Ala Thr Ile Leu Ala Ala Val1 5499PRTArtificial SequenceKSHV.14 peptide 38.0022 49Arg Leu Phe Ala Asp Glu Leu Ala Ala1 5509PRTArtificial SequenceKSHV.65 peptide 38.0024 50Tyr Leu Ser Lys Cys Thr Leu Ala Val1 5519PRTArtificial SequenceKSHV.153 peptide 38.0026 51Leu Val Tyr His Ile Tyr Ser Lys Ile1 5529PRTArtificial SequenceKSHV.208 peptide 38.0029 52Ser Met Tyr Leu Cys Ile Leu Ser Ala1 5539PRTArtificial SequenceKSHV.210 peptide 38.0030 53Tyr Leu Cys Ile Leu Ser Ala Leu Val1 5549PRTArtificial SequenceKSHV.268 peptide 38.0033 54Val Met Phe Ser Tyr Leu Gln Ser Leu1 5559PRTArtificial SequenceKSHV.285 peptide 38.0035 55Arg Leu His Val Tyr Ala Tyr Ser Ala1 5569PRTArtificial SequenceKSHV.98 peptide 38.0039 56Gly Leu Gln Thr Leu Gly Ala Phe Val1 5579PRTArtificial SequenceKSHV.105 peptide 38.0040 57Phe Val Glu Glu Gln Met Thr Trp Ala1 5589PRTArtificial SequenceKSHV.109 peptide 38.0041 58Gln Met Thr Trp Ala Gln Thr Val Val1 5599PRTArtificial SequenceKSHV.130 peptide 38.0042 59Ile Ile Leu Asp Thr Ala Ile Phe Val1 5609PRTArtificial SequenceKSHV.135 peptide 38.0043 60Ala Ile Phe Val Cys Asn Ala Phe Val1 5619PRTArtificial SequenceKSHV.172 peptide 38.0046 61Ala Met Gly Asn Arg Leu Val Glu Ala1 5629PRTArtificial SequenceKSHV.176 peptide 38.0047 62Arg Leu Val Glu Ala Cys Asn Leu Leu1 5639PRTArtificial SequenceKSHV.198 peptide 38.0059 63Thr Leu Ser Ile Val Thr Phe Ser Leu1 5649PRTArtificial SequenceKSHV.292 peptide 38.0063 64Lys Leu Ser Val Leu Leu Leu Glu Val1 5659PRTArtificial SequenceKSHV.296 peptide 38.0064 65Leu Leu Leu Glu Val Asn Arg Ser Val1 5669PRTArtificial SequenceKSHV.78 peptide 38.0068 66Phe Val Ser Ser Pro Thr Leu Pro Val1 5679PRTArtificial SequenceKSHV.281 peptide 38.0070 67Ala Met Leu Val Leu Leu Ala Glu Ile1 5689PRTArtificial SequenceKSHV.1116 peptide 38.0075 68Gln Met Ala Arg Leu Ala Trp Glu Ala1 56910PRTArtificial SequenceKSHV.10 peptide 38.0131 69Val Leu Ala Ile Glu Gly Ile Phe Met Ala1 5 107010PRTArtificial SequenceKSHV.27 peptide 38.0132 70Tyr Leu Tyr His Pro Leu Leu Ser Pro Ile1 5 107110PRTArtificial SequenceKSHV.49 peptide 38.0134 71Ser Leu Phe Glu Ala Met Leu Ala Asn Val1 5 107210PRTArtificial SequenceKSHV.62 peptide 38.0135 72Ser Thr Thr Gly Ile Asn Gln Leu Gly Leu1 5 107310PRTArtificial SequenceKSHV.88 peptide 38.0137 73Leu Ala Ile Leu Thr Phe Gly Ser Phe Val1 5 107410PRTArtificial SequenceKSHV.155 peptide 38.0139 74Ala Leu Leu Gly Ser Ile Ala Leu Leu Ala1 5 107510PRTArtificial SequenceKSHV.161 peptide 38.0141 75Ala Leu Leu Ala Thr Ile Leu Ala Ala Val1 5 107610PRTArtificial SequenceKSHV.162 peptide 38.0142 76Leu Leu Ala Thr Ile Leu Ala Ala Val Ala1 5 107710PRTArtificial SequenceKSHV.14 peptide 38.0143 77Arg Leu Phe Ala Asp Glu Leu Ala Ala Leu1 5 107810PRTArtificial SequenceKSHV.65 peptide 38.0148 78Tyr Leu Ser Lys Cys Thr Leu Ala Val Leu1 5 107910PRTArtificial SequenceKSHV.152 peptide 38.0150 79Leu Leu Val Tyr His Ile Tyr Ser Lys Ile1 5 108010PRTArtificial SequenceKSHV.208 peptide 38.0151 80Ser Met Tyr Leu Cys Ile Leu Ser Ala Leu1 5 108110PRTArtificial SequenceKSHV.68 peptide 38.0153 81His Leu His Arg Gln Met Leu Ser Phe Val1 5 108210PRTArtificial SequenceKSHV.167 peptide 38.0163 82Leu Leu Cys Gly Lys Thr Gly Ala Phe Leu1 5 108310PRTArtificial SequenceKSHV.197 peptide 38.0164 83Glu Thr Leu Ser Ile Val Thr Phe Ser Leu1 5 10849PRTArtificial Sequencemp53.119 peptide 39.0063 84Val Met Cys Thr Tyr Ser Pro Pro Leu1 5859PRTArtificial Sequencemp53.129 peptide 39.0065 85Lys Leu Phe Cys Gln Leu Ala Lys Thr1 5869PRTArtificial Sequencemp53.146 peptide 39.0067 86Ala Thr Pro Pro Ala Gly Ser Arg Val1 58710PRTArtificial Sequencemp53.110 peptide 39.0133 87Phe Leu Gln Ser Gly Thr Ala Lys Ser Val1 5 108810PRTArtificial SequenceKSHV.311 peptide 39.0169 88Cys Met Asp Arg Gly Leu Thr Val Phe Val1 5 108910PRTArtificial SequenceKSHV.327 peptide 39.0170 89Val Leu Leu Asn Trp Trp Arg Trp Arg Leu1 5 10909PRTArtificial SequenceHCV.1565 peptide 40.0070 90Gly Val Phe Thr Gly Leu Thr His Ile1 5919PRTArtificial SequenceHCV.1611 peptide 40.0072 91Gln Met Trp Lys Cys Leu Ile Arg Leu1 5929PRTArtificial SequenceHCV.1650 peptide 40.0074 92Ile Met Thr Cys Met Ser Ala Asp Leu1 5939PRTArtificial SequenceHCV.1674 peptide 40.0076 93Ala Leu Ala Ala Tyr Cys Leu Ser Thr1 5949PRTArtificial SequenceHCV.1692 peptide 40.0080 94Val Leu Ser Gly Lys Pro Ala Ile Ile1 5959PRTArtificial SequenceHCV.1773 peptide 40.0082 95Phe Ile Ser Gly Ile Gln Tyr Leu Ala1 59610PRTArtificial SequenceHCV.1649 peptide 40.0134 96Tyr Ile Met Thr Cys Met Ser Ala Asp Leu1 5 109710PRTArtificial SequenceHCV.1791 peptide 40.0137 97Ala Ile Ala Ser Leu Met Ala Phe Thr Ala1 5 109810PRTArtificial SequenceHCV.1838 peptide 40.0138 98Gly Leu Ala Gly Ala Ala Ile Gly Ser Val1 5 10998PRTArtificial SequenceCEA.692 peptide 41.0058 99Met Ile Gly Val Leu Val Gly Val1 51009PRTArtificial SequenceTRP1 peptide 41.0061 100Val Leu Pro Leu Ala Tyr Ile Ser Leu1 51019PRTArtificial SequenceTRP1 peptide 41.0062 101Ser Leu Gly Cys Ile Phe Phe Pro Leu1 51029PRTArtificial SequenceTRP1 