Expression vectors encoding epitopes of target-associated antigens and methods for their design

Abstract

The invention disclosed herein is directed to methods of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope. The invention further relates to vectors including a housekeeping epitope expression cassette and also vectors including epitope cluster regions. The housekeeping epitope(s) can be derived from a target-associated antigen. The housekeeping epitope can be liberatable, that is capable of liberation, from a translation product of the cassette by immunoproteasome processing. The invention also relates to a method of activating a T cell comprising contacting a substrate polypeptide with an APC and contacting the APC with a T cell.

Description

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention disclosed herein is directed to methods for the design of epitope-encoding vectors, and epitope cluster regions, for use in compositions, including for example, pharmaceutical compositions capable of inducing an immune response in a subject to whom the compositions are administered. The invention is further directed to the vectors themselves. The epitope(s) expressed using such vectors can stimulate a cellular immune response against a target cell displaying the epitope(s).

[0004] 2. Description of the Related Art

[0005] The immune system can be categorized into two discrete effector arms. The first is innate immunity, which involves numerous cellular components and soluble factors that respond to all infectious challenges. The other is the adaptive immune response, which is customized to respond specifically to precise epitopes from infectious agents. The adaptive immune response is further broken down into two effector arms known as the humoral and cellular immune systems. The humoral arm is centered on the production of antibodies by B-lymphocytes while the cellular arm involves the killer cell activity of cytotoxic T Lymphocytes.

[0006] Cytotoxic T Lymphocytes (CTL) do not recognize epitopes on the infectious agents themselves. Rather, CTL detect fragments of antigens derived from infectious agents that are displayed on the surface of infected cells. As a result antigens are visible to CTL only after they have been processed by the infected cell and thus displayed on the surface of the cell.

[0007] The antigen processing and display system on the surface of cells has been well established. CTL recognize short peptide antigens, which are displayed on the surface in non-covalent association with class I major histocompatibility complex molecules (MHC). These class I peptides are in turn derived from the degradation of cytosolic proteins.

SUMMARY OF THE INVENTION

[0008] The invention disclosed herein relates to the identification of epitope cluster regions that are used to generate pharmaceutical compositions capable of inducing an immune response from a subject to whom the compositions have been administered. One embodiment of the disclosed invention relates to an epitope cluster, the cluster being derived from an antigen associated with a target, the cluster including or encoding at least two sequences having a known or predicted affinity for an MHC receptor peptide binding cleft, wherein the cluster is an incomplete fragment of the antigen.

[0009] In one aspect of the invention, the target is a neoplastic cell.

[0010] In another aspect of the invention, the MHC receptor may be a class I HLA receptor.

[0011] In yet another aspect of the invention, the cluster includes or encodes a polypeptide having a length, wherein the length is at least 10 amino acids. Advantageously, the length of the polypeptide may be less than about 75 amino acids.

[0012] In still another aspect of the invention, there is provided an antigen having a length, wherein the cluster consists of or encodes a polypeptide having a length, wherein the length of the polypeptide is less than about 80% of the length of the antigen. Preferably, the length of the polypeptide is less than about 50% of the length of the antigen. Most preferably, the length of the polypeptide is less than about 20% of the length of the antigen.

[0013] Embodiments of the invention particularly relate to epitope clusters identified in the tumor-associated antigen SSX-2 (SEQ ID NO: 40). One embodiment of the invention relates to an isolated nucleic acid containing a reading frame with a first sequence encoding one or more segments of SSX-2, wherein the whole antigen is not encoded, wherein each segment contains an epitope cluster, and wherein each cluster contains at least two amino acid sequences with a known or predicted affinity for a same MHC receptor peptide binding cleft. In various aspects of the invention the epitope cluster can be amino acids 5-28, 16-28, 41-65, 57-67, 99-114, 167-180, and 167-183 of SSX-2. In other aspects the segments can consist of an epitope cluster; the first sequence can be a fragment of SSX-2; the fragment can consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 90, 80, 60, 50, 25, or 10% of the length of SSX-2; the fragment can consist essentially of an amino acid sequence beginning at amino acid 5, 16, 41, 57, or 99 and ending at amino acid 65, 67, 114, 180, or 183 of SSX-2; or the fragment consists of amino acids 15-183 of SSX-2. Further embodiments of the invention include a second sequence encoding essentially a housekeeping epitope. In one aspect of this embodiment the first and second sequences constitute a single reading frame. In aspects of the invention the reading frame is operably linked to a promoter. Other embodiments of the invention include the polypeptides encoded by the nucleic acid embodiments of the invention and immunogenic compositions containing the nucleic acids or polypeptides of the invention.

[0014] Embodiments of the invention provide expression cassettes, for example, for use in vaccine vectors, which encode one or more embedded housekeeping epitopes, and methods for designing and testing such expression cassettes. Housekeeping epitopes can be liberated from the translation product of such cassettes through proteolytic processing by the immunoproteasome of professional antigen presenting cells (pAPC). In one embodiment of the invention, sequences flanking the housekeeping epitope(s) can be altered to promote cleavage by the immunoproteasome at the desired location(s). Housekeeping epitopes, their uses, and identification are described in U.S. patent application Ser. Nos. 09/560,465 and 09/561,074 entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, and METHOD OF EPITOPE DISCOVERY, respectively; both of which were filed on Apr. 28, 2000, and which are both incorporated herein by reference in their entireties.

[0015] Examples of housekeeping epitopes are disclosed in provisional U.S. patent applications entitled EPITOPE SEQUENCES, No. 60/282,211, filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed Mar. 7, 2002; and 60/409,123, filed on Sep. 5, 2002; and U.S. application Ser. No. 10/117,937, filed on Apr. 4, 2002, which is also entitled EPITOPE SEQUENCES; which are all incorporated herein by reference in their entirety.

[0016] In other embodiments of the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites. As used herein the term “arbitrary sequences” refers to sequences chosen without reference to the native sequence context of the epitope, their ability to promote processing, or immunological function. In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. These arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes. Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated. Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes. The term “authentic” refers to natural protein sequences.

[0017] Epitope clusters and their uses are described in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS, filed on Apr. 28, 2000; Ser. No. 10/005,905, entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, filed on Nov. 7, 2001; and Ser. No. 10/026,066, filed on Dec. 7, 2001, also entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS; all of which are incorporated herein by reference in their entirety.

[0018] Embodiments of the invention can encompass screening the constructs to determine whether the housekeeping epitope is liberated. In constructs containing multiple housekeeping epitopes, embodiments can include screening to determine which epitopes are liberated. In a preferred embodiment, a vector containing an embedded epitope can be used to immunize HLA transgenic mice and the resultant CTL can be tested for their ability to recognize target cells presenting the mature epitope. In another embodiment, target cells expressing immunoproteasome can be transformed with the vector. The target cell may express immunoproteasome either constitutively, because of treatment with interferon (IFN), or through genetic manipulation, for example. CTL that recognize the mature epitope can be tested for their ability to recognize these target cells. In yet another embodiment, the embedded epitope can be prepared as a synthetic peptide. The synthetic peptide then can be subjected to digestion by an immunoproteasome preparation in vitro and the resultant fragments can be analyzed to determine the sites of cleavage. Such polypeptides, recombinant or synthetic, from which embedded epitopes can be successfully liberated, can also be incorporated into immunogenic compositions.

[0019] The invention disclosed herein relates to the identification of a polypeptide suitable for epitope liberation. One embodiment of the invention, relates to a method of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope.

[0020] The epitope can be embedded in the substrate polypeptide, and in some aspects the substrate polypeptide can include more than one epitope, for example. Also, the epitope can be a housekeeping epitope.

[0021] In one aspect, the substrate polypeptide can be a synthetic peptide. Optionally, the substrate polypeptide can be included in a formulation promoting protein transfer. Alternatively, the substrate polypeptide can be a fusion protein. The fusion protein can further include a protein domain possessing protein transfer activity. Further, the contacting step can include immunization with the substrate polypeptide.

[0022] In another aspect, the substrate polypeptide can be encoded by a polynucleotide. The contacting step can include immunization with a vector including the polynucleotide, for example. The immunization can be carried out in an HLA-transgenic mouse or any other suitable animal, for example. Alternatively, the contacting step can include transforming a cell with a vector including the polynucleotide. In some embodiments the transformed cell can be a target cell that is targeted by CTL for purposes of assaying for proper liberation of epitope.

[0023] The proteasome processing can take place intracellularly, either in vitro or in vivo. Further, the proteasome processing can take place in a cell-free system.

[0024] The assaying step can include a technique selected from the group including, but not limited to, mass spectrometry, N-terminal pool sequencing, HPLC, and the like. Also, the assaying step can include a T cell target recognition assay. The T cell target recognition assay can be selected from the group including, but not limited to, a cytolytic activity assay, a chromium release assay, a cytokine assay, an ELISPOT assay, tetramer analysis, and the like.

[0025] In still another aspect, the amino acid sequence of the substrate polypeptide including the epitope can be arbitrary. Also, the substrate polypeptide in which the epitope is embedded can be derived from an authentic sequence of a target-associated antigen. Further, the substrate polypeptide in which the epitope is embedded can be conformed to a preferred immune proteasome cleavage site flanking sequence.

[0026] In another aspect, the substrate polypeptide can include an array of additional epitopes. Members of the array can be arranged head-to-tail, for example. The array can include more than one housekeeping epitope. The more than one housekeeping epitope can include copies of the same epitope. The array can include a housekeeping and an immune epitope, or alternating housekeeping and immune epitopes, for example. Also, the array can include a housekeeping epitope positioned between two immune epitopes in an epitope battery. The array can include multiple epitope batteries, so that there are two immune epitopes between each housekeeping epitope in the interior of the array. Optionally, at least one of the epitopes can be truncated distally to its junction with an adjacent epitope. The truncated epitopes can be immune epitopes, for example. The truncated epitopes can have lengths selected from the group including, but not limited to, 9, 8, 7, 6, 5, 4 amino acids, and the like.

[0027] In still another aspect, the substrate polypeptide can include an array of epitopes and epitope clusters. Members of the array can be arranged head-to-tail, for example.

[0028] In yet another aspect, the proteasome can be an immune proteasome.

[0029] Another embodiment of the disclosed invention relates to vectors including a housekeeping epitope expression cassette. The housekeeping epitope(s) can be derived from a target-associated antigen, and the housekeeping epitope can be liberatable, that is capable of liberation, from a translation product of the cassette by immunoproteasome processing.

[0030] In one aspect of the invention the expression cassette can encode an array of two or more epitopes or at least one epitope and at least one epitope cluster. The members of the array can be arranged head-to-tail, for example. Also, the members of the array can be arranged head-to-tail separated by spacing sequences, for example. Further, the array can include a plurality of housekeeping epitopes. The plurality of housekeeping epitopes can include more than one copy of the same epitope or single copies of distinct epitopes, for example. The array can include at least one housekeeping epitope and at least one immune epitope. Also, the array can include alternating housekeeping and immune epitopes. Further, the array includes a housekeeping epitope sandwiched between two immune epitopes so that there are two immune epitopes between each housekeeping epitope in the interior of the array. The immune epitopes can be truncated distally to their junction with the adjacent housekeeping epitope.

[0031] In another aspect, the expression cassette further encodes an authentic protein sequence, or segment thereof, including at least one immune epitope. Optionally, the segment can include at least one epitope cluster. The housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope can be encoded in a single reading frame or transcribed as a single mRNA species, for example. Also, the housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope may not be transcribed as a single mRNA species.

[0032] In yet another aspect, the vector can include a DNA molecule or an RNA molecule. The vector can encode, for example, SEQ ID NO. 4, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 33, and the like. Also, the vector can include SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 30, SEQ ID NO. 34, and the like. Also, the vector can encode SEQ ID NO. 5 or SEQ ID NO. 18, for example.

[0033] In still another aspect, the target-associated antigen can be an antigen derived from or associated with a tumor or an intracellular parasite, and the intracellular parasite can be, for example, a virus, a bacterium, a protozoan, or the like.

[0034] Another embodiment of the invention relates to vectors including a housekeeping epitope identified according to any of the methods disclosed herein, claimed or otherwise. For example, embodiments can relate to vector encoding a substrate polypeptide that includes a housekeeping epitope by any of the methods described herein.

[0035] In one aspect, the housekeeping epitope can be liberated from the cassette translation product by immune proteasome processing

[0036] Another embodiment of the disclosed invention relates to methods of activating a T cell. The methods can include, for example, the steps of contacting a vector including a housekeeping epitope expression cassette with an APC. The housekeeping epitope can be derived from a target-associated antigen, for example, and the housekeeping epitope can be liberatable from a translation product of the cassette by immunoproteasome processing. The methods can further include contacting the APC with a T cell. The contacting of the vector with the APC can occur in vitro or in vivo.

[0037] Another embodiment of the disclosed invention relates to a substrate polypeptide including a housekeeping epitope wherein the housekeeping epitope can be liberated by immunoproteasome processing in a pAPC.

[0038] Another embodiment of the disclosed invention relates to a method of activating a T cell comprising contacting a substrate polypeptide including a housekeeping epitope with an APC wherein the housekeeping epitope can be liberated by immunoproteasome processing and contacting the APC with a T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 depicts the sequence of Melan-A (SEQ ID NO: 2), showing clustering of class I HLA epitopes.

[0040] FIG. 2 depicts the sequence of SSX-2 (SEQ ID NO: 40), showing clustering of class I HLA epitopes.

[0041] FIG. 3 depicts the sequence of NY-ESO (SEQ ID NO: 11), showing clustering of class I HLA epitopes.

[0042] FIG. 4. An illustrative drawing depicting pMA2M.

[0043] FIG. 5. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by mock immunized CTL.

[0044] FIG. 6. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM3 immunized CTL.

[0045] FIG. 7. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM2 immunized CTL.

[0046] FIG. 8. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM1 immunized CTL.

[0047] FIG. 9. Illustrates a sequence of SEQ ID NO. 22 from which the NY-ESO-1157-165 epitope is liberated by immunoproteasomal processing.

[0048] FIG. 10. Shows the differential processing by immunoproteasome and housekeeping proteasome of the SLLMWITQC epitope (SEQ ID NO. 12) in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome.

[0049] FIG. 11. 8A: Shows the results of the human immunoproteasome digest of SEQ ID NO. 31. 8B: Shows the comparative results of mouse versus human immunoproteasome digestion of SEQ ID NO. 31.

[0050] FIG. 12. Shows the differential processing of SSX-231-68 by housekeeping and immunoproteasome.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0051] Definitions

[0052] Unless otherwise clear from the context of the use of a term herein, the following listed terms shall generally have the indicated meanings for purposes of this description.

[0053] PROFESSIONAL ANTIGEN-PRESENTING CELL (PAPC)—a cell that possesses T cell costimulatory molecules and is able to induce a T cell response. Well characterized pAPCs include dendritic cells, B cells, and macrophages.

[0054] PERIPHERAL CELL—a cell that is not a pAPC.

[0055] HOUSEKEEPING PROTEASOME—a proteasome normally active in peripheral cells, and generally not present or not strongly active in pAPCs.

[0056] IMMUNOPROTEASOME—a proteasome normally active in pAPCs; the immunoproteasome is also active in some peripheral cells in infected tissues or following exposure to interferon.

[0057] EPITOPE—a molecule or substance capable of stimulating an immune response. In preferred embodiments, epitopes according to this definition include but are not necessarily limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein the polypeptide is capable of stimulating an immune response. In other preferred embodiments, epitopes according to this definition include but are not necessarily limited to peptides presented on the surface of cells, the peptides being non-covalently bound to the binding cleft of class I MHC, such that they can interact with T cell receptors (TCR). Epitopes presented by class I MHC may be in immature or mature form. “Mature” refers to an MHC epitope in distinction to any precursor (“immature”) that may include or consist essentially of a housekeeping epitope, but also includes other sequences in a primary translation product that are removed by processing, including without limitation, alone or in any combination, proteasomal digestion, N-terminal trimming, or the action of exogenous enzymatic activities. Thus, a mature epitope may be provided embedded in a somewhat longer polypeptide, the immunological potential of which is due, at least in part, to the embedded epitope; or in its ultimate form that can bind in the MHC binding cleft to be recognized by TCR, respectively.

[0058] MHC EPITOPE—a polypeptide having a known or predicted binding affinity for a mammalian class I or class II major histocompatibility complex (MHC) molecule.

[0059] HOUSEKEEPING EPITOPE—In a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which housekeeping proteasomes are predominantly active. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide containing a housekeeping epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid that encodes a housekeeping epitope according to the foregoing definitions. Exemplary housekeeping epitopes are provide in U.S. application Ser. No. 10/117,937, filed on Apr. 4, 2002; and U.S. Provisional Application No. 60/282,211, filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed Mar. 7, 2002; and 60/409,123, filed on Sep. 5, 2002; all of which are entitled EPITOPE SEQUENCES, and all of which above were incorporated herein by reference in their entireties.

[0060] IMMUNE EPITOPE—In a preferred embodiment, an immune epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which immunoproteasomes are predominantly active. In another preferred embodiment, an immune epitope is defined as a polypeptide containing an immune epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide including an epitope cluster sequence, having at least two polypeptide sequences having a known or predicted affinity for a class I MHC. In yet another preferred embodiment, an immune epitope is defined as a nucleic acid that encodes an immune epitope according to any of the foregoing definitions.

[0061] TARGET CELL—a cell to be targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include but are not necessarily limited to: a neoplastic cell and a cell harboring an intracellular parasite, such as, for example, a virus, a bacterium, or a protozoan. Target cells can also include cells that are targeted by CTL as a part of assays to determine or confirm proper epitope liberation and processing by a cell expressing immunoproteasome, to determine T cell specificity or immunogenicity for a desired epitope. Such cells may be transfored to express the substrate or liberation sequence, or the cells can simply be pulsed with peptide/epitope.

[0062] TARGET-ASSOCIATED ANTIGEN (TAA)—a protein or polypeptide present in a target cell.

[0063] TUMOR-ASSOCIATED ANTIGENS (TuAA)—a TAA, wherein the target cell is a neoplastic cell.

[0064] HLA EPITOPE—a polypeptide having a known or predicted binding affinity for a human class I or class II HLA complex molecule.

[0065] ANTIBODY—a natural immunoglobulin (Ig), poly- or monoclonal, or any molecule composed in whole or in part of an Ig binding domain, whether derived biochemically or by use of recombinant DNA. Examples include inter alia, F(ab), single chain Fv, and Ig variable region-phage coat protein fusions.

[0066] ENCODE—an open-ended term such that a nucleic acid encoding a particular amino acid sequence can consist of codons specifying that (poly)peptide, but can also comprise additional sequences either translatable, or for the control of transcription, translation, or replication, or to facilitate manipulation of some host nucleic acid construct.

[0067] SUBSTANTIAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of the sequence. Nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding regions. Amino acid sequences differing only by conservative substitution or minor length variations are substantially similar. Additionally, amino acid sequences comprising housekeeping epitopes that differ in the number of N-terminal flanking residues, or immune epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves also substantially similar.

[0068] FUNCTIONAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of a biological or biochemical property, although the sequences may not be substantially similar. For example, two nucleic acids can be useful as hybridization probes for the same sequence but encode differing amino acid sequences. Two peptides that induce cross-reactive CTL responses are functionally similar even if they differ by non-conservative amino acid substitutions (and thus do not meet the substantial similarity definition). Pairs of antibodies, or TCRs, that recognize the same epitope can be functionally similar to each other despite whatever structural differences exist. In testing for functional similarity of immunogenicity one would generally immunize with the “altered” antigen and test the ability of the elicited response (Ab, CTL, cytokine production, etc.) to recognize the target antigen. Accordingly, two sequences may be designed to differ in certain respects while retaining the same function. Such designed sequence variants are among the embodiments of the present invention.

[0069] EXPRESSION CASSETTE—a polynucleotide sequence encoding a polypeptide, operably linked to a promoter and other transcription and translation control elements, including but not limited to enhancers, termination codons, internal ribosome entry sites, and polyadenylation sites. The cassette can also include sequences that facilitate moving it from one host molecule to another.

[0070] EMBEDDED EPITOPE—an epitope contained within a longer polypeptide, also can include an epitope in which either the N-terminus or the C-terminus is embedded such that the epitope is not in an interior position.

[0071] MATURE EPITOPE—a peptide with no additional sequence beyond that present when the epitope is bound in the MHC peptide-binding cleft.

[0072] EPITOPE CLUSTER—a polypeptide, or a nucleic acid sequence encoding it, that is a segment of a native protein sequence comprising two or more known or predicted epitopes with binding affinity for a shared MHC restriction element, wherein the density of epitopes within the cluster is greater than the density of all known or predicted epitopes with binding affinity for the shared MHC restriction element within the complete protein sequence, and as disclosed in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS.

[0073] SUBSTRATE OR LIBERATION SEQUENCE—a designed or engineered sequence comprising or encoding a housekeeping epitope (according to the first of the definitions offered above) embedded in a larger sequence that provides a context allowing the housekeeping epitope to be liberated by immunoproteasomal processing, directly or in combination with N-terminal trimming or other processes.

[0074] Epitope Clusters

[0075] Embodiments of the invention disclosed herein provide epitope cluster regions (ECRs) for use in vaccines and in vaccine design and epitope discovery. Specifically, embodiments of the invention relate to identifying epitope clusters for use in generating immunologically active compositions directed against target cell populations, and for use in the discovery of discrete housekeeping epitopes and immune epitopes. In many cases, numerous putative class I MHC epitopes may exist in a single target-associated antigen (TAA). Such putative epitopes are often found in clusters (ECRs), MHC epitopes distributed at a relatively high density within certain regions in the amino acid sequence of the parent TAA. Since these ECRs include multiple putative epitopes with potential useful biological activity in inducing an immune response, they represent an excellent material for in vitro or in vivo analysis to identify particularly useful epitopes for vaccine design. And, since the epitope clusters can themselves be processed inside a cell to produce active MHC epitopes, the clusters can be used directly in vaccines, with one or more putative epitopes in the cluster actually being processed into an active MHC epitope.

[0076] The use of ECRs in vaccines offers important technological advances in the manufacture of recombinant vaccines, and further offers crucial advantages in safety over existing nucleic acid vaccines that encode whole protein sequences. Recombinant vaccines generally rely on expensive and technically challenging production of whole proteins in microbial fermentors. ECRs offer the option of using chemically synthesized polypeptides, greatly simplifying development and manufacture, and obviating a variety of safety concerns. Similarly, the ability to use nucleic acid sequences encoding ECRs, which are typically relatively short regions of an entire sequence, allows the use of synthetic oligonucleotide chemistry processes in the development and manipulation of nucleic acid based vaccines, rather than the more expensive, time consuming, and potentially difficult molecular biology procedures involved with using whole gene sequences.

[0077] Since an ECR is encoded by a nucleic acid sequence that is relatively short compared to that which encodes the whole protein from which the ECR is found, this can greatly improve the safety of nucleic acid vaccines. An important issue in the field of nucleic acid vaccines is the fact that the extent of sequence homology of the vaccine with sequences in the animal to which it is administered determines the probability of integration of the vaccine sequence into the genome of the animal. A fundamental safety concern of nucleic acid vaccines is their potential to integrate into genomic sequences, which can cause deregulation of gene expression and tumor transformation. The Food and Drug Administration has advised that nucleic acid and recombinant vaccines should contain as little sequence homology with human sequences as possible. In the case of vaccines delivering tumor-associated antigens, it is inevitable that the vaccines contain nucleic acid sequences that are homologous to those which encode proteins that are expressed in the tumor cells of patients. It is, however, highly desirable to limit the extent of those sequences to that which is minimally essential to facilitate the expression of epitopes for inducing therapeutic immune responses. The use of ECRs thus offers the dual benefit of providing a minimal region of homology, while incorporating multiple epitopes that have potential therapeutic value.

[0078] Note that the following discussion sets forth the inventors' understanding of the operation of the invention. However, it is not intended that this discussion limit the patent to any particular theory of operation not set forth in the claims.

[0079] ECRs are Processed into MHC-Binding Epitopes in pAPCs

[0080] The immune system constantly surveys the body for the presence of foreign antigens, in part through the activity of pAPCs. The pAPCs endocytose matter found in the extracellular milieu, process that matter from a polypeptide form into shorter oligopeptides of about 3 to 23 amino acids in length, and display some of the resulting peptides to T cells via the MHC complex of the pAPCs. For example, a tumor cell upon lysis releases its cellular contents, including various proteins, into the extracellular milieu. Those released proteins can be endocytosed by pAPCs and processed into discrete peptides that are then displayed on the surface of the pAPCs via the MHC. By this mechanism, it is not the entire target protein that is presented on the surface of the pAPCs, but rather only one or more discrete fragments of that protein that are presented as MHC-binding epitopes. If a presented epitope is recognized by a T cell, that T cell is activated and an immune response results.

[0081] Similarly, the scavenger receptors on pAPC can take-up naked nucleic acid sequences or recombinant organisms containing target nucleic acid sequences. Uptake of the nucleic acid sequences into the pAPC subsequently results in the expression of the encoded products. As above, when an ECR can be processed into one or more useful epitopes, these products can be presented as MHC epitopes for recognition by T cells.

[0082] MHC-binding epitopes are often distributed unevenly throughout a protein sequence in clusters. Embodiments of the invention are directed to identifying epitope cluster regions (ECRs) in a particular region of a target protein. Candidate ECRs are likely to be natural substrates for various proteolytic enzymes and are likely to be processed into one or more epitopes for MHC display on the surface of an pAPC. In contrast to more traditional vaccines that deliver whole proteins or biological agents, ECRs can be administered as vaccines, resulting in a high probability that at least one epitope will be presented on MHC without requiring the use of a full length sequence.

[0083] The Use of ECRs in Identifying Discrete MHC-Binding Epitopes

[0084] Identifying putative MHC epitopes for use in vaccines often includes the use of available predictive algorithms that analyze the sequences of proteins or genes to predict binding affinity of peptide fragments for MHC. These algorithms rank putative epitopes according to predicted affinity or other characteristics associated with MHC binding. Exemplary algorithms for this kind of analysis include the Rammensee and NIH (Parker) algorithms. However, identifying epitopes that are naturally present on the surface of cells from among putative epitopes predicted using these algorithms has proven to be a difficult and laborious process. The use of ECRs in an epitope identification process can enormously simplify the task of identifying discrete MHC binding epitopes.

[0085] In a preferred embodiment, ECR polypeptides are synthesized on an automated peptide synthesizer and these ECRs are then subjected to in vitro digests using proteolytic enzymes involved in processing proteins for presentation of the epitopes. Mass spectrometry and/or analytical HPLC are then used to identify the digest products and in vitro MHC binding studies are used to assess the ability of these products to actually bind to MHC. Once epitopes contained in ECRs have been shown to bind MHC, they can be incorporated into vaccines or used as diagnostics, either as discrete epitopes or in the context of ECRs.

[0086] The use of an ECR (which because of its relatively short sequence can be produced through chemical synthesis) in this preferred embodiment is a significant improvement over what otherwise would require the use of whole protein. This is because whole proteins have to be produced using recombinant expression vector systems and/or complex purification procedures. The simplicity of using chemically synthesized ECRs enables the analysis and identification of large numbers of epitopes, while greatly reducing the time and expense of the process as compared to other currently used methods. The use of a defined ECR also greatly simplifies mass spectrum analysis of the digest, since the products of an ECR digest are a small fraction of the digest products of a whole protein.

[0087] In another embodiment, nucleic acid sequences encoding ECRs are used to express the polypeptides in cells or cell lines to assess which epitopes are presented on the surface. A variety of means can be used to detect the epitope on the surface. Preferred embodiments involve the lysis of the cells and affinity purification of the MHC, and subsequent elution and analysis of peptides from the MHC; or elution of epitopes from intact cells; (Falk, K. et al. Nature 351:290, 1991, and U.S. Pat. No. 5,989,565, respectively, both of which references are incorporated herein by reference in their entirety). A sensitive method for analyzing peptides eluted in this way from the MHC employs capillary or nanocapillary HPLC ESI mass spectrometry and on-line sequencing.

[0088] Target-Associated Antigens that Contain ECRs

[0089] TAAs from which ECRs may be defined include those from TuAAs, including oncofetal, cancer-testis, deregulated genes, fusion genes from errant translocations, differentiation antigens, embryonic antigens, cell cycle proteins, mutated tumor suppressor genes, and overexpressed gene products, including oncogenes. In addition, ECRs may be derived from virus gene products, particularly those associated with viruses that cause chronic diseases or are oncogenic, such as the herpes viruses, human papilloma viruses, human immunodeficiency virus, and human T cell leukemia virus. Also ECRs may be derived from gene products of parasitic organisms, such as Trypanosoma, Leishmania, and other intracellular or parasitic organisms.

[0090] Some of these TuAA include α-fetoprotein, carcinoembryonic antigen (CEA), esophageal cancer derived NY-ESO-1, and SSX genes, SCP-1, PRAME, MART-1/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR1 and viral antigens, EBNA1, EBNA2, HPV-E6, -E7; prostate specific antigen (PSA), prostate stem cell antigen (PSCA), MAAT-1, GP-100, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, p185erbB-2, p185erbB-3, c-met, nm-23H1, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p15, and p16.

