HK1093084A - Hpv cd8+ t-cell epitopes - Google Patents

Description

HPV CD8+T cell epitopes

Technical Field

[0001]The present invention provides for the identification of functional CD8 in any protein of interest⁺T cell epitope (CD 8)⁺T-cell epitops). The invention further provides CD8 of various proteins⁺T cell epitope (CD 8)⁺T-cell epitops). In some preferred embodiments, the present invention provides CD8 of human papilloma virus (human papillomavir (HPV))⁺A T cell epitope. In further embodiments, the present invention provides epitopes (epitopes) suitable for use in prophylactic and/or therapeutic vaccines. In some particularly preferred embodiments, the invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines.

Background

[0002] Lymphocytes, particularly B cells and T cells, are two major cell types involved in immune responses in humans and other animals. B cells are involved in the humoral aspect of the immune response, responsible for the production of antibodies, while T cells are involved in the cell-mediated aspect of the immune response. However, these two lymphocyte classes (two lymphocytes) work together through a complex network consisting of recognition factors, cytokines and other components of the immune response (elements).

[0003]Within T cells, there are two major cell types, cytotoxic T-cells (Tc) and helper T-cells (Th). Upon activation, cytotoxic T cells kill infected cells, while helper T cells activate other cells, such as B cells and macrophages. Naive T cellsT-cells) are activated upon exposure to a specific antigen, which binds to a component of the major histocompatibility complex and is presented on the surface of antigen presenting cells, to produce "armed" effector T cells. These two major T cells are often described in terms of their cell surface receptors. Cytotoxic T cells are often referred to as "CD 8" ("CD 8)⁺") cells, helper T cells are often referred to as" CD4 "(" CD4)⁺") cells. Despite their different functions, CD4⁺And CD8⁺And do not operate independently of each other. In fact, CD8 is known⁺Cells often rely on CD4⁺The cells initiate a response to the immunogen (immunogen). Thus, in killing infected cells, CD8⁺Cells often require CD4⁺Activation of the cells. Additionally, in some cases, it appears that CD8 is⁺The cells can effectively kill the infected cells; in some cases, these cells are ineffective. However, despite the increasing understanding of the immune response in recent years, there is still a need to be able to reliably identify effective CD8⁺A method for differentiating between a potent epitope and a null epitope.

Summary of The Invention

[0004]The present invention provides for the identification of functional CD8 on any protein of interest⁺T cell epitope (CD 8)⁺T-cell epitope). The invention further provides CD8 of various proteins⁺T cell epitope (CD 8)⁺T-cell epitope). In some embodiments, the present invention provides CD8 of human papilloma virus (human papillomavir (HPV))⁺A T cell epitope. The invention further provides epitopes (epitopes) suitable for use in prophylactic and/or therapeutic vaccines. In some particularly preferred embodiments, the invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines.

[0005]The invention provides for detecting CD8⁺Means for T cells to react in a functional manner. In particular, the invention provides in vitro methods to assess CD8 in the presence of antibodies⁺T cell responses, which mimic in vivo T-cell activation. In some preferred embodiments, the present invention provides a method for identifying the immunogenicity (immunogenicity) of a protein of interest, comprising the steps of: obtaining a protein of interest; preparing a plurality of amino acid fragments of the protein of interest such that each fragment overlaps in sequence with its adjacent fragments; contacting the amino acid fragments of the protein of interest with a peptide comprising naive human CD8⁺Contacting the T cells with a solution of dendritic cells, wherein the dendritic cells have beenIs differentiated (differentiated), and wherein CD8⁺T cells have been exposed to anti-CD 40 antibodies prior to their contact with dendritic cells and peptides; and identifying an epitope region using the amino acid fragments of the protein of interest, wherein the identifying step comprises measuring the stimulation of the naive human CD8 by the epitope region⁺The ability of T cells to proliferate. In some particularly preferred embodiments, the dendritic cells and CD8⁺The cells are obtained from the same blood source. In a further particularly preferred embodiment, in CD8⁺After the T cells, dendritic cells (dendritic cells) and peptides were combined together, anti-CD 40antibody (anti-CF40antibody) was added to the solution.

[0006]The present invention further provides a method for modifying the immunogenicity (immunogenicity) of a protein of interest, comprising the steps of: obtaining a protein of interest; preparing a plurality of amino acid fragments of the protein of interest such that each fragment overlaps in sequence with its adjacent fragments; so as to contain the original human CD8⁺Contacting a solution of T cells and dendritic cells with the amino acid fragments of the protein of interest, wherein the dendritic cells have been differentiated, and wherein CD8⁺The T cells have been exposed to an anti-CD 40antibody prior to their contact with the dendritic cells and the peptide; and identifying an epitope region using the amino acid fragments of the protein of interest, wherein the identifying step comprises measuring the ability of the epitope region to stimulate proliferation of naive human CD8+ T cells; and then modifying the identified epitope region of the protein of interest such that the modified epitope is more or less immunogenic than the original protein of interest. In some embodiments, multiple epitopes are modified. In some particularly preferred embodiments, the dendritic cells and CD8⁺Cells were obtained from a single blood source. In a further particularly preferred embodiment, CD8 is incorporated⁺After the T cells, dendritic cells and peptide are bound together, anti-CD 40antibody is added to the solution.

[0007]In some embodiments, the present invention provides methods and compositions for identifying epitopes of virusesThese viruses include, but are not limited to, HPV. In particular, the invention provides the use of a modified T cell detection system (i.e., the I-MUNE ® assay) to perform CD8 in a variety of viruses, including HPV⁺Identification of T cell epitopes. In further embodiments, the invention provides methods for identifying HPV epitopes in the sequence of various HPV types, as well as methods for producing peptides; when this peptide is introduced into the HPV sequence, it can elicit CD8⁺T cell responses.

[0008]In some embodiments, the invention provides methods for identifying CD8 in HPV sequences⁺T cell epitope method and method capable of triggering CD8⁺Production of T cell reactive peptides. In particular, the present invention provides methods and compositions for increasing the immunogenicity of HPV epitopes suitable for use in the preparation of HPV vaccines.

[0009] In these embodiments, the invention provides methods for determining the response of human T cells against a variety of epitopes, including proteins of interest. In further embodiments, once a significant epitope is identified by using the I-MUNE ® detection system described herein, the significant epitope (significant epitopes) is altered, thereby generating an epitope that can induce an enhanced immune response against the protein.

[0010] Thus, as noted above, the proteins of the invention exhibit a modified immunogenic response (e.g., antigenicity and/or immunogenicity) when compared to the native proteins encoded by their precursor DNA. For example, HPVs that exhibit enhanced immunogenic responses (e.g., variant HPV epitopes) may find use in therapeutic and prophylactic vaccine compositions.

[0011]The invention also provides CD8 in the E7 protein from two strains (human papillomavir (HPV))⁺A T cell epitope. In some preferred embodiments, the present invention provides methods for developing HPV vaccines, in particular for developing multivalent vaccines (multivalent vaccines) that prevent infection by high-risk HPV strains. In further embodiments, the invention provides for the development ofA method of therapeutic vaccine against high risk HPV types, which vaccine is suitable for use in preventing the development of benign and/or malignant tumours in infected individuals. The invention further provides epitopes suitable for use in prophylactic and/or therapeutic vaccines. In some preferred embodiments, the present invention provides a polypeptide consisting of the sequence seq. 1-25. In a particularly preferred embodiment, the present invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines.

[0012] The present invention further provides compositions and methods for developing vaccine compositions directed against the E7 protein of two high-risk HPV strains (i.e., strains 16 and 18).

In some particularly preferred embodiments, the vaccine composition comprises at least one epitope selected from the group consisting of seq id NOS: 1 to 25. In some alternative preferred embodiments, the vaccine composition comprises epitopes selected from at least one high risk HPV strain and/or at least one intermediate risk HPV strain known in the art. Indeed, it can be considered that: the HPV vaccine of the invention is to be applied to the treatment and prevention of a plurality of HPV virus strains. It is not intended that the present invention be limited to any particular epitope and/or vaccine composition comprising any particular epitope. Thus, in various vaccine embodiments of the invention, any combination of epitopes suitable for the intended use may be applied to the invention.

Brief Description of Drawings

[0013] FIG. 1 provides a graph showing the response to each epitope in the test HPV E7.16.

[0014] FIG. 2 provides a graph showing the response to each epitope in the HPV E7.18 tested.

[0015] FIG. 3 provides a graph showing the response to HPV E7.18 in the presence of anti-CD 40antibody and anti-IgG 1 isotype antibody.

[0016] FIG. 4 provides a graph showing the responses of twenty random donors tested in parallel with an enzyme-linked immunospot assay INF-gamma assay (ELISPOT INF-gamma assay) and a CD 8I-MUNE ® assay (CD 8I-MUNE ® assay) using the HPV 18.E7 peptide.

Description of the invention

[0017]The present invention provides for the identification of functional CD8 in any protein of interest⁺T cell epitope (functional CD 8)⁺T-cell epitops). The invention further provides CD8 of various proteins⁺A T cell epitope. In some preferred embodiments, the present invention provides CD8 of Human Papillomavirus (HPV)⁺T cell epitope (CD 8)⁺T-cell epitops). In other embodiments, the invention provides epitopes suitable for use in prophylactic and/or therapeutic vaccines. In particularly preferred embodiments, the invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines. In some particularly preferred embodiments, the invention provides methods for developing vaccines based on T cell epitopes from various strains of microorganisms, including viruses, and for preventing cancer.

[0018]As set forth herein and in 1998 at 4/15, U.S. patent application serial No. 09/060,872; 09/500,135 on 2/2000; 09/768,080, filed on 23/1/2001; and the I-muscle ® assay described in the related application, were developed to identify functional T cell epitopes in any protein of interest. One feature of this assay is the use of cells obtained from social donors, which may include individuals who have not been exposed to the protein of interest. As described in the cited applications and publications, the assay is for CD4 in a variety of proteins⁺The identification of T cell epitopes has met with great success. However, when studying CD8⁺T cell epitopes, the results are generally not reliable (robust). According to the literature, CD8⁺T cell responses often require reliance on CD4⁺T cells "help" them respond. In part, this help involves the use of antigen-presenting cells (APCs)And (4) activating. When CD4⁺APC activation occurs when T cells interact with APC. CD4⁺The interaction between T cells and APCs is mediated, in part, by the CD40 ligand/CD 40 receptor interaction. Thus, in the development of the present invention, the anti-CD 40 monoclonal antibody was tested as activating CD4⁺Alternatives to the presence of T cells are used.