peptide 41.0063 102Pro Leu Ala Tyr Ile Ser Leu Phe Leu1 51039PRTArtificial SequenceTRP1 peptide 41.0065 103Leu Met Leu Phe Tyr Gln Val Trp Ala1 51049PRTArtificial SequenceTRP1 peptide 41.0071 104Asn Ile Ser Ile Tyr Asn Tyr Phe Val1 51059PRTArtificial SequenceTRP1 peptide 41.0072 105Asn Ile Ser Val Tyr Asn Tyr Phe Val1 51069PRTArtificial SequenceTRP1 peptide 41.0075 106Phe Val Trp Thr His Tyr Tyr Ser Val1 51079PRTArtificial SequenceTRP1 peptide 41.0077 107Phe Leu Thr Trp His Arg Tyr His Leu1 51089PRTArtificial SequenceTRP1 peptide 41.0078 108Leu Thr Trp His Arg Tyr His Leu Leu1 51099PRTArtificial SequenceTRP1 peptide 41.0082 109Met Leu Gln Glu Pro Ser Phe Ser Leu1 51109PRTArtificial SequenceTRP1 peptide 41.0083 110Ser Leu Pro Tyr Trp Asn Phe Ala Thr1 51119PRTArtificial SequenceTRP1 peptide 41.0088 111Arg Leu Pro Glu Pro Gln Asp Val Ala1 51129PRTArtificial SequenceTRP1 peptide 41.0090 112Val Thr Gln Cys Leu Glu Val Arg Val1 51139PRTArtificial SequenceTRP1 peptide 41.0096 113Leu Leu His Thr Phe Thr Asp Ala Val1 51149PRTArtificial SequenceTRP1 peptide 41.0100 114Asn Met Val Pro Phe Trp Pro Pro Val1 51159PRTArtificial SequenceTRP1 peptide 41.0104 115Ala Val Val Gly Ala Leu Leu Leu Val1 51169PRTArtificial SequenceTRP1 peptide 41.0105 116Ala Val Val Ala Ala Leu Leu Leu Val1 51179PRTArtificial SequenceTRP1 peptide 41.0108 117Leu Leu Val Ala Ala Ile Phe Gly Val1 51189PRTArtificial SequenceTRP1 peptide 41.0112 118Ser Met Asp Glu Ala Asn Gln Pro Leu1 51199PRTArtificial SequenceTRP1 peptide 41.0114 119Val Leu Pro Leu Ala Tyr Ile Ser Val1 51209PRTArtificial SequenceTRP1 peptide 41.0115 120Ser Leu Gly Cys Ile Phe Phe Pro Val1 51219PRTArtificial SequenceTRP1 peptide 41.0116 121Pro Leu Ala Tyr Ile Ser Leu Phe Val1 51229PRTArtificial SequenceTRP1 peptide 41.0117 122Leu Leu Leu Phe Gln Gln Ala Arg Val1 51239PRTArtificial SequenceTRP1 peptide 41.0118 123Leu Met Leu Phe Tyr Gln Val Trp Val1 51249PRTArtificial SequenceTRP1 peptide 41.0119 124Leu Leu Pro Ser Ser Gly Pro Gly Val1 51259PRTArtificial SequenceTRP1 peptide 41.0121 125Asn Leu Ser Ile Tyr Asn Tyr Phe Val1 51269PRTArtificial SequenceTRP1 peptide 41.0122 126Asn Leu Ser Val Tyr Asn Tyr Phe Val1 51279PRTArtificial SequenceTRP1 peptide 41.0123 127Phe Leu Trp Thr His Tyr Tyr Ser Val1 51289PRTArtificial SequenceTRP1 peptide 41.0124 128Ser Leu Lys Lys Thr Phe Leu Gly Val1 51299PRTArtificial SequenceTRP1 peptide 41.0125 129Phe Leu Thr Trp His Arg Tyr His Val1 51309PRTArtificial SequenceTRP1 peptide 41.0129 130Met Leu Gln Glu Pro Ser Phe Ser Val1 51319PRTArtificial SequenceTRP1 peptide 41.0130 131Ser Leu Pro Tyr Trp Asn Phe Ala Val1 51329PRTArtificial SequenceTRP1 peptide 41.0131 132Ala Leu Gly Lys Asn Val Cys Asp Val1 51339PRTArtificial SequenceTRP1 peptide 41.0132 133Ser Leu Leu Ile Ser Pro Asn Ser Val1 51349PRTArtificial SequenceTRP1 peptide 41.0133 134Ser Leu Phe Ser Gln Trp Arg Val Val1 51359PRTArtificial SequenceTRP1 peptide 41.0134 135Thr Leu Gly Thr Leu Cys Asn Ser Val1 51369PRTArtificial SequenceTRP1 peptide 41.0136 136Arg Leu Pro Glu Pro Gln Asp Val Val1 51379PRTArtificial SequenceTRP1 peptide 41.0137 137Val Leu Gln Cys Leu Glu Val Arg Val1 51389PRTArtificial SequenceTRP1 peptide 41.0138 138Ser Leu Asn Ser Phe Arg Asn Thr Val1 51399PRTArtificial SequenceTRP1 peptide 41.0139 139Ser Leu Asp Ser Phe Arg Asn Thr Val1 51409PRTArtificial SequenceTRP1 peptide 41.0141 140Phe Leu Asn Gly Thr Gly Gly Gln Val1 51419PRTArtificial SequenceTRP1 peptide 41.0142 141Val Leu Leu His Thr Phe Thr Asp Val1 51429PRTArtificial SequenceTRP1 peptide 41.0145 142Ala Leu Val Gly Ala Leu Leu Leu Val1 51439PRTArtificial SequenceTRP1 peptide 41.0146 143Ala Leu Val Ala Ala Leu Leu Leu Val1 51449PRTArtificial SequenceTRP1 peptide 41.0147 144Leu Leu Val Ala Leu Ile Phe Gly Val1 51459PRTArtificial SequenceTRP1 peptide 41.0148 145Tyr Leu Ile Arg Ala Arg Arg Ser Val1 51469PRTArtificial SequenceTRP1 peptide 41.0149 146Ser Met Asp Glu Ala Asn Gln Pro Val1 514710PRTArtificial SequenceTRP1 peptide 41.0151 147Ser Leu Gly Cys Ile Phe Phe Pro Leu Leu1 5 1014810PRTArtificial SequenceTRP1 peptide 41.0157 148Gly Met Cys Cys Pro Asp Leu Ser Pro Val1 5 1014910PRTArtificial SequenceTRP1 peptide 41.0160 149Ala Ala Cys Asn Gln Lys Ile Leu Thr Val1 5 1015010PRTArtificial SequenceTRP1 peptide 41.0162 150Phe Leu Thr Trp His Arg Tyr His Leu Leu1 5 1015110PRTArtificial SequenceTRP1 peptide 41.0166 151Ser Leu His Asn Leu Ala His Leu Phe Leu1 5 1015210PRTArtificial SequenceTRP1 peptide 41.0174 152Leu Leu Leu Val Ala Ala Ile Phe Gly Val1 5 1015310PRTArtificial SequenceTRP1 peptide 41.0177 153Leu Leu Val Ala Ala Ile Phe Gly Val Ala1 5 1015410PRTArtificial SequenceTRP1 peptide 41.0178 154Ala Leu Ile Phe Gly Thr Ala Ser Tyr Leu1 5 1015510PRTArtificial SequenceTRP1 peptide 41.0180 155Ser Met Asp Glu Ala Asn Gln Pro Leu Leu1 5 1015610PRTArtificial SequenceTRP1 peptide 41.0181 156Leu Leu Thr Asp Gln Tyr Gln Cys Tyr Ala1 5 1015710PRTArtificial SequenceTRP1 peptide 41.0183 157Ser Leu Gly Cys Ile Phe Phe Pro Leu Val1 5

