Abstract:
The present invention relates generally to a methodology for the isolation, purification and identification of peptide ligands presented by MHC positive cells. In particular, the methodology of the present invention relates to the isolation, purification and identification of these peptide ligands from soluble class I and class 11 MHC molecules which may be from uninfected, infected, or tumorigenic cells. The methodology of the present invention broadly allows for these peptide ligands and their cognate source proteins thereof to be identified and used as markers for infected versus uninfected cells and/or tumorigenic versus nontumorigenic cells, with said identification being useful for marking or targeting a cell for therapeutic treatment or priming the immune response against infected cells.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims the benefit under 35 U.S.C. 119(e) of provisional applications U.S. Ser. No. 60732,183, filed Nov. 1, 2005; and U.S. Ser. No. 60800,134, filed May 12, 2006; the contents of each of which are hereby expressly incorporated herein by reference.  
         [0002]     This application is also a continuation-in-part of US Ser. No. 10845,391, filed May 13, 2004; which claims the benefit under 35 U.S.C. 119(e) of provisional applications U.S. Ser. No. 60469,995, filed May 13, 2003; and U.S. Ser. No. 60518,132, filed Nov. 7, 2003; the contents of each of which are hereby expressly incorporated herein by reference in their entirety.  
         [0003]     Said application U.S. Ser. No. 10845,391 is also a continuation-in-part of U.S. Ser. No. 09974,366, filed Oct. 10, 2001, which claims the benefit under 35 U.S.C. 119(e) of provisional applications U.S. Ser. No. 60240,143, filed Oct. 10, 2000; U.S. Ser. No. 60299,452, filed Jun. 20, 2001; U.S. Ser. No. 60256,410, filed Dec. 18, 2000; U.S. Ser. No. 60256,409, filed Dec. 18, 2000; and U.S. Ser. No. 60327,907, filed Oct. 9, 2001; all of which are hereby expressly incorporated herein by reference in their entirety. 
     