[0091] Numerous other TAAs are also contemplated for both pathogens and tumors. In terms of TuAAs, a variety of methods are available and well known in the art to identify genes and gene products that are differentially expressed in neoplastic cells as compared to normal cells. Examples of these techniques include differential hybridization, including the use of microarrays; subtractive hybridization cloning; differential display, either at the level of mRNA or protein expression; EST sequencing; and SAGE (sequential analysis of gene expression). These nucleic acid techniques have been reviewed by Carulli, J. P. et al., J. Cellular Biochem Suppl. 30/31:286-296, 1998 (hereby incorporated by reference). Differential display of proteins involves, for example, comparison of two-dimensional polyacrylamide gel electrophoresis of cell lysates from tumor and normal tissue, location of protein spots unique or overexpressed in the tumor, recovery of the protein from the gel, and identification of the protein using traditional biochemical- or mass spectrometry-based sequencing. An additional technique for identification of TAAs is the Serex technique, discussed in Türeci, Ö., Sahin, U., and Pfreundschuh, M., “Serological analysis of human tumor antigens: molecular definition and implications”, Molecular Medicine Today, 3:342, 1997, and hereby incorporated by reference.

[0092] Use of these and other methods provides one of skill in the art the techniques necessary to identify genes and gene products contained within a target cell that may be used as potential candidate proteins for generating the epitopes of the invention disclosed. However, it is not necessary, in practicing the invention, to identify a novel TuAA or TAA. Rather, embodiments of the invention make it possible to identify ECRs from any relevant protein sequence, whether the sequence is already known or is new.

[0093] Protein Sequence Analysis to Identify Epitope Clusters

[0094] In preferred embodiments of the invention, identification of ECRs involves two main steps: (1) identifying good putative epitopes; and (2) defining the limits of any clusters in which these putative epitopes are located. There are various preferred embodiments of each of these two steps, and a selected embodiment for the first step can be freely combined with a selected embodiment for the second step. The methods and embodiments that are disclosed herein for each of these steps are merely exemplary, and are not intended to limit the scope of the invention in any way. Persons of skill in the art will appreciate the specific tools that can be applied to the analysis of a specific TAA, and such analysis can be conducted in numerous ways in accordance with the invention.

[0095] Preferred embodiments for identifying good putative epitopes include the use of any available predictive algorithm that analyzes the sequences of proteins or genes to predict binding affinity of peptide fragments for MHC, or to rank putative epitopes according to predicted affinity or other characteristics associated with MHC binding. As described above, available exemplary algorithms for this kind of analysis include the Rammensee and NIH (Parker) algorithms. Likewise, good putative epitopes can be identified by direct or indirect assays of MHC binding. To choose “good” putative epitopes, it is necessary to set a cutoff point in terms of the score reported by the prediction software or in terms of the assayed binding affinity. In some embodiments, such a cutoff is absolute. For example, the cutoff can be based on the measured or predicted half time of dissociation between an epitope and a selected MHC allele. In such cases, embodiments of the cutoff can be any half time of dissociation longer than, for example, 0.5 minutes; in a preferred embodiment longer than 2.5 minutes; in a more preferred embodiment longer than 5 minutes; and in a highly stringent embodiment can be longer than 10, or 20, or 25 minutes. In these embodiments, the good putative epitopes are those that are predicted or identified to have good MHC binding characteristics, defined as being on the desirable side of the designated cutoff point. Likewise, the cutoff can be based on the measured or predicted binding affinity between an epitope and a selected MHC allele. Additionally, the absolute cutoff can be simply a selected number of putative epitopes.

[0096] In other embodiments, the cutoff is relative. For example, a selected percentage of the total number of putative epitopes can be used to establish the cutoff for defining a candidate sequence as a good putative epitope. Again the properties for ranking the epitopes are derived from measured or predicted MHC binding; the property used for such a determination can be any that is relevant to or indicative of binding. In preferred embodiments, identification of good putative epitopes can combine multiple methods of ranking candidate sequences. In such embodiments, the good epitopes are typically those that either represent a consensus of the good epitopes based on different methods and parameters, or that are particularly highly ranked by at least one of the methods.

[0097] When several good putative epitopes have been identified, their positions relative to each other can be analyzed to determine the optimal clusters for use in vaccines or in vaccine design. This analysis is based on the density of a selected epitope characteristic within the sequence of the TAA. The regions with the highest density of the characteristic, or with a density above a certain selected cutoff, are designated as ECRs. Various embodiments of the invention employ different characteristics for the density analysis. For example, one preferred characteristic is simply the presence of any good putative epitope (as defined by any appropriate method). In this embodiment, all putative epitopes above the cutoff are treated equally in the density analysis, and the best clusters are those with the highest density of good putative epitopes per amino acid residue. In another embodiment, the preferred characteristic is based on the parameter(s) previously used to score or rank the putative epitopes. In this embodiment, a putative epitope with a score that is twice as high as another putative epitope is doubly weighted in the density analysis, relative to the other putative epitope. Still other embodiments take the score or rank into account, but on a diminished scale, such as, for example, by using the log or the square root of the score to give more weight to some putative epitopes than to others in the density analysis.

[0098] Depending on the length of the TAA to be analyzed, the number of possible candidate epitopes, the number of good putative epitopes, the variability of the scoring of the good putative epitopes, and other factors that become evident in any given analysis, the various embodiments of the invention can be used alone or in combination to identify those ECRs that are most useful for a given application. Iterative or parallel analyses employing multiple approaches can be beneficial in many cases. ECRs are tools for increased efficiency of identifying true MHC epitopes, and for efficient “packaging” of MHC epitopes into vaccines. Accordingly, any of the embodiments described herein, or other embodiments that are evident to those of skill in the art based on this disclosure, are useful in enhancing the efficiency of these efforts by using ECRs instead of using complete TAAs in vaccines and vaccine design.

[0099] Since many or most TAAs have regions with low density of predicted MHC epitopes, using ECRs provides a valuable methodology that avoids the inefficiencies of including regions of low epitope density in vaccines and in epitope identification protocols. Thus, useful ECRs can also be defined as any portion of a TAA that is not the whole TAA, wherein the portion has a higher density of putative epitopes than the whole TAA, or than any regions of the TAA that have a particularly low density of putative epitopes. In this aspect of the invention, therefore, an ECR can be any fragment of a TAA with elevated epitope density. In some embodiments, an ECR can include a region up to about 80% of the length of the TAA. In a preferred embodiment, an ECR can include a region up to about 50% of the length of the TAA. In a more preferred embodiment, an ECR can include a region up to about 30% of the length of the TAA. And in a most preferred embodiment, an ECR can include a region of between 5 and 15% of the length of the TAA.

[0100] In another aspect of the invention, the ECR can be defined in terms of its absolute length. Accordingly, by this definition, the minimal cluster for 9-mer epitopes includes 10 amino acid residues and has two overlapping 9-mers with 8 amino acids in common. In a preferred embodiment, the cluster is between about 15 and 75 amino acids in length. In a more preferred embodiment, the cluster is between about 20 and 60 amino acids in length. In a most preferred embodiment, the cluster is between about 30 and 40 amino acids in length.

[0101] In practice, as described above, ECR identification can employ a simple density function such as the number of epitopes divided by the number of amino acids spanned by the those epitopes. It is not necessarily required that the epitopes overlap, but the value for a single epitope is not significant. If only a single value for a percentage cutoff is used and an absolute cutoff in the epitope prediction is not used, it is possible to set a single threshold at this step to define a cluster. However, using both an absolute cutoff and carrying out the first step using different percentage cutoffs, can produce variations in the global density of candidate epitopes. Such variations can require further accounting or manipulation. For example, an overlap of 2 epitopes is more significant if only 3 candidate epitopes were considered, than if 30 candidates were considered for any particular length protein. To take this feature into consideration, the weight given to a particular cluster can further be divided by the fraction of possible peptides actually being considered, in order to increase the significance of the calculation. This scales the result to the average density of predicted epitopes in the parent protein.

[0102] Similarly, some embodiments base the scoring of good putative epitopes on the average number of peptides considered per amino acid in the protein. The resulting ratio represents the factor by which the density of predicted epitopes in the putative cluster differs from the average density in the protein. Accordingly, an ECR is defined in one embodiment as any region containing two or more predicted epitopes for which this ratio exceeds 2, that is, any region with twice the average density of epitopes. In other embodiments, the region is defined as an ECR if the ratio exceeds 1.5, 3, 4, or 5, or more.

[0103] Considering the average number of peptides per amino acid in a target protein to calculate the presence of an ECR highlights densely populated ECRs without regard to the score/affinity of the individual constituents. This is most appropriate for use of score-based cutoffs. However, an ECR with only a small number of highly ranked candidates can be of more biological significance than a cluster with several densely packed but lower ranking candidates, particularly if only a small percentage of the total number of candidate peptides were designated as good putative epitopes. Thus in some embodiments it is appropriate to take into consideration the scores of the individual peptides. This is most readily accomplished by substituting the sum of the scores of the peptides in the putative cluster for the number of peptides in the putative cluster in the calculation described above.

[0104] This sum of scores method is more sensitive to sparsely populated clusters containing high scoring epitopes. Because the wide range of scores (i.e. half times of dissociation) produced by the BIMAS-NIH/Parker algorithm can lead to a single high scoring peptide dwarfing the contribution of other potential epitopes, the log of the score rather than the score itself is preferably used in this procedure.

[0105] Various other calculations can be devised under one or another condition. Generally speaking, the epitope density function is constructed so that it is proportional to the number of predicted epitopes, their scores, their ranks, and the like, within the putative cluster, and inversely proportional to the number of amino acids or fraction of protein contained within that putative cluster. Alternatively, the function can be evaluated for a window of a selected number of contiguous amino acids. In either case the function is also evaluated for all predicted epitopes in the whole protein. If the ratio of values for the putative cluster (or window) and the whole protein is greater than, for example, 1.5, 2, 3, 4, 5, or more, an ECR is defined.

[0106] Analysis of Target Gene Products for MHC Binding

[0107] Once a TAA has been identified, the protein sequence can be used to identify putative epitopes with known or predicted affinity to the MHC peptide binding cleft. Tests of peptide fragments can be conducted in vitro, or using the sequence can be computer analyzed to determine MHC receptor binding of the peptide fragments. In one embodiment of the invention, peptide fragments based on the amino acid sequence of the target protein are analyzed for their predicted ability to bind to the MHC peptide binding cleft. Examples of suitable computer algorithms for this purpose include that found at the world wide web page of Hans-Georg Rammensee, Jutta Bachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An Internet Database for MHC Ligands and Peptide Motifs (access via hypertext transfer protocol: //134.2.96.221/scripts/hlaserver.dll/EpPredict.htm). Results obtained from this method are discussed in Rammensee, et al., “MHC Ligands and Peptide Motifs,” Landes Bioscience Austin, Tex., 224-227, 1997, which is hereby incorporated by reference in its entirety. Another site of interest is found at hypertext transfer protocol: //bimas.dcrt.nih.gov/molbio/hla_bind, which also contains a suitable algorithm. The methods of this web site are discussed in Parker, et al., “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains,” J. Immunol. 152:163-175, which is hereby incorporated by reference in its entirety.

[0108] As an alternative to predictive algorithms, a number of standard in vitro receptor binding affinity assays are available to identify peptides having an affinity for a particular allele of MHC. Accordingly, by the method of this aspect of the invention, the initial population of peptide fragments can be narrowed to include only putative epitopes having an actual or predicted affinity for the selected allele of MHC. Selected common alleles of MHC I, and their approximate frequencies, are reported in the tables below.

TABLE 1
Estimated gene frequencies of HLA-A antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
A1	15.1843	0.0489	5.7256	0.0771	4.4818	0.0846	7.4007	0.0978	12.0316	0.2533
A2	28.6535	0.0619	18.8849	0.1317	24.6352	0.1794	28.1198	0.1700	29.3408	0.3585
A3	13.3890	0.0463	8.4406	0.0925	2.6454	0.0655	8.0789	0.1019	11.0293	0.2437
A28	4.4652	0.0280	9.9269	0.0997	1.7657	0.0537	8.9446	0.1067	5.3856	0.1750
A36	0.0221	0.0020	1.8836	0.0448	0.0148	0.0049	0.1584	0.0148	0.1545	0.0303
A23	1.8287	0.0181	10.2086	0.1010	0.3256	0.0231	2.9269	0.0628	1.9903	0.1080
A24	9.3251	0.0395	2.9668	0.0560	22.0391	0.1722	13.2610	0.1271	12.6613	0.2590
A9 unsplit	0.0809	0.0038	0.0367	0.0063	0.0858	0.0119	0.0537	0.0086	0.0356	0.0145
A9 total	11.2347	0.0429	13.2121	0.1128	22.4505	0.1733	16.2416	0.1382	14.6872	0.2756
A25	2.1157	0.0195	0.4329	0.0216	0.0990	0.0128	1.1937	0.0404	1.4520	0.0924
A26	3.8795	0.0262	2.8284	0.0547	4.6628	0.0862	3.2612	0.0662	2.4292	0.1191
A34	0.1508	0.0052	3.5228	0.0610	1.3529	0.0470	0.4928	0.0260	0.3150	0.0432
A43	0.0018	0.0006	0.0334	0.0060	0.0231	0.0062	0.0055	0.0028	0.0059	0.0059
A66	0.0173	0.0018	0.2233	0.0155	0.0478	0.0089	0.0399	0.0074	0.0534	0.0178
A10 unsplit	0.0790	0.0038	0.0939	0.0101	0.1255	0.0144	0.0647	0.0094	0.0298	0.0133
A10 total	6.2441	0.0328	7.1348	0.0850	6.3111	0.0993	5.0578	0.0816	4.2853	0.1565
A29	3.5796	0.0252	3.2071	0.0582	1.1233	0.0429	4.5156	0.0774	3.4345	0.1410
A30	2.5067	0.0212	13.0969	0.1129	2.2025	0.0598	4.4873	0.0772	2.5314	0.1215
A31	2.7386	0.0221	1.6556	0.0420	3.6005	0.0761	4.8328	0.0800	6.0881	0.1855
A32	3.6956	0.0256	1.5384	0.0405	1.0331	0.0411	2.7064	0.0604	2.5521	0.1220
A33	1.2080	0.0148	6.5607	0.0822	9.2701	0.1191	2.6593	0.0599	1.0754	0.0796
A74	0.0277	0.0022	1.9949	0.0461	0.0561	0.0096	0.2027	0.0167	0.1068	0.0252
A19 unsplit	0.0567	0.0032	0.2057	0.0149	0.0990	0.0128	0.1211	0.0129	0.0475	0.0168
A19 total	13.8129	0.0468	28.2593	0.1504	17.3846	0.1555	19.5252	0.1481	15.8358	0.2832
AX	0.8204	0.0297	4.9506	0.0963	2.9916	0.1177	1.6332	0.0878	1.8454	0.1925
aGene frequency.
bStandard error.

[0109]

TABLE 2
Estimated gene frequencies for HLA-B antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
B7	12.1782	0.0445	10.5960	0.1024	4.2691	0.0827	6.4477	0.0918	10.9845	0.2432
B8	9.4077	0.0397	3.8315	0.0634	1.3322	0.0467	3.8225	0.0715	8.5789	0.2176
B13	2.3061	0.0203	0.8103	0.0295	4.9222	0.0886	1.2699	0.0416	1.7495	0.1013
B14	4.3481	0.0277	3.0331	0.0566	0.5004	0.0287	5.4166	0.0846	2.9823	0.1316
B18	4.7980	0.0290	3.2057	0.0582	1.1246	0.0429	4.2349	0.0752	3.3422	0.1391
B27	4.3831	0.0278	1.2918	0.0372	2.2355	0.0603	2.3724	0.0567	5.1970	0.1721
B35	9.6614	0.0402	8.5172	0.0927	8.1203	0.1122	14.6516	0.1329	10.1198	0.2345
B37	1.4032	0.0159	0.5916	0.0252	1.2327	0.0449	0.7807	0.0327	0.9755	0.0759
B41	0.9211	0.0129	0.8183	0.0296	0.1303	0.0147	1.2818	0.0418	0.4766	0.0531
B42	0.0608	0.0033	5.6991	0.0768	0.0841	0.0118	0.5866	0.0284	0.2856	0.0411
B46	0.0099	0.0013	0.0151	0.0040	4.9292	0.0886	0.0234	0.0057	0.0238	0.0119
B47	0.2069	0.0061	0.1305	0.0119	0.0956	0.0126	0.1832	0.0159	0.2139	0.0356
B48	0.0865	0.0040	0.1316	0.0119	2.0276	0.0575	1.5915	0.0466	1.0267	0.0778
B53	0.4620	0.0092	10.9529	0.1039	0.4315	0.0266	1.6982	0.0481	1.0804	0.0798
B59	0.0020	0.0006	0.0032	0.0019	0.4277	0.0265	0.0055	0.0028	0c	—
B67	0.0040	0.0009	0.0086	0.0030	0.2276	0.0194	0.0055	0.0028	0.0059	0.0059
B70	0.3270	0.0077	7.3571	0.0866	0.8901	0.0382	1.9266	0.0512	0.6901	0.0639
B73	0.0108	0.0014	0.0032	0.0019	0.0132	0.0047	0.0261	0.0060	0c	—
B51	5.4215	0.0307	2.5980	0.0525	7.4751	0.1080	6.8147	0.0943	6.9077	0.1968
B52	0.9658	0.0132	1.3712	0.0383	3.5121	0.0752	2.2447	0.0552	0.6960	0.0641
B5 unsplit	0.1565	0.0053	0.1522	0.0128	0.1288	0.0146	0.1546	0.0146	0.1307	0.0278
B5 total	6.5438	0.0435	4.1214	0.0747	11.1160	0.1504	9.2141	0.1324	7.7344	0.2784
B44	13.4838	0.0465	7.0137	0.0847	5.6807	0.0948	9.9253	0.1121	11.8024	0.2511
B45	0.5771	0.0102	4.8069	0.0708	0.1816	0.0173	1.8812	0.0506	0.7603	0.0670
B12 unsplit	0.0788	0.0038	0.0280	0.0055	0.0049	0.0029	0.0193	0.0051	0.0654	0.0197
B12 total	14.1440	0.0474	11.8486	0.1072	5.8673	0.0963	11.8258	0.1210	12.6281	0.2584
B62	5.9117	0.0320	1.5267	0.0404	9.2249	0.1190	4.1825	0.0747	6.9421	0.1973
B63	0.4302	0.0088	1.8865	0.0448	0.4438	0.0270	0.8083	0.0333	0.3738	0.0471
B75	0.0104	0.0014	0.0226	0.0049	1.9673	0.0566	0.1101	0.0123	0.0356	0.0145
B76	0.0026	0.0007	0.0065	0.0026	0.0874	0.0120	0.0055	0.0028	0	—
B77	0.0057	0.0010	0.0119	0.0036	0.0577	0.0098	0.0083	0.0034	0c	0.0059
B15 unsplit	0.1305	0.0049	0.0691	0.0086	0.4301	0.0266	0.1820	0.0158	0.0059	0.0206
B15 total	6.4910	0.0334	3.5232	0.0608	12.2112	0.1344	5.2967	0.0835	0.0715	0.2035
									7.4290
B38	2.4413	0.0209	0.3323	0.0189	3.2818	0.0728	1.9652	0.0517	1.1017	0.0806
B39	1.9614	0.0188	1.2893	0.0371	2.0352	0.0576	6.3040	0.0909	4.5527	0.1615
B16 unsplit	0.0638	0.0034	0.0237	0.0051	0.0644	0.0103	0.1226	0.0130	0.0593	0.0188
B16 total	4.4667	0.0280	1.6453	0.0419	5.3814	0.0921	8.3917	0.1036	5.7137	0.1797
B57	3.5955	0.0252	5.6746	0.0766	2.5782	0.0647	2.1800	0.0544	2.7265	0.1260
B58	0.7152	0.0114	5.9546	0.0784	4.0189	0.0803	1.2481	0.0413	0.9398	0.0745
B17 unsplit	0.2845	0.0072	0.3248	0.0187	0.3751	0.0248	0.1446	0.0141	0.2674	0.0398
B17 total	4.5952	0.0284	11.9540	0.1076	6.9722	0.1041	3.5727	0.0691	3.9338	0.1503
B49	1.6452	0.0172	2.6286	0.0528	0.2440	0.0200	2.3353	0.0562	1.5462	0.0953
B50	1.0580	0.0138	0.8636	0.0304	0.4421	0.0270	1.8883	0.0507	0.7862	0.0681
B21 unsplit	0.0702	0.0036	0.0270	0.0054	0.0132	0.0047	0.0771	0.0103	0.0356	0.0145
B21 total	2.7733	0.0222	3.5192	0.0608	0.6993	0.0339	4.3007	0.0755	2.3680	0.1174
B54	0.0124	0.0015	0.0183	0.0044	2.6873	0.0660	0.0289	0.0063	0.0534	0.0178
B55	1.9046	0.0185	0.4895	0.0229	2.2444	0.0604	0.9515	0.0361	1.4054	0.0909
B56	0.5527	0.0100	0.2686	0.0170	0.8260	0.0368	0.3596	0.0222	0.3387	0.0448
B22 unsplit	0.1682	0.0055	0.0496	0.0073	0.2730	0.0212	0.0372	0.0071	0.1246	0.0272
B22 total	2.0852	0.0217	0.8261	0.0297	6.0307	0.0971	1.3771	0.0433	1.9221	0.1060
B60	5.2222	0.0302	1.5299	0.0404	8.3254	0.1135	2.2538	0.0553	5.7218	0.1801
B61	1.1916	0.0147	0.4709	0.0225	6.2072	0.0989	4.6691	0.0788	2.6023	0.1231
B40 unsplit	0.2696	0.0070	0.0388	0.0065	0.3205	0.0230	0.2473	0.0184	0.2271	0.0367
B40 total	6.6834	0.0338	2.0396	0.0465	14.8531	0.1462	7.1702	0.0963	8.5512	0.2168
BX	1.0922	0.0252	3.5258	0.0802	3.8749	0.0988	2.5266	0.0807	1.9867	0.1634
a Gene frequency.
b Standard error.
c The observed gene count was zero.

[0110]

TABLE 3
Estimated gene frequencies of HLA-DR antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
DR1	10.2279	0.0413	6.8200	0.0832	3.4628	0.0747	7.9859	0.1013	8.2512	0.2139
DR2	15.2408	0.0491	16.2373	0.1222	18.6162	0.1608	11.2389	0.1182	15.3932	0.2818
DR3	10.8708	0.0424	13.3080	0.1124	4.7223	0.0867	7.8998	0.1008	10.2549	0.2361
DR4	16.7589	0.0511	5.7084	0.0765	15.4623	0.1490	20.5373	0.1520	19.8264	0.3123
DR6	14.3937	0.0479	18.6117	0.1291	13.4471	0.1404	17.0265	0.1411	14.8021	0.2772
DR7	13.2807	0.0463	10.1317	0.0997	6.9270	0.1040	10.6726	0.1155	10.4219	0.2378
DR8	2.8820	0.0227	6.2673	0.0800	6.5413	0.1013	9.7731	0.1110	6.0059	0.1844
DR9	1.0616	0.0139	2.9646	0.0559	9.7527	0.1218	1.0712	0.0383	2.8662	0.1291
DR10	1.4790	0.0163	2.0397	0.0465	2.2304	0.0602	1.8044	0.0495	1.0896	0.0801
DR11	9.3180	0.0396	10.6151	0.1018	4.7375	0.0869	7.0411	0.0955	5.3152	0.1740
DR12	1.9070	0.0185	4.1152	0.0655	10.1365	0.1239	1.7244	0.0484	2.0132	0.1086
DR5 unsplit	1.2199	0.0149	2.2957	0.0493	1.4118	0.0480	1.8225	0.0498	1.6769	0.0992
DR5 total	12.4449	0.0045	17.0260	0.1243	16.2858	0.1516	10.5880	0.1148	9.0052	0.2218
DRX	1.3598	0.0342	0.8853	0.0760	2.5521	0.1089	1.4023	0.0930	2.0834	0.2037
a Gene frequency.
b Standard error.

[0111] It has been observed that predicted epitopes often cluster at one or more particular regions within the amino acid sequence of a TAA. The identification of such ECRs offers a simple and practicable solution to the problem of designing effective vaccines for stimulating cellular immunity. For vaccines in which immune epitopes are desired, an ECR is directly useful as a vaccine. This is because the immune proteasomes of the pAPCs can correctly process the cluster, liberating one or more of the contained MHC-binding peptides, in the same way a cell having immune proteasomes activity processes and presents peptides derived from the complete TAA. The cluster is also a useful a starting material for identification of housekeeping epitopes produced by the housekeeping proteasomes active in peripheral cells.

[0112] Identification of housekeeping epitopes using ECRs as a starting material is described in copending U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety. Epitope synchronization technology and vaccines for use in connection with this invention are disclosed in copending U.S. patent application Ser. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety. Nucleic acid constructs useful as vaccines in accordance with the present invention are disclosed in copending U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety.

[0113] Vector Design and Vectors

[0114] Degradation of cytosolic proteins takes place via the ubiquitin-dependent multi-catalytic multi-subunit protease system known as the proteasome. The proteasome degrades cytosolic proteins generating fragments that can then be translocated from the cytosol into the endoplasmic reticulum (ER) for loading onto class I MHC. Such protein fragments shall be referred to as class I peptides. The peptide loaded MHC are subsequently transported to the cell surface where they can be detected by CTL.

[0115] The multi-catalytic activity of the proteasome is the result of its multi-subunit structure. Subunits are expressed from different genes and assembled post-translationally into the proteasome complex. A key feature of the proteasome is its bimodal activity, which enables it to exert its protease, or cleavage function, with two discrete kinds of cleavage patterns. This bimodal action of the proteasome is extremely fundamental to understanding how CTL are targeted to recognize peripheral cells in the body and how this targeting requires synchronization between the immune system and the targeted cells.

[0116] The housekeeping proteasome is constitutively active in all peripheral cells and tissues of the body. The first mode of operation for the housekeeping proteasome is to degrade cellular protein, recycling it into amino acids. Proteasome function is therefore a necessary activity for cell life. As a corollary to its housekeeping protease activity, however, class I peptides generated by the housekeeping proteasome are presented on all of the peripheral cells of the body.

[0117] The proteasome's second mode of function is highly exclusive and occurs specifically in pAPCs or as a consequence of a cellular response to interferons (IFNs). In its second mode of activity the proteasome incorporates unique subunits, which replace the catalytic subunits of the constitutive housekeeping proteasome. This “modified” proteasome has been called the immunoproteasome, owing to its expression in pAPC and as a consequence of induction by IFN in body cells.

[0118] APC define the repertoire of CTL that recirculate through the body and are potentially active as killer cells. CTL are activated by interacting with class I peptide presented on the surface of a pAPC. Activated CTL are induced to proliferate and caused to recirculate through the body in search of diseased cells. This is why the CTL response in the body is defined specifically by the class I peptides produced by the pAPC. It is important to remember that pAPCs express the immunoproteasome, and that as a consequence of the bimodal activity of the proteasome, the cleavage pattern of proteins (and the resultant class I peptides produced) are different from those in peripheral body cells which express housekeeping proteasome. The differential proteasome activity in pAPC and peripheral body cells, therefore, is important to consider during natural infection and with therapeutic CTL vaccination strategies.

[0119] All cells of the body are capable of producing IFN in the event that they are infected by a pathogen such as a virus. IFN production in turn results in the expression of the immunoproteasome in the infected cell. Viral antigens are thereby processed by the immunoproteasome of the infected cell and the consequent peptides are displayed with class I MHC on the cell surface. At the same time, pAPC are sequestering virus antigens and are processing class I peptides with their immunoproteasome activity, which is normal for the pAPC cell type. The CTL response in the body is being stimulated specifically by the class I peptides produced by the pAPC. Fortunately, the infected cell is also producing class I peptides from the immunoproteasome, rather than the normal housekeeping proteasome. Thus, virus-related class I peptides are being produced that enable detection by the ensuing CTL response. The CTL immune response is induced by pAPC, which normally produce different class I peptides compared to peripheral body cells, owing to different proteasome activity. Therefore, during infection there is epitope synchronization between the infected cell and the immune system.

[0120] This is not the case with tumors and chronic viruses, which block the interferon system. For tumors there is no infection in the tumor cell to induce the immunoproteasome expression, and chronic virus infection either directly or indirectly blocks immunoproteasome expression. In both cases the diseased cell maintains its display of class I peptides derived from housekeeping proteasome activity and avoids effective surveillance by CTL.

[0121] In the case of therapeutic vaccination to eradicate tumors or chronic infections, the bimodal function of the proteasome and its differential activity in APC and peripheral cells of the body is significant. Upon vaccination with protein antigen, and before a CTL response can occur, the antigen must be acquired and processed into peptides that are subsequently presented on class I MHC on the pAPC surface. The activated CTL recirculate in search of cells with similar class I peptide on the surface. Cells with this peptide will be subjected to destruction by the cytolytic activity of the CTL. If the targeted diseased cell does not express the immunoproteasome, which is present in the pAPC, then the epitopes are not synchronized and CTL fail to find the desired peptide target on the surface of the diseased cell.

[0122] Preferably, therapeutic vaccine design takes into account the class I peptide that is actually present on the target tissue. That is, effective antigens used to stimulate CTL to attack diseased tissue are those that are naturally processed and presented on the surface of the diseased tissue. For tumors and chronic infection this generally means that the CTL epitopes are those that have been processed by the housekeeping proteasome. In order to generate an effective therapeutic vaccine, CTL epitopes are identified based on the knowledge that such epitopes are, in fact, produced by the housekeeping proteasome system. Once identified, these epitopes, embodied as peptides, can be used to successfully immunize or induce therapeutic CTL responses against housekeeping proteasome expressing target cells in the host.

[0123] However, in the case of DNA vaccines, there can be an additional consideration. The immunization with DNA requires that APCs take up the DNA and express the encoded proteins or peptides. It is possible to encode a discrete class I peptide on the DNA. By immunizing with this construct, APCs can be caused to express a housekeeping epitope, which is then displayed on class I MHC on the surface of the cell for stimulating an appropriate CTL response. Constructs for generation of proper termini of housekeeping epitopes have been described in U.S. patent application Ser. No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS, filed on Apr. 28, 2000, which is incorporated herein by reference in its entirety.