1015810PRTArtificial SequenceTRP1 peptide 41.0186 158Phe Leu Met Leu Phe Tyr Gln Val Trp Val1 5 1015910PRTArtificial SequenceTRP1 peptide 41.0189 159Ala Leu Cys Asp Gln Arg Val Leu Ile Val1 5 1016010PRTArtificial SequenceTRP1 peptide 41.0190 160Ala Leu Cys Asn Gln Lys Ile Leu Thr Val1 5 1016110PRTArtificial SequenceTRP1 peptide 41.0191 161Phe Leu Thr Trp His Arg Tyr His Leu Val1 5 1016210PRTArtificial SequenceTRP1 peptide 41.0197 162Ser Leu His Asn Leu Ala His Leu Phe Val1 5 1016310PRTArtificial SequenceTRP1 peptide 41.0198 163Asn Leu Ala His Leu Phe Leu Asn Gly Val1 5 1016410PRTArtificial SequenceTRP1 peptide 41.0199 164Asn Met Val Pro Phe Trp Pro Pro Val Val1 5 1016510PRTArtificial SequenceTRP1 peptide 41.0201 165Ile Leu Val Val Ala Ala Leu Leu Leu Val1 5 1016610PRTArtificial SequenceTRP1 peptide 41.0203 166Leu Leu Val Ala Leu Ile Phe Gly Thr Val1 5 1016710PRTArtificial SequenceTRP1 peptide 41.0205 167Ala Leu Ile Phe Gly Thr Ala Ser Tyr Val1 5 1016810PRTArtificial SequenceTRP1 peptide 41.0206 168Ser Met Asp Glu Ala Asn Gln Pro Leu Val1 5 1016910PRTArtificial SequenceTRP1 peptide 41.0207 169Leu Leu Thr Asp Gln Tyr Gln Cys Tyr Val1 5 1017011PRTArtificial SequenceCEA.107 peptide 41.0212 170Leu Leu Ile Gln Asn Ile Ile Gln Asn Asp Thr1 5 1017111PRTArtificial SequenceCEA.112 peptide 41.0214 171Ile Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu1 5 1017211PRTArtificial SequenceCEA.201 peptide 41.0221 172Thr Leu Phe Asn Val Thr Arg Asn Asp Thr Ala1 5 1017311PRTArtificial SequenceCEA.378 peptide 41.0235 173Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Val1 5 1017411PRTArtificial SequenceCEA.473 peptide 41.0243 174Gly Leu Tyr Thr Cys Gln Ala Asn Asn Ser Ala1 5 1017511PRTArtificial SequenceCEA.687 peptide 41.0268 175Ala Thr Val Gly Ile Met Ile Gly Val Leu Val1 5 1017611PRTArtificial Sequencemp53.184.V3 peptide 44.0075 176Gly Leu Val Pro Pro Gln His Leu Ile Arg Val1 5 1017711PRTArtificial Sequencemp53.184.V6 peptide 44.0087 177Gly Leu Ala Pro Pro Val His Leu Ile Arg Val1 5 1017811PRTArtificial Sequencemp53.184.E6 peptide 44.0092 178Gly Leu Ala Pro Pro Glu His Leu Ile Arg Val1 5 101799PRTArtificial SequenceCEA.691.L2 peptide 1227.10 179Ile Leu Ile Gly Val Leu Val Gly Val1 518010PRTArtificial SequenceHer2/neu.952.L2V10 peptide 1234.26 180Tyr Leu Ile Met Val Lys Cys Trp Met Val1 5 101819PRTArtificial Sequencemp53.261 peptide 1295.06 181Leu Leu Gly Arg Asp Ser Phe Glu Val1 51829PRTArtificial SequenceFLu.RRP2.446 peptide 1319.01 182Phe Met Tyr Ser Asp Phe His Phe Ile1 51839PRTArtificial SequenceFlu.RRP2.446 peptide 1319.06 183Asn Met Leu Ser Thr Val Leu Gly Val1 51849PRTArtificial SequenceFlu.RRP2.446 peptide 1319.14 184Ser Leu Glu Asn Phe Arg Ala Tyr Val1 51859PRTArtificial SequenceMage3.112 peptide 1325.06 185Lys Met Ala Glu Leu Val His Phe Val1 51869PRTArtificial SequenceMage3.112 peptide 1325.07 186Lys Leu Ala Glu Leu Val His Phe Val1 51879PRTArtificial SequenceHer2/neu.153.V9 peptide 1334.01 187Val Leu Ile Gln Arg Asn Pro Gln Val1 51889PRTArtificial SequenceHer2/neu.665.L2V9 peptide 1334.02 188Val Leu Leu Gly Val Val Phe Gly Val1 51899PRTArtificial SequenceHer2/neu.653.L2V9 peptide 1334.03 189Ser Leu Ile Ser Ala Val Val Gly Val1 519010PRTArtificial SequenceHer2/neu.952.B7 peptide 1334.04 190Tyr Met Ile Met Val Lys Asx Trp Met Ile1 5 1019110PRTArtificial SequenceHer2/neu.952.L2B7V10 peptide 1334.05 191Tyr Leu Ile Met Val Lys Asx Trp Met Val1 5 101929PRTArtificial SequenceMage3.220.L2V9 peptide 1334.06 192Lys Leu Trp Glu Glu Leu Ser Val Val1 51939PRTArtificial SequenceHer2/neu.5.M2B3V9 peptide 1334.08 193Ala Met Asx Arg Trp Gly Leu Leu Val1 51949PRTArtificial SequenceCEA.691.J2 peptide 1345.01 194Ile Xaa Ile Gly Val Leu Val Gly Val1 51959PRTArtificial SequenceCEA.687.J6 peptide 1345.02 195Ala Thr Val Gly Ile Xaa Ile Gly Val1 51969PRTArtificial Sequencep53.149.J2 peptide 1345.03 196Ser Xaa Pro Pro Pro Gly Thr Arg Val1 519710PRTArtificial SequenceMAGE3.160.J8 peptide 1345.04 197Leu Val Phe Gly Ile Glu Leu Xaa Glu Val1 5 101988PRTArtificial SequenceFlu.M1.59 peptide 918.12 198Ile Leu Gly Phe Val Phe Thr Leu1 51999PRTArtificial SequenceHer2/neu peptide 1095.22 199Lys Ile Phe Gly Ser Leu Ala Phe Leu1 520010PRTArtificial SequenceMAGE2 peptide 1090.01 200Tyr Leu Gln Leu Val Phe Gly Ile Glu Val1 5 102019PRTArtificial SequencePSM peptide 1126.01 201Met Met Asn Asp Gln Leu Met Phe Leu1 520210PRTArtificial SequencePSM peptide 1126.02 202Ala Leu Val Leu Ala Gly Gly Phe Phe Leu1 5 102039PRTArtificial SequencePSM peptide 1126.03 203Trp Leu Cys Ala Gly Ala Leu Val Leu1 52049PRTArtificial SequencePSM peptide 1126.05 204Met Val Phe Glu Leu Ala Asn Ser Ile1 520510PRTArtificial SequencePSM peptide 1126.06 205Arg Met