# Patent

## Title
Method for isolating hepatitis C virus peptides

## Abstract

Described is a method for isolating Hepatitis C Virus peptides (HPs) which have a binding capacity to a MHC/HLA molecule or a complex comprising said HCV-peptide and said MHC/HLA molecule characterized by the following steps: —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.

## Background
This application 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 Sequence Listing is submitted on one compact disc (Copy 1), together with a duplicate thereof (Copy 2), each created on Aug. 18, 2005, and each containing one 344 kb file entitled “SONN059SEQ.txt.” The material contained on the compact disc is specifically incorporated herein by reference.
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-encoded 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 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*0702 tg 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 100% 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 nM, 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 a was 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-assosiated 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-negativ 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











PEPTIDE



ID
SEQ ID
PEPTIDE







A1
203
MSTNPKPQRKTKRNT



 



A2
204
STNPKPQRKTKRNTN



 



A3
205
TNPKPQRKTKRNTNR



 



A4
206
NPKPQRKTKRNTNRR



 



A5
207
PKPQRKTKRNTNRRP



 



A6
208
KPQRKTKRNTNRRPQ



 



A7
209
PQRKTKRNTNRRPQD



 



A8
210
QRKTKRNTNRRPQDV



 



A9
211
RKTKRNTNRRPQDVK



 



A10
212
KTKRNTNRRPQDVKF



 



A11
213
TKRNTNRRPQDVKFP



 



A12
214
KRNTNRRPQDVKFPG



 



A13
215
RNTNRRPQDVKFPGG



 



A14
216
NTNRRPQDVKFPGGG



 



A15
217
TNRRPQDVKFPGGGQ



 



A16
218
NRRPQDVKFPGGGQI



 



A17
219
RRPQDVKFPGGGQIV



 



A18
220
RPQDVKFPGGGQIVG



 



A19
221
PQDVKFPGGGQIVGG



 



A20
222
QDVKFPGGGQIVGGV



 



A21
223
DVKFPGGGQIVGGVY



 



A22
224
VKFPGGGQIVGGVYL



 



A23
225
KFPGGGQIVGGVYLL



 



A24
226
FPGGGQIVGGVYLLP



 



A25
227
PGGGQIVGGVYLLPR



 



A26
228
GGGQIVGGVYLLPRR



 



A27
229
GGQIVGGVYLLPRRG



 



A28
230
GQIVGGVYLLPRRGP



 



A29
231
QIVGGVYLLPRRGPR



 



A30
232
IVGGVYLLPRRGPRL



 



A31
233
VGGVYLLPRRGPRLG



 



A32
234
GGVYLLPRRGPRLGV



 



A33
235
GVYLLPRRGPRLGVR



 



A34
236
VYLLPRRGPRLGVRA



 



A35
237
YLPRRGPRLGVRAT



 



A36
238
LLPRRGPRLGVRATR



 



A37
239
LPRRGPRLGVRATRK



 



A38
240
PRRGPRLGVRATRKT



 



A39
241
RRGPRLGVRATRKTS



 



A40
242
RGPRLGVRATRKTSE



 



A41
243
GPPWGVRATRKTSER



 



A42
244
PRLGVRATRKTSERS



 



A43
245
RLGVRATRKTSERSQ



 



A44
246
LGVRATRKTSERSQP



 



A45
247
GVRATRKTSERSQPR



 



A46
248
VRATRKTSERSQPRG



 



A47
249
RATRKTSERSQPRGR



 



A48
250
ATRKTSERSQPRGRR



 



A49
251
TRKTSERSQPRGRRQ



 



A50
252
RKTSERSQPRGRRQP



 



A51
253
KTSERSQPRGRRQPI



 



A52
254
TSERSQPRGRRQPIP



 



A53
255
SERSQPRGRRQPIPK



 



A54
256
DPRRRSRNLGKVIDT



 



A55
257
PRRRSRNLGKVIDTL



 



A56
258
RRRSRNLGKVIDTLT



 



A57
259
RRSRNLGKVIDTLTC



 



A58
260
RSRNLGKVIDTLTCG



 



A59
261
SRNLGKVIDTLTCGF



 



A60
262
RNLGKVIDTLTCGFA



 



A61
263
NLGKVIDTLTCGFAD



 



A62
264
LGKVIDTLTCGFADL



 



A63
265
GKVIDTLTCGFADLM



 



A64
266
KVIDTLTCGFADLMG



 