    
     BACKGROUND OF THE INVENTION  
       [0004]     1. Field of the Invention  
         [0005]     The present invention relates generally to a methodology of epitope testing for the identification of peptides that bind to an individual soluble MHC Class I or Class II molecule as well as to peptides identified by such methodology.  
         [0006]     2. Description of the Background Art  
         [0007]     Class I major histocompatibility complex (MHC) molecules, designated HLA class I in humans, bind and display peptide antigen ligands upon the cell surface. The peptide antigen ligands presented by the class I MHC molecule are derived from either normal endogenous proteins (“self”) or foreign proteins (“nonself”) introduced into the cell. Nonself proteins may be products of malignant transformation or intracellular pathogens such as viruses. In this manner, class I MHC molecules convey information regarding the internal fitness of a cell to immune effector cells including but not limited to, CD8 +  cytotoxic T lymphocytes (CTLs), which are activated upon interaction with “nonself” peptides, thereby lysing or killing the cell presenting such “nonself” peptides.  
         [0008]     Class II MHC molecules, designated HLA class II in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC molecules which are expressed on virtually all nucleated cells, class II MHC molecules are normally confined to specialized cells, such as B lymphocytes, macrophages, dendritic cells, and other antigen presenting cells which take up foreign antigens from the extracellular fluid via an endocytic pathway. The peptides they bind and present are derived from extracellular foreign antigens, such as products of bacteria that multiply outside of cells, wherein such products include protein toxins secreted by the bacteria that often times have deleterious and even lethal effects on the host (e.g. human). In this manner, class II molecules convey information regarding the fitness of the extracellular space in the vicinity of the cell displaying the class 11 molecule to immune effector cells, including but not limited to, CD4 +  helper T cells, thereby helping to eliminate such pathogens the examination of such pathogens is accomplished by both helping B cells make antibodies against microbes, as well as toxins produced by such microbes, and by activating macrophages to destroy ingested microbes.  
         [0009]     Class I and class II HLA molecules exhibit extensive polymorphism generated by systematic recombinatorial and point mutation events; as such, hundreds of different HLA types exist throughout the world&#39;s population, resulting in a large immunological diversity. Such extensive HLA diversity throughout the population results in tissue or organ transplant rejection between individuals as well as differing susceptibilities and/or resistances to infectious diseases. HLA molecules also contribute significantly to autoimmunity and cancer. Because HLA molecules mediate most, if not all, adaptive immune responses, large quantities of pure isolated HLA proteins are required in order to effectively study transplantation, autoimmunity disorders, and for vaccine development.  
         [0010]     There are several applications in which purified, individual class I and class II MHC proteins are highly useful. Such applications include using MHC-peptide multimers as immunodiagnostic reagents for disease resistanceautoimmunity; assessing the binding of potentially therapeutic peptides; elution of peptides from MHC molecules to identify vaccine candidates; screening transplant patients for preformed MHC specific antibodies; and removal of anti-HLA antibodies from a patient. Since every individual has differing MHC molecules, the testing of numerous individual MHC molecules is a prerequisite for understanding the differences in disease susceptibility between individuals. Therefore, purified MHC molecules representative of the hundreds of different HLA types existing throughout the world&#39;s population are highly desirable for unraveling disease susceptibilities and resistances, as well as for designing therapeutics such as vaccines.  
         [0011]     Class I HLA molecules alert the immune response to disorders within host cells. Peptides, which are derived from viral- and tumor-specific proteins within the cell, are loaded into the class I molecule&#39;s antigen binding groove in the endoplasmic reticulum of the cell and subsequently carried to the cell surface. Once the class I HLA molecule and its loaded peptide ligand are on the cell surface, the class I molecule and its peptide ligand are accessible to cytotoxic T lymphocytes (CTL). CTL survey the peptides presented by the class I molecule and destroy those cells harboring ligands derived from infectious or neoplastic agents within that cell.  
         [0012]     While specific CTL targets have been identified, little is known about the breadth and nature of ligands presented on the surface of a diseased cell. From a basic science perspective, many outstanding questions have percolated through the art regarding peptide exhibition. For instance, it has been demonstrated that a virus can preferentially block expression of HLA class I molecules from a given locus while leaving expression at other loci intact. Similarly, there are numerous reports of cancerous cells that fail to express class I HLA at particular loci. However, there is no data describing how (or if) the three classical HLA class I loci differ in the immunoregulatory ligands they bind. It is therefore unclear how class I molecules from the different loci vary in their interaction with viral- and tumor-derived ligands and the number of peptides each will present.  
         [0013]     Discerning virus- and tumor-specific ligands for CTL recognition is an important component of vaccine design. Ligands unique to tumorigenic or infected cells can be tested and incorporated into vaccines designed to evoke a protective CTL response. Several methodologies are currently employed to identify potentially protective peptide ligands. One approach uses T cell lines or clones to screen for biologically active ligands among chromatographic fractions of eluted peptides (Cox et al., Science, vol 264, 1994, pages 716-719, which is expressly incorporated herein by reference in its entirety). This approach has been employed to identify peptide ligands specific to cancerous cells. A second technique utilizes predictive algorithms to identify peptides capable of binding to a particular class I molecule based upon previously determined motif and/or individual ligand sequences (De Groot et al., Emerging Infectious Diseases, (7) 4, 2001, which is expressly incorporated herein by reference in its entirety). Peptides having high predicted probability of binding from a pathogen of interest can then be synthesized and tested for T cell reactivity in various assays, such as but not limited to, precursor, tetramer and ELISpot assays.  
         [0014]     However, there has been no readily available source of individual HLA molecules. The quantities of HLA protein available have been small and typically consist of a mixture of different HLA molecules. Production of HLA molecules traditionally involves growth and lysis of cells expressing multiple HLA molecules. Ninety percent of the population is heterozygous at each of the HLA loci; codominant expression results in multiple HLA proteins expressed at each HLA locus. To purify native class I or class II molecules from mammalian cells requires time-consuming and cumbersome purification methods, and since each cell typically expresses multiple surface-bound HLA class I or class II molecules, HLA purification results in a mixture of many different HLA class I or class II molecules. When performing experiments using such a mixture of HLA molecules or performing experiments using a cell having multiple surface-bound HLA molecules, interpretation of results cannot directly distinguish between the different HLA molecules, and one cannot be certain that any particular HLA molecule is responsible for a given result. Therefore, prior to the present invention, a need existed in the art for a method of producing substantial quantities of individual HLA class I or class II molecules so that they can be readily purified and isolated independent of other HLA class I or class II molecules. Such individual HLA molecules, when provided in sufficient quantity and purity as described herein, provides a powerful tool for studying and measuring immune responses.  
         [0015]     Therefore, there exists a need in the art for improved methods of assaying binding of peptides to class I and class II MHC molecules to identify epitopes that bind to specific individual class I and class II MHC molecules. The present invention solves this need by coupling the production of soluble HLA molecules with epitope isolation, discovery, and testing methodology.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]      FIG. 1 . Overview of 2 stage PCR strategy to amplify a truncated version of the human class I MHC.  
         [0017]      FIG. 2 . Flow chart of the epitope discovery of C-terminal-tagged sHLA molecules. Class I positive transfectants are infected with a pathogen of choice, and sHLA is preferentially purified utilizing the tag. Subtractive comparison of MS ion maps yields ions present only in infected cell, which are then MSMS sequenced to derive class I epitopes. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0018]     Before explaining at least one embodiment of the invention in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and/or results. The invention is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary-not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.  
         [0019]     The present invention combines methodologies for assaying the binding of peptide epitopes to individual, soluble MHC molecules with methodologies for the production of individual, soluble MHC molecules and with a method of epitope discovery and comparative ligand mapping (including methods of distinguishing infected/tumor cells from uninfected/non-tumor cells). The method of production of individual, soluble MHC molecules has previously been described in detail in parent application U.S. Publication No. 2003/0166057, filed Dec. 18, 2001, entitled “METHOD AND APPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF,” the contents of which are hereby expressly incorporated herein in their entirety by reference. The method of epitope discovery and comparative ligand mapping has previously been described in detail in parent application U.S. Publication No. 2002/0197672, filed Oct. 10, 2001, entitled “COMPARATIVE LIGAND MAPPING FROM MHC CLASS I POSITIVE CELLS”, the contents of which have previously been expressly incorporated in their entirety by reference. A brief description of each of these methodologies is included herein below for the purpose of exemplification and should not be considered as limiting.  
         [0020]     In addition, the methods of the present invention may be combined with methods of epitope testing as described in U.S. Publication No. 2003/0124613, filed Mar. 11, 2002, entitled “EPITOPE TESTING USING SOLUBLE HLA”, the contents of which are hereby expressly incorporated herein by reference.  
         [0021]     To produce the individual soluble class I molecule-endogenous peptide complexes, genomic DNA or cDNA encoding at least one class I molecule is obtained, and an allele encoding an individual class I molecule in the genomic DNA or cDNA is identified. The allele encoding the individual class I molecule is PCR amplified in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule. The PCR product is then cloned into an expression vector, thereby forming a construct that encodes the individual soluble class I molecule, and the construct is transfected into a cell line to provide a cell line containing a construct that encodes an individual soluble class I molecule. The cell line must be able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules.  
         [0022]     The cell line is then cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and these conditions also allow for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell. The secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are then isolated.  
         [0023]     The construct that encodes the individual soluble class I molecule may further encode a tag, such as a HIS tail or a FLAG tail, which is attached to the individual soluble class I molecule and aids in isolating the individual soluble class I molecule.  
         [0024]     The peptide of interest may be chosen based on several methods of epitope discovery known in the art. Alternatively, the peptide of interest may be identified by a method for identifying at least one endogenously loaded peptide ligand that distinguishes an infected cell from an uninfected cell. Such method includes providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, wherein the uninfected cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules. A portion of the uninfected cell line is infected with at least one of a microorganism (such as HIV, HBV or influenza), a gene from a microorganism or a tumor gene, thereby providing an infected cell line, and both the uninfected cell line and the infected cell line are cultured under conditions which allow for expression of individual soluble class I molecules from the construct. The culture conditions also allow for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell. The secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are isolated from the uninfected cell line and the infected cell line, and the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from both the uninfected cell line and the infected cell line. The endogenously loaded peptide ligands are then isolated from both the uninfected cell line and the infected cell line, and the two sets of endogenously loaded peptide ligands are compared to identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the infected cell line that is not presented by the individual soluble class I molecule on the uninfected cell line, or to identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule in a substantially greater amount on the infected cell line when compared to the uninfected cell line. In addition, the comparison described herein above may also identify at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule on the uninfected cell line that is not presented by the individual soluble class I molecule on the infected cell line, or that is presented in a substantially greater amount on the uninfected cell line when compared to the infected cell line.  