Met Asn Asp Gln Leu Met Phe Leu1 5 102069PRTArtificial SequencePSM peptide 1126.09 206Leu Val Leu Ala Gly Gly Phe Phe Leu1 52079PRTArtificial SequencePSM peptide 1126.10 207Val Leu Ala Gly Gly Phe Phe Leu Leu1 52089PRTArtificial SequencePSM peptide 1126.12 208Leu Leu His Glu Thr Asp Ser Ala Val1 52099PRTArtificial SequencePSM peptide 1126.14 209Leu Met Tyr Ser Leu Val His Asn Leu1 521010PRTArtificial SequencePSM peptide 1126.16 210Gln Leu Met Phe Leu Glu Arg Ala Phe Ile1 5 102119PRTArtificial SequencePSM peptide 1126.17 211Leu Met Phe Leu Glu Arg Ala Phe Ile1 521210PRTArtificial SequencePSM peptide 1126.20 212Lys Leu Gly Ser Gly Asn Asp Phe Glu Val1 5 1021310PRTArtificial SequencePSM peptide 1129.01 213Leu Leu Gln Glu Arg Gly Val Ala Tyr Ile1 5 1021410PRTArtificial SequencePSM peptide 1129.04 214Gly Met Pro Glu Gly Asp Leu Val Tyr Val1 5 1021510PRTArtificial SequencePSM peptide 1129.05 215Phe Leu Asp Glu Leu Lys Ala Glu Asn Ile1 5 102169PRTArtificial SequencePSM peptide 1129.08 216Ala Leu Phe Asp Ile Glu Ser Lys Val1 521710PRTArtificial SequencePSM peptide 1129.10 217Gly Leu Pro Ser Ile Pro Val His Pro Ile1 5 1021810PRTArtificial SequenceHLA-A1 allele-specific motif 218Xaa Xaa Xaa Pro Xaa Xaa Leu Xaa Tyr Lys1 5 1021910PRTArtificial SequenceHDV.nuc.16 peptide 28.0719 219Ile Leu Glu Gln Trp Val Ala Gly Arg Lys1 5 1022010PRTArtificial SequenceHDV.nuc.115 peptide 28.0727 220Leu Ser Ala Gly Gly Lys Asn Leu Ser Lys1 5 1022111PRTArtificial SequenceFlu.HA.29 peptide 1259.02 221Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys1 5 102229PRTArtificial SequenceFlu.HA.63 peptide 1259.04 222Gly Ile Ala Pro Leu Gln Leu Gly Lys1 522310PRTArtificial SequenceFlu.HA.149 peptide 1259.06 223Val Thr Ala Ala Cys Ser His Ala Gly Lys1 5 102249PRTArtificial SequenceFLu.HA.195 peptide 1259.08 224Gly Ile His His Pro Ser Asn Ser Lys1 522510PRTArtificial SequenceFlu.HA.243 peptide 1259.10 225Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys1 5 1022611PRTArtificial SequenceFlu.HA.392 peptide 1259.12 226Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys1 5 1022711PRTArtificial SequenceFlu.HA.402 peptide 1259.13 227Lys Met Asn Ile Gln Phe Thr Ala Val Gly Lys1 5 102289PRTArtificial SequenceFlu.HA.404 peptide 1259.14 228Asn Ile Gln Phe Thr Ala Val Gly Lys1 522911PRTArtificial SequenceFlu.HA.409 peptide 1259.16 229Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys1 5 1023011PRTArtificial SequenceFlu.HA.465 peptide 1259.19 230Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys1 5 1023111PRTArtificial SequenceFlu.HA.495 peptide 1259.20 231Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys1 5 102329PRTArtificial SequenceFlu.VMT1.13 peptide 1259.21 232Ser Ile Ile Pro Ser Gly Pro Leu Lys1 523310PRTArtificial SequenceFlu.VMT1.178 peptide 1259.25 233Arg Met Val Leu Ala Ser Thr Thr Ala Lys1 5 102349PRTArtificial SequenceFlu.VMT1.179 peptide 1259.26 234Met Val Leu Ala Ser Thr Thr Ala Lys1 523510PRTArtificial SequenceFlu.VMT1.243 peptide 1259.28 235Arg Met Gly Val Gln Met Gln Arg Phe Lys1 5 1023610PRTArtificial SequenceFlu.VNUC.22 peptide 1259.33 236Ala Thr Glu Ile Arg Ala Ser Val Gly Lys1 5 1023711PRTArtificial SequenceFlu.VNUC.188 peptide 1259.37 237Thr Met Val Met Glu Leu Val Arg Met Ile Lys1 5 1023810PRTArtificial SequenceFlu.VNUC.342 peptide 1259.43 238Arg Val Leu Ser Phe Ile Lys Gly Thr Lys1 5 102399PRTArtificial SequenceORF3P peptide F119.01 239Met Ser Leu Gln Arg Gln Phe Leu Arg1 52409PRTArtificial SequenceTRP.197 peptide F119.02 240Leu Leu Gly Pro Gly Arg Pro Tyr Arg1 52419PRTArtificial SequenceTRP.197K9 peptide F119.03 241Leu Leu Gly Pro Gly Arg Pro Tyr Lys1 52428PRTArtificial SequenceCEA.139 peptide 34.0019 242Arg Val Tyr Pro Glu Leu Pro Lys1 52438PRTArtificial SequenceCEA.495 peptide 34.0020 243Thr Val Ser Ala Glu Leu Pro Lys1 52448PRTArtificial SequenceCEA.317 peptide 34.0021 244Thr Val Tyr Ala Glu Pro Pro Lys1 52458PRTArtificial SequenceMAGE2.74 peptide 34.0029 245Thr Ile Asn Tyr Thr Leu Trp Arg1 52468PRTArtificial SequenceMAGE2.116 peptide 34.0030 246Leu Val His Phe Leu Leu Leu Lys1 52478PRTArtificial SequenceMAGE2.237 peptide 34.0031 247Ser Val Phe Ala His Pro Arg Lys1 52488PRTArtificial SequenceMAGE3.285 peptide 34.0043 248Lys Val Leu His His Met Val Lys1 52498PRTArtificial Sequencep53.273 peptide 34.0050 249Arg Val Cys Ala Cys Pro Gly Arg1 52508PRTArtificial Sequencep53.132 peptide 34.0051 250Lys Met Phe Cys Gln Leu Ala Lys1 52518PRTArtificial Sequencep53.363 peptide 34.0062 251Arg Ala His Ser Ser His Leu Lys1 52529PRTArtificial SequenceCEA.656 peptide 34.0148 252Phe Val Ser Asn Leu Ala Thr Gly Arg1 52539PRTArtificial SequenceCEA.546 peptide 34.0152 253Arg Leu Gln Leu Ser Asn Gly Asn Lys1 