AU2008229888B2 - Expression vectors encoding epitopes of target-associated antigens and methods for their design - Google Patents

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. 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. 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. figure 1. pMA2M ISS (3) &NIS (7) CMVp (1) pMB ori rev (6) 3315 bp BGH poly A (4) Kan R( Figure Legend: Code in Figure Genetic Element Region I-CMVp Cytomeglovirus Enhancer/Promotor- 63-637 2. SAbs'\ s cot. 696-983 . SeC t.~~I £fTrOPL . 3. ISS Immunostimulatory Sequence 3220-3226 4. BGH poly A' Bovine Growth Hormone 1028-1045 Polyadenylation Signal 5. Kan R Kanamycin Resistance Gene 1431-2225 6. pMB ori rev Bacterial pMB Origin of Replication 3165-2492 7. NIS Nuclear Import sequence from 3227-3304 Simian Virus 40-72bp repeat

Description

P/00/o1I Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT (ORIGINAL) Name of Applicant(s): MannKind Corporation, of 28903 North Avenue Paine, Valencia, California 91355, United States of America Actual Inventor(s): John J.L. SIMARD; David C. DIAMOND; Zhiyong QUI; Xiang Dong LEI Address for Service: DAVIES COLLISON CAVE, Patent & Trademark Attorneys, of I Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM Invention Title: "Expression vectors encoding epitopes of target-associated antigens and methods for their design" The following statement is a full description of this invention, including the best method of performing it known to us: P/00/008b EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN This application is a divisional application of Australian Application No. 5 2002365932 the specification and drawings of which as originally filed are incorporated herein in their entirety by reference. Background of the Invention Field of the Invention 100011 The invention disclosed herein is directed to methods for the design of 10 epitope-encoding vectors 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). 15 Description of the Related Art [00021 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 20 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. [00031 Cytotoxic T Lymphocytes (CTL) do not recognize epitopes on the 25 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. [00041 The antigen processing and display system on the surface of cells has been 30 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. -1 - Summary of the Invention [0005] 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 5 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 10 Application Nos. 09/560,465 and 09/561,074 entitled EPITOPE -la- SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, and METHOD OF EPITOPE DISCOVERY, respectively; both of which were filed on April 28, 2000. (00061 Examples of housekeeping epitopes are disclosed in provisional U.S. Patent Applications entitled EPITOPE SEQUENCES, Nos. 60/282,211, filed on April 6, 2001; 5 60/337,017, filed on November 7, 2001; 60/363210 filed 3/7/02; and 60/409,123, filed on September 5, 2002; and U.S. Application No. 10/117,937, filed on April 4, 2002, which is also entitled EPITOPE SEQUENCES. [00071 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 0 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. 15 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 20 sequences. [00081 Epitope clusters and their uses are described in U.S. Patent application Nos. 09/561,571 entitled EPITOPE CLUSTERS, filed on April 28, 2000; 10/005,905, entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, filed on November 7, 2001; and 10/026,066, filed on December 7, 2001, also entitled EPITOPE SYNCHRONIZATION IN 25 ANTIGEN PRESENTING CELLS. [0009] 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 30 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 35 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 -2immunoproteasome 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. [0010] The invention disclosed herein relates to the identification of a polypeptide 5 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 10 substrate polypeptide by the proteasome; and assaying for liberation of the epitope. 10011] 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. [0012] In one aspect, the substrate polypeptide can be a synthetic peptide. Optionally, 15 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. [00131 In another aspect, the substrate polypeptide can be encoded by a 20 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. 25 [00141 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. [00151 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 30 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. [00161 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 35 substrate polypeptide in which the epitope is embedded can be conformed to a preferred immune proteasome cleavage site flanking sequence. -3- 100171 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 5 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 10 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. [00181 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. [00191 In yet another aspect, the proteasome can be an immune proteasome. 15 [00201 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. 100211 In one aspect of the invention the expression cassette can encode an array of 20 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 25 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. 30 [00221 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 35 cassette and the authentic sequence including at least one immune epitope may not be transcribed as a single mRNA species. -4- [00231 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, 5 and the like. Also, the vector can encode SEQ ID NO. 5 or SEQ ID NO. 18, for example. [00241 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. [00251 Another embodiment of the invention relates to vectors including a 10 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. [0026] In one aspect, the housekeeping epitope can be liberated from the cassette translation product by immune proteasome processing 15 [00271 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 20 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. 100281 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. 25 [00291 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 30 [0030] Figure 1. An illustrative drawing depicting pMA2M. [00311 Figure 2. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by mock immunized CTL. 100321 Figure 3. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM3 immunized CTL. 35 [00331 Figure 4. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM2 immunized CTL. -5- [00341 Figure 5. Assay results showing the % of specific lysis of ELAGIGILTV pulsed and unpulsed T2 target cells by pVAXM 1 immunized CTL. (00351 Figure 6. Illustrates a sequence of SEQ ID NO. 22 from which the NY-ESO 1157.165 epitope is liberated by immunoproteasomal processing. 5 [00361 Figure 7. 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. [00371 Figure 8. 8A: Shows the results of the human immunoproteasome digest of 10 SEQ ID NO. 31. 8B: Shows the comparative results of mouse versus human immunoproteasome digestion of SEQ ID NO. 31. 1[00381 Figure 9. Shows the differential processing of SSX-2 31