A65
267
VIDTLTCGFADLMGY



 



A66
268
IDTLTCGFADLMGYI



 



A67
269
DTLTCGFADLMGYIP



 



A68
270
TLTCCFADLMGYIPL



 



A69
271
LTCGFADLMGYIPLV



 



A70
272
TCGFADLMGYIPLVG



 



A71
273
CGFADLMGYIPLVGA



 



A72
274
GFADLMGYIPLVGAP



 



A73
275
FADLMGYIPLVGAPL



 



A74
276
ADLMGYIPLVGAPLG



 



A75
277
DLMGYIPLVGAPLGG



 



A76
278
DPRHRSRNVGKVIDT



 



A77
279
PRHRSRNVGKVIDTL



 



A78
280
RHRSRNVGKIDTLT



 



B41
281
CECYDAGAAWYELTP



 



B42
282
ECYDAGAAWYELTPA



 



B43
283
CYDAGAAWYELTPAE



 



B44
284
YDAGAAWYELTPAET



 



B45
285
DAGAAWYELTPAETT



 



B46
286
AGAAWYELTPAETTV



 



B47
287
GAAWYELTIPAETTV



 



B48
288
AAWYELTPAETTVRL



 



B49
289
DAGAAWYELTPAETS



 



B50
290
AGAAWYELTPAETSV



 



B51
291
GAAWYELTPAETSVR



 



B52
292
AAWYELTPAETSVRL



 



B53
293
FWAKHMWNFISGIQY



 



B54
294
WAKHMWNFISGIQYL



 



B55
295
AKHMWNFISGIQYLA



 



B56
296
KHMWNFISGIQYLAG



 



B57
297
HMWNFISGIQYLAGL



 



B58
298
MWNFISGIQYLAGLS



 



B59
299
WNFISGIQYLAGLST



 



B60
300
NFISGIQYLAGLSTL



 



B61
301
FISGIQYLAGLSTLP



 



B62
302
ISGIQYLAGLSTLPG



 



B63
303
SGIQYLAGLSTLPGN



 



B64
304
GIQYLAGLSTLPGNP



 



B65
305
IQYLAGLSTLPGNPA



 



B66
306
QYLAGLSTLPGNPAI



 



B67
307
YLAGLSTLPGNPAIA



 



B68
308
LAGLSTLPGNPAIAS



 



B69
309
AGLSTLPGNPAIASI



 



B70
310
GLSTLPGNPAIASLM



 



B71
311
LSTLPGNPAIASLMA



 



B72
312
STLPGNPAIASLMAF



 



B73
313
QYLAGLSTLPGNPAV



 



B74
314
YLAGLSTLPGNPAVA



 



B75
315
LAGLSTLPGNPAVAS



 



B76
316
AGLSTLPGNPAVASM



 



B77
317
GLSTLPGNPAVASMM



 



B78
318
LSTLPGNPAVASMMA



 



B79
319
STLPGNPAVASMMAF



 



B80
320
GAAVGSIGLGKVLVD



 



B81
321
AAVGSIGLGKVLVDI



 



B82
322
AVGSIGLGKVLDIL



 



B83
323
VGSIGLGKVLVDILA



 



B84
324
GSIGLGKVLVDILAG



 



B85
325
SIGLGKVLVDILAGY



 



B86
326
IGLGKVLVDILAGYG



 



B87
327
GLGKVLVDILAGYGA



 



B88
328
LGKVLVDILAGYGAG



 



B89
329
GKVLVDILAGYGAGV



 



B90
330
KVLVDILAGYGAGVA



 



B91
331
VLVDILAGYGAGVAG



 



B92
332
LVDILAGYGAGVAGA



 



B93
333
VDILAGYGAGVAGAL



 



B94
334
DILAGYGAGVAGALV



 



B95
335
ILAGYGAGVAGALVA



 



B96
336
LAGYGAGVAGALVAF



 



B97
337
AGYGAGVAGALVAFK



 



B98
338
GYGAGVAGALVAFKI



 



B99
339
YGAGVAGALVAFKIM



 



B100
340
GAGVAGALVAFKIMS



 



B101
341
AGVAGALVAFKIMSG



 



B102
342
GVAGALVAFKIMSGE



 



B103
343
GYGAGVAGALVAFKV



 



B104
344
YGAGVAGALVAFKVM



 



B105
345
GAGVAGALVAFKVMS



 



B106
346
AGVAGALVAFKVMSG



 