[0019]In addition, it is believed that in some cases, the recovering patient has an immune response against a different epitope than the patient suffering from the chronic disease. Indeed, this condition is manifested in individuals who naturally heal by HCV infection (see Wertheimer et al, hepatol., 37: 577-]) And in having a stronger CD8 despite carrying a high viral load⁺Responding HIV-infected individuals (see Addo et al, J.Virol., 77: 2081-2092[ 2003)]). Thus, identification of epitopes from normal healthy donors, as described herein, could be potentially useful and non-useful for CD8⁺The application of the epitope identification.

[0020]As described herein, in CD8⁺In a repeat experiment (replicate) of the I-MUNE ® assay, anti-CD 40 monoclonal antibody was tested to evaluate its ability to detect CD8⁺Effects of proliferative responses of T cells. As described in more detail in the examples, a narrow concentration range (in vitro) was found to be effective. As indicated, CD8 was upregulated in vitro by the use of anti-CD 40 antibodies⁺Proliferation and IFN-gamma secretion. However, as also described herein, many other antibodies were tried with similar experiments, but they did not induce CD8⁺T cell activation.

[0021]Functional CD8⁺Other methods of T cell epitope identification rely on cells from donors that carry a memory immune response. In these assays, Peripheral Blood Mononuclear Cells (PBMCs) are used, either cultured in vitro prior to use or cloned. Indeed, most current methods for identifying HPV and other Major Histocompatibility Complex (MHC) class I epitope peptides rely on verification of the peptide from a verified sourceThe use of a peripheral blood source of exposed donors who possess an abundance of antigen-specific CD8⁺T cells. For these enriched populations, tetramer staining (tetramerstating) and proliferation methods can be used. Indeed, tetramer analysis using peptide sets in combination with Elispot analysis was used to discover epitope responses to ensure CD8⁺T cells are functional (see, e.g., Terajima et al, J.exp.Med., 197: 927-932[2003 ]]). However, the use of tetramers is somewhat limited because only a few type I constructs are currently available (see Sato et al, J. immunological. meth., 271: 177-184[2002 ]](ii) a And Altman et al, Science 274: 94-96[1996]Reconnaissance in Science 280: 1821[1998])。

[0022]In addition, for CD8⁺Many predictive algorithms for T cell epitopes are known, some of which appear to be very efficient and accurate (see Nussbaum et al, curr. opin. immunol., 15: 69-74[2003 ]]). In some embodiments, these computer algorithms are used to predict MHC class I binding. However, no complete solution is provided by these methods, since the predictive epitopes identified on the basis of computer algorithms must also be functionally validated at the same time.

[0023] The present invention provides significant advantages over currently used methods because the present assay uses unexposed cell donors and does not rely on computer algorithms to assess the relationship between epitopes and immune responses. Importantly, the present invention provides methods that can functionally validate the results obtained from each epitope and sample.

Definition of

[0024] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology second edition, John Wiley and Sons, NY (1994); hale and Marham, The HarperCollins Dictionary of Biology, Harper Perennail, NY (1991), provide those of ordinary skill in The art with a comprehensive Dictionary of many of The words used in The present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. The words defined below are therefore more fully described by reference to the specification as a whole. Also, as used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In order that the invention may be more readily understood, a number of terms are defined below.

[0025] As used herein, "HPV" and "human papillomavirus (humanpapillomavir)" refer to members of the genus papillomavirus (genusapapillomavir) that have the ability to infect humans. There are two major classes of HPV (i.e. reproductive and cutaneous) each containing multiple viral "types" or "strains" (such as HPV16, HPV18, HPV31, HPV32, etc.). Of particular interest in the present invention are HPV types associated with reproductive infections and malignancies.

[0026] As used herein, "prophylactic" and "prophylactic" vaccines refer to vaccines designed and administered to prevent infection, disease and/or any associated sequelae caused by or associated with pathogenic organisms, particularly HPV.

[0027] As used herein, a "therapeutic" vaccine refers to a vaccine designed and administered to a patient already infected with a pathogenic organism, such as by at least one strain of HPV virus. Therapeutic vaccines (e.g., HPV therapeutic vaccines) are used to prevent and/or treat the development of benign or malignant tumors in these infected individuals.

[0028] As used herein, "antigen presenting cells" (APCs) refer to cells of the immune system that present antigens on their surface. This antigen is recognized by T cells. Dendritic cells, inter-dendritic cells, activated B cells and macrophages are examples of antigen presenting cells.

[0029] The term "lymphoid", when used in reference to a cell line or cell, means that the cell line or cell is derived from the lymphoid lineage (line) and includes cells of the B and T lymphocyte lineages.

[0030]As used herein, the terms "T lymphocyte (Tlymphocyte)" and "T cell (T-cell)" include reference to a precursor T cell (including a TCR without a rearranged T cell receptor)]Gene Thy positive cells) to mature T cells (i.e., to CD4)⁺Or CD8⁺Single positive, surface TCR positive cells) of any cell belonging to the T lymphocyte lineage.

[0031]As used herein, the terms "B lymphocyte (Blymphocyte)" and "B cell (B-cell)" include cells derived from B cell precursors, such as pre-B cells (B220 which begin rearranging Ig heavy chain genes)⁺Cells) to mature B cells and plasma cells.

[0032]As used herein, "CD 4⁺T cell (CD 4)⁺T-cell) "and" CD4T cell (CD4T-cell) "refer to helper T cells, and" CD8⁺T cell (CD 8)⁺T-cell) "and" CD8T cell (CD 8T-cell) "refer to cytotoxic T cells.

[0033] As used herein, "B-cell proliferation" refers to an increase in the number of B cells produced during the culture of B cells with or without antigen-presenting cells.

[0034] As used herein, "baseline B-cell proliferation," as used herein, refers to the degree of B-cell proliferation; in the absence of peptide or protein antigens, B cell proliferation in response to exposure to antigen presenting cells is typically observed in individuals. For purposes herein, a baseline level of B cell proliferation is determined as B cell proliferation in the absence of antigen, and is determined on a per sample basis for each individual.

[0035] As used herein, "B-cell epitope" refers to a characteristic of a peptide or protein that is recognized by a B-cell receptor in an immune response to a peptide (i.e., an immunogen) containing the antigen.

[0036] As used herein, "altered B-cell epitope" refers to an epitope amino acid sequence that differs from the precursor peptide or peptide of interest such that the variant peptide of interest produces a different (i.e., altered) immunogenic response in humans or other animals. Altered immunogenic responses including altered immunogenicity and/or allergenicity (i.e., increased or decreased overall immunogenic response) are also contemplated. In some embodiments, the altered B cell epitope comprises a substitution and/or deletion of an amino acid selected from those residues located within the identified epitope. In alternative embodiments, the altered B cell epitope comprises the addition of one or more residues to the epitope.

[0037] As used herein, "T-cell epitope (T-cell epitope)" refers to a characteristic of a peptide or protein that is recognized by a T-cell receptor during the initiation of an immunogenic response to the peptide containing the antigen. T cell recognition of T cell epitopes is generally thought to occur by a mechanism in which T cells recognize antigenic peptide fragments that bind to class I or class II Major Histocompatibility Complex (MHC) molecules expressed on antigen presenting cells (see Moeller (ed), Immunol. Rev., 98: 187[1987 ]). In some embodiments of the invention, the epitopes or epitope fragments identified as described herein are used to detect cellular antigen presenting cells having MHC molecules that can bind to or display the epitope or fragment. In some embodiments, the epitope/epitope fragment further comprises a detectable label (i.e., a label) that facilitates identification of the cell that binds to or displays the epitope/epitope fragment of interest.

[0038] As used herein, "T-cell proliferation" refers to the number of T-cells produced during T-cell culture with or without antigen presenting cells.

[0039] As used herein, "baseline T-cell proliferation" refers to the degree of T-cell proliferation, which is typically observed in an individual in response to exposure to antigen presenting cells in the absence of peptide or protein antigens. For purposes herein, a baseline level of T cell proliferation is determined as T cell proliferation in response to antigen presenting cells in the absence of antigen, and is determined on a per sample basis for each individual.

[0040] As used herein, "altered immunogenic response" refers to an increased or decreased immunogenic response. Proteins and peptides exhibit an "increased immunogenic response" when they elicit a greater T cell and/or B cell response than the response elicited by their parent (e.g., precursor) protein or peptide (e.g., a protein of interest). Generally, the net result of this higher response is an increased antibody response directed against the anti-variant protein or peptide. Proteins and peptides exhibit a "reduced immunogenic response" when the T cell and/or B cell response elicited by the protein or peptide is less than the response elicited by its parent (e.g., precursor) protein or peptide. In some embodiments, the net result of this lower response is a reduction in antibody responses directed against the variant protein or peptide. In some preferred embodiments, the parent protein is a wild-type protein or peptide.

[0041] With respect to a particular amino acid sequence, an "epitope" refers to a group of amino acid residues that are involved in recognition by a particular immunoglobulin or, in the context of T cells, are essential for recognition by a T cell receptor protein and/or Major Histocompatibility Complex (MHC) receptor. In the context of the immune system, whether in vivo or in vitro, epitopes are a collective feature of molecules, such as primary, secondary and tertiary peptide structures, and charges, which together form sites recognized by immunoglobulins, T cell receptors or HLA molecules. Throughout this disclosure, "epitope" and "peptide" may be used interchangeably.

[0042] As used herein, the term "major epitope" refers to an epitope (i.e., a T cell and/or B cell epitope) in which the response rate in the measured donor pool is at least three standard deviations above the average background response rate.

[0043] As used herein, the term "moderate epitope" refers to an epitope (i.e., a T cell and/or B cell epitope) in which the rate of response in the measured donor pool exceeds the mean by at least two standard deviations or by at least three times the background value.

[0044] As used herein, the term "minor epitope" refers to an epitope (i.e., a T cell and/or B cell epitope) in which the rate of response in the measured donor pool is at least twice that of the background.

[0045] As used herein, the term "significant epitope" refers to an epitope (i.e., a T cell and/or B cell epitope) wherein the rate of response in the donor pool being tested is equal to or greater than about three times the background rate of response.