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6 s by housekeeping and immunoproteasome. Detailed Description of the Preferred Embodiment 15 Definitions [00391 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. [00401 PROFESSIONAL ANTIGEN-PRESENTING CELL (pAPC) - a cell that possesses T cell costimulatory molecules and is able to induce a T cell response. Well 20 characterized pAPCs include dendritic cells, B cells, and macrophages. 100411 PERIPHERAL CELL - a cell that is not a pAPC. [00421 HOUSEKEEPING PROTEASOME - a proteasome normally active in peripheral cells, and generally not present or not strongly active in pAPCs. [00431 IMMUNOPROTEASOME - a proteasome normally active in pAPCs; the 25 immunoproteasome is also active in some peripheral cells in infected tissues or following exposure to interferon. 100441 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 30 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 35 ("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 -6limitation, 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 5 by TCR, respectively. [00451 MIHC EPITOPE - a polypeptide having a known or predicted binding affinity for a mammalian class I or class II major histocompatibility complex (MHC) molecule. [00461 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 .0 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 15 are provide in U.S. Application No. 10/117,937, filed on April 4, 2002; and U.S. Provisional Application Nos. 60/282,211, filed on April 6, 2001; 60/337,017, filed on November 7, 2001; 60/363210 filed 3/7/02; and 60/409,123, filed on September 5, 2002; all of which are entitled EPITOPE SEQUENCES. [00471 IMMUNE EPITOPE - In a preferred embodiment, an immune epitope is 20 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, 25 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. (0048] 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 30 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 35 pulsed with peptide/epitope. -7- 100491 TARGET-ASSOCIATED ANTIGEN (TAA) - a protein or polypeptide present in a target cell. 100501 TUMOR-ASSOCIATED ANTIGENS (TuAA) - a TAA, wherein the target cell is a neoplastic cell. 5 [00511 HLA EPITOPE - a polypeptide having a known or predicted binding affinity for a human class I or class 11 HLA complex molecule. 100521 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 .0 region-phage coat protein fusions. 100531 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. 15 100541 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 20 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. 25 100551 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 30 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.) 35 to recognize the target antigen. Accordingly, two sequences may be designed to differ in certain -8respects while retaining the same function. Such designed sequence variants are among the embodiments of the present invention. [00561 EXPRESSION CASSETTE - a polynucleotide sequence encoding a polypeptide, operably linked to a promoter and other transcription and translation control elements, 5 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. 100571 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 10 that the epitope is not in an interior position. 100581 MATURE EPITOPE - a peptide with no additional sequence beyond that present when the epitope is bound in the MHC peptide-binding cleft. [00591 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 15 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 No. 09/561,571 entitled EPITOPE CLUSTERS. [00601 Substrate or liberation sequence- a designed or engineered sequence 20 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. [00611 Degradation of cytosolic proteins takes place via the ubiquitin-dependent 25 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. 30 [00621 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 35 recognize peripheral cells in the body and how this targeting requires synchronization between the immune system and the targeted cells. -9- [00631 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 5 generated by the housekeeping proteasome are presented on all of the peripheral cells of the body. 100641 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 0 the immunoproteasome, owing to its expression in pAPC and as a consequence of induction by IFN in body cells. [00651 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 15 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 20 differential proteasome activity in pAPC and peripheral body cells, therefore, is important to consider during natural infection and with therapeutic CTL vaccination strategies. [00661 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 25 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 30 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. 35 [00671 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 -10immunoproteasome 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. [00681 In the case of therapeutic vaccination to eradicate tumors or chronic infections, 5 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 10 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. [00691 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 1 5 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 20 as peptides, can be used to successfully immunize or induce therapeutic CTL responses against housekeeping proteasome expressing target cells in the host. [00701 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 25 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 No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS, filed on April 28, 2000. 30 100711 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 35 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 -11embodiments 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 5 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 10 immune epitopes. [00721 Several disclosures make reference to polyepitopes or string-of-bead arrays. See, for example, W00119408Al, March 22, 2001; W09955730A2, November 4, 1999; W00040261A2, July 13, 2000; W09603144A1, February 8, 1996; EPI181314A1, February 27, 2002; WO0123577A3, April 5; US6074817, June 13, 2000; US5965381, October 12, 1999; 15 WO9741440AI, November 6, 1997; US6130066, October 10, 2000; US6004777, December 21, 1999; US5990091, November 23, 1999; W09840501A1, September 17, 1998; W09840500A1, September 17, 1998; WO0118035A2, March 15, 2001; W002068654A2, September 6, 2002; W00189281A2, November 29, 2001; W00158478A, August 16, 2001; EP1118860Al, July 25, 2001; WO0111040AI, February 15, 2001; W00073438A1, December 7, 2000; WO0071158A1, 20 November 30, 2000; W00066727AI, November 9, 2000; W00052451A1, September 8, 2000; W00052157A1, September 8, 2000; W00029008A2, May 25, 2000; W00006723Al, February 10, 2000. Additional disclosures, include Palmowski MJ, et al - J Immunol 2002;168(9):4391-8; Fang ZY, et al - Virology 2001;291(2):272-84; Firat H, et al - J Gene Med 2002;4(l):38-45; Smith SG, et al - Clin Cancer Res 2001;7(12):4253-61; Vonderheide RH, et al - Clin Cancer Res 2001; 25 7(11):3343-8; Firat H, et al - Eur J Immunol 2001;31(10):3064-74; Le TT, et al - Vaccine 2001;19(32):4669-75; Fayolle C, et al - J Virol 2001;75(16):7330-8; Smith SG - 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 MH, et al - Cell Immunol 1999;195(l):10-7; Woodberry T, et al - J Virol 1999;73(7):5320-5; Hanke T, et al - Vaccine 1998;16(4):426-35; 30 Thomson SA, et al - J Immunol 1998;160(4):1717-23; Toes RE, et al - Proc Natl Acad Sci USA 1997;94(26):14660-5; Thomson SA, et al - J Immunol 1996;157(2):822-6; Thomson SA, et al Proc Natl Acad Sci USA 1995;92(13):5845-9; Street MD, et al - Immunology 2002;106(4):526-36; Hirano K, et al - Histochem Cell Biol 2002;1 17(l):41-53; Ward SM, et al - Virus Genes 2001;23(1):97-104; Liu WJ, et al - Virology 2000;273(2):374-82; Gariglio P, et al - Arch Med Res 35 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 LL, et al - J Virol 1997;71(3):2292-302; Whitton JL, et al - J -12- 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. 100731 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 5 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 10 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 15 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 tlie 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 20 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 25 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 30 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 35 from others. Some embodiments relate to repeated administration of vectors. In some of those embodiments, nonviral and nonbacterial vectors are included. Likewise, some embodiments -13include 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. [00741 Embodiments of the present invention also include methods, uses, therapies, 5 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 1-5, 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 1-5, or in any of 10 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, lyniphangiosarcoma, 15 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 20 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 25 (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 30 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 B1 9, 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, 35 Mycobacterium, Leishmania, Trypanasoma, Toxoplasma, Plasmodium, and the like. 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Res., 270:35-49 (1988); Falk, K. et al., Nature, 351:290, (1991); Ausubel et al., Short Protocols in Molecular Biology, Third Edition, Unit 11.2 (1997); hypertext transfer protocol address 10 134.2.96.221/scripts/hlaserver.dll/EpPredict.htm; Levy, Morel, S. et al., Immunity 12:107-117 (2000); Seipelt et al., Virus Research, 62:159-68, 1999; Storkus et al., U.S. Patent No. 5,989,565, issued November 23, 1999; Morton, U.S. Patent No. 5,993,828, issued November 30, 1999; Virus Research 62:159-168, (1999); Simard et al., U.S. Patent Application No. 10/026066, filed December 07, 2001; Simard et al., U.S. Patent Application No. 09/561571, filed April 28, 2000; 15 Simard et al., U.S. Patent Application No. 09/561572, filed April 28, 2000; Miura et al., WO 99/01283, January 14, 1999; Simard et al., U.S. Patent Application No. 09/561074, filed April 28, 2000; Simard et al., U.S. Patent Application No. 10/225568, filed August 20, 2002; Simard et al., U.S. Patent Application No. 10/005905, filed November 07, 2001; Simard et al., U.S. Patent Application No. 09/561074, filed April 28, 2000. 20 [00761 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 1-5, 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. 25 Other embodiments may exclude any combination or all of those antigens. Several examples of such antigens include MelanA (MART-I), gplOO (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, 30 MAGE-6, p185erbB2, pl80erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CAM 17.1, NuMa, K-ras, $ Catenin, CDK4, Mum-1, p16, as well as any of those set forth in the above mentioned references. Other antigens are included in Tables 1-4 below. [00771 Further embodiments include methods, uses, compositions, and therapies relating to epitopes, including, for example those epitopes listed in Tables 1-5. These epitopes can 35 be useful to flank housekeeping epitopes in screening vectors, for example. Some embodiments -17include one or more epitopes from Tables 1-5, while other embodiments specifically exclude one or more of such epitopes or combinations thereof. Table 1 Virus Protein AA T cell epitope MHC MHC molecule Position ligand (Antigen) 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 ElB 192-200 VNIRNCCY 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 d/d polyprotein I (SEQ. ID NO.:48) Dengue virus 4 NS3 500-508 TPEGIIPTL HLA-B*3501 (SEQ. ID NO.:49) EBV LMP-2 426-434 CLGGLLTMV HLA-A*0201 (SEQ. ID NO.:50) EBV EBNA-l 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-l 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 RMLWMANY HLA-A*0201 (SEQ. ID NO.:59) EBV EBNA-2 133-141 MLWMANYIV HL.A-A*0201 (SEQ. ID NO.:60) EBV EBNA-2 151-159 ILPQGPQTA HLA-A*0201 (SEQ. ID NO.:61) 18 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 BZLFI 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 IDNO.:81) EBV EBNA-3 379-387 RPPIFIRRI. HLA-B7 (SEQ. ID NO.:82) EBV EBNA-1 426-434 EPDVPPGAI HLA-B7 I _(SEQ. ID NO.:83) EBV EBNA-1 228-236 IPQCRLTPL HLA-B7 19 (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 I (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 BZLFI 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 Y1KSFVSDA 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 20 ________(SEQ. ID NO.: 106) __________ HCV NSI 77-85 PPLTDFDQGW HLA-B*5301 (SEQ. ID NO.: 107)_________ HCV core 18-27 LMGYIPL VGA 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 NSI 205-213 KIIPDATYSR Papa-A06 ____ ____ __ ____ ___ ___ (SEQ. ID NO.: I11) HCV-1 NS3 400-409 KLVALGINAV HLA-A*0201 S______ ~~(SEQ. ID NO.: 112)_ _ _ _ _ _ _ _ HCV-1 NS3 440-448 GDFDSVIDC Patr-B 16 S______ ~~(SEQ. ID NO.: 113) _ _ _ _ _ _ _ HCV- 1 env E 118-126 GNASRCWVA Patr-B16 S_________ ~~(SEQ. ID NO.: 114) __________ HCV-1 NSI 159-167 TRPPLGNWF Patr-B 13 S_______(SEQ. ID NO.: 115) HCV-1 NS3 351-359 VPHPNIEEV Patr-B 13 S(SEQ. ID NO.: 116) HCV-1 NS3 438-446 YTGDFDSVI Patr-BO I S___ ___(SEQ. ID NO.: 117) HCV-1 NS4 328-335 SWAIKWEY Patr-A1 1 S(SEQ. ID NO.: 118) HCV-1 NSI 205-213 KHPDATYSR Patr-A04 S________(SEQ. ID NO.: 119) HCV-1 NS3 440-448 GDFDSVIDC Patr-A04 S~~~~~~(SEQ. ID NO.: 120) __________ HIV gp4l 583-59 1 RYLKDQQLL HLA A24 S________ ~(SEQ. ID NO.: 12 1)_ _ _ _ _ _ _ _ _ HIV gagp24 267-275 IVGLNKIVR HLA-A*3302 S~~~~~~~(SEQ. ID NO.:122) __________ HIV gagp24 262-270 EIYKRWIIL HLA-B8 S________ ~~(SEQ. ID NO.: 123)_ _ _ _ _ _ _ _ _ HIV gagp24 261-269 GE1YKRWII HLA-B8 S_________ ~~(SEQ. ID NO.: 124) _ ________ HIV gagp17 93-101 EIKDTKEAL HLA-B8 _______(SEQ. ID NO.: 125)__________ HLV gp4l 586-593 YLKDQQLL HLA-B8 ________(SEQ. ID NO.: 126) __________ HIV gagp24 267-277 ILGLNKIVRMY HLA-B* 1501 ____________ ________ I (SEQ. ID NO.: 127) _ _ _ _ _ _ _ _ _ _ 21 HIV gp4I 584-592 ERYLKDQQL HLA-B 14 ____________ ~~(SEQ. ID NO.: 128) __________ HIV nef 115-125 YHTQGYFPQWQ HLA-B 17 ____________ ________(SEQ. ID NO.: 129) _ _ _ _ _ _ _ _ _ HIV nef 117-128 TQGYFPQWQNYT HLA-B 17 ___ ___ __ ___ __(SEQ. ID NO.: 130) HIV gp]20 3 14-322 GRAFVT1GK HLA-B*27O5 _________ ___ ___(SEQ. ID NO.: 131) HIV gagp24 263-27 1 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) __________ H-IV gagp24 143-151 HQAISPRTI, HLA-Cw*O301 ________(SEQ. ID NO.: 13 5) __________ HIVgag24 40-5 1 QMVHQAISPRTL HLA-Cw*O301 _____ ____ _____ _ ___ ___ SEQ. ID NO.: 136)_ _ _ _ _ _ _ _ _ HIV gp120 431-440 MYAPPIGGQI H2-Kd __________ ___ ___ _ _______(SEQ. ID NO.: 13 7) HIV gp160 318-327 RGPGRAFVTI H2-Dd __________ ___ ___ _ _______(SEQ. ID NO.: 13 8) HIV gp120 17-29 MIPGRAFVTI H2-Ld ____ ____ __ ____ ___ ___(SEQ. ID NO.: 139) HIV-1 RI 476-484 ILKEPVHGV HLA-A*02O1 ____ ____ __ ________ ____ ___(SEQ. ID NO.: 140) _ _ _ _ _ _ _ _ _ _ HIV-1 nef 190-198 AFHHVAREL HLA-A*0201 __________ _______ (SEQ. ID NO.: 14 1)_ _ _ _ _ _ _ _ _ HIV-1 gp160 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*020 1 ___________ _________(SEQ. ID NO.: 145) _ _ _ _ _ _ _ _ _ _ HIV-1 gp160 315-329 1RGPGRAFVT I llLA-A*0201 ___________ ________(SEQ. ID NO.: 146) _ _ _ _ _ _ _ _ _ HIV-1 gp4l 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 120-29 RLRPGGKKK HLA-A3 22 ________________(SEQ. ID NO.: 150) HIV-1 gp120 38-46 VYYGVPVWK HLA-A3 ___________ ~(SEQ. ID NO.: 15 1)__________ HIV- I nef 74-82 VPLRPMTYK HLA-a* 10 1 __________ ________ (SEQ. ID NO.: 152) _ _ _ _ _ _ _ _ _ HIV-1 gagp24 325-333 AIFQSSMTK HLA-A*1 101 _______ __ ___ ____ _ ______(SEQ. ID NO.: 153) _ _ _ _ _ _ _ _ _ HIV-1I nef 73-82 QVPLRPMTYK HLA-A*1 101 __________ _______(SEQ. IDNO.: 154) HIV- I nef 83-94 AAVDLSHFLKEK HLA-A*1 101 __________ ______(SEQ. ID NO.: 15 5) HIV-1 gagp24 349-359 ACQGVGGPGGHK HLA-A* I 10 1 ____________ __________(SEQ. ID 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 24-3 1 GGKKKYKL HLA-B8 ________(SEQ. ID NO.: 159) HIV-l gp120 2-10 RVKEKYQHL HLA-B8 _______I (SEQ. ID NO.: 160) HIV-1 gagp24 298-306 DRFYKTLRA HLA-B 14 ____ _ __ ___(SEQ. INO.: 16 1) HIV-1 NEF 132-147 GVRYPLTFGWCYKLVP HLA-B 18 ________ _______(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 33 5-343 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-B5 I ___________ _________(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 -s~ 348-357 1GLSPTVWLSV HLA-A*0201 __________(SEQ. ID NO.: 170) i-mv S ~ 335-343 WLSLLVPFV HLA-A*O201 __________I (SEQ. ID NO.: 17 1) ________ HBV Ic~ 18-27 FLPSDFFPSV HLA-A*O201 __ _ _ __ _ _ __ _ __I___ (SEQ. ID NO.: 172) _ _ _ _ _ _ _ _ _ _ 23 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.:18 1) HBV 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) 1 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 sAg 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. ID NO.: 194) HCMV El 978-989 SDEEFAIVAYTL HLA-B18 24 (SEQ. ID NO.:195) HCMV pp65 397-411 DDVWTSGSDSDEELV HLA-b35 (SEQ. ID NO.:196) HCMV pp 6 5 123-131 IPSINVHHY HLA-B*3501 (SEQ. ID NO.:197) HCMV pp65 495-504 NLVPMVATVO HLA-A*0201 (SEQ. ID NO.:198) HCMV pp 6 5 415-429 RKTPRVTOGGAMAGA HLA-B7 (SEQ. ID 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) HCV NS2 234-246 GLRDLAVAVEPVV HLA-A*0201 (SEQ. ID 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 CINGVCWTV HLA-A*0201 (SEQ. ID NO.:216) HCV NS3 163-171 LLCPAGHAV HLA-A*0201 25 ____ ___ __ _______ ___ ___(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 28 1-290 TGAPVTYSTY HLA-A*0201 ________(SEQ. ID NO. :22 1) __________ 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*1 101 (SEQ. ID NO. :224) __________ HCV NS2 109-116 YISWCLWW HLA-A23 __________ (SEQ. ID NO. :22 5)___________ HCV core 40-48 GPRLGVRAT HLA-B7 protein_____________ _________ ___ __________ (SEQ. ID NO. :226) _ _________ HIV-1 gp 12 0 380-388 SFNCGGEFF HLA-Cw*0401 _________ (SEQ. ID NO. :227) __________ HIV-1 RT 206-214 TEMEKEGKI H2-Kk __________ (SEQ. ID NO. :22 8)___________ HIV-1 p17l 18-26 KRLRPGGK HLA-A*0301 (SEQ. ID NO. :229) __________ HIV-1 P 17 20-29 RLRPGGKKKY HLA-A*0301 (SQCDNO __________ HIV- I RT 325-333 AIFQSSMTK HLA-A*0301 (SEQ. ID NO.:23 1) __________ HIV-1 p17 84-92 TLYCVHQRI HLA-AlI1 ____________ ~~(SEQ. ID NO. :23 2) __________ HIV-1 RT 508-5 17 IYQEPFKNLK HLA-AI1 S_________ ~~(SEQ. ID NO.:233) __________ HIV-1 p7 28-36 KYKLKHIVW HLA-A24 ____________ ~~(SEQ. ID NO.:234) __________ HIV-1 gp120 53-62 LFCASDAKAY HLA-A24 S_________ ~~(SEQ. ID NO.:23 5) __________ HIV-1 gagp24 145-155 QAISPRTLNAW HLA-A25 S_________ ~~~(SEQ. ID NO. :236) __________ HIV-1 gagp24 167-175 IEVIPMFSAL HLA-A26 (SEQ. ID NO.:237) __________ HIV-1 RT 593-603 ETFYVDGAANR HLA-A26 ___________ ________(SEQ. ID NO.:238) _ _ _ _ _ _ _ _ _ HIV-1 gp4l 775-785 RLRDLLLIVTR JHLA-A31 26 (SEQ. ID NO.:239) HIV-1 RT 559-568 PIQKETWETW HLA-A32 (SEQ. ID NO.:240) HIV-1 120 419-427 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 gag.p2_4_1816 SRLAVHA HIV-1 gagp24 179-187 ATPQDLNTM _ _ __HLAB7 (SEQ. ID NO.:246) HIV-1 gpI20 303-312 RPNNNTRKSI HLA-B7 (SEQ. ID NO.:247) HIV-1 gp41 843-851 IPRRIRQGL HLA-B7 (SEQ. ID NO.:248) HIV-1 _17 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 gag p24 183-191 DLNTMLNTV HLA-B14 (SEQ. ID NO.:252) HyV-~ P17 18-27 KIRLRPGGKK HLA-B27 (SEQ. ID NO.:253) HIV-1 p 17 19-27 IRLRPGGKK HLA-B27 (SEQ. ID NO.:254) HIV-1 gp41 791-799 GRRGWEALKY HLA-B27 (SEQ. ID NO.:25 5) HIV-1 nef 73-82 QVPLRPMTYK HLA-B27 i f (SEQ. ID NO.:256) _________ HIV-1 GP41 590-597 RYLKDQQL HLA-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 17 36-44 WASRELERF HLA-B35 (SEQ. ID NO.:260) HIV-1 GAG P24 262-270 TVLDVGDAY HLA-B35 _ I(SEQ. IDNO.:261) 27 HIV-1 _IELO_ 42-52 VPVWKEATTTL HLA-B35 (SEQ. ID NO.:262) HIV-1 P17 36-44 NSSKVSQNY HLA-B35 (SEQ. ID NO.:263) HIV-1 gag p24 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 _______ 120__128 _(SEQ. ID NO.:267) HIV-1 nef 120-128 YFPDWQNYT HLA-B37 (SEQ. ID NO.:268) HIV-1 gag p 2 4 193-201 GHQAAMQML HLA-B42 (SEQ. ID NO.:269) HIV-1 p 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 gag p 2 4 325-333 NANPDCKTI HLA-B51 (SEQ. ID NO.:273) HIV-1 gag p 24 275-282 RMYSPTSI HLA-B52 -~ _-- _~ _(SE Q . ID N O .:274)_ HIV-1 42-51 VPVWKEATTT HLA-B*5501 (SEQ. ID NO.:275) HIV-1 gag p 24 147-155 ISPRTLNAW HLA-B57 (SEQ. ID NO.:276) HIV-1 gag p 2 4 240-249 TSTLQEQIGW HLA-B57 (SEQ. ID NO.:277) HIV-1 gag p 2 4 162-172 KAFSPEVIPMF HLA-B57 (SEQ. ID NO.:278) HIV-1 gag p 2 4 311-319 QASQEVKNW HLA-B57 _(SEQ. ID NO.:279) HIV-1 ga3p 24 11-319 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 gag p 2 4 240-249 TSTLQEQIGW HLA-B58 (SEQ. ID NO.:283) HIV-1 p 1 7 20-29 RLRPGGKKKY HLA-B62 28 (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 gag p 2 4 168-175 VIPMFSAL HLA-Cw*0102 (SEQ. ID NO.:290) HIV-1 gp120 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 gag p24 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 p24 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 gp12 312-320 IGPGRAFHT H2-Dd (SEQ. ID NO.:305) HIV-1 5F2 pol 25-33 NPDIVIYQY HLA-B*3501 (SEQ. ID NO.:306) | 29 HIV-15F2 pol 432-441 EPIVGAETFY HLA-B*3501 ____ ____ _ __ ____ ___ ____(SEQ. ID NO.:307)_ _ _ _ _ _ _ _ _ HIV-1 S F2 pol 432-440 EPIVGAETF HLA-B*3501 ____ ____ __ _ ___ ___ _______(SEQ. ID NO. :308) _ _ _ _ _ _ _ _ _ _ HIV-1 S F2 pol 6-14 SPAIFQSSM HLA-B*3501 _________ ___ ____(SEQ. ID NO.:309) HIV-1 ISF2 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) HIV-1 5F2 nef 69-79 RPQVPLRPMTY HLA-B*3501 __________ ________ _______(SEQ. ID NO. :312) H-IV-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) HLV-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 SF2 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 gp4l 593-607 GIWGCSGKLICTITAV 14LA-B 17 ____________ __________(SEQ. ID NO.: 32 1) _________ HIV-1 5F2 gp4l 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) _ _________ HPY1 I E7 4-12 RLVTLKDIV HLA-A*0201 ________(SEQ. ID NO. :325) HPV 16 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) _ _ _ _ _ _ _ _ _ _ HPVI6 JE7 1-0 MLLQPETT IHLA-A*0201 30 ___________(SEQ. ID NO. :3 29) HPV16 E6 15-22 RPRKLPQL HLA-B7 ____________ ~~(SEQ. ID NO.:330) __________ HPV16 E6 49-57 RAHYNIVTF HW-Db __________ ~(SEQ. ID NO.:33 1) _ _ _ _ _ _ _ _ HSV gp 498-505 SSIEFARL H2-Kb (SEQ. ID NO.:332) _ _ _ _ _ _ _ _ HSV-1 g 480-488 GIGIGVLAA HLA-A*02O1 ________(SEQ. ID NO. :33 3) __________ HSV-1 ICP27 448-456 DYATLGVGV H2-Kd ________ SEQ.I N . :3 34) _ _ _ _ _ _ _ _ _ _ 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 g 446-454 GAGIGVAVL HLA-A*O201 ____ ___ ____ ___ ___ __ _ ___ ___ (SEQ. ID NO. :33 7)_ _ _ _ _ _ _ _ _ _ _ HLTV-1I 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.: 34 1) Influenza NP 91-99 KTGGPIYKR HLA-A*6801 _____________ ~~(SEQ. ID NO. :342) _ _________ Influenza NP 3 80-388 ELRSRYWAI HLA-B8 .I: *, (SEQ. ID NO. :343) ___________ Influenza NP381-388 LRSRYWAI HLA-B*2702 (SE . I N .:344) __________ Influenza NP 33-347 EDLRVLSFI HLA-B*3701 ____ ____ __ _ ____ ___ ____ ___(SEQ. ID NO. :345) _ _ _ _ _ _ _ _ _ _ Influenza NSI 158-166 GEISPLPSL HLA-B44 ____ ___ ____ ___ ___ __ _ ___ ___ (SEQ. ID NO. :346) _ _ _ _ _ _ _ _ _ _ Influenza NP 3846 FEDLRVLSF HLA-B44 ________ _______(SEQ. ID NO. :347) _ _ _ _ _ _ _ _ _ _ Influenza NI 158-166 GEISPLPSL HLA-B*4402 _______(SEQ. ID NO. :34 8) _ _ _ _ _ _ _ _ Influenza NP 338-346 FEDLRVLSF HLA-B*4402 ________(SEQ. ID NO.:349)___________ Influenza PBI 591-599 VSDGGPKLY HLA-A1 ___ ___ _ _______(SEQ. ID NO.:350) _ _ _ _ _ _ _ _ _ _ 31 Influenza A NP 44-52 CTELKLSDY HLA-Al (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 IEGGWTGM1 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 ~_ _-- --- _~_~_~_ (S E Q . ID N O .:3 6 9 ) 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 cA 318-326 APTAGAFFF H2-Ld (SEQ. ID NO.:372) LCMV NP 118-126 RPQASGVYM H2-Ld 32 (SEQ. ID NO.:373) 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 pp 89 168-176 YPHFMPTNL H2-Ld (SEQ. ID NO.:377) MHV spike 510-518 CLSWNGPHL H2-Db protein (SEQ. ID NO.:378) MMTV env gp 36 474-482 SFAVATTAL H2-Kd (SEQ. ID NO.:379) MMTV gag p 27 425-433 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 gp76 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 VPI 208-217 FYDGFSKVPL H2-Kd (SEQ. ID NO.:392) Pseudorabies GI1 455-463 IAGIGILAI HLA-A*0201 virus __ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ (SEQ. ID NO.:393) 33 Rabiesvirus NS 197-205 VEAEIAHQI H2-Kk (SEQ. ID NO.:394) Rotavirus VP7 33-40 IIYRFLLI 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 gagpllC 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) V40 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._IDNO. :406) SV40 T 560-568 SEFLLEKRI H2-Kk (SEQ. ID NO.:407) VSV NP 52-59 RGYVYQGL H2-Kb (SEQ. ID NO.:408) 34 Table 2 HLA-Al Position (Antigen) Source T cell epitopes EADPTGHSY MAGE-1 161-169 (SEQ. ID NO.:409) VSDGGPNLY Influenza A PB 1591-599 (SEQ. ID NO.:4 10) CTELKLSDY Influenza A NP 44-52 (SEQ. ID NO.:41 1) EVDPIGHLY MAGE-3 168-176 (SEQ. ID NO.:412) HLA-A201 MLLSVPLLLG Calreticulin signal sequence I-10 (SEQ, ID NO.:413) STBXQSGXQ HBV PRE-S PROTEIN 141-149 (SEQ. ID NO.:414) 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) CG___CLGOLLTMV EBV LMP-2 426-434 (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 35 _____ _____ (SEQ. ID NO.:429)_ _ _ _ _ _ _ _ _ _ _ _ _ _ __________YLEPGPVTA Pmel 17/gp 100 480-488 ____ ____ ___ (SEQ. ID NO.:430)_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __________ILDGTATLRL PmeI 17/ gp 100 457-466 ______ _____ (SEQ. ID NO.:43 1) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _________LLDGTATLRL PmeI gpIOO 457-466 ITDQVPFSV Pmel gp 100 208-2 17 (SEQ. ID NO.:433) _______________ AFHIIVARL WV-I nef 105-18 (SEQ. ID NO.:436) YLITQNPSL P.e facpau 1 S0 2 4-342 (SEQ. ID NO.:437) MMHRLHIV P. fau CS90-10 (SEQ. ID NO.:436) 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.:44 1) _______________ ALAII'QCRL EBNA-1 525-533 (SEQ. ID NO.:442) ________________ VLKDAIKDL EBNA-1 575-582 (SEQ. ID NO. :443) _________ MY2!R_________ EBNA-I 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.:45 1)_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 36 PLPPATLTV EBNA-2 205-2 13 (SEQ. ID NO. :452) ________RMHLPVLHV 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)________ ________ ________DLDESWDY1 EBNA-2 453-46 1 ____________ (SEQ. ID NO. :45 7) ________________ PLPCVLWPVV BZLFI 43-51 ___________ (SQ. D NO.:458) _________SLEECDSEL BZLFI 167-175 ____________ (SEQ, ID NO. :459) _________EIKRYKNRV BZLFI 176-184 ____________ (SEQ. ID NO. :460) _________QLLQ IYREV BZLF1 195-203 ____________ (SEQ. ID NO.:461) _________LLQHYREVA BZLFI 196-204 (SEQ. ID NO. :462) _________LLKQMCPSL BZLFI 217-225 ____________ (SEQ. ID NO. :463) _________SIIPRTPDV BZLFI 229-23 7 ____________ (SEQ. ID NO. :464) ___________AIMDKNIIL Influenza ANSI 122-130 ____________ (SEQ. ID NO. :465) ___________IMDKNIILKA Influenza A NSI 123-132 ____________(SEQ. ID NO. :466) _________LLALLSCLTV HCV MP 63-72 ____________ (SEQ. ID NO. :467) ________________ ________ILHTPGCV HCV MP 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)_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ________HLH NVDV HCV NSI 308-3 16 ____________ (SEQ. ID NO. :472) ________________ _________FLLLADARV HCV NSI 340-348 ____ ____ ____ (SEQ. ID NO. :473) ________________ _________GLRDLAVAVEPVV HCV NS2 234-246 37 (SEQ. ID NO.:474) SLLAPGAKQN'. HCV NS1 18-28 (SEQ. ID NO.:475) LLAPGAKQNV HCV NS 119-28 (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 gplOO 476-485 (SEQ. ID NO.:484) YIGEVLVSV Non-filament forming class I myosin family (HA-2)** (SEQ. ID NO.:485) 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 34-43 (SEQ. ID NO.:491) 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 38 (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 decarboxylase 114-123 (SEQ. ID NO.:499_ ILTVILGVL Melan A/Mart- 32-40 (SEQ. ID NO.:500)_ FLWGPRALV _MAGE-3 271-279 (SEQ. ID NO.:501)_ LLCPAGHAV HCV NS3 163-171 (SEQ. ID NO.:502) ILDSFDPLV HCV NSS 239-247 SEO. ID NO.:503) LLLCLIFLL HBV env 250-258 (SEQ. ID NO.:504) LIDYQMLPV HBV env 260-269 SE .IDNO.:508 SIVSPFIPLL HBV env 370-379 (SEQ. ID NO.:506) FLLTRILTI HBV env 183-191 (SEQ. ID NO.:507) HLGNVKYLV P. faciparu TRAP 3 -11 (SEQ. ID) NO.:508 ~GIAGGLALL P. faciparum TRAP 500-508 SEQ. ID NO.:50119 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 I)