B107
347
GVAGALVAFKVMSGE



 



B108
348
GKVLVDILAGYGAGI



 



B109
349
KVLVDILAGYGAGIS



 



B110
350
VLVDILAGYGAGISG



 



B111
351
LVDILAGYGAGISGA



 



B112
352
VDILAGYGAGISGAL



 



B113
353
DILAGYGAGISGALV



 



B114
354
ILAGYGAGISGALVA



 



B115
355
LAGYGAGISGALVAF



 



B116
356
AGYGAGISGALVAFK



 



B117
357
GYGAGISGALVAFKI



 



B118
358
YGAGISGALVAFKIM



 



B119
359
GAGISGALVAFKIMS



 



A79
360
HRSRNVGKVIDTLTC



 



A80
361
RSRNVGKVIDTLTCG



 



A81
362
SRNVGKVIDTLTCGF



 



A82
363
RNVGKVIDTLTCGFA



 



A83
364
NVGKVIDTLTCGFAD



 



A84
365
VGKVIDTLTCGFADL



 



A85
366
TLTCGFADLMGYIPV



 



A86
367
LTCGFADLMGYIPVV



 



A87
368
TCGFADLMGYIPVVG



 



A88
369
CGFADLMGYIPVVGA



 



A89
370
GFADLMGYIPVVGAP



 



A90
371
FADLMGYIPVVGAPL



 



A91
372
ADLMGYIPVVCAPLG



 



A92
373
DLMGYIPVVGAPLGG



 



A93
374
ARALAHGVRVLEDGV



 



A94
375
RALAHGVRVLEDGVN



 



A95
376
ALAHGVRVLEDGVNY



 



A96
377
LAHGVRVLEDGVNYA



 



A97
378
AHGVRVLEDGVNYAT



 



A98
379
HGVRVLEDGVNYATG



 



A99
380
GVRVLEDGVNYATGN



 



A100
381
VRVLEDGVNYATGNL



 



A101
382
RVLEDGVNYATGNLP



 



A102
383
VLEDGVNYATGNLPG



 



A103
384
LEDGGVNYATGNLPGC



 



A104
385
EDGVNYATGNLPGCS



 



A105
386
DGVNYATGNLPGCSF



 



A106
387
GVNYATGNLPGCSES



 



A107
388
VNYATGNLPGCSFSI



 



A108
389
NYATGNLPGCSFSIF



 



A109
390
YATGNLPGCSFSIFL



 



A110
391
ATGNLPGCSFSIFLL



 



A111
392
TGNLPGCSFSIFLLA



 



A112
393
GNLPGCSFSIFLLAL



 



A113
394
NLPGCSFSIFLLALL



 



A114
395
LPGCSFSIFLLALLS



 



A115
396
PGCSFSIFLLALLSC



 



A116
397
IQLINTNGSWHINRT



 



A117
398
QLINTNGSWHINRTA



 



A118
399
LINTNGSWHINRTAL



 



A119
400
INTNGSWINRTALN



 



A120
401
NTNGSWHINRTALNC



 



A121
402
TNGSWHINRTALNCN



 



A122
403
NGSWHINRTALNCND



 



A123
404
GSWHINRTALNCNDS



 



A124
405
SWHINRTALNCNDSL



 



A125
406
IQLVNTNGSWHINRT



 



A126
407
QLVNTNGSWHINRTA



 



A127
408
LVNTNGSWHINRTAL



 



A128
409
VNTNGSWHINRTALN



 



A129
410
VDYPYRLWHYPCTVN



 



A130
411
DYPYRLWHYPCTVNF



 



A131
412
YPYRLWHYPCTVNFT



 



A132
413
PYRLWHYPCTVNFTI



 



A133
414
YRLWHYPCTVNFTIF



 



A134
415
RLWHYPCTVNFTIFK



 



A135
416
LWHYPCTVNFTIFKV



 



A136
417
WHYPCTVNFTIFKVR



 



A137
418
HYPCTVNFTIFKVRM



 



A138
419
YPCTVNFTIFKVRMY



 



A139
420
PCTVNFTIFKVRMYV



 



A140
421
CTVNFTIFKVRMYVG



 



A141
422
TVNFTIFKVRMYVGG



 



A142
423
VNFTIFKVRMYVGGV



 



A143
424
NFTIFKVRMYVGGVE



 



A144
425
FTIFKVRMYVGGVEH



 