         [0025]     The term “substantially greater amount” as used herein refers to an amount that is detectably greater than another amount; for example, the term “presented in a substantially greater amount” as used herein refers to an at least 1-fold increase in a first amount of presentation when compared to a second amount of presentation. The tables provided herein disclose “Fold Increase” amounts for the peptides identified by the methods of the present invention.  
         [0026]     Optionally, proteomics may eventually allow for sequencing all epitopes from a diseased cell so that comparative mapping, i.e., comparison of infected cells to healthy cells, would no longer be required. Microarrays and other proteomic data should provide insight as to the healthy cell.  
         [0027]     Following identification of the peptide ligand that distinguishes an infected cell from an uninfected cell, a source protein from which the endogenously loaded peptide ligand is obtained can be identified. Such source protein may be encoded by at least one of the microorganism, the gene from a microorganism or the tumor gene with which the cell line was infected to form the infected cell line, or the source protein may be encoded by the uninfected cell line. When the source protein is encoded by the uninfected cell line, such protein may also demonstrate increased expression in a tumor cell line.  
         [0028]     Therefore, the present invention is also directed to isolated peptide ligands for an individual class I molecule isolated by the methods described herein. In one embodiment, the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 1-315. In another embodiment, the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 99-301. In yet another embodiment, the isolated peptide ligand has a length of from about 7 to about 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS: 302-315.  
         [0029]     The isolated peptide ligand described herein above may be an endogenously loaded peptide ligand presented by an individual class I molecule in a substantially greater amount on an infected cell when compared to an uninfected cell.  
         [0030]     The peptide ligands of the present invention may be isolated by a method that includes providing a cell line containing a construct that encodes an individual soluble class I molecule, wherein the cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules. The cell line is cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell. Secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are then isolated, and the peptide ligands are then separated from the individual soluble class I molecules.  
         [0031]     In another embodiment, the isolated peptide ligands of the present invention may be identified by a method that includes providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, wherein the cell line is able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules. A portion of the uninfected cell line is infected with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing an infected cell line. The uninfected cell line and the infected cell line are cultured under conditions which allow for expression of the individual soluble class I molecules from the construct, and also allow for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I molecule prior to secretion of the individual soluble class I molecules from the cell. The secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto are isolated from both the uninfected cell line and the infected cell line; then, the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from the uninfected cell, and the endogenously loaded peptide ligands are separated from the individual soluble class I molecules from the infected cell. The endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line are then isolated and compared. Finally, at least one endogenously loaded peptide ligand presented by the individual soluble class I molecule in a substantially greater amount on the infected cell line when compared to the uninfected cell line is identified.  
         [0032]     The uninfected cell line containing the construct that encodes the individual soluble class I molecule may be produced by a method that includes obtaining genomic DNA or cDNA encoding at least one class I molecule and identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA. The allele encoding the individual class I molecule is PCR amplified in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule. The PCR product is cloned into an expression vector to form a construct that encodes the individual soluble class I molecule, and the construct is tranfected into an uninfected cell line. The construct may further encode a tag, such as but not limited to, a HIS tail or a FLAG tail, which is attached to the individual soluble class I molecule, and the tag aids in isolating the individual soluble class I molecule. The tag may be encoded by a PCR primer utilized in the PCR step, or the tag may be encoded by the expression vector into which the PCR product is cloned.  
         [0033]     The at least one endogenously loaded peptide ligand may be obtained from a protein encoded by at least one of the microorganism, the gene from the microorganism or the tumor gene with which the portion of the uninfected cell line is infected to form the infected cell line. Alternatively, the at least one endogenously loaded peptide ligand may be obtained from a protein encoded by the uninfected cell line.  
       Production of Individual, Soluble MHC Molecules  
       [0034]     The methods of the present invention may, in one embodiment, utilize a method of producing MHC molecules (from genomic DNA or cDNA) that are secreted from mammalian cells in a bioreactor unit. Substantial quantities of individual MHC molecules are obtained by modifying class I or class II MHC molecules so that they are capable of being secreted, isolated, and purified. Secretion of soluble MHC molecules overcomes the disadvantages and defects of the prior art in relation to the quantity and purity of MHC molecules produced. Problems of quantity are overcome because the cells producing the MHC do not need to be detergent lysed or killed in order to obtain the MHC molecule. In this way the cells producing secreted MHC remain alive and therefore continue to produce MHC. Problems of purity are overcome because the only MHC molecule secreted from the cell is the one that has specifically been constructed to be secreted. Thus, transfection of vectors encoding such secreted MHC molecules into cells which may express endogenous, surface bound MHC provides a method of obtaining a highly concentrated form of the transfected MHC molecule as it is secreted from the cells. Greater purity is assured by transfecting the secreted MHC molecule into MHC deficient cell lines.  
         [0035]     Production of the MHC molecules in a hollow fiber bioreactor unit allows cells to be cultured at a density substantially greater than conventional liquid phase tissue culture permits. Dense culturing of cells secreting MHC molecules further amplifies the ability to continuously harvest the transfected MHC molecules. Dense bioreactor cultures of MHC secreting cell lines allow for high concentrations of individual MHC proteins to be obtained. Highly concentrated individual MHC proteins provide an advantage in that most downstream protein purification strategies perform better as the concentration of the protein to be purified increases. Thus, the culturing of MHC secreting cells in bioreactors allows for a continuous production of individual MHC proteins in a concentrated form.  
         [0036]     The method of producing MHC molecules utilized in the present invention and described in detail in U.S. Ser. No. 10/022,066 begins by obtaining genomic or complementary DNA which encodes the desired MHC class I or class II molecule. Alleles at the locus which encode the desired MHC molecule are PCR amplified in a locus specific manner. These locus specific PCR products may include the entire coding region of the MHC molecule or a portion thereof. In one embodiment a nested or hemi-nested PCR is applied to produce a truncated form of the class I or class II gene so that it will be secreted rather than anchored to the cell surface.  FIG. 1  illustrates the PCR products resulting from such nested PCR reactions. In another embodiment the PCR will directly truncate the MHC molecule.  
         [0037]     Locus specific PCR products are cloned into a mammalian expression vector and screened with a variety of methods to identify a clone encoding the desired MHC molecule. The cloned MHC molecules are DNA sequenced to ensure fidelity of the PCR. Faithful truncated clones of the desired MHC molecule are then transfected into a mammalian cell line. When such cell line is transfected with a vector encoding a recombinant class I molecule, such cell line may either lack endogenous class I MHC molecule expression or express endogenous class I MHC molecules. One of ordinary skill of the art would note the importance, given the present invention, that cells expressing endogenous class I MHC molecules may spontaneously release MHC into solution upon natural cell death, infection, transformation, etc. In cases where this small amount of spontaneously released MHC is a concern, the transfected class I MHC molecule can be “tagged” such that it can be specifically purified away from spontaneously released endogenous class I molecules in cells that express class I molecules. For example, a DNA fragment encoding a HIS tail may be attached to the protein by the PCR reaction or may be encoded by the vector into which the PCR fragment is cloned, and such HIS tail, therefore, further aids in the purification of the class I MHC molecules away from endogenous class I molecules. Tags beside a histidine tail have also been demonstrated to work, and one of ordinary skill in the art of tagging proteins for downstream purification would appreciate and know how to tag a MHC molecule in such a manner so as to increase the ease by which the MHC molecule may be purified.  
         [0038]     Cloned genomic DNA fragments contain both exons and introns as well as other non-translated regions at the 5′ and 3′ termini of the gene. Following transfection into a cell line which transcribes the genomic DNA (gDNA) into RNA, cloned genomic DNA results in a protein product thereby removing introns and splicing the RNA to form messenger RNA (mRNA), which is then translated into an MHC protein. Transfection of MHC molecules encoded by gDNA therefore facilitates reisolation of the gDNA, mRNA/cDNA, and protein. Production of MHC molecules in non-mammalian cell lines such as insect and bacterial cells requires cDNA clones, as these lower cell types do not have the ability to splice introns out of RNA transcribed from a gDNA clone. In these instances the mammalian gDNA transfectants of the present invention provide a valuable source of RNA which can be reverse transcribed to form MHC cDNA. The cDNA can then be cloned, transferred into cells, and then translated into protein. In addition to producing secreted MHC, such gDNA transfectants therefore provide a ready source of mRNA, and therefore cDNA clones, which can then be transfected into non-mammalian cells for production of MHC. Thus, the present invention which starts with MHC genomic DNA clones allows for the production of MHC in cells from various species.  
         [0039]     A key advantage of starting from gDNA is that viable cells containing the MHC molecule of interest are not needed. Since all individuals in the population have a different MHC repertoire, one would need to search more than 500,000 individuals to find someone with the same MHC complement as a desired individual—such a practical example of this principle is observed when trying to find a donor to match a recipient for bone marrow transplantation. Thus, if it is desired to produce a particular MHC molecule for use in an experiment or diagnostic, a person or cell expressing the MHC allele of interest would first need to be identified. Alternatively, in the method of the present invention, only a saliva sample, a hair root, an old freezer sample, or less than a milliliter (0.2 ml) of blood would be required to isolate the gDNA. Then, starting from gDNA, the MHC molecule of interest could be obtained via a gDNA clone as described herein, and following transfection of such clone into mammalian cells, the desired protein could be produced directly in mammalian cells or from cDNA in several species of cells using the methods of the present invention described herein.  