52549PRTArtificial SequenceCEA.628 peptide 34.0153 254Arg Ile Asn Gly Ile Pro Gln Gln Lys1 52559PRTArtificial SequenceHER2/neu.681 peptide 34.0154 255Lys Ile Arg Lys Tyr Thr Met Arg Lys1 52569PRTArtificial SequenceMAGE2.116 peptide 34.0155 256Leu Val His Phe Leu Leu Leu Lys Lys1 52579PRTArtificial SequenceMAGE2.226 peptide 34.0156 257Ser Met Leu Glu Val Phe Glu Gly Lys1 52589PRTArtificial SequenceMAGE2.69 peptide 34.0157 258Ser Ser Phe Ser Thr Thr Ile Asn Lys1 52599PRTArtificial SequenceMAGE2.281 peptide 34.0158 259Thr Ser Tyr Val Lys Val Leu His Lys1 52609PRTArtificial SequenceMAGE2.149 peptide 34.0159 260Val Ile Phe Ser Lys Ala Ser Glu Lys1 52619PRTArtificial SequenceMAGE3.130 peptide 34.0160 261Gly Ser Val Val Gly Asn Trp Gln Lys1 52629PRTArtificial SequenceMAGE3.69 peptide 34.0161 262Ser Ser Leu Pro Thr Thr Met Asn Lys1 52639PRTArtificial SequenceMAGE3.226 peptide 34.0162 263Ser Val Leu Glu Val Phe Glu Gly Lys1 52649PRTArtificial Sequencep53.240 peptide 34.0171 264Ser Ser Asx Met Gly Gly Met Asn Lys1 52659PRTArtificial Sequencep53.240 peptide 34.0172 265Ser Ser Cys Met Gly Gly Met Asn Lys1 526610PRTArtificial SequenceCEA.554 peptide 34.0211 266Arg Thr Leu Thr Leu Phe Asn Val Thr Lys1 5 1026710PRTArtificial SequenceCEA.241 peptide 34.0212 267Thr Ile Ser Pro Leu Asn Thr Ser Tyr Lys1 5 1026810PRTArtificial SequenceMAGE2.72 peptide 34.0214 268Ser Thr Thr Ile Asn Tyr Thr Leu Trp Lys1 5 1026910PRTArtificial SequenceMAGE3.68 peptide 34.0215 269Ala Ser Ser Leu Pro Thr Thr Met Asn Lys1 5 1027010PRTArtificial Sequencep53.101 peptide 34.0225 270Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Lys1 5 1027110PRTArtificial Sequencep53.172 peptide 34.0226 271Val Val Arg Arg Asx Pro His His Glu Lys1 5 1027210PRTArtificial Sequencep53.187 peptide 34.0228 272Gly Leu Ala Pro Pro Gln His Leu Ile Lys1 5 1027310PRTArtificial Sequencep53.239 peptide 34.0229 273Asn Ser Ser Cys Met Gly Gly Met Asn Lys1 5 1027410PRTArtificial Sequencep53.240 peptide 34.0230 274Ser Ser Asx Met Gly Gly Met Asn Arg Lys1 5 1027510PRTArtificial Sequencep53.273 peptide 34.0232 275Arg Val Cys Ala Cys Pro Gly Arg Asp Lys1 5 1027611PRTArtificial SequenceCEA.492 peptide 34.0295 276Lys Thr Ile Thr Val Ser Ala Glu Leu Pro Lys1 5 1027711PRTArtificial SequenceCEA.314 peptide 34.0296 277Thr Thr Ile Thr Val Tyr Ala Glu Pro Pro Lys1 5 1027811PRTArtificial SequenceCEA.418 peptide 34.0298 278Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg1 5 1027911PRTArtificial SequenceMAGE2.188 peptide 34.0301 279Gly Leu Leu Gly Asp Asn Gln Val Met Pro Lys1 5 1028011PRTArtificial SequenceMAGE2.113 peptide 34.0306 280Met Val Glu Leu Val His Phe Leu Leu Leu Lys1 5 1028111PRTArtificial SequenceMAGE2.71 peptide 34.0308 281Phe Ser Thr Thr Ile Asn Tyr Thr Leu Trp Arg1 5 1028211PRTArtificial SequenceMAGE3.188 peptide 34.0311 282Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys1 5 1028311PRTArtificial Sequencep53.110 peptide 34.0317 283Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys1 5 1028411PRTArtificial Sequencep53.129 peptide 34.0318 284Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys1 5 1028511PRTArtificial Sequencep53.273 peptide 34.0323 285Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg1 5 1028611PRTArtificial Sequencep53.14 peptide 34.0324 286Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys1 5 1028711PRTArtificial Sequencep53.363 peptide 34.0328 287Arg Ala His Ser Ser His Leu Lys Ser Lys Lys1 5 1028811PRTArtificial Sequencep53.122 peptide 34.0329 288Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys1 5 1028911PRTArtificial Sequencep53.154 peptide 34.0330 289Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys1 5 1029011PRTArtificial Sequencep53.376 peptide 34.0332 290Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys1 5 102919PRTArtificial SequenceHer2/neu.846 peptide 40.0107 291Leu Ala Ala Arg Asn Val Leu Val Lys1 52929PRTArtificial SequenceHer2/neu.889 peptide 40.0109 292Met Ala Leu Glu Ser Ile Leu Arg Arg1 529310PRTArtificial SequenceHer2/neu.450 peptide 40.0145 293Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg1 5 1029410PRTArtificial SequenceHer2/neu.727 peptide 40.0147 294Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys1 5 1029510PRTArtificial SequenceHer2/neu.997 peptide 40.0153 295Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg1 5 102968PRTArtificial SequenceCEA.680I8 peptide 40.0013 296Ser Pro Gly Leu Ser Ala Gly Ile1 52978PRTArtificial SequenceHer2/neu.921 peptide 40.0022 297Lys Pro Tyr Asp Gly Ile Pro Ala1 52988PRTArtificial SequenceHer2/neu.921I8 peptide 40.0023 298Lys Pro Tyr Asp Gly Ile Pro Ile1 52998PRTArtificial Sequencep53.63 peptide 40.0050 299Ala Pro Arg Met Pro Glu Ala Ala1