[0124] Embodiments of the invention provide expression cassettes that encode one or more embedded housekeeping epitopes, and methods for designing and testing such expression cassettes. The expression cassettes and constructs can encode epitopes, including housekeeping epitopes, derived from antigens that are associated with targets. Housekeeping epitopes can be liberated from the translation product(s) of the cassettes. For example, in some embodiments of the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites. In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. In some embodiments, these arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes. Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated. In some preferred embodiments, each housekeeping epitope can be flanked on either side by an immune epitope, such that an array of such arrangements has two immune epitopes between each housekeeping epitope. Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes.

[0125] Several disclosures make reference to polyepitopes or string-of-bead arrays. See, for example, WO0119408A1, Mar. 22, 2001; WO9955730A2, Nov. 4, 1999; WO0040261A2, Jul. 13, 2000; WO9603144A1, Feb. 8, 1996; EP1181314A1, Feb. 27, 2002; WO0123577A3, April 5; U.S. Pat. No. 6,074,817, Jun. 13, 2000; U.S. Pat. No. 5,965,381, Oct. 12, 1999; WO9741440A1, Nov. 6, 1997; U.S. Pat. No. 6,130,066, Oct. 10, 2000; U.S. Pat. No. 6,004,777, Dec. 21, 1999; U.S. Pat. No. 5,990,091, Nov. 23, 1999; WO9840501A1, Sep. 17, 1998; WO9840500A1, Sep. 17, 1998; WO0118035A2, Mar. 15, 2001; WO02068654A2, Sep. 6, 2002; WO0189281A2, Nov. 29, 2001; WO0158478A, Aug. 16, 2001; EP1118860A1, Jul. 25, 2001; WO0111040A1, Feb. 15, 2001; WO0073438A1, Dec. 7, 2000; WO0071158A1, Nov. 30, 2000; WO0066727A1, Nov. 9, 2000; WO0052451A1, Sep. 8, 2000; WO0052157A1, Sep. 8, 2000; WO0029008A2, May 25, 2000; WO0006723A1, Feb. 10, 2000; all of which are incorporated by reference in their entirety. Additional disclosures, all of which are hereby incorporated by reference in their entirety, include Palmowski M J, et al—J Immunol 2002;168(9):4391-8; Fang Z Y, et al—Virology 2001;291(2):272-84; Firat H, et al—J Gene Med 2002;4(1):38-45; Smith S G, et al—Clin Cancer Res 2001;7(12):4253-61; Vonderheide R H, et al—Clin Cancer Res 2001; 7(11):3343-8; Firat H, et al—Eur J Immunol 2001;31(10):3064-74; Le T T, et al—Vaccine 2001;19(32):4669-75; Fayolle C, et al—J Virol 2001;75(16):7330-8; Smith S G—Curr Opin Mol Ther 1999;1(1):10-5; Firat H, et al—Eur J Immunol 1999;29(10):3112-21; Mateo L, et al—J Immunol 1999;163(7):4058-63; Heemskerk M H, et al—Cell Immunol 1999;195(1):10-7; Woodberry T, et al—J Virol 1999;73(7):5320-5; Hanke T, et al—Vaccine 1998;16(4):426-35; Thomson S A, et al—J Immunol 1998;160(4):1717-23; Toes R E, et al—Proc Natl Acad Sci USA 1997;94(26):14660-5; Thomson S A, et al—J Immunol 1996;157(2):822-6; Thomson S A, et al—Proc Natl Acad Sci USA 1995;92(13):5845-9; Street M D, et al—Immunology 2002;106(4):526-36; Hirano K, et al—Histochem Cell Biol 2002;117(1):41-53; Ward S M, et al—Virus Genes 2001;23(1):97-104; Liu W J, et al—Virology 2000;273(2):374-82; Gariglio P, et al—Arch Med Res 1998;29(4):279-84; Suhrbier A—Immunol Cell Biol 1997;75(4):402-8; Fomsgaard A, et al—Vaccine 1999;18(7-8):681-91; An L L, et al—J Virol 1997;71(3):2292-302; Whitton J L, et al—J Virol 1993;67(1):348-52; Ripalti A, et al—J Clin Microbiol 1994;32(2):358-63; and Gilbert, S. C., et al., Nat. Biotech. 15:1280-1284, 1997.

[0126] One important feature that the disclosures in the preceding paragraph all share is their lack of appreciation for the desirability of regenerating housekeeping epitopes when the construct is expressed in a pAPC. This understanding was not apparent until the present invention. Embodiments of the invention include sequences, that when processed by an immune proteasome, liberate or generate a housekeeping epitope. Embodiments of the invention also can liberate or generate such epitopes in immunogenically effective amounts. Accordingly, while the preceding references contain disclosures relating to polyepitope arrays, none is enabling of the technology necessary to provide or select a polyepitope capable of liberating a housekeeping epitope by action of an immunoproteasome in a pAPC. In contrast, embodiments of the instant invention are based upon a recognition of the desirability of achieving this result. Accordingly, embodiments of the instant invention include any nucleic acid construct that encodes a polypeptide containing at least one housekeeping epitope provided in a context that promotes its generation via immunoproteasomal activity, whether the housekeeping epitope is embedded in a string-of-beads array or some other arrangement. Some embodiments of the invention include uses of one or more of the nucleic acid constructs or their products that are specifically disclosed in any one or more of the above-listed references. Such uses include, for example, screening a polyepitope for proper liberation context of a housekeeping epitope and/or an immune epitope, designing an effective immunogen capable of causing presentation of a housekeeping epitope and/or an immune epitope on a pAPC, immunizing a patient, and the like. Alternative embodiments include use of only a subset of such nucleic acid constructs or a single such construct, while specifically excluding one or more other such constructs, for any of the purposes disclosed herein. Some preferred embodiments employ these and/or other nucleic acid sequences encoding polyepitope arrays alone or in combination. For example, some embodiments exclude use of polyepitope arrays from one or more of the above-mentioned references. Other embodiments may exclude any combination or all of the polyepitope arrays from the above-mentioned references collectively. Some embodiments include viral and/or bacterial vectors encoding polyepitope arrays, while other embodiments specifically exclude such vectors. Such vectors can encode carrier proteins that may have some immunostimulatory effect. Some embodiments include such vectors with such immunostimulatory/immunopotentiating effects, as opposed to immunogenic effects, while in other embodiments such vectors may be included. Further, in some instances viral and bacterial vectors encode the desired epitope as a part of substantially complete proteins which are not associated with the target cell. Such vectors and products are included in some embodiments, while excluded from others. Some embodiments relate to repeated administration of vectors. In some of those embodiments, nonviral and nonbacterial vectors are included. Likewise, some embodiments include arrays that contain extra amino acids between epitopes, for example anywhere from 1-6 amino acids, or more, in some embodiments, while other embodiments specifically exclude such arrays.

[0127] Embodiments of the present invention also include methods, uses, therapies, and compositions directed to various types of targets. Such targets can include, for example, neoplastic cells such as those listed below, for example; and cells infected with any virus, bacterium, protozoan, fungus, or other agents, examples of which are listed below, in Tables 4-8, or which are disclosed in any of the references listed above. Alternative embodiments include the use of only a subset of such neoplastic cells and infected cells listed below, in Tables 4-8, or in any of the references disclosed herein, or a single one of the neoplastic cells or infected cells, while specifically excluding one or more other such neoplastic cells or infected cells, for any of the purposes disclosed herein. The following are examples of neoplastic cells that can be targeted: human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, hepatocellular cancer, brain cancer, stomach cancer, liver cancer, and the like. Examples of infectious agents that infect the target cells can include the following: adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, human T cell leukemia virus II, Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coxiella, Rickettsia, Mycobacterium, Leishmania, Trypanasoma, Toxoplasma, Plasmodium, and the like. Exemplary infectious agents and neoplastic cells are also included in Tables 4-8 below.

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No. 10/225,568, filed Aug. 20, 2002; Simard et al., U.S. patent application Ser. No. 10/005,905, filed Nov. 7, 2001; Simard et al., U.S. patent application Ser. No. 09/561,074, filed Apr. 28, 2000.

[0129] Additional embodiments of the invention include methods, uses, therapies, and compositions relating to a particular antigen, whether the antigen is derived from, for example, a target cell or an infective agent, such as those mentioned above. Some preferred embodiments employ the antigens listed herein, in Tables 4-8, or in the list below, alone, as subsets, or in any combination. For example, some embodiments exclude use of one or more of those antigens. Other embodiments may exclude any combination or all of those antigens. Several examples of such antigens include MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, CEA, RAGE, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, as well as any of those set forth in the above mentioned references. Other antigens are included in Tables 4-7 below.

[0130] Further embodiments include methods, uses, compositions, and therapies relating to epitopes, including, for example those epitopes listed in Tables 4-8. These epitopes can be useful to flank housekeeping epitopes in screening vectors, for example. Some embodiments include one or more epitopes from Tables 4-8, while other embodiments specifically exclude one or more of such epitopes or combinations thereof.