         [0040]     Current methodologies used by others to obtain an MHC allele for protein expression typically start from mRNA, which requires a fresh sample of mammalian cells that express the MHC molecule of interest. Working from gDNA does not require gene expression or a fresh biological sample. It is also important to note that RNA is inherently unstable and is not as easily obtained as is gDNA. Therefore, if production of a particular MHC molecule starting from a cDNA clone is desired, a person or cell line that is expressing the allele of interest must traditionally first be identified in order to obtain RNA. Then a fresh sample of blood or cells must be obtained; experiments using the methodology of the present invention show that ≧5 milliliters of blood that is less than 3 days old is required to obtain sufficient RNA for MHC cDNA synthesis. Thus, by starting with gDNA, the breadth of MHC molecules that can be readily produced is expanded. This is a key factor in a system as polymorphic as the MHC system; hundreds of MHC molecules exist, and not all MHC molecules are readily available. This is especially true of MHC molecules unique to isolated populations or of MHC molecules unique to ethnic minorities. Starting class I or class II MHC molecule expression from the point of genomic DNA simplifies the isolation of the gene of interest and insures a more equitable means of producing MHC molecules for study; otherwise, one would be left to determine whose MHC molecules are chosen and not chosen for study, as well as to determine which ethnic population from which fresh samples cannot be obtained and therefore should not have their MHC molecules included in a diagnostic assay.  
         [0041]     While cDNA may be substituted for genomic DNA as the starting material, production of cDNA for each of the desired HLA class I types will require hundreds of different, HLA typed, viable cell lines, each expressing a different HLA class I type. Alternatively, fresh samples are required from individuals with the various desired MHC types. The use of genomic DNA as the starting material allows for the production of clones for many HLA molecules from a single genomic DNA sequence, as the amplification process can be manipulated to mimic recombinatorial and gene conversion events. Several mutagenesis strategies exist whereby a given class I gDNA clone could be modified at either the level of gDNA or at the cDNA resulting from this gDNA clone. The process of producing MHC molecules utilized in the present invention does not require viable cells, and therefore the degradation which plagues RNA is not a problem.  
       Methods of Epitope Discovery and Comparative Ligand Mapping  
       [0042]     Peptide epitopes unique to infected and cancerous cells can be directly identified by the methods of the present invention, which include producing sHLA molecules in cancerous and infected cells and then sequencing the epitopes unique to the cancerous or infected cells. Such epitopes can then be tested for their binding to various HLA molecules to see how many HLA molecules these epitopes might bind. This direct method of epitope discovery is described in detail in U.S. Ser. No. 09/974,366 and is briefly described herein below.  
         [0043]     The method of epitope discovery included in the present invention (and described in detail in U.S. Ser. No. 09/974,366) includes the following steps: (1) providing a cell line containing a construct that encodes an individual soluble class I or class II MHC molecule (wherein the cell line is capable of naturally processing self or nonself proteins into peptide ligands capable of being loaded into the antigen binding grooves of the class I or class II MHC molecules); (2) culturing the cell line under conditions which allow for expression of the individual soluble class I or class II MHC molecule from the construct, with such conditions also allowing for the endogenous loading of a peptide ligand (from the self or non-self processed protein) into the antigen binding groove of each individual soluble class I or class II MHC molecule prior to secretion of the soluble class I or class II MHC molecules having the peptide ligands bound thereto; and (3) separating the peptide ligands from the individual soluble class I or class II MHC molecules.  
         [0044]     Class I and class II MHC molecules are really a trimolecular complex consisting of an alpha chain, a beta chain, and the alphabeta chain&#39;s peptide cargo (i.e. the peptide ligand) which is presented on the cell surface to immune effector cells. Since it is the peptide cargo, and not the MHC alpha and beta chains, which marks a cell as infected, tumorigenic, or diseased, there is a great need to identify and characterize the peptide ligands bound by particular MHC molecules. For example, characterization of such peptide ligands greatly aids in determining how the peptides presented by a person with MHC-associated diabetes differ from the peptides presented by the MHC molecules associated with resistance to diabetes. As stated above, having a sufficient supply of an individual MHC molecule, and therefore that MHC molecule&#39;s bound peptides, provides a means for studying such diseases. Because the method of the present invention provides quantities of MHC protein previously unobtainable, unparalleled studies of MHC molecules and their important peptide cargo can now be facilitated and utilized to distinguish infected/tumor cells from uninfected/non-tumor cells by unique epitopes presented by MHC molecules in the disease or non-disease state.  
         [0045]     The method of the present invention includes the direct comparative analysis of peptide ligands eluted from class I HLA molecules (as described previously in U.S. Publication No. 2002/097672). The teachings of U.S. Publication No. 2002/097672 demonstrates that the addition of a C-terminal epitope tag (such as a 6-HIS or FLAG tail) to transfected class I molecules has no effects on peptide binding specificity of the class I molecule and consequently has no deleterious effects on direct peptide ligand mapping and sequencing, and also does not disrupt endogenous peptide loading.  
         [0046]     The method described in parent application U.S. Publication No. 2002/097672 further relates to a novel method for detecting those peptide epitopes which distinguish the infected/tumor cell from the uninfected/non-tumor cell. The results obtained from the present inventive methodology cannot be predicted or ascertained indirectly; only with a direct epitope discovery method can the unique epitopes described therein be identified. Furthermore, only with this direct approach can it be ascertained that the source protein is degraded into potentially immunogenic peptide epitopes. Finally, this unique approach provides a glimpse of which proteins are uniquely up and down regulated in infected/tumor cells.  
         [0047]     The utility of such HLA-presented peptide epitopes which mark the infected/tumor cell are three-fold. First, diagnostics designed to detect a disease state (i.e., infection or cancer) can use epitopes unique to infected/tumor cells to ascertain the presence/absence of a tumor/virus. Second, epitopes unique to infected/tumor cells represent vaccine candidates. For example, the present invention describes and claims epitopes which arise on the surface of cells infected with HIV. Such epitopes could not be predicted without natural virus infection and direct epitope discovery. The epitopes detected are derived from proteins unique to virus infected and tumor cells. These epitopes can be used for virus/tumor vaccine development and virus/tumor diagnostics. Third, the process indicates that particular proteins unique to virus infected cells are found in compartments of the host cell they would otherwise not be found in. Thus, uniquely upregulated or trafficked host proteins are identified for drug targeting to kill infected cells.  
         [0048]     While the epitopes detected as unique to infected/tumor cells may serve as direct targets (i.e., through diagnostic, vaccine or therapeutic means), such epitopes may also be utilized to influence the environment around a diseased cell so that these treatments and therapies are effective, and thus allowing the immune responses to see the diseased cell.  
         [0049]     The presently disclosed and claimed invention, as well as the parent application U.S. Publication No. 2002/097672, describe, in particular, peptide epitopes unique to HIV infected cells. Peptide epitopes unique to the HLA molecules of HIV infected cells were identified by direct comparison to HLA peptide epitopes from uninfected cells by the method illustrated in the flow chart of  FIG. 2 . Such method has been shown to be capable of identifying: (1) HLA presented peptide epitopes, derived from intracellular host proteins, that are unique to infected cells but not found on uninfected cells, and (2) that the intracellular source-proteins of the peptides are uniquely expressed/processed in HIV infected cells such that peptide fragments of the proteins can be presented by HLA on infected cells but not on uninfected cells.  
         [0050]     The method of epitope discovery and comparative ligand mapping also, therefore, describes the unique expression of proteins in infected cells or, alternatively, the unique trafficking and processing of normally expressed host proteins such that peptide fragments thereof are presented by HLA molecules on infected cells. These HLA presented peptide fragments of intracellular proteins represent powerful alternatives for diagnosing virus infected cells and for targeting infected cells for destruction (i.e., vaccine development).  
         [0051]     A group of the host source-proteins for HLA presented peptide epitopes unique to HIV infected cells represent source-proteins that are uniquely expressed in cancerous cells. For example, through using the methodology of the present invention a peptide fragment (SEQ ID NO:12) of reticulocalbin is uniquely found on HIV infected cells. A literature search indicates that the reticulocalbin gene is uniquely upregulated in cancer cells (breast cancer, liver cancer, colorectal cancer). Thus, the HLA presented peptide fragment of reticulocalbin which distinguishes HIV infected cells from uninfected cells can be inferred to also differentiate tumor cells from healthy non-tumor cells. Thus, HLA presented peptide fragments of host genes and gene products that distinguish the tumor cell and virus infected cell from healthy cells have been directly identified. The epitope discovery method is also capable of identifying host proteins that are uniquely expressed or uniquely processed on virus infected or tumor cells. HLA presented peptide fragments of such uniquely expressed or uniquely processed proteins can be used as vaccine epitopes and as diagnostic tools.  
         [0052]     The methodology of targeting and detecting virus infected cells is not meant to target the virus-derived peptides. Rather, the methodology of the present invention indicates that the way to distinguish infected cells from healthy cells is through alterations in host encoded protein expression and processing. This is true for cancer as well as for virus infected cells. The methodology according to the present invention results in data which indicates, without reservation, that proteins/peptides distinguish virus/tumor cells from healthy cells.  
         [0053]     In a brief example of the methodology of comparative ligand mapping utilized in the methods of the present invention, a cell line producing individual, soluble MHC molecules is constructed as described herein before and in US Publication No. 2003/0166057. A portion of the transfected cell line is cocultured with a virus of interest, resulting in high-titre-virus and providing infected cells. In the case of influenza virus, the infection is not productive in the bioreactor and does not result in the production of high titer virus. Because of this, fresh influenza virus was added to the coculture. In the example provided herein and in detail in US Publication No. 2003/0166057, the viruses of interest are HIV, influenza and WNV. Alternatively, a portion of the cell line producing individual, soluble MHC molecules may be transformed to produce a tumor cell line.  
         [0054]     The non-infected cell line and the cell line infected with HIV are both cultured in hollow-fiber bioreactors as described herein above and in detail in US Publication No. 2003/0166057, and the soluble HLA-containing supernatant is then removed from the hollow-fiber bioreactors. The uninfected and infected harvested supernatants were then treated in an identical manner post-removal from the cell-pharm.  
         [0055]     MHC class I-peptide complexes were affinity purified from the infected and uninfected supernatants using W6/32 antibody. Following elution, peptides were isolated from the class I molecules and separated by reverse phase HPLC fractionation. Separate but identical (down to the same buffer preparations) peptide purifications were done for each peptide-batch from uninfected and infected cells.  
         [0056]     Fractionated peptides were then mapped by mass spectrometry to generate fraction-based ion maps. Spectra from the same fraction in uninfected/infected cells were manually aligned and visually assessed for the presence of differences in the ions represented by the spectra. Ions corresponding to the following categories were selected for MSMS sequencing: (1) upregulation in infected cells (at least 1.5 fold over the same ion in uninfected cells), (2) downregulation in infected cells (at least 1.5 fold over the same ion in the uninfected cells), (3) presence of the ion only in infected cells, or (4) absence of ion in infected cells that is present in uninfected cells. In addition, multiple parameters were established before peptides were assigned to one of the above categories, including checking the peptide fractions preceding and following the peptide fraction by MS/MS to ensure that the peptide of interest was not present in an earlier or later fraction as well as generation of synthetic peptides and subjection to MSMS to check for an exact match. In addition, one early quality control step involves examining the peptide&#39;s sequence to see if it fits the “predicted motif” defined by sequences that were previously shown to be presented by the MHC molecule utilized.  