53008PRTArtificial Sequencep53.63I8 peptide 40.0051 300Ala Pro Arg Met Pro Glu Ala Ile1 53018PRTArtificial Sequencep53.76I8 peptide 40.0055 301Ala Pro Ala Ala Pro Thr Pro Ile1 53028PRTArtificial Sequencep53.79I8 peptide 40.0057 302Ala Pro Thr Pro Ala Ala Pro Ile1 53038PRTArtificial Sequencep53.81I8 peptide 40.0059 303Thr Pro Ala Ala Pro Ala Pro Ile1 53048PRTArtificial Sequencep53.84I8 peptide 40.0061 304Ala Pro Ala Pro Ala Pro Ser Ile1 53058PRTArtificial Sequencep53.127 peptide 40.0062 305Ser Pro Ala Leu Asn Lys Met Phe1 53068PRTArtificial Sequencep53.127I8 peptide 40.0063 306Ser Pro Ala Leu Asn Lys Met Ile1 53079PRTArtificial SequenceCEA.3I9 peptide 40.0117 307Ser Pro Ser Ala Pro Pro His Arg Ile1 53089PRTArtificial SequenceCEA.7I9 peptide 40.0119 308Pro Pro His Arg Trp Cys Ile Pro Ile1 53099PRTArtificial SequenceCEA.92 peptide 40.0120 309Gly Pro Ala Tyr Ser Gly Arg Glu Ile1 531011PRTArtificial SequenceHer2/neu.706I10 peptide 40.0156 310Met Pro Asn Gln Ala Gln Met Arg Ile Leu Ile1 5 1031111PRTArtificial SequenceHer2/neu.801I10 peptide 40.0157 311Met Pro Tyr Gly Cys Leu Leu Asp His Val Ile1 5 1031210PRTArtificial SequenceCEA.6 peptide 40.0161 312Ala Pro Pro His Arg Trp Cys Ile Pro Trp1 5 1031310PRTArtificial SequenceCEA.6I10 peptide 40.0162 313Ala Pro Pro His Arg Trp Cys Ile Pro Ile1 5 1031410PRTArtificial SequenceCEA.13 peptide 40.0163 314Ile Pro Trp Gln Arg Leu Leu Leu Thr Ala1 5 1031510PRTArtificial SequenceCEA.13I10 peptide 40.0164 315Ile Pro Trp Gln Arg Leu Leu Leu Thr Ile1 5 1031610PRTArtificial SequenceCEA.58I10 peptide 40.0166 316Leu Pro Gln His Leu Phe Gly Tyr Ser Ile1 5 1031711PRTArtificial SequenceHer2/neu.966 peptide 40.0201 317Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe1 5 1031811PRTArtificial SequenceHer2/neu.966I11 peptide 40.0202 318Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Ile1 5 1031911PRTArtificial SequenceHer2/neu.1149 peptide 40.0205 319Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro Ala1 5 1032011PRTArtificial SequenceHer2/neu.1149I11 peptide 40.0206 320Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro Ile1 5 1032111PRTArtificial SequenceHer2/neu.1155 peptide 40.0207 321Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala1 5 1032211PRTArtificial SequenceHer2/neu.1155I11 peptide 40.0208 322Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ile1 5 1032311PRTArtificial Sequencep53.74 peptide 40.0231 323Ala Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala1 5 1032411PRTArtificial Sequencep53.74I11 peptide 40.0232 324Ala Pro Ala Pro Ala Ala Pro Thr Pro Ala Ile1 5 1032511PRTArtificial Sequencep53.76 peptide 40.0233 325Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala1 5 1032611PRTArtificial Sequencep53.76I11 peptide 40.0234 326Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ile1 5 103278PRTArtificial SequenceCEA.13.I8 peptide 45.0003 327Ile Pro Trp Gln Arg Leu Leu Ile1 53288PRTArtificial SequenceCEA.58.I8 peptide 45.0004 328Leu Pro Gln His Leu Phe Gly Ile1 53298PRTArtificial SequenceCEA.428.I8 peptide 45.0007 329Arg Pro Gly Val Asn Leu Ser Ile1 53308PRTArtificial SequenceCEA.632.I8 peptide 45.0010 330Ile Pro Gln Gln His Thr Gln Ile1 53318PRTArtificial SequenceCEA.646.I8 peptide 45.0011 331Thr Pro Asn Asn Asn Gly Thr Ile1 53328PRTArtificial SequenceHer2/neu.315.I8 peptide 45.0016 332Cys Pro Leu His Asn Gln Glu Ile1 53338PRTArtificial SequenceHer2/neu.336.I8 peptide 45.0017 333Lys Pro Cys Ala Arg Val Cys Ile1 53348PRTArtificial SequenceHer2/neu.415.I8 peptide 45.0019 334Trp Pro Asp Ser Leu Pro Asp Ile1 53358PRTArtificial SequenceHer2/neu.779.I8 peptide 45.0023 335Ser Pro Tyr Val Ser Arg Leu Ile1 53368PRTArtificial SequenceHer2/neu.884.I8 peptide 45.0024 336Val Pro Ile Lys Trp Met Ala Ile1 53378PRTArtificial SequenceHer2/neu.966I8 peptide 45.0026 337Arg Pro Arg Phe Arg Glu Leu Ile1 53388PRTArtificial SequenceHer2/neu.1036.I8 peptide 45.0028 338Ala Pro Gly Ala Gly Gly Met Ile1 53398PRTArtificial SequenceHer2/neu.1174.I8 peptide 45.0031 339Ser Pro Gly Lys Asn Gly Val Ile1 53408PRTArtificial SequenceMAGE3.64.I8 peptide 45.0037 340Ser Pro Gln Gly Ala Ser Ser Ile1 53418PRTArtificial SequenceMAGE3.77.I8 peptide 45.0038 341Tyr Pro Leu Trp Ser Gln Ser Ile1 53428PRTArtificial Sequencep53.33.I8 peptide 45.0044 342Ser Pro Leu Pro Ser Gln Ala Ile1 53438PRTArtificial Sequencep53.66.I8 peptide 45.0046 343Met Pro Glu Ala Ala Pro Pro Ile1 53448PRTArtificial Sequencep53.86.I8 peptide 45.0047 344Ala Pro Ala Pro Ser Trp Pro Ile1 53459PRTArtificial SequenceCEA.155.I9 peptide 45.0051 345Lys Pro Val Glu Asp Lys Asp Ala Ile1 53469PRTArtificial SequenceCEA.632.I9 peptide 45.0054 346Ile Pro Gln Gln His Thr Gln Val Ile1 53479PRTArtificial Sequencep53.70.I9 peptide 45.0060 347Ala Pro Pro Val Ala Pro Ala Pro Ile1 53489PRTArtificial Sequencep53.76.I9 peptide 45.0062 348Ala Pro Ala Ala Pro Thr Pro Ala Ile1 53499PRTArtificial Sequencep53.152.I9 peptide 45.0064 349Pro Pro Gly Thr Arg Val Arg Ala Ile1 53509PRTArtificial Sequencep53.189.I9 peptide 45.0065 350Ala Pro Pro Gln His Leu Ile Arg Ile1 535110PRTArtificial SequenceCEA.632.I10 peptide 45.0071 351Ile Pro Gln Gln His Thr Gln Val Leu Ile1 5 1035210PRTArtificial SequenceCEA.680.I10 peptide 45.0072 352Ser Pro Gly Leu Ser Ala Gly Ala Thr Ile1 5 1035310PRTArtificial SequenceHer2/neu.196.I10 peptide 45.0073 353Ser Pro Met Cys Lys Gly Ser Arg Cys Ile1 5 1035410PRTArtificial SequenceHer2/neu.282.I10 peptide 45.0074 354Met Pro Asn Pro Glu Gly Arg Tyr Thr Ile1 5 1035510PRTArtificial SequenceHer2/neu.315.I10 peptide 45.0076 355Cys Pro Leu His Asn Gln Glu Val Thr Ile1 5 1035610PRTArtificial SequenceHer2/neu.605.I10 peptide 45.0079 356Lys Pro Asp Leu Ser Tyr Met Pro Ile Ile1 5 1035710PRTArtificial SequenceHer2/neu.701.I10 peptide 45.0080 357Thr Pro Ser Gly Ala Met Pro Asn Gln Ile1 5 1035810PRTArtificial SequenceHer2/neu.995.I10 peptide 45.0084 358Gly Pro Ala Ser Pro Leu Asp Ser Thr Ile1 5 1035910PRTArtificial Sequencep53.70.I10 peptide 45.0091 359Ala Pro Pro Val Ala Pro Ala Pro Ala Ile1 5 1036010PRTArtificial Sequencep53.74.I10 peptide 45.0092 360Ala Pro Ala Pro Ala Ala Pro Thr Pro Ile1 5 1036110PRTArtificial Sequencep53.79.I10 peptide 45.0093 361Ala Pro Thr Pro Ala Ala Pro Ala Pro Ile1 5 1036210PRTArtificial Sequencep53.88.I10 peptide 45.0094 362Ala Pro Ser Trp Pro Leu Ser Ser Ser Ile1 5 1036311PRTArtificial SequenceCEA.239.I11 peptide 45.103 363Ala Pro Thr Ile Ser Pro Leu Asn Thr Ser Ile1 5 1036411PRTArtificial SequenceCEA.421.I11 peptide 45.0108 364Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Ile1 5 1036511PRTArtificial SequenceHer2/neu.600.I11 peptide 45.0117 365Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Ile1 5 1036611PRTArtificial SequenceHer2/neu.649.I11 peptide 45.0118 366Ser Pro Leu Thr Ser Ile Ile Ser Ala Val Ile1 5 1036711PRTArtificial SequenceHer2/neu.740.I11 peptide 45.0119 367Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Ile1 5 1036811PRTArtificial SequenceHer2/neu.998.I11 peptide 45.0124 368Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Ile1 5 1036911PRTArtificial SequenceHer2/neu.1157.I11 peptide 45.0128 369Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Ile1 5 1037011PRTArtificial SequenceMAGE2.241.I11 peptide 45.0134 370His Pro Arg Lys Leu Leu Met Gln Asp Leu Ile1 5 1037111PRTArtificial SequenceMAGE2.274.I11 peptide 45.0135 371Gly Pro Arg Ala Leu Ile Glu Thr Ser Tyr Ile1 5 1037211PRTArtificial SequenceMAGE3.274.I11 peptide 45.0139 372Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Ile1 5 1037311PRTArtificial Sequencep53.63.I11 peptide 45.0140 373Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Ile1 5 1037411PRTArtificial Sequencep53.97.I11 peptide 45.0141 374Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Ile1 5 103759PRTArtificial SequenceHBV POL 541 analog peptide 1145.10 375Phe Pro His Cys Leu Ala Phe Ala Tyr1 53769PRTArtificial SequenceHBV POL 541 analog peptide 1145.09 376Phe Pro Val Cys Leu Ala Phe Ser Tyr1 537711PRTArtificial SequenceHBV.pol645 peptide 26.0570 377Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile1 5 1037810PRTArtificial SequenceHLA-A3,2 allele-specific motif 378Xaa Xaa Xaa Xaa Xaa Xaa Ile Xaa Lys Lys1 5 1037910PRTArtificial SequenceHLA-A11 allele-specific motif 379Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Lys Lys1 5 1038010PRTArtificial SequenceHLA-A24.1 allele-specific motif 380Xaa Tyr Xaa Xaa Xaa Val Xaa Xaa Xaa Xaa1 5 10

