METHOD FOR ISOLATING HEPATITIS C VIRUS PEPTIDES

Abstract

Described are methods for isolating Hepatitis C Virus peptides (HPs) that have a binding capacity to a MHC/HLA molecule or a complex comprising the HCV-peptide and the MHC/HLA molecule. In one aspect, these methods include providing a pool of HCV-peptide, said pool containing HCV-peptides which bind to said MHC/HLA molecule and HCV-peptides which do not bind to said MHC/HLA molecule, contacting said MHC/HLA molecule with said pool of HCV-peptides whereby a HCV-peptide which has a binding capacity to said MHC/HLA molecule binds to said MHC/HLA molecule and a complex comprising said HCV-peptide and said MHC/HLA molecule is formed, detecting and optionally separating said complex from the HCV-peptide which do not bind to said MHC/HLA molecule and optionally isolating and characterizing the HCV-peptide from said complex. Also described are immunogenic compositions that stimulate a specific T cell response to hepatitis C virus when administered to a subject in an effective amount.

Description

This application is a continuation of U.S. application Ser. No. 10/512,885, filed Oct. 28, 2004, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2003/009482 filed 27 Aug. 2003, which claims priority to Austrian Application No. A 1376/2002 filed 13 Sep. 2002, International Application No. PCT/EP03/02005 filed 27 Feb. 2003, and European Patent Application No. 03450171.8 filed 11 Jul. 2003, the contents of all of which applications are incorporated herein by reference in their entirety.

The present invention relates to a method for isolating HCV-peptides, especially for isolating HCV T cell epitopes which have a binding capacity to a MHC/HLA molecule.

The immune system is a complex network of inter-related cell types and molecules, which has evolved in order to protect multicellular organisms from infectious microorganisms. It can be divided into the evolutionary older innate (or natural) immunity and adaptive (or acquired) immunity. The innate immune system recognizes patterns, which are usually common and essential for pathogens. For this limited number of molecular structures germ-line encoded receptors have evolved. By contrast, cells of the adaptive immune system—B and T lymphocytes—can recognize a huge variety of antigenic structures. The receptors, termed according to the cell types expressing them, B cell receptor (BCR, its soluble versions are called antibodies) and T cell receptor (TCR, only cell-surface associated forms) are generated by somatic recombination and show a clonal distribution. Thus, initially there is only small number of cells with a certain specificity. Upon antigen encounter these cells start to divide (clonal expansion) to generate an effector population able to cope with the antigen. After elimination of antigen a specialized sub-population of cells specifically recognizing this antigen remains as immunological memory. Taken together the adaptive immune system is slow (compared to innate immunity), however specific and it improves upon repeated exposure to a given pathogen/antigen.

T cells have a central role in adaptive immunity. Their receptors (TCRs) recognize “major histocompatibility complex” (MHC or HLA):peptide complexes on the surface of cells. These peptides are called T cell epitopes and represent degradation products of antigens. There are two major classes of T cells: CD8-positive cytotoxic T cells (CTL) are restricted to MHC class I. CD4-positive helper T cells (HTL) are restricted to MHC class II. HTL are essential for many features of adaptive immunity: activation of so called “professional antigen-presenting cells” (APCs), immunoglobulin (Ig) class switch, the germinal center reaction and Ig affinity maturation, activation of CTL, immunological memory, regulation of the immune response and others.

MHC molecules collect peptides inside the cell and present them on the cell surface to TCRs of T cells. There are two major classes of MHC, class I recognized by CD8-positive CTL and class II recognized by CD4-positive HTL.

MHC class I molecules consist of a membrane-anchored alpha-chain of 45 kDa and the non-covalently attached b2-microglobulin (b2m) of 12 kDA. Resolution of the 3-dimensional structure by X-ray crystallography (Stern and Wiley 1994) revealed that the alpha-chain possesses a cleft, which is closed at both ends and accommodates peptides from 8 to 11 amino acids length. Class I molecules are ubiquitously expressed, and the peptides they present originate from cytoplasmic proteins. These are degraded by the proteasome, and the resulting peptides are actively transported into the endoplasmatic reticulum (ER). There, with the help of several chaperones, MHC:peptide complexes are formed and transported to the cell surface (Heemels 1995). Thus, MHC class I mirrors the proteome of a cell on its surface and allows T cells to recognize intracellular pathogens or malignant cells.

MHC class II molecules consist of two membrane-anchored proteins (alpha- and beta-chain) of 35 kDa and 30 kDa, respectively. These together form a cleft, open at both ends, which can accommodate peptides of variable length, usually from 12 to 25 amino acids. Despite these differences, class I and II molecules share surprising structural similarity (Stern and Wiley 1994). Class II molecules are only expressed on professional APC including dendritic cells (DC), B-cells and macrophages/monocytes. These cells are specialized in taking up and processing antigens in the endosomal pathway. Immediately after their biosynthesis, class II molecules are complexed by the so-called invariant chain (Ii), which prevents binding of peptides in the ER. When vesicles containing class II:Ii complexes fuse with endosomes containing degradation products of exogenous antigen, Ii is degraded until the MHC binding cleft is only complexed by the so-called CLIP peptide. The latter is with the help of chaperones like HLA-DM exchanged by antigenic peptides (Villadangos 2000). Finally, MHC:peptide complexes are again presented on the surface of APCs, which interact in numerous ways with HTL.

Being both polygenic and extremely polymorphic, the MHC system is highly complex. For the class I alpha-chain in humans there are three gene loci termed HLA-A, -B and -C. Likewise, there are three class II alpha-chain loci (DRA, DQA, DPA); for class II beta-chain loci the situation is even more complex as there are four different DR beta-chains (DRB1,2,3,5) plus DQB and DPB. Except the monomorphic DR alpha-chain DRA, each gene locus is present in many different alleles (dozens to hundreds) in the population (Klein 1986). Different alleles have largely distinct binding specificities for peptides. Alleles are designated, for example, HLA-A*0201 or HLA-DRB1*0401 or HLA-DPA*0101/DPB*0401.

T cell epitopes have been identified by a variety of approaches (Van den Eynde 1997). T cell lines and clones have for instance been used to screen cDNA expression libraries for instance in the context of COS cells transfected with the appropriate HLA-molecule. Alternatively, biochemical approaches have been pursued. The latter involved elution of natural ligands from MHC molecules on the surface of target cells, the separation of these peptides by several chromatography steps, analysis of their reactivity with lymphocytes in epitope reconstitution assays and sequencing by mass spectrometry (Wölfel et al. 1994, Cox et al. 1994).

Recently the advent of highly sensitive cytokine detection assays like the IFN-gamma ELIspot allowed using lymphocytes directly ex vivo for screening of overlapping synthetic peptides (Maecker 2001, Kern 2000, Tobery 2001). Primarily, Kern et al. (1999&2000) used arrays of pools of overlapping 9mer peptides to map CD8+ T cell epitopes in vitro. Later, Tobery et al., 2001 modified this approach and demonstrated that pools containing as many as 64 20mer peptides may be used to screen for both CD8+ and CD4+ T cell epitopes in mice. Both these methods were based on the monitoring of antigen-specific response by measuring INF-gamma production either by intracellular staining (Kern et al 2000) or in ELIspot assay (Tobery et al., 2001). By use of mixtures of 15-mers the CD4+ T cell responses are approximately equal to those detected when whole soluble protein was used as an antigen, while—not surprising—the CD8+ T cell responses are significantly higher than the often negligible responses detected with soluble protein stimulation. Furthermore, the CD8+ T cell responses to a mixture of 15 amino acid peptides are similar to those obtained with a mix of 8-12 amino acid peptides, selected to represent known MHC class I minimal epitopes. Most probably peptidases associated with the cell membrane are responsible for “clipping” peptides to optimal length under these circumstances (Maecker et al, 2001).

An interesting alternative is to screen synthetic combinatorial peptide libraries with specific lymphocytes. For instance, a decapeptide library consisting of 200 mixtures arranged in a positional scanning format, has been successfully used for identification of peptide ligands that stimulate clonotypic populations of T cells (Wilson, et al., J. Immunol., 1999, 163:6424-6434).

Many T cell epitopes have been identified by so called “Reverse immunological approaches” Rammensee 1999). In this case the protein giving rise to a potential T cell epitope is known, and its primary sequence is scanned for HLA binding motifs. Typically dozens to hundreds of candidate peptides or even a full set of overlapping peptides are synthesized and tested for binding to HLA molecules. Usually, the best binders are selected for further characterization with regard to their reactivity with T cells. This can for instance be done by priming T cells in vitro or in vivo with the help of HLA transgenic mice.

Hepatitis C Virus (HCV) is a member of the flaviviridiae chronically infecting about 170 million people worldwide. There are at least 6 HCV genotypes and more than 50 subtypes have been described. In America, Europe and Japan genotypes 1, 2 and 3 are most common. The geographic distribution of HCV genotypes varies greatly with genotype 1a being predominant in the USA and parts of Western Europe, whereas 1b predominates in Southern and Central Europe (Bellentani 2000).

HCV is transmitted through the parenteral or percutan route, and replicates in hepatocytes. About 15% of patients experience acute self-limited hepatitis associated with viral clearance and recovery. About 80% of infected persons become chronic carriers. Infection often persists asymptomatically with slow progression for years, however ultimately HCV is a major cause of cirrhosis, end-stage liver disease and liver cancer (Liang 2000). Strength and quality of both HTL and CTL responses determine whether patients recover (spontaneously or as a consequence of therapy) or develop chronic infection (Liang 2000).

Standard therapy of HCV comprises a combination of pegylated interferon-alpha and the antiviral ribavirin. Virologic responses are, depending on the genotype, achieved in about 50% of HCV patients. The low tolerability and the considerable side effects of this therapy clearly necessitate novel therapeutic intervention including therapeutic vaccines (Cornberg 2002). However, presently the detailed understanding of which epitopes in which MHC combination lead to successful immune responses is lacking (Ward 2002). Therefore, a comprehensive analysis of the T-cell response against the entire HCV is required for development of therapeutic epitope-based vaccines.

The HCV virion contains a 9.5-kilobase positive single-strand RNA genome encoding a large single polyprotein of about 3000 amino acids. The latter is processed to at least 10 proteins by both host and HCV-enoded proteolytic activities (Liang 2000). Importantly, the HCV RNA-dependent RNA polymerase is error prone giving rise to the evolution of viral quasispecies and contributing to immune-escape variants (Farci 2000).

It is an object of the present invention to provide a method for screening HCV-peptides for specific MHC molecules, preferably for delivering suitable and specific HCV T cell epitopes selected from a variety of HCV-peptides having unknown specificity for a given MHC molecule and thereby to provide efficient means for preventing and combatting HCV infections.

Therefore the present invention provides a method for isolating HCV-peptides which have a binding capacity to a MHC/HLA molecule or a complex comprising said HCV-peptide and said MHC/HLA molecule which method comprises the following steps:

• • providing a pool of HCV-peptides, said pool containing HCV-peptides which bind to said MHC/HLA molecule and HCV-peptides which do not bind to said MHC/HLA molecule,
  • contacting said MHC/HLA molecule with said pool of HCV-peptides whereby a HCV-peptide which has a binding capacity to said MHC/HLA molecule binds to said MHC/HLA molecule and a complex comprising said HCV-peptide and said MHC/HLA molecule is formed,
  • detecting and optionally separating said complex from the HCV-peptides which do not bind to said MHC/HLA molecule and
  • optionally isolating and characterising the HCV-peptide from said complex.

The present invention also provides a method for isolating HCV T cell epitopes which have a binding capacity to a MHC/HLA molecule or a complex comprising said epitope and said MHC/HLA molecule which method comprises the following steps:

• • providing a pool of HCV-peptides, said pool containing HCV-peptides which bind to a MHC/HLA molecule and HCV-peptides which do not bind to said MHC/HLA molecule,
  • contacting said MHC/HLA molecule with said pool of HCV-peptides whereby a HCV-peptide which has a binding capacity to said MHC/HLA molecule binds to said MHC/HLA molecule and a complex comprising said HCV-peptide and said MHC/HLA molecule is formed,
  • detecting and optionally separating said complex from the HCV-peptides which do not bind to said MHC/HLA molecule,
  • optionally isolating and characterising the HCV-peptide from said complex,
  • assaying said optionally isolated HCV-peptide or said complex in a T cell assay for T cell activation capacity and
  • providing the optionally isolated HCV-peptide with a T cell activation capacity as HCV T cell epitope or as complex.

The method according to the present invention enables a screening system for screening binding capacity to specific MHC/HLA molecules. Identifying MHC binding molecules is an important tool for molecular characterisation of pathogens, tumors, etc. It is therefore possible with the present invention to screen a variety (a “pool”) of potential HCV-peptides as ligands at once for their functional affinity towards MHC molecules. Binding affinity towards MHC molecules is also a necessary prerequisite for HCV-peptides intended to be used as T cell epitopes, although not a sufficient one. Suitable HCV T cell epitope candidates have also to be screened and assayed with respect to their T cell activation capacity. The combination of the screening method for binding according to the present invention with a suitable T cell assay therefore provides the method for isolating HCV T cell epitopes according to the present invention wherein such T cell epitopes are identifiable out of a pool of potential HCV-peptides using an MHC binding assay.

In contrast to the prior art, where such assays have always been performed on ligands with known binding/MHC specificity, the methods according to the present invention provide such assays as a screening tool for pools with ligands of unknown specificity. In the prior art such assays have been typically performed on individual single ligands, to test their binding affinity to MHC/HLA molecules. In Kwok et al. (2001) pools of maximally up to 5 overlapping synthetic peptides were used to generate MHC class II tetramers; the latter were then used to stain PBMC for T cells specific for particular MHC class II:peptide complexes which were generated in the binding reaction with the pools of 5 peptides. However, an increase in the number of ligands per pool in such an approach was not regarded as being possible, both for sensitivity and specificity reasons (Novak et al. 2001). A problem with regard to specificity would be the generation of MHC tetramers with more then one binder per tetramer, if more than one binder would be present in the pool. This would preclude staining of T cells, which is used for identification of epitopes in the approach described in the prior art. In strong contrast to that the approach according to the present invention allows the identification of more than on binder out of highly complex mixtures containing more than one binder.

The nature of the pool to be screened with the present invention is not critical: the pools may contain any naturally or not naturally occurring HCV-peptide which a) binds specifically to MHC/HLA molecules and/or b) may be specifically recognized by T cells. The binding properties of the set of HCV-peptides of the pool with respect to MHC molecules is not known; therefore, usually binders and at least a non-binder for a given MHC molecule are contained in the pool. The pool therefore comprises at least ten different HCV-peptides. Practically, pools are used according to the present invention containing significantly more different HCV-peptide species, e.g. 20 or more, 100 or more, 1.000 or more or 10.000 or more. It is also possible to screen larger libraries (with e.g. more than 10⁶, more than 10⁸ or even more than 10¹⁰ different HCV-peptide species). This, however, is mainly dependent on the availability of such HCV-peptide libraries.

Preferred pools of ligands to be used in the method according to the present invention are selected from the group consisting of a pool of peptides, especially overlapping peptides, a pool of protein fragments, a pool of modified peptides, a pool obtained from antigen-presenting cells, preferably in the form of total lysates or fractions thereof, especially fractions eluted from the surface or the MHC/HLA molecules of these cells, a pool comprised of fragments of cells, especially HCV containing cells, tumor cells or tissues, especially from liver, a pool comprised of peptide libraries, pools of (poly)-peptides generated from recombinant DNA libraries, especially derived from pathogens or (liver) tumor cells, a pool of proteins and/or protein fragments from HCV or mixtures thereof.

The HCV-peptides of the pools may be derived from natural sources (in native and/or derivatised form) but also be produced synthetically (e.g. by chemical synthesis or by recombinant technology). If (poly)peptide ligands are provided in the pools, those peptides are preferably generated by peptide synthesizers or by recombinant technology. According to a preferred embodiment, a pool of (poly)peptides may be generated from recombinant DNA libraries, e.g. derived from HCV or HCV containing (tumor) cells, by in vitro translation (e.g. by ribosome display) or by expression through heterologous hosts like E. coli or others.

The nature of the specific MHC molecules (of course also MHC-like molecules are encompassed by this term) to be selected for the present methods is again not critical. Therefore, these molecules may be selected in principle from any species, especially primates like humans (HLA, see below), chimpanzees, other mammals, e.g. maquaques, rabbits, cats, dogs or rodents like mice, rats, guinea pigs and others, agriculturally important animals like cattle, horses, sheep and fish, although human (or “humanized”) molecules are of course preferred for providing vaccines for humans. For providing vaccines for specific animals, especially agriculturally important animals, like cattle, horses, sheep and fish, the use of MHC molecules being specific for these animals is preferred.

Preferred HLA molecules therefore comprise Class I molecules derived from the HLA-A, -B or -C loci, especially A1, A2, A3, A24, A11, A23, A29, A30, A68; B7, B8, B15, B16, B27, B35, B40, B44, B46, B51, B52, B53; Cw3, Cw4, Cw6, Cw7; Class II molecules derived from the HLA-DP, -DQ or -DR loci, especially DR1, DR2, DR3, DR4, DR7, DR8, DR9, DR11, DR12, DR13, DR51, DR52, DR53; DP2, DP3, DP4; DQ1, DQ3, DQ5, DQ6; and non-classical MHC/HLA and MHC/HLA-like molecules, which can specifically bind ligands, especially HLA-E, HLA-G, MICA, MICB, Qa1, Qa2, T10, T18, T22, M3 and members of the CD1 family.

According to a preferred embodiment, the methods according to the present invention is characterised in that said MHC/HLA molecules are selected from HLA class I molecules, HLA class II molecules, non classical MHC/HLA and MHC/HLA-like molecules or mixtures thereof, or mixtures thereof.

Preferably, the optional characterising step of the HCV-peptides of the complex is performed by using a method selected from the group consisting of mass spectroscopy, polypeptide sequencing, binding assays, especially SDS-stability assays, identification of ligands by determination of their retention factors by chromatography, especially HPLC, or other spectroscopic techniques, especially violet (UV), infra-red (IR), nuclear magnetic resonance (NMR), circular dichroism (CD) or electron spin resonance (ESR), or combinations thereof.

According to a preferred embodiment the method of the present invention is characterised in that it is combined with a cytokine secretion assay, preferably with an Elispot assay, an intracellular cytokine staining, FACS or an ELISA (enzyme-linked immunoassays) (see e.g. Current Protocols in Immunology).