         [0057]     After identification of the epitopes, literature searches were performed on source proteins to determine their function within the infected cell, and the source proteins were classified into groups according to functions inside the cell. Secondly, source proteins were scanned for other possible epitopes which may be bound by other MHC class I alleles. Peptide binding predictions were employed to determine if other peptides presented from the source proteins were predicted to bind, and proteasomal prediction algorithms were likewise employed to determine the likelihood of a peptide being created by the proteasome.  
         [0058]     In accordance with the present invention, Table I lists peptide ligands that have been identified as being presented by the B*0702 and A*0201 or B*1801 class I MHC molecule in cells infected with the HIV MN-1 virus but not in uninfected cells, and also lists one peptide ligand that has been identified as not being presented by the B*0702 class I MHC molecule in cells infected with the HIV MN-1 virus that is presented in uninfected cells. One of ordinary skill in the art can appreciate the novelty and usefulness of the present methodology in directly identifying such peptide ligands and the importance such identification has for numerous therapeutic (vaccine development, drug targeting) and diagnostic tools.  
         [0059]     As stated above, Table I identifies the sequences of peptide ligands identified to date as being unique to HIV infected cells. Class I sHLA B*0702, A*0201 or B*1801 was harvested from T cells infected and not infected with HIV. Peptide ligands were eluted from B*0702, A*0201 or B*1801 and comparatively mapped on a mass spectrometer so that ions unique to infected cells were apparent. Ions unique to infected cells (and one ligand unique to uninfected cells) were subjected to mass spectrometric fragmentation for peptide sequencing.  
                                                                                 TABLE I                           Peptides Identified on Infected Cells that are not Present on Uninfected Cells           Restricting allele for Sequences marked with a (•) is HLA-B*0702.       Restricting allele for Sequences marked with a (□) is HLA-A*0201 or HLA-B*1801.                Seq               ID                    Sequence   Source Protein   Category   No                    •   EQMFEDIISL   HIV MN-1, ENV   HIV-DERIVED   1                   •   IPCLLISFL   Cholinergic Receptor, alpha-3 polypeptide   Signal transduction; ion channel   2               •   STTAICATGL   Ubiquitin-specific protease 3   Ubiquitin-protease activity; hydrolase   3                   activity               •   APAQNPEL   HLA-B associated transcript 3 (BAT3)   MHC gene product   4               •   LVMAPRTVL   HLA-B heavy chain leader sequence   MHC gene product   5               •   APFI[NS]PADX   Unknown, close to several cDNA&#39;s   UNKNOWN   6               •   TPQSNRPVm   RNA polymerase II, polypeptide A   DNA binding; protein binding;   7                   transcription               •   AARPATSTL   Eukaryotic translation iniation factor 4GI   RNA binding; translation initiation   8                   factor               •   MAMMAALMA   Sparc-likek protein 1   calcium ion binding; extracellular space   9               •   IATVDSYVI   Tenascin   protein binding; extracellular space   10               •   SPNQARAQAAL   Polypyrimidine tract binding protein 1   RNA binding   11               •   GPRTAALGLL   Reticulocalbin 2   calcium ion binding; protein binding   12               •   NPNQNKNVAL   ELAV (HuR)   RNA binding; RNA catabolism   13               •   RPYSNVSNL   Set-binding factor 1   protein phosphatase activity   14               •   LPQANRDTL   Rac GTPase activating protein 1   electron transporter; iron binding;   15                   intracellular signalling               •   QPRYPVNSV   TCP-1 alpha   ATP binding; chaperone activity   16               •   APAYSRAL   Heat shock protein 27   protein binding; chaperone   17               •   APKRPPSAF   High mobility group protein 1 or 2   DNA binding; DNA unwinding   18               •   AASKERSGVSL   Histone H1 family member   DNA binding   19               □   FIISRTQAL   karyopherin beta 2; importin beta 2;   intracellular protein transport; nuclear   20               transportin   import               □   SLAGSLRSV   FLJ00164 protein   no description   21               □   YGMPRQIL   similar to  Homo sapiens  mRNA for KIAA0120   muscle development   22               gene with GenBank Accession Number               D21261.1               □   MIIINKFV   hypothetical protein XP_103946   no description   23               □   ALWDIETGQQTV   G protein beta subunit   GTPase activity; signal transducer   24               □   VLMTEDIKL   eukaryotic translation initiation factor   calcium ion binding; extracellular space   25               4 gamma, 1               □   YIYDKDMEII   usp22   Ubiquitin-protease activity; hydrolase   26                   activity               □   ALMPVLNQV   homolog of yeast mRNA transport regulator   exosome constituent   27               3               □   DLIIKGISV   TAR DNA binding protein   RNA binding; transcription factor   28                   activity               □   QLVDIIEKV   proteasome activator 28-gamma; 11S   proteasome activator activity   29               regulator complex gamma subunit;               proteasome activator subunit 3 isoform 2;               Ki nuclear autoantigen               □   IMLEALERV   snRNP polypeptide G   RNA binding; RNA splicing; spliceosome   30                   assembly               □   DAYIRIVL   engulfment and cell motility 1 isoform 1;   signal transduction; cell motility   31               ced-12 homolog 1               □   ILDPHVVLL   nucleoporin 88 kDa   transporter activity; nuclear pore   32                   transport               □   DAKIRIFDL   laminin receptor homolog or   ribosome constituent   33               ribosomal protein L10               □   ALLDKLYAL   brms2 or mitochondrial ribosomal protein   RNA binding; ribosome constituent   34               S4 or               □   FMFDEKLVTV   serine/threonine protein phosphatase   hydrolase activity; manganese ion binding   35               catalytic subunit               □   SLAQYLINV   hnRNP E2   DNA binding; RNA binding   36               □   SLLQTLYKV   Similar to RAN GTPase activating protein   GTPase activator activity; signal   37               1   transducer               □   YMAELIERL   Geminin   cell cycle; DNA replication inhibitor   38               □   FLYLIIISY   HIV-1 TAR RNA-binding protein B   no description   39               □   SLLENLEKI   hnrnpC1/C2   MHC gene product   40               □   FLFNKVVNL   yippee protein   no description   41               □   VLWDRTFSL   STAT-1   transcription factor activity; signal   42                   transduction               □   SLASVFVRL   Similar to histone deacetylase 4   no description   43               □   FLMDFIHQV   Nuclear pore complex protein Nup133   transporter activity; nuclear pore   44               (Nucleoporin Nup133)   transport               □   FLWDEGFHQL   glucosidase I   carbohydrate metabolism   45               □   TALPRIFSL   TAP   ABC transporter   46               □   KLWEMDNMLI   T-cell activation protein   ribosome constituent   47               □   MVDGTLLLL   HLA-E leader sequence   MHC gene product   48               □   SLLDEFYKL   membrane component, chromosome 11, surface   integral to plasma membrane   49               marker 1               □   YLLPAIVHI   P68 RNA helicase   ATP binding; RNA binding; RNAhelicase   50                   activity               □   SLASLHPSV   PLAG-LIKE 1 or ZAC delta 2 protein or   nucleic acid binding; zinc ion binding   51               zinc finger protein or lost on               transformation LOT1               □   KLWDIINVNI   steroid-dehydrogenase like   oxidoreductase activity; metabolism   52               □   KYPENFFLL   protein phosphatase I   protein phosphatase activity   53               □   YLLIEEDIRDLAA   TdT binding protein   TdT binding   54               □   DELQQPLEL   signal transducer and activator of   transcription factor acivity; signal   55               transcription 2; signal transducer and   transduction               activator of transcription 2, 113 kD;               interferon alpha induced transcriptional               activator               □   DEYEKLQVL   Dynein heavy chain, cytosolic (DYHC)   ATP binding; nucleic acid binding;   56               (Cytoplasmic dynein heavy chain 1)   mitotic spindle assembly               (DHC1)               □   EEYQSLIRY   Protein CGI-126 (Protein HSPC155)   ubiquitin-conjugating enzyme activity   57               □   DDWKVIANY   c-myb protein   DNA binding   58               □   DELLNKFV   adaptor-related protein complex 2, alpha   protein transporter   59               1 subunit isoform 1; adaptin, alpha A;               clathrin-associated/assembly/adaptor               protein               □   DEFKVVVV   COPG protein   vesicle coat complex   60               □   LEGLTVVY   CGI-120 protein; likely ortholog of mouse   protein transporter activity   61               coatomer protein complex, subunit zeta 1               □   VEEILSVAY   RNA helicase II/Gu protein   ATP binding; RNA binding   62               □   DEDVLRYQF   cyclophilin 60 kDa; peptidylprolyl   isomerase activity; protein   63               isomerase-like 2 isoform b; cyclophilin-   folding               like protein CyP-60; peptidylprolyl cis-               trans isomerase;               □   DEGTAFLVY   butyrylcholinesterase precursor   enzyme binding; hydrolase   64                   activity               □   MEQVIFKY   ARP3 actin-related protein 3 homolog;   constituent of cytoskeleton; cell   65               ARP3 (actin-related protein 3, yeast)   motility               homolog               □   NEQAFEEVF   replication protein A1, 70 kDa; replication   DNA binding; DNA recombination   66               protein A1 (70 kD)               □   VEEYVYEF   heat shock 105 kD; heat shock 105 kD   ATP binding; chaperone activity   67               alpha; heat shock 105 kD beta; heat shock               105 kDa protein 1               □   DEIQVPVL   rab3-GAP regulatory domain   GTPase activator; intracellular protein   68                   transporter               □   DEYQFVERL   mitochondrial ribosomal protein L49;   structural constituent of ribosomes   69               neighbor of FAU; next to FAU               [ Homo sapiens ]               □   DEYSIFPQTY   ras-related GTP-binding protein   GTP binding; signal tranducer   70               □   DEYSLVREL   talin   actin binding; cytoskeleton   71               □   EEVETFAF   HSP 90   chaperone activity   72               □   NENDIRVMF   elav-type RNA-binding protein; RNA-   RNA binding; RNA processing   73               binding protein BRUNOL3               □   DEYDFYRSF   polymyositis/scleroderma autoantigen 2,   RNA binding; hydrolase activity   74               100 kDa; autoantigen PM-SCL;               polymyositis/scleroderma autoantigen 2               (100 kD)               □   DEFQLLQAQY   AES-1 or AES-2   transcription factor activity   75               □   DEFEFLEKA   zinc finger protein 147 (estrogen-   transcription factor activity   76               responsive finger protein)               □   DEMKVLVL   beta-fodrin   actin binding   77               □   DERVFVALY   similar to source of immunodominant MHC-   no description   78               associated peptides               □   IENPFGETF   integral inner nuclear membrane protein   integral to inner nuclear membrane   79               □   SEFELLRSY   sorting nexin 4   protein transporter; intracellular   80                   signalling               □   DEGRLVLEF   Acyl-coA/cholesterol acyltransferase   no description   81               □   DEGWFLIL   RNA helicase family   ATP binding; nucleic acid binding;   82                   hydrolase activity               □   DEISFVNF   structure specific recognition protein 1;   DNA binding; transcription regulator   83               recombination signal sequence recognition   activity               protein; chromatin-specific transcription               elongation factor 80 kDa subunit               □   SEVLSWQF   signal transducer and activator of   transcription factor activity; signal   84               transcription-1;   transduction               □   YEILLGKATLY   T cell receptor beta-chain   MHC binding; receptor activity   85               □   YENLLAVAF   unnamed protein product   protein modification   86               □   DETQIFSYF   nucleolar phosphoprotein Nopp34   RNA binding; protein binding   87               □   MEPLRVLEL   DNA methyltransferase 2 isoform d; DNA   DNA binding; DNA methylation   88               methyltransferase-2; DNA methyltransferase               homolog HsaIIP; DNA MTase homolog HsaIIP               □   MPLGKTLPC   laminin   protein binding; structural molecule   89                   activity               □   VYMDWYEKF   U5 snrnp 200 kDa helicase   ATP binding; nucleic acid binding; RNA   90                   splicing               □   SELLIHVF   protein kinase c-iota   ATP binding; protein binding   91               □   DEHLITFF   U5 snrnp 200 kDa helicase   ATP binding; nucleic acid binding; RNA   92                   splicing               □   DEFKIGELF   DNA-PKcs   DNA binding; transferase activity   93               □   DELEIIEGMKF   (Heat shock protein 60) (HSP-60)   ATP binding; chaperone activity   94               □   KYLLSATKLR   melanoma-derived leucine zipper, extra-   no description   95               nuclear factor               □   SEIELFRVF   U5 small nuclear ribonucleoprotein 200   ATP binding; nucleic acid binding; RNA   96               kDa helicase   splicing               □   LEDVLPLAF   HP1-BP74   DNA binding; nucleosome assembly   97                  
 