[0046] As used herein, "weakly significant epitope" refers to an epitope (i.e., a T cell and/or B cell epitope) in which the rate of response in the donor pool being tested is greater than the background rate of response, but less than about three times the background rate of response.

[0047] As used herein, "background level" and "background response" refer to the average percentage of a responder to any particular peptide in a data set of proteins being tested. This value was determined by averaging the percent of responders for all peptides in the group, which was summarized based on all donors tested. For example, a background response of 3% indicates: when the experiment was performed on 100 donors, there were an average of three positive (SI greater than 2.95) responses to any peptide in the data set.

[0048] As used herein, the term "sample" is used in its broadest sense. However, in a preferred embodiment, the term is used to refer to a sample (e.g., an aliquot) containing a peptide (i.e., a peptide within a pepset comprising the sequence of a protein of interest) that is analyzed, identified, modified, and/or (or) compared to other peptides. Thus, in most cases, the term is used to refer to a substance that includes a protein or peptide of interest.

[0049] As used herein, "protein of interest" refers to a protein that is analyzed, identified, and/or modified. Naturally occurring as well as recombinant proteins, synthetically prepared, variants and derived proteins find use in the present invention.

[0050] As used herein, "protein" refers to any composition comprising amino acids and recognized as a protein by one of ordinary skill in the art. The terms "protein", "peptide" and polypeptide (polypeptide) are used interchangeably herein. Amino acids may be referred to by their full name (e.g., alanine), or by a commonly recognized letter (e.g., a), or by a three-letter abbreviation (e.g., ala). Where peptides are part of a protein, those of ordinary skill in the art understand this concept used in context. The expression "protein" encompasses the mature form of the protein, as well as the precursor (pro-) and prepro-precursor forms (prepro-) of the protein of interest. The prepro form of a protein comprises the mature form of a protein having a prepro sequence operably linked to the amino terminus of the protein, and a "prepro" or "signal" sequence operably linked to the amino terminus of the prepro sequence.

[0051] As used herein, functionally similar proteins are considered "related proteins". In some embodiments, these proteins are derived from different genera and/or species, including differences between classes of organisms such as bacterial proteins and fungal proteins). In other embodiments, the related proteins are from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source.

[0052] As used herein, the term "derivative" refers to a protein derived from a precursor protein in such a way that: adding one or more amino acids to either or both of the N-and C-termini, substituting one or more amino acids at one or more different positions in the amino acid sequence, and/or deleting one or more amino acids at either or both ends of the protein or at one or more positions in the amino acid sequence, and/or inserting one or more amino acids at one or more positions in the amino acids. The preparation of the protein derivatives is preferably obtained by: modifying a DNA sequence encoding a native protein, transforming the DNA sequence into a suitable host, and forming a derivative protein by modifying expression of the DNA sequence.

[0053] A "variant protein" is a type of related protein (and derived proteins). In a preferred embodiment, the variant protein differs from the parent protein and the variant proteins differ from each other by a small number of amino acid residues. The number of different amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In a preferred embodiment, the number of different amino acids between variants is 1 to 10. In particularly preferred embodiments, the related proteins and the specific (particulate) variant proteins comprise at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence homology. In addition, a related protein or a variant protein, as used herein, refers to a protein that differs in the number of significant regions (protein regions) from another related protein or a parent protein. For example, in some embodiments, a variant protein has 1, 2, 3, 4,5, or 10 corresponding significant regions that differ from its parent protein.

[0054] In one embodiment, the significant corresponding region of the variant produces only a background level of immunogenic response. Some of the residues identified for substitution, insertion and deletion are conserved residues, while others are not. Where residues are not conserved, substitution of one or more amino acids is limited to the generation of variant substitutions having an amino acid sequence that does not correspond to a naturally occurring sequence. Where the residues are conserved, such substitutions should not result in a naturally occurring sequence.

[0055] In some embodiments, the following cassette mutagenesis method is used in the construction of protein variants of the invention, although other methods may be used. First, a naturally occurring gene encoding a protein is obtained and sequenced in whole or in part. The sequence is then scanned for a site at which mutation (deletion, insertion or substitution) of one or several amino acids in the encoded protein is desired. Assessing the sequences flanking the site for the presence of a restriction site to replace the short segment of the gene with a pool of oligonucleotides; when expressed, it will encode a variety of mutants. Such restriction sites are preferably the only sites located within the gene of the protein to facilitate replacement of the gene segment. However, any convenient restriction site that is not overly redundant in the protein gene may be used, provided that the gene fragments resulting from restriction digestion can be reassembled (reassortable) in the appropriate order. If the restriction site is not present at a position within a distance from the selected point (from 10 to 15 nucleotides), such a site is created by substituting nucleotides in the gene in such a way that neither the reading frame nor the encoded amino acid is changed in the final construction. Mutation of the gene to bring the gene sequence into agreement with the desired sequence is carried out by extension of the M13 primer according to a generally known method. The tasks of locating the appropriate flanking regions and assessing the need for alterations to reach the two convenient restriction enzyme site sequences are routinely accomplished by the redundant sequences encoded by the gene, the restriction enzyme map of the gene, and the large number of different restriction enzymes. It is noted that the above method needs to be used only in connection with flanking regions that do not contain sites, if appropriate flanking restriction sites are present that can be exploited.

[0056] Upon cloning of naturally occurring or synthetic DNA, the restriction sites flanking the positions to be mutated are digested with cognate restriction enzymes (cognate restriction enzymes) and multiple end-complementary oligonucleotide cassettes (end-complementary oligonucleotide cassettes) are ligated into the gene. Mutagenesis is simplified by this method because all oligonucleotides can be synthesized so as to have the same restriction sites, without the need for synthetic linkers to generate the restriction sites.

[0057] As used herein, "corresponding" refers to a residue at the enumerated position in a protein or peptide, or a residue that is similar, homologous, or equivalent to the enumerated residue in the protein or peptide.

[0058] As used herein, "corresponding region" generally refers to a similar position along a related protein or a parent protein.

[0059] As used herein, the term "analogous sequence" refers to a sequence that provides similar function, tertiary structure, and/or conserved residues in a protein as the protein of interest (i.e., typically the original protein of interest). In a particularly preferred embodiment, the analogous sequence comprises a sequence at or near the epitope. For example, in epitope regions comprising alpha helix or beta sheet structures, the replacement amino acids in similar sequences preferably retain the same specific structure. The term also refers to nucleotide sequences, as well as amino acid sequences. In some embodiments, similar sequences are developed such that the replacement amino acid exhibits similar function, tertiary structure, and/or conserved residues as amino acids on or near the epitope in the protein of interest. Thus, where the epitope comprises a structure such as an alpha helix or a beta sheet, the replacement amino acid preferably retains its particular structure.

[0060] As used herein, "homologous protein" refers to a protein that has a similar effect, structure, antigenic and/or immunogenic response to a protein of interest. It does not mean: it is necessary that the homologue and the protein of interest be evolutionarily related. Thus, it is intended that the term encompass proteins of the same function from different species. In some preferred embodiments, it is desirable to identify homologues having similar tertiary and/or primary structure to the protein of interest, since replacing an epitope in the protein of interest with a similar segment from the homologue reduces the disruption of the change. Thus, in most cases, closely related homologous proteins provide the most desirable source of epitope substitution. Alternatively, it is advantageous to find human analogues for a given protein. For example, in some embodiments, replacement of a particular epitope in one human HPV type with a sequence from another HPV or other species of papillomavirus will result in increased immunogenicity to a level suitable for vaccine preparation.

[0061] As used herein, "homologous genes" refers to at least one pair of genes from different, but usually related, species, which correspond to each other and are identical or very similar to each other. The term includes genes that are isolated as a result of speciation (i.e., development of new species) (e.g., orthologous genes), as well as genes that are isolated by genetic duplication (e.g., paralogous genes). These genes encode "homologous proteins".

[0062] As used herein, "ortholog" and "orthologous gene" refer to genes that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation and that exist in different species. In general, orthologs retain the same function during evolution. The identification of orthologs can be applied in the reliable prediction of gene function in newly sequenced genomes.

[0063] As used herein, "paralogs" and "paralogous genes (pathlogous genes)" refer to genes that are linked by duplication in the genome. Although orthologs retain the same function during evolution, paralogs develop new functions even though some are often related to the original function. Examples of paralogs include, but are not limited to, genes encoding trypsin, chymotrypsin, elastase and thrombin, which are serine proteases and occur together within the same species.

[0064] As used herein, "wild-type" and "native" proteins refer to those proteins found in nature. The terms "wild-type sequence" and "wild-type gene" are used interchangeably herein to refer to a sequence that occurs naturally or naturally in a host cell. In some embodiments, a wild-type sequence refers to a sequence of interest as a starting point in a genetic engineering project. Genes encoding naturally occurring (i.e., precursor) proteins can be obtained according to the use of general methods known to those of ordinary skill in the art. The method comprises synthesizing a labeled probe having a putative sequence encoding a region of the protein of interest, preparing a genomic library from an organism expressing the protein, and screening the library for the gene of interest by hybridization to the probe. Positively hybridizing clones were then mapped and sequenced.

[0065] As used herein, the term "recombinant DNA molecule" refers to a DNA molecule comprised of segments of DNA joined together by molecular biological techniques.

[0066] The degree of homology between sequences (degree of homology), may be determined by any suitable method known in the art (see Smith and waterman, adv. appl. Math., 2: 482[1981 ]; Needleman and Wunsch, J.mol. biol., 48: 443[1970 ]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444[1988 ]; Software in Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis), such as GAP, BESTFIT, FASTA and TFASTA; and Devereux et al, Nucl. Acr. Res., 12: 387 395[1984 ]).