Preferred T cell assays comprise the mixing and incubation of said complex with isolated T cells and subsequent measuring cytokine secretion or proliferation of said isolated T cells and/or the measuring up-regulation of activation markers, especially CD69, CD38, or down-regulation of surface markers, especially CD3, CD8 or TCR and/or the measuring up-/down-regulation of mRNAs involved in T cell activation, especially by real-time RT-PCR (see e.g. Current Protocols in Immunology, Current Protocols in Molecular Biology).

Further preferred T cell assays are selected from T cell assays measuring phosphorylation/de-phosphorylation of components downstream of the T cell receptor, especially p56 lck, ITAMS of the TCR and the zeta chain, ZAP70, LAT, SLP-76, fyn, and lyn, T cell assays measuring intracellular Ca⁺⁺ concentration or activation of Ca⁺⁺-dependent proteins, T cell assays measuring formation of immunological synapses, T cell assays measuring release of effector molecules, especially perforin, granzymes or granulolysin or combinations of such T cell assays (see e.g. Current Protocols in Immunology, Current Protocols in Cell Biology).

In order to identify the molecular determinants of immune-protection against HCV a specific method of epitope capturing was applied using synthetic peptides representing the conserved parts of HCV genotypes 1, 2 and 3. Focusing on conserved regions ensures broad applicability of the epitopes. Moreover, these regions probably cannot easily be mutated by the virus, thus minimizing the danger of evolution of immune-escape variants.

With the methods of the present invention novel HCV-epitopes are detected. According to a further aspect, the present invention therefore also provides HCV T cell epitopes identifiable by a method according to the present invention, said T cell epitopes preferably being selected from the group consisting of polypeptides comprising the peptides A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0401, *0404, *0701 and thus covering at least 45-55% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides 1630, C97, 1547, B94-B98, A272-A276 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0401, *0701 and thus covering at least 40-50% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides B120, B122, C108, C134, C152 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0404, *0701 and thus covering at least 45% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides 1606, 1607, 1577, 1578 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0401, *0404, *0701 and thus covering at least 45% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides B50-52, 1623, C130 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0401, *0404 and thus covering at least 40% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides 1603, C96 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0701 and thus covering at least 40% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides C191 according to Table 1, being a novel ligand for at least HLA-DRB1*0401, *0701 and thus covering at least 40% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides A216-A224, A242-A244, C92-C93 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0401 and thus covering at least 35% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptide A174 according to Table 1 or 2, being a novel ligand for at least HLA-DRB1*0404, *0701 and thus covering at least 25-30% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides B32-B38, B100-B102, C135 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101, *0404 and thus covering at least 20-25% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptide C162 according to Table 1 or 2, being a novel ligand for at least HLA-DRB1*0401, *0404 and thus covering at least 20-25% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides 1618, 1622, 1624, 1546, 1556 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0701 and thus covering at least 25% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, C174, 1558, 1581 according to Table 1 or 2. These peptides are novel ligands for at least HLA-DRB1*0101 and thus covering at least 20% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptide C95, being a novel ligand for at least HLA-DRB1*0401 and thus covering at least 20% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides C129, C157-C158, A254-A258, 1605, C109, C161 according to Table 1 or 2. These peptides comprising novel ligands for at least HLA-DRB1*0404 and thus covering at least 5% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising the peptides 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1604, 1605, 1616, 1621, 1623, 1625, 1627, 1630, 1649, 1650, 1651, 1652, 1654, 1655, 1656 according to Table 1 or 2, these peptides displaying immunogenicity in HLA-DRB1*0401 transgenic mice (see Example II) and thus representing or containing a confirmed HLA class II T-cell epitope binding to at least HLA-DRB1*0401 (see Tab. 3).

Preferred polypeptides are selected from the group comprising the peptides 1545, 1552, 1555, 1558, 1559, 1560, 1577, 1592, 1604, 1605, 1615, 1617, 1621, 1627, 1631, 1632, 1641, 1647, 1650, 1651, 1652, 1653, 1654, 1655 according to Table 1 or 2, these peptides displaying immunogenicity in HLA-A*0201 transgenic mice (see Example II) and thus representing or containing a confirmed HLA class I T-cell epitope binding to at least HLA-A*0201 (see Tab. 3).

Preferred polypeptides which are shown to be HLA-B*0702 epitopes with T-cell activating capacity are selected from the group consisting of polypeptides 1506, 1526, 1547, 1552, 1553, 1555, 1558, 1562, 1563, 1565, 1577, 1578, 1580, 1587, 1592, 1604, 1605, 1621, 1623, 1624, 1627, 1628, 1647, 1650, 1651, 1843 with sequence LPRRGPRL (SEQ ID NO:163) (contained in 1506) and 1838 with sequence SPGALVVGVI (SEQ ID NO:164) (contained in 1587) as minimal HLA-B*0702 epitopes.

Peptides 1526, 1565, 1631 are also shown to be immunogenic in HLA-DRB1*0401 transgenic mice contain known class II epitopes. Peptides 1526, 1553, 1565, 1587, 1623, 1630 are also shown to be immunogenic in HLA-A*0201 transgenic mice contain known A2 epitopes.

Preferred polypeptides are selected from the group comprising the peptides listed in tables 3, 5 and the bold peptides in 7 (“hotspots”).

The preferred polypeptides mentioned above also include all fragments containing the minimal sequence of the epitope, i.e. the 8- or 9-mer being necessary for binding to MHC/HLA molecules.

Preferably, the epitopes or peptides according to the present invention further comprises 1 to 30, preferably 2 to 10, especially 2 to 6, naturally occurring amino acid residues at the N-terminus, the C-terminus or at the N- and C-terminus. For the purposes of the present invention the term “naturally occurring” amino acid residue relates to amino acid residues present in the naturally occurring protein at the specific position, relative to the epitope or peptide. For example, for the HLA-A2 epitope with the amino acid sequence HMWNFISGI (SEQ ID NO:192) contained within peptide ID 1565 (Tab. 1), the naturally occurring amino acid residue at the N-terminus is -K; the three naturally occurring amino acid residues at the C-terminus are -QYL. A “non-naturally occurring” amino acid residue is therefore any amino acid residue being different as the amino acid residue at the specific position relative to the epitope or peptide.

According to a preferred embodiment of the present invention, the present epitopes or peptides further comprise non-naturally occurring amino acid(s), preferably 1 to 1000, more preferred 2 to 100, especially 2 to 20 non-naturally occurring amino acid residues, especially at the N-terminus, the C-terminus or at the N- and C-terminus. Also combinations of non-naturally and naturally occurring amino acid residues are possible under this specific preferred embodiment. The present epitope may also contain modified amino acids (i.e. amino acid residues being different from the 20 “classical” amino acids, such as D-amino acids or S-S bindings of Cys) as additional amino acid residues or in replacement of a naturally occurring amino acid residue.

It is clear that also epitopes or peptides derived from the present epitopes or peptides by amino acid exchanges improving, conserving or at least not significantly impeding the T cell activating capability of the epitopes are covered by the epitopes or peptides according to the present invention. Therefore, the present epitopes or peptides also cover epitopes or peptides, which do not contain the original sequence as derived from a specific strain of HCV, but trigger the same or preferably an improved T cell response. These epitopes are referred to as “heteroclitic”. These include any epitope, which can trigger the same T cells as the original epitope and has preferably a more potent activation capacity of T cells preferably in vivo or also in vitro. Also the respective homologous epitopes from other strains of HCV are encompassed by the present invention.

Heteroclitic epitopes can be obtained by rational design i.e. taking into account the contribution of individual residues to binding to MHC/HLA as for instance described by Ramensee et al. 1999 or Sturniolo et al. 1999, combined with a systematic exchange of residues potentially interacting with the TCR and testing the resulting sequences with T cells directed against the original epitope. Such a design is possible for a skilled man in the art without much experimentation.

Another possibility includes the screening of peptide libraries with T cells directed against the original epitope. A preferred way is the positional scanning of synthetic peptide libraries. Such approaches have been described in detail for instance by Blake et al 1996 and Hemmer et al. 1999 and the references given therein.

As an alternative to epitopes represented by the cognate HCV derived amino acid sequence or heteroclitic epitopes, also substances mimicking these epitopes e.g. “peptidemimetica” or “retro-inverso-peptides” can be applied.

Another aspect of the design of improved epitopes is their formulation or modification with substances increasing their capacity to stimulate T cells. These include T helper cell epitopes, lipids or liposomes or preferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes is their formulation with immune stimulating substances for instance cytokines or chemokines like interleukin-2, -7, -2, -18, class I and II interferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligand and others.

According to a further aspect, the present invention is drawn to the use of a HCV epitope or HCV peptide according to the present invention for the preparation of a HLA restricted vaccine for treating or preventing hepatitis C virus (HCV) infections.

The invention also encompasses the use of an epitope according to the present invention for the preparation of a vaccine for treating or preventing hepatitis C virus (HCV) infections.

Consequently, the present invention also encompasses a vaccine for treating or preventing hepatitis C virus (HCV) infections comprising an epitope according to the present invention.

Furthermore, also a HLA specific vaccine for treating or preventing hepatitis C virus (HCV) infections comprising the epitopes or peptides according to the present invention is an aspect of the present invention.

Preferably, such a vaccine further comprises an immunomodulating substance, preferably selected from the group consisting of polycationic substances, especially polycationic polypeptides, immunomodulating nucleic acids, especially deoxyinosine and/or deoxyuracile containing oligodeoxynucleotides, or mixtures thereof.

Preferably the vaccine further comprises a polycationic polymer, preferably a polycationic peptide, especially polyarginine, polylysine or an antimicrobial peptide.

The polycationic compound(s) to be used according to the present invention may be any polycationic compound, which shows the characteristic effect according to the WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyaminoacids or mixtures thereof. These polyaminoacids should have a chain length of at least 4 amino acid residues. Especially preferred are substances containing peptidic bounds, like polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% of basic amino acids in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantly or may be derived from natural sources.

Cationic (poly)peptides may also be polycationic anti-bacterial microbial peptides. These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly. Peptides may also belong to the class of defensines. Such host defense peptides or defensines are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substance in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (WO 02/13857), incorporated herein by reference), especially antimicrobial peptides derived from mammal cathelicidin, preferably from human, bovine or mouse, or neuroactive compounds, such as (human) growth hormone (as described e.g. in WO01/24822).

Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are cathelin or related or derived substances from cathelin, especially mouse, bovine or especially human cathelins and/or cathelicidins. Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino acids, which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen/vaccine composition according to the present invention. However, these cathelin molecules surprisingly have turned out to be also effective as an adjuvant for a antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without further immunactivating substances.

Another preferred polycationic substance to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids, especially L (WO 02/32451, incorporated herein by reference).

The immunomodulating (or:immunogenic) nucleic acids to be used according to the present invention can be of synthetic, prokaryotic and eukaryotic origin. In the case of eukaryotic origin, DNA should be derived from, based on the phylogenetic tree, less developed species (e.g. insects, but also others). In a preferred embodiment of the invention the immunogenic oligodeoxynucleotide (ODN) is a synthetically produced DNA-molecule or mixtures of such molecules. Derivatives or modifications of ODNs such as thiophosphate substituted analogues (thiophosphate residues substitute for phosphate) as for example described in US patents U.S. Pat. No. 5,723,335 and U.S. Pat. No. 5,663,153, and other derivatives and modifications, which preferably stabilize the immunostimulatory composition(s) but do not change their immunological properties, are also included. A preferred sequence motif is a six base DNA motif containing an (unmethylated) CpG dinucleotide flanked by two 5′ purines and two 3′ pyrimidines (5′-Pur-Pur-C-G-Pyr-Pyr-3′). The CpG motifs contained in the ODNs according to the present invention are more common in microbial than higher vertebrate DNA and display differences in the pattern of methylation. Surprisingly, sequences stimulating mouse APCs are not very efficient for human cells. Preferred palindromic or non-palindromic ODNs to be used according to the present invention are disclosed e.g. in Austrian Patent applications A 1973/2000, A 805/2001, EP 0 468 520 A2, WO 96/02555, WO 98/16247, WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581, WO 98/52962, WO 99/51259 and WO 99/56755 all incorporated herein by reference. Apart from stimulating the immune system certain ODNs are neutralizing some immune responses. These sequences are also included in the current invention, for example for applications for the treatment of autoimmune diseases. ODNs/DNAs may be produced chemically or recombinantly or may be derived from natural sources. Preferred natural sources are insects.

Alternatively, also nucleic acids based on inosine and cytidine (as e.g. described in the WO 01/93903) or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and PCT/EP 02/05448, incorporated herein by reference) may preferably be used as immunostimulatory nucleic acids for the present invention.

Of course, also mixtures of different immunogenic nucleic acids may be used according to the present invention.

Preferably, the present vaccine further comprises a pharmaceutically acceptable carrier.

According to a further preferred embodiment, the present vaccine comprises an epitope or peptide which is provided in a form selected from peptides, peptide analogues, proteins, naked DNA, RNA, viral vectors, virus-like particles, recombinant/chimeric viruses, recombinant bacteria or dendritic cells pulsed with protein/peptide/RNA or transfected with DNA comprising the epitopes or peptides.

According to a further aspect, the present invention is drawn to T cells, a T cell clone or a population (preparation) of T cells specifically recognizing any HCV epitope or peptide according to the present invention, especially a HCV epitope as described above. A preferred application of such T cells is their expansion in vitro and use for therapy of patients e.g. by adoptive transfer. Therefore, the present invention also provides the use of T cells, a T cell clone or a population (preparation) of T cells for the preparation of a composition for the therapy of HCV patients.

Such T cells (clones or lines) according to the present invention, specifically those recognizing the aforementioned HCV peptides are also useful for identification of heteroclitic epitopes, which are distinct from the originally identified epitopes but trigger the same T cells.

Such cells, compositions or vaccines according to the present invention are administered to the individuals in an effective amount.

According to a further aspect, the present invention also relates to the use of the peptides with formulae QRKTKRNTN (SEQ ID NO:167), QRKTKRNT (SEQ ID NO:166), or 1615, 1616, 1617 in particular 9meric peptides derived from the latter 3 peptides with formulae SAKSKFGYG (SEQ ID NO:193), SAKSKYGYG (SEQ ID NO:194), or SARSKYGYG (SEQ ID NO:195) as HLA-B*08 epitopes, especially for the preparation of a pharmaceutical preparation for a HLA-B*08 specific vaccine; the use of the peptides with the formulae RKTKRNTNR (SEQ ID NO:170) as HLA-B*2705 epitope, especially for the preparation of a pharmaceutical preparation for a HLA-B*2705 specific vaccine; and the use of the peptides with the formulae ARLIVFPDL (SEQ ID NO:1053) as HLA-B*2705 and HLA-B*2709 specific vaccine. Further, it also relates to the use of the hotspot epitopes selected from the group of peptides 1835, 84EX, 87EX, 89EX, 1426, 1650, 1836, 1846, 1651, 1800, 1799, C114, 1827, C112, C114EX, 1827EX, 1798, 1604, 1829, 1579, 1624, 1848, 1547, A1A7, A122EX, A122, 1825, A241, B8B38, C70EX, C92, C97, C106, and C134 according to table 7 for the preparation of a vaccine comprising synthetic peptides, recombinant protein and/or DNA constitutes of such epitopes.

In particular, two or more epitope hotspots can be combined, with or without linker sequences. Preferred linker sequences consist for instance of 3 to 5 glycine, or alanine or lysine residues. This may be achieved by peptide synthesis, However, combination of hotspots may result in quite long polypeptides. In this case, cloning DNA encoding for such constructs and expressing and purifying the corresponding recombinant protein

is an alternative. Such recombinant proteins can be used as antigens, which in combination with the right adjuvant (IC31, pR, . . . ) can elicit T-cell responses against all the epitopes they harbor. At the same time, such artificial polypeptides are devoid of the activities (enzymatic, toxic, immuno-suppressive, . . . ), the natural HCV antigens may possess.

There are several other ways of delivering T-cell epitope hotspots or combinations thereof. These include: recombinant viral vectors like vaccinia virus, canary pox virus, adenovirus; self-replicating RNA vectors; “naked DNA” vaccination with plasmids encoding the hotspots or combination thereof; recombinant bacteria (e.g. Salmonella); dendritic cells pulsed with synthetic peptides, or recombinant protein, or RNA or transfected with DNA, each encoding T-cell epitope hotspots or combinations thereof.

The invention will be explained in more detail by way of the following examples and drawing figures, to which, however it is not limited.

FIG. 1 shows 40 peptide mixtures each containing up to 20 HCV derived 15- to 23mer peptides.

FIG. 2 shows the Epitope Capture approach using peptide pools and empty DRB1*0401 molecules.

FIG. 3 shows the Epitope Capture approach using peptide pools and empty DRB1*0404 molecules.

FIG. 4 shows binding of individual peptides to DRB1*0401.

FIG. 5 shows binding of individual peptides to DRB1*0404.

FIG. 6 shows binding of individual peptides to DRB1*0101.

FIG. 7 shows peptides binding to DRB1*0701.

FIG. 8 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+ or CD4+ cells from HLA-DRB1*0401 tg mice vaccinated with Ipep1604+IC31.

FIG. 9 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+ or CD4+ cells from HLA-A*0201 tg mice vaccinated with Ipep1604+IC31.

FIG. 10 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+ or CD4+ cells from HLA-B*0702tg mice vaccinated with Ipep1604+IC31.

EXAMPLES

General Description of the Examples

The present examples show the performance of the present invention on a specific pathogen hepatitis C virus (HCV).

In the first part the method according to the present invention was applied, which is based on the use of “empty HLA molecules”. These molecules were incubated with mixtures of potential HCV derived peptide ligands, screening for specific binding events. The possibility to use highly complex mixtures allows a very quick identification of the few binders out of hundreds or even thousands of potential ligands. This is demonstrated by using HLA-DRB1*0101, -DRB1*0401, -DRB1*0404, -DRB1*0701 molecules and pools of overlapping 15- to 23mers. Importantly, this analysis using multiple different HLA-alleles allows identifying promiscuous ligands capable to binding to more than one HLA allele. Promiscuous T-cell epitopes are particularly valuable components of epitope-based vaccines. They enable treating a higher portion of a population than epitopes restricted to one HLA allele.