         [0060]     In order to provide an analysis of peptides after HIV-infection under as-close-as possible conditions as those that would occur inside an infected person, a human T cell line was utilized for infection with HIV. This cell line, Sup-T1, possesses its own class I; HLA-A and -B types are A*2402, A*6801, B*0801, and B*1801. Although only the soluble class I specifically introduced into the cell should be secreted, under some conditions shedding of full-length class I molecules has been observed. It is believed that HLA-B*1801 is shed after HIV infection.  
         [0061]     Analysis of soluble A*0201 produced a number of ligands that did not appear to fit the A*0201 peptide motif (an indication of which amino acids are preferred at particular positions of the peptide). For instance, A*0201 prefers peptides with an L at position 2 (P2) and an L or V at P9. Most of the peptides that did not match the A*0201 motif had an E at P2 and a Y or F at P9.  
         [0062]     Upon inspection, these peptides were most likely derived from B*1801. To confirm, several peptides from B*1801 molecules in a class I negative cell line were sequenced, and several overlapping peptides were identified. Therefore, at this point, the peptides are labeled as either A*0201 or B*1801 restricted. Tests are currently being performed to delineate which of the two molecules binds each peptide. However, simple analysis of the peptide sequence (P2 and P9 amino acids) should be sufficient to determine the restricting molecule, and such simple analysis is within the ability of a person having ordinary skill in the art.  
         [0063]     The methodology used herein is to use sHLA to determine what is unique to unhealthy cells as compared to healthy cells. Using sHLA to survey the contents of a cell provides a look at what is unique to unhealthy cells in terms of proteins that are processed into peptides. The data summarized in TABLE I shows that the epitope discovery technique described herein is capable of identifying sHLA bound epitopes and their corresponding source proteins which are unique to infected/unhealthy cells.  
         [0064]     Likewise, peptide ligands presented in individual class I MHC molecules in an uninfected cell that are not presented by individual class I MHC molecules in an uninfected cell can also be identified. The peptide “GSHSMRY” (SEQ ID NO:98), for example, was identified by the method of the present invention as being an individual class I MHC molecule which is presented in an uninfected cell but not in an infected cell. The source protein for this peptide is MHC Class I Heavy Chain, which could be derived from multiple alleles, i.e., HLA-B*0702 or HLA-G, etc.  
         [0065]     The utility of this data is at least threefold. First, the data indicates what comes out of the cell with HLA. Such data can be used to target CTL to unhealthy cells. Second, antibodies can be targeted to specifically recognize HLA molecules carrying the ligand described. Third, realization of the source protein can lead to therapies and diagnostics which target the source protein. Thus, an epitope unique to unhealthy cells also indicates that the source protein is unique in the unhealthy cell.  
         [0066]     The methods of epitope discovery and comparative ligand mapping described herein are not limited to cells infected by a microorganism such as HIV. Unhealthy cells analyzed by the epitope discovery process described herein can arise from virus infection or also from cancerous transformation. Unhealthy cells may also be produced following treatment of healthy cells with a cancer causing agent, such as but not limited to, nicotine, or by a disease state cytokine such as IL4. In addition, the status of an unhealthy cell can also be mimicked by transfecting a particular gene known to be expressed during viral infection or tumor formation. For example, particular genes of HIV can be expressed in a cell line as described (Achour, A., et al.,  AIDS Res Hum Retroviruses,  1994. 10(1): p. 19-25; and Chiba, M., et al.,  CTL. Arch Virol,  1999. 144(8): p.1469-85, all of which are expressly incorporated herein by reference) and then the epitope discovery process performed to identify how the expression of the transferred gene modifies epitope presentation by sHLA. In a similar fashion, genes known to be upregulated during cancer (Smith, E. S., et al.,  Nat Med, . 2001. 7(8): p. 967-72, which is expressly incorporated herein by reference) can be transferred in cells with sHLA and epitope discovery then completed. Thus, epitope discovery with sHLA as described herein can be completed on cells infected with intact pathogens, cancerous cells or cell lines, or cells into which a particular cancer, viral, or bacterial gene has been transferred. In all these instances the sHLA described here will provide a means for detecting what changes in terms of epitope presentation and the source proteins for the epitopes.  
         [0067]     The methods of the present invention have also been applied to identifying epitopes unique or upregulated in influenza infected cells as well as West Nile virus infected cells. The methods for obtaining soluble HLA form cells infected with Influenza and West Nile Virus (WNV) are similar to those described hereinabove for HIV infection, except as described herein below. During the course of both the Influenza and WNV infection in the bioreactor, the viral infection was monitored to ensure that the cells secreting the HLA molecules were infected. For Influenza, this was accomplished by measuring intracellular infection using antibody staining combined with flow cytometry. For West Nile virus (WNV), this was accomplished by: (1) measuring viral titer in supernatant using reverse transcriptase real-time PCR; and/or (2) measuring intracellular infection using antibody staining and fluorescence in situ hybridization combined with flow cytometry.  
         [0068]     Table II lists unique and upregulated peptide epitopes that have been identified by the A*0201 and B*0702 class I MHC molecules in cells infected with the PR8 strain of influenza A virus.  
         [0069]     Table III lists unique peptide epitopes that have been identified by the A*0201 class I MHC molecules in cells infected with the West Nile virus. Both self and viral epitopes have been identified.  
                                                                                                                                                     TABLE II                           Peptides Identified on Influenza-Infected Cells.                    SEQ                    Fold       ID                Peptide   Source Protein   Increase   Gene   NO:                    PR8 A0201                NDHFVKL   Uracil DNA glycosylase/   7.75   GAPDH   99               GAPDH               GLMTTVHAIT   Uracil DNA glycosylase/   2.5   GAPDH   100           GAPDH               ALNDHFVKL   Uracil DNA glycosylase/   23.02   GAPDH   101           GAPDH               RLTPKLMEV   eIF3-gamma   2.2   EIF3S3   102               KLEEIIHQI   Hypothetical protein   2.08       103               KLLEGEESRISL   Vimentin   2.1   VIM   104               ALNEKLVNL   eIF3-epsilon   1.52   EIF3S5   105               LLDVPTAAV   GILT   5.18   IF130   106               AVGKVIPEL   Uracil DNA glycosylase/   12.46   GAPDH   107           GAPDH               GLMTTVHAITA   Uracil DNA glycosylase/   3.2   GAPDH   108           GAPDH               TLAEVERLKGL   U2 snRNP   Unique   SNRPA1   109               GLMTTVHAITATQ   Uracil DNA glycosylase/   Unique   GAPDH   110           GAPDH               GVLDNIQAV   Histone   Unique   HIST1H2AE   111               ALDKATVLL   Programmed cell death 4   2.13   PDCD4   112           isoform 2               KVPEWVDTV   Ribosomal protein S19   5.94   RPS19   113               KMLEKLPEL   ABCF3 protein   2.14   ABCF3   114               FLGRINEI   Suppressor of K+ transport   1.99   CLPB   115           defect-3               GLIEKNIEL   DNA methyl transferase   1.58   DNMT1   116               KVFDPVPVGV   DEAH box polypeptide 9   1.74   DHX9   117               GLMTTVHAITAT   Uracil DNA glycosylase/   Unique   GAPDH   118           GAPDH               FAITAIKGV   ribosomal protein S18   3.49   RPS18   119               SMTLAIHEI   Sphingolipid delta 4   2.11   DEGS1   120           desaturase protein DES1               LLDANLNIKI   KIAA0999   2.78       121               TLWDIQKDLK   Lactate dehydrogenase   1.64   LDHB   122               KMYEEFLSKV   c-AMP dependent protein   1.8   PRKAR1B   123           kinase type 1 β regulatory           subunit               FLASESLIKQIPR   Ribosomal Protein L10a   Unique   RPL10A   124               KLFDDDETGKISF   Caltractin   Unique   CETN2   125               SLDQPTQTV   eIF3 subunit 8   9.84   EIF3S8   126               GIDSSSPEV   poly(rc) binding protein   Unique   PCBP1   127               KAPPAPLAA   Inner nuclear membrane   Unique   MAN1   128           protein               ILDKKVEKV   HSP90   Unique   HSP90AB1   129               KLDEGNSL   DNA topisomerase II   4.32   TOP2A   130               VVQDGIVKA   Peroxiredoxin 5   Unique   PRDX5   131               ALGNVRTV   Unknown protein           132               YLEAGGTKV   Homolog of yeast mRNA           133           Transport Regulator               ALSDGVHKI   Fas apoptotic inhibitory   1.88   FAIM   134           molecule               GLAEDSPKM   Chromosome 17 open reading   2   c17orf27   135           frame 27               EAAHVAEQL   MHC A2 antigen           136               AQAPDLQRV   Nol1       NOL1   137               GVYGDVHRV   Rod 1 regulator of   2.9   ROD1   138           differentiation               YLTHDSPSV   sNRPC       snRPC   139               RLDDVSNDV   Heat repeat containing 2   2.55   HEATR2   140               KLMELHGEGSS   Ribosomal protein S3A   Unique       141               KMWDPHNDPNA   U1 small ribonucleoprotein   Unique   SNRP70   142           70 kDa               ALSDGVHKI   Fas apoptotic inhibitory   2.36   FAIM   143           molecule               KLDPTKTTL   n-Myc downstream regulated   2.93   DRG1   144           gene 1               RVPPPPPIA   hnRPC   6.54   HNRPC   145               FIQTQQLHAA   Pyruvate kinase   Unique   PKM2   146               SLTGHISTV   Pleiotropic Regulator 1   3.12   PLRG1   147               KIAPNTPQL   Pm5 protein   2.63   PM5   148               NLDPAVHEV   ATP(GTP) binding protein       XAB1   149               NMVAKVDEV   Ribosomal protein L10a           150               YLEDSGHTL   Peroxiredoxin 4       PRDX4   151               TLDEYTTRV   Nuclear respiratory factor   3.74   NRF1   152           