[0067] For example, PILEUP is a useful program for determining homology levels. PILEUP uses progressive pair-wise alignment to generate multiple sequence alignments (multiple sequence alignment) from a set of related sequences. It may also draw a tree diagram showing the clustering relationships (relations) used to generate the alignment. PILEUP uses a simplified method of Feng and Doolittle progressive alignment (Feng and Doolittle, J.mol.Evol., 35: 351-360[1987 ]). This method is similar to the method described by Higgins and Sharp (Higgins and Sharp, CABIOS 5: 151-153[1989 ]). Useful PILEUP parameters include: the default gap weight (gap weight) is 3.00, the default gap length weight (gap length weight) is 0.10, and the weighted end gaps (weighted end gaps). Another example of a useful algorithm is the BLAST algorithm described by Altschul et al (Altschul et al, J.Mol.biol., 215: 403 + 410[1990 ]; and Karlin et al, Natl.Acad.Sci.USA 90: 5873 + 5787[1993 ]). A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al, meth. enzymol., 266: 460-. The parameters "W", "T" and "X" determine the sensitivity and speed of alignment (alignment). The BLAST program defaults to a word length (W) of 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915[1989]) alignment value (B) of 50, expectation value (E) of 10, M '5, N' 4 and comparison of the two strands.

[0068] As used herein, "percent (%) nucleic acid identity" refers to the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues in the sequence.

[0069] As used herein, the term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand of nucleic acid by base pairing, as is known in the art.

[0070] As used herein, "maximum stringency" refers to the level of hybridization that typically occurs at about Tm-5 deg.C (a temperature 5 deg.C below the melting temperature (Tm) of the probe); "high stringency" is at about 5 ℃ to 10 ℃ below the Tm (melting temperature); "intermediate stringency" is about 10 ℃ to 20 ℃ below the Tm (melting temperature); "Low stringency (low stringency)" is about 20 ℃ to 25 ℃ below the Tm (melting temperature). As will be appreciated by those skilled in the art, maximum stringency hybridization can be used to identify or detect identical polynucleotides, while medium or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.

[0071] In the context of two nucleic acids or polypeptides, the phrases "substantially similar" and "substantially identical" generally mean: the polynucleotide or polypeptide comprises a sequence which has at least 75% sequence identity, preferably at least 80%, more preferably at least 90%, more preferably 95%, most preferably 97% sequence identity, sometimes up to 98% and 99% sequence identity, to a reference sequence (wild type). Sequence identity can be determined by known programs, such as BLAST, ALIGN and CLUSTAL, using standard parameters. (see Altschul et al, J.mol.biol.215: 403- & 410[1990 ]; Henikoff et al, Proc.Natl.Acad Sci.USA 89: 10915[1989 ]; Karin et al, Proc.Natl Acad.Sci USA 90: 5873[1993 ]; and Higgins et al, Gene 73: 237- & 244[1988 ]). Software for performing BLAST analysis is publicly available from the National center for Biotechnology Information. Furthermore, the database can be searched using FASTA (Pearson et al, Proc. Natl. Acad. Sci. USA 85: 2444-.

[0072] As used herein, "equivalent residues" refers to proteins that share specific amino acid residues. For example, equivalent residues can be identified by determining homology at the level of tertiary structure of a protein (e.g., IFN- β) whose tripolar structure has been determined by X-ray diffraction techniques. Equivalent residues are defined as residues: the atomic coordinates of two or more backbone atoms of one particular amino acid residue of the protein with putative equivalent residues and the protein of interest (N and N, CA and CA, C and C, O and O) after alignment, lie within 0.13nm, and preferably within 0.1 nm. After the optimal model is oriented and positioned to produce the maximum overlap of the non-hydrogen protein atomic coordinates of the analyzed protein, the comparison is complete. The preferred model is a crystal model that provides the smallest R-factor to the experimental diffraction data at the highest resolution available, as determined using methods known to those of ordinary skill in the crystallographic and protein identification/analysis arts.

[0073] In some embodiments, it is preferred that the "precursor DNA sequence" encoding the amino acid sequence of the precursor enzyme is modified, but this can be done by manipulating the precursor protein. In the case where the residues are non-conserved, substitution of one or more amino acids is limited to substitutions that result in variants: the variant contains an amino acid sequence that does not correspond to the naturally found amino acid sequence. In the case of conserved residues, such substitutions should not result in a naturally occurring sequence. The derivatives provided by the present invention further include chemical modifications that alter the properties of the protease.

[0074] In some preferred embodiments, the protein gene is ligated into a suitable expression plasmid. The cloned protein gene is then used to transform or transfect a host cell for the purpose of expressing the protein gene. The plasmid can replicate in the host cell, in the sense that it contains well known components required for plasmid replication; or the plasmid is designed to integrate into the host chromosome. The necessary elements are provided for efficient gene expression (e.g., a promoter operably linked to the gene of interest). In some embodiments, these essential elements are supplied as the homologous promoter of the gene itself, if it is recognized, (i.e., transcribed by the host); a transcription terminator (the polyadenylation region of a eukaryotic host cell) which is exogenous or is provided by an endogenous terminator region of the protein gene. In some embodiments, selection genes are also included, such as antibiotic resistance genes; the gene allows plasmid transfected host cells to be maintained in continuous culture by growth in a medium containing an antimicrobial substance.

[0075] The invention includes proteins with altered immunogenicity, which are equivalents. Is "equivalent," meaning that under medium to high stringency conditions, a protein is encoded by a polynucleotide that can hybridize to the polynucleotide, the nucleotide having a sequence as set forth in any one of the sequences provided herein; and still maintain altered immunogenic reactivity to human T cells. Is "equivalent", meaning: the protease has at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with respect to the epitope sequence; and variant proteases comprising such epitopes (e.g., having modified amino acid sequences).

[0076] As used herein, the terms "hybrid proteins" and "fusion proteins" refer to proteins engineered from at least two different or "parent" proteins. In a preferred embodiment, these parent proteins are homologues of each other. For example, in some embodiments, preferred hybrid proteases or fusion proteins comprise the N-terminus of a protein and the C-terminus of a homolog of the protein. In some preferred embodiments, both ends are bound to correspond to the full-length active protein. In other alternative preferred embodiments, these homologues share substantial similarity but do not share the same T-cell epitope. Thus, in one embodiment, the invention provides a protease of interest which: it has one or more T cell epitopes at its C-terminus, but in which the C-terminus is replaced by the C-terminus of a homologue having less active T cell epitopes, or fewer or no T cell epitopes at its C-terminus. Thus, one of ordinary skill will appreciate that by being able to identify T cell epitopes in a homolog, a variety of variants can be formed that produce different immunogenic responses. Furthermore, it should be understood that: the internal part and more than one homologue may be used to generate variants of the invention.

[0077] "operable linked" and "operable combination" when describing the relationship between two DNA regions, only mean: which are functionally connected to each other. For example, a pre-sequence is operably linked to a peptide if it functions as a signal sequence that is involved in the secretion of the mature form of the protein that is most likely to involve cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; if the ribose binding site is positioned to allow translation, the ribose binding site is operably linked to the coding sequence.

[0078] DNA molecules are described as having a "5 'end" and a "3' end" because mononucleotides are reacted in such a way to form oligonucleotides such that the 5 'phosphate on the pentose ring of one mononucleotide is linked to the 3' oxygen of its neighbor in one direction by phosphodiester bonds. Thus, if the 5 ' phosphate of an oligonucleotide is not linked to the 3 ' oxygen on the pentose ring of a single nucleotide, its end is referred to as the "5 ' end"; this end is called the "3 ' end" if the 3 ' oxygen of the oligonucleotide is not ligated to the 5 ' phosphate of the subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even within a large oligonucleotide, may be said to have a 5 'end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as "downstream" or "upstream" or 5 'ends of 3' elements. This term reflects the fact that the transcription process proceeds along the DNA strand, from 5 'to 3'. Promoter and enhancer elements that direct the transcription of linked genes are typically located 5 'or upstream of the coding region (enhancers may play a role even when located 3' to the promoter and coding regions). The transcription endpoint or polyadenylation signal is located 3' or downstream of the coding region.

[0079] The term "oligonucleotide having a nucleotide sequence encoding a gene" means a DNA sequence containing the coding region of the gene, or in other words, a DNA sequence encoding the gene product. The coding region may be present in the form of cDNA or genomic DNA. If desired, appropriate control elements such as enhancers/promoters, splice junctions (splice junctions), polyadenylation signals, and the like can be placed in close proximity to the coding region of the gene to allow proper initiation of transcription and/or proper processing of the primary RNA transcript. Alternatively, the coding region used in the expression vectors of the invention may comprise endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of endogenous and exogenous control elements.

[0080] The term "recombinant oligonucleotide" refers to an oligonucleotide created using molecular biological manipulations, including but not limited to: ligation of two or more oligonucleotides by restriction digestion of a polynucleotide sequence, oligonucleotide synthesis (e.g., synthesis of primers or oligonucleotides), and the like.

[0081] As used herein, the term "transcription unit" refers to a segment of DNA between the initiation and termination sites of transcription, and the regulatory elements necessary for efficient initiation and termination. For example, a DNA segment comprising an enhancer/promoter, a coding region, and termination and polyadenylation sequences constitutes a transcription unit.

[0082] As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (as described below).

[0083] As used herein, the term "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid sequences necessary for expression of an operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used forms of vectors at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.

[0084] A "host cell" as used in the present invention is typically a prokaryotic or eukaryotic host containing an expression vector and/or a gene of interest. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are competent to replicate the vector encoding the protein variant or to express the desired protein variant. In the case of vectors encoding a precursor (pre-) or prepro-precursor (prepro-) form of a protein variant, such variant, when expressed, is typically secreted from the host cell into the host cell medium.

[0085] The term "promoter/enhancer" refers to a segment of DNA that includes sequences that provide promoter and enhancer functions (e.g., the long terminal repeat of a retrovirus includes both promoter and enhancer functions). Enhancers/promoters may be "endogenous" or "exogenous" or "heterologous". Endogenous enhancers/promoters are enhancers/promoters naturally linked to a particular gene in the genome. Exogenous (heterologous) enhancers/promoters are enhancers/promoters that are placed in a gene-juxtaposed position by means of genetic manipulation (i.e., molecular biology techniques).

[0086] The presence of "splicing signals" (on the expression vector) results in higher levels of expression of the recombinant transcript. The splicing signal mediates the removal of introns from nascent RNA transcripts, which consists of splice donor and acceptor sites. (Sambrook et al, molecular cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989], pp.16.7-16.8). A commonly used splice donor and acceptor site is the splice junction (splice junction) of 16S RNA from SV 40.