TABLE 4
		AA	T cell epitope MHC
Virus	Protein	Position	ligand (Antigen)	MHC molecule
Adenovirus 3	E3 9Kd	30-38	LIVIGILIL	HLA-A*0201
			(SEQ. ID NO.:44)
Adenovirus 5	EIA	234-243	SGPSNTPPEI	H2-Db
			(SEQ. ID NO.:45)
Adenovirus 5	EIB	192-200	VNIRNCCYI	H2-Db
			(SEQ. ID NO.:46)
Adenovirus 5	EIA	234-243	SGPSNIPPEI (T>I)	H2-Db
			(SEQ. ID NO.:47)
CSFV	NS	2276-2284	ENALLVALF	SLA, haplotype
	polyprotein		(SEQ. ID NO.:48)	d/d
Dengue virus 4	NS3	500-508	TPEGIIPTL	HLA-B*3501
			(SEQ. ID NO.:49)
EBV	LMIP-2	426-434	CLGGLLTMV	HLA-A*0201
			(SEQ. ID NO.:50)
EBV	EBNA-1	480-484	NIAEGLRAL	HLA-A*0201
			(SEQ. ID NO.:51)
EBV	EBNA-1	519-527	NLRRGTALA	HLA-A*0201
			(SEQ. ID NO.:52)
EBV	EBNA-1	525-533	ALAIPQCRL	HLA-A*0201
			(SEQ. ID NO.:53)
EBV	EBNA-1	575-582	VLKDAIKDL	HLA-A*0201
			(SEQ. ID NO.:54)
EBV	EBNA-1	562-570	FMVFLQTHI	HLA-A*0201
		(SEQ. ID NO.:55)
EBV	EBNA-2	15-23	HLIVDTDSL	HLA-A*0201
			(SEQ. ID NO.:56)
EBV	EBNA-2	22-30	SLGNPSLSV	HLA-A*0201
			(SEQ. ID NO.:57)
EBV	EBNA-2	126-134	PLASAMRML	HLA-A*0201
			(SEQ. ID NO.:58)
EBV	EBNA-2	132-140	RMLWMANYI	HL.A-A*0201
			(SEQ. ID NO.:59)
EBV	EBNA-2	133-141	MLWMANYIV	HLA-A*0201
			SEQ. ID NO.:60)
EBV	EBNA-2	151-159	ILPQGPQTA	HLA-A*0201
			(SEQ. ID NO.:61)
EBV	EBNA-2	171-179	PLRPTAPTI	HLA-A*0201
			(SEQ. ID NO.:62)
EBV	EBNA-2	205-213	PLPPATLTV	HLA-A*0201
			(SEQ. ID NO.:63)
EBV	EBNA-2	246-254	RMHLPVLHV	HLA-A*0201
			(SEQ. ID NO.:64)
EBV	EBNA-2	287-295	PMPLPPSQL	HLA-A*0201
			(SEQ. ID NO.:65)
EBV	EBNA-2	294-302	QLPPPAAPA	HLA-A*0201
			(SEQ. ID NO.:66)
EBV	EBNA-2	381-389	SMPELSPVL	HLA-A*0201
			(SEQ. ID NO.:67)
EBV	EBNA-2	453-461	DLDESWDYI	HLA-A*0201
			(SEQ. ID NO.:68)
EBV	BZLF1	43-51	PLPCVLWPV	HLA-A*0201
			(SEQ. ID NO.:69)
EBV	BZLF1	167-175	SLEECDSEL	HLA-A*0201
			(SEQ. ID NO.:70)
EBV	BZLF1	176-184	EIKRYKNRV	HLA-A*0201
			(SEQ. ID NO.:71)
EBV	BZLF1	195-203	QLLQHYREV	HLA-A*0201
			(SEQ. ID NO.:72)
EBV	BZLF1	196-204	LLQHYREVA	HLA-A*0201
			(SEQ. ID NO.:73)
EBV	BZLFI	217-225	LLKQMCPSL	HLA-A*0201
			(SEQ. ID NO.:74)
EBV	BZLF1	229-237	SIIPRTPDV	HLA-A*0201
			(SEQ. ID NO.:75)
EBV	EBNA-6	284-293	LLDFVRFMGV	HLA-A*0201
			(SEQ. ID NO.:76)
EBV	EBNA-3	464-472	SVRDRLARL	HLA-A*0203
			(SEQ. ID NO.:77)
EBV	EBNA-4	416-424	IVTDFSVIK	HLA-A*1101
			(SEQ. ID NO.:78)
EBV	EBNA-4	399-408	AVFDRKSDAK	HLA-A*0201
			(SEQ. ID NO.:79)
EBV	EBNA-3	246-253	RYSIFFDY	HLA-A24
			(SEQ. ID NO.:80)
EBV	EBNA-6	881-889	QPRAPIRPI	HLA-B7
			(SEQ. ID NO.:81)
EBV	EBNA-3	379-387	RPPIFIRRI.	HLA-B7
			(SEQ. ID NO.:82)
EBV	EBNA-1	426-434	EPDVPPGAI	HLA-B7
			(SEQ. ID NO.:83)
EBV	EBNA-1	228-236	IPQCRLTPL	HLA-B7
			(SEQ. ID NO.:84)
EBV	EBNA-1	546-554	GPGPQPGPL	HLA-B7
			(SEQ. ID NO.:85)
EBV	EBNA-1	550-558	QPGPLRESI	HLA-B7
			(SEQ. ID NO.:86)
EBV	EBNA-1	72-80	R.PQKRPSCI	HLA-B7
			(SEQ. ID NO.:87)
EBV	EBNA-2	224-232	PPTPLLTVL	HLA-B7
			(SEQ. ID NO.:88)
EBV	EBNA-2	241-249	TPSPPRMHL	HLA-B7
			(SEQ. ID NO.:89)
EBV	EBNA-2	244-252	PPRMHLPVL	HLA-B7
			(SEQ. ID NO.:90)
EBV	EBNA-2	254-262	VPDQSMHPL	HLA-B7
			(SEQ. ID NO.:91)
EBV	EBNA-2	446-454	PPSIDPADL	HLA-B7
			(SEQ. ID NO.:92)
EBV	BZLFI	44-52	LPCVLWPVL	HLA-B7
			(SEQ. ID NO.:93)
EBV	BZLF1	222-231	CPSLDVDSII	HLA-B7
			(SEQ. ID NO.:94)
EBV	BZLFI	234-242	TPDVLHEDL	HLA-B7
			(SEQ. ID NO.:95)
EBV	EBNA-3	339-347	FLRGRAYGL	HLA-B8
			(SEQ. ID NO.:96)
EBV	EBNA-3	26-34	QAKWRLQTL	HLA-B8
			(SEQ. ID NO.:97)
EBV	EBNA-3	325-333	AYPLHEQHG	HLA-B8
			(SEQ. ID NO.:98)
EBV	EBNA-3	158-166	YIKSFVSDA	HLA-B8
			(SEQ. ID NO.:99)
EBV	LMP-2	236-244	RRRWRRLTV	HLA-B*2704
			(SEQ. ID NO.:100)
EBV	EBNA-6	258-266	RRIYDLIEL	HLA-B*2705
			(SEQ. ID NO.:101)
EBV	EBNA-3	458-466	YPLHEQHGM	HLA-B*3501
			(SEQ. ID NO.:102)
EBV	EBNA-3	458-466	YPLHEQHGM	HLA-B*3503
			(SEQ. ID NO.:103)
HCV	NS3	389-397	HSKKKCDEL	HLA-B8
			(SEQ. ID NO.:104)
HCV	env E	44-51	ASRCWVAM	HLA-B*3501
			(SEQ. ID NO.:105)
HCV	core	27-35	GQIVGGVYL	HLA-B*40012
	protein		(SEQ. ID NO.:106)
HCV	NSI	77-85	PPLTDFDQGW	HLA-B*5301
			(SEQ. ID NO.:107)
HCV	core	18-27	LMGYIPLVGA	H2-Dd
	protein		(SEQ. ID NO.:108)
HCV	core	16-25	ADLMGYIPLV	H2-Dd
	protein		(SEQ. ID NO.:109)
HCV	NS5	409-424	MSYSWTGALVTPCAEE	H2-Dd
			(SEQ. ID NO.:110)
HCV	NS1	205-213	KHPDATYSR	Papa-A06
			(SEQ. ID NO.:111)
HCV-1	NS3	400-409	KLVALGINAV	HLA-A*0201
			(SEQ. ID NO.:112)
HCV-1	NS3	440-448	GDFDSVIDC	Patr-B16
			(SEQ. ID NO.:113)
HCV-1	env E	118-126	GNASRCWVA	Patr-B16
			(SEQ. ID NO.:114)
HCV-1	NS1	159-167	TRPPLGNWF	Patr-B13
			(SEQ. ID NO.:115)
HCV-1	NS3	351-359	VPHPNIEEV	Patr-B13
			(SEQ. ID NO.:116)
HCV-1	NS3	438-446	YTGDFDSVI	Patr-B01
			(SEQ. ID NO.:117)
HCV-1	NS4	328-335	SWAIKWEY	Patr-Al 1
			(SEQ. ID NO.:118)
HCV-1	NSI	205-213	KHPDATYSR	Patr-A04
			(SEQ. ID NO.:119)
HCV-1	NS3	440-448	GDFDSVIDC	Patr-A04
			(SEQ. ID NO.:120)
HIV	gp41	583-591	RYLKDQQLL	HLA A24
			(SEQ. ID NO.:121)
HIV	gagp24	267-275	IVGLNKIVR	HLA-A*3302
			(SEQ. ID NO.:122)
HIV	gagp24	262-270	EIYKRWIIL	HLA-B8
			(SEQ. ID NO.:123)
HIV	gagp24	261-269	GElYKRWI1	HLA-B8
			(SEQ. ID NO.:124)
HIV	gagp17	93-101	EIKDTKEAL	HLA-B8
			(SEQ. ID NO.:125)
HTV	gp41	586-593	YLKDQQLL	HLA-B8
			(SEQ. ID NO.:126)
HIV	gagp24	267-277	ILGLNKIVRMY	HLA-B* 1501
			(SEQ. ID NO.:127)
HIV	gp41	584-592	ERYLKDQQL	HLA-B14
			(SEQ. ID NO.:128)
HIV	nef	115-125	YHTQGYFPQWQ	HLA-B17
			(SEQ. ID NO.:129)
HIV	nef	117-128	TQGYFPQWQNYT	HLA-B17
			(SEQ. ID NO.:130)
HIV	gp120	314-322	GRAFVTIGK	HLA-B*2705
			(SEQ. ID NO.:131)
HIV	gagp24	263-271	KRWIILGLN	HLA-B*2702
			(SEQ. ID NO.:132)
HIV	nef	72-82	QVPLRPMTYK	HLA-B*3501
			(SEQ. ID NO.:133)
HIV	nef	117-125	TQGYFPQWQ	HLA-B*3701
			(SEQ. ID NO.:134)
HIV	gagp24	143-151	HQAISPRTI,	HLA-Cw*0301
			(SEQ. ID NO.:135)
HIV	gagp24	140-151	QMVHQAISPRTL	HLA-Cw*0301
			(SEQ. ID NO.:136)
HIV	gp120	431-440	MYAPPIGGQI	H2-Kd
			(SEQ. ID NO.:137)
HIV	gp160	318-327	RGPGRAFVTI	H2-Dd
			(SEQ. ID NO.:138)
HIV	gp120	17-29	MPGRAFVTI	H2-Ld
			(SEQ. ID NO.:139)
HIV-1	RT	476-484	ILKEPVHGV	HLA-A*0201
			(SEQ. ID NO.:140)
HIV-1	nef	190-198	AFHHVAREL	HLA-A*0201
			(SEQ. ID NO.:141)
HIV-1	gpI60	120-128	KLTPLCVTL	HLA-A*0201
			(SEQ. ID NO.:142)
HIV-1	gp]60	814-823	SLLNATDIAV	HLA-A*0201
			(SEQ. ID NO.:143)
HIV-1	RT	179-187	VIYQYMDDL	HLA-A*0201
			(SEQ. ID NO.:144)
HIV-1	gagp 17	77-85	SLYNTVATL	HLA-A*0201
			(SEQ. ID NO.:145)
HIV-1	gp160	315-329	RGPGRAFVT1	HLA-A*0201
			(SEQ. ID NO.:146)
HIV-1	gp41	768-778	RLRDLLLIVTR	HLA-A3
			(SEQ. ID NO.:147)
HIV-1	nef	73-82	QVPLRPMTYK	HLA-A3
			(SEQ. ID NO.:148)
HIV-1	gp120	36-45	TVYYGVPVWK	HLA-A3
			(SEQ. ID NO.:149)
HIV-1	gagp17	20-29	RLRPGGKKK	HLA-A3
			(SEQ. ID NO.:150)
HIV-1	gp120	38-46	VYYGVPVWK	HLA-A3
			(SEQ. ID NO.:151)
HIV-1	nef	74-82	VPLRPMTYK	HLA-a*1101
			(SEQ. ID NO.:152)
HIV-1	gagp24	325-333	AIFQSSMTK	HLA-A*1101
			(SEQ. ID NO.:153)
HIV-1	nef	73-82	QVPLRPMTYK	HLA-A*1101
			(SEQ. ID NO.:154)
HIV-1	nef	83-94	AAVDLSHFLKEK	HLA-A*1101
			(SEQ. ID NO.:155)
HIV-1	gagp24	349-359	ACQGVGGPGGHK	HLA-A*1101
			(SEQ. 110 NO.:156)
HIV-1	gagp24	203-212	ETINEEAAEW	HLA-A25
			(SEQ. ID NO.:157)
HIV-1	nef	128-137	TPGPGVRYPL	HLA-B7
			(SEQ. ID NO.:158)
HIV-1	gagp 17	24-31	GGKKKYKL	HLA-B8
			(SEQ. ID NO.:159)
HIV-1	gp120	2-10	RVKEKYQHL	HLA-B8
			(SEQ. ID NO.:160)
HIV-1	gagp24	298-306	DRFYKTLRA	HLA-B 14
			(SEQ. ID NO.:161)
HIV-1	NEF	132-147	GVRYPLTFGWCYKLVP	HLA-B18
			(SEQ. ID NO.:162)
HIV-1	gagp24	265-24	KRWIILGLNK	HLA-B*2705
			(SEQ. ID NO.:163)
HIV-1	nef	190-198	AFHHVAREL	HLA-B*5201
			(SEQ. ID NO.:164)
EBV	EBNA-6	335-343	KEHVIQNAF	HLA-B44
			(SEQ. ID NO.:165)
EBV	EBNA-6	130-139	EENLLDFVRF	HLA-B*4403
			(SEQ. ID NO.:166)
EBV	EBNA-2	42-51	DTPLIPLTIF	HLA-B51
			(SEQ. ID NO.:167)
EBV	EBNA-6	213-222	QNGALAINTF	HLA-1362
			(SEQ. ID NO.:168)
EBV	EBNA-3	603-611	RLRAEAGVK	HLA-A3
			(SEQ. ID NO.:169)
HBV	sAg	348-357	GLSPTVWLSV	HLA-A*0201
			(SEQ. ID NO.:170)
HBV	SAg	335-343	WLSLLVPFV	HLA-A*0201
			(SEQ. ID NO.:171)
HBV	cAg	18-27	FLPSDFFPSV	HLA-A*0201
			(SEQ. ID NO.:172)
HBV	cAg	18-27	FLPSDFFPSV	HLA-A*0202
			(SEQ. ID NO.:173)
HBV	cAg	18-27	FLPSDFFPSV	HLA-A*0205
			(SEQ. ID NO.:174)
HBV	cAg	18-27	FLPSDFFPSV	HLA-A*0206
			(SEQ. ID NO.:175)
HBV	pol	575-583	FLLSLGIHL	HLA-A*0201
			(SEQ. ID NO.:176)
HBV	pol	816-824	SLYADSPSV	HLA-A*0201
			(SEQ. ID NO.:177)
HBV	pol	455-463	GLSRYVARL	HLA-A*0201
			(SEQ. ID NO.:178)
HBV	env	338-347	LLVPFVQWFV	HLA-A*0201
			(SEQ. ID NO.:179)
HBV	pol	642-650	ALMPLYACI	HLA-A*0201
			(SEQ. ID NO.:180)
HBV	env	378-387	LLPIFFCLWV	HLA-A*0201
			(SEQ. ID NO.:181)
HEy	pol	538-546	YMDDVVLGA	HLA-A*0201
			(SEQ. ID NO.:182)
HBV	env	250-258	LLLCLIFLL	HLA-A*0201
			(SEQ. ID NO.:183)
HBV	env	260-269	LLDYQGMLPV	HLA-A*0201
			(SEQ. ID NO.:184)
HBV	env	370-379	SIVSPFIPLL	HLA-A*0201
			(SEQ. ID NO.:185)
HBV	env	183-191	FLLTRILTI	HLA-A*0201
			(SEQ. ID NO.:186)
HBV	cAg	88-96	YVNVNMGLK	HLA-A* 1101
			(SEQ. ID NO.:187)
HBV	cAg	141-151	STLPETTVVRR	HLA-A*3101
			(SEQ. ID NO.:188)
HBV	cAg	141-151	STLPETTVVRR	HLA-A*6801
			(SEQ. ID NO.:189)
HBV	cAg	18-27	FLPSDFFPSV	HLA-A*6801
			(SEQ. ID NO.:190)
HBV	cAg	28-39	IPQSLDSWWTSL	H2-Ld
			(SEQ. ID NO.:191)
HBV	cAg	93-100	MGLKFRQL	H2-Kb
			(SEQ. ID NO.:192)
HBV	preS	141-149	STBXQSGXQ	HLA-A*0201
			(SEQ. ID NO.:193)
HCMV	gp B	618-628	FIAGNSAYEYV	HLA-A*0201
			(SEQ. lID NO.:194)
HCMV	E1	978-989	SDEEFAIVAYTL	HLA-B18
			(SEQ. ID NO.:195)
HCMV	pp65	397-411	DDVWTSGSDSDEELV	HLA-b35
			(SEQ. ID NO.:196)
HCMV	pp65	123-131	IPSINVHHY	HLA-B*3501
			(SEQ. ID NO.:197)
HCMV	pp65	495-504	NLVPMVATVO	HLA-A*0201
			(SEQ. ID NO.:198)
HCMV	pp65	415-429	RKTPRVTOGGAMAGA	HLA-B7
			(SEQ. lID NO.:199)
HCV	MP	17-25	DLMGYIPLV	HLA-A*0201
			(SEQ. ID NO.:200)
HCV	MP	63-72	LLALLSCLTV	HLA-A*0201
			(SEQ. ID NO.:201)
HCV	MP	105-112	ILHTPGCV	HLA-A*0201
			(SEQ. ID NO.:202)
HCV	env E	66-75	QLRRHIDLLV	HLA-A*0201
			(SEQ. ID NO.:203)
HCV	env E	88-96	DLCGSVFLV	HLA-A*0201
			(SEQ. ID NO.:204)
HCV	env E	172-180	SMVGNWAKV	HLA-A*0201
			(SEQ. ID NO.:205)
HCV	NSI	308-316	HLIIQNIVDV	HLA-A*0201
			(SEQ. ID NO.:206)
HCV	NSI	340-348	FLLLADARV	HLA-A*0201
			(SEQ. ID NO.:207) (SEQ. liD NO.:208)
HCV	NSI	18-28	SLLAPGAKQNV	HLA-A*0201
			(SEQ. ID NO.:209)
HCV	NSI	19-28	LLAPGAKQNV	HLA-A*0201
			(SEQ. ID NO.:210)
HCV	NS4	192-201	LLFNILGGWV	HLA-A*0201
			(SEQ. ID NO.:211)
HCV	NS3	579-587	YLVAYQATV	HLA-A*0201
			(SEQ. ID NO.:212)
HCV	core	34-43	YLLPRRGPRL	HLA-A*0201
	protein		(SEQ. ID NO.:213)
HCV	MP	63-72	LLALLSCLTI	HLA-A*0201
			(SEQ. ID NO.:214)
HCV	NS4	174-182	SLMAFTAAV	HLA-A*0201
			(SEQ. ID NO.:215)
HCV	NS3	67-75	CINGVCTV	HLA-A*0201
			(SEQ. ID NO.:216)
HCV	NS3	163-171	LLCPAGHAV	HLA-A*0201
			(SEQ. ID NO.:217)
HCV	NS5	239-247	ILDSFDPLV	HLA-A*0201
			(SEQ. ID NO.:218)
HCV	NS4A	236-244	ILAGYGAGV	HLA-A*0201
			(SEQ. ID NO.:219)
HCV	NS5	714-722	GLQDCTMLV	HLA-A*0201
			(SEQ. ID NO.:220)
HCV	NS3	281-290	TGAPVTYSTY	HLA-A*0201
			(SEQ. ID NO.:221)
HCV	NS4A	149-157	HMWNFISGI	HLA-A*0201
			(SEQ. ID NO.:222)
HCV	NS5	575-583	RVCEKMALY	HLA-A*0201-A3
			(SEQ. ID NO.:223)
HCV	NS1	238-246	TINYTIFK	HLA-A*1101
			(SEQ. ID NO.:224)
HCV	NS2	109-116	YISWCLWW	HLA-A23
			(SEQ. ID NO.:225)
HCV	core	40-48	GPRLGVRAT	HLA-B7
	protein		(SEQ. ID NO.:226)
HIV-1	gp120	380-388	SFNCGGEFF	HLA-Cw*0401
			(SEQ. ID NO.:227)
HIV-1	RT	206-214	TEMEKEGKI	H2-Kk
			(SEQ. ID NO.:228)
HIV-1	p17	18-26	KIRLRPGGK	HLA-A*0301
			(SEQ. ID NO.:229)
HIV-1	P17	20-29	RLRPGGKKKY	HLA-A*0301
			(SEQ. ID NO.:230)
HIV-I	RT	325-333	AIFQSSMTK	HLA-A*0301
			(SEQ. ID NO.:231)
HIV-1	p17	84-92	TLYCVHQRI	HLA-A11
			(SEQ. ID NO.:232)
HIV-1	RT	508-517	IYQEPFKNLK	HLA-A11
			(SEQ. ID NO.:233)
HIV-1	p17	28-36	KYKLKHIVW	HLA-A24
			(SEQ. ID NO.:234)
HIV-1	gp120	53-62	LFCASDAKAY	HLA-A24
			(SEQ. ID NO.:235)
HIV-1	gagp24	145-155	QAISPRTLNAW	HLA-A25
			(SEQ. ID NO.:236)
HIV-1	gagp24	167-175	EVIPMFSAL	HLA-A26
			(SEQ. ID NO.:237)
HIV-1	RT	593-603	ETFYVDGAANR	HLA-A26
			(SEQ. ID NO.:238)
HIV-1	gp41	775-785	RLRDLLLIVTR	HLA-A31
			(SEQ. ID NO.:239)
HIV-1	RT	559-568 PIQKETWETW	HLA-A32
			(SEQ. ID NO.:240)
HIV-1	gp120	419-427	RIKQIINMW	HLA-A32
			(SEQ. ID NO.:241)
HIV-1	RT	71-79	ITLWQRPLV	HLA-A*6802
			(SEQ. ID NO.:242)
HIV-1	RT	85-93	DTVLEEMNL	HLA-A*6802
			(SEQ. ID NO.:243)
HIV-1	RT	71-79	ITLWQRPLV	HLA-A*7401
			(SEQ. ID NO.:244)
HIV-1	gagp24	148-156	SPRTLNAWV	HLA-B7
			(SEQ. ID NO.:245)
HIV-1	gagp24	179-187	ATPQDLNTM	HLA-B7
			(SEQ. ID NO.:246)
HIV-1	gp120	303-312	RPNNNTRKSI	HLA-B7
			(SEQ. ID NO.:247)
HIV-1	gp41	843-851	IPRRIRQGL	HLA-B7
			(SEQ. ID NO.:248)
HIV-1	p17	74-82	ELRSLYNTV	HLA-B8
			(SEQ. ID NO.:249)
HIV-1	nef	13-20	WPTVRERM	HLA-B8
			(SEQ. ID NO.:250)
HIV-1	nef	90-97	FLKEKGGL	HLA-B8
			(SEQ. ID NO.:251)
HIV-1	gagp24	183-191	DLNTMLNTV	HLA-B14
			(SEQ. iD NO.:252)
HIV-1	P17	18-27	KIRLRPGGKK	HLA-B27
			(SEQ. ID NO.:253)
HIV-1	p17	19-27	IRLRPGGKK	HLA-B27
			(SEQ. ID NO.:254)
HIV-1	gp41	791-799	GRRGWEALKY	HLA-B27
			(SEQ. ID NO.:255)
HIV-1	nef	73-82	QVPLRPMTYK	HLA-B27
			(SEQ. ID NO.:256)
HIV-1	GP41	590-597	RYLKDQQL	11LA-B27
			(SEQ. ID NO.:257)
HIV-1	nef	105-114	RRQDILDLWI	HLA-B*2705
			(SEQ. ID NO.:258)
HIV-1	nef	134-141	RYPLTFGW	HLA-B*2705
			(SEQ. ID NO.:259)
HIV-1	p17	36-44	WASRELERF	HLA-B35
			(SEQ. ID NO.:260)
HIV-1	GAGP24	262-270	TVLDVGDAY	HLA-B35
			(SEQ. ID NO.:261)
HIV-1	gp120	42-52	VPVWKEATTTL	HLA-B35
			(SEQ. ID NO.:262)
HIV-1	P17	36-44	NSSKVSQNY	HLA-B35
			(SEQ. ID NO.:263)
HIV-1	gagp24	254-262	PPIPVGDIY	HLA-B35
			(SEQ. ID NO.:264)
HIV-1	RT	342-350	HPDIVIYQY	HLA-B35
			(SEQ. ID NO.:265)
HIV-1	gp41	611-619	TAVPWNASW	HLA-B35
			(SEQ. ID NO.:266)
HIV-1	gag	245-253	NPVPVGNIY	HLA-B35
			(SEQ. ID NO.:267)
HIV-1	nef	120-128	YFPDWQNYT	HLA-B37
			(SEQ. ID NO.:268)
HIV-1	gagp24	193-201	GHQAAMQML	HLA-B42
			(SEQ. ID NO.:269)
HIV-1	p17	20-29	RLRPGGKKKY	HLA-B42
			(SEQ. ID NO.:270)
HIV-1	RT	438-446	YPGIKVRQL	HLA-B42
			(SEQ. ID NO.:271)
HIV-1	RT	591-600	GAETFYVDGA	HLA-B45
			(SEQ. ID NO.:272)
HIV-1	gagp24	325-333	NANPDCKTI	HLA-B51
			(SEQ. ID NO.:273)
HIV-1	gagp24	275-282	RMYSPTSI	HLA-B52
			(SEQ. ID NO.:274)
HLV-1	gp120	42-51	VPVWKEATTT	HLA-B*5501
			(SEQ. iD NO.:275)
HIV-1	gagp24	147-155	ISPRTLNAW	HLA-B57
			(SEQ. ID NO.:276)
HIV-1	gagp24	240-249	TSTLQEQIGW	HLA-B57
			(SEQ. ID NO.:277)
HIV-1	gagp24	162-172	KAFSPEVIPMF	HLA-B57
			(SEQ. ID NO.:278)
HIV-1	gagp24	311-319	QASQEVKNW	HLA-B57
			(SEQ. ID NO.:279)
HIV-1	gagp24	311-319	QASQDVKNW	HLA-B57
			(SEQ. ID NO.:280)
HIV-1	nef	116-125	HTQGYFPDWQ	HLA-B57
			(SEQ. ID NO.:281)
HIV-1	nef	120-128 YFPDWQNYT	HLA-B57
			(SEQ. ID NO.:282)
HIV-1	gagp24	240-249	TSTLQEQIGW	HLA-B58
			(SEQ. ID NO.:283)
HIV-1	p17	20-29	RLRPGGKKKY	HLA-B62
			(SEQ. ID NO.:284)
HIV-1	P24	268-277	LGLNKJVRMY	HLA-B62
			(SEQ. ID NO.:285)
HIV-1	RT	415-426	LVGKLNWASQIY	HLA-B62
			(SEQ. ID NO.:286)
HIV-1	RT	476-485	ILKEPVHGVY	HLA-B62
			(SEQ. ID NO.:287)
HIV-1	nef	117-127	TQGYFPDWQNY	HLA-B62
			(SEQ. ID NO.:288)
HIV-1	nef	84-91	AVDLSHFL	HLA-B62
			(SEQ. ID NO.:289)
HIV-1	gagp24	168-175	VIPMFSAL	HLA-Cw*0102
			(SEQ. ID NO.:290)
HLV-1	p120	376-384	FNCGGEFFY	HLA-A29
			(SEQ. ID NO.:291)
HIV-1	gp120	375-383	SFNCGGEFF	HLA-B15
			(SEQ. ID NO.:292)
HIV-1	nef	136-145	PLTFGWCYKL	HLA-A*0201
			(SEQ. ID NO.:293)
HIV-1	nef	180-189	VLEWRFDSRL	HLA-A*0201
			(SEQ. ID NO.:294)
HIV-1	nef	68-77	FPVTPQVPLR	HLA-B7
			(SEQ. ID NO.:295)
HIV-1	nef	128-137	TPGPGVRYPL	HLA-B7
			(SEQ. ID NO.:296)
HIV-1	gagp24	308-316	QASQEVKNW	HLA-Cw*0401
			(SEQ. ID NO.:297)
HIV-1 IIIB	RT	273-282	VPLDEDFRKY	HLA-B35
			(SEQ. ID NO.:298)
HIV-1 IIIB	RT	25-33	NPDIVIYQY	HLA-B35
			(SEQ. ID NO.:299)
HIV-1 IIIB	gp41	557-565 RAIEAQAHL	HLA-B51
			(SEQ. ID NO.:300)
HIV-1 IIIB	RT	231-238 TAFTIPSI	HLA-B51
			(SEQ. ID NO.:301)
HIV-I IIIB	pol	215-223	VHPVHAGPIA	HLA-B*5501
			(SEQ. ID NO.:302)
HIV-1 IIIB	gp120	156-165	NCSFNISTSI	HLA-Cw8
			(SEQ. ID NO.:303)
HIV-I IIIB	gp120	241-249	CTNVSTVQC	HLA-Cw8
			(SEQ. ID NO.:304)
HIV-1 5F2	gp120	312-320	IGPGRAFHT	H2-Dd
			(SEQ. ID NO.:305)
HIV-1 5F2	pol	25-33	NPDIVIYQY	HLA-B*3501
			(SEQ. ID NO.:306)
HIV-1 5F2	pol	432-441	EPIVGAETFY	HLA-B*3501
			(SEQ. ID NO.:307)
HIV-1 5F2	pol	432-440	EPIVGAETF	HLA-B*3501
			(SEQ. ID NO.:308)
HLV-1 5F2	pol	6-14	SPAIFQSSM	HLA-B*3501
			(SEQ. ID NO.:309)
HIV-1 5F2	pol	59-68	VPLDKDFRKY	HLA-B*3501
			(SEQ. ID NO.:310)
HIV-1 5F2	pol	6-14	IPLTEEAEL	HLA-B*3501
			(SEQ. ID NO.:311)
HlV-1 5F2	nef	69-79	RPQVPLRPMTY	HLA-B*3501
			(SEQ. ID NO.:312)
HIV-1 5F2	nef	66-74	FPVRPQVPL	HLA-B*3501
			(SEQ. ID NO.:313)
HIV-1 5F2	env	10-18	DPNPQEVVL	HLA-B*3501
			(SEQ. ID NO.:314)
HIV-1 5F2	env	7-15	RPIVSTQLL	HLA-B*3501
			(SEQ. ID NO.:315)
HIV-1 5F2	pol	6-14	IPLTEEAEL	HLA-B51
			(SEQ. ID NO.:316)
HIV-1 5F2	env	10-18	DPNPQEVVL	HLA-B51
			(SEQ. ID NO.:317)
HIV-1 5F2	gagp24	199-207	AMQMLKETI	H2-Kd
			(SEQ. ID NO.:318)
HIV-2	gagp24	182-190	TPYDrNQML	HLA-B*5301
			(SEQ. ID NO.:319)
HIV-2	gag	260-269	RRWIQLGLQKV	HLA-B*2703
			(SEQ. ID NO.:320)
HIV-1 5F2	gp41	593-607	GIWGCSGKLICTTAV	HLA-B17
			(SEQ. ID NO.:321)
HIV-1 5F2	gp41	753-767	ALIWEDLRSLCLFSY	HLA-B22
			(SEQ. ID NO.:322)
HPV 6b	E7	21-30	GLHCYEQLV	HLA-A*0201
			(SEQ. ID NO.:323)
HPV 6b	E7	47-55	PLKQHFQIV	HLA-A*0201
			(SEQ. ID NO.:324)
HPV11	E7	4-12	RLVTLKDIV	HLA-A*0201
			(SEQ. ID NO.:325)
HPV16	E7	86-94	TLGIVCPIC	HLA-A*0201
			(SEQ. ID NO.:326)
HPV16	E7	85-93	GTLGIVCPI	HLA-A*0201
			(SEQ. ID NO.:327)
HPV16	E7	12-20	MLDLQPETT	HLA-A*0201
			(SEQ. ID NO.:328)
HPV16	E7	11-20	YMLDLQPETT	HLA-A*0201
			(SEQ. ID NO.:329)
HPV16	E6	15-22	RPRKLPQL	HLA-B7
			(SEQ. ID NO.:330)
HPV16	E6	49-57	RAHYNIVTF	HW-Db
			(SEQ. ID NO.:331)
HSV-1	gp B	498-505	SSIEFARL	H2-Kb
			(SEQ. ID NO.:332)
HSV-1	gp C	480-488	GIGIGVLAA	HLA-A*0201
			(SEQ. ID NO.:333)
HSV-1	ICP27	448-456	DYATLGVGV	H2-Kd
			(SEQ. ID NO.:334)
HSV-1	ICP27	322-332	LYRTFAGNPRA	H2-Kd
			(SEQ. ID NO.:335)
HSV-1	UL39	822-829	QTFDFGRL	H2-Kb
			(SEQ. ID NO.:336)
HSV-2	gpC	446-454	GAGIGVAVL	HLA-A*0201
			(SEQ. ID NO.:337)
HLT V-i	TAX	11-19	LLFGYPVYV	HLA-A*0201
			(SEQ. ID NO.:338)
Influenza	MP	58-66	GILGFVFTL	HLA-A*0201
			(SEQ. ID NO.:339)
Influenza	MP	59-68	ILGFVFTLTV	HLA-A*0201
			(SEQ. ID NO.:340)
Influenza	NP	265-273	ILRGSVAHK	HLA-A3
			(SEQ. ID NO.:341)
Influenza	NP	91-99	KTGGPIYKR	HLA-A*6801
			(SEQ. ID NO.:342)
Influenza	NP	380-388	ELRSRYWAI	HLA-B8
			(SEQ. ID NO.:343)
Influenza	NP	381-388	LRSRYWAI	HLA-B*2702
			(SEQ. ID NO.:344)
Influenza	NP	339-347	EDLRVLSFI	HLA-B*3701
			(SEQ. ID NO.:345)
Influenza	NSI	158-166	GEISPLPSL	HLA-B44
			(SEQ. ID NO.:346)
Influenza	NP	338-346	FEDLRVLSF	HLA-B44
			(SEQ. ID NO.:347)
Influenza	NSI	158-166	GEISPLPSL	HLA-B*4402
			(SEQ. ID NO.:348)
Influenza	NP	338-346	FEDLRVLSF	HLA-B*4402
			(SEQ. ID NO.:349)
Influenza	PBI	591-599	VSDGGPKLY	HLA-A1
			(SEQ. ID NO.:350)
Influenza A	NP	44-52	CTELKLSDY	HLA-A1
			(SEQ. ID NO.:351)
Influenza	NSI	122-130	AIMDKNIIL	HLA-A*0201
			(SEQ. ID NO.:352)
Influenza A	NSI	123-132	IMDKNIILKA	HLA-A*0201
			(SEQ. ID NO.:353)
Influenza A	NP	383-391	SRYWAIRTR	HLA-B*2705
			(SEQ. ID NO.:354)
Influenza A	NP	147-155	TYQRTRALV	H2-Kd
			(SEQ. ID NO.:355)
Influenza A	HA	210-219	TYVSVSTSTL	H2-Kd
			(SEQ. ID NO.:356)
Influenza A	HA	518-526	IYSTVASSL	H2-Kd
			(SEQ. ID NO.:357)
Influenza A	HA	259-266	FEANGNLI	H2-Kk
			(SEQ. ID NO.:358)
Influenza A	HA	10-18	IEGGWTGMl	H2-Kk
			(SEQ. ID NO.:359)
Influenza A	NP	50-57	SDYEGRLI	H2-Kk
			(SEQ. ID NO.:360)
Influenza A	NSI	152-160	EEGAIVGEI	H2-Kk
			(SEQ. ID NO.:361)
Influenza A34	NP	336-374	ASNENMETM	H2Db
			(SEQ. ID NO.:362)
Influenza A68	NP	366-374	ASNENMDAM	H2Db
			(SEQ. ID NO.:363)
Influenza B	NP	85-94	KLGEFYNQMM	HLA-A*0201
			(SEQ. ID NO.:364)
Influenza B	NP	85-94	KAGEFYNQMM	HLA-A*0201
			(SEQ. ID NO.:365)
Influenza JAP	HA	204-212	LYQNVGTYV	H2Kd
			(SEQ. ID NO.:366)
Influenza JAP	HA	210-219	TYVSVGTSTL	H2-Kd
			(SEQ. ID NO.:367)
Influenza JAP	HA	523-531	VYQILATYA	H2-Kd
			(SEQ. ID NO.:368)
Influenza JAP	HA	529-537	IYATVAGSL	H2-Kd
			(SEQ. ID NO.:369)
Influenza JAP	HA	210-219	TYVSVGTSTI(L>I)	H2-Kd
			(SEQ. ID NO.:370)
Influenza JAP	HA	255-262	FESTGNLI	H2-Kk
			(SEQ. ID NO.:371)
JHMV	cAg	318-326	APTAGAFFF	H2-Ld
			(SEQ. ID NO.:372)
LCMV	NP	118-126	RPQASGVYM	H2-Ld
			(SEQ. ID NO.:373)	(SEQ. ID NO.:374)
LCMV	NP	396-404	FQPQNGQFI	H2-Db
			(SEQ. ID NO.:374)
LCMV	GP	276-286	SGVENPGGYCL	H2-Db
			(SEQ. ID NO.:375)
LCMV	GP	33-42	KAVYNFATCG	H2-Db
			(SEQ. ID NO.:376)
MCMV	pp89	168-176	YPHFMPTNL	H2-Ld
			(SEQ. ID NO.:377)
MHV	spike	510-518	CLSWNGPHL	H2-Db
	protein		(SEQ. ID NO.:378)
MMTV	env gp36	474-482	SFAVATTAL	H2-Kd
			(SEQ. ID NO.:379)
MMTV	gagp27	425-433	SYETFISRL	H2-Kd
			(SEQ. ID NO.:380)
MMTV	env gp73	544-551	ANYDFICV	H2-Kb
			(SEQ. ID NO.:381)
MuLV	env p15E	574-581	KSPWFTTL	H2-Kb
			(SEQ. ID NO.:382)
MuLV	env gp70	189-196	SSWDFITV	H2-Kb
			(SEQ. ID NO.:383)
MuLV	gag 75K	75-83	CCLCLTVFL	H2-Db
			(SEQ. ID NO.:384)
MuLV	env gp70	423-431	SPSYVYHQF	H2Ld
			(SEQ. ID NO.:385)
MV	F protein	437-447	SRRYPDAVYLH	HLA-B*2705
			(SEQ. ID NO.:386)
Mv	F protein	438-446	RRYPDAVYL	HLA-B*2705
			(SEQ. ID NO.:387)
Mv	NP	281-289	YPALGLHEF	H2-Ld
			(SEQ. ID NO.:388)
Mv	HA	343-351	DPVIDRLYL	H2-Ld
			(SEQ. ID NO.:389)
MV	HA	544-552	SPGRSFSYF	H2-Ld
			(SEQ. ID NO.:390)
Poliovirus	VP1	111-118	TYKDTVQL	H2-kd
			(SEQ. ID NO.:391)
Poliovirus	VP1	208-217	FYDGFSKVPL	H2-Kd
			(SEQ. ID NO.:392)
Pseudorabies	G111	455-463	IAGIGILAI	HLA-A*0201
virus gp			(SEQ. ID NO.:393)
Rabiesvirus	NS	197-205	VEAEIAHQI	H2-Kk
			(SEQ. ID NO.:394)
Rotavirus	VP7	33-40	llYRFLLl	H2-Kb
			(SEQ. ID NO.:395)
Rotavirus	VP6	376-384	VGPVFPPGM	H2-Kb
			(SEQ. ID NO.:396)
Rotavirus	VP3	585-593	YSGYIFRDL	H2-Kb
			(SEQ. ID NO.:397)
RSV	M2	82-90	SYIGSINNI	H2-Kd
			(SEQ. ID NO.:398)
SIV	gagp11C	179-190	EGCTPYDTNQML	Mamu-A*01
			(SEQ. ID NO.:399)
SV	NP	324-332	FAPGNYPAL	H2-Db
			(SEQ. ID NO.:400)
SV	NP	324-332	FAPCTNYPAL	H2-Kb
			(SEQ. ID NO.:401)
SV40	T	404-411	VVYDFLKC	H2-Kb
			(SEQ. ID NO.:402)
SV40	T	206-215	SAINNYAQKL	H2-Db
			(SEQ. ID NO.:403)
SV40	T	223-231	CKGVNKEYL	H2-Db
			(SEQ. ID NO.:404)
SV40	T	489-497	QGINNLDNL	H2-Db
			(SEQ. ID NO.:405)
SV40	T	492-500	NNLDNLRDY(L)	H2-Db
		(501)	(SEQ. ID NO.:406)
SV40	T	560-568	SEFLLEKRI	H2-Kk
			(SEQ. ID NO.:407)
VSV	NP	52-59	RGYVYQGL	H2-Kb
			(SEQ. ID NO.:408)

[0131]