A145
426
TIFKVRMYVGGVEHR



 



A146
427
IFKVRMYVGGVEHRL



 



A147
428
DYPYRLWHYPCTVNY



 



A148
429
YPYRLWHYPCTVNYT



 



A149
430
PYRLWHYPCTVNYTI



 



A150
431
YRLWHYPCTVNYTIF



 



A151
432
RLWHYPCTVNYTIFK



 



A152
433
LWHYPCTVNYTIFKI



 



A153
434
WHYPCTVNYTIFKIR



 



A154
435
HYPCTVNYTIFKIRM



 



A155
436
YPCTVNYTIFKIRMY



 



A156
437
PCTVNYTIFKIRMYV



 



B120
438
AGISGALVAFKIMSG



 



B121
439
GISGALVAFKIMSGE



 



B122
440
VNLLPAILSPGALVV



 



B123
441
NLLPAILSPGALWG



 



B124
442
LLPAILSPGALVVGV



 



C1
443
LPAILSPGALVVGVV



 



C2
444
PAILSPGALVVGVVC



 



C3
445
AILSPGALVVGVVCA



 



C4
446
ILSPGALVVGVVCAA



 



C5
447
LSPGALVVGVVCAAI



 



C6
448
SPGALVVGVVCAAIL



 



C7
449
PGALVVGVVCAAILR



 



C8
450
GALVVGVV0AAILRR



 



C9
451
ALVVGVVCAAILRRH



 



C10
452
LVVGWCAAILRRHV



 



C11
453
VVGVVCAAILRRHVG



 



C12
454
VGVVCAAILRRHVGP



 



C13
455
GVVCAAILRRHVGPG



 



C14
456
VVCAAILRRHVGPGE



 



C15
457
VCAAILRRHVGPGEG



 



C16
458
CAAILRRHVGPGEGA



 



C17
459
AAILRRHVGPGEGAV



 



C18
460
AILRRHVGPGEGAVQ



 



C19
461
ILRRHVGPGEGAVQW



 



C20
462
LRRHVGPGEGAVQWM



 



C21
463
RRHVGPGEGAVQWMN



 



C22
464
RHVGPGEGAVQWMNR



 



C23
465
HVGPGEGAVQWMNRL



 



C24
466
VGPGEGAVQWMNRLI



 



C25
467
GPGEGAVQWMNRLIA



 



C26
468
PGEGAVQWMNRLIAF



 



C27
469
GEGAVQWMNRLIAFA



 



C28
470
EGAVQWMNRLIAFAS



 



C29
471
GAVQWMNRLIAFASR



 



C30
472
AVQWMNRLIAFASRG



 



C31
473
VQWMNRLIAFASRNGN



 



C32
474
QWMNRLIAFASRGNH



 



C33
475
WMNRLIAFASRGNHV



 



C34
476
MNRLAFASRGNHVS



 



C35
477
NRLIAFASRGNHVSP



 



C36
478
RLIAFASRGNHVSPT



 



C37
479
LIAFASRGNHVSPTH



 



C38
480
IAFASRGNHVSPTHY



 



C39
481
AFASRGNHVSPTHYV



 



C40
482
VNLLPGILSPGALVV



 



C41
483
NLLPGILSPGALVVG



 



C42
484
LLPGILSPGALVVGV



 



C43
485
LPGILSPGALVVGVI



 



C44
486
PGILSPGALVVGVIC



 



C45
487
GILSPGALVVGVICA



 



C46
488
ILSPGALVVGVICAA



 



C47
489
LSPGALVVGVICAAI



 



C48
490
SPGALVVGVICAAIL



 



C49
491
PGALVVGVICAAILR



 



C50
492
GALVVGVICAAILRR



 



C51
493
ALVVGVICAAILRRH



 



C52
494
LVVGVICAAILRRHV



 



C53
495
VVGVI0AAILRRHVG



 



C54
496
VGVIOAAILRRHVGP



 



C55
497
GVICAAILRRHVGPG



 



C56
498
VICAAILRRHVGPGE



 



C57
499
CAAILRRHVGPGEG



 



C58
500
MNRLIAFASRGNHVA



 



C59
501
NRLIAFASRGNHVAP



 



C60
502
RLIAFASRGNHVAPT



 



C61
503
LIAFASRGNHVAPTH



 



C62
504
IAFASRGNHVAPTHY



 



C63
505
AFASRGNHVAPTHYV



 



C64
506
KGGRKPARLIVFPDL



 