[0087] Efficient expression of recombinant DNA sequences in eukaryotic cells requires signals that direct efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are typically found downstream of polyadenylation signals, being several hundred nucleotides in length. The term "polyadenylation site" or "polyadenylation sequence," as used herein, refers to a DNA sequence that directs the termination and polyadenylation of a nascent RNA transcript. Efficient polyadenylation or polyadenylation (polyadenylation) of recombinant transcripts is desirable because transcripts lacking a poly A tail are unstable and will be degraded quickly. The polyadenylation signal utilized by the expression vector may be "heterologous" or "endogenous". The endogenous polyadenylation signal is a polyadenylation signal which is naturally present at the 3' -end of the coding region of a particular gene on the genome. A heterologous polyadenylation signal is a polyadenylation signal which is isolated from one gene and placed 3' to another gene. A commonly used heterologous polyadenylation signal is the SV40 polyadenylation signal.

[0088] The terms "stable transfection" and "stably transfected" refer to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectants" refers to cells that have stably integrated foreign DNA into genomic DNA.

[0089] The terms "selectable marker" and "selectable gene product", as used herein, refer to the use of a gene that encodes an enzymatic activity that provides resistance to an antibiotic or drug once a cell expresses the selectable marker.

[0090] As used herein, the terms "amplification" and "gene amplification" refer to a process by which a particular DNA sequence is disproportionately replicated so that the amplified gene is present in the genome at a higher copy number than it was originally present. In some embodiments, cells are selected by growth in the presence of a drug (e.g., an inhibitor of a suppressible enzyme), which results in amplification of an endogenous gene encoding a gene product desired for growth in the presence of the drug, or results in amplification of an exogenous (i.e., import) sequence encoding such gene product, or both. Gene amplification naturally occurs during the development of a particular gene, such as the amplification of ribosomal genes in amphibian oocytes. Gene amplification can be induced by treating cultured cells with a drug. An example of drug-induced amplification is methotrexate-induced amplification of the endogenous dfhr gene in mammalian cells (Schmike et al, Science 202: 1051[1978 ]). Selection of cells by growth in the presence of a drug, such as an inhibitor of an inhibitable enzyme, may result in amplification of the endogenous gene encoding the gene product required for growth in the presence of the drug, or by amplification of an exogenous (i.e., input) gene encoding the gene product, or both.

[0091] Amplification is a special case of nucleic acid replication, involving template specificity. It contrasts with non-specific template replication (i.e., replication that relies on template but not on a specific template). Template specificity here is distinguished from the fidelity of replication (i.e., the synthesis of the correct polynucleotide sequence) and nucleotide (either ribose or deoxyribose) specificity. Frequently, template specificity is described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be selected from other nucleic acids. Amplification techniques are primarily designed to perform this selection.

[0092] As used herein, the term "co-amplification" refers to the introduction of an amplifiable marker into a single cell in combination with other gene sequences (i.e., comprising one or more non-selectable genes, such as those contained in an expression vector), and the application of appropriate selection pressure to cause the cell to amplify the amplifiable marker and the other non-selectable gene sequences. Amplifiable markers (amplifiable markers) can be physically linked to other sequences; or alternatively, two separate pieces of DNA may be introduced into the same cell, one containing the amplifiable marker and the other containing the non-selectable marker.

[0093] As used herein, the terms "amplifiable marker", "amplifiable gene" and "amplification vector" refer to a gene or vector that: the gene or vector encodes a gene that allows amplification of the gene under suitable growth conditions.

[0094] As used herein, the term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification means. It is contemplated that "amplifiable nucleic acids" typically include "sample templates".

[0095] As used herein, the term "sample template" refers to nucleic acid derived from a sample that is analyzed for the presence of a "target" (defined below). In contrast, "background template" is used to refer to nucleic acids other than a sample template, which may or may not be present in a sample. Background templates are often ignored. This may be the result of sample transfer, or may be due to the presence of nucleic acid contaminants attempting to be purified from the sample. For example, nucleic acids from organisms in a test sample may be present as background, in addition to those from the organism to be detected.

[0096] "template specificity" is achieved in most amplification techniques by selection of the enzyme. The amplification enzyme is an enzyme: under the conditions under which they are used, they process only specific sequences of nucleic acids which are in a heterogeneous mixture consisting of nucleic acids. For example: in the case of Q.beta.replicase, MDV-1 RNA is the specific template for the replicase (see Kacian et al, Proc. Natl. Acad. Sci. USA 69: 3038[1972 ]). Other nucleic acids are not replicated by the amplidase. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has strict specificity for its own promoter (see Chamberlin et al, Nature 228: 117[1970 ]). In the case of T4 DNA ligase, the enzyme does not ligate two oligonucleotides or polynucleotides whose substrates are mismatched to the template at the point of ligation (see Wu and Wallance, Genomics 4: 560[1989 ]). Finally, it was found that Taq and Pfu polymerases, by virtue of their ability to function at high temperatures, have high specificity for sequences bound by and thus defined by primers; the high temperature results in thermodynamic conditions that favor hybridization of the primer to the target sequence, but not to non-target sequences.

[0097] As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or produced synthetically, that functions as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase, at a suitable temperature and pH). For maximum efficiency in amplification, the primer is preferably single stranded, but alternatively may be double stranded. If double stranded, the primer is first treated to separate its double strands before being used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleic acid. The primer must be long enough to prime the synthesis of extension products in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, primer source and method use.

[0098] As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides) that is capable of hybridizing to another oligonucleotide of interest, whether the oligonucleotide is naturally occurring in a purified restriction digest or is produced synthetically, recombinantly or by PCR amplification. The probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of specific gene sequences. It is also contemplated that any probe used in the present invention will be labeled with a "reporter molecule" so that it is detectable in any detection system, including but not limited to enzymatic (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent detection systems. It is not intended that the present invention be limited to any particular detection system or label.

[0099] As used herein, the term "target", when used in reference to a polymerase chain reaction, refers to a region of nucleic acid bound by a primer for use in a polymerase chain reaction. Thus, the "target" is intended to be selected from other nucleic acid sequences. A "segment or fragment" is defined as a region of a nucleic acid in a sequence of interest.

[0100] As used herein, the phrase "polymerase chain reaction" (PCR) refers to U.S. Pat. nos. 4,683,195; 4,683,202 and 4,965,188, which are incorporated herein by reference, include methods for increasing the concentration of target sequence fragments in a mixture of genomic DNA without cloning or purification. The process of amplifying the target sequence comprises: a large excess of two oligonucleotide primers is introduced into a DNA mixture containing the desired target sequence, followed by a precise continuous thermal cycling process in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and then the primers are annealed to their complementary sequences within the target molecule. After the annealing process, the primers are extended with a polymerase, thereby forming a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated multiple times (i.e., denaturation, annealing and extension constitute one "cycle", and such a cycle can be performed multiple times) to obtain a high concentration of amplified segments of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers to each other, and thus, is a controllable parameter. Because of the reproducibility of this process, the method is called "polymerase chain reaction" (hereinafter, "PCR"). Because the target sequences are expected to amplify fragments that become dominant sequences (in terms of concentration) in the mixture, they are referred to as "PCR amplified.

[0101] As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleoside triphosphates, buffers, etc.) required for amplification in addition to primers, nucleic acid template, and amplification enzyme. Generally, amplification reagents are placed and contained in reaction vessels (test tubes, microwell plates, etc.) along with other reaction components.

[0102]By means of PCR, using a labeled probe,by several different methods (e.g., hybridization to a labeled probe; incorporation of biotinylated primers followed by detection of avidin-protease conjugates; incorporation³²P-labeled deoxyribonucleotide triphosphates, such as dCTP or dATP, into the amplified fragment), it is possible to amplify a single copy of a particular target sequence in genomic DNA to detectable levels. In addition to genomic DNA, any oligonucleotide or polynucleotide sequence may be amplified using a collection of suitable primer molecules. In particular, the amplified segment generated by the PCR process itself is itself a valid template for subsequent PCR amplification.

[0103] As used herein, the terms "PCR product", "PCR fragment (PCRfragment)" and "amplification product (amplification product)" refer to: after the end of two or more cycles of the denaturation, annealing and extension PCR steps, a mixture of compounds is produced. These terms include the following: wherein one or more fragments of one or more target sequences are amplified.

[0104] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cleave double-stranded DNA at or near a specific nucleotide sequence.

[0105] The terms "nucleic acid molecule coding", "DNA sequence coding", and "DNA coding" refer to a sequence or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes an amino acid sequence.

[0106]The peptides of the invention, as well as pharmaceutical and vaccine compositions thereof, are useful for administration to mammals, particularly humans, for the treatment and/or prevention of HPV infections. Vaccines comprising an immunogenically effective amount of one or more peptides as described herein are the inventionAccording to further embodiments of the present invention. Once suitable immunogenic epitopes have been identified, they, referred to herein as "vaccine" compositions, can be delivered by a variety of means. For example, such vaccine compositions may include lipopeptides (lipopeptids) (e.g., Vitiello et al, J Clin. Invest., 95: 341[1995 ]]) (ii) a And PCTUSOO/P17842; peptide compositions encapsulated in poly (DL-lactide-co-glycolide) ("TLG") microspheres (see Eldridge et al, mol. Immunol., 28: 287-294[1991 ]](ii) a Alonso et al, Vaccine 12: 299-306[1994](ii) a Jonse et al, Vaccine 13: 675-681[1995]) Peptide compositions contained in immune-stimulating complexes (ISCOMS) (see Takahashi et al, Nature 344: 873-875[1990](ii) a Hu et al, clin. exp. immunol., 113: 235-243[1998]) Multiple Antigenic Peptide Systems (MAPs) (see Tam, proc.natl.acad.sci.u.s.a.85: 5409-5413[1988]；Tam，J.Immunol.Meth.，196：17-32[1996]) Viral delivery vectors (Perkus et al, inConcepts in Vaccine Development，Kaufinann(ed.)，p.379[1996](ii) a Chakrabarti et al, Nature 320: 535[1986](ii) a Hu et al, Nature 320: 537[1986](ii) a Kieny et al, AIDSBi/Technol, 4: 790[1986](ii) a Top et al, J infect.dis., 124: 148[1971](ii) a Chanda et al, virol, 175: 535[1990]) Microparticles of viral or synthetic origin (e.g., Kofler et al, jimmunol, meth., 192: 25[1996](ii) a Eldridge et al, sem, hemtool, 30: 16[1993](ii) a Falo et al, Nature Med., 7: 649[1995]) Adjuvant (Warren et al, ann.rev.immunol., 4: 369[1986](ii) a Gupta et al, Vaccine 11: 293[1993](ii) a Liposomes (liposomes) (Reddy et al, J Immunol., 148: 1585[ 1992)]；Rock，Immunol.Today 17：131[1996]) (ii) a Alternatively, naked DNA and particles adsorb DNA (Ulmer et al, Science 259: 1745[ 1993)](ii) a Robinson et al, Vaccine 11: 957[1993]) (ii) a Shiver et al in Concepts in Vaccine Development, Kaufinann (ed.), p.423[1996 ]]；Cease and Berzofsky，Ann.Rev.Immunol.，12：923[1994](ii) a And Eldridge et al, sem, hemtool, 30: 16[1993]). Toxin-directed delivery techniques, also known as receptor-mediated targeting, such as those of Avant Immunotherapeutics, Inc (needleham, Massachusetts), may also be used.