TABLE 5
HLA-A1	Position (Antigen)	Source
T cell	EADPTGHSY	MAGE-1 161-169
epitopes	(SEQ. ID NO.:409)
	VSDGGPNLY	Influenza A PB
	(SEQ. ID NO.:410)	1591-599
	CTELKLSDY	Influenza A NP
	(SEQ. ID NO.:411)	44-52
	EVDPIGHLY	MAGE-3 168-176
	(SEQ. ID NO.:412)
HLA-A201	MLLSVPLLLG	Calreticulin signal
	(SEQ, ID NO.:413)	sequence I-10
	STBXQSGXQ	HBV PRE-S PROTEIN
	(SEQ. ID NO.:414)	141-149
	YMDGTMSQV	Tyrosinase 369-377
	(SEQ. ID NO.:415)
	ILKEPVHGV	HIV - I RT 476-484
	(SEQ. ID NO.:416)
	LLGFVFTLTV	Influenza MP 59-68
	(SEQ. ID NO.:417)
	LLFGYPVYVV	HTLV-1 tax 11-19
	(SEQ. ID NO.:418)
	GLSPTVWLSV	HBV sAg 348-357
	(SEQ. ID NO.:419)
	WLSLLVPFV	HBV sAg 335-343
	(SEQ. ID NO.:420)
	FLPSDFFPSV	HBV cAg 18-27
	(SEQ. ID NO.:421)
	C L G 0 L L T M V	EBVLMP-2426-434
	(SEQ. ID NO.:422)
	FLAGNSAYEYV	HCMV gp 618-628B
	(SEQ. ID NO.:423)
	KLGEFYNQMM	Influenza BNP 85-94
	(SEQ. ID NO.:424)
	KLVALGINAV	HCV-1 NS3 400-409
	(SEQ. ID NO.:425)
	DLMGYIPLV	HCV MP 17-25
	(SEQ. ID NO.:426)
	RLVTLKDIV	HPV 11 EZ 4-12
	(SEQ. ID NO.:427)
	MLLAVLYCL	Tyrosinase 1-9
	(SEQ. ID NO.:428)
	AAGIGILTV	Melan A\Mart-127-35
	(SEQ. ID NO.:429)
	YLEPGPVTA	Pmel 17/gp 100 480-488
	(SEQ. ID NO.:430)
	ILDGTATLRL	Pmel 17/gp 100 457-466
	(SEQ. ID NO.:431)
	LLDGTATLRL	Pmel gp1OO 457-466
	(SEQ. ID NO.:432)
	ITDQVPFSV	Pmel gp 100 209-217
	(SEQ. ID NO.:433)
	KTWGQYWQV	Pmel gp 100 154-162
	(SEQ. ID NO.:434)
	TITDQVPFSV	Pmel gp 100 208-217
	(SEQ. ID NO.:435)
	AFHIIVAREL	HIV-I nef 190-198
	(SEQ. ID NO.:436)
	YLNKIQNSL	P. falciparum CSP 334-342
	(SEQ. ID NO.:437)
	MMRKLAILSV	P. falciparum CSP 1-10
	(SEQ. ID NO.:438)
	KAGEFYNQMM	Influenza BNP 85-94
	(SEQ. ID NO.:439)
	NIAEGLRAL	EBNA-1 480-488
	(SEQ. ID NO.:440)
	NLRRGTALA	EBNA-1 519-527
	(SEQ. ID NO.:441)
	ALAIPQCRL	EBNA-1 525-533
	(SEQ. ID NO.:442)
	VLKDAIKDL	EBNA-1 575-582
	(SEQ. ID NO.:443)
	FMVFLQTHI	EBNA-1 562-570
	(SEQ. ID NO.:444)
	HLIVDTDSL	EBNA-2 15-23
	(SEQ. ID NO.:445)
	SLGNPSLSV	EBNA-2 22-30
	(SEQ. ID NO.:446)
	PLASAMRML	EBNA-2 126-134
	(SEQ. ID NO.:447)
	RMLWMANYI	EBNA-2 132-140
	(SEQ. ID NO.:448)
	MLWMANYIV	EBNA-2 133-141
	(SEQ. ID NO.:449)
	ILPQGPQTA	EBNA-2 151-159
	(SEQ. ID NO.:450)
	PLRPTAPTTI	EBNA-2 171-179
	(SEQ. ID NO.:451)
	PLPPATLTV	EBNA-2 205-213
	(SEQ. ID NO.:452)
	R M H L P V L H V	EBNA-2 246-254
	(SEQ. ID NO.:453)
	PMPLPPSQL	EBNA-2 287-295
	(SEQ. ID NO.:454)
	QLPPPAAPA	EBNA-2 294-302
	(SEQ. ID NO.:455)
	SMPELSPVL	EBNA-2 381-389
	(SEQ. ID NO.:456)
	DLDESWDYl	EBNA-2 453-461
	(SEQ. ID NO.:457)
	P L P C V L W P VV	BZLFl 43-51
	(SEQ. ID NO.:458)
	SLEECDSEL	BZLFl 167-175
	(SEQ. ID NO.:459)
	EIKRYKNRV	BZLFI 176-184
	(SEQ. ID NO.:460)
	QLLQFIYREV	BZLFl 195-203
	(SEQ. ID NO.:461)
	LLQHYREVA	BZLFI 196-204
	(SEQ. ID NO.:462)
	LLKQMCPSL	BZLFI 217-225
	(SEQ. ID NO.:463)
	SIIPRTPDV	BZLFI 229-237
	(SEQ. ID NO.:464)
	AIMDKNIIL	Influenza A NS1 122-130
	(SEQ. ID NO.:465)
	IMDKNIILKA	Influenza A NS1 123-132
	(SEQ. ID NO.:466)
	LLALLSCLTV	HCV MP 63-72
	(SEQ. ID NO.:467)
	ILHTPGCV	HCVMP 105-112
	(SEQ. ID NO.:468)
	QLRRHIDLLV	HCV env E 66-75
	(SEQ. ID NO.:469)
	DLCGSVFLV	HCV env E 88-96
	(SEQ. ID NO.:470)
	SMVGNWAKV	HCV env E 172-180
	(SEQ. ID NO.:471)
	HLHQNIVDV	HCV NSI 308-316
	(SEQ. ID NO.:472)
	FLLLADARV	HCV NSI 340-348
	(SEQ. ID NO.:473)
	GLRDLAVAVEPVV	HCV NS2 234-246
	(SEQ. ID NO.:474)
	SLLAPGAKQNV	HCV NS1 18-28
	(SEQ. ID NO.:475)
	LLAPGAKQNV	HCV NS1 19-28
	(SEQ. ID NO.:476)
	FLLSLGIHL	HBV pol 575-583
	(SEQ. ID NO.:477)
	SLYADSPSV	HBV pol 816-824
	(SEQ. ID NO.:478)
	GLSRYVARL	HBV POL 455-463
	(SEQ. ID NO.:479)
	KIFGSLAFL	HER-2 369-377
	(SEQ. ID NO.:480)
	ELVSEFSRM	HER-2 971-979
	(SEQ. ID NO.:481)
	KLTPLCVTL	HIV-I gp 160 120-128
	(SEQ. ID NO.:482)
	SLLNATDIAV	HIV-I GP 160 814-823
	(SEQ. ID NO.:483)
	VLYRYGSFSV	Pmel gp100 476-485
	(SEQ. ID NO.:484)
	YIGEVLVSV	Non-filament forming
	(SEQ. ID NO.:485)	class I myosin family
		(HA-2)**
	LLFNILGGWV	HCV NS4 192-201
	(SEQ. ID NO.:486)
	LLVPFVQWFW	HBV env 338-347
	(SEQ. ID NO.:487)
	ALMPLYACI	HBV pol 642-650
	(SEQ. ID NO.:488)
	YLVAYQATV	HCV NS3 579-587
	(SEQ. ID NO.:489)
	TLGIVCPIC	HIPV 16 E7 86-94
	(SEQ. ID NO.:490)
	YLLPRRGPRL	HCV core protein
	(SEQ. ID NO.:491)	34-43
	LLPIFFCLWV	HBV env 378-387
	(SEQ. ID NO.:492)
	YMDDVVLGA	HBV Pol 538-546
	(SEQ. ID NO.:493)
	GTLGIVCPI	HPV16 E7 85-93
	(SEQ. ID NO.:494)
	LLALLSCLTI	HCV MP 63-72
	(SEQ. ID NO.:495)
	MLDLQPETT	HPV 16 E7 12-20
	(SEQ. ID NO.:496)
	SLMAFTAAV	HCV NS4 174-182
	(SEQ. ID NO.:497)
	CINGVCWTV	HCV NS3 67-75
	(SEQ. ID NO.:498)
	VMNILLQYVV	Glutarnic acid
	(SEQ. ID NO.:499)	decarboxylase 114-123
	ILTVILGVL	Melan A/Mart - 32-40
	(SEQ. ID NO.:500)
	FLWGPRALV	MAGE-3 271-279
	(SEQ. ID NO.:501)
	L L C P A G H A V	HCV NS3 163-171
	(SEQ. ID NO.:502)
	ILDSFDPLV	HGV NSS 239-247
	(SEQ. ID NO.:503)
	LLLCLIFLL	HBV env 250-258
	(SEQ. ID NO.:504)
	LIDYQGMLPV	HBV env 260-269
	(SEQ. ID NO.:505)
	SIVSPFIPLL	HBV env 370-379
	(SEQ. ID NO;:506)
	FLLTRILTI	HBV env 183-191
	(SEQ. ID NO.:507)
	HLGNVKYLV	P. faciparum TRAP
	(SEQ. ID NO.:508)	3-11
	GIAGGLALL	P. faciparum TRAP
	(SEQ. ID NO.:509)	500-508
	ILAGYGAGV	HCV NS S4A 236-244
	(SEQ. ID NO.:510)
	GLQDCTMLV	HCV NS5 714-722
	(SEQ. ID NO.:511)
	TGAPVTYSTY	HCV NS3 281-290
	(SEQ. ID NO.:512)
	VIYQYMDDLV	HIV-1RT 179-187
	(SEQ. ID NO.:513)
	VLPDVFIRCV	N-acetylglucosaminyl-
	(SEQ. ID NO.:514)	transferase V Gnt-V
		intron
	VLPDVFIRC	N-acetylglucosaminyl-
	(SEQ. ID NO.:515)	transferase V Gnt-V
		intron
	AVGIGIAVV	Human CD9
	(SEQ. ID NO.:516)
	LVVLGLLAV	Human glutamyl-
	(SEQ. ID NO.:517)	transferase
	ALGLGLLPV	Human G protein coupled receptor
	(SEQ. ID NO.:518)
	164-172
	GIGIGVLAA	HSV-I gp C 480-488
	(SEQ. ID NO.:519)
	GAGIGVAVL	HSV-2 gp C 446-454
	(SEQ. ID NO.:520)
	IAGIGILAI	Pseudorabies gpGN
	(SEQ. ID NO.:521)	455-463
	LIVIGILIL	Adenovirus 3 E3 9kD
	(SEQ. ID NO.:522)	30-38
	LAGIGLIAA	S. Lincolnensis ImrA
	(SEQ. ID NO.:523)
	VDGIGILTI	Yeast ysa-1 77-85
	(SEQ. ID NO.:524)
	GAGIGVLTA	B. polymyxa,
	(SEQ. ID NO.:525)	βcndoxylanase 149-157
	157
	AAGIGIIQI	E. coli methionine
	(SEQ. ID NO.:526)	synthase 590-598
	QAGIGILLA	E. coli hypothetical
	(SEQ. ID NO.:527)	protein 4-12
	KARDPHSGHFV	CDK4w1 22-32
	(SEQ. ID NO.:528)
	KACDPI-ISGIIFV	CDK4-R24C 22-32
	(SEQ. ID NO.:529)
	ACDPFISGHFV	CDK4-R24C 23-32
	(SEQ. ID NO.:530)
	SLYNTVATL	HIV-I gag p 17 77-85
	(SEQ. ID NO.:531)
	ELVSEFSRV	HER-2, m>V
	(SEQ. lD NO.:532)	substituted 971-979
	RGPGRAFVTI	HIV-I gp 160 315-329
	(SEQ. ID NO.:533)
	HMWNFISGI	HCV NS4A 149-157
	(SEQ. ID NO.:534)
	NLVPMVATVQ	HCMV pp65 495-504
	(SEQ. ID NO.:535)
	GLHCYEQLV	HPV 6b E7 21-30
	(SEQ. ID NO.:536)
	PLKQHFQIV	HPV 6b E7 47-55
	(SEQ. ID NO.:537)
	LLDFVRFMGV	EBNA-6 284-293
	(SEQ. ID NO.:538)
	AIMEKNIML	Influenza Alaska NS 1
	(SEQ. ID NO.:539)	122-130
	YLKTIQNSL	P. falciparum cp36
	(SEQ. ID NO.:540)	CSP
	YLNKIQNSL	P. falciparum cp39
	(SEQ. ID NO.:541)	CSP
	YMLDLQPETT	HPV 16 E7 11-20*
	(SEQ. ID NO.:542)
	LLMGTLGIV	HPV 16 E7 82-90**
	(SEQ. ID NO.:543)
	TLGIVCPI	HPV 16 E7 86-93
	SEQ ID NO.:544
	TLTSCNTSV	HIV-1 gp120 197-205
	(SEQ. ID NO.:545)
	KLPQLCTEL	HPV 16 E6 18-26
	(SEQ. ID NO.:546)
	TIHDIILEC	HPV16 E6 29-37
	(SEQ. ID NO.:547)
	LGIVCPICS	HPV16 E7 87-95
	(SEQ. ID NO.:548)
	VILGVLLLI	Melan A/Mart-1 35-43
	(SEQ. ID NO.:549)
	ALMDKSLHV	Melan A/Mart-1 56-64
	(SEQ. ID NO.:550)
	GILTVILGV	Melan A/Mart-1 31-39
	(SEQ. ID NO.:551)
T cell	MINAYLDKL	P. Falciparum STARP
epitopes	(SEQ. ID NO.:552)	523-531
	AAGIGILTV	Melan A/Mart-1 27-35
	(SEQ. ID NO.:553)
	FLPSDFFPSV	HBV cAg 18-27
	(SEQ. ID NO.:554)
Motif	SVRDRLARL	EBNA-3 464-472
unknown	(SEQ. ID NO.:555)
T cell
epitopes
T cell	AAGIGILTV	Melan A/Mart-1 27-35
epitopes	(SEQ. ID NO.:556)
	FAYDGKDYI	Human MHC I-ot
	(SEQ. ID NO.:557)	140-148
T cell	AAGIGILTV	Melan A/Mart-1 27-35
epitopes	(SEQ. ID NO.:558)
	FLPSDFFPSV	HBV cAg 18-27
	(SEQ. ID NO.:559)
Motif	AAGIGILTV	Meland A/Mart-1 27-35
unknown	(SEQ. ID NO.:560)
T cell
epitopes
	FLPSDFFPSV	HBV cAg 18-27
	(SEQ. ID NO.:561)
	AAGIGILTV	Melan A/Mart-1 27-35
	(SEQ. ID NO.:562)
	ALLAVGATK	Pmel17 gp 100 17-25
	(SEQ. ID NO.:563)
T cell	R L R D L L L I V T R	HIV-1 gp41 768-778
epitopes	(SEQ. ID NO.:564)
	QVPLRPMTYK	HIV-1 nef 73-82
	(SEQ. ID NO.:565)
	TVYYGVPVWK	HIV-1 gp120 - 36-45
	(SEQ. ID NO.:566)
	RLRPGGKKK	HIV-1 gag p 17 20-29
	(SEQ. ID NO.:567)
	ILRGSVAHK	Influenza NP 265-273
	(SEQ. ID NO.:568)
	RLRAEAGVK	EBNA-3 603-611
	(SEQ. ID NO.:569)
	RLRDLLLIVTR	HIV-1 gp41 770-780
	(SEQ. ID NO.:570)
	VYYGVPVWK	HIV-I GP 120 38-46
	(SEQ. ID NO.:571)
	RVCEKMALY	HCV NS5 575-583
	(SEQ. ID NO.:572)
Motif	KIFSEVTLK	Unknown; muta
unknown	(SEQ. ID NO.:573)	melanoma peptide ted
T cell		(p I 83L) 175-183
epitope
	YVNVNMGLK*	HBV cAg 88-96
	(SEQ. ID NO.:574)
T cell	IVTDFSVIK	EBNA-4 416-424
epitopes	(SEQ. ID NO.:575)
	ELNEALELK	P53 343-351
	(SEQ. ID NO.:576)
	VPLRPMTYK	HIV-1 NEF 74-82
	(SEQ. ID NO.:577)
	AIFQSSMTK	HIV-I gagp24 325-333
	(SEQ. ID NO.:578)
	QVPLRPMTYK	HIV-1 nef 73-82
	(SEQ. ID NO.:579)
	TINYTIFK HCV	NSI 238-246
	(SEQ. ID NO.:580)
	AAVDLSHFLKEK	HIV-1 nef 83-94
	(SEQ. ID NO.:581)
	ACQ G V G G P G G H K	HIV-1 II 1B p24
	(SEQ. ID NO.:582)	349-359
HLA-A24	S Y L D S G I H F*	β-catenin, mutated
	(SEQ. ID NO.:583)	(proto-onocogen)
		29-37
T cell	RYLKDQQLL	HIV GP 41 583-591
epitopes	(SEQ. ID NO.:584)
	AYGLDFYIL	P15 melanoma Ag 10-18
	(SEQ. ID NO.:585)
	AFLPWHRLFL	Tyrosinase 206-215
	(SEQ. ID NO.:586)
	AFLPWHRLF	Tyrosinase 206-214
	(SEQ. ID NO.:587)
	RYSIFFDY	Ebna-3 246-253
	(SEQ. ID NO.:588)
T cell	ETINEEAAEW	HIV-1 gagp24 203-212
epitope	(SEQ. ID NO.:589)
T cell	STLPETTVVRR	HBV cAg 141-151
epitopes	(SEQ. ID NO.:590)
	MSLQRQFLR	ORF 3P-gp75 294-321
	(SEQ. ID NO.:591)	(bp)
	LLPGGRPYR	TRP (tyrosinase rel.)
	(SEQ. ID NO.:592)	197-205
T cell	IVGLNKIVR	HIV gagp24
epitope	(SEQ. ID NO.:593)	267-267-275
	AAGIGILTV	Melan A/Mart-1 27 35
	(SEQ. ID NO.:594)

[0132] Table 6 sets forth additional antigens useful in the invention that are available from the Ludwig Cancer Institute. The Table refers to patents in which the identified antigens can be found and as such are incorporated herein by reference. TRA refers to the tumor-related antigen and the LUD No. refers to the Ludwig Institute number.

TABLE 6
TRA	LUD No.	Patent No.	Date Patent Issued	Peptide (Antigen)	HLA
MAGE-4	5293	5,405,940	11 Apr. 1995	EVDPASNTY	HLA-A1
				(SEQ. ID NO.:979)
MAGE-41	5293	5,405,940	11 Apr. 1995	EVDPTSNTY	HLA-AI
				(SEQ ID NO:595)
MAGE-5	5293	5,405,940	11 Apr. 1995	EADPTSNTY	HLA-AI
				(SEQ ID NO:596)
MAGE-51	5293	5,405,940	11 Apr. 1995	EADPTSNTY	HLA-AI
				(SEQ ID NO:597)
MAGE-6	5294	5,405,940	11 Apr. 1995	EVDPIGHVY	HLA-A1
				(SEQ ID NO:598)
	5299.2	5,487,974	30 Jan. 1996	MLLAVLYCLL	HLA-A2
				(SEQ ID NO:599)
	5360	5,530,096	25 Jun. 1996	MLLAVLYCL	HLA-B44
				(SEQ ID NO:600)
Tyrosinase	5360.1	5,519,117	21 May 1996	SEIWRDIDFA	HLA-B44
				(SEQ ID NO:601)
				SEIWRDIDF
				(SEQ ID NO:602)
Tyrosinase	5431	5,774,316	28 Apr. 1998	XEIWRDIDF	HLA-B44
				(SEQ ID NO:603)
MAGE-2	5340	5,554,724	10 Sep. 1996	STLVEVTLGEV	HLA-A2
				(SEQ ID NO:604)
				LVEVTLGEV
				(SEQ ID NO:605)
				VIFSKASEYL
				(SEQ ID NO:606)
				IIVLAIIAl
				(SEQ ID NO:607)
				KIWEELSMLEV
				(SEQ ID NO:608)
				LIETSYVKV
				(SEQ ID NO:609)
	5327	5,585,461	17 Dec. 1996	FLWGPRALV	HLA-A2
				(SEQ ID NO:610)
				TLVEVTLGEV
				(SEQ ID NO:611)
				ALVETSYVKV
				(SEQ ID NO:612)
MAGE-3	5344	5,554,506	10 Sep. 1996	KIWEELSVL	HLA-A2
				(SEQ ID NO:613)
MAGE-3	5393	5,405,940	11 Apr. 1995	EVDPIGHLY	HLA-A1
				(SEQ ID NO:614)
MAGE	5293	5,405,940	11 Apr. 1995	EXDX5Y	HLA-A1
				(SEQ. ID NO.:615)
				(but not EADPTGHSY)
				SEQ. ID NO.:616)
				E (A/V) D X5 Y
				(SEQ. ID NO.:617)
				E (A/V) D P X4 Y
				(SEQ. ID NO.:618)
				E (A/V) D P (I/A/T) X3 Y
				(SEQ. ID NO.:619)
				E (A/V) D P (I/A/T) (G/S) X2 Y
				(SEQ. ID NO.:620)
				E (A/V) D P (I/A/T) (G/S) (H/N) X Y
				(SEQ. ID NO.:621)
				E (A/V) DP (I/A/T) (G/S) (H/N)
				(L/T/V) Y
				(SEQ. 11) NO.:622)
MAGE-1	5361	5,558,995	24 Sep. 1996	ELHSAYGEPRKLLTQD	HLA-C
				(SEQ ID NO:623)	Clone 10
				EHSAYGEPRKLL
				(SEQ ID NO:624)
				SAYGEPRKL
				(SEQ ID NO:625)
MAGE-1	5253.4	TBA	TBA	EADPTGHSY	HLA-AI
				(SEQ ID NO:626)
BAGE	5310.1	TBA	TBA	MAARAVFLALSAQLLQARLMKE	HLA-C
				(SEQ ID NO:627)	Clone 10
				MAARAVFLALSAQLLQ	HLA-C
				(SEQ ID NO:628)	Clone 10
				AARAVFLAL	HLA-C
				(SEQ ID NO:629)	Clone 10
GAGE	5323.2	5,648,226	15 Jul. 1997	YRPRPRRY	HLA-CW6
				(SEQ. ID NO.:630)

[0133]

TABLE 7
				T cell epitope
		AA	MHC	MHC ligand	SEQ.
Source	Protein	Position molecules	(Antigen)	ID NO.:	Ref.
synthetic	synthetic	synthetic	HLA-A2	ALFAAAAAV	631	Parker, et al., “Scheme for
peptides	peptides	peptides				ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIFGGVGGV	632	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLDKGGGV	633	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGFGGV	634	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGAGV	635	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGEGV	636	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGFGV	637	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGL	638	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGV	639	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGVGV	640	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGVGGV	641	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGVGKV	642	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFKGVGGV	643	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGGGFGV	644	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLLGGGVGV	645	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLYGGGGGV	646	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GMFGGGGGV	647	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GMFGGVGGV	648	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GQFGGVGGV	649	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GVFGGVGGV	650	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLFGGGGGV	651	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLFGGVGGV	652	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				AILGFVFTL	653	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GAIGFVFTL	654	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GALGFVFTL	655	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GELGFVFTL	656	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIAGFVFTL	657	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIEGFVFTL	658	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILAFVFTL	659	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGAVFTL	660	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGEVFTL	661	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILFGAFTL	662	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFEFTL	663	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFKFTL	664	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVATL	665	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVETL	666	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFAL	667	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFEL	668	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFKL	669	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFTA	670	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFTL	671	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVFVL	672	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGFVKTL	673	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILGKVFTL	674	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILKFVFTL	675	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GILPFVFTL	676	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIVGFVFTL	677	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GKLGFVFTL	678	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLLGFVFTL	679	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GQLGFVFTL	680	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KALGFVFTL	681	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KILGFVFTL	682	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KILGKVFTL	683	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				AILLGVFML	684	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				AIYKRWIIL	685	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ALFFFDIDL	686	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ATVELLSEL	687	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				CLFGYPVYV	688	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				FIFPNYTIV	689	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				IISLWDSQL	690	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILASLFAAV	691	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILESLFAAV	692	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLGEFFNQM	693	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLGEFYNQM	694	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LLFGYPVYV	695	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LLWKGEGAV	696	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LMFGYPVYV	697	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LNFGYPVYV	698	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LQFGYPVYV	699	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				NIVAHTFKV	700	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				NLPMVATV	701	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				QMLLAIARL	702	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				QMWQARLTV	703	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				RLLQTGIHV	704	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				RLVNGSLAL	705	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				SLYNTVATL	706	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				TLNAWVKVV	707	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				WLYRETCNL	708	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				YLFKRMIDL	709	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GAFGGVGGV	710	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GAFGGVGGY	711	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GEFGGVGGV	712	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GGFGGVGGV	713	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIFGGGGGV	714	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIGGFGGGL	715	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIGGGGGGL	716	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLDGGGGGV	717	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLDGKGGGV	718	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLDKKGGGV	719	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGFGF	720	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGFGG	721	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGFGN	722	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGFGS	723	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGI	724	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGM	725	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGT	726	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGY	727	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGFGGGGV	728	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGFGGGV	729	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGGFGGV	730	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGGGGFV	731	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGGGGGY	732	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLGGGVGGV	733	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLLGGGGGV	734	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLPGGGGGV	735	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GNFGGVGGV	736	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GSFGGVGGV	737	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GTFGGVGGV	738	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				AGNSAYEYV	739	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFPGQFAY	740	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				HILLGVFML	741	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILESLFRAV	742	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KKKYKLKHI	743	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				MLASIDLKY	744	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				MLERELVRK	745	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLFGFVFTV	746	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILDKKVEKV	747	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILKEPVHGV	748	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ALFAAAAAY	749	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIGFGGGGL	750	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GKFGGVGGV	751	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GLFGGGGGK	752	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				EILGFVFTL	753	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GIKGFVFTL	754	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				GQLGFVFTK	755	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				ILGFVFTLT	756	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KILGFVFTK	757	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KKLGFVFTL	758	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				KLFEKVYNY	759	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
				LRFGYPVYV	760	Parker, et al., “Scheme for
						ranking potential HLA-A2
						binding peptides based on
						independent binding of
						individual peptide side-
						chains,” J. Immunol.
						152:163-175
Human	HSP60	140-148	HLA-B27	IRRGVMLAV	761	Rammensee et al. 1997
						160
″	″	369-377	″	KRIQEIIEQ	762	Rammensee et al. 1997
						160
″	″	469-477	″	KRTLKIPAM	763	Rammensee et al. 1997
						160
Yersinia	HSP6O	35-43	GRNVVLDKS	764	Rammensee et al. 1997
						160
″	″	117-125	KRGIDKAVI	765	Rammensee et al. 1997
						160
″	″	420-428	IRAASAITA	766	Rammensee et al. 1997
						160
″	HSP 60	284-292	HLA-	RRKAMFEDI	767	169
			B*2705
P.	LSA-1	1850-1857	HLA-B3501	KPKDELDY	768	170
falciparum
Influenza		379-387	HLA-	LELRSRYWA	769	183
NP			B*4402
	Tum-P35B	4-13	HLA-Dd	GPPHSNNFGY	770	230
Rotavirus	VP7	33-40		IIYRFLLI	771	262
	OGDH	104-112	H2-Ld	QLSPYPFDL	772	253
	(F108Y)
	TRP-2	181-188	p287	VYDFFVWL	773	284
	DEAD box	547-554	p287	SNFVFAGI	774	283
	p 68
	Vector		p287	SVVEFSSL	775	260
	“artefact”
	Epitope		p287	AHYLFRNL	776	278
	mimic of
	tumor Ag
			″	THYLFRNL	777	″
	Epitope mimic		″	LIVIYNTL	778	279
	of H-3 miHAg”
			″	LIYEFNTL	779	″
			″	IPYIYNTL	780	″
			″	IIYIYHRL	781	″
			″	LIYIFNTL	782	″
	HBV cAg	93-100	″	MGLKFRQL	783	280
Human	autoantigen	51-58	″	IMIKFRNRL	784	281
	LA
Mouse	UTY protein		H2Db	WMHHNMDLI	785	303
Mouse	p53	232-240	″	KYMCNSSCM	786	302
MURINE	MDM2	441-449	″	GRPKNGCIV	787	277
	Epitope mimic		″	AQHPNAELL	788	278
	of natural
	MuLV	75-83	″	CCLCLTVFL	789	301
	gag75K
P.	CSP	375-383	p290	YENDIEKK	790	315
Falciparum
P.	″	371-379	″	DELDYENDI	791	315
Falciparum
HIV	−1RT	206-214	″	TEMEKEGKI	792	316
Rabies	NS	197-205		VEAEIAHQI	793	309, 310
Influenza	NS1	152-160	″	EEGAIVGEI	794	304
A
Murine	SMCY		p291	TENSGKDI	795	317
	MHC class	3-11	p293	AMAPRTLLL	796	318
	1 leader
	ND1alpha	1-12	p293	FFINILTLLVP	797	323
	ND Beta	1-12	p293	FFINILTLLVP	798	323
	ND alpha	1-17	″	FFINILTLLVPI	799	324
				LIAM
	ND Beta	1-17	″	FFINALTLLVPI	800	″
				LIAM
	COI	1-6	″	FINRW	801	325
	mitochondrial
L.	LemA	1-6	″	IGWII	802	326
monocyto-
genes
	SIV gag	179-190	Mamu-	EGCTPYDINQ	803	334
	p11C		A*01	ML
	MAGE-3		HLA-A2	ALSRKVAEL	804	5,554,506
			″	IMPKAGLLI	805	″
			″	KIWEELSVL	806	″
			″	ALVETSYVKV	807	″
			″	ThrLeuValGluVal	808	″
				ThrLeuGlyGluVal
			″	AlaLeuSerArgLys	809	″
				ValAlaGluLeu
			″	IleMetProLysAla	810	″
				GlyLeuLeuIle
			″	LysIleTrpGluGlu	811	″
				LeuSerValLeu
			″	AlaLeuValGluThr	812	″
				SerTyrValLysVal
	peptides		HLA-A2	Lys Gly Ile Leu	813	5,989,565
	which bind			Gly Phe Val Phe
	to MHCs			Thr Leu Thr Val	Thr Leu Thr Val
			″	Gly Ile Ile Gly	814	″
				Phe Val Phe Thr
				Ile
			″	Gly Ile Ile Gly	815	″
				Phe Val Phe Thr
				Leu
			″	Gly Ile Leu Gly	816	″
				Phe Val Phe Thr
				Leu
			″	Gly Leu Leu Gly	817	″
				Phe Val Phe Thr
				Leu
			″	XXTVXXGVX,	818	″
				X = Leu or Ile (6-37)
			″	Ile Leu Thr Val	819	″
				Ile Leu Gly Val
				Leu
			″	Tyr Leu Glu Pro	820	″
				Gly Pro Val Thr
				Ala
			″	Gln Val Pro Leu	821	″
				Arg Pro Met Thr
				Tyr Lys
			″	Asp Gly Leu Ala	822	″
				Pro Pro Gln His
				Leu Ile Arg
			″	Leu Leu Gly Arg	823	″
				Asn Ser Phe Glu
				Val
	Peptides from		HLA-C	GluHisSerAlaTyr	824	5,558,995
	MAGE-1		clone 10	GlyGluProArgLys
				LeuLeuThrGlnAsp
				Leu
			HLA-C	GluHisSerAlaTyr	825	″
			clone 10	GlyGluProArgLys
				LeuLeuThrGlnAsp
				Leu
			HLA-C	SerAlaTyrGlyGlu	826	″
			clone 10	ProArgLysLeu
	GAGE		HLA-Cw6	TyrArgProArgPro	827	5,648,226 ‘
				ArgArgTyr
			″	ThrTyrArgProArg	828	″
				ProArgArgTyr
			″	TyrArgProArgPro	829	″
				ArgArgTyrVal
			″	ThrTyrArgProArg	830	″
				ProArgArgTyrVal
			″	ArgProArgProArg	831	″
				ArgTyrValGlu
			″	MetSerTrpArgGly	832	″
				ArgSerThrTyrArg
				ProArgProArgArg
			″	ThrTyrArgProArg	833	″
				ProArgArgTyrVal
				GluProProGluMet
				Ile
	MAGE		HLA-A1,	Isolated nonapeptide	834	5,405,940
			primarily	having Glu at its N
				terminal, Tyr at its
				C-terminal, and Asp at
				the third residue from
				its N terminal, with
				the proviso that said
				isolated nonapeptide is
				not Glu Ala Asp Pro Thr
				Gly His Ser Tyr (SEQ ID
				NO: 1), and wherein
				said isolated nona-
				peptide binds to a
				human leukocyte antigen
				molecule on a cell to
				form a complex, said
				complex provoking lysis
				of said cell by a
				cytolytic T cell
				specific to said
				complex
			HLA-A1,	GluValValProIle	835	″
			primarily	SerHisLeuTyr
			HLA-A1,	GluValValArgIle	836	″
			primarily	GlyHisLeuTyr
			HLA-A1,	GluValAspProIle	837	″
			primarily	GlyHisLeuTyr
			HLA-A1,	GluValAspProAla	838	″
			primarily	SerAsnThrTyr
			HLA-A1,	GluValAspProThr	839	″
			primarily	SerAsnThrTyr
			HLA-A1,	GluAlaAspProThr	840	″
			primarily	SerAsnThrTyr
			HLA-A1,	GluValAspProIle	841	″
			primarily	GlyHisValTyr	″
			HLA-A1,	GAAGTGGTCC	842	″
			primarily	CCATCAGCCA
				CTTGTAC
			HLA-A1,	GAAGTGGTCC	843	″
			primarily	GCATCGGCCA
				CTTGTAC
			HLA-A1,	GAAGTGGAC	844	″
			primarily	CCCATCGGCC
				ACTTGTAC
			HLA-A1,	GAAGTGGAC	845	″
			primarily	CCCGCCAGCA
				ACACCTAC
			HLA-A1,	GAAGTGGAC	846	″
			primarily	CCCACCAGCA
				ACACCTAC
			HLA-A1,	GAAGCGGAC	847	″
			primarily	CCCACCAGCA
				ACACCTAC
			HLA-A1,	GAAGCGGAC	848	″
			primarily	CCCACCAGCA
				ACACCTAC
			HLA-A1,	GAAGTGGAC	849	″
			primarily	CCCATCGGCC
				ACGTGTAC
			HLA-A1,	GluAlaAspProThr	850	″
			primarily	GlyHisSer
			HLA-A1,	AlaAspProTrpGly	851	″
			primarily	HisSerTyr
	MAGE peptides		HLA-A2	SerThrLeuValGlu	852	5,554,724
				ValThrLeuGly
				GluVal
	″		″	LeuValGluValThr	853	″
				LeuGlyGluVal
	″		″	LysMetValGluLeu	854	″
				ValHisPheLeu
	″		″	ValIlePheSerLys	855	″
				AlaSerGluTyrLeu
	″		″	TyrLeuGlnLeuVal	856	″
				PheGlyIleGluVal
	″		″	GlnLeuValPheGly	857	″
				IleGluValVal
	″		″	GlnLeuValPheGly	858	″
				IleGluValValGlu
				Val
	″		″	IleIleValLeuAla	859	″
				IleIleAlaIle
	″		″	LysIleTrpGluGlu	860	″
				LeuSerMetLeuGlu
				Val
	″		″	AlaLeuIleGluThr	861	″
				SerTyrValLysVal
	″		″	LeuIleGluThrSer	862	″
				TyrValLysVal
	″		″	GlyLeuGluAlaArg	863	″
				GlyGluAlaLeuGly
				GlyLeu
	″		″	GlyLeuGluAlaArg	864	″
				GlyGluAlaLeu
	″		″	AlaLeuGlyLeuVal	865	″
				GlyAlaGlnAla
	″		″	GlyLeuValGlyAla	866	″
				GlnAlaProAla
	″		″	AspLeuGluSerGlu	867	″
				PheGlnAlaAla
	″		″	AspLeuGluSerGlu	868	″
				PheGlnAlaAlaIle
	″		″	AlaIleSerArgLys	869	″
				MetValGluLeuVal
	″		″	AlaIleSerArgLys	870	″
				MetValGluLeu
	″		″	LysMetValGluLeu	871	″
				ValHisPheLeuLeu
	″		″	LysMetValGluLeu	872	″
				ValHisPheLeu
				LeuLeu
	″		″	LeuLeuLeuLysTyr	873	″
				ArgAlaArgGlu
				ProVal
	″		″	LeuLeuLysTyrArg	874	″
				AlaArgGluProVal
	″		″	ValLeuArgAsnCys	875	″
				GlnAspPhePhe
				ProVal
	″		″	TyrLeuGlnLeuVal	876	″
				PheGlyIleGlu
				ValVal
	″		″	HisLeuTyrIleLeu	879	″
				ValThrCysLeu
	″		″	HisLeuTyrIleLeu	880	″
				ValThrCysLeuGly
				Leu
	″		″	TyrIleLeuValThr	881	″
				CysLeuGlyLeu
				CysLeuGlyLeuSer	882	″
				TyrAspGlyLeu
	″		″	CysLeuGlyLeuSer	883	″
				TyrAspGlyLeuLeu
	″		″	ValMetProLysThr	884	″
				GlyLeuLeuIle
	″		″	ValMetProLysThr	885	″
				GlyLeuLeuIleIle
	″		″	ValMetProLysThr	886	″
				GlyLeuLeuIleIle
				Val
	″		″	GlyLeuLeuIleIle	887	″
				ValLeuAlaIle
	″		″	GlyLeuLeuIleIle	888	″
				ValLeuAlaIleIle
	″		″	GlyLeuLeuIleIle	889	″
				ValLeuAlaIleIle
				Ala
	″		″	LeuLeuIleIleVal	890	″
				LeuAlaIleIle
	″		″	LeuLeuIleIleVal	891	″
				LeuAlaIleIleAla
	″		″	LeuLeuIleIleVal	892	″
				LeuAlaIleIleAlaIle
	″		″	LeuIleIleValLeu	893	″
				AlaIleIleAla
	″		″	LeuIleIleValLeu	894	″
				AlaIleIleAlaIle
	″		″	IleIleAlaIleGluGly	895	″
				AspCysAla
	″		″	LysIleTrpGluGlu	896	″
				LeuSerMetLeu
	″		″	LeuMetGlnAspLeu	897	″
				ValGlnGluAsn
				TyrLeu
	″		″	PheLeuTrpGlyPro	898	″
				ArgAlaLeuIle
	″		″	LeuIleGluThrSer	899	″
				TyrValLysVal
	″		″	AlaLeuIleGluThr	900	″
				SerTyrValLysVal
				Leu
	″		″	ThrLeuLysIleGly	901	″
				GlyGluProHisIle
	″		″	HisIleSerTyrPro	902	″
				ProLeuHisGluArg
				Ala
	″		″	GlnThrAlaSerSer	903	″
				SerSerThrLeu
	″		″	GlnThrAlaSerSer	904	″
				SerSerThrLeuVal
	″		″	ValThrLeuGlyGlu	905	″
				ValProAlaAla
	″		″	ValThrLysAlaGlu	906	″
				MetLeuGluSerVal
	″		″	ValThrLysAlaGlu	907	″
				MetLeuGluSer
				ValLeu
	″		″	ValThrCysLeuGly	908	″
				LeuSerTyrAsp
				GlyLeu
	″		″	LysThrGlyLeuLeu	909	″
				IleIleValLeu
	″		″	LysThrGlyLeuLeu	910	″
				IleIleValLeuAla
	″		″	LysThrGlyLeuLeu	911	″
				IleIleValLeuAla
				Ile
	″		″	HisThrLeuLysIle	912	″
				GlyGlyGluProHis
				Ile
	″		″	MetLeuAspLeu	913	″
				GlnProGluThrThr
	Mage-3		HLA-A2	GlyLeuGluAlaArg	914	5,585,461
	peptides			GlyGluAlaLeu
	Mage-3		″	AlaLeuSerArgLys	915	″
	peptides			ValAlaGluLeu
	Mage-3		″	PheLeuTrpGlyPro	916	″
	peptides			ArgAlaLeuVal
	Mage-3		″	ThrLeuValGluVal	917	″
	peptides			ThrLeuGlyGluVal
	Mage-3		″	AlaLeuSerArgLys	918	″
	peptides			ValAlaGluLeuVal
	Mage-3		″	AlaLeuValGluThr	919	″
	peptides			SerTyrValLysVal
	Tyrosinase		HLA-A2	TyrMetAsnGlyThr	920	5,487,974
				MetSerGlnVal
			″	MetLeuLeuAlaVal	921	″
				LeuTyrCysLeuLeu
	Tyrosinase		HLA-A2	MetLeuLeuAlaVal	922	5,530,096
				LeuTyrCysLeu
	″		″	LeuLeuAlaValLeu	923	″
				TyrCysLeuLeu
	Tyrosinase		HLA-A2 and	SerGluIleTrpArg	924	5,519,117
			HLA-B44	AspIleAspPheAla
				HisGluAla
	″		HLA-A2 and	SerGluIleTrpArg	925	″
			HLA-B44	AspIleAspPhe
	″		HLA-A2 and	GluGluAsnLeuLeu	926	″
			HLA-B44	AspPheValArg
				Phe
	Melan			EAAGIGILTV	927	Jäger, E. et al.
	A/MART-1					Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	Tyrosinase			MLLAVLYCL	928	Jäger, E. et al.
						Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	″			YMDGTMSQV	929	Jäger, E. et al.
						Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	gp100/Pme			YLEPGPVTA	930	Jäger, E. et al.
	117					Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	gp100/Pme			LLDGTATLRL	931	Jäger, E. et al.
	117					Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	Influenza			GILGFVFTL	932	Jäger, E. et al.
	matrix					Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	MAGE-1			EADPTGHSY	933	Jäger, E. et al.
						Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	MAGE-1		HLA-A1	EADPTGHSY	934
	BAGE		HLA-C	MAARAVFLAL	935	Jäger, E. et al.
				SAQLLQARLM		Granulocyte-macrophage-
				KE	colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	″		″	MAARAVFLAL	936	Jäger, E. et al.
				SAQLLQ		Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
	″		″	AARAVFLAL	937	Jäger, E. et al.
						Granulocyte-macrophage-
						colony-stimulating Factor
						Enhances Immune Responses
						To Melanoma-′associated
						Peptides in vivo Int. J
						Cancer 67, 54-62 (1996)
Influenza	PRS NP	147-154 Kd	IYQRIRALV	938	Falk et al., Allele-
						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
SELF	P815		″	SYFPEITHI	939	Falk et al., Allele-
PEPTIDE						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
Influenza	Jap HA		″	IYATVAGSL	940	Falk et al., Allele-
	523-549					specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
″	Jap HA		″	VYQILAIYA	941	Falk et al., Allele-
	523-549					specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
″	Jap HA		″	IYSTVASSL	942	Falk et al., Allele-
	523-549					specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
″	JAP HA		″	LYQNVGTYV	943	Falk et al., Allele-
	202-221					specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
	HLA-A24		″	RYLENQKRT	944	Falk et al., Allele-
						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
	HLA-Cw3		″	RYLKNGKET	945	Falk et al., Allele-
						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
	P815		″	KYQAVTTTL	946	Falk et al., Allele-
						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
Plasmodium	CSP		″	SYIPSAEKI	947	Falk et al., Allele-
berghei						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
Plasmodium	CSP		″	SYVPSAFQI	948	Falk et al., Allele-
yoehi						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
Vesicular	NP 52-59		Kb	RGYVYQGL	949	Falk et al., Allele-
stomatitis						specific motifs revealed
viruse						by sequencing of self-
						peptides eluted from
						MHC molecules
Ovalbumin			″	SIINFEKL	950	Falk et al., Allele-
						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
Sendai	NP 321-332	APGNYPAL	951	Falk et al., Allele-
virus						specific motifs revealed
						by sequencing of self-
						peptides eluted from
						MHC molecules
				VPYGSFKHV	952	Morel et al., Processing
						of some antigens by the
						standard proteasome but not
						by the immunoproteasome
						results in poor
						presentation by dendritic
						cells, Immunity, vol.
						12:107-117, 2000.
			MOTIFS
influenza	PRS NP		Kd	TYQRTRALV	953	5,747,269
			restricted
			peptide
			motif
self peptide	P815		Kd	SYFPEITHI	954	″
			restricted
			peptide
			motif
influenza	JAP HA		Kd	IYATVAGSL	955	″
			restricted
			peptide
			motif
influenza	JAP HA		Kd	VYQILAIYA	956	″
			restricted
			peptide
			motif
influenza	PR8 HA		Kd	IYSTVASSL	957	″
			restricted
			peptide
			motif
influenza	JAP HA		Kd	LYQNVGTYV	958	″
			restricted
			peptide
			motif
			HLA-A24	RYLENGKETL	959	″
			HLA-Cw3	RYLKNGKETL	960	″
	P815 tumour		″	KYQAVTTTL	961	″
	antigen
Plasmodium	CSP		″	SYIPSAEKI	962	″
berghei
Plasmodium	CSP		″	SYVPSAEQI	963	″
yoelii
influenza	NP		Db -	ASNENMETM	964	″
			restricted
			peptide
			motif
adenovirus	EIA		Db -	SGPSNTPPEI	965	″
			restricted
			peptide
			motif
lymphocytic			Db -	SGVENPGGYC	966	″
chorio-			restricted	L
meningitis			peptide
			motif
simian virus	40 T		Db -	SAINNY...	967	″
			restricted
			peptide
			motif
HIV	reverse		HLA-A2.1 -	ILKEPVHGV	968	″
	transcriptase		restricted
			peptide
			motif
	influenza		HLA-A2.1 -	GILGFVFTL	969	″
	matrix		restricted
	protein		peptide
			motif
influenza	influenza		HLA-A2.1 -	ILGFVFTLTV	970	″
	matrix		restricted
	protein		peptide
			motif
HIV	Gag protein			FLQSRPEPT	971	″
HIV	Gag protein			AMQMLKE..	972	″
HIV	Gag protein			PIAPGQMRE	973	″
HIV	Gag protein			QMKDCTERQ	974	″
			HLA-	VYGVIQK	975	″
			A*0205 -
			restricted
			peptide
			motif