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VIYYMDDLV HIV-IRT 179-187 SE .ID NO.:51G VLPDVFIRCV N-acetylglucosamiyltranfera se intron__... - . (SEQ. ID NO.:514 ~VLPDVFIR3C N-acetylglucosaminyltransferase V Gnt-V mntron (SEQ. ID NO.:515') AVGIGIAVV Human CD9 ---- LVVLGLLAV Human glutamyltransferase (E.ID NO.: 517) 39 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 HCVNSa 149-1 46 (SEQ. ID NO.:521) LIVIGILIL Aeoiu (SEQ. IDNO.:522) LAGIGLIAAS.Lncnessmr (SEQ. IDNO.:523) _ VDG GIL YIs-1 77-5 (SEQ. ID NO.:524) GAGIGVLTA B. lmyxa,pcndo lanase149 SE. IDNO.:3Il525) AGIIIQ E. coli methionine synthase 590-598 AAIMEIML 0oli hypothetical protein 4-12 ~ (SEQ. ID NO.:527') -XO IRPSGHIFV CDIK4wl 22.32 (SE .ID 0.52 8) KACDPI-ISGIIFV CDK4-R24C 22-32 (SEQ. ID NO.:529) ACDPFISGHFV CDK4-R24C 23-32 (SEQ. ID NO.:530) SLYNTVATL _HHIV- I gag p 17 77-85 ---- ELQ NO.531 HER-2, m>Vi substituted 971-979 (SEQ. ID NO.:532) RGPGRAFVTI HIV- I gp 160 315-329 ----- ~---- ~~ N O .:533) HWNFISGI _HCV NS4A 149-157 NLVPMVATVQ ----------- 95