The same process can be applied for class I molecules and peptides of appropriate length i.e. 8 to 11-mers. The ligand-pools can be synthetic overlapping peptides. Another possibility is to digest the antigen in question enzymatically or non-enzymatically. The latter achieved by alkali-hydrolysis generates all potential degradation products and has been successfully used to identify T cell epitopes (Gavin 1993). Enzymatic digestions can be done with proteases. One rational way would further be to use proteases involved in the natural antigen-processing pathway like the proteasome for class I restricted epitopes (Heemels 1995) or cathepsins for class II restricted epitopes (Villadangos 2000). Ligand pools could also be composed of naturally occurring ligands obtained for instance by lysis of or elution from cells carrying the respective epitope. In this regard it is important to note that also non-peptide ligands like for instance glycolipids can be applied. It is known that nonclassical class I molecules, which can be encoded by the MHC (e.g. HLA-G, HLA-E, MICA, MICB) or outside the MHC (e.g. CD1 family) can present various non-peptide ligands to lymphocytes (Kronenberg 1999). Use of recombinant “empty” nonclassical class I molecules would allow binding reactions and identification of binders in similar manner as described here.

After rapid identification of ligands capable of binding to HLA molecules the process according to the present invention also offers ways to characterize directly specific T cell responses against these binders. One possibility is to directly use the isolated HLA:ligand complex in a so called “synthetic T cell assay”. The latter involves antigen-specific re-stimulation of T cells by the HLA:ligand complex together with a second signal providing co-stimulation like activation of CD28 by an activating antibody. This assay can be done in an ELIspot readout.

Another possibility is the immunization of HLA-transgenic mice to prove immunogenicity of ligands identified by the Epitope Capture approach as demonstrated in Example II.

Materials & Methods

Peptides

In order to identify conserved regions between HCV genotypes 1, 2 and 3, about 90 full genomes publicly available through Genebank were aligned. In total, 43% of the coding region of HCV was found to be conserved in at least 80% of clinical isolates. In cases, where at a certain position consistently two distinct amino acids (eg. arginine or lysine) were found, both variants were considered for analysis. Altogether 148 conserved regions, longer than 8 amino acids were identified. Conserved region were spanned by ˜500 fifteen amino acid residue (15mer) peptides, each peptide overlapping its precursor by 14 out of 15 amino acids. Conserved regions between 8 and 14 amino acids long were covered by further 80 (non-overlapping) 15mers. 15mers were synthesized using standard F-moc chemistry in parallel (288 at a time) on a Syro II synthesizer (Multisyntech, Witten, Germany). Each fourth 15mer was checked by mass spectrometry. 15mers were applied for experiments without further purification. In addition 63 peptides of 16-xx aa were synthesized using standard F-moc chemistry on an ABI 433A synthesizer (Applied Biosystems, Weiterstadt, Germany) and purified by RP-HPLC (Biocut 700E, Applied Biosystems, Langen, Germany) using a C18 column (either ODS ACU from YMC or 218TP, Vydac). Purity and identity were characterized by MALDI-TOF on a Reflex III mass-spectrometer (Bruker, Bremen, Germany). Peptides were solubilized in 100% DMSO at ˜10 mg/ml (˜5 mM). Stocks of peptide pools (20 peptides each) were made in 1000 DMSO at a final concentration of 0.5 mg/ml (˜0.25 mM) for each peptide. All peptides used in the present invention are listed in Table 1. Peptides YAR (YARFQSQTTLKQKT (SEQ ID NO:196)), HA (PKYVKQNTLKLAT (SEQ ID NO:197)), P1 (GYKVLVLNPSVAAT (SEQ ID NO:198)), P2 (HMWNFISGIQYLAGLSTLPGNPA (SEQ ID NO:199)), P3 (KFPGGGQIVGVYLLPRRRGPRL (SEQ ID NO:200)), P4 (DLMGYIPAV (SEQ ID NO:201)) and CLIP (KLPKPPKPVSKMRMATPLLMQALPM (SEQ ID NO:202)) were used as control peptides in binding assays.

Epitope Capture and Peptide Binding Assay

Soluble HLA class II DRA1*0101/DRB1*0101/Ii, DRA1*0101/DRB1*0401/Ii, DRA1*0101/DRB1*0404/Ii and DRA1*0101/DRB1*0701/Ii molecules were expressed in SC-2 cells and purified as described in Aichinger et al., 1997. In peptide binding reactions soluble DRB1*0101, DRB1*0401, DRB1*0404 molecules were used in a concentration of ˜0.5 μM, and each single peptide was added in 10-fold molar excess (5 μM) if not mentioned differently. The concentration of DMSO in the binding reaction did not exceed 4%. The reaction was performed in PBS buffer (pH 7.4) at room temperature for 48 hours in the presence of a protease inhibitor cocktail (Roche) and 0.1% octyl-beta-D-glucopyranoside (Sigma). Peptide binding was evaluated in an SDS-stability assay (Gorga et al., 1987): trimeric HLA class II alpha:beta:peptide complexes are resistant to SDS and consequently appear as ˜60 kDa band in SDS-PAGE Western blot analysis. Individual HLA class II alpha- and beta-chains not stabilized by bound peptide migrate as ˜35 kDa and 25 kDa bands, respectively. Briefly, HLA-peptide complexes were treated with 1% SDS at room temperature and resolved by SDS-PAGE run with 20 mA for approximately 2.5 hours at room temperature. Protein was transferred onto PVDF membrane by electroblotting, and stained with anti-alpha-chain TAL.1B5 or/and beta-chain MEM136 antibodies. For detection of Western-blot signals ECL solutions (Amersham) were used. For DRB1*0101 molecules HA and P1 peptides were used as controls for evaluation of strong binding, P2 peptide for intermediate binding and YAR as a negative control. For DRB1*0401 the strongest binding controls were YAR and HA peptides, while P1 and P2 served as an intermediate and weak binder, respectively. In the case of DRB1*0404 molecules P1 and P2 peptides were used to estimate strong binding, YAR peptide to control intermediate binding and HA peptide as an negative control. The binding affinities to DRB1*0701 were test by a peptide-competition assay (Reay et al., 1992). Briefly, binding of the biotinylated CLIP peptide with high affinity (reference peptide) has been used for monitoring of HLA:peptide complex formation. A testing peptide added to the binding reaction at an equimolar concentration to CLIP peptide could compete out CLIP when its affinity is higher or inhibit binding for 50% if its affinity is equal to affinity of CLIP. In the case of lower affinity peptides they should be added in excess to the reference peptide to compete for occupancy of HLA binding grove. The values of the concentration of competitor peptides required for 50% inhibition of reference peptide (biotinylated CLIP) binding (IC₅₀) can be used for evaluation of peptide binding affinities. Alternatively, comparing of the amount of reference peptide bound to HLA molecules in the presence or absence of competitor peptide one can determine the binding activity of the peptide of interest. In the present peptide-competition assay conditions of peptide binding were similar to described above. DRB1*0701 molecules were used in a concentration of ˜0.5 μM and biotinylated CLIP was added to all samples in the final concentration of 2 μM. Competitor peptides were added in three different concentrations: 2 μM, 20 μM and 200 μM. Binding reaction was performed in PBS buffer (pH 7.4) for 18 hours at 37° C. The amount of biotinylated CLIP associated with soluble DRB1*0701 molecules was determined by ELISA. Briefly, MaxiSorp 96-well plates (Nunc, Denmark) were coated with mouse anti-DR antibody L243 by overnight incubation with 50 μl of 10 μg/ml dilution in PBS at 4° C. Non-specific binding to wells awas blocked by incubation with T-PBS containing 3% of BSA for 2 hours at 37° C. and binding reactions were then “captured” for 2 hours at room temperature. Following extensive washing, HLA-associated peptide complexes were detected using alkaline phosphatase-streptavidin (Dako) and Sigma 104 phosphatase substrate. A microplate reader (VICTOR) was used to monitor optical density at 405 nm. Non-biotinylated CLIP, P1 and P2 peptides were used as positive controls to evaluate strong binding. Peptide P3 and P4 served as a weakly binding and non-binding control, respectively.

Immunization of HLA-Transgenic Mice

Immunogenicity of synthetic HCV-derived peptides was tested in HLA-DRB1*0401- and HLA-A*0201-transgenic mice as follows: Groups of 3 mice (female, 8 weeks of age) were injected subcutaneously into the flank (in total 100 μg of peptide+30 μg oligodinucleotide CpI (Purimex, Göttingen, Germany) per mouse). One week after the vaccination, spleens were removed and the splenocytes were activated ex vivo with the peptide used for vaccination and an irrelevant negative control peptide to determine IFN-gamma-producing specific cells (mouse ELISpot assay).

Mouse splenocyte ELIspot assay for single cell IFN-gamma release ELISpot plates (MAHA S4510, Millipore, Germany) were rinsed with PBS (200 μl/well), coated with anti-mouse IFN-gamma mAb (clone R46A2; 100 μl/well of 5 μg/ml in 0.1 M NaHCO₃, pH 9.2-9.5) and incubated overnight at 4° C. Plates were washed four times with PBS/0.1% Tween 20 and incubated with PBS/1% BSA (200 μl/well) at room temperature for 2 h to block nonspecific binding. Spleen cells from vaccinated mice were prepared and plated at 1×10⁶-3×10⁵ cells/well and incubated overnight at 37° C./5% CO₂ either in the presence of the immunizing antigen (peptide), control peptides or with medium alone. Subsequently, plates were washed four times and incubated with biotinylated anti-mouse IFN-gamma mAb (clone AN18.17.24, 100 μl/well of 2 μg/ml in PBS/1% BSA) for 2 h at 37° C. After washing, streptavidin-peroxidase (Roche Diagnostics, Vienna, Austria) was added ( 1/5000 in PBS, 100 μl/well) and plates were incubated at room temperature for 2 additional hours. Subsequently, substrate was added to the washed plates (100 μl/well of a mixture of 10 ml 100 mM Tris pH 7.5 supplemented with 200 μl of 40 mg/ml DAB stock containing 50 μl of 80 mg/ml NiCl₂ stock and 5 μl of 30% H₂O₂). The reaction was stopped after 20-30 minutes by washing the plates with tap water. Dried plates were evaluated with an ELISpot reader (BIOREADER 2000, BioSys, Karben, Germany).

IFN-Gamma ELIspot with Human PBMC

PBMC from HCV RNA-negative therapy responders or subjects spontaneously recovered were collected and HLA-typed serologically. Whole blood was collected in ACD Vacutainer tubes (Becton Dickinson Europe, Erembodegem, Germany). PBMC were isolated on Lymphoprep (Nycomed Pharma AS, Oslo, Norway) using Leuco-sep tubes (Greiner, Frickenhausen, Germany), washed 3× with PBS (Invitrogen Life Technologies (formerly GIBCOBRL), Carlsbad, Calif., USA) and resuspended at a concentration of 2×10⁷/ml in freezing medium consisting of 4 parts RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM 2-mercaptoethanol (all from Invitrogen Life Technologies), 9 parts foetal bovine serum (FCS; from PAA, Linz, Austria) and 1 part DMSO (SIGMA, Deisenhofen, Germany). PBMC were stored over night in 1° C. freezing containers (Nalgene Nunc International, Rochester, N.Y., USA) at −80° C. and then transferred into liquid nitrogen. The ELIspot assay was essentially done as described (Lalvani et al.). Briefly, Multi Screen 96-well filtration plates MAIP S4510 (Millipore, Bedford, Mass.) were coated with 10 μg/ml (0.75 μg/well) anti-human IFN-g monoclonal antibody (Mab) B140 (Bender Med Systems, Vienna, Austria) over night at 4° C. Plates were washed 2 times with PBS (Invitrogen Life Technologies) and blocked with ELIspot medium (RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM 2-mercaptoethanol (all from Invitrogen Life Technologies) and 10% human serum type AB (PAA, Linz, Austria). Cryo-preserved PBMC were thawed quickly in a 37° C. water bath, washed 1× with ELISPOT medium and incubated overnight (37° C., 5% CO₂). The next day cells were plated at 200,000 PBMC/well and co-cultivated with either individual peptides (10 μg/ml) or peptide pools (each peptide at a final concentration of 5 μg/ml) for 20 hrs. After removing cells and washing 6 times with wash buffer (PBS; 0.1% Tween 20 from SIGMA), 100 μl of a 1:10000 dilution (0.015 μg/well) of the biotinylated anti-human IFN-γ MAb B308-BT2 (Bender Med Systems), was added for an incubation of 2 hrs at 37° C. or alternatively for over night at 4° C. After washing, Streptavidin-alkaline phosphatase (DAKO, Glostrup, Denmark) was added at 1.2 μg/ml for 1 hr at 37° C. The assay was developed by addition of 100 μl/well BCIP/NBT alkaline phosphatase substrate (SIGMA).

In Vitro Priming of Human PBMCs

Human PBMCs are repeatedly stimulated with antigen (peptide or peptide mixture) in the presence of IL-2 and IL-7. This leads to the selective oligoclonal expansion of antigen-specific T cells. Responses against individual epitopes can be assessed for instance by IFN-γ ELIspot assays. Freshly thawed PBMCs were cultured in 6 well plates (2-4×10⁶/mL viable cells) in RPMI-1640 (GibcoBRL), 1% non-essential amino acids (GibcoBRL, cat# 11140-035), 1% Penicillin (10,000 U/ml)-Streptomycin (10,000 μg/ml) (GibcoBRL, cat#15140-122), 1% L-Glutamine (GibcoBRL), 0.1% beta-mercapto-ethanol (GibcoBRL), 1% Na-pyruvate (GibcoBRL), plus 10% Human AB serum (PAA, Linz, Austria). Peptides (10 μM each) were added to each well. rhIL-7 (Strathmann Biotech) was added at 10 ng/mL final concentration. 20-30 U/mL rhIL-2 (Strathmann Biotech) were added on day 4. On day 10, all cells were removed from plates, washed once in media (as above), and counted. For the next cycle of in vitro priming, viable cells were co-cultivated with autologous gamma irradiated (1.2 gray/min, for 20 minutes) PBMC as feeders (plated at 100,000 per well) and peptides, rh-IL-2 as described above. ELIspot was done as described above, except that 200,000 responder cells (pre-stimulated for 2 rounds of in vitro priming) were used together with 60,000 autologous irradiated responder cells.

Example I

Rapid Identification of Promiscuous HLA-Binding Peptides from HCV by Measuring Peptide Pools Arrayed in Matrix Format

To span conserved regions within the HCV polyprotein more than 640 peptides were synthesized (Table 1). For rapid identification of HLA ligands and novel T-cell epitopes, 40 peptide pools each containing 20 single peptides were prepared. The pools were constructed in a way that each peptide was present in 2 pools (matrix format). This allows identification of reactive peptides at the crossover points of row- and column mixtures (FIG. 1 HCV peptide matrix).