[0107] The vaccine compositions of the invention comprise a nucleic acid-mediated modality. DNA or RNA encoding one or more peptides of the invention may also be administered to the patient. For example, the method is described in Wolff et al, Science 247: 1465 (1990); and U.S. Pat. nos. 5,580,859; 5,589,466; 5,804,566, respectively; 5,739,118, respectively; 5,736,524; 5,679,647, respectively; as described in WO 98/04720; and is described in more detail below. Examples of DNA-based delivery techniques include "naked DNA", facilitated (procaine (bupivicaine), polymer, peptide-mediated) delivery, cationic lipid complexes, and microparticle-mediated ("gene gun") or pressure-mediated delivery (see U.S. Pat. No. 5,922,687).

[0108] For therapeutic or prophylactic immunization purposes, the peptides of the invention may be expressed from viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox. For example, the present approach involves the use of vaccinia virus as a vector for expressing nucleotide sequences encoding the peptides of the invention. Upon introduction into an acutely or chronically infected or uninfected host, the recombinant vaccinia virus expresses immunogenic peptides, thereby eliciting a CTL and/or HTL response in the host. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacillus Calmette Guerin). BCG has been reported in Stover et al, Nature 351: 456-460(1991) are described. A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as adenoviral and adeno-associated viral vectors, retroviral vectors, Salmonella typhi (Salmonella typhi) vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those of ordinary skill in the art from the description herein.

[0109] Furthermore, a vaccine according to the invention may comprise one or more of the peptides of the invention. Thus, the peptide may be present in the vaccine alone. Alternatively, the peptide may be separately attached to its own carrier (carrier); alternatively, the peptide may be present as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of multiple peptides. The polymer has the advantages that: it has an enhanced immunological response and, in the case of polymers composed of a plurality of different peptide epitopes, it has the additional capacity to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism targeted in the immune response. The components may be naturally occurring regions of the antigen or may be prepared, for example, recombinantly or by chemical synthesis.

[0110] Vectors (carriers) that can be used with the vaccines of the present invention are well known in the art and include: for example, thyroglobulin; albumins, such as human serum albumin; tetanus toxoid; poly-amino acids such as poly-L-lysine, poly-L-glutamic acid; influenza virus, hepatitis B virus, core protein (core protein) and the like. The vaccine may comprise a physiologically tolerable (i.e. acceptable) diluent, such as water or saline, preferably phosphate buffered saline. Vaccines may also typically include an adjuvant. Such as incomplete freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum, are examples of materials well known in the art. In addition, a CTL response may be initiated by coupling the peptide of the invention with a lipid, such as tripalmitoyl-S-glycerocysteinyl-serine (P3 CSS).

[0111]By injection, aerosol, oral, transdermal, transmucosal, pleural, intrathecal and other suitable routes, the immune system of the host following immunization with the peptide composition according to the invention is activated by CD8⁺T-cell responses in response to the vaccine.

[0112] As a result, the host becomes at least partially immune to subsequent infection; or at least partially resistant to developing an ongoing chronic infection; alternatively, where the antigen is tumor-associated, at least some therapeutic benefit is obtained.

[0113] In some embodiments, the component that induces a T-cell response is combined with a component that induces an antibody response to a target antigen of interest. Preferred embodiments of such compositions comprise both class I and class II epitopes according to the invention.

[0114]For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual already infected with HPV. Individuals in the latent or acute phase of infection may be treated by the use of immunogenic peptides alone or in combination with other therapeutic methods where appropriate. In therapeutic applications, the composition is administered to a patient at a dosage such that: the dose is sufficient to elicit effective CD8 against the virus⁺T cell responses and is sufficient to cure or at least partially arrest symptoms and/or complications. An amount sufficient to achieve this effect is defined as a "therapeutically effective dose". For this use, the effective amount depends on a number of factors, including but not limited to: peptide composition, mode of administration, stage and severity of the disease being treated, weight and general health of the patient, the judgment of the treating physician, but for a patient weighing 70kg, the range of first immunization (i.e., for therapeutic or prophylactic administration) is generally from about 1.0ug to about 50,000ug of peptide, followed by measurement of the specific CD8 in the patient's blood over several weeks to months, depending on the patient's response and status⁺T cell activity was boosted at a peptide dose of about 1.0ug to about 50,000ug, according to a boosting regimen.

[0115] An immunizing dose and subsequent booster doses at defined intervals (e.g., from one week to four weeks) may be required, possibly for an extended period of time, to effectively immunize an individual. In the case of chronic infection, administration should be continued for a period of time both before and after at least clinical symptoms or laboratory tests indicate that viral infection has been eliminated or substantially reduced.

[0116] Pharmaceutical compositions for therapeutic treatment are intended to be administered parenterally (parenteral), topically (topical), orally (oral) or topically (local). Preferably, the pharmaceutical composition is administered by injection (e.g., intravenously, subcutaneously, intradermally, intramuscularly). Accordingly, the present invention provides a composition for injectable administration comprising an immunogenic peptide dissolved or suspended in an acceptable carrier solution, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged in the state in which they are used, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable excipients to achieve a desired approximate physiological environment, for example, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate (sorbate), triethanolamine oleate, etc.

[0117]The present invention provides methods for identifying HPV epitopes in the sequence of various HPV types, as well as methods for producing peptides; when the peptide is introduced into the HPV sequence, it can initiate CD8⁺T cell responses.

[0118]In some embodiments, the invention provides methods for identifying CD8 in HPV sequences⁺T cell epitopes and production can be used to elicit CD8⁺T cell reactive peptides. In particular, the present invention provides methods and compositions suitable for increasing the immunogenicity of HPV epitopes, which epitopes have utility in the preparation of HPV vaccines.

[0119]In these embodiments, the present invention provides means for determining resistance to human CD8 comprising multiple epitopes of a protein of interest⁺T cell responses. In additional embodiments, once a significant epitope is identified using the modified I-MUNE ® assay system described herein, the significant epitope is subsequently altered to produce an epitope that induces an enhanced immune response against the protein.

[0120] Thus, as indicated above, the proteins of the invention exhibit a modified immunogenic response (e.g., antigenicity and/or immunogenicity) as compared to the native protein encoded by their precursor DNA. For example, HPVs that exhibit enhanced immunogenic responses (e.g., variant HPV epitopes) may be used in therapeutic and prophylactic vaccine compositions.

Detailed description of the invention

[0121]The present invention provides for the identification of functional CD8 in any protein of interest⁺T cell epitopes. The invention further provides CD8 of various proteins⁺A T cell epitope. In some preferred embodiments, the present invention provides CD8 of human papilloma virus (human papillomavir (HPV))⁺A T cell epitope. In further embodiments, the invention provides epitopes suitable for use in prophylactic/therapeutic vaccines. In a particularly preferred embodiment, the present invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines.

[0122]In a development of the invention, it was determined that: addition of anti-CD 40antibody to the test system provides an evaluable CD8⁺Methods of T cell response to various peptides. Although not intending to limit the present invention to any particular mechanism, it is believed that: incorporation of anti-CD 40 antibodies in activated CD4⁺CD40 ligand was mimicked on T cells. It attaches to the CD40 receptor present on the surface of APCs (e.g., dendritic cells) and stimulates the associated CD8 by increasing MHC and B7 expression⁺Activation of T cells.

[0123] In preliminary experiments, anti-CD 40 antibodies were tested in the I-MUNE ® assay system using the HLA-A2 restricted peptide. When the anti-CD 40antibody was not used, no response was observed. In contrast, when anti-CD 40antibody was added, a significant increase in response was observed. In these early experiments, 5 concentrations (10ug/ml, 5ug/ml, 2ug/ml, 1ug/ml and 0.5ug/ml) of anti-CD 40 were tested. The concentration of 10ug/ml was determined to be too high because it killed the cells. The concentration of 0.5ug/ml is slightly too low, although it does work in case the cell donor has a higher background value than usual. Therefore, six comparisons were made with concentrations of 5ug/ml, 2ug/ml and 1 ug/ml. The results were statistically analyzed using Stat-Ease DX6.1 software (Stat-Ease, inc., Minneapolis, MN). From these results, a concentration of 1ug/ml was chosen, since: this concentration resulted in the highest proliferative response compared to background. A concentration of 1ug/ml also helps to maintain a lower background and thus more of the reaction detected compared to concentrations of 2.5ug/ml and 5 ug/ml. Nevertheless, it should be understood that: in other assay systems, different antibody concentrations will be used.

[0124]The invention further provides CD8 in the E7 protein from two strains of Human Papilloma Virus (HPV)⁺A T cell epitope. In some preferred embodiments, the present invention provides means for the development of HPV vaccines, in particular multivalent vaccines for the prevention of high risk HPV strain infection. In additional embodiments, the invention provides methods for developing therapeutic vaccines against high risk HPV types suitable for use in preventing the development of benign and/or malignant tumors in infected individuals. The invention further provides epitopes suitable for use in prophylactic and/or therapeutic vaccines. In a particularly preferred embodiment, the present invention provides modified epitopes suitable for use in prophylactic and/or therapeutic vaccines.