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(SEQ. ID NO.: 53 5) ____ P bE 13 GLHCYEQLV V------------0 (SE_Q ID NO).:536)HP6bE475 PLKQH4FQIV P6bE475 (SEQ. ID NO.:537) LLDFVRFMGV EBNA-6 284-293 A E .DN .5 8 Influenza Alaska NS1_122-130 40 (SEQ. ID NO.:53) YLKTIQNSL P. falciparum ep36 CSP (SEQ. ID NO.:540) YLNKIQNSL P. falciparurn cp39 CSP (SEQ. ID NO.:541) YMLDLPETTHPV 16 E7 11-20* (SEQ . ID NO.:542) LLMGTLGIV HV6 E7 82-90** (SEQ. ID NO.:543 I) TLGIVCPI HPV 16 E7 86-93 (SEQ. IDNO.:544 ) TLTSCTV HIV-1 gp120 197-205 (SEQ._ID NO.:545') ~ -KLPQLCTEL HPV 16 E6 18-26 (SEQ . ID NO.:546) TIHDIILEC HPV 16 E6 29-3 7 (SEQ. ID NO.: 547) LGIVCPICS HPV 16 E7 87-95 (SEQ. ID NO.:548)MlnAMr- 54 VILG!VLLLI -----------------

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(SEQ. ID NO.:549) ALMDKSLHV Melan A/Mart- 1 56-64 (SEQ. ID NO.:5_50) GILTVILGV Melan A/Mart- 1 31-39 (SEQ. ID N~O.: 551) T cell epitopes MINAYLDKL ' .P. Falciparum STARP 523-531 ~ (SEQ . ID NO.:552') -AAGIGIL TV Melan A/Mart- 127-3 5 (SEQ. ID NO.:553) FLPSDFFPSV HBV cAg 18-27 (SEQ. ID NO.- 554) Motif unknown SVRDRLARL EBNA-3 464-472 T cell epitopes (E.I O:5)MlnAMr- 73 T cell epitopes AAGIGILTV MliAMit 2 (SEQ. ID NO.:556) FAYDKY _Human MHC I-ot 140-148 T cell epitopes AAGIGIL TV _ea /ar- 73 (SEQ. ID NO.:558) FLPSDFFPSV HBV cAg 18-27 (SEQ. ID NO.:559) _MeadAar-273 Motif unknown AAGIGILTV Mln /atI2 T cell epitopes (SEQ ID NO.:560)___..HVc1 82 FLPSDFFPSV ( SEQD ID NO.:561') 41 AAGIGILTV ___________ ______27-35 (SEQ. ID NO.:562) ALLAVGATK Pine! 17 gp 100 17-25 ____________ (SEQ ID NO. :563) _________________ T cell epitopes RLRDLLLIVTR HIV.1 gp4l 768-778 ____________(SEQ. ID NO. .564) ________QVPLRPMTYK HIV-1 nef 73-82 ______ _____ _ (SE . ID NO. :565) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ TVYYGVPVWK HIV-1 gp] 2

O-

3 6

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45 _____________ (SEQ. ID NO. :566') _______________ RLRPGGKKK HIV- 1 gag p 17 20-29 (SEQ.ID NO:5 42 (SEQ. ID NO.:583) __ _ _GP _41_583-591 T cell epitopes RYLKDQQLL HIV GP 41 583-591 (SEQ. ID NO.:584) .__ _ AYGLDFYIL P15 melanoma Ag 10- 18 (SEQ. ID NO.:585) AFLPWIHRLFL Tyrosinase 206-215 (SEQ. ID NO.:586) __ _ _ __ _ _ _ __206_214 AFLPWHRLF Tyrosinase 206-214 (S E Q . ID N O .:5 8 7 ) E b na -3 2 4 6 -2 5 3 RYSIFFDY Ebna-3 246-253 (SEQ. ID NO.:588) T cell epitope ETINEEAAEW HIV- I gag p 24 203-212 (SEQ. ID NO.:589) T cell epitopes STLPETTVVRR HBV cAg 141 -151 (SEQ. ID NO.:590) MSLQRQFLR ORF 3P-gp75 294-321 (bp) (SEQ. ID NO.:591) LLPGGRPYR TRP tyrosinasee rel.) 197-205 (SE Q . ID N O .:592) ___ _gag _p24 _267 -267 -275 T cell epitope IVGLNKIVR HIy gag p 24 267-267-275 (SEQ. ID NO.:593) AAGIGILTV Melan A/Mart- 127 35 (SEQ. ID NO.:594) 43 Table 3 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. TRA refers to the tumor-related antigen and the LUD No. refers to the Ludwig Institute number. Table 3 -TRA LUD Patent No. Date Patent Issued Peptide (Antigen)

HLA

MAGE-4 5293 5,405,940 11 April 1995 EVDPASNTY HLA-Al (SEQ. ID NO.:979) MAGE-41 5293 5,405,940 11 April 1995 EVDPTSNTY HLA-A I (SEQ ID NO:595) MAGE-5 52-93 5,405,940 11 April 1995 EADPTSNTY H LA-A I (SEQ ID NO:596) MAGE-51 5431 5,4,340 2 April 1995 EADPTSNTY HLA-A I MAGE 72-53 5,505,74 1 etme 96 SLETGVHA (SEQ ID NO:597) M-6 524 54590 11 April 1995 EVDPIGHVY H LA-Al (SEQ ID NO:598) 2 5,487,974 3Jauary 1996 MLVYLL H LA-A2

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(SEQ ID NO:599) 536 5,30,096 25 June 1996 MLLAVLYCL HLA-B44 (SEQ ID~NO:600) 7360.1 SEWRDIDFA HLA-B44 (4 QIDNO:60) SEIWREMb (EIDNO:602) Tyrsiase 54 1 ,74,16XEIWRD 198IDF HLA-B44 (SEQ ID NO:603) MAE- _340-554,24 1 Sptebe196 STLVEVTLGEV HLA-A2 (SQID NO:604) LVEVTLGEV (SEQ ID NO:605) - VIF5KASEYL (SEQ ID~NO:606) IIVLAIIAl (SQ ID NO:607) KIWEELSMLEV (5EQ ID NO:608)

LIETSYVKV

(SEQ ID NO:609) 5327 5,585,461 17 December 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 September 199 6 KIWEELSVL HLA-A2 (SEQ ID NO:613) MAGE-3 5393 5,405,940 11 April 1995 EVDPIGHLY H LA-Al (SEQ ID NO:614) IAGE 5293 5,405,940 11 April 1995 EXDX5Y HLA-Al (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 (1/Afm X3 Y (SEQ. ID NO.:619) E (A/V) D P (I/AfT) (G/S) X2 Y (SEQ. ID NO.:620) E (A/V) D P (/AlT) (G/S) (H/N) X Y (SEQ. ID NO.:621) E (AIV) DP (I/Aff) (G/S) (H/N) (IJT/N) Y (SEQ. 11) NO.:622) MAGE-1 5361 5,558.995 24 September 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-A 1 (SEQ ID NO:626) BAGE 5310.1 TBA TBA MAARAVFLALSAQLLQARLMKE HLA-C (SEQ ID NO:627) Clone 10 MAARAVFLALSAQLLQ H LA-C 45 (SEQ ID NO:628) Clone 10 AARAVFLAL HLA-C (SEQ ID NO:629) Clone 10 3AGE 5323.2 5,648,226 15 July 1997 YRPRPRRY HLA-CW6 (SEQ. .... .. 630) 46 Table 4 Source Protein AA MHC T cell epitope SEQ. Ref. Position molecules MHC ligand ID (Antigen) NO.: synthetic synthetic synthetic HLA-A2 ALFAAAAAV 631 Parker, et al., "Scheme ,eptides peptides peptides for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side chains," J. Immunol. 152:163-175 GIFGGVGGV 632 " GLDKGGGV 633 GLFGGFGGV 634 GLFGGGAGV 635 GLFGGGEGV 636 " GLFGGGFGV 637 " "_ GLFGGGGGL 638 GLFGGGGGV 639 GLFGGGVGV 640 GLFGGVGGV 641 __ GLFGGVGKV 642 " GLFKGVGGV 643 GLGGGGFGV 644 " GLLGGGVGV 645 " GLYGGGGGV 646 " GMFGGGGGV 647 " GMFGGVGGV 648 _____ "_ GQFGGVGGV 649 GVFGGVGGV 650 " KLFGGGGGV 651 KLFGGVGGV 652 AILGFVFTL _ 653 GAIGFVFTL 654 -- GALGFVFTL 655 " GELGFVFTL 656 _ GIAGFVFTL 657 " GIEGFVFTL 658__ GILAFVFTL 659 " GILGAVFTL 660 " GILGEVFTL 661 " GILFGAFTL 662 " 47 GILGFEFTL _ 663 _________ GILGFKFTL 664 ________ GILGFVATL 665 __________ GILGVETL 666 ____________ GILGFVFAL 667 GILTGFVFEL 668 i GILGFVFKL 669 6 GILGFVFTA 670 GILGFVFTL 671 __________ GILGFVYVL 672 ________ GILGFVKTL 673 ________ it GILGKVFTL 674 ________ GILKFVFTL 675 6 46 GILPFVFTL 676 ___ ______ GIVGFVFTL 677 _________ GKLGFVFTL 678 GLLGFVFTL 679 GQLGFVFTL 680 KALGFVFTL 681 ________ KILGFVFTL 682 it________ KILGKVFTL 683 AILLGVFML 684 ________ AIYKR WIIL 685 ____ ______ ALFFFDIDL 686 ___ ______ ATVELLSEL 687 CLFGYPVYV 688 FIFPNYTIV 689 6 IISLWDSQL 690 ____ ______ ILASLFAAV 691 ___ ______ ILESLFAAV 692 ___ ______ KLGEFFNQM 693 ________ KLGEFYNQM 694________ LLFGYPVYY 695 _________ LLWKGEGAV 696 LMFGYPVYV 697 i LNFGYPVYV 698 1_______ LQFGYPVYV 699 _______ NIVAI-TFKV 700 __ _______ NLPMVATV 701_________ QMLLAIARL 702_________ QMWQARL-TV 703__________ it RLLQTGIHV 704 ;t_______ RLVNGSLAL 705 ____ ______ -lSLYNTVATL 706 ________ 48 TLNAWVKVV 707 WLYRETCNL 708 YLFKRMIDL 709 GAFGGVGGV 710 GAFGGVGGY 711 GEFGGVGGV 712 " GGFGGVGGV 713 GIFGGGGGV 714 " GIGGFGGGL 715 " GIGGGGGGL 716" "E GLDGGGGGV 717 " GLDGKGOGV 718" GLDKKGGGV 719 " GLFGGGFGF 720 " GLFGGGFGG 721 " GLFGGGFGN 722 " GLFGGGFGS 723 " GLFGGGGGI 724 " GLFGGGGGM 725 GLFGGGGGT 726 GLFGGGGGY 727 GLGFGGGGV 728 GLGGFGGGV 729 " GLGGGFGGV 730 GLGGGGGFV 731 GLGGGGGGY 732 _ GLGGGVGGV 733 GLLGGGGGV 734 GLPGGGGGV 735 " GNFGGVGGV 736 " GSFGGVGGV 737 " GTFGGVGGV 738 __ AGNSAYEYV 739 GLFPGQFAY 740 HILLGVFML 741 ILESLFRAV 742 "KKKYKLKHI 743_ MLASIDLKY 744 "MLERELVRK 745 KLFGFVFTV 746 ILDKKVEKV 747 ILKEPVHGV 748 ALFAAAAAY 749 GIGFGGGGL 750 " 49 "_ GKFGGVGGV _1 ____751 GLFGGGGGK 752 _ "__ __ " EILGFVFTL 753 "_ GIKGFVFTL 754 "c " GQLGFVFTK 755 "_ " ILGFVFTLT 756 "_ KILGFVFTK 757 " KKLGFVFTL 758 " KLFEKVYNY 759 " LRFGYPVYV 760 "t Human HSP60 140-148 HLA-B27 IRRGVMLAV 761 Rammensee et al. 1997 160 " 369-377 " KRIQEIIEQ 762 "6 469-477 " KRTLKIPAM 763 "_ Yersinia HSP60 35-43 " GRNVVLDKS 764 "C " _ 117-125 " KRGIDKAVI 765 "4 420-428 IRAASAITA 766 " HSP 60 284-292 HLA- RRKAMFEDI 767 169 B*2705 P. L ~A- 1850- HLA- KPKDELDY 768 170 falciparumn 1857 B3501 _______ Influenza 379-387 HLA- LELRSRYWA 769 183 NP B*4402 Tum-P35B]4-13 ______ GPPHSNNFGY 770 230 Rotavirus VP7 33-40 IIYRFLLI 771 262 OGDH 104-112 H-12-L QLSPYPFDL 772 253 (F108Y) TRP-2 181-188 p 2 8 7 VYDFFVWL 773 284 DEAD box 547-554 p 2 8 7 SNFVFAGI 774 283 p68 ___ V-ctor p287 SVVEFSSL 775 260 "artefact" pitope p2 8 7 AHYLFRNL 776 278 mimic of tumor Ag THYLFRNL 777 Epitope LIVIYNTL 778 279 mimic of H-3 miHA" LIYEFNTL 779 __ IPYIYNTL 780 " IIYIYHRL 781 " LIYIFNTL 782 " 50 CA 93-100 KFRL 783 280 -luman autoantigen 51-58 IMIKFRNRL 784 281 LA e -UTY--~H2D WM-HNMDLI 785 303 --- ~~-V-~75-83 " CCLCLTVFL 78930 a 75K _ H__ - --- ~- 3 15 p. CSP 375-383 p 2 9 0 YENDIEKK 790 315 FU N . _ __----1-379 -ELDYENDI 791_ 3 HIV -iRT 206-214 TEMEKEGKI 792 316 NS 17-60 VIAHQI 789 325 P.NS1 1752-160 "29 EGAIVE I 794 1 Fal~jp jal SMC P291 T-379EENDI 795 _1 M I n e M H C cl a s s - 21 4 p 2 3M A P R T L L L 7 9 6 3 1 8 _N aph 1-12 __p293 _ -~-i NDS et 19-20 p293 FF I ILV 79_3 L LeA A1-16 " FFINIVI 799 324 - LIAM