TABLE 1

Synthetic peptides derived from conserved regions of HCV

PEP-

PEP-

PEP-

PEP-

TIDE

SEQ

TIDE

SEQ

TIDE

SEQ

TIDE

SEQ

ID

ID

PEPTIDE

ID

ID

PEPTIDE

ID

ID

PEPTIDE

ID

ID

PEPTIDE

A1

203

MSTNPKPQRKTKRNT

A79

360

HRSRNVGKVIDTLTC

A157

517

CTVNYTIFKIRMYVG

A235

674

ADGGCSGGAYDIIIC

A2

204

STNPKPQRKTKRNTN

A80

361

RSRNVGKVIDTLTCG

A158

518

TVNYTIFKIRMYVGG

A236

675

DGGCSGGAYDIIICD

A3

205

TNPKPQRKTKRNTNR

A81

362

SRNVGKVIDTLTCGF

A159

519

VNYTIFKIRMYVGGV

A237

676

GGCSGGAYDIIICDE

A4

206

NPKPQRKTKRNTNRR

A82

363

RNVGKVIDTLTCGFA

A160

520

NYTIFKIRMYVGGVE

A238

677

GCSGGAYDIIICDEC

A5

207

PKPQRKTKRNTNRRP

A83

364

NVGKVIDTLTCGFAD

A161

521

YTIFKIRMYVGGVEH

A239

678

CSGGAYDIIICDECH

A6

208

KPQRKTKRNTNRRPQ

A84

365

VGKVIDTLTCGFADL

A162

522

TIFKIRMYVGGVEHR

A240

679

SGGAYDIIICDECHS

A7

209

PQRKTKRNTNRRPQD

A85

366

TLTCGFADLMGYIPV

A163

523

IFKIRMYVGGVEHRL

A241

680

TTILGIGTVLDQAET

A8

210

QRKTKRNTNRRPQDV

A86

367

LTCGFADLMGYIPVV

A164

524

LPALSTGLIHLHQNI

A242

681

TILGIGTVLDQAETA

A9

211

RKTKRNTNRRPQDVK

A87

368

TCGFADLMGYIPVVG

A165

525

PALSTGLIHLEQNIV

A243

682

ILGIGTVLDQAETAG

A10

212

KTKRNTNRRPQDVKF

A88

369

CGFADLMGYIPVVGA

A166

526

ALSTGLIHLHQNIVD

A244

683

LGIGTVLDQAETAGA

A11

213

TKRNTNRRPQDVKFP

A89

370

GFADLMGYIPVVGAP

A167

527

LSTGLIHLHQNIVDV

A245

684

GIGTVLDQAETAGAR

A12

214

KRNTNRRPQDVKFPG

A90

371

FADLMGYIPVVGAPL

A168

528

STGLIHLHQNIVDVQ

A246

685

IGTVLDQAETAGARL

A13

215

RNTNRRPQDVKFPGG

A91

372

ADLMGYIPVVGAPLG

A169

529

TGLIHLHQNIVDVQY

A247

686

GTVLDQAETAGARLV

A14

216

NTNRRPQDVKFPGGG

A92

373

DLMGYIPVVGAPLGG

A170

530

GLIHLHQNIVDVQYL

A248

687

TVLDQAETAGARLVV

A15

217

TNRRPQDVKFPGGGQ

A93

374

ARALAHGVRVLEDGV

A171

531

LIHLHQNIVDVQYLY

A249

688

VLDQAETAGARLVVL

A16

218

NRRPQDVKFPGGGQI

A94

375

RALAHGVRVLEDGVN

A172

532

IHLHQNIVDVQYLYG

A250

689

LDQAETAGARLVVLA

A17

219

RRPQDVKFPGGGQIV

A95

376

ALAHGVRVLEDGVNY

A173

533

LPALSTGLLHLHQNI

A251

690

DQAETAGARLVVLAT

A18

220

RPQDVKFPGGGQIVG

A96

377

LAHGVRVLEDGVNYA

A174

534

PALSTGLLHLHQNIV

A252

691

QAETAGARLVVLATA

A19

221

PQDVKFPGGGQIVGG

A97

378

AHGVRVLEDGVNYAT

A175

535

ALSTGLLHLHQNIVD

A253

692

AETAGARLVVLATAT

A20

222

QDVKFPGGGQIVGGV

A98

379

HGVRVLEDGVNYATG

A176

536

LSTGLLHLHQNIVDV

A254

693

ETAGARLVVLATATP

A21

223

DVKFPGGGQIVGGVY

A99

380

GVRVLEDGVNYATGN

A177

537

STGLLHLHQNIVDVQ

A255

694

TAGARLVVLATATPP

A22

224

VKFPGGGQIVGGVYL

A100

381

VRVLEDGVNYATGNL

A178

538

TGLLHLHQNIVDVQY

A256

695

AGARLVVLATATPPG

A23

225

KFPGGGQIVGGVYLL

A101

382

RVLEDGVNYATGNLP

A179

539

GLLHLHQNIVDVQYM

A257

696

GARLVVLATATPPGS

A24

226

FPGGGQIVGGVYLLP

A102

383

VLEDGVNYATGNLPG

A180

540

LLHLHQNIVDVQYMY

A258

697

ARLVVLATATPPGSV

A25

227

PGGGQIVGGVYLLPR

A103

384

LEDGGVNYATGNLPGC

A151

541

LHLHQNIVDVQYMYG

A259

698

RLVVLATATPPGSVT

A26

228

GGGQIVGGVYLLPRR

A104

385

EDGVNYATGNLPGCS

A182

542

HLHAPTGSGKSTKVP

A260

699

TSILGIGTVLDQAET

A27

229

GGQIVGGVYLLPRRG

A105

386

DGVNYATGNLPGCSF

A183

543

LHAPTGSGKSTKVPA

A261

700

SILGIGTVLDQAETA

A28

230

GQIVGGVYLLPRRGP

A106

387

GVNYATGNLPGCSFS

A184

544

HAPTGSGKSTKVPAA

A262

701

LGIGTVLDQAETAGV

A29

231

QIVGGVYLLPRRGPR

A107

388

VNYATGNLPGCSFSI

A185

545

APTGSGKSTKVPAAY

A263

702

GIGTVLDQAETAGVR

A30

232

IVGGVYLLPRRGPRL

A108

389

NYATGNLPGCSFSIF

A186

546

PTGSGKSTKVPAAYA

A264

703

IGTVLDQAETAGVRL

A31

233

VGGVYLLPRRGPRLG

A109

390

YATGNLPGCSFSIFL

A187

547

TGSGKSTKVPAAYAA

A265

704

GTVLDQAETAGVRLT

A32

234

GGVYLLPRRGPRLGV

A110

391

ATGNLPGCSFSIFLL

A188

548

GSGKSTKVPAAYAAQ

A266

705

TVLDQAETAGVRLTV

A33

235

GVYLLPRRGPRLGVR

A111

392

TGNLPGCSFSIFLLA

A189

549

SGKSTKVPAAYAAQG

A267

706

VLDQAETAGVRLTVL

A34

236

VYLLPRRGPRLGVRA

A112

393

GNLPGCSFSIFLLAL

A190

550

GKSTKVPAAYAAQGY

A268

707

LDQAETAGVRLTVLA

A35

237

YLPRRGPRLGVRAT

A113

394

NLPGCSFSIFLLALL

A191

551

KSTKVPAAYAAQGYK

A269

708

DQAETAGVRLTVLAT

A36

238

LLPRRGPRLGVRATR

A114

395

LPGCSFSIFLLALLS

A192

552

STKVPAAYAAQGYKV

A270

709

QAETAGVRLTVLATA

A37

239

LPRRGPRLGVRATRK

A115

396

PGCSFSIFLLALLSC

A193

553

TKVPAAYAAQGYKVL

A271

710

AETAGVRLTVLATAT

A38

240

PRRGPRLGVRATRKT

A116

397

IQLINTNGSWHINRT

A194

554

KVPAAYAAQGYKVLV

A272

711

ETAGVRLTVLATATP

A39

241

RRGPRLGVRATRKTS

A117

398

QLINTNGSWEINRTA

A195

555

VPAAYAAQGYKVLVL

A273

712

TAGVRLTVLATATPP

A40

242

RGPRLGVRATRKTSE

A118

399

LINTNGSWHINRTAL

A196

556

PAAYAAQGYKVLVLN

A274

713

AGVRLTVLATATPPG

A41

243

GPRLGVRATRKTSER

A119

400

INTNGSWINRTALN

A197

557

AAYAAQGYKVLVLNP

A275

714

GVRLTVLATATPPGS

A42

244

PRLGVRATRKTSERS

A120

401

NTNGSWHINRTALNC

A198

558

AYAAQGYKVLVLNPS

A276

715

VRLTVLATATPPGSV

A43

245

RLCVRATRKTSERSQ

A121

402

TNGSWHINRTALNCN

A199

559

YAAQGYKVLVLNPSV

B5

716

SGMFDSSVLCECYDA

A44

246

LGVRATRKTSERSQP

A122

403

NGSWHINRTALNCND

A200

560

AAQGYKVLVLNPSVA

B6

717

GMEDSSVLCECYDAG

A45

247

GVRATRKTSERSQPR

A123

404

GSWHINRTALNCNDS

A201

561

AQGYKVLVLNPSVAA

B7

718

MFDSSVLCECYDAGC

A46

248

VRATRKTSERSQPRC

A124

405

SWHINRTALNCNDSL

A202

S62

QCYKVLVLNPSVAAT

B8

719

FDSSVLCECDAGCA

A47

249

RATRKTSERSQPRGR

A125

406

IQLVNTNGSWHINRT

A203

563

GYKVLVLNPSVAATL

B9

720

DSSVLCECYDAGCAW

A48

250

ATRKTSERSQPRGRR

A126

407

QLVNTNGSWHINRTA

A204

564

YKVLVLNPSVAATLG

B10

721

SSVLCECYDAGCAWY

A49

251

TRKTSERSQPRGRRQ

A127

408

LVNTNGSWHINRTAL

A205

565

KVLVLNPSVAATLGF

B11

722

SVLCECYDAGCAWYE

A50

252

RKTSERSQPRGRRQP

A128

409

VNTNGSWHINRTALN

A206

566

VLVLNPSVAATLGFG

B12

723

VLCECYDAGCAWYEL

A51

253

KTSERSQPRGRRQPI

A129

410

VDYPYRLWHYPCTVN

A207

567

LVLNPSVAATLGFGA

B13

724

LCECYDAGCAWYELT

A52

254

TSERSQPRGRRQPIP

A130

411

DYPYRLWHYPCTVNF

A208

568

VLNPSVAATLGFGAY

B14

725

CECYDAGCAWYELTP

A53

255

SERSQPRGRRQPIPK

A131

412

YPYRLWHYPCTVNFT

A209

569

YLHAPTGSGKSTKVP

B15

726

ECYDAGCAWYELTA

A54

256

DPRRRSRNLGKVIDT

A132

413

PYRLWHYPCTVNFTI

A210

570

LHAPTGSGKSTKVPV

B16

727

CYDAGCAWYELTPAE

A55

257

PRRRSRNLGKVIDTL

A133

414

YRLWHYPCTVNFTIF

A211

571

HAPTGSGKSTKVPVA

B17

728

YDAGCAWYELTPAET

A56

258

RRRSRNLGKVIDTLT

A134

415

RLWHYPCTVNFTIFK

A212

572

APTGSGKSTKVPVAY

B18

729

DAGCAWYELTPAETT

A57

259

RRSRNLGKVIDTLTC

A135

416

LWHYPCTVNFTIFKV

A213

573

PTGSGKSTKVPVAYA

B19

730

AGCAWYELTPAETTV

A58

260

RSRNLGKVIDTLTCG

A136

417

WHYPCTVNFTIFKVR

A214

574

TGSGKSTKVPVAYAA

B20

731

GCAWYELTPAETTVR

A59

261

SRNLGKVIDTLTCGF

A137

418

HYPCTVNFTIFKVRM

A215

575

GSGKSTKVPVAYAAQ

B21

732

CAWYELTPAETTVRL

A60

262

RNLGKVIDTLTCGFA

A138

419

YPCTVNFTIFKVRMY

A216

576

SGKSTKVPVAYAAQG

B22

733

AWYELTPAETTVRLR

A61

263

NLGKVIDTLTCGFAD

A139

420

PCTVNFTIFKVRMYV

A217

577

GKSTKVPVAYAAQGY

B23

734

WYELTPAETTVRL˜A

A62

264

LGKVIDTLTCGFADL

A140

421

CTVNFTIFKVRMYVG

A218

578

KSTKVPVAYAAQGYK

B24

735

YELTPAETTVRLRAY

A63

265

GKVIDTLTCGFADLM

A141

422

TVNFTIFKVRMYVGG

A219

579

STKVPVAYAAQGTKV

B25

736

DAGCAWYELTPAETS

A64

266

KVIDTLTCGFADLMG

A142

423

VNFTIFKVRMYVGGV

A220

580

TKVPVAYAAQGYKVL

B26

737

AGCAWYELTPAETSV

A65

267

VIDTLTCGFADLMGY

A143

424

NFTIFKVRMYVGGVE

A221

581

KVPVAYAAQGYKVLV

B27

738

GCAWYELTPAETSVR

A66

268

IDTLTCGFADLMGYI

A144

425

FTIFKVRMYVGGVEH

A222

582

VPVAYAAQGYKVLVL

B28

739

CAWYELTPAETSVRL

A67

269

DTLTCGFADLMGYIP

A145

426

TIFKVRMYVGGVEHR

A223

583

PVAYAAQGYKVLVLN

B29

740

AWYELTPAETSVRLR

A68

270

TLTCGFADLMGYIPL

A146

427

IFKVRMYVGGVEHRL

A224

584

VAYAAQGYKVLVLNP

B30

741

WYELTPAETSVRLPA

A69

271

LTCGFADLMGYIPLV

A147

428

DYPYRLWHYPCTVNY

A225

585

ITSTYGKFLADGGC

B31

742

GMFDSVVLCECYDAG

A70

272

TCGFADLMGYIPLVG

A148

429

YPYRLWHYPCTVNYT

A226

586

TYSTYGKFLADGGCS

B32

743

SGMFDSVVLCECYDA

A71

273

CGFADLMGYIPLVGA

A149

430

PYRLWHYPCTVNYTI

A227

587

YSTYGKFLADGGCSG

B33

744

GMFDSVVLCECYDAG

A72

274

GFADLMGYIPLVGAP

A150

431

YRLWHYPCTVNYTIF

A228

588

STYGKFLADGGCSGG

B34

745

MFDSVVLCECYDAGA

A73

275

FADLMGYIPLVGAPL

A151

432

RLWHYPCTVNYTIFK

A229

589

TYGKFLADGGCSGGA

B35

746

FDSVVLCECYDAGAA

A74

276

ADLMGYIPLVGAPLG

A152

433

LWHYPCTVNYTIFKI

A230

590

YGKFLADGGCSGGAY

B36

747

DSVVLCECYDAGAAW

A75

277

DLMGYIPLVGAPLGG

A153

434

WHYPCTVNYTIFKIR

A231

591

GKFLADGGCSGGAYD

B37

748

SVVLCECYDAGAAWY

A76

278

DPRHRSRNVGKVIDT

A154

435

HYPCTVNYTIFKIRM

A232

592

KFLADGGCSGGAYDI

B38

749

VVLCECYDAGAAWYE

A77

279

PRHRSRNVGKVIDTL

A155

436

YPCTVNYTIFKIRMY

A233

593

FLADGGCSGGAYDII

B39

750

VLCECYDAGAAWYEL

A78

280

RHRSRNVGKIDTLT

A156

437

PCTVNYTIFKIRMYV

A234

594

LADGGCSGGAYDIII

B40

751

LCECYDAGAAWYELT

B41

281

CECYDAGAAWYELTP

B120

438

AGISGALVAFKIMSG

C75

595

FPDLGVRVCEKMALY

C154

752

DCTMLVNGDDLVVIC

B42

282

ECYDAGAAWYELTPA

B121

439

GISGALVAFKIMSGE

C76

596

PDLGVRVCEKMALYD

C155

753

DPTMLVCGDDLVVIC

B43

283

CYDAGAAWYELTPAE

B122

440

VNLLPAILSPGALVV

C77

597

DLGVRVCEKMALYDV

C156

754

DPTMLVNGDDLWIC

B44

284

YDAGAAWYELTPAET

B123

441

NLLPAILSPGALWG

C78

598

KGGKKAARLIVYPDL

C157

755

LWARMILMTHFFSIL

B45

285

DAGAAWYELTPAETT

B124

442

LLPAILSPGALVVGV

C79

599

GGKKAARLIVYPDLG

C158

756

LWVRMVLMTHFFSIL

B46

286

AGAAWYELTPAETTV

C1

443

LPAILSPGALVVGVT

C80

600

GKKAARLIVYPDLGV

C159

757

DLPQIIEELHGLSAF

B47

287

GAAWYELTIPAETTV

C2

444

PAILSPGALVVGVTC

C81

601

KKAARLIVYPDLGVR

C160

758

DLPQIIQRLHGLSAF

B48

288

AAWYELTPAETTVRL

C3

445

AILSPGALVVGVVCA

C82

602

KAARLIVYPDLGVEV

C161

759

AVRTKLKLTPIPAAS

B49

289

DAGAAWYELTPAETS

C4

446

ILSPGALVVGVVCAA

C83

603

AARLIVYPDLGVEVC

C162

760

AVRTKLKLTPLPAAS

B50

290

AGAAWYELTPAETSV

C5

447

LSPGALVVGVVCAAI

C84

604

ARLIVYPDLGVRVCE

C163

761

SGGDIYESLSRARPR

B51

291

GAAWYELTPAETSVR

C6

448

SPGALVVGVVCAAIL

C85

605

RLIVYPDLGVRVCEK

C164

762

SGGDIYHSVSRARPR

B52

292

AAWYELTPAETSVRL

C7

449

PGALVVGVVCAAILR

C86

606

LIVYPDLGVRVCEKM

C165

763

WGENETDVLLLNNTR

B53

293

FWAKHMWNFISGIQY

C8

450

GALVVGVVCAAILRR

C87

607

IVPDLGVRVCEKMA

C166

764

GENETDVLLLNNTRP

B54

294

WAKHMWNFISGIQYL

C9

451

ALVVGVVCAAILRRE

C88

608

VYPDLGVEVCEKMAL

C167

765

WFGCTWMNSTGFTKT

B55

295

AKHMWNFISGIQYLA

C10

452

LVVGWCAAILREEV

C89

609

YPDLGVRVCEKMALY

C168

766

FGCTWMNSTGFTKTC

B56

296

KHMENFISGIQYLAG

C11

453

VVGVVCAAILEEHVG

C90

610

AQPGYPWPLYGNEGL

C169

767

GCTWMNSTGFTKTCG

B57

297

HMWNFISGIQYLAGL

C12

454

VGVVCAAILRRHVGP

C91

611

GQPGYPWPLYGNEGL

C170

768

GLPVSARRGREILLG

B58

298

MWNFISGIQYLAGLS

C13

455

GVVCAAILREHVGPG

C92

612

AFCSAMYVGDLCGSV

C171

769

LPVSARRGREILLGP

B59