[0134]

TABLE 8
VSV-NP peptide (49-62)
LCMV-NP peptide (118-132)
LCMV glycoprotein peptide. 33-41

[0135] Still further embodiments are directed to methods, uses, therapies and compositions related to epitopes with specificity for MHC, including, for example, those listed in Tables 9-13. Other embodiments include one or more of the MHCs listed in Tables 9-13, including combinations of the same, while other embodiments specifically exclude any one or more of the MHCs or combinations thereof. Tables 11-13 include frequencies for the listed HLA antigens.

TABLE 9
Class I MHC Molecules
Class I
Human
HLA-A1
HLA-A*0101
HLA-A*0201
HLA-A*0202
HLA-A*0203
HLA-A*0204
HLA-A*0205
HLA-A*0206
HLA-A*0207
HLA-A*0209
HLA-A*0214
HLA-A3
HLA-A*0301
HLA-A*1101
HLA-A23
HLA-A24
HLA-A25
HLA-A*2902
HLA-A*3101
HLA-A*3302
HLA-A*6801
HLA-A*6901
HLA-B7
HLA-B*0702
HLA-B*0703
HLA-B*0704
HLA-B*0705
HLA-B8
HLA-B13
HLA-B14
HLA-B*1501 (B62)
HLA-B17
HLA-B18
HLA-B22
HLA-B27
HLA-B*2702
HLA-B*2704
HLA-B*2705
HLA-B*2709
HLA-B35
HLA-B*3501
HLA-B*3502
HLA-B*3701
HLA-B*3801
HLA-B*39011
HLA-B*3902
HLA-B40
HLA-B*40012 (B60)
HLA-B*4006 (B61)
HLA-B44
HLA-B*4402
HLA-B*4403
HLA-B*4501
HLA-B*4601
HLA-B51
HLA-B*5101
HLA-B*5102
HLA-B*5103
HLA-B*5201
HLA-B*5301
HLA-B*5401
HLA-B*5501
HLA-B*5502
HLA-B*5601
HLA-B*5801
HLA-B*6701
HLA-B*7301
HLA-B*7801
HLA-Cw*0102
HLA-Cw*0301
HLA-Cw*0304
HLA-Cw*0401
HLA-Cw*0601
HLA-Cw*0602
HLA-Cw*0702
HLA-Cw8
HLA-Cw*1601 M
HLA-G
Murine
H2-Kd
H2-Dd
H2-Ld
H2-Kb
H2-Db
H2-Kk
H2-Kkml
Qa-1a
Qa-2
H2-M3
Rat
RT1.Aa
RT1.Al
Bovine
Bota-A11
Bota-A20
Chicken
B-F4
B-F12
B-F15
B-F19
Chimpanzee
Patr-A*04
Patr-A*11
Patr-B*01
Patr-B*13
Patr-B*16
Baboon
Papa-A*06
Macaque
Mamu-A*01
Swine
SLA (haplotype d/d)
Virus homolog
hCMV class I homolog UL18

[0136]

TABLE 10
Class I MHC Molecules
Class I
Human
HLA-A1
HLA-A*0101
HLA-A*0201
HLA-A*0202
HLA-A*0204
HLA-A*0205
HLA-A*0206
HLA-A*0207
HLA-A*0214
HLA-A3
HLA-A*1101
HLA-A24
HLA-A*2902
HLA-A*3101
HLA-A*3302
HLA-A*6801
HLA-A*6901
HLA-B7
HLA-B*0702
HLA-B*0703
HLA-B*0704
HLA-B*0705
HLA-B8
HLA-B14
HLA-B*1501 (B62)
HLA-B27
HLA-B*2702
HLA-B*2705
HLA-B35
HLA-B*3501
HLA-B*3502
HLA-B*3701
HLA-B*3801
HLA-B*39011
HLA-B*3902
HLA-B40
HLA-B*40012 (B60)
HLA-B*4006 (B61)
HLA-B44
HLA-B*4402
HLA-B*4403
HLA-B*4601
HLA-B51
HLA-B*5101
HLA-B*5102
HLA-B*5103
HLA-B*5201
HLA-B*5301
HLA-B*5401
HLA-B*5501
HLA-B*5502
HLA-B*5601
HLA-B*5801
HLA-B*6701
HLA-B*7301
HLA-B*7801
HLA-Cw*0102
HLA-Cw*0301
HLA-Cw*0304
HLA-Cw*0401
HLA-Cw*0601
HLA-Cw*0602
HLA-Cw*0702
HLA-G
Murine
H2-Kd
H2-Dd
H2-Ld
H2-Kb
H2-Db
H2-Kk
H2-Kkml
Qa-2
Rat
RT1.Aa
RT1.Al
Bovine
Bota-A11
Bota-A20
Chicken
B-F4
B-F12
B-F15
B-F19
Virus homolog
hCMV class I homolog UL18

[0137]

TABLE 11
Estimated gene frequencies of HLA-A antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
A1	15.1843	0.0489	5.7256	0.0771	4.4818	0.0846	7.4007	0.0978	12.0316	0.2533
A2	28.6535	0.0619	18.8849	0.1317	24.6352	0.1794	28.1198	0.1700	29.3408	0.3585
A3	13.3890	0.0463	8.4406	0.0925	2.6454	0.0655	8.0789	0.1019	11.0293	0.2437
A28	4.4652	0.0280	9.9269	0.0997	1.7657	0.0537	8.9446	0.1067	5.3856	0.1750
A36	0.0221	0.0020	1.8836	0.0448	0.0148	0.0049	0.1584	0.0148	0.1545	0.0303
A23	1.8287	0.0181	10.2086	0.1010	0.3256	0.0231	2.9269	0.0628	1.9903	0.1080
A24	9.3251	0.0395	2.9668	0.0560	22.0391	0.1722	13.2610	0.1271	12.6613	0.2590
A9 unsplit	0.0809	0.0038	0.0367	0.0063	0.0858	0.0119	0.0537	0.0086	0.0356	0.0145
A9 total	11.2347	0.0429	13.2121	0.1128	22.4505	0.1733	16.2416	0.1382	14.6872	0.2756
A25	2.1157	0.0195	0.4329	0.0216	0.0990	0.0128	1.1937	0.0404	1.4520	0.0924
A26	3.8795	0.0262	2.8284	0.0547	4.6628	0.0862	3.2612	0.0662	2.4292	0.1191
A34	0.1508	0.0052	3.5228	0.0610	1.3529	0.0470	0.4928	0.0260	0.3150	0.0432
A43	0.0018	0.0006	0.0334	0.0060	0.0231	0.0062	0.0055	0.0028	0.0059	0.0059
A66	0.0173	0.0018	0.2233	0.0155	0.0478	0.0089	0.0399	0.0074	0.0534	0.0178
A10 unsplit	0.0790	0.0038	0.0939	0.0101	0.1255	0.0144	0.0647	0.0094	0.0298	0.0133
A10 total	6.2441	0.0328	7.1348	0.0850	6.3111	0.0993	5.0578	0.0816	4.2853	0.1565
A29	3.5796	0.0252	3.2071	0.0582	1.1233	0.0429	4.5156	0.0774	3.4345	0.1410
A30	2.5067	0.0212	13.0969	0.1129	2.2025	0.0598	4.4873	0.0772	2.5314	0.1215
A31	2.7386	0.0221	1.6556	0.0420	3.6005	0.0761	4.8328	0.0800	6.0881	0.1855
A32	3.6956	0.0256	1.5384	0.0405	1.0331	0.0411	2.7064	0.0604	2.5521	0.1220
A33	1.2080	0.0148	6.5607	0.0822	9.2701	0.1191	2.6593	0.0599	1.0754	0.0796
A74	0.0277	0.0022	1.9949	0.0461	0.0561	0.0096	0.2027	0.0167	0.1068	0.0252
A19 unsplit	0.0567	0.0032	0.2057	0.0149	0.0990	0.0128	0.1211	0.0129	0.0475	0.0168
A19 total	13.8129	0.0468	28.2593	0.1504	17.3846	0.1555	19.5252	0.1481	15.8358	0.2832
AX	0.8204	0.0297	4.9506	0.0963	2.9916	0.1177	1.6332	0.0878	1.8454	0.1925
a Gene frequency.
b Standard error.

[0138]

TABLE 12
Estimated gene frequencies for HLA-B antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
B7	12.1782	0.0445	10.5960	0.1024	4.2691	0.0827	6.4477	0.0918	10.9845	0.2432
B8	9.4077	0.0397	3.8315	0.0634	1.3322	0.0467	3.8225	0.0715	8.5789	0.2176
B13	2.3061	0.0203	0.8103	0.0295	4.9222	0.0886	1.2699	0.0416	1.7495	0.1013
B14	4.3481	0.0277	3.0331	0.0566	0.5004	0.0287	5.4166	0.0846	2.9823	0.1316
B18	4.7980	0.0290	3.2057	0.0582	1.1246	0.0429	4.2349	0.0752	3.3422	0.1391
B27	4.3831	0.0278	1.2918	0.0372	2.2355	0.0603	2.3724	0.0567	5.1970	0.1721
B35	9.6614	0.0402	8.5172	0.0927	8.1203	0.1122	14.6516	0.1329	10.1198	0.2345
B37	1.4032	0.0159	0.5916	0.0252	1.2327	0.0449	0.7807	0.0327	0.9755	0.0759
B41	0.9211	0.0129	0.8183	0.0296	0.1303	0.0147	1.2818	0.0418	0.4766	0.0531
B42	0.0608	0.0033	5.6991	0.0768	0.0841	0.0118	0.5866	0.0284	0.2856	0.0411
B46	0.0099	0.0013	0.0151	0.0040	4.9292	0.0886	0.0234	0.0057	0.0238	0.0119
B47	0.2069	0.0061	0.1305	0.0119	0.0956	0.0126	0.1832	0.0159	0.2139	0.0356
B48	0.0865	0.0040	0.1316	0.0119	2.0276	0.0575	1.5915	0.0466	1.0267	0.0778
B53	0.4620	0.0092	10.9529	0.1039	0.4315	0.0266	1.6982	0.0481	1.0804	0.0798
B59	0.0020	0.0006	0.0032	0.0019	0.4277	0.0265	0.0055	0.0028	0c	—
B67	0.0040	0.0009	0.0086	0.0030	0.2276	0.0194	0.0055	0.0028	0.0059	0.0059
B70	0.3270	0.0077	7.3571	0.0866	0.8901	0.0382	1.9266	0.0512	0.6901	0.0639
B73	0.0108	0.0014	0.0032	0.0019	0.0132	0.0047	0.0261	0.0060	0c	—
B51	5.4215	0.0307	2.5980	0.0525	7.4751	0.1080	6.8147	0.0943	6.9077	0.1968
B52	0.9658	0.0132	1.3712	0.0383	3.5121	0.0752	2.2447	0.0552	0.6960	0.0641
B5 unsplit	0.1565	0.0053	0.1522	0.0128	0.1288	0.0146	0.1546	0.0146	0.1307	0.0278
B5 total	6.5438	0.0435	4.1214	0.0747	11.1160	0.1504	9.2141	0.1324	7.7344	0.2784
B44	13.4838	0.0465	7.0137	0.0847	5.6807	0.0948	9.9253	0.1121	11.8024	0.2511
B45	0.5771	0.0102	4.8069	0.0708	0.1816	0.0173	1.8812	0.0506	0.7603	0.0670
B12 unsplit	0.0788	0.0038	0.0280	0.0055	0.0049	0.0029	0.0193	0.0051	0.0654	0.0197
B12 total	14.1440	0.0474	11.8486	0.1072	5.8673	0.0963	11.8258	0.1210	12.6281	0.2584
B62	5.9117	0.0320	1.5267	0.0404	9.2249	0.1190	4.1825	0.0747	6.9421	0.1973
B63	0.4302	0.0088	1.8865	0.0448	0.4438	0.0270	0.8083	0.0333	0.3738	0.0471
B75	0.0104	0.0014	0.0226	0.0049	1.9673	0.0566	0.1101	0.0123	0.0356	0.0145
B76	0.0026	0.0007	0.0065	0.0026	0.0874	0.0120	0.0055	0.0028	0	—
B77	0.0057	0.0010	0.0119	0.0036	0.0577	0.0098	0.0083	0.0034	0c	0.0059
B15 unsplit	0.1305	0.0049	0.0691	0.0086	0.4301	0.0266	0.1820	0.0158	0.0059	0.0206
B15 total	6.4910	0.0334	3.5232	0.0608	12.2112	0.1344	5.2967	0.0835	0.0715	0.2035
									7.4290
B38	2.4413	0.0209	0.3323	0.0189	3.2818	0.0728	1.9652	0.0517	1.1017	0.0806
B39	1.9614	0.0188	1.2893	0.0371	2.0352	0.0576	6.3040	0.0909	4.5527	0.1615
B16 unsplit	0.0638	0.0034	0.0237	0.0051	0.0644	0.0103	0.1226	0.0130	0.0593	0.0188
B16 total	4.4667	0.0280	1.6453	0.0419	5.3814	0.0921	8.3917	0.1036	5.7137	0.1797
B57	3.5955	0.0252	5.6746	0.0766	2.5782	0.0647	2.1800	0.0544	2.7265	0.1260
B58	0.7152	0.0114	5.9546	0.0784	4.0189	0.0803	1.2481	0.0413	0.9398	0.0745
B17 unsplit	0.2845	0.0072	0.3248	0.0187	0.3751	0.0248	0.1446	0.0141	0.2674	0.0398
B17 total	4.5952	0.0284	11.9540	0.1076	6.9722	0.1041	3.5727	0.0691	3.9338	0.1503
B49	1.6452	0.0172	2.6286	0.0528	0.2440	0.0200	2.3353	0.0562	1.5462	0.0953
B50	1.0580	0.0138	0.8636	0.0304	0.4421	0.0270	1.8883	0.0507	0.7862	0.0681
B21 unsplit	0.0702	0.0036	0.0270	0.0054	0.0132	0.0047	0.0771	0.0103	0.0356	0.0145
B21 total	2.7733	0.0222	3.5192	0.0608	0.6993	0.0339	4.3007	0.0755	2.3680	0.1174
B54	0.0124	0.0015	0.0183	0.0044	2.6873	0.0660	0.0289	0.0063	0.0534	0.0178
B55	1.9046	0.0185	0.4895	0.0229	2.2444	0.0604	0.9515	0.0361	1.4054	0.0909
B56	0.5527	0.0100	0.2686	0.0170	0.8260	0.0368	0.3596	0.0222	0.3387	0.0448
B22 unsplit	0.1682	0.0055	0.0496	0.0073	0.2730	0.0212	0.0372	0.0071	0.1246	0.0272
B22 total	2.0852	0.0217	0.8261	0.0297	6.0307	0.0971	1.3771	0.0433	1.9221	0.1060
B60	5.2222	0.0302	1.5299	0.0404	8.3254	0.1135	2.2538	0.0553	5.7218	0.1801
B61	1.1916	0.0147	0.4709	0.0225	6.2072	0.0989	4.6691	0.0788	2.6023	0.1231
B40 unsplit	0.2696	0.0070	0.0388	0.0065	0.3205	0.0230	0.2473	0.0184	0.2271	0.0367
B40 total	6.6834	0.0338	2.0396	0.0465	14.8531	0.1462	7.1702	0.0963	8.5512	0.2168
BX	1.0922	0.0252	3.5258	0.0802	3.8749	0.0988	2.5266	0.0807	1.9867	0.1634
a Gene frequency.
b Standard error.
c The observed gene count was zero.

[0139]

TABLE 13
Estimated gene frequencies of HLA-DR antigens
	CAU	AFR	ASI	LAT	NAT
Antigen	Gfa	SEb	Gf	SE	Gf	SE	Gf	SE	Gf	SE
DR1	10.2279	0.0413	6.8200	0.0832	3.4628	0.0747	7.9859	0.1013	8.2512	0.2139
DR2	15.2408	0.0491	16.2373	0.1222	18.6162	0.1608	11.2389	0.1182	15.3932	0.2818
DR3	10.8708	0.0424	13.3080	0.1124	4.7223	0.0867	7.8998	0.1008	10.2549	0.2361
DR4	16.7589	0.0511	5.7084	0.0765	15.4623	0.1490	20.5373	0.1520	19.8264	0.3123
DR6	14.3937	0.0479	18.6117	0.1291	13.4471	0.1404	17.0265	0.1411	14.8021	0.2772
DR7	13.2807	0.0463	10.1317	0.0997	6.9270	0.1040	10.6726	0.1155	10.4219	0.2378
DR8	2.8820	0.0227	6.2673	0.0800	6.5413	0.1013	9.7731	0.1110	6.0059	0.1844
DR9	1.0616	0.0139	2.9646	0.0559	9.7527	0.1218	1.0712	0.0383	2.8662	0.1291
DR10	1.4790	0.0163	2.0397	0.0465	2.2304	0.0602	1.8044	0.0495	1.0896	0.0801
DR11	9.3180	0.0396	10.6151	0.1018	4.7375	0.0869	7.0411	0.0955	5.3152	0.1740
DR12	1.9070	0.0185	4.1152	0.0655	10.1365	0.1239	1.7244	0.0484	2.0132	0.1086
DR5 unsplit	1.2199	0.0149	2.2957	0.0493	1.4118	0.0480	1.8225	0.0498	1.6769	0.0992
DR5 total	12.4449	0.0045	17.0260	0.1243	16.2858	0.1516	10.5880	0.1148	9.0052	0.2218
DRX	1.3598	0.0342	0.8853	0.0760	2.5521	0.1089	1.4023	0.0930	2.0834	0.2037
a Gene frequency.
b Standard error.

[0140] It can be desirable to express housekeeping peptides in the context of a larger protein. Processing can be detected even when a small number of amino acids are present beyond the terminus of an epitope. Small peptide hormones are usually proteolytically processed from longer translation products, often in the size range of approximately 60-120 amino acids. This fact has led some to assume that this is the minimum size that can be efficiently translated. In some embodiments, the housekeeping peptide can be embedded in a translation product of at least about 60 amino acids, in others 70, 80, 90 amino acids, and in still others 100, 110 or 120 amino acids, for example. In other embodiments the housekeeping peptide can be embedded in a translation product of at least about 50, 30, or 15 amino acids.

[0141] Due to differential proteasomal processing, the immunoproteasome of the pAPC produces peptides that are different from those produced by the housekeeping proteasome in peripheral body cells. Thus, in expressing a housekeeping peptide in the context of a larger protein, it is preferably expressed in the pAPC in a context other than its full-length native sequence, because, as a housekeeping epitope, it is generally only efficiently processed from the native protein by the housekeeping proteasome, which is not active in the pAPC. In order to encode the housekeeping epitope in a DNA sequence encoding a larger polypeptide, it is useful to find flanking areas on either side of the sequence encoding the epitope that permit appropriate cleavage by the immunoproteasome in order to liberate that housekeeping epitope. Such a sequence promoting appropriate processing is referred to hereinafter as having substrate or liberation sequence function. Altering flanking amino acid residues at the N-terminus and C-terminus of the desired housekeeping epitope can facilitate appropriate cleavage and generation of the housekeeping epitope in the pAPC. Sequences embedding housekeeping epitopes can be designed de novo and screened to determine which can be successfully processed by immunoproteasomes to liberate housekeeping epitopes.

[0142] Alternatively, another strategy is very effective for identifying sequences allowing production of housekeeping epitopes in APC. A contiguous sequence of amino acids can be generated from head to tail arrangement of one or more housekeeping epitopes. A construct expressing this sequence is used to immunize an animal, and the resulting T cell response is evaluated to determine its specificity to one or more of the epitopes in the array. These immune responses indicate housekeeping epitopes that are processed in the pAPC effectively. The necessary flanking areas around this epitope are thereby defined. The use of flanking regions of about 4-6 amino acids on either side of the desired peptide can provide the necessary information to facilitate proteasome processing of the housekeeping epitope by the immunoproteasome. Therefore, a substrate or liberation sequence of approximately 16-22 amino acids can be inserted into, or fused to, any protein sequence effectively to result in that housekeeping epitope being produced in an APC. In some embodiments, a broader context of a substrate sequence can also influence processing. In such embodiments, comparisons of a liberaton sequence in a variety of contexts can be useful in further optimizing a particular substrate sequence. In alternate embodiments the whole head-to-tail array of epitopes, or just the epitopes immediately adjacent to the correctly processed housekeeping epitope can be similarly transferred from a test construct to a vaccine vector.