-

NI ag 1-190 Mam- EGCITPN 803 3234 Nlpha 1-17 FMLTLP7932 ND Beta 1-17 FlNMATLLVP 800 ILIAM . CoI 1-6 FKW 801 325 mitochondr AA - WV 802 326 IMPKuAGLI 808 alThrLeuGlyGlu Val lL~euerAr L 809 51 ysVaIAlaGluLeu IleMetProLysAl 810 aGlyLeuLeulle LysIleTrpGluGI 811 uLeuSerValLeu "6 AlaLeuValGluT 812 hrSerTyrValLys Val peptides HLA-A2 Lys Gly Ile Leu 813 5,989,565 which bind Gly Phe Val Phe to MHCs 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 Gin Val Pro Leu 821 Arg Pro Met Thr Tyr Lys_ Asp Gly Leu Ala 822 Pro Pro Gin His Leu Ile Arg Leu Leu Gly 823 Arg Asn Ser Phe Glu Val Peptides HLA-C GluHisSerAlaTy 824 5,558,995 from clone 10 rGlyGluProArgL 52 MAGE-1 ysLeuLeuThrGl nAspLeu GluHisSerAlaTy 825 rGlyGluProArgL ysLeuLeu SerAlaTyrGlyGl 826 uProArgLysLeu GAGE HLA-Cw6 TyrArgProArgPr 827 5,648,226 oArgArgTyr __ ThrTyrArgProAr 828 gProArgArgTyr _ TyrArgProArgPr 829 oArgArgTyrVal ThrTyrArgProAr 830 gProArgArgTyr Val ArgProArgProA 831 rgArgTyrValGlu _ MetSerTrpArgG 832 lyArgSerThrTyr ArgProArgProA rgArg ThrTyrArgProAr 833 gProArgArgTyr ValGluProProGl uMetIle _ MAGE HLA-A1, Isolated 834 5,405,940 primarily nonapeptide 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 53 nonapeptide 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 GluValValProIle 835 SerHisLeuTyr GluValValArgIl 836 eGlyHisLeuTyr GluValAspProIl 837 eGlyHisLeuTyr GluValAspProA 838 laSerAsnThrTyr GluValAspProT 839 hrSerAsnThrTyr GluAlaAspProT 840 hrSerAsnThrTyr GluValAspProIl 841 eGlyHisValTyr GAAGTGGTCC 842 CCATCAGCCA CTTGTAC GAAGTGGTCC 843 GCATCGGCCA CTTGTAC GAAGTGGAC 844 CCCATCGGCC ACTTGTAC GAAGTGGAC 845 CCCGCCAGCA ACACCTAC GAAGTGGAC 846 CCCACCAGCA ACACCTAC GAAGCGGAC 847 CCCACCAGCA ACACCTAC GAAGCGGAC 848 54 CCCACCAGCA ACACCTAC GAAGTGGAC 849 CCCATCGGCC ACGTGTAC GluAlaAspProT 850 ________ rGlyHisSer AlaAspProTrpGl 851 ________ yHisSerTyr__ _________ MAGE HRLA-A2 SerThrLeuValGl 8-g52 5,554,724 peptides uVaiThrLeuGly GluVal LeuVaIGluValT 853 ______ _______hrLeuGlyGluVal __________ LysMetValGluL 854 euValHisPhel ell__________ VallePheSerLys 855 AlaSerGiuTyrLe U__ _ _ _ _ _ _ _ _ _ TyrLeuGlnLeuV 856 alPheGlylleGlu Val GInLeuVaiPheG 857 lyIyleGluValVal___________ GInLeuVaiPheG 858 lylleGluValVal GluVal__________ IleIleValLeuAlal 859 lelleAlalle LysIeTrpGluGI 860 uLeuSerMetLeu GluVal AlaLeulleGluTh 861 rSerTyrValLysV al__ _ _ _ _ _ _ _ _ _ _ LeulleGluThrSer 862 TyrValLysVal __________ GlyLeuGluAlaA 863 rgGlyGluAlaLeu GlyLeu __ GlyLeuGluAlaA 864 rgGlyGluAlaLeu AlaLeuGlyLeuV 865 alGlyAlaGlnAla __________ GlyLeuVaG1YA 866 55 laGInAlaProAla AspLeuGluSerG 867 IuPheGlnAlaAla AspLeuGluSerG 868 luPheGlnAlaAla le AlalleSerArgLys 869 MetValGluLeuV al_____________ AlalleSerArgLys 80 MetValGluLeu__________ LysMetValGlL 87 euValHisPheLeu Leu LysMetValGluL 872 euValHisPheLeu LeuLeu _________ LeuLeuLeuLysT 873 yrArgAlaArgGlu ProVal LeuLeuLysTyrA 874 rgAlaArgGluPro Val ValLeuArgAsnC 875 ysGlnAspPhePh eProVal __________ TyrLeuGlnLeuV 876 alPheGlyIleGlu ValVal GlylleGluValVa 877 iGluValVaiProIl e ProlleSerHisLeu 878 TyrIleLeuVal ___________ HisLeuTyrleLe 879 uyalThrCysLeu _________ HisLeuTyrleLe 880 uValThrCysLeu GlyLeu TyrleLeuVaiTh 881 rCysLeuGlyLeu __________ CysLeuGlyLeuS 882 erTyrAspGi Leu _________ CysLeuGlyLeuS 883 erT rAs GI Leu ________ 56 Leu84 hrGlyLeuLeulle ValMetProLysT 885 hrGlyLeuLeullel le ____________ ValMetProLysT 886 brGlyLeuLeullel leVal GlyLeuLeullelle 887 VaiLeuAlafle___________ GlyLeuLeullelle 888 ValLeuAlaIlefle 66 GlyLeuLeullelle 889 F - ValLeuAlaIlelle Ala______ _ LeuLeullelleVal 890 LeuAlaIlelle____________ LeuLeullelle Val 891 LeuAlaIlelleAla LeuLeullelleVal 892 LeuAlaIlelleAlaI le LeulleIleValLeu 893 AlallelleAla LeullelleValLeu 894 AlallelleAlalle ____________ 6( 66 IlelleAlalleGluG 895 lyAspCysAia ___________ LysIeTrpGluGI 896 uLeuSerMetLeu LeuMetGlnAspL 897 F euValGInGluAs PheLeuTrpGlyPr 898 LeulleGluThrSer 899 AlaLeulleGluTh 900 rSerTyrValLysV alLeu ThrLeuLysIleGl 901 yGlyGluProHisl e ...... His~leSerT YrPro 902 ___________ 57 ProLeuHisGluAr _________ _______gAla GinThrAlaSerSe 903 rSerSerThrLeu __________ GInThrAlaSerSe 94 rSerSerThrLeuV al ValThrLeuGlyG 905 luValProAlaAla __________ ValThrLysAlaGl 906 uMetLeuGluSer Val__ _ _ _ _ _ ValThrLysAlaGl 907 uMetLeuGluSer ValLeu __________ ValThrCysLeuG 908 lyLeuSerTyrAsp GlLysheyu U 0 LysThrGlyLeuL 909 LysThrGlyLeuL 911 eullelleValLeuA la~l __ HisThrLeuLysIl 912 eGlyGlyGluPro HisIle ____________ MetLeuAspLeu 913 GlnProGluThrT hr Mage-3 HfL-A-A2 GlyLeuGluAlaA 914 -5,585,461 ve tides rgGlyGluAlaLeu AlaLeuSerArgL *915 ,rsValAlaGluLeu PheLeuTrpGlyPr 916 oArgAlaLeuVal__________ ThrLeuValGluV 917 alTbrLeuGlyGlu Val AlaLeuSerArgb 918 ysVaIAlaGluLeu Val AlaLeuVaIGIUT 919 58 hrSerTyrValLys Val Tyrosinase HLA-A2 TyrMetAsnGlyT 920 5,487,974 hrMetSerGlflVal MetLeuLeuAlaV 921 alLeuTyrCysLeu Leu Tyrosinase HLA-A2 MetLeuLeuAlaV 922 5,530,096 alLeuTyrCysLeu LeuLeuAlaValL 923 euTyrCysLeuLe Tyrosinase HLA-A2 'SerGlulleTrpArg 9 24 5,519,117 and HLA- AspIleAspPheAl B44 allisGluAla_ SerGlulleTrpArg 925 " AspIleAspPhe SGluGluAsnLeuL 926 euAspPheValAr gPhe Me-lan EAAGIGILTV 927 Jager, E. et al. AIMART- 1 Granulocyte macrophage-colony stimulating Factor Enhances Immune Responses To Melanoma-'associated Peptides in vivo Int. J Cancer 67, 54-62 (1996) T rosinase MLLAVLYCL 928. YMDGTMS V 929 gp100/Pme YLEPGPVTA 930 117- ~ ~ LLDGTATLRL 931 Influenza GILGFVFTL 932 matrix MAGE-1 EADPTGHSY 933 MAGE-1 HLA-Al EADPTGHSY 934 DIRECTLY FROM DAVID'S LIST BAGE HLA-C MAARAVFLA 935 LSAQLLQARL 59 MKE MAARAVFLA 936 LSAQLLQ AARAVFLAL 937 influenzaa PR8 NP 147-154 K IYQRIRALV 938 Falk et al., Allele specific motifs revealed by sequencing of self-peptides eluted from MHC molecules SELF P815 SYFPEITHI 939 PEPTIDE Influenza Jap HA IYATVAGS 940 523-549 VYQILAIYA 941 IYSTVASSL 942 JAP HA LYQNVGTYV 943 202-221 -.