299

WNFISGIQYLAGLST

C14

456

VVCAAILRRHVGPGE

C93

613

AFCSALYVGDLCGSV

C172

770

PVSARRGREILLGPA

B60

300

NFISGIQYLAGLSTL

C15

457

VCAAILRRHVGPGEG

C94

614

ETVQDCNCSIYPGHV

C173

771

VSARRGEEILLGPAD

B61

301

FISGIQYLAGLSTLP

C16

458

CAAILRRHVGPGEGA

C95

615

EFVQDCNCSIYPGHV

C174

772

GLPVSALEGREILLG

B62

302

ISGIQYLAGLSTLPG

C17

459

AAILRRHVGPGEGAV

C96

616

GVLAGLAYYSMVGNW

C175

773

LPVSALRGREILLGP

B63

303

SGIQYLAGLSTLPGN

C18

460

AILRRHVGPGEGAVQ

C97

617

GVLFGLAYFSMVGNW

C176

774

PVSALRGREILLGPA

B64

304

GIQYLAGLSTLPGNP

C19

461

ILRRHVGPGEGAVQW

C98

618

DQRPYCWHYAPRPCG

C177

775

VSALRGREILLGPAD

B65

305

IQYLAGLSTLPGNPA

C20

462

LRRHVGPGEGAVQWM

C99

619

DQRPYCWYPPRPCG

C178

776

PDREVLYREFDEMEE

B66

306

QYLAGLSTLPGNPAI

C21

463

RRHVGPGEGAVQWMN

C100

620

TCPTDCFRKHPEATY

C179

777

DREVLYREFDEMEEC

B67

307

YLAGLSTLPGNPAIA

C22

464

RHVGPGEGAVQWMNR

C101

621

YTKCGSGPWLTPRCL

C180

778

REVLYREFDEMEECA

B68

308

LAGLSTLPGNPAIAS

C23

465

HVGPGEGAVQWMNRL

C102

622

LNAACNWTRGERCDL

C181

779

EVLYREFDEMEECAS

B69

309

AGLSTLPGNPAIASI

C24

466

VGPGEGAVQWMNRLI

C103

623

LNAACNFTRGERCDL

C182

780

VLYREFDEMEECASS

B70

310

GLSTLPGNPAIASLM

C25

467

GPGEGAVQWMNRLIA

C104

624

IAQAEAALENLVVLN

C183

781

LYREFDEMEECASHL

B71

311

LSTLPGNPAIASLMA

C26

468

PGEGAVQWMNRLIAF

C105

625

IAQAEAALEKLVVLH

C184

782

YYLTRDPTTPLARAA

B72

312

STLPGNPAIASLMAF

C27

469

GEGAVQWMNRLIAFA

C106

626

TRVPYFVRAQGLIRA

C185

783

YLTRDPTTPLARAAW

B73

313

QYLAGLSTLPGNPAV

C28

470

EGAVQWMNRLIAFAS

C107

627

TRVPYFVRAHGLIPA

C186

784

LTRDPTTPLARAAWE

B74

314

YLAGLSTLPGNPAVA

C29

471

GAVQWMNRLIAFASR

C108

628

HAGLRDLAVAVEPVV

C187

785

TRDPTTPLARAAWET

B75

315

LAGLSTLPGNPAVAS

C30

472

AVQWMNRLIAFASRG

C109

629

AAGLRDLAVAVEPIV

C188

786

RDPTTPLARAAWETA

B76

316

AGLSTLPGNPAVASM

C31

473

VQWMNRLIAFASRNGN

C110

630

ITWGADTAACGDIIL

C189

787

DPTTPLARAAWETAR

B77

317

GLSTLPGNPAVASMM

C32

474

QWMNRLIAFASRGNH

C111

631

ITWGAETAACGDIIL

C190

788

PTTPLARAWETARH

B78

318

LSTLPGNPAVASMMA

C33

475

WMNRLIAFASRGNHV

C112

632

GQGWRLLAPITAYSQ

C191

789

YYLTRDPTTPLARAA

B79

319

STLPGNPAVASMMAF

C34

476

MNRLAFASRGNHVS

C113

633

TAYSQQTRGLLGCII

C192

790

YLTRDPTTPLARAAW

B80

320

GAAVGSIGLGKVLVD

C35

477

NRLIAFASRGNHVSP

C114

634

TAYSQQTRGLLGCIV

C193

791

LTRDPTTPLAPAAWE

B81

321

AAVGSIGLGKVLVDI

C36

478

RLIAFASRGNHVSPT

C115

635

GCIITSLTGRDKNQV

C194

792

TRDPTTPLARAAWET

B82

322

AVGSIGLGKVLDIL

C37

479

LIAFASRGNHVSPTH

C116

636

GCIVVSMTGRDKTQV

C195

793

RDPTTPLARAAWETV

B83

323

VGSIGLGKVLVDILA

C38

480

IAFASRGNHVSPTHY

C117

637

VNGVCWTVYHGAGSK

C196

794

DPTTPLARAAWETVR

B84

324

GSIGLGKVLVDILAG

C39

481

AFASRGNHVSPTHYV

C118

638

KGPITQMYTNVDQDL

C197

795

PTTPLARAAWETVRH

B85

325

SIGLGKVLVDILAGY

C40

482

VNLLPGILSPGALVV

C119

639

KGPITQMYSSAEQDL

B86

326

IGLGKVLVDILAGYG

C41

483

NLLPGILSPGALVVG

C120

640

GDSRGSLLSPRPVSY

B87

327

GLGKVLVDILAGYGA

C42

484

LLPGILSPGALVVGV

C121

641

GDSRGALLSPRPVSY

B88

328

LGKVLVDILAGYGAG

C43

485

LPGILSPGALVVGVI

C122

642

SYLKGSSGGPLLCPS

B89

329

GKVLVDILAGYGAGV

C44

486

PGILSPGALVVGVIC

C123

643

SYLKGSSGGPVLCPS

B90

330

KVLVDILAGYGAGVA

C45

487

GILSPGALVVGVICA

C124

644

GHAVGIFRAAVCTRG

B91

331

VLVDILAGYGAGVAG

C46

488

ILSPGALVVGVICAA

C125

645

GVDPNIRTGVRTITT

B92

332

LVDILAGYGAGVAGA

C47

489

LSPGALVVGVICAAI

C126

646

VPHPNIEEVALSNTG

B93

333

VDILAGYGAGVAGAL

C48

490

SPGALVVGVICAAIL

C127

647

TGEIPFYGKAIPIEV

B94

334

DILAGYGAGVAGALV

C49

491

PGALVVGVICAAILR

C128

648

TGEIPFYGKAIPLEV

B95

335

ILAGYGAGVAGALVA

C50

492

GALVVGVICAAILRR

C129

649

PTSGDVVVVATDALM

B96

336

LAGYGAGVAGALVAF

C51

493

ALVVGVICAAILRRH

C130

650

QTVDFSLDPTFTIET

B97

337

AGYGAGVAGALVAFK

C52

494

LVVGVICAAILRRHV

C131

651

TLHGPTPLLYRLGAV

B98

338

GYGAGVAGALVAFKI

C53

495

VVGVICAAILERHVG

C132

652

VQNEVTLTHPITKYI

B99

339

YGAGVAGALVAFKIM

C54

496

VGVICAAILERHVGP

C133

653

LYREFDEMEECASHL

B100

340

GAGVAGALVAFKIMS

C55

497

GVICAAILRRHVGPG

C134

654

TTLLFNILGGWVAAQ

B101

341

AGVAGALVAFKIMSG

C56

498

VICAAILRRHVGPGE

C135

655

TTLLNILGGWLAAQ

B102

342

GVAGALVAFKIMSGE

C57

499

CAAILRRHVGPGEG

C136

656

PSAASAFVGAGIAGA

B103

343

GYGAGVAGALVAEKV

C58

500

MNRLIAFASRGNHVA

C137

657

PSAATGEVVSGLAGA

B104

344

YGAGVAGALVAFKVM

C59

501

NRLIAFASEGNHVAP

C138

658

TPCSGSWLRDVWDWI

B105

345

GAGVAGALVAFKVMS

C60

502

RLIAFASEGNRVAPT

C139

659

VAAEEYVEVTRVGDF

B106

346

AGVAGALVAFKVMSG

C61

503

LIAFASEGNHVAPTH

C140

660

VAAEEYAEVTRHGDF

B107

347

GVAGALVAFKVNSGE

C62

504

IAFASRGNHVAPTHY

C141

661

FFTEVDGVRLHRYAP

B108

348

GKVLVDILAGYGAGI

C63

505

AFASRGNHVAPTHYV

C142

662

FFTELDGVRLHRYAP

B109

349

KVLVDILAGYGAGIS

C64

506

KGGRKPARLIVFPDL

C143

663

FFTWVDGVQIHRYAP

B110

350

VLVDILAGYGAGISG

C65

507

GGRKPARLIVFPDLG

C144

664

FFTWLDGVQIHRYAP

B111

351

LVDILAGYGAGISGA

C66

508

GRKPARLIVFPDLGV

C145

665

YLVGSQLPCEPEPDV

B112

352

VDILAGYGAGISGAL

C67

509

RKPARLIVFPDLGVR

C146

666

YLVGSQLPCDPEPDV

B113

353

DILAGYGAGISGALV

C68

510

KPARLIVFPDLGVRV

C147

667

LPTWARPDYNPPLLE

B114

354

ILAGYGAGISGALVA

C69

511

PARLIVFPDLGVRVC

C148

668

ASLRQKKVTFDRLQV

B115

355

LAGYGAGISGALVAF

C70

512

ARLIVFPDLGVRVCE

C149

669

ASLRAKKVTFDRLQV

B116

356

AGYGAGISGALVAFK

C71

513

RLIVFPDLGVRVCEK

C150

670

HIRSVWKDLLEDTET

B117

357

GYGAGISGALVAFKI

C72

514

LIVFPDLGVRVCEKM

C151

671

IDTTIMAKNEVFCVQ

B118

358

YGAGISGALVAFKIM

C73

515

IVFPDLGVRVCEKNA

C152

672

VMGSSYGFQYSPGQR

B119

359

GAGISGALVAFKIMS

C74

516

VFPDLGVRVCEKMAL

C153

673

DCTMLVCGDDLVVIC

Peptide ID

SEQ ID

(Ipep)

NO:

1506

796

MSTNPKPQRKTKRNTNRRPQDVKFFGCGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPK

1526

797

VNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRCNHVSPTHYV

1545

798

IKGGRHLIFCHSKKKCDELA

1546

799

TVPQDAVSRSQRRGRTGRGR

1547

800

YLVAYQATVCARAQAPPPSWD

1551

801

HLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAY

1552

802

YLHAPTCSGKSTKVPVAYAAQGYKVLVLNPSVAATLGFGAY

1553

803

GAAVGSIGLGKVLVDILAGYGAGVAGALVAFKIMSGE

1554

804

GAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVNSGE

1555

805

GAAVGSIGLGKVLVDILAGYGAGISGALVAFKIMSGE

1556

806

FTEAMTRYSAPPGDPP

1557

807

SSMPPLEGEPGDFDL

1558

808

CGYRRCRASGVLTTS

1559

809

PVNSWLGNIIMYAPT

1560

810

PVNSWLGNIIQYAPT

1561

811

SGMFDSSVLCECYDAGCAWYELTPAETTVRLRAY

1562

812

SGMFDSSVLCECYDAGCAWYELTPAETSVRLRAY

1563

813

SGMFDSVVLCECYDAGAAWYELTPAETTVRLRAY

1564

814

SGMFDSVVLCECYDAGAAWYELTPAETSVRLRAY

1565

815

FWAKHMWNFISCIQYLAGLSTLPGNPAIASLMAF

1577

816

GEVQVVSTATQSFLAT

1578

817

GEVQVLSTVTQSFLGT

1579

818

FTDNSSPPAVPQTFQV

1580

819

FSDNSTPPAVPQTYQV

1581

820

NAVAYYRGLDVSVIPT

1587

821

VNLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIAFASRGNHVAPTHYV

1588

822

TTILGIGTVLDQAETAGARLVVLATATPPGSVT

1589

823

TSILGIGTVLDQAETAGARLVVLATATPPGSVT

1590

824

TSILGIGTVLDQAETAGVRLTVLATATPPGSVT

1591

825

TTILGIGTVLDQAETAGVRLTVLATATPPGSVT

1592

826

FWAKHMWNFISGIQYLAGLSTLPGNPAVASMMAF

1603

827

VFTGLTHIDAHFLSQTKQ

1604

828

VVCCSMSYTWTGALITPC

1605

829

VVCCSMSYSWTGALITPC

1606

830

VLTSMLTDPSHITAETA

1607

831

VLTSMLTDPSHITAEAA

1613

832

ASSSASQLSAPSLRATCTT

1614

833

LTPPHSARSKFGYGAKDVR

1615

834

LTPPHSADSKFGYGAKDVR

1616

835

LTPPHSAKSKYGYGAKEVR

1617

836

LTPPHSARSKYGYGAKEVR

1618

837

PMGFSYDTRCFDSTVTE

1619

838

PMGFAYDTRCFDSTVTE

1620

839

TGDFDSVIDCNTCVTQ

1621

840

TGDFDSVIDCNVAVTQ

1622

841

NTPGLPVCQDHLEFWE

1623

842

YLVAYQATVCARAXAPPPSWD

1624

843

LEDRDRSELSPLLLSTTEW

1625

844

LEDRDRSQLSPLLHSTTEW

1626

845

ASSSASQLSAPSLKATCTT

1627

846

PEYDLELITSCSSNVSVA

1628

847

VCGPVYCFTPSPVVVGTTDR

1629

848

GWAGWLLSPRGSRPSWGP

1630

849

LLFLLLADARVCACLWM

1631

850

SGHRMAWDMMMNWSPT

1632

851

TGHRMAWDMMMNWSPT

1641

852

ITYSTYGKFLADGGCSGGAYDIIICDECHS

1647

853

ARALAHGVRVLEDGVNYATGNLPGCSFSIFLIALLSC

1648

854

DPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGG

1649

855

DPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLGG

1650

856

VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL

1651

857

VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL

1652

858

XGGRKPARLIVFPDLGVRVCEKMALYDV

1653

859

KGGKKAARLIVYPDLGVRVCEKMALYDV

1654

860

IQLTNTNGSWHTNRTALNCNDSL

1655

861

IQLVNTNGSWHINRTALNCNDSL

1656

862

LPALSTGLIHLHQNIVDVQYLYG

For epitope capture, each peptide pool was incubated with soluble recombinant HLA-class II molecules and specific binding was assessed by an SDS-stability assay. The results using the HLA molecules DRB1*0401, DRB1*0404 and DRB1*0101 are shown in FIGS. 2 and 3 respectively: 28 peptide pools were found which bind to DRB1*0401 molecules: no. 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20 from “row” pools and no. 23, 25, 26, 27, 29, 30, 31, 34, 36, 38, 39 and 40 from “column” pools (FIG. 2). 35 peptide pools out of 40 tested were positive in binding to DRB1*0404 molecules (FIG. 3), while all peptide pools showed binding activity to DRB1*0101 molecules. By finding the intersections of reactive pools in the array, potential individual binders were determined and re-checked for binding affinity individually.

All individually confirmed peptides are summarized in Table 2. Binding to DRB1*0401 is shown in FIG. 4: 54 individual peptides were identified as ligands of this HLA-type. Often several overlapping 15mers in a row bound to HLA allowing identification of their core binding regions. Peptide differing only by one or two amino acids representing variants (see Table 1) usually bound both to soluble HLA class II molecules. Such “duplicates” were considered to represent the same epitope. Thus, 31 ligands capable to bind to DRB1*0401 were identified, including 11 previously known class II epitopes. From the latter, however, only two (A202-A206 and B60-B68) had been known to be restricted to DR4 (see Table 2). 20 ligands are candidates for novel epitopes. For DRB1*0404, 64 binders designated as 28 potential epitopes were determined, 4 of them belong to already known epitopes (FIG. 5, Table 2). For DRB1*0101, 83 peptides representing 44 potential epitopes were identified (FIG. 6, Table 2). Of those, 7 had been described previously but with different HLA restriction.

All individually confirmed peptides binding to at least one of the 3 above mentioned HLA types were also tested for affinity to molecules in a peptide-competition assay (FIG. 7, Table 2). Here, 50 ligands were identified. Of those, 7 correspond to already known class II epitopes, but only one was described as DRB1*0701 epitope (A202-A206).