[0143] In a preferred embodiment, the housekeeping epitopes can be embedded between known immune epitopes, or segments of such, thereby providing an appropriate context for processing. The abutment of housekeeping and immune epitopes can generate the necessary context to enable the immunoproteasome to liberate the housekeeping epitope, or a larger fragment, preferably including a correct C-terminus. It can be useful to screen constructs to verify that the desired epitope is produced. The abutment of housekeeping epitopes can generate a site cleavable by the immunoproteasome. Some embodiments of the invention employ known epitopes to flank housekeeping epitopes in test substrates; in others, screening as described below is used, whether the flanking regions are arbitrary sequences or mutants of the natural flanking sequence, and whether or not knowledge of proteasomal cleavage preferences are used in designing the substrates.

[0144] Cleavage at the mature N-terminus of the epitope, while advantageous, is not required, since a variety of N-terminal trimming activities exist in the cell that can generate the mature N-terminus of the epitope subsequent to proteasomal processing. It is preferred that such N-terminal extension be less than about 25 amino acids in length and it is further preferred that the extension have few or no proline residues. Preferably, in screening, consideration is given not only to cleavage at the ends of the epitope (or at least at its C-terminus), but consideration also can be given to ensure limited cleavage within the epitope.

[0145] Shotgun approaches can be used in designing test substrates and can increase the efficiency of screening. In one embodiment multiple epitopes can be assembled one after the other, with individual epitopes possibly appearing more than once. The substrate can be screened to determine which epitopes can be produced. In the case where a particular epitope is of concern, a substrate can be designed in which it appears in multiple different contexts. When a single epitope appearing in more than one context is liberated from the substrate additional secondary test substrates, in which individual instances of the epitope are removed, disabled, or are unique, can be used to determine which are being liberated and truly confer substrate or liberation sequence function.

[0146] Several readily practicable screens exist. A preferred in vitro screen utilizes proteasomal digestion analysis, using purified immunoproteasomes, to determine if the desired housekeeping epitope can be liberated from a synthetic peptide embodying the sequence in question. The position of the cleavages obtained can be determined by techniques such as mass spectrometry, HPLC, and N-terminal pool sequencing; as described in greater detail in U.S. patent application Ser. Nos. 09/561,074, 09/560,465 and 10/117,937, and Provisional U.S. Patent Application Nos. 60/282,211, 60/337,017, and 60/363, 210, which were all cited and incorporated by reference above.

[0147] Alternatively, in vivo and cell-based screens such as immunization or target sensitization can be employed. For immunization a nucleic acid construct capable of expressing the sequence in question is used. Harvested CTL can be tested for their ability to recognize target cells presenting the housekeeping epitope in question. Such targets cells are most readily obtained by pulsing cells expressing the appropriate MHC molecule with synthetic peptide embodying the mature housekeeping epitope. Alternatively, immunization can be carried out using cells known to express housekeeping proteasome and the antigen from which the housekeeping epitope is derived, either endogenously or through genetic engineering. To use target sensitization as a screen, CTL, or preferably a CTL clone, that recognizes the housekeeping epitope can be used. In this case it is the target cell that expresses the embedded housekeeping epitope (instead of the pAPC during immunization) and it must express immunoproteasome. Generally, the cell or target cell can be transformed with an appropriate nucleic acid construct to confer expression of the embedded housekeeping epitope. Loading with a synthetic peptide embodying the embedded epitope using peptide loaded liposomes, or complexed with cationic lipid protein transfer reagents such as BIOPORTER™ (Gene Therapy Systems, San Diego, Calif.), represents an alternative.

[0148] Once sequences with substrate or liberation sequence function are identified they can be encoded in nucleic acid vectors, chemically synthesized, or produced recombinantly. In any of these forms they can be incorporated into immunogenic compositions. Such compositions can be used in vitro in vaccine development or in the generation or expansion of CTL to be used in adoptive immunotherapy. In vivo they can be used to induce, amplify or sustain and active immune response. The uptake of polypeptides for processing and presentation can be greatly enhanced by packaging with cationic lipid, the addition of a tract of cationic amino acids such as poly-L-lysine (Ryser, H. J. et al., J. Cell Physiol. 113:167-178, 1982; Shen, W. C. & Ryser, H. J., Proc. Natl. Aced. Sci. USA 75:1872-1876, 1978), the incorporation into branched structures with importation signals (Sheldon, K. et al., Proc. Natl. Aced. Sci. USA 92:2056-2060, 1995), or mixture with or fusion to polypeptides with protein transfer function including peptide carriers such as pep-1 (Morris, M. C., et al., Nat. Biotech. 19:1173-1176, 2001), the PreS2 translocation motif of hepatitis B virus surface antigen, VP22 of herpes viruses, and HIV-TAT protein (Oess, S. & Hildt, E., Gene Ther. 7:750-758, 2000; Ford, K. G., et al., Gene Ther. 8:1-4, 2001; Hung, C. F. et al., J. Virol. 76:2676-2682, 2002; Oliveira, S. C., et al. Hum. Gene Ther. 12:1353-1359, 2001; Normand, N. et al., J. Biol. Chem. 276:15042-15050, 2001; Schwartz, J. J. & Zhang, S., Curr. Opin. Mol. Ther. 2:162-167, 2000; Elliot G., 7 Hare, P. Cell 88:223-233, 1997), among other methodologies. Particularly for fusion proteins the immunogen can be produced in culture and the purified protein administered or, in the alternative, the nucleic acid vector can be administered so that the immunogen is produced and secreted by cells transformed in vivo. In either scenario the transport function of the fusion protein facilitates uptake by pAPC.

[0149] The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.

EXAMPLES

Example 1

[0150] A recombinant DNA plasmid vaccine, pMA2M, which encodes one polypeptide with an HLA A2-specific CTL epitope ELAGIGILTV (SEQ ID NO. 1) from melan-A (26-35A27L), and a portion (amino acids 31-96) of melan-A (SEQ ID NO. 2) including the epitope clusters at amino acids 31-48 and 56-69, was constructed. These clusters were previously disclosed in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS incorporated by reference above. Flanking the defined melan-A CTL epitope are short amino acid sequences derived from human tyrosinase (SEQ ID NO. 3) to facilitate liberation of the melan-A housekeeping epitope by processing by the immunoproteasome. In addition, these amino acid sequences represent potential CTL epitopes themselves. The cDNA sequence for the polypeptide in the plasmid is under the control of promoter/enhancer sequence from cytomegalovirus (CMVp) (see FIG. 4), which allows efficient transcription of messenger for the polypeptide upon uptake by APCs. The bovine growth hormone polyadenylation signal (BGH polyA) at the 3′ end of the encoding sequence provides a signal for polyadenylation of the messenger to increase its stability as well as for translocation out of nucleus into the cytoplasm for translation. To facilitate plasmid transport into the nucleus after uptake, a nuclear import sequence (NIS) from simian virus 40 (SV40) has been inserted in the plasmid backbone. The plasmid carries two copies of a CpG immunostimulatory motif, one in the NIS sequence and one in the plasmid backbone. Lastly, two prokaryotic genetic elements in the plasmid are responsible for amplification in E. coli, the kanamycin resistance gene (Kan R) and the pMB1 bacterial origin of replication.

[0151] Substrate or Liberation Sequence

[0152] The amino acid sequence of the encoded polypeptide (94 amino acid residues in length) (SEQ ID NO. 4) containing a 28 amino acid substrate or liberation sequence at its N-terminus (SEQ ID NO. 5) is given below:

MLLAVLYCL-ELAGIGILTV-YMDGTMSQV-
GILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFD
HRDSKVSLQEKNCEPV

[0153] The first 9 amino acid residues are derived from tyrosinase1-9 (SEQ ID NO. 6), the next ten constitute melan-A (26-35A27L) (SEQ ID NO. 1), and amino acid residues 20 to 29 are derived from tyrosinase369-377 (SEQ ID NO. 7). These two tyrosinase nonamer sequences both represent potential HLA A2-specific CTL epitopes. Amino acid residues 10-19 constitute melan-A (26-35A27L) an analog of an HLA A2-specific CTL epitope from melan-A, EAAGIGILTV (SEQ ID NO. 8), with an elevated potency in inducing CTL responses during in vitro immunization of human PBMC and in vivo immunization in mice. The segment of melan-A constituting the rest of the polypeptide (amino acid residues 30 to 94) contain a number of predicted HLA A2-specific epitopes, including the epitope clusters cited above, and thus can be useful in generating a response to immune epitopes as described at length in the patent applications ‘Epitope Synchronization in Antigen Presenting Cells’ and ‘Epitope Clusters’ cited and incorporated by reference above. This region was also included to overcome any difficulties that can be associated with the expression of shorter sequences. A drawing of pMA2M is shown in FIG. 4.

[0154] Plasmid Construction

[0155] A pair of long complementary oligonucleotides was synthesized which encoded the first 30 amino acid residues. In addition, upon annealing, these oligonucleotides generated the cohensive ends of Afl II at the 5′ end and that of EcoR I at the 3′ end. The melan A31-96 region was amplified with PCR using oligonucleotides carrying restriction sites for EcoR I at the 5′ end and Not I at the 3′ end. The PCR product was digested with EcoR I and Not I and ligated into the vector backbone, described in Example 1, that had been digested with Afl II and Not I, along with the annealed oligonucleotides encoding the amino terminal region in a three-fragment ligation. The entire coding sequence was verified by DNA sequencing. The sequence of the entire insert, from the Afl II site at the 5′ end to the Not I site at the 3′ end is disclosed as SEQ ID NO. 9. Nucleotides 12-293 encode the polypeptide.

Example 2

[0156] Three vectors containing melan-A (26-35A27L) (SEQ ID NO. 1) as an embedded housekeeping epitope were tested for their ability to induce a CTL response to this epitope in HLA-A2 transgenic HHD mice (Pascolo et al. J. Exp. Med. 185:2043-2051, 1997). One of the vectors was pMA2M described above (called pVAXM3 in FIG. 6). In pVAXM2 the same basic group of 3 epitopes was repeated several times with the flanking epitopes truncated by differing degrees in the various repeats of the array. Specifically the cassette consisted of:

M-Tyr(5-9)-ELA-Tyr(369-373)-Tyr(4-9)-ELA-
Tyr(369-374)-Tyr(3-9)-ELA-Tyr(369-375)-
Tyr(2-9)-ELA (SEQ ID NO. 10)

[0157] where ELA represents melan-A (26-35A27L) (SEQ ID NO. 1). This cassette was inserted in the same plasmid backbone as used for pVAXM3. The third, pVAXM1 is identical to pVAXM2 except that the epitope array is followed by an IRES (internal ribosome entry site for encephalomyocarditis virus) linked to a reading frame encoding melan-A 31-70.

[0158] Four groups of three HHD A2.1 mice were injected intranodally in surgically exposed inguinal lymph nodes with 25 μl of 1 mg/ml plasmid DNA in PBS on days 0, 3, and 6, each group receiving one of the three vectors or PBS alone. On day 14 the spleens were harvested and restimulated in vitro one time with 3-day LPS blasts pulsed with peptide (melan-A (26-35A27L) (SEQ ID NO. 1)). The in vitro cultures were supplemented with Rat T-Stim (Collaborative Biomedical Products) on the 3rd day and assayed for cytolytic activity on the 7th day using a standard 51Cr-release assay. FIGS. 5 to 8 show % specific lysis obtained using the cells immunized with PBS, pVAXM1, pVAXM2, and pVAXM3, respectively on T2 target cells and T2 target cells pulsed with melan-A (26-35A27L) (ELA) (SEQ ID NO. 1). All three vectors generated strong CTL responses. These data indicated that the plasmids have been taken up by APCs, the encoded polypeptide has been synthesized and proteolytically processed to produce the decamer epitope in question (that is, it had substrate or liberation sequence function), and that the epitope became HLA-A2 bound for presentation. Also, an isolated variant of pVAXM2, that terminates after the 55th amino acid, worked similarly well as the full length version (data not shown). Whether other potential epitopes within the expression cassette can also be produced and be active in inducing CTL responses can be determined by testing for CTL activity against target cells pulsed with corresponding synthetic peptides.

Example 3

[0159] An NY-ESO-1 (SEQ ID NO. 11) Substrate/Liberation Sequence

[0160] Six other epitope arrays were tested leading to the identification of a substrate/liberation sequence for the housekeeping epitope NY-ESO-1157-165 (SEQ ID NO. 12). The component epitopes of the arrays were:

SSX-241-49:
KASEKIFYV	(SEQ ID NO. 13)	Array element A
NY-ESO-1157-165:
SLLMWITQC	(SEQ ID NO. 12)	Array element B
NY-ESO-1163-171:
TQCFLPVFL	(SEQ ID NO. 14)	Array element C
PSMA288-297:
GLPSIPVHPI	(SEQ ID NO. 15)	Array element D
TYR4-9:
AVLYCL	(SEQ ID NO. 16)	Array element E

[0161] The six arrays had the following arrangements of elements after starting with an initiator methionine:

pVAX-PC-A: B-A-D-D-A-B-A-A
pVAX-PC-B: D-A-B-A-A-D-B-A
pVAX-PC-C: E-A-D-B-A-B-E-A-A
pVAX-BC-A: B-A-C-B-A-A-C-A
pVAX-BC-B: C-A-B-C-A-A-B-A
pVAX-BC-C: E-A-A-B-C-B-A-A

[0162] These arrays were inserted into the same vector backbone described in the examples above. The plasmid vectors were used to immunize mice essentially as described in Example 2 and the resulting CTL were tested for their ability to specifically lyse target cells pulsed with the peptide NY-ESO-1 157-165, corresponding to element B above. Both pVAX-PC-A and pVAX-BC-A were found to induce specific lytic activity. Comparing the contexts of the epitope (element B) in the various arrays, and particularly between pVAX-PC-A and pVAX-BC-A, between pVAX-PC-A and pVAX-PC-B, and between pVAX-BC-A and pVAX-BC-C, it was concluded that it was the first occurrence of the epitope in pVAX-PC-A and pVAX-BC-A that was being correctly processed and presented. In other words an initiator methionine followed by elements B-A constitute a substrate/liberation sequence for the presentation of element B. On this basis a new expression cassette for use as a vaccine was constructed encoding the following elements:

[0163] An initiator methionine,

[0164] NY-ESO-1157-165 (bold)—a housekeeping epitope,

[0165] SSX241-49 (italic)—providing appropriate context for processing, and

[0166] NY-ESO-177-180—to avoid “short sequence” problems and provide immune epitopes.

[0167] Thus the construct encodes the amino acid sequence:

M-SLLMWITQC-KASEKIFYV-
RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEF
TVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPS
GQRR (SEQ ID NO. 17)

[0168] and MSLLMWITQCKASEKIFYV (SEQ ID NO. 18) constitutes the liberation or substrate sequence. A polynucleotide encoding SEQ ID NO. 17 (SEQ ID NO. 19: nucleotides 12-380) was inserted into the same plasmid backbone as used for pMA2M generating the plasmid pN157.

Example 4

[0169] A construct similar to pN157 containing the whole epitope array from pVAX-PC-A was also made and designated pBPL. Thus the encoded amino acid sequence in pBPL is:

M-SLLMWITQC-KASEKIFYV-GLPSIPVHPI-GLPSIPVHIPI-
KASEKIFYV-SLLMWITQC-KASEKIFYV-KASEKIFYV-
RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEF
TVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPS
GQRR. (SEQ ID NO. 20)

[0170] SEQ ID NO. 21 is the polynucleotide encoding SEQ ID NO. 20 used in pBPL.

[0171] A portion of SEQ ID NO. 20, IKASEKIFYVSLLMWITQCKASEKIFYVK (SEQ ID NO. 22) was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing. The identification of a cleavage after the C residue indicates that this segment of the construct can function as a substrate or liberation sequence for NY-ESO-1157-165 (SEQ ID NO. 12) epitope (see FIG. 9). FIG. 10 shows the differential processing of the SLLMWITQC epitope (SEQ ID NO. 12) in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome. The immunoproteasome also produces a major cleavage internal to the epitope, between the T and the Q when the epitope is in its native context, but not in the context of SEQ ID NO. 22 (compare FIGS. 6 and 7).

Example 5

[0172] Screening of further epitope arrays led to the identification of constructs promoting the expression of the epitope SSX-241-49 (SEQ ID NO. 13). In addition to some of the array elements defined in Example 3, the following additional elements were also used:

SSX-457-65:
VMTKLGFKV	(SEQ ID NO. 23)	Array element F.
PSMA730-739:
RQIYVAAFTV	(SEQ ID NO. 24)	Array element G.

[0173] A construct, denoted CTLA02, encoding an initiator methionine and the array F-A-G-D-C-F-G-A, was found to successfully immunize HLA-A2 transgenic mice to generate a CTL response recognizing the peptide SSX-241-49 (SEQ ID NO. 13).

[0174] As described above, it can be desirable to combine a sequence with substrate or liberation sequence function with one that can be processed into immune epitopes. Thus SSX-215-183 (SEQ ID NO. 25) was combined with all or part of the array as follows:

CTLS1:	F-A-G-D-C-F-G-A-SSX-215-183	(SEQ ID NO. 26)
CTLS2:	SSX-215-183-F-A-G-D-C-F-G-A	(SEQ ID NO. 27)
CTLS3:	F-A-G-D-SSX-215-183	(SEQ ID NO. 28)
CTLS4:	SSX-215-183-C-F-G-A.	(SEQ ID NO. 29)

[0175] All of the constructs except CTLS3 were able to induce CTL recognizing the peptide SSX-241-49 (SEQ ID NO. 13). CTLS3 was the only one of these four constructs which did not include the second element A from CTLA02 suggesting that it was this second occurrence of the element that provided substrate or liberation sequence function. In CTLS2 and CTLS4 the A element is at the C-terminal end of the array, as in CTLA02. In CTLS1 the A element is immediately followed by the SSX-215-183 segment which begins with an alanine, a residue often found after proteasomal cleavage sites (Toes, R. E. M., et al., J. Exp. Med. 194:1-12, 2001). SEQ ID NO. 30 is the polynucleotide sequence encoding SEQ ID NO. 26 used in CTLS1, also called pCBP.

[0176] A portion of CTLS1 (SEQ ID NO. 26), encompassing array elements F-A-SSX-215-23 with the sequence RQIYVAAFTV-KASEKIFYV-AQIPEKIQK (SEQ ID NO. 31), was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing. The observation that the C-terminus of the SSX-241-49 epitope (SEQ ID NO. 13) was generated (see FIG. 11) provided further evidence in support of substrate or liberation sequence function. The data in FIG. 12 showed the differential processing of the SSX-241-49 epitope, KASEKIFYV (SEQ ID NO. 13), in its native context, where the cleavage following the V was the predominant cleavage produced by housekeeping proteasome, while the immunoproteasome had several major cleavage sites elsewhere in the sequence. By moving this epitope into the context provided by SEQ ID NO. 31 the desired cleavage became a major one and its relative frequency compared to other immunoproteasome cleavages was increased (compare FIGS. 11 and 12). The data in FIG. 1B also showed the similarity in specificity of mouse and human immunoproteasome lending support to the usefulness of the transgenic mouse model to predict human antigen processing.

Example 6

[0177] Screening also revealed substrate or liberation sequence function for a tyrosinase epitope, Tyr207-215 (SEQ ID NO. 32), as part of an array consisting of the sequence [Tyr1-17-Tyr207-215]4, [MLLAVLYCLLWSFQTSA-FLPWHRLFL]4, (SEQ ID NO. 33). The same vector backbone described above was used to express this array. This array differs from those of the other examples in that the Tyr1-17 segment, which was included as a source of immune epitopes, is used as a repeated element of the array. This is in contrast with the pattern shown in the other examples where sequence included as a source of immune epitopes and/or length occurred a single time at the beginning or end of the array, the remainder of which was made up of individual epitopes or shorter sequences.

[0178] Plasmid Construction

[0179] The polynucleotide encoding SEQ ID NO. 33 was generated by assembly of annealed synthetic oligonucleotides. Four pairs of complementary oligonucleotides were synthesized which span the entire coding sequence with cohesive ends of the restriction sites of Afl II and EcoR I at either terminus. Each complementary pair of oligonucleotides were first annealed, the resultant DNA fragments were ligated stepwise, and the assembled DNA fragment was inserted into the same vector backbone described above pre-digested with Afl II/EcoR I. The construct was called CTLT2/pMEL and SEQ ID NO. 34 is the polynucleotide sequence used to encode SEQ ID NO. 33.

Example 7

[0180] Administration of a DNA Plasmid Formulation of a Immunotherapeutic for Melanoma to Humans.

[0181] An MA2M melanoma vaccine with a sequence as described in Example 1 above, was formulated in 1% Benzyl alcohol, 1% ethyl alcohol, 0.5 mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 200, 400, and 600 μg DNA/ml were prepared for loading into MINIMED 407C infusion pumps. The catheter of a SILHOUETTE infusion set was placed into an inguinal lymph node visualized by ultrasound imaging. The pump and infusion set assembly was originally designed for the delivery of insulin to diabetics. The usual 17 mm catheter was substituted with a 31 mm catheter for this application. The infusion set was kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 μl/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion was approximately 500, and 1000 μg; and can be 1500 μg, respectively, for the three concentrations described above. Following an infusion, subjects were given a 10 day rest period before starting a subsequent infusion. Given the continued residency of plasmid DNA in the lymph node after administration and the usual kinetics of CTL response following disappearance of antigen, this schedule will be sufficient to maintain the immunologic CTL response.

Example 8

[0182] SEQ ID NO. 22 is made as a synthetic peptide and packaged with a cationic lipid protein transfer reagent. The composition is infused directly into the inguinal lymph node (see example 7) at a rate of 200 to 600 μg of peptide per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted.

Example 9

[0183] A fusion protein is made by adding SEQ ID NO. 34 to the 3′ end of a nucleotide sequence encoding herpes simplex virus 1 VP22 (SEQ ID NO. 42) in an appropriate mammalian expression vector; the vector used above is suitable. The vector is used to transform HEK 293 cells and 48 to 72 hours later the cells are pelleted, lysed and a soluble extract prepared. The fusion protein is purified by affinity chromatagraphy using an anti-VP22 monoclonal antibody. The purified fusion protein is administered intranodally at a rate of 10 to 100 μg per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted.

Examples 10-13

[0184] The following examples, Examples 10-13, all concern the prediction of 9-mer epitopes presented by HLA-A2.1, although the procedure is equally applicable to any HLA type, or epitope length, for which a predictive algorithm or MHC binding assay is available.

Example 10

Melan-A/MART-1 (SEQ ID NO: 2)

[0185] This melanoma tumor-associated antigen (TuAA) is 118 amino acids in length. Of the 110 possible 9-mers, 16 are given a score≧16 by the SYFPEITHI/Rammensee algorithm. (See Table 14). These represent 14.5% of the possible peptides and an average epitope density on the protein of 0.136 per amino acid. Twelve of these overlap, covering amino acids 22-49 of SEQ ID NO: 2 resulting in an epitope density for the cluster of 0.428, giving a ratio, as described above, of 3.15. Another two predicted epitopes overlap amino acids 56-69 of SEQ ID NO: 2, giving an epitope density for the cluster of 0.143, which is not appreciably different than the average, with a ratio of just 1.05. See FIG. 1.

TABLE 14
SYFPEITHI (Rammensee algorithm) Results for
Melan-A/MART-1 (SEQ ID NO: 2)
Rank	Start	Score
1	31	27
2	56	26
3	35	26
4	32	25
5	27	25
6	29	24
7	34	23
8	61	20
9	33	19
10	22	19
11	99	18
12	36	18
13	28	18
14	87	17
15	41	17
16	40	16

[0186] Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm leaves only 5. (See Table 15). The average density of epitopes in the protein is now only 0.042 per amino acid. Three overlapping peptides cover amino acids 31-48 of SEQ ID NO: 2 and the other two cover 56-69 of SEQ ID NO: 2, as before, giving ratios of 3.93 and 3.40, respectively. (See Table 16).

TABLE 15
BIMAS-NIH/Parker algorithm Results for
Melan-A/MART-1 (SEQ ID NO: 2)
	Rank	Start	Score	Log(Score)
	1	40	1289.01	3.11
	2	56	1055.104	3.02
	3	31	81.385	1.91
	4	35	20.753	1.32
	5	61	4.968	0.70

[0187]

TABLE 16
Predicted Epitope Clusters for Melan-A/MART-1
(SEQ ID NO: 2)
Calculations(Epitopes/AAs)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	31-48	3, 4, 1	0.17	0.042	3.93
2	56-69	2, 5	0.14	0.042	3.40

Example 11

SSX-2/HOM-MEL-40 (SEQ ID NO: 40)

[0188] This melanoma tumor-associated antigen (TuAA) is 188 amino acids in length. Of the 180 possible 9-mers, 11 are given a score≧16 by the SYFPEITHI/Rammensee algorithm. These represent 6.1% of the possible peptides and an average epitope density on the protein of 0.059 per amino acid. Three of these overlap, covering amino acids 99-114 of SEQ ID NO: 40 resulting in an epitope density for the cluster of 0.188, giving a ratio, as described above, of 3.18. There are also overlapping pairs of predicted epitopes at amino acids 16-28, 57-57, and 167-183 of SEQ ID NO: 40, giving ratios of 2.63, 3.11, and 2.01, respectively. There is an additional predicted epitope covering amino acids 5-28. Evaluating the region 5-28 of SEQ ID NO: 40 containing three epitopes gives an epitope density of 0.125 and a ratio 2.14.

[0189] Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm leaves only 6. The average density of epitopes in the protein is now only 0.032 per amino acid. Only a single pair overlap, at 167-180 of SEQ ID NO: 40, with a ratio of 4.48. However the top ranked peptide is close to another single predicted epitope if that region, amino acids 41-65 of SEQ ID NO: 40, is evaluated the ratio is 2.51, representing a substantial difference from the average. See FIG. 2.

TABLE 17
SYFPEITHI/Rammensee algorithm for
SSX-2/HOM-MEL-40 (SEQ ID NO: 40)
Rank	Start	Score
1	103	23
2	167	22
3	41	22
4	16	21
5	99	20
6	59	19
7	20	17
8	5	17
9	175	16
10	106	16
11	57	16

[0190]

TABLE 18
Calculations(Epitopes/AAs) (SEQ ID NO: 40)
Calculations(Epitopes/AAs)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	5 to 28	8, 4, 7	0.125	0.059	2.14
2	16-28	4, 7	0.15	0.059	2.63
3	57-67	11, 6	0.18	0.059	3.11
4	99-114	5, 1, 10	0.19	0.059	3.20
5	167-183	2, 9	0.12	0.059	2.01

[0191]

TABLE 19
BIMAS-NIH/Parker algorithm (SEQ ID NO: 40)
	Rank	Start	Score	Log(Score)
	1	41	1017.062	3.01
	2	167	21.672	1.34
	3	57	20.81	1.32
	4	103	10.433	1.02
	5	172	10.068	1.00
	6	16	6.442	0.81

[0192]

TABLE 20
Calculations(Epitopes/AAs) (SEQ ID NO: 40)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	41-65	1, 3	0.08	0.032	2.51
2	167-180	2, 5	0.14	0.032	4.48

Example 12

NY-ESO (SEQ ID NO: 11)

[0193] This tumor-associated antigen (TuAA) is 180 amino acids in length. Of the 172 possible 9-mers, 25 are given a score≧16 by the SYFPEITHI/Rammensee algorithm. Like Melan-A above, these represent 14.5% of the possible peptides and an average epitope density on the protein of 0.136 per amino acid. However the distribution is quite different. Nearly half the protein is empty with just one predicted epitope in the first 78 amino acids. Unlike Melan-A where there was a very tight cluster of highly overlapping peptides, in NY-ESO the overlaps are smaller and extend over most of the rest of the protein. One set of 19 overlapping peptides covers amino acids 108-174 of SEQ ID NO: 11, resulting in a ratio of 2.04. Another 5 predicted epitopes cover 79-104 of SEQ ID NO: 11, for a ratio of just 1.38.

[0194] If instead one takes the approach of considering only the top 5% of predicted epitopes, in this case 9 peptides, one can examine whether good clusters are being obscured by peptides predicted to be less likely to bind to MHC. When just these predicted epitopes are considered we see that the region 108-140 of SEQ ID NO: 11 contains 6 overlapping peptides with a ratio of 3.64. There are also 2 nearby peptides in the region 148-167 of SEQ ID NO: 11 with a ratio of 2.00. Thus the large cluster 108-174 of SEQ ID NO: 11 can be broken into two smaller clusters covering much of the same sequence.

[0195] Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm brings 14 peptides into consideration. The average density of epitopes in the protein is now 0.078 per amino acid. A single set of 10 overlapping peptides is observed, covering amino acids 144-171 of SEQ ID NO: 11, with a ratio of 4.59. All 14 peptides fall in the region 86-171 of SEQ ID NO: 11 which is still 2.09 times the average density of epitopes in the protein. While such a large cluster is larger than we consider ideal it still offers a significant advantage over working with the whole protein. See FIG. 3.