[0125] Because of its widespread expression in cervical cancer cases, the E7 oncoprotein from HPV is a particularly attractive target for DNA vaccines. The E7 protein and E6 protein are responsible for the oncogenic characteristics of HPV (Finzer et al, Cancer Lett., 188: 15-24[2002 ]). Continuous proliferation and survival of cervical Cancer cells requires continuous expression of both proteins (von Knebel-Doeberitz et al, Cancer Res., 48: 3780-6[1988 ]). E6 and E7 are responsible for the transformation and inhibition of apoptosis in cervical lesions. Several studies have shown that: the immunological response against these proteins is protective with respect to cervical cancer. HPV16 positive women without squamous intraepithelial neoplasms (SIL) showed more general natural CTL responses against E6 and E7 relative to HPV16 positive women with squamous intraepithelial neoplasias (SIL). (Nakagawa et al, J.Infece.Dis., 175: 927-. In addition, the cell-mediated immune response to specific protective peptides of E6 and E7 is also associated with disease regression and resolution of viral infections. (Kadish et al, Cancer epidemic. Biomarkers prev., 11: 483-488[2002 ]). Furthermore, it has been demonstrated that: vaccination with E7DNA was highly effective in eliciting cytotoxic T cell responses (Osen et al, Vaccine 19: 4276-4286[2001 ]).

[0126]The use of epitope vaccines in the present invention, rather than full length vaccines, makes the present invention attractive because it avoids the problem of administering carcinogenic products. Also, due to the size limitations of DNA vaccines, the inclusion of only the immunogenic region of E7 makes it possible to cover more high risk virus strains. Patients with HPV infection often carry more than one strain of HPV virus, and individuals who have cleared one strain of HPV infection may be reinfected by another strain. Although CTL epitopes are generally used in combination with antiviral vaccines, in some preferred embodiments of the invention, there are several reasons for binding CD8⁺Epitope CD4⁺A T cell epitope. For example, antigen-specific CD4⁺Helper cells, in general, activate CD8 by cross-priming of antigen presenting cells⁺Cytolytic activity is required (see Bennett et al, Nature 393: 478-480[1998 ]](ii) a Schoenberger et al, Nature 393: 480-483[1998](ii) a And Ridge et al, Nature 393: 474-478[1998]). Furthermore, studies in animal models demonstrate that: vaccines comprising CD4 and CD8 epitopes derived from alloantibodies induce strong protective responses (see Ossendrop et al, J.Exp.Med., 187: 693-H702 [1998 ]](ii) a DeVeermann et al, J.Immunol., 162: 144-151[1999](ii) a And Zwaveling et al, j.immunol., 169: 350-8[2002])。

[0127]In some preferred embodiments, the present invention provides compositions and methods for developing vaccine compositions against HPV strains, particularly those associated with higher risk malignancies. Thus, in some particularly preferred embodiments, the present invention provides vaccine compositions and methods for developing E7 protein directly against two high-risk HPV strains (i.e., 16 and 18 strains). Importantly, the emergence of DNA from these HPV strains and cervical lesions and carcinomaAnd (4) correlating. (Lorincz et al, ObstetGynecol., 79: 328-337.[1992 ]]). MHC class II helper epitopes in the E6 and E7 proteins of a number of high and moderate risk HPV strains were identified as described in co-pending U.S. patent No. 60/466,235, set forth at 28/4/2003. Thus, in addition to the helper epitopes previously described, reference is made to CD8 of the present invention⁺It is also contemplated that the compositions and methods of epitopes will find use in therapeutic and/or prophylactic vaccine compositions.

Experiment of

[0128] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

[0129] In the following experimental disclosure, the following abbreviations apply: m (mole/liter); mM (millimole/liter); μ M (micromole/liter); nM (nanomole/liter); mol (mole); mmol (millimole); μ mol (micromolar); nmol (nanomole); gm (gram); mg (milligrams); μ g (μ g); pg (picogram); l (liter); ml (milliliters); μ l (microliter); cm (centimeters); mm (millimeters); μ m (micrometers); nm (nanometers); deg.C (degrees Celsius); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single-stranded DNA); dsDNA (double stranded DNA); dntps (deoxyribonucleic acid triphosphates); RNA (ribonucleic acid); HRP (horseradish peroxidase); AEC substrate (a solution of sodium acetate, dimethyl sulfoxide, methanol, and urea peroxide); AEC chromogen (solution of 3-amino-9-ethylcarbazole (2% w/v) and N, N dimethylformamide); PBS (phosphate buffered saline); g (gravity); DC (dendritic cells); PHA (phytohemagglutinin); OD (optical density); dulbecco's Phosphate Buffer (DPBS); HEPES (N- [ 2-hydroxyethyl ] piperazine-N- [ 2-ethanesulfonic acid ]); HBS (HEPES buffered saline); SDS (sodium dodecyl sulfate); Tris-HCL (Tris [ hydroxymethyl ] aminomethane-hydrochloric acid); DMSO (dimethyl sulfoxide); EGTA (ethylene glycol-bis (β -aminoethylether) N, N' -tetraacetic acid); EDTA (ethylenediaminetetraacetic acid); DPBS (Dulbecco's phosphate buffer); bla (beta-lactamase or ampicillin resistance gene); endogen (Endogen, Woburn, MA); CytoVax (CytoVax by Edmonton, canada); Wyeth-Ayerst (Wyeth-Ayerst, Philadelphia, PA); NEN (NEN Life Science Products, Boston, MA); wallace Oy (Wallace Oy of Turku, finland); pharma AS (Pharma AS by Oslo, norway); dynal (Dynal of Oslo norway); Bio-Synthesis (Bio-Synthesis, Lewis ville, TX); mimotopes (Mimotopes inc., San Diego, CA); ATCC (American Type Culture, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, NY); sigma (Sigma Chemical co., st. louis, MO); pharmacia (Pharmacia Biotech, Pisacataway, NJ); invitrogen (Invitrogen, inc., Grand Island, NY); abbott (Abbott Laboratories, Abbott Park, IL); list (List Biological Laboratories, Campbell, Calif.); perkin Elmer (Perkin Elmer Life Sciences, Boston, MA); eBioscience (eBioscience, san diego, CA); BD Bioscience (BD Bioscience); cellular Technology (Cellular Technology, Cleveland, OH); and Stratagene (Stratagene, La Jolla, Calif.).

Example 1

Preparation of E7 epitope

[0130] The full-length amino acid sequence of the E7 protein from HPV16 and 18 was used to generate a 9-mer (9-mer) peptide set. SwissProt.P03129 corresponds to HPV16E7 and SwissProt.P06788 corresponds to HPV18E 7. These variant peptides are synthesized from Mimotopes (Mimotopes) using multi-needle synthesis techniques known in the art (see, e.g., Maeji et al, j. immunological. meth., 134: 23-33[1990 ]). The 9-mer peptide was generated such that the sequence shared 8 amino acids with the adjacent peptide (i.e., each peptide was offset by one amino acid). Peptides were diluted with DMSO to provide a stock concentration of approximately 2 mg/ml. The final peptide concentration used in each experiment was 5. mu.g/ml.

Example 2

Preparation of peptide T cell epitopes for identification of HPV using human T cells

In a test system

[0131] Fresh human peripheral blood cells were collected from a human of unknown HPV exposure status. These cells were tested to determine antigenic epitopes in HPV16 and HPV18, e.g., in HPV16 and HPV18

As described in example 3.

[0132]Peripheral mononuclear blood cells (perpheral mononeuclear blood cells) (stored at room temperature for up to twenty-four hours) were prepared as follows. PBMC were separated from pale yellow surface material (buffy coat material) by centrifugation at 1000 Xg for 30 min on a Lymphoprep pad. The interface layer was collected and washed and counted using Cell-Dyn 3700System (Abbott). Then, a catalyst containing 10 is prepared⁸The suspension of PBMCs of (1) was resuspended in 30ml of AIM-5(invitrogen) and then allowed to adhere to a plastic T-75 flask for two hours. The remaining cells were frozen in 45% FCS (Gibco/BRL), 45% PBS w/o Ca&Mg (Mediatech) and 10% DMSO (Sigma), at 5X 10⁷Individual cells/ml were frozen. After two hours of PBMC culture, the non-adherent cells were removed from the culture flask. Adherent cells were cultured in culture flasks using 800 units/ml recombinant human GM-CSF (R)&D System) and 100 units/ml of recombinant human IL-4(Endogen) at 37 ℃ with 5% CO₂The process is carried out as follows. On day 5 of culture, 50 units/ml of recombinant human Il-1 α (Endogen) and 0.2 units/ml of recombinant human TNF- α were added to the culture. On day seven, adherent and non-adherent dendritic cells were harvested, washed and counted after one hour treatment with 30mg/ml mitomycin C (Sigma) and 10mM EDTA.

[0133]Preparation of autologous CD8 from frozen aliquots of PBMCs⁺T cells. After thawing and washing in DPBS, CD8 was isolated using a commercially available CD8 negative selection kit (Dynal) according to the manufacturer's instructions⁺T cells. Cells were counted using the Abbott Cell-Dyn 3700 System. The purity obtained using these methods has generally been foundGreater than 90%.

Example 3

T cell proliferation assay

[0134]This example describes an assay system used in the present invention. The basic test system is also called the "I-MUNE ®" assay system. The basic I-MUNE ® assay system was modified as described herein to target CD8⁺Analysis of T cell responses is more convenient. As described in more detail below, modifications used in the development of the invention involve: on PBMC, CD8 negative selection beads (CD8 negative selection beads) were used (i.e. instead of CD 4). In addition, when CD8 cells were resuspended, DCs (dendritic cells) and peptide were added at 1.5X 10 before plating onto plates⁵ml to 2.5X 10⁵Between ml of 2. mu.g/ml anti-CD 40 solution (final concentration of anti-CD 40 of 1 ug/ml); and 1. mu.l of 1ug/ml PHA was used in place of tetanus toxoid as a positive control.