TABLE 2
HCV derived peptides binding to soluble HLA class II molecules.
About 400 15- to 23-mer peptides derived from conserved
regions of HCV were analyzed by the Epitope Capture Method using
pools of up to 20 peptides arrayed in matrix format (see FIG. 1)
and four different HLA class II molecules. Specific binding was
confirmed for individual peptides.
				Known/new
	SEQ			potential
	ID		Binding to DRB1	epitope, HLA
ID	NO:	Peptide Sequences	*0101	*0401	*0404	*0701	coverage
A120	863	NTNGSWHINRTALNC	*			nb	new DRB1*0101,
A122	864	NGSWHINRTALNCND	*		*	*	*0404, *0701: 45-
A124	865	SWHINRTALNCNDSL	*				55%
B25	866	DAGCAWYELTPAETS	***	*	***		new DRB1*0101,
B26	867	AGCAWYELTPAETSV	***	***	***	nb	*0404, *0701: 45-
B28	868	CAWYELTPAETSVRL	***	**	***	*	55%
B30	869	WYELTPAETSVRLRA	**	*	**	nb
B46	870	AGAAWYELTPAETTV	***		***	nb	new DRB1*0101,
B48	871	AAWYELTPAETTVRL	***		***	**	*0404, *0701: 45-
							55%
B84	872	GSIGLGKVLVDILAG	*	*	*		new DRB1*0101,
B86	873	IGLGKVLVDILAGYA	*	**	*	**	*0404, *0701: 45-
B88	874	LGKVLVDILAGYGAG	*	**	*		55%
B92	875	LVDILAGYGAGVAGA	*	nb
C106	876	TRVPYFVRAQGLIRA	*	*	*	*	new DRB1*0101,
							*0404, *0701: 45-
							55%
C113	877	TAYSQQTRGLLGCII	***	*			new DRB1*0101,
C114	878	TAYSQQTRGLLGCIV	***	*	*	*	*0404, *0701: 45-
							55%
1627	879	PEYDELELITSCSSNVSVA	***		***	**	new DRB1*0101,
							*0404, *0701: 45-
							55%
1628	880	VCGPVYCFTPSPVVVGTTDR	***	**	*	*	new DRB1*0101,
							*0404, *0701: 45-
							55%
1629	881	GWAGWLLSPRGSPRPSWGP	*	*	**	*	new DRB1*0101,
							*0404, *0701: 45-
							55%
1604	882	VVCCSMSYTWTGALITPC	*		**	***	new DRB1*0101,
							*0404, *0701: 45-
							55%
1630	883	LLFLLLADARVCACLWM	*		nb	***	new DRB1*0101,
							*0701: 40-50%
C97	884	GVLFGLAYFSMVGNW	**	**	nb	**	new DRB1*0101,
							*0701: 40-50%
1547	885	YLVAYQATVCARAQAPPPSWD	***		NB	**	new DRB1*0101,
							*0701: 40-50%
B94	886	DILAGYGAGVAGALV	*	nb	nb	nb	new DRB1*0101,
B95	887	ILAGYGAGVAGALVA	*	nb	nb		*0404, *0701: 40-
B96	888	LAGYGAGVAGALVAF	**	**	nb	*	50%
B97	889	AGYGAGVAGALVAFK	*	nb	nb
B98	890	GYGAGVAGALVAKFI	*	nb	nb	nb
A272	891	ETAGVRLTVLATATT	*	*	nb		new DRB1*0101,
A274	892	AGVRLTVLATATPPG	*	nb		nb	*0404, *0701: 40-
A276	893	VRLTVLATATPPGSV		nb		*	50%
B120	894	AGISGALVAFKIMSG	*	nb	*	***	new DRB1*0101,
							*0404, *0701:~45
B122	895	VNLLPAILSPGALVV	*	nb	*	*	new DRB1*0101,
							*0404, *0701:~45
C108	896	HAGLRDLAVAVEPVV	*	nb	*	*	new DRB1*0101,
							*0404 *0701:~45
C134	897	TTLLFNILGGWVAAQ	*	nb	*	**	new DRB1*0101,
							*0404, *0701:~45
C152	898	VMGSSYGFQYSPGQR	*	nb	*	*	new DRB1*0101,
							*0404, *0701:~45
1606	899	VLTSMLTDPSHITAETA	nb	***	**	**	new DRB1*0401,
							*0404, *0701:~45
1607	900	VLTSMLTDPSHITAEAA	nb	***	**	*	new DRB1*0401,
							*0404, *0701:~45
1577	901	GEVQVVSTATQSFLAT	nb	*	***	***	new DRB1*0401,
							*0404, *0701:~45
1578	902	GEVQVLSTVTQSFLGT	nb	*	***	***	new DRB1*0401,
							*0404, *0701:~45
B50	903	AGAAWYBLTPAETSV	***	***	***		new DRB1*0101,
B52	904	AAWYELTPAETSVRL	***	***	***	nb	*0401, *0404:~40
1623	905	YLVAYQATVCARAKAPPPSWD	*		**		new DRB1*0101,
							*0401, *0404:~40
C130	906	QTVDFSLDPTFTTET	**	***	**	nb	new DRB1*0101,
							*0401, *0404:~40
1603	907	VFTGLTHIDAHFLSQTKQ	***	nb	nb	*	new DRB1*0101,
							*0701: ~-40
C96	908	GVLAGLAYYSMVGNW	**	nb	nb	*	new DRB1*0101,
							*0701: ~-40
C191	909	YYLTRDPTTPLAPAA	nb	***	nb		new DRB1*0401,
							*0701:~40
A216	910	SGKSTKVPVAYAAQG	nb	*	nb	nb	new DRB1*0101,
A218	911	KSTKVPVAYAAQGYK	nb	*	nb		*0401:~35
A220	912	TKVPVAYAAQGYKVL	*	**	nb
A222	913	VPVAYAAQGYKVLVL	*	nb	nb	nb
A224	914	VAYAAQGYKVLVLNP	*	nb	nb
A242	915	TILGIGTVLDQAETA	nb	nb	*		new DRB1*0101,
A244	916	LGIGTVLDQAETAGA	*	*	nb	nb	*0401:~35
C92	917	AFCSANYVGDLCGSV	*	**	nb	nb	new DRB1*0101,
C93	918	AECSALYVGDLCGSV	*	*	nb		*0401:~35
A174	919	PALSTGLLHLHQNTV		nb	*	*	new DRB1*0404,
							*0701:~25-30
B32	920	SGMFDSVVLCECYDA	*	nb	***		new DRB1*0101,
B34	921	MFDSVVLCECYDAGA	*	nb	***	nb	*0404:~20-25
B36	922	DSVVLCECYDAGAAW	*	nb	***
B38	923	VVLCECYDAGAAWYE	nb	nb	*	nb
B100	924	GAGVAGALVAFKIMS	**	nb	**		new DRB1*0101,
B102	925	GVAGALVAFKIMSGE	***	nb	***		*0404:~20-25
C135	926	TTLLLNILGGWLAAQ	**	nb	*		new DRB1*0101,
							*0404:~20-25
C162	927	AVRTKLKLTPLPAAS	nb	*	*	nb	new DRB1*0401,
							*0404:~20-25
1618	928	PMGFSYDTRCFDSTVTE	nb	nb		**	new DRB1*0701:~25
1622	929	NTPGLPVCQDHLEFWE	nb	nb		***	new DRB1*0701:~25
1624	930	LEDRDRSELSPLLLSTTEW	nb	nb		*	new DRB1*0701:~25
1546	931	TVPQDAVSRSQRRGRTGRGR	nb	nb	nb	*	new DRB1*0701:~25
1556	932	FTEAMTRYSAPPGDPP	nb	nb	nb	*	new DRB1*0701:~25
A114	933	LPGCSFSIFLLALLS	**	nb	nb	nb	new DRB1*0101:~20
B58	934	MWNFISGTQYLAGLS	*	nb	nb		new DRB1*0101:~20
B112	935	VDTLAGYGAGISGAL	*				new DRB1*0101:~20
B114	936	ILAGYGAGISGALVA	***
B116	937	AGYGAGISGALVAFK	***		nb	nb
B118	938	YGAGISGALVAFKIM	***		nb
B18	939	DAGCAWYELTPAETT	***		nb	nb	new DRB1*0101:~20
B20	940	GCAWYELTPAETTVR	***	nb
B22	941	AWYELTPAETTVRLR	**		nb	nb
C112	942	GQGWRLLAPTTAYSQ	**	nb			new DRB1*0101:~~20
C116	943	GCIVVSMTGRDKTQV	*	nb	nb		new DRB1*0101:~20
C122	944	SYLKGSSGGPLLCPS	*	nb	nb		new DRB1*01O1:~20
C127	945	TGEIPFYGKAIPTEV	*	nb			new DRB1*0101:~20
C144	946	FFTWLDGVQIHRYAP	**	nb	nb		new DRB1*0101:.~20
C159	947	DLPQIIERLHGLSAF	*	nb	nb	nb	new DRB1*0101:~2
C160	948	DLPQIIQRLHGLSAF	*	nb
C174	949	GLPVSALRGRETLLG	*	nb	nb		new DRB1*0101:~20
1558	950	CGYRRCRASGVLTTS	***		nb	nb	new DRB1*0101:~20
1581	951	NAVAYYRGLDVSVIPT	**	nb	nb	nb	new DRB1*0101:~20
C95	952	EFVQDCNCSIYPGHV	nb	**	nb	nb	new DRB1*0401:~20
C129	953	PTSGDVVVVATDALM	nb	nb	**		new DRB1*0404:~5
C157	954	LWARMILMTHFFSIL	nb	nb	*	nb	new DRB1*0404:~5
C158	955	LWVRMVLMTHFFSIL		nb	*
A254	956	ETAGARLVVLATATP	nb	nb	*		new DRB1*0404:~5
A256	957	AGARLVVLATATPPG	nb	nb	*
A258	958	ARLVVLATATPPGSV	nb	nb	**
1605	959	VVCCSMSYSWTGALITPC	nb	nb	*	nb	new DRB1*0404:~5
C109	960	AAGLRDLAVAVEPIV		nb	*		new DRB1*0404:~5
C161	961	AVRTKLKLTPIPAAS		nb	*		new DRB1*0404:~5
A60	962	LGKVIDTLTCGFA	**	nb	nb	**	known DR4, DR8,
							DR15
A61	963	GKVIDTLTCGFAD	**				new DR*0101, 0701
A70	964	TCGFADLMGYIPLVG	*	nb	nb		known class II
A72	965	GFADLMGYIPLVGAP	***	*	**		DR*0101, 0404,
A74	966	DLMGYIPVVGAPLGG	***		***	**	0701
A88	967	CGFADLMGYIPVVGA	**		**	*	known class II
A90	968	FADLMGYIPVVGAPL	***		***		DR*0101, 0404,
A92	969	DLMGYIPVVGAPLGG	***		***	***	0701
A96	970	LAHGVRVLEDGVNYA	nb	***	nb		known DR11
A98	971	HGVRVLEDGVNYATG	nb	***	***	**	new DR*0401,
A100	972	VRVLEDGVNYATGNL	nb	***	***	*	0404, 0701
A102	973	VLEDGVNYATGNLPG	nb	*
A104	974	EDGVNYATGNLPGCS	nb	*	nb	nb
A200	975	AAQGYKVLVLNPSVA		nb	***	**	known DRB1*0401,
A202	976	QGYKVLVLNPSVAAT	***	***	***	***	0701, DR11, DR15
A204	977	YKVLVLNPSVAATLG	***	***	***	***	new DR*0101
A206	978	VLVLNPSVAATLGFG	***	***	***	***
C30	979	AVQWMNRLIAFASRG	*		nb		known DR11, DQ5,
							also DR*0101
B60	980	NFISGIQYLAGLSTL	**		nb	8	known DR#40401,
B62	981	ISGIQYLAGLSTLPG	***		***	**	1101
B64	982	GIQYLAGLSTLPGNP	***		***	**	new 0101, 0404,
B66	983	QYLAGLSTLPGNPAI	*		***	**	0701
B68	984	LAGLSTLPGNPAIAS	*		***	**
C124	985	GHAVGIFRAAVCTRG	**	*	nb	**	known DR*0101,
							0401, 0701
1620	986	TGDFDSVIDCNTCVTQ	nb	nb	*		new DR*0404
1621	987	TGDFDSVIDCNVAVTQ	*		nb	nb	known DR13, also
							DR*0101, 0401
1631	988	SGHRMAWDMMMNWSPT	nb			nb	known class II,
							also DR*0401
1632	989	TGHRMAWDMMMNWSPT	nb	*			known class II,
							also DR*0401
*** strong binding
** intermediate binding
* weak binding
nb no binding
Boldface peptide IDs indicates HLAligands with confirmed immunogenicity in HLA-transgenic mice
Boldface peptide sequences indicate putative core binding regions based on prediction algorithms as descnbed in the test.
1)immunogenic in DRB1 *0401 transgenic mice.

Some of the highly promiscuous peptides and/or with computer algorithm (SYFPEITHI (SEQ ID NO:203), TEPITOPE (SEQ ID NO:204))-predicted affinities were checked for binding to soluble HLA-DRB1*1101 molecules in a peptide-competition assay as it is described for HLA-DRB1*0701. Several known DR11 epitopes were used as controls and were confirmed to bind HLA-DRB1*1101 molecules in vitro. Among newly identified HLA-DRB1*1101 binders, there are peptides with IDs A120, A122, A141, C114, C134, 1426, 1628, 1629 of high affinity, 5 peptides with IDs C106, C135, 1578, 1547, 1604 of moderate affinity and 4 peptides with IDs B46, B48, B86, B96 of weak affinity ligands.

In summary eight novel ligands binding at least to HLA-DRB1*0101, *0401, *0404, *0701 and *1101 (Tab. 2: peptide Ids A120, A122, A141, 1604, 1547, 1628, 1629, and Tab. 6: peptide ID 1426); novel 10 ligands binding at least to HLA-DRB1*0101, *0401, *0404 and *0701 (Tab. 2: peptide IDs A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604); 5 novel ligands binding at least to HLA-DRB1*0101, *0401 and *0701, 5 novel ligands binding at least to HLA-DRB1*0101, *0404 and *0701, 4 novel ligands binding at least to HLA-DRB1*0401, *0404 and *0701, 3 novel ligands binding at least to HLA-DRB1*0101, *0401 and *0404, 2 novel ligands binding at least to HLA-DRB1*0101 and *0701, 1 novel ligand binding at least to HLA-DRB1*0401 and *0701, 3 novel ligands binding at least to HLA-DRB1*0101, *0401, 1 novel ligand binding at least to HLA-DRB1*0404 and *0701, 4 novel ligand binding at least to HLA-DRB1*0101 and *0404, 5 novel ligands binding at least to HLA-DRB1*0701, 13 novel ligands binding at least to HLA-DRB1*0101, 1 novel ligand binding at least to HLA-DRB1*0401, and 6 novel ligands binding at least to HLA-DRB1*0404.

Moreover, 12 known HLA class II epitopes were confirmed, in several cases binding to alleles not reported yet was demonstrated (Tab. 2, last group).

Having established physical binding too HLA class II it is straightforward to verify immunogenicity for a given ligand: for instance peptide IDs A120-A124, B46-B48, 1627, 1604, 1630, 1547, 1623, B112-118, 1558, all binding to one or more HLA class II alleles were also shown to be immunogenic in HLA-DRB1*0401 transgenic mice (see Example II).

To determine the optimal epitope within a longer polypeptide, mice can be vaccinated with a longer polypeptide incorporating the candidate epitope sequences. Generation of specific CD4+ T cell responses against naturally processed and presented epitopes can then be assayed by re-stimulation of murine splenocytes or lymph node cells with overlapping 15-mers and IFN-gamma ELIspot. Final confirmation/validation of the newly identified HLA-ligands can be achieved by testing these peptides with T-cells from humans. Ideally, these comprise therapy responders or subjects spontaneously recovered from infection.

Example II

Immunogenicity of HCV-Derived Peptides in HLA-Transgenic Mice

Synthetic HCV-derived peptides (from conserved regions) were investigated for immunogenicity in HLA-transgenic mice: 36 of 68 peptides tested were found to induce peptide-specific IFN-gamma-producing cells in vaccination experiments. As summarized in Table 3, some peptides were either immunogenic (+, less than 100 peptide-specific cells among a million splenocytes) or even strongly immunogenic (++, more than 100 peptide-specific cells among a million splenocytes) in DR4- and/or A*0201-transgenic mice.

TABLE 3
	DRB1*			SEQ ID
Ipep	0401	A*0201	B*0702	NO	Sequence
1506			+	990	MSTNPKPQRKTKTNRRPQD
					VKFPGGGQIVGGVYLLPRR
					GPRLGVATRKTSERSQPRG
					RRQPIPK
1526	+	+	+	991	VNLLPAILSPGALVVGVVC
					AAILRRHVGPGEGAVQWMN
					RLIAFASRGNHVSPTHYV
1545		+		992	IKGGRHLIFCHSKKKCDEL
					A
1547	++		+++	993	YLVAYQATVCARAQAPPPS
					WD
1552		+	+	994	YLHAPTGSGKSTKVPVAYA
					AQGYKVLVLNPSVAATLGF
					GAY
1553		+	+	995	GAAVGSIGLGKVLVDILAG
					YGAGVAGALVAFKIMSGE
1555	+	+	+	996	GAAVGSIGLGKVLVDILAG
					YGAGISGALVAFKIMSGE
1558	++	+	++	997	GGYRRCRASGVLTTS
1559	+	++		998	PVNSWLGNIIMYAPT
1560	+	++		999	PVNSWLGNIIQYAPT
1562			+	1000	SGMFDSSVLCECYDAGCAW
					YELTPAETSVRLRAY
1563	+		+	1001	SGMFDSVVLCECYDAGAAW
					YELTPAETTVRLRAY
1565	++	++	+	1002	FWAKHMWNFISGIQYLAGL
					STLPGNPAIASLMAF
1577		+	+	1003	GEVQVVSTATQSFLAT
1578			+	1004	GEVQVLSTVTQSFLGT
1580			+	1005	FSDNSTPPAVPQTYQV
1587		+	+	1006	VNLLPGILSPGALVVGVIC
					AAILRRHVGPGEGAVQWMN
					RLTAFASRGNHVAPTHYV
1592	++	++	+	1007	FWAKHMWNFISGIQYLACL
					STLPGNPAVASMMAF
1604	++	++	++	1008	VVCCSMSYTWTGALITPC
1605	+	+	+	1009	VVCCSMSYSWTGALITPC
1615		+		1010	LTPPHSAKSKFGYGAKDVR
1616	+			1011	LTPPHSAKSKYGYGAKEVR
1617		+		1012	LTPPHSARSKYGYGAKEVR
1621	+	+	+	1013	TGDFDSVIDCNVAVTQ
1623	+	+	+	1014	YLVAYQATVCARAKAPPPS
					WD
1624			+	1015	LEDRDRSELSPLLLSTTEW
1625	+			1016	LEDRDRSQLSPLLHSTTEW
1627	+	+	++	1017	PEYDLELITSCSSNVSVA
1628			++	1018	VCGPVYCFTPSPVVVGTTDR
1630	++	++		1019	LLFLLLADARVCACLWM
1631	+	+		1020	SGHRMAWDMMMNWSPT
1632		+		1021	TGHRMAWDMMMNWSPT
1641		+		1022	ITYSTYGKFLADGGCSGGA
					YDIIICDECHS
1647		+	+	1023	ARALAHGVRVLEDGVNYAT
					GNLPGCSFSIFLLALLSC
1649	+			1024	DPRHRSRNVGKVIDTLTCG
					FADLMGYIPVVGAPLGG
1650	++	++	+	1025	VDYPYRLWHYPCTVNFTIF
					KVRNYVGGVEHRL
1651	++	++	+	1026	VDYPYRLNHYPCTVNYTIF
					KTRNYVGGVEHRL
1652	++	+		1027	KGGRKPARLIVFPDLGVRV
					CEKMALYDV
1653		+		1028	KGGKKAARLIVYPDLGVRV
					CEKMALYDV
1654	++	+		1029	IQLINTNGSWHINRTALNC
					NDSL
1655	++	+		1030	IQLVNTNGSWHTNRTALNC
					NDSL
1656	+			1031	LPALSTGLIHLHQNIVDYQ
					YLYG

Peptide 1526, 1565, 1631, also shown to be immunogenic in HLA-DRB1*0401 transgenic mice contain known class II epitopes. Peptide IDs 1526, 1553, 1565, 1587, 1623, 1630 also shown to be immunogenic in HLA-A*0201 transgenic mice contain known A2 epitopes.

For further characterizing the novel epitopes provided herewith, one may define the exact HLA restriction of these epitopes and the minimal epitopes within the sequences recognized by T cells. Both can be done by a variety of well-established approaches known to the one skilled in the art (Current Protocols in Immunology, John Wiley& Sons, Inc.).

First, publicly available programs can be used to predict T cell epitopes on the basis of binding motifs. These include for instance: http://bimas.dcrt.nih.gov/molbio/hla_bind/ (Parker et al. 1994), http://134.2.96.221/scripts/MHCServer.dll/home.htm (Rammensee at al. 1999), http://mypage.ihost.com/usinet.hamme76/(Sturniolo et al. 1999). The latter prediction algorithm offers the possibility to identify promiscuous T helper-epitopes, i.e. peptides that bind to several HLA class II molecules. These predictions can be verified by testing of binding of the peptide to the respective HLA.

A way of quickly discerning whether the response towards a peptide is class I or class II restricted is to repeat the ELIspot assay with pure CD4+ or CD8+ T cell effector populations. This can for instance be achieved by isolation of the respective subset by means of magnetic cell sorting. Pure CD8+ T cells can also be tested in ELIspot assays together with artificial antigen-presenting-cells, expressing only one HLA molecule of interest. One example are HLA-A*0201 positive T2 cells (174CEM.T2, Nijman et al., 1993). Alternatively, one can use ELIspot assays with whole PBMCs in the presence of monoclonal antibodies specifically blocking either the CD4+ or CD8+ T cell sub-population. Exact HLA restriction can be determined in a similar way, using blocking monoclonal antibodies specific for a certain allele. For example the response against an HLA-A24 restricted epitope can be specifically blocked by addition of an HLA-A24 specific monoclonal antibody.

For definition of the minimal epitopes within the peptide sequences recognized by T cells, one can test overlapping and truncated peptides (e.g. 8-, 9-, 10-mers) with splenocytes from immunized transgenic mice or T-cells from humans recognizing the respective epitope.

Example III

HLA Restriction of Immunogenic HCV-Derived Peptides Investigated in Transgenic Mice

Groups of 5 mice (HLA-A*0201-, HLA-DRB1*0401- and HLA-B*0702 transgenic mice, male, 8-14 weeks of age) were injected subcutaneous into the hind footpads with 100 μg of peptide+IC31 per mouse (50 μg per footpad). (PCT/EP01/12041, WO 02/32451 A1 and PCT/EP01/06433, WO 01/93905 A1; IC31 is a combination of the immunizer disclosed in WO 01/93905 and WO 02/32451).