Claims

1. A composition comprising an immunogenic peptide having an HLA-A2.1 binding motif, which immunogenic peptide is selected from a group consisting of: TABLE-US-00014 AILTFGSFV, HLRDFALAV, ALLGSIALL, ALLATILAA, LLATILAAV, RLFADELAA, YLSKCTLAV, LVYHIYSKI, SMYLCILSA, YLCILSALV, VMFSYLQSL, RLHVYAYSA, GLQTLGAFV, FVEEQMTWA, QMTWAQTVV, IILDTAIFV, AIFVCNAFV, AMGNRLVEA, RLVEACNLL TLSIVTFSL, KLSVLLLEV, LLLEVNRSV, FVSSPTLPV, AMLVLLAEI, QMARLAWEA, VLAIEGIFMA, YLYHPLLSPI, SLFEAMLANV, STTGTNQLGL, LAILTFGSFV, ALLGSIALLA, ALLATILAAV, LLATILAAVA, RLFADELAAL, YLSKCTLAVL, LLVYHIYSKI, SMYLCILSAL, HLHRQMLSFV, LLCGKTGAFL, ETLSIVTFSL, VMCTYSPPL, KLFCQLAKT, ATPPAGSRV, FLQSGTAKSV, CMDRGLTVFV, VLLNWWRWRL, GVFTGLTHI, QMWKCLIRL, IMTCMSADL, ALAAYCLST, VLSGKPAII, FISGIQYLA, YIMTCMSADL, AIASLMAFTA, GLAGAAIGSV, MIGVLVGV, VLPLAYISL, SLGCIFFPL, PLAYISLFL, LMLFYQVWA, NISIYNYFV, NISVYNYFV, FVWTHYYSV, FLTWHRYHL, LTWHRYHLL, MLQEPSFSL, SLPYWNFAT, RLPEPQDVA, VTQCLEVRV, LLHTFTDAV, NMVPFWPPV, AVVGALLLV, AVVAALLLV, LLVAAIFGV, SMDEANQPL, VLPLAYISV, SLGCIFFPV, PLAYISLFV, LLLFQQARV, LMLFYQVWV, LLPSSGPGV, NLSIYNYFV, NLSVYNYFV, FLWTHYYSV, SLKKTFLGV, FLTWHRYHV, MLQEPSFSV, SLPYWNFAV, ALGKNVCDV, SLLISPNSV, SLFSQWRVV, TLGTLCNSV, RLPEPQDVV, VLQCLEVRV, SLNSFRNTV, SLDSFRNTV FLNGTGGQV VLLHTFTDV ALVGALLLV ALVAALLLV, LLVALIFGV, YLIRARRSV, SMDEANQPV, SLGCIFFPLL, GMCCPDLSPV, AACNQKILTV FLTWHRYHLL, SLHNLAHLFL LLLVAAIFGV LLVAAIFGVA, ALIFGTASYL, SMDEANQPLL, LLTDQYQCYA, SLGCIFFPLV, FLMLFYQVWV, ALCDQRVLIV, ALCNQKILTV, FLTWHRYHLV, SLHNLAHLFV, NLAHLFLNGV, NMVPFWPPVV, ILVVAALLLV, LLVALIFGTV, ALIFGTASYV,

SMDEANQPLV, LLTDQYQCYV, LLIQNIIQNDT, IIQNDTGFYTL, TLFNVTRNDTA LTLLSVTRNDV GLYTCQANNSA, ATVGIMIGVLV, GLVPPQHLIRV, GLAPPVHLIRV, GLAPPEHLIRV, ILIGVLVGV, YLIMVKCWMV, LLGRDSFEV, FMYSDFHFI, NMLSTVLGV SLENFRAYV, KMAELVHFV, KLAELVHFV VLIQRNPQV, VLLGVVFGV, SLISAVVGV, YMIMVKBWMI, YLIMVKBWMV, KLWEELSVV, AMBRWGLLV, IJIGVLVGV, ATVGIJIGV, SJPPPGTRV, LVFGIELJEV, ILGFVFTL, KIFGSLAFL, YLQLVFGIEV, MMNDQLMFL, ALVLAGGFFL, WLCAGALVL, MVFELANSI, RMMNDQLMFL, LVLAGGFFL, VLAGGFFLL, LLHETDSAV, LMYSLVHNL, QLMFLERAFI, LMFLERAFI, KLGSGNDFEV, LLQERGVAYI, GMPEGDLVYV, FLDELKAENI, ALFDIESKV, and GLPSIPVHPI.

2. A method of inducing a cytotoxic T cell response against a preselected antigen in a patient expressing an HLA-A2.1 MHC product, the method comprising contacting cytotoxic T cells from the patient with a composition comprising an immunogenic peptide selected from the group consisting of: TABLE-US-00015 AILTFGSFV, HLRDFALAV, ALLGSIALL, ALLATILAA, LLATILAAV, RLFADELAA, YLSKCTLAV, LVYHIYSKI, SMYLCILSA, YLCILSALV, VMFSYLQSL, RLHVYAYSA, GLQTLGAFV, FVEEQMTWA, QMTWAQTVV, IILDTAIFV, AIFVCNAFV, AMGNRLVEA, RLVEACNLL TLSIVTFSL, KLSVLLLEV, LLLEVNRSV, FVSSPTLPV, AMLVLLAEI, QMARLAWEA, VLAIEGIFMA, YLYHPLLSPI, SLFEAMLANV, STTGINQLGL, LAILTFGSFV, ALLGSIALLA, ALLATILAAV, LLATILAAVA, RLFADELAAL, YLSKCTLAVL, LLVYHIYSKI, SMYLCILSAL, HLHRQMLSFV, LLCGKTGAFL, ETLSIVTFSL, VMCTYSPPL, KLFCQLAKT, ATPPAGSRV, FLQSGTAKSV, CMDRGLTVFV, VLLNWWRWRL, GVFTGLTHI, QMWKCLIRL, IMTCMSADL, ALAAYCLST, VLSGKPAII, FISGIQYLA, YIMTCMSADL, AIASLMAFTA, GLAGAAIGSV, MIGVLVGV, VLPLAYISL, SLGCIFFPL, PLAYISLFL, LMLFYQVWA, NISIYNYFV, NISVYNYFV, FVWTHYYSV, FLTWHRYHL, LTWHRYHLL, MLQEPSFSL, SLPYWNFAT, RLPEPQDVA, VTQCLEVRV, LLHTFTDAV, NMVPFWPPV, AVVGALLLV, AVVAALLLV, LLVAAIFGV, SMDEANQPL, VLPLAYISV, SLGCIFFPV, PLAYISLFV, LLLFQQARV, LMLFYQVWV, LLPSSGPGV, NLSIYNYFV, NLSVYNYFV, FLWTHYYSV, SLKKTFLGV, FLTWHRYHV, MLQEPSFSV, SLPYWNFAV, ALGKNVCDV, SLLISPNSV, SLFSQWRVV, TLGTLCNSV, RLPEPQDVV, VLQCLEVRV, SLNSFRNTV, SLDSFRNTV FLNGTGGQV VLLHTFTDV ALVGALLLV ALVAALLLV, LLVALIFGV, YLIRARRSV, SMDEANQPV, SLGCIFFPLL, GMCCPDLSPV, AACNQKILTV FLTWHRYHLL, SLHNLAHLFL LLLVAAIFGV LLVAAIFGVA, ALIFGTASYL, SMDEANQPLL, LLTDQYQCYA, SLGCIFFPLV, FLMLFYQVWV, ALCDQRVLIV, ALCNQKILTV, FLTWHRYHLV, SLHNLAHLFV, NLALHLFLNGV, NMVPFWPPVV, ILVVAALLLV, LLVALIFGTV,