[0135]Autologous dendritic cells and CD8 were plated in 96-well round-bottom plates⁺T cells were mixed with test peptides. More specifically, in a volume of 100. mu.l/well, 2X 10 in AIM V will be⁴Individual dendritic cells were combined with individual peptides (final peptide concentration of 5. mu.g/ml, final DMSO concentration of 0.25%). At 37 deg.C, 5% CO₂Next, after one hour of cultivation, 2X 10 cells were cultured⁵An individual CD8⁺T cells were added to the culture with 2. mu.g/ml of anti-CD 40 (eBioscience; Clone 5C3 mouse IgG1, kappa (Clone 5C3 mouse IgG1, kappa)) in a total volume of 200. mu.l and a final concentration of anti-CD 40 of 1. mu.g/ml per well. Negative control wells contained dendritic cells, CD8⁺T cells and 0.25% DMSO. Positive control wells contained dendritic cells, CD8⁺T cells (at the same concentration as the test wells) and 0.25% DMSO, with 5. mu.g/ml PHA (Sigma) (List). In some experiments, 1ug/ml of anti-IgG 1 (eBioscience; Clone P3 mouseIgG1, kappa) was used as an isotype control for comparison purposes. Individual peptides were tested in duplicate or triplicate for each donor.

[0136]At 37 deg.C, 5% CO₂After 5 days of incubation, cultures were pulsed with 0.25. mu. Ci/well tritiated thymidine (Perkin Elmer). After a subsequent 24-hour incubation, the plates were harvested and tritiated thymidine incorporation was assessed using a Wallac Microbeta TriLux liquid scintillation counter (Perkin Elmer). The reactions were averaged by repeating the experiment for each sample. A positive reaction is defined as having a reaction at least 2.95 times background. Based on the results obtained with the anti-CD 40antibody and the anti-IgG 1 isotype antibody, the effect of anti-CD 40 was found to be specific (see fig. 3).

[0137] One set of data was accumulated for both proteins in at least 45 donor assays. The percent response rate for each peptide was determined for the entire population of donors. In this assay system, "mean background response rate for the donor population (donors)" is defined as the mean percent response to all peptides in a collection. In this assay system, a "major epitope(s)" is defined as having a response rate that is at least three standard deviations above the average background response rate. "moderate epitopes" are those epitopes that produce results at least two standard deviations above the average background or three times the background. "minor epitopes" are those epitopes that have a response rate of at least twice the background value. As described herein, this assay identified several epitopes in the two HPV strains tested.

A.HPV E7.16

[0138]For this antigen, 45 donors were tested in the I-MUNE ® assay to determine the epitope for HPV E7.16. Figure 1 provides a diagram showing the response to each epitope. As also indicated in table 1, 19 valuable epitopes were identified on this antigen.

Table 1 valuable HPV E7.16 epitope
				Peptide numbering	Epitope classification	Epitope sequences	SEQ ID NO：
1	Minor epitopes	MHGDTPTLH	SEQ ID NO：1
				4	Mesogenic epitopes	DTPTLHEYM	SEQ ID NO：2
5	Minor epitopes	TPTLHEYML	SEQ ID NO：3
				7	Minor epitopes	TLHEYMLDL	SEQ ID NO：4
8	Minor epitopes	LHEYMLDLQ	SEQ ID NO：5
				9	Minor epitopes	HEYMLDLQP	SEQ ID NO：6
12	Minor epitopes	MLDLQPETT	SEQ ID NO：7
				21	Major epitopes	DLYCYEQLN	SEQ ID NO：8
36	Mesogenic epitopes	DEIDGPAGO	SEQ ID NO：9
				43	Minor epitopes	GQAEPDRAH	SEQ ID NO：10
54	Minor epitopes	IVTFCCKCD	SEQ ID NO：11
				76	Minor epitopes	IRTLEDLLM	SEQ ID NO：12
79	Minor epitopes	LEDLLMGTL	SEQ ID NO：13
				80	Minor epitopes	EDLLMGTLG	SEQ ID NO：14
81	Minor epitopes	DLLMGTLGI	SEQ ID NO：15
				82	Minor epitopes	LLMGTLGIV	SEQ ID NO：16
83	Minor epitopes	LMGTLGIVC	SEQ ID NO：17
				84	Minor epitopes	MGTLGIVCP	SEQ ID NO：18
89	Minor epitopes	IVCPICSQK	SEQ ID NO：19

B.HPV ET.18

[0139]For this antigen, 58 donors were tested in an I-MUNE ® assay to determine the valuable epitope for HPV E7.18. Figure 2 provides a graph showing the response to each epitope. Also, as is evident in table 2,6 valuable epitopes were identified in this antigen.

TABLE 2 valuable HPV E7.18 epitopes
				Peptide numbering	Epitope classification	Epitope sequences	SEQ ID NO
12	Major epitopes	VLHLEPQNE	SEQ ID NO：20
				17	Minor epitopes	PQNEIPVDL	SEQ ID NO：21
51	Minor epitopes	ARRAEPQRH	SEQ ID NO：22
				66	Minor epitopes	CKCEARIKL	SEQ ID NO：23
71	Minor epitopes	RIKLVVESS	SEQ ID NO：24
				95	Minor epitopes	SFVCPWCAS	SEQ ID NO：25

[0140]The results shown above provide valuable epitopes in the HPV16 and HPV18E7 proteins. Thus, the present invention not only provides a means to evaluate CD8 against an epitope of a protein of interest⁺T cell responses, and provides epitopes suitable for modification and use in such compositions as vaccines.

Example 4

INF-gamma enzyme-linked immunospot assay (ELISPOT)

[0141]In this example, an enzyme linked immunospot assay (BDBiosciences) was used to determine whether the epitopes identified in the previous examples are effector epitopes. In these experiments, INF- γ ELISPOT (INF- γ enzyme linked immunospot assay) assays were performed with the epitopes identified as described above, together with low and no reactive peptides from the HPV E7 peptide set (HPV 18E7 pepset). These assays were compared to CD8⁺I-MUNE ® epitope mapping assay (CD 8)⁺I-MUNE ® epitope mappingassay) were performed in parallel, with 20 donors.

[0142]In these experiments, CD8⁺T cells and dendritic cells were plated in round-bottomed 96-well format plates with 100 μ L of each cell mix in each well. Each peptide of interest in 0.25% DMSO was added to the wells at a final concentration of 5. mu.g/ml. Control wells contained DMSO, but no peptide. To each well, 1. mu.g/ml of anti-human CD40 Ab (eBioscience) was added. Each peptide was tested twice. At 37 deg.C, 5% CO₂Next, the culture was cultured for 5 days.

[0143]On the fifth day of the culture process, the cells were resuspended by pipetting, and then the cell suspension was transferred to elispot (bd biosciences) plates, which had been pre-coated with purified a-human IFN- γ antibody. Plates were incubated at 37 ℃ with 5% CO₂The cells were cultured for 24 hours. The plates were washed and then incubated with biotinylated a-human IFN- γ detection antibody for two hours. Dot reduction was performed using Avidin-HRP (Avidin-HRP) and AEC substrate and chromogen. Spots were quantified using an ImmunoSpot ® analyzer (cell Technology) according to the manufacturer's instructions. A positive reaction is defined as a reaction at least three times above background level.

[0144]In fig. 4, the assembly result is provided. As shown in this figure, there is a strong correlation between INF- γ production and epitope proliferation (r 0.67; p ═0.0019). In INF-gamma secretion and CD8⁺This correlation between T cell proliferation, which is well documented with CD8 in proliferation⁺T cells are indeed effector cells. CD8⁺Comparison of proliferation with INF- γ/production shows that the epitopes found are effector epitopes. Thus, CD8 in the I-MUNE ® assay⁺Proliferation is attributed to effector cells, not to anergic cells (anergic cells).

[0145] As indicated above, there are some peptides that are more effective against INF- γ than proliferation. The production of cytokines without proliferation is not an uncommon phenomenon, particularly as a memory cell response. In the case where proliferation was observed without INF-gamma production, INF-gamma was detected at twice the background level, but this did not meet the "positive" criteria that had been established in the development of the present invention. However, this can be considered positive by many other researchers. In summary, the results provided herein demonstrate that: the epitope identified was determined by I-MUNE ® and CTL activity could be confirmed by a commercially available assay system such as the INF- γ ELISPOT assay.

Claims (12)

1. For identifying CD8⁺Methods for producing epitopes.

2. A method for determining a CD8+ epitope in a protein, comprising the steps of:

(a) obtaining a solution of dendritic cells and naive CD8 from a single human blood source⁺A solution of T cells;

(b) differentiating said dendritic cells in said solution of dendritic cells to produce a solution of differentiated dendritic cells;

(c) preparing a peptide set from the protein, wherein the peptide set comprises the T cell epitopes;

(d) combining the solution of the CD8+ T cells with an anti-CD 40antibody to provide a solution of T cells and antibody;

(e) combining said differentiated dendritic cells and said peptide set with a solution of said T cells and antibodies; and

(f) measuring proliferation of said T cells in said step (e).

3. The method of claim 1, wherein said protein is selected from the group consisting of viral proteins, bacterial proteins, parasitic proteins, fungal proteins and tumor-associated proteins.

4. A method of reducing the allergenicity of a protein comprising the steps of:

(a) identifying a T cell epitope in the protein as set forth in claim 1; and

(b) modifying said protein so as to neutralize said T cell epitope such that the modified protein induces a baseline proliferation that is less than or substantially equal to that of said naive T cell.

5. The method of claim 4, wherein the T cell epitope is modified by a substitution selected from the group consisting of:

(a) replacing the amino acid sequence of said T cell epitope with an analogous sequence from a homologue of the protein of interest; and

(b) replacing the amino acid sequence of said T cell epitope with a sequence that substantially mimics the major tertiary structural attributes of the epitope.

6. A method of increasing the immunogenicity of a protein comprising the steps of:

(a) identifying a T cell epitope in the protein as set forth in claim 1; and

(b) modifying the protein so as to produce a modified protein such that the modified protein induces a proliferative response greater than the baseline proliferation of the naive T cell.

7. The method of claim 6, wherein the T cell epitope is modified by a substitution selected from the group consisting of:

(a) replacing the amino acid sequence of said T cell epitope with an analogous sequence from a homologue of the protein of interest; and

(b) replacing the amino acid sequence of said T cell epitope with a sequence that substantially mimics the major tertiary structural attributes of the epitope.

8. A human papillomavirus CD4+ T cell epitope identified using the method of claim 1.

9. A composition comprising the human papillomavirus CD4+ T cell epitope of claim 8.

10. The method of claim 1, further comprising the step of modifying the human papillomavirus to produce a variant human papillomavirus, wherein the variant protein exhibits an altered immunogenic response compared to the human papillomavirus.

11. A variant human papilloma virus, produced according to the method of claim 8.

12. A composition comprising the variant protein of claim 9.