6 days after vaccination single cell suspension of pooled spleens were prepared and additionally pure fractions of CD8+ in the case of A2 and B7 μg mice (CD8+ fraction for B7 mice containing 97% of CD8 and 1.5% of CD4 cells and for A2tg mice 83% of CD8 and 8% of CD4 cells) and CD4+ for DR4tg mice (CD4+ fraction for DR4tg mice containing 98% of CD4 cells and 0.2% of CD8 cells) were separated from the spleen cell suspension using MACS separating kit (Miltenyi, Germany). All cells (not separated cells, positive and corresponding negative fractions) were re-stimulated ex vivo with relevant peptide (for instance Ipep1604) and irrelevant peptides as negative control (known HLA-DRB1*0401 CMV-derived epitope Ipep 1505, HLA-B*0702 HIV-derived epitope Ipep 1787, or HLA-A*0201 tyrosinase-derived epitope Ipep1124) to detect INF-γ producing cells in ELISpot assay.

As an example shown in FIG. 8-10 the Ipep1604 (VVCCSMSYTWTGALITPC (SEQ ID NO:30), in combination with immunizer IC31) was able to induce high numbers of specific INF-γ producing T cells in all three transgenic class I and II mouse strains. This was shown not only with whole spleen derived cells but also with enriched fractions of CD8+ cells correspondingly for A2 and B7 and CD4+ cells for DR4tg mice. Similar, albeit weaker responses were seen with Ipep1605 (VVCCSMSYSWTGALITPC (SEQ ID NO:126)), a sequence variant with a serine instead of a threonine.

Thus, Ipep1604 contains class I epitopes for HLA-A*0201 and HLA-B*0702 and a class II epitope for HLA-DRB1*0401 molecules.

As shown in Tables 2 and 6, Ipep 1604 binds to class II molecules in a promiscuous manner. Thus, it contains further epitopes, at least for HLA-DRB1*0101, DRB1*0404, DRB1*0701 and DRB1*1101.

Other peptides were analysed in a similar way:

Ipeps 1605, 1623, 1547, 1558, 1559, 1560, 1565, 1592, 1650, 1654 and 1655 were confirmed to contain human HLA-DRB1*0401 epitopes. Again, for most of these epitopes binding is not limited to HLA-DRB1*0401 as shown in Tables 2 and 6.

Ipeps 1565, 1605 and 1650 were confirmed to contain human HLA-A*0201 epitopes.

Ipeps 1506, 1587 were confirmed to contain human HLA-B*0702 epitopes.

Ipep 1843 with sequence LPRRGPRL (SEQ ID NO:1046) was shown to be the HLA-B*0702 minimal epitope contained in 1506:

FIG. 10 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+ or CD4+ cells from HLA-A*0702 transgenic mice vaccinated with Ipep1506+IC31 or Ipep1835+IC31.

FIGS. 10 A) and B) shows that after a single vaccination with either Ipep 1506+IC31 or Ipep1835+IC31, upon restimulation with overlapping 15mers, the 15mers A30 to A37 (see Tab.1) react. The common sequence of these 15mers is LPRRGPRL (SEQ ID NO:1046) (Ipep 1843, see Tab.4).

FIG. 10 C) confirms these findings: after a single vaccination with either Ipep1506+IC31 or Ipep1835+IC31, significant interferon-gamma induction against Ipep1843 can be detected. In both cases Ipep 1790 an HIV NEF-derived HLA-B*0702 epitope (sequence RPMTYKAAL (SEQ ID NO:1032)) was used as negative control for restimulation.

Ipep 1838 with sequence SPGALVVGVI (SEQ ID NO:1045) (see Tab.4) was shown to be an HLA-B*0702 minimal epitope contained in 1587: In the case of Ipep1587 a different approach was taken: the sequence of Ipep1587 was inspected for HLA-B*0702 binding motifs and a couple of short peptides were synthesized accordingly. These were tested in a competition-type peptide binding assay using soluble HLA-B*0702 and the FITC-labelled reference peptide LPCVLWPVL (SEQ ID NO:1033), which is a known HLA-B*0702 epitope derived from EBV (Stuber et al., 1995). Peptide Ipep1838 showed ˜30% competition when used in 80-fold molar excess for 48 h at 37° C. Thus it is likely to present the minimal HLA-B*0702 epitope contained in Ipep 1587.

Example IV

Identification and Confirmation of Novel HCV Peptides Reactive in IFN-Gamma ELIspot with Human PBMC from HCV Therapy Responders or Patients with Spontaneous Recovery

40 peptide mixtures in matrix format (FIG. 1) containing synthetic peptides derived from conserved regions of HCV (Table 1) were screened in IFN-gamma ELIspot using PBMC from more than 50 individuals who were either responders to interferon/ribavirin standard therapy, or, who had spontaneously cleared HCV (i.e. all subjects were HCV antibody positive, but HCV-RNA negative). PBMC from such individuals are supposed to contain the relevant T-cell populations responsible for clearing HCV. Thus, peptides discovered or confirmed by using these PBMC are likely to represent the structural determinants of immune protection against/clearance of HCV. Based on the results from this primary matrix-screen, a number of peptides were chosen for individual re-testing in IFN-gamma ELIspot using PBMC from selected donors. In addition, several new peptides incorporating sequences from overlapping reactive peptides or avoiding critical residues like cystein were synthesized. These are summarized in Table 4.

TABLE 4
additional peptides derived
from conserved regions of HCV.
	Peptide sequence
Peptide ID	(1 amino acid code)	SEQ ID NO:
1006	MWNFISGIQYLAGLSTLPGN	1034
1334	HMWNFISGI	1035
1425	NFISGIQYLAGLSTLPGNPA	1036
1426	HMWNFISGIQYLAGLSTLPGNPA	1037
1798	IGLGKVLVDILAGYGAGVAGALVAFK	1038
1799	AAWYELTPAETTVRLR	1039
1800	DYPYRLWHYPCTVNYTIFKI	1040
1836	DYPYRLWHYPCTVNFTIFKI	1041
1801	AYSQQTRGLL	1042
1827	TAYSQQTRGLLG	1043
1829	SMSYTWTGALITP	1044
1838	SPGALVVGVI	1045
1843	LPRRGPRL	1046

Results of the secondary screening with individual peptides are summarized in Table 5. Altogether ˜20% of subjects (G05, G18, H02, H03, H04, H10, H12, H19, H32, H38) showed a significant IFN-gamma T-cell response against one or more of the peptides. In some cases the observed number of ELIspots was clearly elevated, but not statistically significant above background. In these cases, PBMC (donors H03, H10, H33, H38) were stimulated with the respective peptides in vitro (2 rounds of in vitro priming, see Material & Methods) in order to increase the peptide specific response. Several peptides were confirmed in this way, results are again summarized in Table 5.

Peptides A3-A7 represent overlapping 15mers spanning the sequence TNPKPQRKTKRNTNRRPQD (SEQ ID NO:1047). Since they all react with PBMC from donor H03, the minimal sequence of the epitope is located within the sequence PQRKTKRNTNR (SEQ ID NO:1048). Prediction algorithms indicate that QRKTKRNTN (SEQ ID NO:1049) and QRKTKRNT (SEQ ID NO:1050) represent ligands of HLA-B*08, whereas RKTKRNTNR (SEQ ID NO:1051) most probably binds to HLA-B*2705.

Peptides C64-C70 represent overlapping 15mers spanning the sequence KGGRKPARLIVFPDLGVRVCE (SEQ ID NO:1052). C64 and C70 react with PBMC from donor H32 and H38, respectively. The minimal sequence of the epitope is therefore located within the sequence ARLIVFPDL (SEQ ID NO:1053). Prediction algorithms indicate that ARLIVFPDL (SEQ ID NO:1053) represents a ligand of HLA-HLA-B*2705 and HLA-B*2709.

TABLE 5
Summary of HCV peptides reactive with PBMC.
	Donors reactive directly ex vivo in IFN-gamma ELIspot with	. . . reactive after 2 rounds
	HCV-derived peptides	of in vitro stimulation
Peptide									H32				H33
ID	G05	G18	H02	H03	H04	H10	H12	H19	SPR	H38	H03	H10	SPR	H38
1557				20							75
1577			15		30								165
1579			45		35						75
1605					25
1615				30							325
1624	30		55		30			420
1628			40		45		25			20				100
1629			30		70		15
1798					25							115
1799							20					90
1800										35		95
1801						20				20
A3											80
A4				15
A5											110
A7											70
A78					25
A170					35
A212					60
A241					35
B08					55
B38									30
B76					35
C64									30
C70										20
C92					25
C94					25
C97					35
C98					70
C100		60			70
C101					50
C102				20
C106					45
C112					20
C118				35
C120				25	45						105
C134					20
C138													30
	Donors reactive directly ex vivo in IFN-gamma ELIspot	. . . reactive after 2
	with HCV-derived peptides	rounds of in vitro stimulation
Peptide									H32				H33
ID	G05	G18	H02	H03	H04	H10	H12	H19	SPR	H38	H03	H10	SPR
1557				20							75
1577			15		30								165
1579			45		35						75
1605					25
1615				30							325
1624	30		55		30			420
1628			40		45		25			20
1629			30		70		15
1798					25							115
1799							20					90
1800										35		95
1801						20				20
A3											80
A4				15
A5											110
A7											70
A78					25
A170					35
A212					60
A241					35
B08					55
B38									30
B76					35
C64									30
C70										20
C92					25
C94					25
C97					35
C98					70
C100		60			70
C101					50
C102				20
C106					45
C112					20
C118				35
C120				25	45						105
Numbers represent peptide-specific IFN-gamma secreting T-cells/106 PBMC calculated from ELIspot results (duplicate determinations); values > 8 (>3× over background) were regarded statistically significant. Donors H32 and H33 are spontaneously recovered patients.

Example V

Binding of HCV Derived Peptides to HLA Class II Molecules

In addition to the peptides listed in Table 1, several new peptides incorporating sequences from overlapping reactive peptides or avoiding critical residues like cystein were synthesized (Table 4). These were retested for their affinities to class II soluble HLA molecules, and results were compared to those obtained with the original (Table 6).

TABLE 6
Binding of selected HCV-derived peptides and their 15-mer
counterparts to soluble HLA class II molecules
SEQ ID	Peptide		Binding to soluble HLA-DRB1*
NO:	ID	Peptide sequences	0101	0401	0404	0701	1101
1054	1798	IGLGKVLVDILAGYGAGVAGALVAFK	−	−	+	++	+/−
1055	B84	GSIGLGKVLVDILAG	+	+	+
1056	B86	IGLGKVLVDILAGYG	+	++	+	+
1057	B88	LGKVLVDILAGYGAG	+	++	+
1058	B92	LVDILAGYGAGVAGA	+	−
1059	B94	DILAGYGAGVAGALV	+	−	−	−
1060	B96	LAGYGAGVAGALVAF	++	++	−	+/−	+/−
1061	1799	AAWYELTPAETTVRLR	++	+	+	−	+/−
1062	B46	AGAAWYELTPALETTV	+++	+++	+++	−	+/−
1063	B48	AAWYELTPAETTVRL	+++	+++	+++	−	+/−
1064	1827	TAYSQQTRGLLG	++	−	+/−	+	+
1065	C114	TAYSQQTRGLLGCIV	+++	+/−	+/−	+	++
1066	1829	SMSYTWTGALITP	+	−	−	+	+/−
1067	1604	VVCCSMSYTWTGALITPC	+	+	++	++	+
1068	1650	VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL
1069	A130	DYPYRLWHYPCTVNF	+	++	+/−
1070	A131	YPYRLWHYPCTVNFT		−
1071	A135	LWHYPCTVNFTIFKV	−	−		++
1072	A141	TVNFTIFKVRMYVGG	−	−	+/−		++
1073	A145	TIFKVRMYVGGVEHR		+/−	−
1074	1651	VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL
1075	1800	DYPYRLWHYPCTVNYTIFKI	−	−	+/−	++	−
1076	A147	DYPYRLWHYPCTVNY	−	−
1077	A152	LWHYPCTVNYTIFKI	−	−
1078	A158	TVNYTIFKIRMYVGG −	−	+/−
1079	A162	TIFKIRMYVGGVEHR		+/−	−
1080	1817	RMYVGGVEHRL		−	−	+/−
1081	1426	HMWNFISGIQYLAGLSTLPGNPA	+	+	++	++	+
1082	1425	NFISGIQYLAGLSTLPGNPA	++	++	++	nd	nd
1083	1006	MWNFISGIQYLAGLSTLPGN	++	+	++	nd	nd
(“+++” strong affinity, “++” intermediate affinity, “+” weak affinity, “−+ no affinity, “nd” not done; core binding motifs are underlined)

Abolished affinities to DRB1*0101 and DRB1*0401 molecules in the case of peptide 1798 in comparison with its shorter counterparts (B84-B96) is probably due to the long sequence (26 amino acids) which can have a secondary structure that prevents binding. It is to be expected that in vivo, upon proteolytic cleavage, peptide 1798 will give rise to two shorter class II epitopes. Removed cystein (C) residues in peptides 1827 and 1829 (derivatives of peptides C114 and 1604, respectively) seem to be crucial for binding to DRB1*0401 molecules but do not essentially change affinities to other tested DR subtypes.

Example VI

Identification and Characterization of HCV-Epitope Hotspots

Here, a T-cell epitope hotspot (thereafter referred to as “hotspot”) is defined as a short peptide sequence at least comprising more than one T-cell epitope. For example, two or more epitopes may be located shortly after each other (shortly being defined as less than 5-10 amino acids), or directly after each other, or partially or even fully over-lapping. Hotspots may contain only class I or class II epitopes, or a combination of both. Epitopes in hotspots may have different HLA restrictions.

Due to the highly complex and selective pathways of class I and class II antigen processing, referred to in the introduction, T-cell epitopes cannot be easily predicted within the sequence of a polypeptide. Though widely used, computer algorithms for T-cell epitope prediction have a high rate of both false-negatives and false-positives.

Thus, as even individual T-cell epitopes are not obvious within the sequence of a polypeptide, the same is even more the case for hotspots. Several radically different experimental approaches are combined according to the present invention for T-cell epitope identification, including epitope capture, HLA-transgenic animals and in vitro stimulation of human mononuclear cells. All three approaches are systematically applied on overlapping peptides spanning the antigen of interest, enabling comprehensive identification of epitopes (refer to CMV Epitope Capture patent). Upon such a comprehensive analysis, not limited to a particular HLA allele, but rather unravelling all possibly targeted epitopes within a population, epitope hotspots may become apparent. Within an antigen, only few if any sequences show characteristics of hotspots. Thus the identification of a hotspot is always a surprising event.

T-cell epitope hotspots offer important advantages: Hotspots can activate and can be recognized by different T-cell clones of a subject. Hotspots (when comprising epitopes with different HLA restriction) can interact with T-cells from different non HLA-matched individuals.

Epitope-based vaccines, so far have aimed at selected prevalent HLA-alleles, for instance HLA-A2, which is expressed in about half of Caucasians. Since other alleles are less frequent, epitope-based vaccines with broad worldwide population coverage will have to comprise many different epitopes. The number of chemical entities (for instance peptides) of a vaccine is limited by constraints originating from manufacturing, formulation and product stability.

Hotspots enable such epitope-based vaccines with broad worldwide population coverage, as they provide a potentially high number of epitopes by a limited number of peptides.