ALIFGTASYV, SMDEANQPLV, LLTDQYQCYV, LLIQNIIQNDT, IIQNDTGFYTL, TLFNVTRNDTA LTLLSVTRNDV GLYTCQANNSA, ATVGIMIGVLV, GLVPPQHLIRV, GLAPPVHLIRV, GLAPPEHLIRV, ILIGVLVGV, YLIMVKCWMV, LLGRDSFEV, FMYSDFHFI, NMLSTVLGV SLENFRAYV, KMAELVHFV, KLAELVHFV VLIQRNPQV, VLLGVVFGV, SLISAVVGV, YMIMVKBWMI, YLIMVKBWMV, KLWEELSVV, AMBRWGLLV, IJIGVLVGV, ATVGIJIGV, SJPPPGTRV, LVFGIELJEV, ILGFVFTL, KIFGSLAFL, YLQLVFGIEV, MMNDQLMFL, ALVLAGGFFL, WLCAGALVL, MVFELANSI, RMMNDQLMFL, LVLAGGFFL, VLAGGFFLL, LLHETDSAV, LMYSLVHNL, QLMFLERAFI, LMFLERAFI, KLGSGNDFEV, LLQERGVAYI, GMPEGDLVYV, FLDELKAENI, ALFDIESKV, and GLPSIPVHPI.

3. A composition comprising an immunogenic peptide selected from a group consisting of: TABLE-US-00016 RVYPELPK, TVSAELPK, TVYAEPPK, TINYTLWR, LVHFLLLK, SVFAHPRK, KVLHHMVK, RVCACPGR, KMFCQLAK, RAHSSHLK, FVSNLATGR, RLQLSNGNK, RINGIPQQK, KIRKYTMRK, LVHFLLLKK, SMLEVFEGK, SSFSTTINK, TSYVKVLHK, VIFSKASEK, GSVVGNWQK, SSLPTTMNK, SVLEVFEGK, SSBMGGMNK, SSCMGGMNK, RTLTLFNVTK, TISPLNTSYK, STTINYTLWK, ASSLPTTMNK, KTYQGSYGFK, VVRRBPHHEK, GLAPPQHLIK, NSSCMGGMNK, SSBMGGMNRK, RVCACPGRDK, KTITVSAELPK, TTITVYAEPPK, PTISPSYTYYR, GLLGDNQVMPK, MVELVHFLLLK, FSTTINYTLWR, GLLGDNQIMPK, RLGFLHSGTAK, ALNKMFCQLAK, RVCACPGRDRR, LSQETFSDLWK, RAHSSHLKSKK, VTCTYSPALNK, GTRVRAMAIYK, STSRHKKLMFK, LAARNVLVK, MALESILRR, ISWLGLRSLR, GSGAFGTVYK, and ASPLDSTFYR.

4. A method of inducing a cytotoxic T cell response against a preselected antigen in a patient, the method comprising contacting cytotoxic T cells from the patient with a composition comprising an immunogenic peptide selected from the group consisting of: TABLE-US-00017 RVYPELPK, TVSAELPK, TVYAEPPK, TINYTLWR, LVHFLLLK, SVFAHPRK, KVLHHMVK, RVCACPGR, KMFCQLAK, RAHSSHLK, FVSNLATGR, RLQLSNGNK, RINGIPQQK, KIRKYTMRK, LVHFLLLKK, SMLEVFEGK, SSFSTTINK, TSYVKVLHK, VIFSKASEK, GSVVGNWQK, SSLPTTMNK, SVLEVFEGK, SSBMGGMNK, SSCMGGMNK, RTLTLFNVTK, TISPLNTSYK, STTINYTLWK, ASSLPTTMNK, KTYQGSYGFK, VVRRBPHHEK, GLAPPQHLIK, NSSCMGGMNK, SSBMGGMNRK, RVCACPGRDK, KTITVSAELPK, TTITVYAEPPK, PTISPSYTYYR, GLLGDNQVMPK, MVELVHFLLLK, FSTTINYTLWR, GLLGDNQIMPK, RLGFLHSGTAK, ALNKMFCQLAK, RVCACPGRDRR, LSQETFSDLWK, RAHSSHLKSKK, VTCTYSPALNK, GTRVRAMAIYK, STSRHKKLMFK, LAARNVLVK, MALESILRR, ISWLGLRSLR, GSGAFGTVYK, and ASPLDSTFYR.

5. A composition comprising an immunogenic peptide selected from a group consisting of the peptides listed in Tables 3, 12 and 13.

6. A method of inducing a cytotoxic T cell response against a preselected antigen in a patient, the method comprising contacting cytotoxic T cells from the patient with a composition comprising an immunogenic peptide selected from the group consisting of the peptides listed in Tables 3, 12 and 13.

7. A composition comprising an immunogenic peptide, wherein said immunogenic peptide consists of a sequence selected from a group consisting of SEQ ID NOs: 1-377.

8. A composition comprising an immunogenic peptide of less than about 15 amino acids in length, wherein said immunogenic peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-377.

9. An isolated peptide less than about 15 amino acids in length, wherein said peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-377.

10. An isolated peptide having a sequence selected from a group consisting of SEQ ID NOs: 1-377.

11. A method of inducing a cytotoxic T cell response against a preselected antigen in a patient, comprising contacting cytotoxic T cells from the patient with the composition of claim 7 or 8.

12. The composition of claim 7, wherein said immunogenic peptide comprises the sequence KLBPVQLWV (SEQ ID NO:9).

13. The composition of claim 8, wherein said immunogenic peptide comprises the sequence KLBPVQLWV (SEQ ID NO:9).

14. The isolated peptide of claim 9, wherein said peptide comprises the sequence KLBPVQLWV (SEQ ID NO:9).

15. The isolated peptide of claim 10, wherein said peptide consists of the sequence KLBPVQLWV (SEQ ID NO:9).

16. The composition of claim 7, wherein said immunogenic peptide comprises the sequence SMPPPGTRV (SEQ ID NO:4).

17. The composition of claim 8, wherein said immunogenic peptide comprises the sequence SMPPPGTRV (SEQ ID NO:4).

18. The isolated peptide of claim 9, wherein said peptide comprises the sequence SMPPPGTRV (SEQ ID NO:4).

19. The isolated peptide of claim 10, wherein said peptide consists of the sequence SMPPPGTRV (SEQ ID NO:4).