TABLE 21
SYFPEITHI (Rammensee algorithm) Results for
NY-ESO (SEQ ID NO: 11)
Rank	Start	Score
1	108	25
2	148	24
3	159	21
4	127	21
5	86	21
6	132	20
7	122	20
8	120	20
9	115	20
10	96	20
11	113	19
12	91	19
13	166	18
14	161	18
15	157	18
16	151	18
17	137	18
18	79	18
19	139	17
20	131	17
21	87	17
22	152	16
23	144	16
24	129	16
25	15	16

[0196]

TABLE 22
Calculations(Epitopes/AAs) (SEQ ID NO: 11)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	108-140	1, 9, 8, 7, 4, 6	0.18	0.05	3.64
2	148-167	2, 3	0.10	0.05	2.00
3	79-104	5 12, 10, 18, 21	0.19	0.14	1.38
4	108-174	1, 11, 9, 8, 7, 4, 6, 17, 2, 16, 15, 3,	0.28	0.14	2.04
		14, 13, 24, 20, 19, 23, 22

[0197]

TABLE 23
BIMAS-NIH/Parker algorithm Results for
NY-ESO (SEQ ID NO: 11)
	Rank	Start	Score	Log(Score)
	1	159	1197.321	3.08
	2	86	429.578	2.63
	3	120	130.601	2.12
	4	161	83.584	1.92
	5	155	52.704	1.72
	6	154	49.509	1.69
	7	157	42.278	1.63
	8	108	21.362	1.33
	9	132	19.425	1.29
	10	145	13.624	1.13
	11	163	11.913	1.08
	12	144	11.426	1.06
	13	148	6.756	0.83
	14	152	4.968	0.70

[0198]

TABLE 24
Calculations(Epitopes/AAs) (SEQ ID NO: 11)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	86-171	2, 8, 3, 9, 10, 12,	0.163	0.078	2.09
		13, 14, 6, 5, 7, 1,
		4, 11
2	144-171	10, 12, 13, 14, 6,	0.36	0.078	4.59
		5, 7, 1, 4, 11

Example 13

Tyrosinase (SEQ ID NO: 3)

[0199] This melanoma tumor-associated antigen (TuAA) is 529 amino acids in length. Of the 521 possible 9-mers, 52 are given a score≧16 by the SYFPEITHI/Rammensee algorithm. These represent 10% of the possible peptides and an average epitope density on the protein of 0.098 per amino acid. There are 5 groups of overlapping peptides containing 2 to 13 predicted epitopes each, with ratios ranging from 2.03 to 4.41, respectively. There are an additional 7 groups of overlapping peptides, containing 2 to 4 predicted epitopes each, with ratios ranging from 1.20 to 1.85, respectively. The 17 peptides in the region 444-506 of SEQ ID NO: 3, including the 13 overlapping peptides above, constitutes a cluster with a ratio of 2.20.

[0200] Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm brings 28 peptides into consideration. The average density of epitopes in the protein under this condition is 0.053 per amino acid. At this density any overlap represents more than twice the average density of epitopes. There are 5 groups of overlapping peptides containing 2 to 7 predicted epitopes each, with ratios ranging from 2.22 to 4.9, respectively. Only three of these clusters are common to the two algorithms. Several, but not all, of these clusters could be enlarged by evaluating a region containing them and nearby predicted epitopes.

TABLE 25
SYFPEITHI/Rammensee algorithm
Results for Tyrosinase (SEQ ID NO: 3)
Rank	Start	Score
1	490	34
2	491	31
3	487	28
4	1	27
5	2	25
6	482	23
7	380	23
8	369	23
9	214	23
10	506	22
11	343	22
12	207	22
13	137	22
14	57	22
15	169	20
16	118	20
17	9	20
18	488	19
19	483	19
20	480	19
21	479	19
22	478	19
23	473	19
24	365	19
25	287	19
26	200	19
27	5	19
28	484	18
29	476	18
30	463	18
31	444	18
32	425	18
33	316	18
34	187	18
35	402	17
36	388	17
37	346	17
38	336	17
39	225	17
40	224	17
41	208	17
42	186	17
43	171	17
44	514	16
45	494	16
46	406	16
47	385	16
48	349	16
49	184	16
50	167	16
51	145	16
52	139	16

[0201]

TABLE 26
Calculations(Epitopes/AAs) (SEQ ID NO: 3)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	1 to 17	4, 5, 27, 17	0.24	0.098	2.39
2	137-153	13, 52, 51	0.18	0.098	1.80
3	167-179	15, 43, 50	0.23	0.098	2.35
4	184-195	34, 42, 49	0.25	0.098	2.54
5	200-222	26, 41, 9, 12	0.17	0.098	1.77
6	224-233	39, 40	0.20	0.098	2.03
7	336-357	38, 11, 37, 48	0.18	0.098	1.85
8	365-377	24, 8	0.15	0.098	1.57
9	380-396	7, 47, 36	0.18	0.098	1.80
10	402-414	35, 46	0.15	0.098	1.57
11	473-502	29, 28, 23, 22,	0.43	0.098	4.41
		21, 20, 6, 19, 3,
		18, 1, 2, 45
12	506-522	10, 44	0.12	0.098	1.20
	444-522	31, 30, 23, 29,	0.22	0.098	2.20
		22, 21, 20, 6,
		19, 28, 3, 18,
		1, 2, 45, 10,
		44

[0202]

TABLE 27
BIMAS-NIH/Parker algorithm Results (SEQ ID NO: 3)
	Rank	Start	Score	Log(Score)
	1	207	540.469	2.73
	2	369	531.455	2.73
	3	1	309.05	2.49
	4	9	266.374	2.43
	5	490	181.794	2.26
	6	214	177.566	2.25
	7	224	143.451	2.16
	8	171	93.656	1.97
	9	506	87.586	1.94
	10	487	83.527	1.92
	11	491	83.527	1.92
	12	2	54.474	1.74
	13	137	47.991	1.68
	14	200	30.777	1.49
	15	208	26.248	1.42
	16	460	21.919	1.34
	17	478	19.425	1.29
	18	365	17.14	1.23
	19	380	16.228	1.21
	20	444	13.218	1.12
	21	473	13.04	1.12
	22	57	10.868	1.04
	23	482	8.252	0.92
	24	483	7.309	0.86
	25	5	6.993	0.84
	26	225	5.858	0.77
	27	343	5.195	0.72
	28	514	5.179	0.71

[0203]

TABLE 28
Calculations(Epitopes/AAs) (SEQ ID NO: 3)
Cluster	AA	Peptides	Cluster	Whole protein	Ratio
1	1 to 17	3, 12, 25, 4	0.24	0.053	4.45
2	200-222	14, 1, 15, 6	0.17	0.053	3.29
3	224-233	7, 26	0.20	0.053	3.78
4	365-377	18, 2	0.15	0.053	2.91
5	473-499	21, 17, 23, 24,	0.26	0.053	4.90
		10, 5, 11
6	506-522	9, 28	0.12	0.053	2.22
7	365-388	18, 2, 19	0.13	0.053	2.36
8	444-499	20, 16, 21, 17,	0.16	0.053	3.03
		23, 24, 10, 5,
		11
9	444-522	20, 16, 21, 17,	0.14	0.053	2.63
		23, 24, 10, 5,
		11, 9, 28
10	200-233	14, 1, 15, 6,	0.18	0.053	3.33
		7, 26

[0204] All references mentioned herein are hereby incorporated by reference in their entirety. Further, the present invention can utilize various aspects of the following, which are all incorporated by reference in their entirety: U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, entitled A METHOD OF INDUCING A CTL RESPONSE; Ser. No. 09/776,232, filed on Feb. 2, 2001, entitled METHOD OF INDUCING A CTL RESPONSE; Ser. No. 09/715,835, filed on Nov. 16, 2000, entitled AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPOGATION; Ser. No. 09/999,186, filed on Nov. 7, 2001, entitled METHODS OF COMMERCIALIZING AN ANTIGEN; and Provisional U.S. Patent Application No. 60/274,063, filed on Mar. 7, 2001, entitled ANTI-NEOVASCULAR VACCINES FOR CANCER.

TABLE 29
Partial listing of SEQ ID NOS.
1	ELAGIGILTV	melan-A 26-35 (A27L)
2	Melan-A protein	Accession number:
		NP_005502
3	Tyrosinase protein	Accession number:
		P14679
4	MLLAVLYCLELAGIGILTVYMDGTMSQVGILTVILGVL	pMA2M expression
	LLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEG	product
	FDHRDSKVSLQEKNCEPV
5	MLLAVLYCLELAGIGILTVYMDGTMSQV	Liberation or substrate
		sequence for SEQ ID
		NO. 1 from pMA2M
6	MLLAVLYCL	tyrosinase 1-9
7	YMDGTMSQV	tyrosinase 369-377
8	EAAGIGILTV	melan-A 26-35
9	cttaagccaccatgttactagctgttttgtactgcctg	pMA2M insert
	gaactagcagggatcggcatattgacagtgtatatgga
	tggaacaatgtcccaggtaggaattctgacagtgatcc
	tgggagtcttactgctcatcggctgttggtattgtaga
	agacgaaatggatacagagccttgatggataaaagtct
	tcatgttggcactcaatgtgccttaacaagaagatgcc
	cacaagaagggtttgatcatcgggacagcaaagtgtct
	cttcaagagaaaaactgtgaacctgtgtagtgagcggc
	cgc
10	MVLYCLELAGIGILTVYMDGTAVLYCLELAGIGILTVY	Epitope array from
	MDGTMLAVLYCLELAGIGILTVYMDGTMSLLAVLYCLE	pVAXM2 and pVAXM1
	LAGIGILTV
11	NY-ESO-1 protein	Accession number:
		P78358
12	SLLMWITQC	NY-ESO-1 157-165
13	KASEKIFYV	SSX-2 41-49
14	TQCFLPVFL	NY-ESO-1 163-171
15	GLPSIPVHPI	PSMA 288-297
16	AVLYCL	tyrosinase 4-9
17	MSLLMWITQCKASEKIFYVRCGARGPESRLLEFYLAMP	pN157 expression
	FATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNIL	product
	TIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFL
	AQPPSGQRR
18	MSLLMWITQCKASEKIFYV	liberation or substrate
		sequence for SEQ ID
		NO. 12 from pN157
19	cttaagccaccatgtccctgttgatgtggatcacgcag	Insert for pN157
	tgcaaagcttcggagaaaatcttctacgtacggtgcgg
	tgccagggggccggagagccgcctgcttgagttctacc
	tcgccatgcctttcgcgacacccatggaagcagagctg
	gcccgcaggagcctggcccaggatgccccaccgcttcc
	cgtgccaggggtgcttctgaaggagttcactgtgtccg
	gcaacatactgactatccgactgactgctgcagaccac
	cgccaactgcagctctccatcagctcctgtctccagca
	gctttccctgttgatgtggatcacgcagtgctttctgc
	ccgtgtttttggctcagcctccctcagggcagaggcgc
	tagtgagaattc
20	MSLLMWITQCKASEKIFYVGLPSIPVHPIGLPSIPVHP	pBPL expression
	IKASEKIFYVSLLMWITQCKASEKIFYVKASEKIFYVR	product
	CGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPP
	LPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCL
	QQLSLLMWITQCFLPVFLAQPPSGQRR
21	atgtccctgttgatgtggatcacgcagtgcaaagcttc	pBPL insert coding
	ggagaaaatcttctatgtgggtcttccaagtattcctg	region
	ttcatccaattggtcttccaagtattcctgttcatcca
	attaaagcttcggagaaaatcttctatgtgtccctgtt
	gatgtggatcacgcagtgcaaagcttcggagaaaatct
	tctatgtgaaagcttcggagaaaatcttctacgtacgg
	tgcggtgccagggggccggagagccgcctgcttgagtt
	ctacctcgccatgcctttcgcgacacccatggaagcag
	agctggcccgcaggagcctggcccaggatgccccaccg
	cttcccgtgccaggggtgcttctgaaggagttcactgt
	gtccggcaacatactgactatccgactgactgctgcag
	accaccgccaactgcagctctccatcagctcctgtctc
	cagcagctttccctgttgatgtggatcacgcagtgctt
	tctgcccgtgtttttggctcagcctccctcagggcaga
	ggcgctagtga
22	IKASEKIFYVSLLMWITQCKASEKIFYVK	Substrate in FIG. 9
23	VMTKLGFKV	SSX-457-65
24	RQIYVAAFTV	PSMA730-739
25	AQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYMK	SSX-215-183
	RKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNR
	GNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVPE
	ASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWTH
	RLRERKQLVIYEEISDP
26	MVMTKLGFKVKASEKIFYVRQIYVAAFTVGLPSIPVHP	CTLS 1/pCBP
	ITQCFLPVFLVMTKLGFKVRQIYVAAFTVKASEKIFYV	expression product
	AQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYMK
	RKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNR
	GNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVPE
	ASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWTH
	RLRERKQLVIYEEISDP
27	MAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYM	CTLS2 expression
	KRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPN	product
	RGNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVP
	EASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWT
	HRLRERKQLVIYEEISDPVMTKLGFKVKASEKIFYVRQ
	IYVAAFTVGLPSIPVHPITQCFLPVFLVMTKLGFKVRQ
	IYVAAFTVKASEKIFYV
28	MVMTKLGFKVKASEKIFYVRQIYVAAFTVGLPSIPVHP	CTLS3 expression
	IAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYM	product
	KRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPN
	RGNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVP
	EASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWT
	HRLRERKQLVIYEEISDP
29	MAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYM	CTLS4 expression
	KRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPN	product
	RGNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVP
	EASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWT
	HRLRERKQLVIYEEISDPTQCFLPVFLVMTKLGFKVRQ
	IYVAAFTVKASEKIFYV
30	atggtcatgactaaactaggtttcaaggtcaaagcttc	pCBP insert coding
	ggagaaaatcttctatgtgagacagatttatgttgcag	region
	ccttcacagtgggtcttccaagtattcctgttcatcca
	attacgcagtgctttctgcccgtgtttttggtcatgac
	taaactaggtttcaaggtcagacagatttatgttgcag
	ccttcacagtgaaagcttcggagaaaatcttctacgta
	gctcaaataccagagaagatccaaaaggccttcgatga
	tattgccaaatacttctctaaggaagagtgggaaaaga
	tgaaagcctcggagaaaatcttctatgtgtatatgaag
	agaaagtatgaggctatgactaaactaggtttcaaggc
	caccctcccacctttcatgtgtaataaacgggccgaag
	acttccaggggaatgatttggataatgaccctaaccgt
	gggaatcaggttgaacgtcctcagatgactttcggcag
	gctccagggaatctccccgaagatcatgcccaagaagc
	cagcagaggaaggaaatgattcggaggaagtgccagaa
	gcatctggcccacaaaatgatgggaaagagctgtgccc
	cccgggaaaaccaactacctctgagaagattcacgaga
	gatctggacccaaaaggggggaacatgcctggacccac
	agactgcgtgagagaaaacagctggtgatttatgaaga
	gatcagcgacccttagtga
31	RQIYVAAFTVKASEKIFYVAQIPEKIQK	FIG. 11 substrate/
		CTLS1-2
32	FLPWHRLFL	TYR207-215
33	MLLAVLYCLLWSFQTSAFLPWHRLFLMLLAVLYCLLWS	CTLT2/pMEL
	FQTSAFLPWHRLFLMLLAVLYCLLWSFQTSAFLPWHRL	expression product
	FLMLLAVLYCLLWSFQTSAFLPWHRLFL
34	atgctcctggctgttttgtactgcctgctgtggagttt	CTLT2/pMEL insert
	ccagacctccgcttttctgccttggcatagactcttct	coding region
	tgatgctcctggctgttttgtactgcctgctgtggagt
	ttccagacctccgcttttctgccttggcatagactctt
	cttgatgctcctggctgttttgtactgcctgctgtgga
	gtttccagacctccgcttttctgccttggcatagactc
	ttcttgatgctcctggctgttttgtactgcctgctgtg
	gagtttccagacctccgcttttctgccttggcatagac
	tcttcttgtagtga
35	MELAN-A cDNA	Accession number:
		NM_005511
36	Tyrosinase cDNA	Accession number:
		NM_000372
37	NY-ESO-1 cDNA	Accession number:
		U87459
38	PSMA protein	Accession number:
		NP_004467
39	PSMA cDNA	Accession number:
		NM_004476
40	SSX-2 protein	Accession number:
		NP_003138
41	SSX-2 cDNA	Accession number:
		NM_003147
42	atgacctctcgccgctccgtgaagtcgggtccgcggga	From accession number:
	ggttccgcgcgatgagtacgaggatctgtactacaccc	D10879
	cgtcttcaggtatggcgagtcccgatagtccgcctgac	Herpes Simplex virus 1
	acctcccgccgtggcgccctacagacacgctcgcgcca	UL49 coding sequence
	gaggggcgaggtccgtttcgtccagtacgacgagtcgg	(VP22)
	attatgccctctacgggggctcgtcatccgaagacgac
	gaacacccggaggtcccccggacgcggcgtcccgtttc
	cggggcggttttgtccggcccggggcctgcgcgggcgc
	ctccgccacccgctgggtccggaggggccggacgcaca
	cccaccaccgccccccgggccccccgaacccagcgggt
	ggcgactaaggcccccgcggccccggcggcggagacca
	cccgcggcaggaaatcggcccagccagaatccgccgca
	ctcccagacgcccccgcgtcgacggcgccaacccgatc
	caagacacccgcgcaggggctggccagaaagctgcact
	ttagcaccgcccccccaaaccccgacgcgccatggacc
	ccccgggtggccggctttaacaagcgcgtcttctgcgc
	cgcggtcgggcgcctggcggccatgcatgcccggatgg
	cggcggtccagctctgggacatgtcgcgtccgcgcaca
	gacgaagacctcaacgaactccttggcatcaccaccat
	ccgcgtgacggtctgcgagggcaaaaacctgcttcagc
	gcgccaacgagttggtgaatccagacgtggtgcaggac
	gtcgacgcggccacggcgactcgagggcgttctgcggc
	gtcgcgccccaccgagcgacctcgagccccagcccgct
	ccgcttctcgccccagacggcccgtcgag
43	MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPD	Accession number:
	TSRRGALFTQTRSRQRGEVRFVQYDESDYALYGGSSSE	P10233
	DDEHPEVPRTRRPVSGAVLSGPGPARAPPPFTPAGSGG	Herpes Simplex virus 1
	AGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQP	UL49/VP22 protein
	ESAALPDAPASTAPTFTRSKTPAQGLARKLHFSTAPPN	sequence
	PDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWD
	FTMSRPRTDEDLNELLGITTRIVTVCEGKNLLQRANEL
	VNPDVVQDVDAATATRGRSAASRFTPTERPRAPARSAS
	RPRRPVE

[0205] Melan-A mRNA Sequence

LOCUS      NM_005511  1524 bp  mRNA  PRI  14 OCT. 2001
DEFINITION Homo sapiens melan-A (MLANA), mRNA.
ACCESSION  NM_005511
VERSION    NM_005511.1 GI: 5031912
(SEQ ID NO. 2)
/translation = “MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNG
YRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP”
(SEQ ID NO. 35)
ORIGIN
1          agcagacaga ggactctcat taaggaaggt gtcctgtgcc ctgaccctac aagatgccaa
61         gagaagatgc tcacttcatc tatggttacc ccaagaaggg gcacggccac tcttacacca
121        cggctgaaga ggccgctggg atcggcatcc tgacagtgat cctgggagtc ttactgctca
181        tcggctgttg gtattgtaga agacgaaatg gatacagagc cttgatggat aaaagtcttc
241        atgttggcac tcaatgtgcc ttaacaagaa gatgcccaca agaagggttt gatcatcggg
301        acagcaaagt gtctcttcaa gagaaaaact gtgaacctgt ggttcccaat gctccacctg
361        cttatgagaa actctctgca gaacagtcac caccacctta ttcaccttaa gagccagcga
421        gacacctgag acatgctgaa attatttctc tcacactttt gcttgaattt aatacagaca
481        tctaatgttc tcctttggaa tggtgtagga aaaatgcaag ccatctctaa taataagtca
541        gtgttaaaat tttagtaggt ccgctagcag tactaatcat gtgaggaaat gatgagaaat
601        attaaattgg gaaaactcca tcaataaatg ttgcaatgca tgatactatc tgtgccagag
661        gtaatgttag taaatccatg gtgttatttt ctgagagaca gaattcaagt gggtattctg
721        gggccatcca atttctcttt acttgaaatt tggctaataa caaactagtc aggttttcga
781        accttgaccg acatgaactg tacacagaat tgttccagta ctatggagtg ctcacaaagg
841        atacttttac aggttaagac aaagggttga ctggcctatt tatctgatca agaacatgtc
901        agcaatgtct ctttgtgctc taaaattcta ttatactaca ataatatatt gtaaagatcc
961        tatagctctt tttttttgag atggagtttc gcttttgttg cccaggctgg agtgcaatgg
1021       cgcgatcttg gctcaccata acctccgcct cccaggttca agcaattctc ctgccttagc
1081       ctcctgagta gctgggatta caggcgtgcg ccactatgcc tgactaattt tgtagtttta
1141       gtagagacgg ggtttctcca tgttggtcag gctggtctca aactcctgac ctcaggtgat
1201       ctgcccgcct cagcctccca aagtgctgga attacaggcg tgagccacca cgcctggctg
1261       gatcctatat cttaggtaag acatataacg cagtctaatt acatttcact tcaaggctca
1321       atgctattct aactaatgac aagtattttc tactaaacca gaaattggta gaaggattta
1381       aataagtaaa agctactatg tactgcctta gtgctgatgc ctgtgtactg ccttaaatgt
1441       acctatggca atttagctct cttgggttcc caaatccctc tcacaagaat gtgcagaaga
1501       aatcataaag gatcagagat tctg

[0206] Tyrosinase mRNA Sequence

LOCUS      NM_000372  1964 bp  mRNA  PRI  31 OCT. 2000
DEFINITION Homo sapiens tyrosinase (oculocutaneous albinism IA) (TYR), mRNA.
ACCESSION  NM_000372
VERSION    NM_000372.1 GI: 4507752
(SEQ ID NO. 3)
/translation = “MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRG
SCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRN
IFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGG
SEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNP
NLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYES
GSMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFE
QWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSY
LEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLP EEKQPLLMEKEDYHSLYQSHL”
(SEQ ID NO. 36)
ORIGIN
1          atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccag ttcctgcaga
61         ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgc tgtggagttt
121        ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacc tgatggagaa
181        ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagcttt caggcagagg
241        ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttc ccttcacagg
301        ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg
361        caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggac caaactgcac
421        agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccag agaaggacaa
481        attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatg tcatccccat
541        agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgaca tcaatattta
601        tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttg ggggatctga
661        aatctggaga gacattgatt ttgcccatga agcaccagct tttctgcctt ggcatagact
721        cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaa acttcactat
781        tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatg agtacatggg
841        aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttct cctcttggca
901        gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgca atggaacgcc
961        cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccc caaggctccc
1021       ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctg gttccatgga
1081       taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtc cacttactgg
1141       gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctata tgaatggaac
1201       aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcacc atgcatttgt
1261       tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaag tttatccaga
1321       agccaatgca cccattggac ataaccggga atcctacatg gttcctttta taccactgta
1381       cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactata gctatctaca
1441       agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaac aagcgagtcg
1501       gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactg ccctgctggc
1561       agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaag aaaagcagcc
1621       actcctcatg gagaaagagg attaccacag cttgtatcag agccatttat aaaaggctta
1681       ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaat gtccaggttc
1741       ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgt aacctaatac
1801       aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttg ctgttttcac
1861       tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta
1921       atgaggaact gttatttgta tgtgaattaa agtgctctta tttt

[0207]

(SEQ ID NO. 36)
ORIGIN
1    atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccag ttcctgcaga
61   ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgc tgtggagttt
121  ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacc tgatggagaa
181  ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagcttt caggcagagg
241  ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttc ccttcacagg
301  ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg
361  caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggac caaactgcac
421  agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccag agaaggacaa
481  attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatg tcatccccat
541  agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgaca tcaatattta
601  tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttg ggggatctga
661  aatctggaga gacattgatt ttgcccatga agcaccagct tttctgcctt ggcatagact
721  cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaa acttcactat
781  tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatg agtacatggg
841  aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttct cctcttggca
901  gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgca atggaacgcc
961  cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccc caaggctccc
1021 ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctg gttccatgga
1081 taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtc cacttactgg
1141 gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctata tgaatggaac
1201 aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcacc atgcatttgt
1261 tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaag tttatccaga
1321 agccaatgca cccattggac ataaccggga atcctacatg gttcctttta taccactgta
1381 cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactata gctatctaca
1441 agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaac aagcgagtcg
1501 gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactg ccctgctggc
1561 agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaag aaaagcagcc
1621 actcctcatg gagaaagagg attaccacag cttgtatcag agccatttat aaaaggctta
1681 ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaat gtccaggttc
1741 ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgt aacctaatac
1801 aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttg ctgttttcac
1861 tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta
1921 atgaggaact gttatttgta tgtgaattaa agtgctctta tttt

[0208] NY-ESO-1 mRNA Sequence

LOCUS      HSU87459  752 bp  mRNA  PRI  22 DEC. 1999
DEFINITION Human autoimmunogenic cancer/testis antigen NY-ESO-1 mRNA,
           complete cds.
ACCESSION  U87459
VERSION    U87459.1 GI: 1890098
(SEQ ID NO. 11)
/translation = “MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARA
SGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVL
LKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR”
(SEQ ID NO. 37)
ORIGIN
1          atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gccatgcagg
61         ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga ggccctggca
121        ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca
181        gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg
241        gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc
301        cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag
361        agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc
421        tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc
481        gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca
541        cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaagccc
601        agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc agcacgagtg
661        gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt acatgtttgt
721        ttctgtagaa aataaaactg agctacgaaa aa

[0209] PSMA cDNA Sequence

LOCUS      NM_004476  2653 bp  mRNA  PRI  01 NOV. 2000
DEFINITION Homo sapiens folate hydrolase (prostate-specific membrane
           antigen) 1 (FOLH1), mRNA.
ACCESSION  NM_004476
VERSION    NM_004476.1 GI: 4758397
(SEQ ID NO. 38)
/translation = “MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNIT
PKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNK
THPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERD
MKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILN
LNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNV
GPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEI
VRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCT
PLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRAR
YTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLR
KYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERA
FIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAA
ETLSEVA”

[0210]

(SEQ ID NO. 39)
ORIGIN
1    ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg
61   attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga
121  gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac
181  cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag
241  gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc
301  accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt
361  ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact
421  ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc
481  ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca
541  aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat
601  gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa
661  gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat
721  gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat
781  ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa
841  atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag
901  gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac
961  tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc
1021 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca
1081 gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct
1141 gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca
1201 ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt
1261 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca
1321 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt
1381 ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct
1441 gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga
1501 agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag
1561 tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac
1621 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg
1681 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt
1741 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc
1801 aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc
1861 agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac
1921 agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac
1981 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc
2041 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt
2101 atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt
2161 tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt
2221 gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga
2281 gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct
2341 ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt
2401 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat
2461 gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat
2521 tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt
2581 atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa
2641 aaaaaaaaaa aaa

[0211]

NM_003147  Homo sapiens                   synovial sarcoma, X breakpoint 2 (SSX2), mRNA
LOCUS      NM_003147  766 bp  mRNA  PRI   14 MAR. 2001
DEFINITION Homo sapiens                   synovial sarcoma, X breakpoint 2 (SSX2), mRNA.
ACCESSION  NM_003147
VERSION    NM_003147.1 GI: 10337582
SEQ ID NO. 40
/translation = “MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYMKR
KYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNRGNQVERPQMTFGRLQGISPKIMPKKPAEEGND
SEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWTHRLRERKQLVIYEEISDPEEDDE”
SEQ ID NO. 41
1          ctctctttcg attcttccat actcagagta cgcacggtct gattttctct ttggattctt
61         ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcct ttgcaaggag
121        acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatg atattgccaa
181        atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatct tctatgtgta
241        tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccc tcccaccttt
301        catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatg accctaaccg
361        tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaa tctccccgaa
421        gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgc cagaagcatc
481        tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaacta cctctgagaa
541        gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccaca gactgcgtga
601        gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatg acgagtaact
661        cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgac ctttcacgaa
721        catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg

[0212]

Claims

1. An isolated nucleic acid comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen SSX-2 (SEQ ID NO: 40), wherein the first sequence does not encode the complete SSX-2 antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC receptor peptide binding cleft.

2. The nucleic acid of claim 1, wherein said epitope cluster is chosen from the group consisting of amino acids 5-28, 16-28, 41-65, 57-67, 99-114, 167-180, and 167-183 of SSX-2.

3. The nucleic acid of claim 1, wherein said one or more segments consist of said epitope cluster.

4. The nucleic acid of claim 1, wherein said first sequence encodes a fragment of SSX-2.

5. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 90% of the length of SSX-2.

6. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 80% of the length of SSX-2.

7. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 60% of the length of SSX-2.

8. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 50% of the length of SSX-2.

9. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 25% of the length of SSX-2.

10. The nucleic acid of claim 4, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 10% of the length of SSX-2.

11. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of amino acids 5-65, 5-67, or 5-114.

12. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of amino acids 16-65, 16-67, 16-114, 16-180, or 16-183.

13. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of amino acids 41-67, 41-114, 41-180, or 41-183.

14. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of amino acids 57-114, 57-180, or 57-183.

15. The nucleic acid of claim 4, wherein said encoded fragment consists essentially amino acids 99-180 or 99-183.

16. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of amino acids 16-183 of SSX-2.

17. The nucleic acid of claim 16, wherein said first sequence encodes exactly amino acids 15-183 of SSX-2.

18. The nucleic acid of claim 4, wherein said encoded fragment consists essentially of an amino acid sequence beginning at one of amino acids selected from the group consisting of 5, 16, 41, 57, and 99 of SSX-2, and ending at one of the amino acids selected from the group consisting of amino acid 65, 67, 114, 180, and 183 of SSX-2

19. The nucleic acid of claim 1, further comprising a second sequence, wherein the second sequence encodes essentially a housekeeping epitope.

20. The nucleic acid of claim 1, wherein said reading frame is operably linked to a promoter.

21. The nucleic acid of claim 19, wherein said first and second sequences constitute a single reading frame.

22. The nucleic acid of claim 21, wherein said reading frame is operably linked to a promoter.

23. An isolated polypeptide comprising the amino acid sequence encoded in said reading frame of claim 22.

24. An immunogenic composition comprising the nucleic acid of claim 22.

25. An immunogenic composition comprising the polypeptide of claim 23.