HLA-A24 RLN 4 HLA-Cw3 RLNKT 945 _ P815 " KYQAVTTTL 946 Plasmodium CSP " SYIPSAEKI 947 berghei - ---- N PAQ 4 Plasmodiumm CSP948 Vesicular NP52-59 ~~ ipVQGLo stpomnatitis Ovalbumin ___1.EK -9- Sendai virus NP 321-e 332 --- 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 11762000. 60 MOTIFS nfluenza PR8 NP K TYQRTRALV 953 5,747,269 restricted peptide motifs ;elfapeptide P815 SYFPEITHI 954 influenza JAP HASYISAEI 962 influenza JAP HA " VYILAIYA 956 influenza PR8 HA IYSTVASSL 957 influenza JAP HA " LYNVGTYV 958 HLA-A24 RYLENGKETL 959_ HLA-Cw3 RYLKNGKETL 90 P815 " KYQAVTTTL 961 tumour antigen _" SYPAK 96 Plasmiodium CSP YPAK 96 rfluenza NPIF___AS___________ restricted peptide HIV Ga roteinotfLSPT 97 Hd e o-i~ - ~ - Ot i P I G P P E I 9 6 5 lymphocytic LGEPGY 6 choriomeniL -ngitis .- " S IN 6 sim ian v irus 4 0 T _ _ _ _ _W -.- ... IR f G 9 6 8 HIV reversein KDCTER 974 transHiptas A2.I e restricted peptide -motif influenza " GILGFVFTL 969 matrix Protein influenza influenza IGVT 97 matrix rotein-- - EP 7 HIV Ga rotein FLQSRPEP 7 HIV _Ga rotein M LK. 97 HIV Gag protein PIAPGQMRE 973_ T41V lap, poteiQMKDCTERQ_ 974 HAVYGVIQK ,97 61 A*0205 restricted peptide motif Table 5 SEQ. ID NO.:976 VSV-NP peptide (49-62) SEQ. ID NO.:977 LCMV-NP peptide (118-132) SE .ID NO.:978 LCMV gl coprotein eptide. 33-41 [00781 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 6-10. Other embodiments include one or more of the MHCs listed in Tables 6-10, including combinations of the same, while other embodiments specifically exclude any one or more of the MHCs or combinations thereof. Tables 8-10 include frequencies for the listed HLA antigens. Table 6 62 Class I MHk Molecules Class I Human HLA-Al HLA-A*0101 HLA-A*O2O1 HLA-A*O2O2 HLA-A*O2O3 HLA-A*O204 HLA-A*O2O5 HLA-A*0206 HLA-A*02O7 HLA-A*O2O9 HLA-A*02 14 HLA-A3 HLA-A*0301 HLA-A* 10 1 HLA-A23 IILA-A24 HLA-A25 HLA-A*29O2 HLA-A*31O1 HLA-A*3302 HLA-A*68O 1 HLA-A*69O1 HLA-B7 HLA-B*07O2 HLA-B*07O3 HfLA-B*07O4 H~LA-B*07O5 HLA-B8 HLA-BI 3 RLA-B1 4 HLA-B*1501 (B62) HLA-B 17 H-LA-B18 HLA-B22 HLA-B27 HLA-B*2702 HLAB*2704 HLA-B*2705 HLA-B*2709 HLA-B35 HLA-B*35OI HLA-B*35O2 HLA-B '3701 HLA-B*3801 HLA-B*3901 I HLA-B*3902 63 HLA-B40 HLA-B*4012 (B 60) HLA-B*4OO6 (B61) HLA-B44 HLA-B*4402 HLA-B*4403 HLA-B*4501 HLA-B*460 1 HLA-B5 I HLA-B*5:101 HLA-B*5 102 HLA-B*5 103 HLA-B*5201 HiLA-B*53OI HLA-B*540 1 IILA-B*550 1 HLA-B*5601 HLA-B*5801 HLA-B*6701 HLA-B*730 1 HLA-B *7 801 HLA-Cw*0 102 HLA-Cw*0301 HLA-Cw*0304 HLA-Cw* 040 1 HLA-Cw*060 I HLA-Cw'0602 HLA-Cw*0702 HLA-Cw8 HLA-Cw*1601 M HLA-G Murine H2-K H2-D H2-0d H2-Kb H2-&b H2-Kk Qa-l* Qa-2 H2-M3 Rat RTI.Aa RTI.A' 64 Bovine Bota-Al II Bota-A20 Chicken B-F4 B-F12 B-F15 B-F19 Chimpanzee Patr-A*04 Patr-A *I I Patr-B01 Patr-B* 13 Patr-B* 16 Baboon Papa-A*06 Macaque Mamu-A*01 Swine SLA (haplotype did) Virus homolog hCMV class I homolog ULI 8 Table 7 Class I MHC Molecules Class I Human ElLA-Al HLA-A*O 101 HLA-A*0201 HLA-A*0202 HLA-AM0204 HLA-A*020S HLA-AM0206 HLA-A*0207 HLA-A*0214 -BLA-A3 HLA-A* I 10 1 HLA-A24 HLA-A*2902 HLA-A*3 101 HLA-A*3302 BLA-A*6801 lILA-A' 6901 65 HLA-B7 HLA-B*0702 HLA-B*0703 HLA-B*0704 H LA-B *0705 .HLA-B8 HLA-B 14 HLA-B*15OI (B62) HLA-B27 HLA-B*2702 HLA-B*2705 HLA-B35 HLA-B*350 I HLA-B*3502 HLA-B*3701 HLA-B*3 801 HLA-B*390I I HLA-B*3902 HLA-B40 HLA-B*40012 (B60) HLA-B*4006 (B61) HLA-B44 HLA-B*4402 HLA-B*44O3 HLA-B*460 I HLA-B5 I HLA-B*5 101 ILLA-B*5 102 HLA-B*5 103 H7LA-B*520 I HLA-B*530 I HLA-B*540 I HLA-B*550 I HLA-B*5502 HLA-B*5601 1LLA-B*5 801 HLA-B*6701 HLA-B* 7301 HLA-B*7801 HLA-Cw*0 102 HLA-Cw*O301 HLA-Cw*0304 HLA-Cw*'0401 HLA-Cw*0601 HLA-Cw*0602 HLA-Cw*0702 HLA-G Murine H2.Kd 66 H2-Dd H2-L H2-K' H2-Db H2-Kk H2-Kkmil Qa-2 Rat RT1.A* RTI.AI Bovine Bota-AI 1 Bota-A20 Chicken B-F4 B-F12 B-Fl 5 B-Fl 9 Virus homolog hCMV class I homolog UL 18 67 Table 8 100791 Estimated gene frequencies of HLA-A antigens CAU AFR AS] LAT NAT Antigen G - SEb Gf SE Gf SE Gf SE Gf SE Al 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 "Gene frequency. bStandard error. - Table 9 Estimated gene frequencies for HLA-B antigens Antigen CAU AFR ASI LAT NAT AGf" SE- _ 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 68 CAU AFR ASI LAT NAT Antigen Gf' SEb Gf SE Gf SE Gf SE Gf SE B59 0.0020 0.0006 0.0032 0.0019 0.4277 0.0265 0.0055 0.0028 O' 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 O' 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 0 1973 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 0.0356 0.0471 B75 0.0104 0.0014 0.0226 0.0049 1.9673 0.0566 0.1101 0.0123 0 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 0.0059 0.0059 B15 unsplit 0.1305 0.0049 0.0691 0.0086 0.4301 0.0266 0.1820 0.0158 0.0715 0.0206 B15 total 6.4910 0.0334 3.5232 0.0608 12.2112 0.1344 5.2967 0.0835 7.4290 0.2035 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 0.0-252 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 16"~52 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 ~ .0922 0.0252 3.5258 0.0802 3.8749 0.0988 12.5266 0.0807 1.9867 0.1634 'Gene frequency. bStandard error. *The observed gene count was zero. 69 Table 10. Estimated gene frequencies of HLA-DR antigens CAU AFR ASI LAT NAT Antigen Gf" SE Gf SE Gf SE Gf SE Gf SE DR) 10.2279 0.0413 6.8200 0.0832 3.4628 0.0747 7.9859 0.1013 8.2512 0.21391 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 DRID 1.4790 0.0163 2.0397 0.0465 2.2304 0.0602 1.8044 0.0495 1.0896 0.0801 DRl1 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 DRS 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 "Gene frequency. bStandard error. 100801 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. 100811 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 70 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. [00821 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. [00831 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. [00841 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 71 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. [00851 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. 100861 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 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. 100871 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 72 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 BIOPORTERm (Gene Therapy Systems, San Diego, CA), represents an alternative. 100881 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. Natil. Aced. Sci. USA 92:2056-2060, 1995), or mixture with or fusion to polypeptides with protein transfer function including peptide carriers such as pep-I (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 a;. 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. EXAMPLES Example 1 [00891 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 No. 09/561,571 entitled EPITOPE CLUSTERS. 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. 73 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 Figure 1), 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 innunostimulatory 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 pMB I bacterial origin of replication. SUBSTRATE or LIBERATION sequence 10090) 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: 100911 MLLAVLYCL-ELAGIGILTV-YMDGTMSQV GILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEK NCEPV 100921 The first 9 amino acid residues are derived from tyrosinase.9 (SEQ ID NO. 6), the next ten constitute melan-A (26-35A27L) (SEQ ID NO. 1), and amino acid residues 20 t6 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'. This region was also included to overcome any difficulties 74 that can be associated with the expression of shorter sequences. A drawing of pMA2M is shown in Figure 1. Plasmid construction [00931 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 I at the 5' end and that of EcoR I at the 3' end. The melan A 31

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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 100941 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 Figure 3). In pVAXM2 the .same basic group of 3 epitopes was repeated several times with the flanking epitopes tnmncated by differing degrees in the various repeats of the array. Specifically the cassette consisted of: [00951 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) 100961 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, pVAXMI 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. (00971 Four groups of three HHD A2.1 mice were injected intranodally in surgically exposed inguinal lymph nodes with 25 pl of I mg/mi 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 75 35A27L)(SEQ ID NO. 1)). The in vitro cultures were supplemented with Rat T-Stim (Collaborative Biomedical Products) on the 3 'd day and assayed for cytolytic activity on the 7 h day using a standard s1Cr-release assay. Figures 2 to 5 show % specific lysis obtained using the cells immunized with PBS, pVAXMJ, 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 5 5 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 An NY-ESO-l (SEO ID NO. 11) SUBSTRATE/LIBERATION Sequence [00981 Six other epitope arrays were tested leading to the identification of a substrate/liberation sequence for the housekeeping epitope NY-ESO-l 157-65 (SEQ ID NO. 12). The component epitopes of the arrays were: 100991 SSX-2 41 -49: KASEKIFYV (SEQ ID NO. 13) Array element A [01001 NY-ESO- 1157-165: SLLMWITQC (SEQ ID NO. 12) Array element B 101011 NY-ESO-1I63-171: TQCFLPVFL (SEQ ID NO. 14) Array element C 101021 PSMA 28 8-297: GLPSIPVHPI (SEQ ID NO. 15) Array element D [0103] TYR4-9: AVLYCL (SEQ ID NO. 16) Array element E [01041 The six arrays had the following arrangements of elements after starting with an initiator methionine: 101051 pVAX-PC-A: B-A-D-D-A-B-A-A [01061 pVAX-PC-B: D-A-B-A-A-D-B-A 101071 pVAX-PC-C: E-A-D-B-A-B-E-A-A 10108] pVAX-BC-A: B-A-C-B-A-A-C-A [01091 pVAX-BC-B: C-A-B-C-A-A-B-A 76 101101 pVAX-BC-C: E-A-A-B-C-B-A-A 10111] 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-l 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: [01121 An initiator methionine, 10113] NY-ESO-l 157-165 (bold) - a housekeeping epitope, 10114] SSX2 4 1 .49 (italic) - providing appropriate context for processing, and [01151 NY-ESO-177-180 - to avoid "short sequence" problems and provide immune epitopes. [0116] Thus the construct encodes the amino acid sequence: 10117] M-SLLMWITQC-KASEKIFYV RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTA ADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO. 17) 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 101181 A construct similar to pNl57 containing the whole epitope array from pVAX PC-A was also made and designated pBPL. Thus the encoded amino acid sequence in pBPL is: 10119] M-SLLMWITQC-KASEKIFYV-GLPSIPVHPI-GLPSIPVHPI-KSEKIFYV

SLLMWITQC-KASEKFYV-KASEKIFYV

RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTA ADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO. 20). [0120] SEQ ID NO. 21 is the polynucleotide encoding SEQ ID NO. 20 used in pBPL. 77 101211 A portion of SEQ ID NO. 20, IK4SEKIFYVSLLMWITQCKASEKIFYVK (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-l 157-165 (SEQ ID NO. 12) epitope (see Figure 6). Figure 7 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 fig. 6 and 7). Example 5 [01221 Screening of further epitope arrays led to the identification of constructs promoting the expression of the epitope SSX-2 41

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49 (SEQ ID NO. 13). In addition to some of the array elements defined in Example 3, the following additional elements were also used: [01231 SSX-4 57 -6 5 :VMTKLGFKV (SEQ ID NO. 23) Array element F. 101241 PSMA 73 0-739: RQIYVAAFTV (SEQ ID NO. 24) Array element G. 10125] 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-2 4 1 -49 (SEQ ID NO. 13). [01261 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-2 15 183 (SEQ ID NO. 25) was combined with all or part of the array as follows: 101271 CTLSI: F-A-G-D-C-F-G-A- SSX-2 1 5

-

1 83 (SEQ ID NO. 26) 101281 CTLS2: SSX-2 1 5 -1 83 - F-A-G-D-C-F-G-A (SEQ ID NO. 27) 10129] CTLS3: F-A-G-D- SSX-2 1 5- 183 (SEQ ID NO. 28) 101301 CTLS4: SSX-2 1 5

-

183 -C-F-G-A (SEQ ID NO. 29). [01311 All of the constructs except CTLS3 were able to induce CTL recognizing the peptide SSX-2 4 1 -4 9 (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 78 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 CTLSI the A element is immediately followed by the SSX-2 1 5-183 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. 101321 A portion of CTLSI (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-2 41 .4 9 epitope (SEQ ID NO. 13) was generated (see Figure 8) provided further evidence in support of substrate or liberation sequence function. The data in Figure 9 showed the differential processing of the SSX-2 41

.