1               TLYEHNNEL   AAAS       AAAS   153               GLATDVQTV   Proteasome subunit HsC 10-II   3.5   PSMB3   154               QLLGSAHEV   Non-erythroid alpha-spectrin   4.98   SPTAN1   155               GLDKQIQEL   ATP dependent 26s proteasome   4.09   PSMC3   156           regulatory subunit               YAYDGKDYIA   MHC-B antigen   1.6       157               AVSDGVIKV   Cofilin 1   8.98   CFL1   158               VLEDPVHAV   Hypothetical protein   3.91       159               VMDSKIVQV   Karyopherin alpha 1   22.84   KPNA5   160               ILGYTEHQV   GAPDH   23.91   GAPDH   161               SMMDVDHQI   Chaperonin containing   3.58   CCT5   162           TCP-1 subunit 5               YAYDGKDYI   MHC-B antigen   Unique       163               LMTTVHAITAT   GAPDH   Unique   GAPDH   164               AIVDKVPSV   Coatomer protein complex   1.88   COPG   165           subunit gamma 1               SLAKIYTEA   H1 histone family member X   5.38   H1FX   166               SMLEDVQRA   RNA binding motif protein   2.4   RBM28   167           28               VLLSDSNLHDA   Cytokine induced apoptosis   10.95   CIAPIN1   168           inhibitor 1               YLDKVRALE   Keratin   Unique   KRT1   169               LLDVVHPA   TCP-1   33.09   CCT7   170               LLDVVHPAA   TCP-1   3.43   CCT7   171               ALASHLIEA   EH domain containing 2   1.67   EHD2   172               ALMDEVVKA   Phosphoglycerate kinase   2.59   PGK1   173               ILSGVVTKM   Ribosomal protein S11   1.74   RPS11   174               ILMEHIHKL   Ribosomal protein L19   5.46   RPL19   175               YMEEIYHRI   Farnesyl-diphosphate   3.98   FDFT1   176           farnesyltransferase               FLLEKGYEV   GDP-mannose-4,6-   1.81   GMDS   177           dehydratase               TLLEDGTFKV   NmrA-like family domain   1.67   NMRAL1   178               GLGPTFKL   BBS1 protein   Unique   BBS1   179               GLIDGRLTI   SPCS2 protein   1.67   SPCS2   180               ALDEKLLNI   CPSF   1.61   CPSF3   181               VLMTEDIKL   eIF4G   1.69   EIF4G   182               SLYEMVSRV   SSRP1   1.87   SSRP1   183               TLAEIAKVEL   p54nrb   3.32   NONO   184               GLDIDGIYRV   ARHGAP12 protein   1.95   ARHGAP12   185               LLLDVPTAAVQA   GILT   6.24   IF130   186               AIIGGTFTV   ERGIC1   4.17   ERGIC1   187               GMASVISRL   Tubulin gamma complex   Unique   TUBGCP2   188           associated protein 2               TIAQLHAV   Unknown protein   Unique       189               RLWPKIQGL   Unknown protein   Unique       190               ALQELLSKGL   similar to 40s ribosomal   2.8   RPS25   191           protein s25               TLWGIQKEL   Lactate dehydrogenase   3.27   LDHA   192               TLWPEVQKL   STATIP1 (signal transducer   2.97   STATIP1   193           and activator of           transcription 3           interacting protein 1)               FLFNTENKL   Isopentenyl-diphosphate-   1.85   IDI1   194           delta-isomerase 1               ALLSAVTRL   Alpha catenin   Unique   CTNNA1   195               SLLEKSLGL   eukaryotic translation   1.64   EEF1E1   196           elongation factor 1           epsilon 1               KIADFGWSV   Aurora kinase C   2.26   AURKC   197               KLQEFLQTL   Unknown protein   2.3       198               ALWEAKEGGLL   Hypothetical protein   1.54       199               KLIGDPNLEFV   Ras-related nuclear   2.82   RAN   200           protein               GLIENDALL   Unknown protein   1.71       201               GLAKLIADV   Flap structure-specific   2.91   FEN1   202           endonuclease 1               TLIGLSIKV   Hypothetical protein   2.28       203               LLLDVPTAAV   GILT   1.95   IF130   204               IMLEALERV   SNRPG   1.64   SNRPG   205               TLIDLPGITKV   Dynamin   6.48   DNM2   206               ALLAGSEYLKL   eIF3 zeta   1.51   EIF3S7   207               KIIDEDGLLNL   replication factor C Irg   1.56   LLDBP   208           subunit               TLQEVFERATF   Nucleolin   Unique   NCL   209               RLIDLGVGL   Hypothetical protein   2.03       210               GIVEGLMTTV   Uracil DNA glycosylase   3.1   HNG   211               SMPDFDLHL   AHNAK nucleoprotein   1.83   AHNAK   212           isoform 1               VLFDVTGQVRL   Major vault protein   2.48   MVP   213               FLAEEGFYKF   Integral membrane protein   2.98   STT3A   214           1               ALVSSLHLL   Coatomer protein complex   1.51   IMP3   215           subunit gamma 1               ALLDKLYAL   U3 snoRNP protein 3   3.1       216           homolog               GMYVFLHAV   ORMDL1 protein   2.73   ORMLD1   217               AMIELVERL   DIPB protein   1.81   TRIM44   218               VINDVRDIFL   TFIIA   1.71   GTF2A1   219               FMFDEKLVTV   Protein phosphatase 6   1.99   PPP6C   220               GVAESIHLWEV   WDR18   2.89   WDR18   221               GMYIFLHTV   ORM1-like 3   2.32   ORMDL3   222               GLLDPSVFHV   Noc4L protein   2.17   NOC4L   223               GLWDKFSEL   human retinoic acid   2.59   RARB   224           receptor gamma bound               KLLDFGSLSNL   40s ribosomal protein S17   3.57   RPS17   225               RLYPWGVVEV   Septin 2   2.79   (SEPT2)   226               KLFPDTPLAL   ILF3   Unique   ILF3   227               GLQDFDLLRV   Protein kinase C iota   2.29       228               ILYDIPDIRL   Phenylalanyl-tRNA   5.99   FARS1   229           synthetase alpha chain               LLDVTPLSL   HSP 70   9.68   HSPA2   230               TLAKYLMEL   Cyclin B1   6.81       231               ALVEIGPRFVL   Brix   10.83   BRIX   232               GIWGFIKGV   Hypothetical protein   6.1       233               ILCPMIFNL   Unamed protein product   2.51       234               FLPSYIIDV   CPSF-1   2.57   CPSF1   235               NLAEDIMRL   Vimentin   2.02   VIM   236               YLDIKGLLDV   Skp1   2.44   SKP1A   237               IIMLEALERV   SNRPG   13.68   SNRPG   238               SIIGRLLEV   Protein phosphatase 1   56.92       239           catalytic subunit alpha 1               SLLDIIEKV   Tuberin   2.56   TSC2   240               KIFEMGPVFTL   Cytochrome C oxidase   6.45   COX2   241           subunit II               GVIAEILRGV   Serine   1.56   SHMT2   242           hyroxymethyltransferase               SLWSIISKV   Transmembrane protein 49EG   3.06   TMEM49/   243                   TDC1               SLFEGTWYL   3-hydroxy-3-methylglutaryl   2.36   HMGCS1   244           CoA synthase                    PR8 B0702                RPKANSA   Unknown protein product   1.8       245                   APRPPPKM   Ribosomal protein S26   2.9       246               KPQDYKKR   Catenin beta-1   2.9       247               RPTGGVGAV   Hydroxymethyl glutanyl CoA   2.7       248           synthase               ARPATSL   eIF4G   2.2       249               NLGSPRPL   Tripeptidyl peptidase II   5.6       250               AARPATSTL   eIF4G   5.1       251               RPGLKNNL   Unknown protein product   1.5       252               SPGPPTRKL   c14orf12   1.9       253               IPSIQSRGL   Influenza A/PR8/34   1.6       254           Hemagglutinin               LPFDRTTVM   Influenza A/PR8/34   1.3       255           Nucleoprotein               GPPGTGKTAL   TATA binding protein   1.5   RPS2   256           interacting protein               APRGTGIVSA   RPS2 protein   2.2   RPL8   257               APAGRKVGL   RPL8 protein   1.5   NGRN   258               APGAPPRTL   Mesenchymal stem cell   1.5       259           protein               APPPPPKAL   MHC HLA B associated   2.29   BAG3   260           transcript 2               LPSSGRSSL   BAG family molecular   2   FBXL6   261           chaperone regulator 3       LPKPPGRGV   FBOX protein Fb16   1.9       262               NLPLSNLAI   Phosphatidylinositol   4.3   TYMS   263           phospholipase X domain           containing 2       EPRPPHGEL   Thymidylate Synthase   2.7       264               APNRPPAAL   MHC antigen   1.5   HMGB1   265               APKRPPSAF   HMG213   1.82   TERF2   266               SPPSKPTVL   Telomeric repeat factor 2   1.9   CDKN1C   267               APRPVAVAV   p57 KIP2   1.5   MCL1   268               RPPPIGAEV   MC-1 delta SITM   2.9   CPNE3   269               RPAGKGSITI   Copine III   1.8   GH2   270               SPGIPNPGAPL   hGH-V2 human growth   1.84   RUVBL1   271           factor hormone varient               RPQGGQDIL   TATA binding protein   2.24   ATP5J   272           interacting protein               PKFEVIEKPQA   ATP synthase H+   3.6       273           Transporting mitochondrial           F0 comlex subunit F6           isoform A precursor               VFLKPWI   Hypothetical protein   1.62   SCD   274               ITAPPSRVL   SCD Protein   1.98       275               TPEQIFQN   Hypothetical protein   1.51   TGIF2   276               LPRGSSPSVL   TGFB-induced factor 2   1.57       277               GPREAFRQL   SCAN related protein RAZ   6.03       278               KPVIKKTL   Hypothetical protein   U       279               SPRSGLIRV   glycyl-tRNA synthetase   1.53   SMG1   280               LLPGENINLL   PI-3 kinases related   7.13       281           kinase               HLNEKRRF   HPV-18 E6 Protein   2.02       282               TQFVRFDSD   MHC I antigen   1.64   DYNC1H1   283               RVEPLRNEL   Dynein   1.95       284               YQFTGIKKY   HCV F-Transactivated   2.3   SF3B3   285           Protein 2               GPRSSLRVL   Splicing factor 3B subunit   3.16   HNRPL   286           3               GPYPYTL   Human hnRPL protein   2.01   SND1   287               SPAKIHVF   100 kDA coativator   2.8   SRP9   288               DPMKARVVL   SRP9 protein   1.87       289               SPQEDKEVI   Novel protein   4.19   CLTC   290               NPASKVIAL   Clathrin heavy chain I   1.64       291               RPSGKGIVEF   human mRNA gene product   13.7       292               SPVPSRPL   putative GTP-binding   2.91   ACTG1   293           protein Ray-like variant               APEEHPVLL   Actin-like Protein   1.92       294               SPKIRRL   Similar to putative   1.63   PFKM   295           membrane bound dipeptidase           2               LVFQPVAEL   Phosphofructokinase   4.33   CDADR   296               GPLDIEWLI   Coxsackie-adenovirus   2.2       297           receptor isoform CA R217       RIVPRFSEL   Unknown protein product   1.54   DDX3X   298               YPKRPLLGL   DEAD box polypeptide 24   1.61   UBE2D3   299           variant               YPFKPPKVAF   Ubiquitin conjugating   3.27   RPL12   300           enzyme 1               APKIGPLGL   60s Ribosomal protein L12   1.54       301           LIKE protein                  
 