TABLE 7
T-cell epitope hotspots in conserved regions of HCV.
peptide
ID	peptide sequence	Class I	class II	source data
1835	KFPGGGQIVGGVYLLPRRGPRLGVRATRK	(SEQ ID NO:1084)	A2, A3, B7	DR11	Example III, VI
83	KFPGGGQIVGGVYLLPRRGPRL	(SEQ ID NO:1085)	A2 B7	DR11	Example VI
1051	YLLPRRGPRL	(SEQ ID NO:1086)	A2		Bategay 1995
1843	LPRRGPRL	(SEQ ID NO:1087)	B7		Example III
	GPRLGVRAT	(SEQ ID NO:1088)	B7		Koziel 1993
	RLGVRATRK	(SEQ ID NO:1089)	A3		Chang 1999
84	GYKVLVLNPSVAAT	(SEQ ID NO:1090)		DR1, 4, 7, 11	Tab.2:A200-A206
	AYAAQGYKVL (SEQ ID NO:1091)	A24		prediction
84EX	AYAAQGYKVLVLNPSVAAT	(SEQ ID NO:1092) A24		DR1, 4, 7, 11	Example VI
87	DLMGYIPAV	(SEQ ID NO:1093)	A2		Sarobe 1998
	GYIPLVGAPL	(SEQ ID NO:1094)	A24		prediction
87EX	DLMGYIPLVGAPL	(SEQ ID NO:1095)	A2, A24		Example VI
89	CINGVCWTV	(SEQ ID NO:1096)	A2		Koziel 1995
1577	GEVQVVSTATQSFLLAT	(SEQ ID NO:1097)		DR 4, 7	Tab.2
89EX	GEVQVVSTATQSFLATCINGVCWTV	(SEQ ID NO:1098)	A2	DR 4, 7	Example VI
1426	HMWNFISGIQYLAGLSTLPGNPA	(SEQ ID NO:1099)	A2	DR1, 4, 7, 11	Example VII
1006	MWNFISGIQYLAGLSTLPGN	(SEQ ID NO:1100)			Example VII
1425	NFISGIQYLAGLSTLPGNPA	(SEQ ID NO:1101)			Example VII
	QYLAGLSTL	(SEQ ID NO:1102)	A24		prediction
1334	HMWNFISGI	(SEQ ID NO:1103)	A2		Wentworth 1996
1650	VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL	(SEQ ID NO:1104)	Cw7 , A2 , A24,	DR1, 4,7,11	Tab.2, 3, 6
			A11, A3		Example III
1836	DYPYRLWHYPCTVNFTIFKI	(SEQ ID NO:1105)	Cw7, A2; A24,	DR1, 4, 7, 11	Tab.2, 3, 6
			A11
1846	DYPYRLWHYPCTVNFTIFKV	(SEQ ID NO:1106)	Cw7, A2; A24,	DR1, 4, 7, 11	Tab.2, 3, 6
			A11		Example III
1651	VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL	(SEQ ID NO:1107)			Tab.2, 3, 6
1800	DYPYRLWHYPCTVNYTIFKI	(SEQ ID NO:1108)	Cw7, A24, A11	DR7	Tab.2, 5, 6
1754	DYPYRLWHY	(SEQ ID NO:1109)	Cw7		Lauer 2002
1815	TVNYTIFKI	(SEQ ID NO:1110)	A11		prediction
1816	TINYTIFK	(SEQ ID NO:1111)	A11		Koziel 1995
	TVNFTIFKV	(SEQ ID NO:1112)	A11		prediction
	HYPCTVNYTI	(SEQ ID NO:1113)	A24		prediction
	HYPCTVNFTI	(SEQ ID NO:1114)	A24		prediction
	RMYVGGVEHR	(SEQ ID NO:1115)	A3		Chang 1999
1799	AAWYELTPAETTVRLR	(SEQ ID NO:1116)	B7? B35	DR1, 4	Tab.2, 5, 6
1818	TPAETTVRL	(SEQ ID NO:1117)	B7? B35		Ibe 1998
1827EX	GWRLLAPITAYSQQTRGLLGCIV	(SEQ ID NO:1118)	A2 ,A3, A24,	DR1, 4, 7, 11	Example VI
			B8
C114	TAYSQQTRGLLGCIV	(SEQ ID NO:1119)	A24, B8?	DR1, 4, 7, 11	Tab.2, 6
1827	TAYSQQTRGLLG	(SEQ ID NO:1120)	A24, B8?	DR1, 7, 11	Tab.6
C112	GQGWRLLAPITAYSQ	(SEQ ID NO:1121)	A3?, A2?,	DR1	Tab.2, 5
	RLLAPITAY	(SEQ ID NO:1122)	A3		prediction
C114EX	GQGWRLLAPITAYSQQTRGLLGCIV	(SEQ ID NO:1123)	A24, A3?, A2?,	DR1, 4, 7, 11	Tab.2, 5, 6
			B8?
1827EX	GQGWRLLAPITAYSQQTRGLLG	(SEQ ID NO:1124)	A24, A3?, A2?,	DR1, 7, 11	Tab.2, 5, 6
			B8?
1801	AYSQQTRGLL	(SEQ ID NO:1125)	A24		Tab.5
1819	AYSQQTRGL	(SEQ ID NO:1126)	A24		Kurokohchi 2001
1798	IGLGKVLVDILAGYGAGVAGALVAFK	(SEQ ID NO:1127)	A2, 24, 3, 11	DR1, 4, 7	Tab.2, 3, 5, 6
1820	ILAGYGAGV	(SEQ ID NO:1128)	A2		Bategay 1995
1821	VAGALVAFK	(SEQ ID NO:1129)	A3, 11		Chang 1999
	GYGAGVAGAL	(SEQ ID NO:1130)	A24		prediction
1604	VVCCSMSYTWTGALITPC	(SEQ ID NO:1131)	A2, A24, B7	DR1, 4, 7, 11	Tab.2, 3, 6
1829	SMSYTWTGALITP	(SEQ ID NO:1132)	A2, A24, B7,	DR1, 7, 11	Tab.6
	SMSYTWTGAL	(SEQ ID NO:1133)	A2, B7		prediction
	SYTWTGALI	(SEQ ID NO:1134)	A24		prediction
1579	FTDNSSPPAVPQTFQV	(SEQ ID NO:1135)	A1, 2; B7, 51	DR53 = B4*01	Tab.5
1624	LEDRDRSELSPLLLSTTEW	(SEQ ID NO:1136)	A1, 2, 3, 26	DR7	Tab.2, 3, 5
			B8, 27, 4402, 60
1848	LEDRDRSELSPLLLST	(SEQ ID NO:1137)	A1, 2, 3, 26,	DR7	Example VI
			B8, 27, 44 02, 60
	RSELSPLLL	(SEQ ID NO:1138)	A1		prediction
	ELSPLLLST	(SEQ ID NO:1139)	A2, A3		prediction
	DRDRSELSPL	(SEQ ID NO:1140)	A26, B27		prediction
	LEDRDRSEL	(SEQ ID NO:1141)	B08, B4402		prediction
1824	LEDRDRSEL	(SEQ ID NO:1142)	B60		Wang 2001
1547	YLVAYQATVCAEAQAPPPSWD	(SEQ ID NO:1143)	A2	DR1, 4, 7, 11	Tab.2, 3
1822	YLVAYQATV	(SEQ ID NO:1144)	A2		Wentworth 1996
A1A7	MSTNPKPQRKTKRNTNR	(SEQ ID NO:1145)	A11, B08, B27		Tab.5
A3A7	PQRKTKRNTNR	(SEQ ID NO:1146)	B08, B27		Tab.5
	QRKTKRNTN	(SEQ ID NO:1147)	B08		prediction
	RKTKRNTNR	(SEQ ID NO:1148)	B2705		prediction
	MSTNPKPQR	(SEQ ID NO:1149)	A11		prediction
	MSTNPKPQK	(SEQ ID NO:1150)	A11		Wong 1998
A122EX	LINTNGSWHINRTALNCNDSL	(SEQ ID NO:1151)	A2, 2, 3, B8	DR1, 4, 7, 11	Tab.2, 3
A122	NGSWHINRTALNCNDSL	(SEQ ID NO:1152)	A2	DR1, 4, 7, 11	Tab.2, 3
	LINTNGSWHI	(SEQ ID NO:1153)	A2, 3		prediction
	RTALNCNDSL	(SEQ ID NO:1154)	A2		prediction
1825	LINTNGSWHINRTALN	(SEQ ID NO:1155)	A2, 3, B8		prediction
1826	SWHINRTALN	(SEQ ID NO:1156)	B8		prediction
A241	TTILGIGTVLDQAET	(SEQ ID NO:1157)	A2, A3	DR1, 4	Tab.2, 5
	TTILGIGTV	(SEQ ID NO:1158)	A2		prediction
	TILGIGTVL	(SEQ ID NO:1159)	A3		prediction
B8B38	FDSSVLCECYDAGAAWYE	(SEQ ID NO:1160)	A1, 2, 3, 26		Tab.5
B8	FDSSVLCECYDAGCA	(SEQ ID NO:1161)	A3, A26		Tab.5
	VLCECYDAGA	(SEQ ID NO:1162)	A2		prediction
B38	VVLCECYDAGAAWYE	(SEQ ID NO:1163)	A1		Tab.5
C70EX	ARLIVFPDLGVRVCEKMALY	(SEQ ID NO:1164)	A2, A3, B27		Tab.5
C64-C70	ARLIVFPDL	(SEQ ID NO:1165)	B*2705?, *2709?		Tab.5
1831	RLIVFPDLGV	(SEQ ID NO:1166)	A2		Gruener 2000
1832	RVCEKMALY	(SEQ ID NO:1167)	A3		Wong 1998
C92	AFCSAMYVGDLCGSV	(SEQ ID NO:1168)	A2, B51	DR1, 4	Tab.2, 5
C97	GVLFGLAYFSHVGNW	(SEQ ID NO:1169)	A2, 3, 26,	DR1, 4, 7	Tab.5
			B2705, 51
C106	TRVPYFVRAQGLIRA	(SEQ ID NO:1170)	A3, 24,	DR1, 4, 7	Tab.2, 5
			B7 ,B8, B2705
C134	TTLLFNILGGWVAAQ	(SEQ ID NO:1171)	A2	DR1, 7, 11	Tab.2, 5
1823	LLFNILGGWV	(SEQ ID NO:1172)	A2		Bategay 1995
Hotspots (incl. some variations) are shown in bold, epitopes contained within the hotspots in normal font. Peptide number and sequence, as well as HLAclass I and class II coverage are given. Source data refers to Examples and Tables within this specification, or literature references.

Example VII

HCV Epitope Hotspot Ipep 1426 Contains at Least HLA-A*0201 and Several Promiscuous Class II T-Cell Epitopes

The major objective of this experiment was to compare the immunogenicity of the “hospot” Ipep 1426, which contains at least one HLA-A*0201 epitope (Ipep 1334) and 2 promiscuous class II epitopes (Ipeps 1006 and 1425), to the individual epitopes. To this end peripheral blood mononuclear cells (PBMC) from several healthy HLA-typed blood donors were stimulated in vitro either with 1426 or a mixture of 1334, 1006, 1425. Three rounds of stimulation were performed resulting in oligoclonal T cell lines. Then, responses against all four peptides were assessed by interferon-gamma (IFN-γ) ELIspot analysis.

Peptide 1426, induces T cell responses similarly well as individual epitopes comprised within its sequence. In particular, CD8 positive T cells directed against the HLA-A*0201 restricted epitope 1334 were successfully generated.

TABLE 8
peptide induced IFN-γ secretion of oligoclonal T cell lines.
	Donor HLA
	A*0201, A*03, B7, B60;	A*0206, A*01, B27, B50;
	DRB1*1501, −B1*1302	DRB1*0401, −B1*1402
	line raised	line raised	line raised	line raised
	against	against 1006 +	against	against 1006 +
Peptide ID	1426	1425 + 1334	1426	1425 + 1334
1006	++	++	++	++
1425	+++	+++	+++	++
1334	+	+	−	−
1006 +	++	++	++	++
1425 +
1334
1426	+++	+++	+++	++
84 (HCV	−	−	−	−
derived
negative
control)
Lines were generated from two HLA-typed healthy individuals by 3 rounds of in vitro priming with either peptide 1426 or a mixture of peptides 1006 + 1425 + 1334. The reactivity of CD4 and CD8 positive T cells in these lines was assessed by IFN-γ ELIspot (“+++” very strong, “++” strong, “+” significant, “−” no IFN-gamma secretion).

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Claims

1. An immunogenic composition comprising a pharmaceutically acceptable carrier and one or more epitopes from polypeptides A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604, 1630, C97, 1547, B94-B98, A272-A276, B120, B122, C108, C134, C152, 1606, 1607, 1577, 1578, B50-52, 1623, C130, 1603, C96, C191, A216-A224, A242-A244, C92-C93, A174, B32-B38, B100-B102, C135, C162, 1618, 1622, 1624, 1546, 1556, A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, C174, 1558, 1581, C95, C129, C157-C158, A254-A258, 1605, C109, C161, 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1605, 1616, 1621, 1623, 1625, 1649, 1650, 1651, 1652, 1654, 1655, 1656, 1545, 1552, 1557, 1615, 1617, 1631, 1632, 1641, 1647, 1653, A141, C114, C134, C135, 1426, 1506, 1526, 1553, 1562, 1565, 1580, 1587, 1624, 1627, 1843, or 1838, wherein the immunogenic composition stimulates a specific T cell response to hepatitis C virus when administered to a subject in an effective amount.

2. The immunogenic composition of claim 1, wherein the epitope further comprises 1 to 30 naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

3. The immunogenic composition of claim 1, wherein the epitope further comprises 2 to 10 naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

4. The immunogenic composition of claim 1, wherein the epitope further comprises 2 to 6 naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

5. The immunogenic composition of claim 1, wherein the epitope further comprises 2 to 100 non-naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

6. The immunogenic composition of claim 1, wherein the epitope further comprises 2 to 20 non-naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

7. The immunogenic composition of claim 1, wherein the epitope further comprises 1 to 1000 non-naturally occurring amino acid residues at its N-terminus, its C-terminus or at its N- and C-terminus.

8. The immunogenic composition of claim 1, wherein at least one of the epitopes is an HLA A0201 binding epitope from polypeptide 1545, 1552, 1555, 1558, 1559, 1560, 1577, 1592, 1604, 1605, 1615, 1617, 1621, 1627, 1631, 1632, 1641, 1647, 1650, 1651, 1652, 1653, 1654, or 1655.

9. The immunogenic composition of claim 1, wherein at least one of the epitopes is an HLA-B*0702 binding epitope from polypeptide 1506, 1526, 1547, 1552, 1553, 1555, 1558, 1562, 1563, 1565, 1577, 1578, 1580, 1587, 1592, 1604, 1605, 1621, 1623, 1624, 1627, 1628, 1647, 1650, 1651, 1843 or 1838.

10. The immunogenic composition of claim 1, further comprising an immunomodulating substance.

11. The immunogenic composition of claim 1, wherein the immuno-modulating substance is a polycationic polypeptide, an immunomodulating nucleic acid, or mixtures thereof.

12. The immunogenic composition of claim 1, wherein immunomodulating nucleic acid is a deoxyinosine and/or deoxyuracil containing oligodeoxynucleotide.

13. The immunogenic composition of claim 1, wherein the epitope is provided in a dendritic cell pulsed with a protein, peptide, or RNA or transfected with a DNA comprising the epitope.

14. A method for stimulating a specific immune response against hepatitis C virus (HCV) in a subject comprising administering to the subject an effective amount of an immunogenic composition comprising one or more epitopes from polypeptides A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604, 1630, C97, 1547, B94-B98, A272-A276, B120, B122, C108, C134, C152, 1606, 1607, 1577, 1578, B50-52, 1623, C130, 1603, C96, C191, A216-A224, A242-A244, C92-C93, A174, B32-B38, B100-B102, C135, C162, 1618, 1622, 1624, 1546, 1556, A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, C174, 1558, 1581, C95, C129, C157-C158, A254-A258, 1605, C109, C161, 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1605, 1616, 1621, 1623, 1625, 1649, 1650, 1651, 1652, 1654, 1655, 1656, 1545, 1552, 1557, 1615, 1617, 1631, 1632, 1641, 1647, 1653, A141, C114, C134, C135, 1426, 1506, 1526, 1553, 1562, 1565, 1580, 1587, 1624, 1627, 1843, or 1838, wherein the specific immune response to hepatitis C virus is stimulated in the subject.

15. The method of claim 14, wherein the immunogenic composition further comprising an immunomodulating substance.

16. The method of claim 15, wherein the immunomodulating substance is a polycationic polypeptide, an immunomodulating nucleic acid, or mixtures thereof.

17. The method of claim 16, wherein immunomodulating nucleic acid is a deoxyinosine and/or deoxyuracile containing oligodeoxynucleotide.

18. The method of claim 14, wherein the epitope is provided in a dendritic cell pulsed with a protein, peptide, or RNA or transfected with a DNA comprising the epitope.

19. The method of claim 14, wherein at least one of the epitopes is an HLA A0201 binding epitope from polypeptide 1545, 1552, 1555, 1558, 1559, 1560, 1577, 1592, 1604, 1605, 1615, 1617, 1621, 1627, 1631, 1632, 1641, 1647, 1650, 1651, 1652, 1653, 1654, or 1655.

20. The method of claim 14, wherein at least one of the epitopes is an HLA-B*0702 binding epitope from polypeptide 1506, 1526, 1547, 1552, 1553, 1555, 1558, 1562, 1563, 1565, 1577, 1578, 1580, 1587, 1592, 1604, 1605, 1621, 1623, 1624, 1627, 1628, 1647, 1650, 1651, 1843 or 1838.

21. An isolated T cell that specifically recognizes an epitope of polypeptide A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604, 1630, C97, 1547, B94-B98, A272-A276, B120, B122, C108, C134, C152, 1606, 1607, 1577, 1578, B50-52, 1623, C130, 1603, C96, C191, A216-A224, A242-A244, C92-C93, A174, B32-B38, B100-B102, C135, C162, 1618, 1622, 1624, 1546, 1556, A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, C174, 1558, 1581, C95, C129, C157-C158, A254-A258, 1605, C109, C161, 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1605, 1616, 1621, 1623, 1625, 1649, 1650, 1651, 1652, 1654, 1655, 1656, 1545, 1552, 1557, 1615, 1617, 1631, 1632, 1641, 1647, 1653, A141, C114, C134, C135, 1426, 1506, 1526, 1553, 1562, 1565, 1580, 1587, 1624, 1627, 1843, or 1838.

22. A method of treating a hepatitis C virus infection in a subject comprising administering to the subject an effective amount of a composition comprising a T cell that specifically recognizes an epitope of polypeptide A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629, 1604, 1630, C97, 1547, B94-B98, A272-A276, B120, B122, C108, C134, C152, 1606, 1607, 1577, 1578, B50-52, 1623, C130, 1603, C96, C191, A216-A224, A242-A244, C92-C93, A174, B32-B38, B100-B102, C135, C162, 1618, 1622, 1624, 1546, 1556, A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, C174, 1558, 1581, C95, C129, C157-C158, A254-A258, 1605, C109, C161, 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1605, 1616, 1621, 1623, 1625, 1649, 1650, 1651, 1652, 1654, 1655, 1656, 1545, 1552, 1557, 1615, 1617, 1631, 1632, 1641, 1647, 1653, A141, C114, C134, C135, 1426, 1545, 1552, 1555, 1558, 1559, 1560, 1577, 1592, 1604, 1605, 1615, 1617, 1621, 1627, 1631, 1632, 1641, 1647, 1650, 1651, 1652, 1653, 1654, 1655, 1506, 1526, 1552, 1553, 1562, 1565, 1577, 1578, 1580, 1587, 1623, 1624, 1628, 1843, or 1838, wherein the hepatitis C virus infection is treated in the subject.

23. A method of stimulating an HLA-B*08 specific immune response against a hepatitis C virus in a subject comprising administering to the subject an effective amount of a composition comprising one or more epitopes of peptide sequences QRKTKRNTN (SEQ ID NO: 167), QRKTKRNT (SEQ ID NO: 166), SAKSKFGYG (SEQ ID NO: 193), SAKSKYGYG (SEQ ID NO: 194), or SARSKYGYG (SEQ ID NO: 195), wherein the HLA-B*08 specific immune response against the hepatitis C virus is stimulated in the subject.

24. A method of stimulating an HLA-B*2705 specific immune response against a hepatitis C virus in a subject comprising administering to the subject an effective amount of a composition comprising a peptide having sequence RKTKRNTNR (SEQ ID NO: 170), wherein the HLA-B*2705 specific immune response against the hepatitis C virus in the subject.

25. A method of stimulating an HLA-B*2705 and HLA-B*2709 specific immune responses against a hepatitis C virus in a subject comprising administering to the subject an effective amount of a composition comprising a peptide having sequence ARLIVFPDL (SEQ ID NO: 1053), wherein the HLA-B*2705 and HLA-B*2709 specific immune responses against the hepatitis C virus in the subject.

26. A method for stimulating a specific immune response against hepatitis C virus (HCV) in a subject comprising administering to the subject an effective amount of an immunogenic composition comprising one or more epitopes from polypeptides 1835, 84EX, 87EX, 89EX, 1426, 1650, 1836, 1846, 1651, 1800, 1799, C114, 1827, C112, C114EX, 1827EX, 1798, 1604, 1829, 1579, 1624, 1848, 1547, A1A7, A122EX, A122, 1825, A241, B8B38, C70EX, C92, C97, C106, or C134, wherein the specific immune response against the hepatitis C virus (HCV) is stimulated in the subject.