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. 8 and 9). The data in figure 8B 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 [01331 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 [Tyr 1

.

17 - Tyr 2 07 21514, [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 Tyri.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. 79 Plasmid construction 101341 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 Afil I/EcoR I. The construct was called CTLT2/pMEL and SEQ 1D NO. 34 is the polynucleotide sequence used to encode SEQ ID NO. 33. Example 7 Administration of a DNA plasmid formulation of a immunotherapeutic for melanoma to humans. [01351 - An MA2M melanoma vaccine with a sequence as described in Example 1 above, was formulated in 1% Benzyl alcohol, 1% ethyl alcohol, 0.5mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 200, 400, and 600 pg 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 17mm catheter was substituted with a 31mm catheter for this application. The infusion set was kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 iL/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion was approximately 500, and 1000 pg; and can be 1500 pg, 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 [01361 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 pg of peptide per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted. 80 Example 9 101371 A fusion protein is made by adding SEQ ID NO. 34 to the 3' end of a nucleotide sequence encoding herpes simplex virus I 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 pg per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted. 101381 Further, the present invention can utilize various aspects of the following: U.S. Patent Application Nos. 09/380,534, filed on September 1, 1999, entitled A METHOD OF INDUCING A CTL RESPONSE; 09/776,232, filed on February 2, 2001, entitled METHOD OF INDUCING A CTL RESPONSE; 09/715,835, filed on November 16, 2000, entitled AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPOGATION; 09/999,186, filed on November 7, 2001, entitled METHODS OF COMMERCIALIZING AN ANTIGEN; and Provisional U.S. Patent Application No 60/274,063, filed on March 7, 2001, entitled ANTI-NEOVASCULAR VACCINES FOR CANCER. Table 11 Partial listing of SEQ ID NOS. I ELAGIGILTV melan-A 26-35 (A27L) 2 Melan -A protein Accession number: NP 005502 3 Tyrosinase protein Accession number: P14679 4 MLLAVLYCLELAGIGILTVYMDGTMSQVGILT pMA2M expression product 'VILGVLLLIGCWYCRRRNGYRALMDKSLHVG TQCALTRRCPQEGFDHRDSKVSLQEKNCEPV 5 MLLAVLYCLELAGIGILTVYMDGTMSQV Liberation or substrate sequence for SEQ ID NO. I from pMA2M 6 MLLAVLYCL tyrosinase 1-9 7 YMDGTMSQV ' tyrosinase 369-377 8 EAAGIGILTV melan-A 26-35 9 cttaagccaccatgttactagctgttttgtactgcctggaact pMA2M insert agcagggatcggcatattgacagtgtatatgga tggaacaatgtcccaggtaggaattctgacagtgatcctggga gtcttactgetcatcggctgttggtattgtaga agacgaaatggatacagagccttgatggataaaagtcttcatg ttggcactcaatgtgccttaacaagaaga tgcc cacaagaagggtttgatcatcgggacagcaaagtgtctcttca agagaaaaactgtgaacctgtgtagtgagcggc cgc 10 MVLYCLELAGIGILTVYMDGTAVLYCLELAGI Epitope array from pVAXM2 and 81 GILTVYMDGTMLAVLYCLELAGIGILTVYMD pVAXM1 GTMSLLAVLYCLELAGIGILTV 11 NY-ESO-1 protein Accession number: P78358 12 SLLMWITQC NY-ESO-l 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 MSLLMWITQCKASEKIFYVRCGARGPESRLLE pNl 57 expression product FYLAMPFATPMEAELARRSLAQDAPPLPVPGV LLKEFTVSGNILTIRLTAADHRQLQLSISSCLQ QLSLLMWITQCFLPVFLAQPPSGQRR 18 MSLLMWITQCKASEKIFYV liberation or substrate sequence for SEQ ID NO. 12 from pN 57 19 cttaagccaccatgtccctgttgatgtggatcacgcagtgcaa Insert for pN 157 agcttcggagaaaatcttctacgtacggtgcgg tgccagggggccggagagccgcctgcttgagttctacctcgcc atgcctttcgcgacacccatggaagcagagctg gcccgcaggagcctggcccaggatgccccaccgcttcccgtgc caggggtgcttctgaaggagttcactgtgtccg gcaacatactgactatccgactgactgctgcagaccaccgcca actgcagctctccatcagctcctgtctccagca gctttccctgttgatgtggatcacgcagtgctttctgcccgtg tttttggtcagcctccctcagggcagaggcgc tagtgagaattc 20 MSLLMWITQCKASEKIFYVGLPSIPVHPIGLPSI pBPL expression product PVHPIKASEKIFYVSLLMWITQCKASEKIFYVK ASEKIFYVRCGARGPESRLLEFYLAMPFATPM EAELARRSLAQDAPPLPVPGVLLKEFTVSGNIL TIRLTAADHRQLQLSISSCLQQLSLLMWITQCF LPVFLAQPPSGQRR 21 atgtccctgttgatgtggatcacgcagtgcaaagcttcggaga pBPL insert coding region aaatcttctatgtgggtcttccaagtattcctg . ttcatccaattggtcttccaagtattcctgttcatccaattaa agcttcggagaaaatcttctatgtgtccctgtt gatgtggatcacgcagtgcaaagcttcggagaaaatcttctat gtgaaagcttcggagaaaatcttctacgtacgg tgcggtgccagggggccggagagccgcctgcttgagttctacc tcgccatgcctttcgcgacacccatggaagcag agctggcccgcaggagcctggcccaggatgccccaccgcttcc cgtgccaggggtgcttctgaaggagttcactgt gtccggcaacatactgactatccgactgactgctgcagaccac cgccaactgcagctctccatcagctcctgtctc cagcagctttccctgttgatgtggatcacgcagtgctttctgc ccgtgtttttggctcagcctccctcagggcaga ggcgctagtga 22 IKASEKIFYVSLLMWITQCKASEKIFYVK Substrate in Fig. 6 23 VMTKLGFKV SSX4 57 -65 24 RQIYVAAFTV

PSMA

73 0- 73 9 25 AQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIF SSX-2 1 5-183 YVYMKRKYEAMTKLGFKATLPPFMCNKRAE DFQGNDLDNDPNRGNQVERPQMTFGRLQGIS PKIMPKKPAEEGNDSEEVPEASGPQNDGKELC PPGKPTTSEKIHERSGPKRGEHAWTHRLRERK QLVIYEEISDP 82 26 MVMTKLGFKVKASEKIFYVRQIYVAAFTV CTLSI/pCBP expression product GLPSIPVHPITQCFLPVFLVMTKLGFKVRQIYV AAFTVKASEKIFYVAQIPEKIQKAFDDIAKYFS KEEWEKMKASEKIFYVYMKRKYEAMTKLGF KATLPPFMCNKRAEDFQGNDLDNDPNRGNQ VERPQMTFGRLQGISPKIMPKKPAEEGNDSEE VPEASGPQNDGKELCPPGKPTTSEKIHERSGPK RGEHAWTHRLRERKQLVIYEEISDP 27 MAQIPEKIQKAFDDIAKYFSKEEWEKMKASE CTLS2 expression product KIFYVYMKRKYEAMTKLGFKATLPPFMCNKR AEDFQGNDLDNDPNRGNQVERPQMTFGRLQ GISPKIMPKKPAEEGNDSEEVPEASGPQNDGK ELCPPGKPTTSEKIHERSGPKRGEHAWTHRLR ERKQLVIYEEISDPVMTKLGFKVKASEKIFYV RQIYVAAFTVGLPSIPVHPITQCFLPVFLVMTK LGFKVRQIYVAAFTVKASEKIFYV 28 MVMTKLGFKVKASEKIFYVRQIYVAAFTV CTLS3 expression product GLPSIPVHPIAQIPEKIQKAFDDIAKYFSKEEWE KMKASEKIFYVYMKRKYEAMTKLGFKATLPP FMCNKRAEDFQGNDLDNDPNRGNQVERPQM TFGRLQGISPKIMPKKPAEEGNDSEEVPEASGP QNDGKELCPPGKPTTSEKIHERSGPKRGEHAW THRLRERKQLVIYEEISDP 29 MAQIPEKIQKAFDDIAKYFSKEEWEKMKASE CTLS4 expression product KIFYVYMKRKYEAMTKLGFKATLPPFMCNKR AEDFQGNDLDNDPNRGNQVERPQMTFGRLQ GISPKIMPKKPAEEGNDSEEVPEASGPQNDGK ELCPPGKPITTSEKIHERSGPKRGEHAWTHRLR ERKQLVIYEEISDPTQCFLPVFLVMTKLGFKVR QIYVAAFFVKASEKIFYV 30 atggtcatgactaaactaggttcaaggtcaaagttggaga pCBP insert coding region aaatcttctatgtgagacagatttatgttgcag ccttcacagtgggtcttccaagtattcctgttcatccaattac gcagtgctttctgcccgtgtttttggtcatgac taaactaggtttcaaggtcagacagatttatgttgcagccttc acagtgaaagcttcggagaaaatcttctacgta gctcaaataccagagaagatccaaaaggccttcgatgatattg ccaaatacttctctaaggaagagtgggaaaaga tgaaagcctcggagaaaatcttctatgtgtatatgaagagaaa gtatgaggctatgactaaactaggtttcaaggc caccctcccacctttcatgtgtaataaacgggccgaagacttc caggggaatgatttggataatgaccctaaccgt gggaatcaggttgaacgtcctcagatgactttggcaggctcc agggaatctccccgaagatcatgcccaagaagc cagcagaggaaggaaatgattggaggaagtgccagaagcatc tggcccacaaaatgatgggaaagagctgtgccc cccgggaaaaccaactacctctgagaagattcacgagagatct ggacccaaaaggggggaacatgcctggacccac agactgcgtgagagaaaacagctggtgatttatgaagagatca gcgaccttagtga 31 RQIYVAAFTVKASEKIFYVAQIPEKIQK Fig. 8 substrate/ CTLS1-2 32 FLPWHRLFL TYR207-2 1 5 33 MLLAVLYCLLWSFQTSAFLPWHRLFLMLLAV CTLT2/pMEL expression product LYCLLWSFQTSAFLPWHRLFLMLLAVLYCLL 83 WSFQTSAFLPWHRLFLMLLAVLYCLLWSFQT SAFLPWHRLFL 34 atgctcctggctgttttgtactgcctgetgtggagtttccaga CTLT2/pMEL insert coding region cctccgcttttctgcct tggcatagactcttct tgatgctcctggctgttttgtactgcctgctgtggagtttcca gacctccgcttttctgccttggcatagactctt cttgatgctcctggctgttttgtactgcctgctgtggagtttc cagacctccgcttttctgccttggcatagactc ttcttgatgctcctggctgttttgtactgcctgctgtggagtt tccagacctccgcttttctgccttggcatagac tcttcttgtaqtga 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 atgacctctcgccgctccgtgaagtcgggtcggggaggttccgcgc From accession number: D 10879 gatgagtacgaggatctgtactacaccccgtttcaggtatggcgagtcc Herpes Simplex virus 1 ULA9 cgatagtccgcctgaacctcccgcgtggcgccctacagacacgctc coding sequence (VP22) gcgccagaggggcgaggtccgtttcgtcagtacgacgagtcggattat gccctctacgggggctcgtcatccgaagacgacgaacacccggaggt cccccggacgcggcgtcccgtttccggggcggttttgtccggcccggg gcctgcgcgggcgcctccgcacccgctgggtceggaggggccgga cgcacacccaccaccgccccccgggccccccgaacccagegggtgg cgactaaggcccccgcggccccggcggCggagaccacccgcggca ggaaatcggcccagccagaatccgccgcactccagacgcccccgcg tcgacggcgccaacccgatccaagacacccgcgcaggggctggcca gaaagctgcactttagcaccgcccccccaaaccccgacggccatgga .ccccccgggtggccggctttaacaagcgcgttttgcgccgcggtcg ggcgcetggcggccatgatgcccggatggggggtcagtctgg gacatgtcgcgtccgcgcacagacgaagactcaacgaactcttggC atcaccaccatccgcgtgacggtctgcgagggcaaaaacctgcttcag cgcgccaacgagttggtgaatccagacgtggtgcaggacgtcgacgcg gccacggcgadtcgagggcgttctgcggcgtcgcgccccaccgagcg acctcgagccccagcccgctccgtttcgccccagacggcccgtcga 43 MTSRRSVKSGPREVPRDEYEDLYYTPSSGMAS Accession number P10233 PDSPPDTSRRGALFTQTRSRQRGEVRFVQYDE Herpes Simplex virus I SDYALYGGSSSEDDEHPEVPRTRRPVSGAVLS UL49NP22 protein sequence GPGPARAPPPFTPAGSGGAGRTPTTAPRAPRT QRVATKAPAAPAAETTRGRKSAQPESAALPD APASTAPTFTRSKTPAQGLARKLHFSTAPPNP DAPWTPRVAGFNKRVFCAAVGRLAAMHARM AAVQLWDFTMSRPRTDEDLNELLGITTIRVTV CEGKNLLQRANELVNPDVVQDVDAATATRG RSAASRFTPTERPRAPARSASRPRRPVE 84 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="MPREDAHFIYGYPKKGHGHSYrrAEEAAGIGILTVILGVLLLIGCWYCRRRNGY RALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPP YSP" (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 gggtatctg 721 gggccatcca atttctcttt acttgaaatt tggctaataa caaactagtc aggttttcga 781 accttgaccg acatgaactg tacacagaat tgttccagta ctatggagtg ctcacaaagg 841 atacttttac aggttaagac aaagggttga ctggcctatt tatctgatca agaacaigtc 901 agcaatgtct ctttgtgctc taaaattcta ttatactaca ataatatatt gtaaagatcc 961 tatagctctt tttttttgag atggagtttc gettttgttg cccaggctgg agtgcaatgg 1021 cgcgatcttg gctcaccata acctccgcct cccaggttca agcaattctc ctgccttagc 1081 ctcctgagta gctgggatta caggcgtgcg ccactatgcc tgactaattt tgtagtttta 1141 gtagagacgg ggtttctcca tgttggtcag gctggtctca aactcctgac ctcaggtgat 85 1201 ctgcccgcct cagcctccca aagtgctgga attacaggcg tgagccacca cgcctggctg 1261 gatcctatat cttaggtaag acatataacg cagtctaatt acattcact tcaaggctca 1321 atgctattct aactaatgac aagtattttc tactaaacca gaaattggta gaaggattta 1381 aataagtaaa agctactatg tactgccita gtgctgatgc ctgtgtactg ccttaaatgt 1441 acctatggca atttagctct cttgggttcc caaatccctc tcacaagaat gtgcagaaga 1501 aatcataaag gatcagagat tctg Tyrosinase mnRNA sequence LOCUS NM_000372 1964 bp mRNA PR! 3 1 -OCT-2000 DEFINIION Homo sapiens tyrosinase (oculocutaneous albinismn IA) (TYR), iR.NA. ACCESSION NM_000372 VERSION NM_000372.1 GI45 07752 (SEQ ED NO. 3) /translation="MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRS PCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFG FWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHI-SSDYVIPIGTYGQMKNGSTPMFND DiNYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENF TIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTP EGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIAI) ASQSSMHNALHIYMNGTMSQVQGSA1NDPEFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAP GHI4RESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASR~rWSWLLGA AMVGAVLTALLAGLVSLLCRHXRKQLP EEKQPLLMEKEDYHSLYQSHL" *(SEQ I0D NO. 36) ORIGIN * I 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 ccftcacagg 30 1..gggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg 86 361 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 tggctecttg 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 taacatttte ccctaagccc atatgtctaa ggaaaggatg ctatttggta 1921 atgaggaact gttatttgta tgtgaattaa agtgctctta tttt NY-ESO-I mRNA sequence LOCUS HSU87459 752 bp mRNA PRI 22-DEC-1999 DEFINITION Human autoimmunogenic cancer/testis antigen NY-ESO-1 mRNA, complete 87 cds. ACCESSION U87459 VERSION U87459.1 GI:1890098 (SEQ ID NO. 11) /translation="MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAAR ASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDA PPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLM WITQCFLPVFLAQPPSGQRR" (SEQ ID NO. 37) ORIGIN I 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 PSMA cDNA sequence LOCUS NM_004476 2653 bp mRNA PRI O1-NOV-2000 DEFINITION Homo sapiens folate hydrolase (prostate-specific membrane antigen) I (FOLH1), mRNA. ACCESSION NM_004476 88 VERSION NM_004476.1 GI:4758397 (SEQ ID NO. 38) /translation="MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNIT PKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYD VLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNY ARTEDFFKLERDMKJNCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVK SYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDA QKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFrGNFSTQKVKMHiHSTNEVTRIYNVIGTLRG AVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEF GLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGF EGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPL YHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIY SISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAF IDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFT VQAAAETLSEVA" (SEQ ID NO. 39) ORIGIN I ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctetc 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 gatgtcetgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa 661 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcetccagg atatgaaaat 721 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 89 841 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 aaagtgccet 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 gtacagettg 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 tccaagttea 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 90 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 5 knowledge in the field of endeavour to which this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or 10 steps. -91-

