Comparative ligand mapping from MHC class I positive cells

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

BACKGROUND OF THE INVENTION

1. Field of the Invention

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.

2. Description of the Background Art

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.

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.

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'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.

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's population are highly desirable for unraveling disease susceptibilities and resistances, as well as for designing therapeutics such as vaccines.

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'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.

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.

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.

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.

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

FIG. 1. Overview of 2 stage PCR strategy to amplify a truncated version of the human class I MHC.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

Class I and class II MHC molecules are really a trimolecular complex consisting of an alpha chain, a beta chain, and the alphabeta chain'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'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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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'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.

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.

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.

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'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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

Claims

1. An isolated peptide ligand for an individual class I molecule, the isolated peptide ligand having a length of from 7 to 13 amino acids and consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 99-315, the isolated peptide ligand isolated by a method comprising the steps of: providing a cell line containing a construct that encodes an individual soluble class I molecule, the cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules; culturing the cell line under conditions which allow for expression of the individual soluble class I molecules from the construct, such conditions 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; isolating the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto; and separating the peptide ligands from the individual soluble class I molecules.

2. An isolated peptide ligand for an individual class I molecule, the isolated peptide ligand having a length of from 7 to 13 amino acids and consisting essentially of a sequence selected from the group consisting of SEQ ID NOS:99-315.

3. An isolated peptide ligand for an individual class I molecule, wherein the isolated peptide ligand is 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, wherein the isolated peptide ligand has a length of from 7 to 13 amino acids and consists essentially of a sequence selected from the group consisting of SEQ ID NOS:99-315.

4. An isolated peptide ligand presented by an individual class I molecule in a substantially greater amount on an infected cell when compared to an uninfected cell, the peptide ligand identified by a method comprising the steps of: providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, the cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules; infecting a portion of the uninfected cell line with at least one of a microorganism, a gene from a microorganism or a tumor gene, thereby providing-an infected cell line; culturing the uninfected cell line and the infected cell line under conditions which allow for expression of the individual soluble class I molecules from the construct, such conditions also allowing 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; isolating the secreted individual soluble class I molecules having the endogenously loaded peptide ligands bound thereto from the uninfected cell line and the infected cell line; separating the endogenously loaded peptide ligands from the individual soluble class I molecules from the uninfected cell and the endogenously loaded peptide ligands from the individual soluble class I molecules from the infected cell; isolating the endogenously loaded peptide ligands from the uninfected cell line and the endogenously loaded peptide ligands from the infected cell line; comparing the endogenously loaded peptide ligands isolated from the infected cell line to the endogenously loaded peptide ligands isolated from the uninfected cell line; and identifying 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.

5. The isolated peptide ligand of claim 4 wherein, in the step of providing an uninfected cell line containing a construct that encodes an individual soluble class I molecule, the uninfected cell line containing the construct that encodes the individual soluble class I molecule is produced by a method comprising the steps of: obtaining genomic DNA or cDNA encoding at least one class I molecule; identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA; PCR amplifying the allele encoding the individual class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule; cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble class I molecule; and transfecting the construct into an uninfected cell line.

6. The isolated peptide ligand of claim 5, wherein the construct further encodes a tag which is attached to the individual soluble class I molecule and aids in isolating the individual soluble class I molecule.

7. The isolated peptide ligand of claim 6, wherein the tag is selected from the group consisting of a HIS tail and a FLAG tail.

8. The isolated peptide ligand of claim 6, wherein the tag is encoded by a PCR primer utilized in the step of PCR amplifying the allele encoding the individual class I molecule.

9. The isolated peptide ligand of claim 6, wherein the tag is encoded by the expression vector into which the PCR product is cloned.

10. The isolated peptide ligand of claim 4, wherein the at least one endogenously loaded peptide ligand is 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.

11. The isolated peptide ligand of claim 4, wherein the at least one endogenously loaded peptide ligand is obtained from a protein encoded by the uninfected cell line.

12. The isolated peptide ligand of claim 4, wherein the portion of the uninfected cell line is infected with influenza.

13. The isolated peptide ligand of claim 4, wherein the portion of the uninfected cell line is infected with West Nile virus.