         [0070]    
       
         
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE III 
               
             
             
               
                   
               
               
                   
               
               
                 Peptides Identified on West Nile Virus 
                   
               
               
                 Infected Cells. 
               
             
          
           
               
                   
                 SEQ 
                   
               
             
          
           
               
                   
                 Fold 
                 ID 
                   
               
             
          
           
               
                 Species 
                 Sequence 
                 Protein 
                 increase 
                 NO: 
               
               
                   
               
             
          
           
               
                 SELF EPITOPES 
                   
               
             
          
           
               
                 Human 
                 AVLDELKVA 
                 carbamoyl-phosphate 
                 Unique 
                 302 
                   
               
               
                   
                   
                 synthase 
               
               
                   
               
               
                 Human 
                 NLMHISYEA 
                 Argininosuccinate 
                 Unique 
                 303 
               
               
                   
                   
                 synthase 
               
               
                   
               
               
                 Human 
                 LLDVPTAA 
                 Ifn-g inducable 
                 Unique 
                 304 
               
               
                   
                   
                 protein 30 Kda 
               
               
                   
               
               
                 Human 
                 FLKEPALNEA 
                 Proteosome 
                 Unique 
                 305 
               
               
                   
                   
                 activaing factor 
               
               
                   
                   
                 PA28 a-chain 
               
               
                   
               
               
                 Human 
                 SLDQSVTHL 
                 Intestinal alkaline 
                 Unique 
                 306 
               
               
                   
                   
                 phosphatase 
               
               
                   
               
               
                 Human 
                 KIVVVTAGV 
                 Lactate 
                 Unique 
                 307 
               
               
                   
                   
                 dehydrogenase B 
               
               
                   
               
               
                 Human 
                 HLIEQDFPGM 
                 HPAST 
                   
                 308 
               
               
                   
               
               
                 Human 
                 FGVEQDVDMV 
                 Pyruvate kinase M2 
                   
                 309 
               
               
                   
               
             
          
           
               
                 Viral Epitopes 
                   
               
             
          
           
               
                 WNV 
                 RLDDDGNFQL 
                 NS2b 
                 Unique 
                 310 
                   
               
               
                   
               
               
                 WNV 
                 ATWAENIQV 
                 NS5 
                 Unique 
                 311 
               
               
                   
               
               
                 WNV 
                 SVGGVFTSV 
                 Env 
                 Unique 
                 312 
               
               
                   
               
               
                 WNV 
                 YTMDGEYRL 
                 NS3 
                 Unique 
                 313 
               
               
                   
               
               
                 WNV 
                 SLTSINVQA 
                 NS4b 
                 Unique 
                 314 
               
               
                   
               
               
                 WNV 
                 SLFGQRIEN 
                 NS4b 
                 Unique 
                 315 
               
               
                   
               
             
          
         
       
     
         [0071]     Thus, in accordance with the present invention, there has been provided a method of epitope discovery and comparative ligand mapping that includes methodology for producing and manipulating Class I and Class II MHC molecules from gDNA as well as methodology for directly discovering epitopes unique to infected or tumor cells that fully satisfies the objectives and advantages set forth herein above. Although the invention has been described in conjunction with the specific drawings, experimentation, results and language set forth herein above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the invention.