Claims (43)

1. A vector comprising a housekeeping epitope expression cassette, wherein the housekeeping epitope is derived from a target-associated antigen, and wherein the housekeeping epitope is liberatable from a translation product of the cassette by immunoproteasome processing.

2. The vector of Claim 1, wherein the expression cassette encodes an array of two or more epitopes, or at least one epitope and at least one epitope cluster.

3. The vector of Claim 1, wherein the target-associated antigen is an antigen derived from or associated with a tumor or an intracellular parasite.

4. A method of activating a T cell comprising contacting the vector of Claim I with an APC and contacting said APC with a T cell.

5. An engineered substrate polypeptide comprising a housekeeping epitope wherein the housekeeping epitope can be liberated by immunoproteasome processing in a pAPC.

6. The vector of Claim I comprising a housekeeping epitope expression cassette comprising a sequence selected from SEQ ID NO: 9, SEQ ID NO: 21 and SEQ ID NO: 30.

7. The vector of Claim I wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 4.

8. The vector of Claim 1, wherein a housekeeping epitope having the sequence of SEQ ID NO: 13 is liberatable from a translation product of the cassette by immunoproteasome processing.

9. The vector of Claim 8, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising a sequence selected from SEQ ID NO: 31 and SEQ ID NO: 26. - 92 - C:\NRPonbl\DCC\$CG 392671 DOC-I S16/2U12

10. The vector of Claim 8, wherein the epitope expression cassette encodes a polypeptide comprising the sequence of MVMTKLGFKVKASEKIFYVRQIYVAAFTVG LPSIPVHPITQCFLPVFLVMTKLGFKVRQIYVAAFTVKASEKIFYV.

11. The vector of Claim 10, wherein the housekeeping epitope expression cassette further encodes at least one epitope cluster.

12. The vector of either Claim 10 or 11, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 26.

13. The vector of Claim 10, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 27.

14. The vector of Claim 8, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising a sequence selected from SEQ ID NO: 29, SEQ ID NO: 17 and SEQ ID NO: 20.

15. The vector of Claim I wherein a housekeeping epitope having the sequence of SEQ ID NO: 12 is liberatable from a translation product of the cassette by immunoproteasome processing.

16. The vector of Claim 15, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 18.

17. The vector of Claim 16, wherein the housekeeping epitope expression cassette further encodes at least one epitope cluster.

18. The vector of Claim 16, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 17.

19. The vector of Claim 16, wherein the epitope expression cassette encodes a -93 - C:\NRPonb\DCCSCGUI9K267_ I DOC-I5/I6/2112 polypeptide comprising the sequence of MSLLMWITQCKASEKIFYVGLPSIPVHPIGLP SIPVHPIKASEKIFYVSLLMWITQCKASEKIFYVKASEKIFYV.

20. The vector of Claim 16, wherein the epitope expression cassette encodes a polypeptide comprising the sequence of MSLLMWITQCKASEKIFYVTQCFLPVFLSLL MWITQCKASEKIFYVKASEKIFYVTQCFLPVFLKASEKIFYV.

21. The vector of Claim 15, wherein the housekeeping epitope expression cassette encodes a polypeptide comprising the sequence of SEQ ID NO: 22.

22. The vector of Claim 21, wherein the housekeeping epitope expression cassette further encodes at least one epitope cluster.

23. The vector of either Claim 21 or Claim 22, wherein the housekeeping epitope expression cassette encodes a polynucleotide comprising the sequence of SEQ ID NO: 20.

24. The vector of Claim 1, wherein the housekeeping epitope has a sequence that is identical, cross-reactive with, or substantially similar to a segment of the antigen, and wherein the epitope is not ELAGIGILTV (SEQ ID NO: 1), AAGIGILTV (SEQ ID NO: 429), or EAAGIGILTV (SEQ ID NO: 8), and wherein the housekeeping epitope expression cassette does not comprise the whole antigen.

25. The vector of Claim 24, wherein the expression cassette encodes an array of two or more epitopes or at least one epitope and at least one epitope cluster.

26. The vector of Claim 24, wherein the target-associated antigen is an antigen derived from or associated with a tumor or an intracellular parasite, or a tumor-associated antigen.

27. The vector of Claim 26, wherein the tumor-associated antigen is associated with neovasculature of the tumor, or is a differentiation antigen, or is a cancer-testis antigen.

28. The vector of Claim 27, wherein the antigen associated with the neovasculature of -94- C:\NRPorbrlDCC\SCGCW K267_ L.DOC-5I 5A6/20I2 the tumor is PSMA.

29. The vector of Claim 27, wherein the differentiation antigen is tyrosinase or PSMA.

30. The vector of Claim 27, wherein the cancer-testis antigen is selected from the group consisting of SSX-2, NY-ESO-l and PRAME.

31. An immunogenic composition comprising the vector of Claim 24.

32. The vector of Claim 1, wherein the housekeeping epitope has a sequence that is identical, cross-reactive, or substantially similar to a segment of the antigen, further comprising a nucleic acid sequence encoding an epitope cluster, the cluster being derived from a second target-associated antigen, the cluster comprising at least two putative epitopes, each of the putative epitopes having a known or predicted affinity for a same class I MHC receptor peptide binding cleft, wherein the cluster is a fragment of the second antigen.

33. The vector of Claim 32, wherein the first and the second target-associated antigens are the same.

34. The vector of Claim 32, wherein the cluster encoding sequence is contained within the housekeeping epitope expression cassette.

35. The vector of Claim 32, wherein the second antigen is selected from the group consisting of SSX-2, NY-ESO-1, FRAME and PSMA.

36. The vector of Claim 32, wherein the MHC is an MHC recited in Table 7.

37. The vector of Claim 1, wherein the housekeeping epitope has a sequence that is identical, cross-reactive with, or substantially similar to a segment of the antigen, and wherein the housekeeping epitope expression cassette does not express a polypeptide comprising a complete target-associated antigen. - 95 - C:\RPonbl\DCC\SCG39X261 DOC-I 5/12012

38. The vector of Claim 37, wherein the housekeeping epitope has a known or predicted affinity for a class I MHC receptor peptide binding cleft, wherein the MHC is an MIC recited in Appendix C.

39. The vector of Claim 1, wherein the housekeeping epitope has a sequence corresponding to a segment of the antigen, and wherein the housekeeping epitope expression cassette does not express a polypeptide comprising the complete target associated antigen.

40. A vector comprising a promoter and means for encoding an immunogen, said immunogen being capable, when expressed in a pAPC, of inducing a CTL response recognizing a housekeeping epitope, wherein the housekeeping epitope is derived from a target-associated antigen, and wherein the epitope is not ELAGIGILTV (SEQ ID NO: I), AAGIGILTV (SEQ ID NO: 429), or EAAGIGILTV (SEQ ID NO:8).

41. The vector of Claim 1, wherein the housekeeping epitope has a sequence that is identical, cross-reactive with, or substantially similar to a segment of the antigen, and wherein the epitope is not ELAGIGILTV (SEQ ID NO: 1), AAGIGILTV (SEQ ID NO: 429), or EAAGIGILTV (SEQ ID NO: 8), and wherein the expression cassette encodes an array of two or more epitopes, or at least one epitope and at least one epitope cluster.

42. The vector of any one of Claims I to 3 and 6 to 41, wherein the vector is not a viral vector.

43. A vector according to any one of Claims I to 3, 6 to 30, 32 to 42 or a method according to Claim 4 or an engineered substrate according to Claim 5 or an immunogenic composition according to Claim 31 substantially as hereinbefore described with reference to the Figures and/or Examples. - 96 -