C65
507
GGRKPARLIVFPDLG



 



C66
508
GRKPARLIVFPDLGV



 



C67
509
RKPARLIVFPDLGVR



 



C68
510
KPARLIVFPDLGVRV



 



C69
511
PARLIVFPDLGVRVC



 



C70
512
ARLIVFPDLGVRVCE



 



C71
513
RLIVFPDLGVRVCEK



 



C72
514
LIVFPDLGVRVCEKM



 



C73
515
IVFPDLGVRVCEKMA



 



C74
516
VFPDLGVRVCEKMAL



 



A157
517
CTVNYTIFKIRMYVG



 



A158
518
TVNYTIFKIRMYVGG



 



A159
519
VNYTIFKIRMYVGGV



 



A160
520
NYTIFKIRMYVGGVE



 



A161
521
YTIFKIRMYVGGVEH



 



A162
522
TIFKIRMYVGGVEHR



 



A163
523
IFKIRMYVGGVEHRL



 



A164
524
LPALSTGLIHLHQNI



 



A165
525
PALSTGIHLHQNIV



 



A166
526
ALSTGLIHLHQNIVD



 



A167
527
LSTGIHLHQNIVDV



 



A165
528
STGLIHLHQNIVDVQ



 



A169
529
TQLIHLHQNIVDVQY



 



A170
530
GLIHLHQNIVDVQYL



 



A171
531
LIHLHQNIVDVQYLY



 



A172
532
IHLHQNIVDVQYLYG



 



A173
533
LPALSTGLLHLHQNI



 



A174
534
PALSTGLLHLHQNIV



 



A175
535
ALSTGLLHLHQNIVD



 



A176
536
LSTGLLHLHQNIVDV



 



A177
537
STGLLHLHQNIVDVQ



 



A178
538
TGLLHLHQNIVDVQY



 



A179
539
GLLHLHQNIVDVQYM



 



A180
540
LLHLHQNIVDVQYMY



 



A181
541
LHLHQNIVDVQYMYG



 



A182
542
HLHAPTGSGKSTKVP



 



A183
543
LHAPTGSGKSTKVPA



 



A184
544
HAPTGSGKSTKVPAA



 



A185
545
APTGSGKSTKVPAAY



 



A186
546
PTGSGKSTKVPAAYA



 



A187
547
TGSGKSTKVPAAYAA



 



A188
548
GSGKSTKVPAAYAAQ



 



A189
549
SGKSTKVPAAYAAQG



 



A190
550
GKSTKVPAAYAAQGY



 



A191
551
KSTKVPAAYAAQGYK



 



A192
552
STKVPAAYAAQGYKV



 



A193
553
TKVPAAYAAQGYKVL



 



A194
554
KVPAAYAAQGYKVLV



 



A195
555
VPAAYAAQGYKVLVL



 



A196
556
PAAYAAQGYKVLVLN



 



A197
557
AAYAAQGYKVLVLNP



 



A198
558
AYAAQGYKVLVLNPS



 



A199
559
YAAQGYKVLVLNPSV



 



A200
560
AAQGYKVLVLNPSVA



 



A201
561
AQGYKVLVLNPSVAA



 



A202
562
QGYKVLVLNPSVAAT



 



A203
563
GYKVLVLNPSVAATL



 



A204
564
YKVLVLNPSVAATLG



 



A205
565
KVLVLNPSVAATLGF



 



A206
566
VLVLNPSVAATLGFG



 



A207
567
LVLNPSVAATLGFGA



 



A208
568
VLNPSVAATLGFGAY



 



A209
569
YLHAPTGSGKSTKVP



 



A210
570
LHAPTGSGKSTKVPV



 



A211
571
HAPTGSGKSTKVPVA



 



A212
572
APTGSGKSTKVPVAY



 



A213
573
PTGSGKSTKVPVAYA



 



A214
574
TGSGKSTKVPVAYAA



 



A215
575
GSGKSTKVPVAYAAQ



 



A216
576
SGKSTKVPVAYAAQG



 



A217
577
GKSTKVPVAYAAQGY



 



A218
578
KSTKVPVAYAAQGYK



 



A219
579
STKVPVAYAAQGTKV



 



A220
580
TKVPVAYAAQGYKVL



 



A221
581
KVPVAYAAQGYKVLV



 



A222
582
VPVAYAAQGYKVLVL



 



A223
583
PVAYAAQGYKVLVLN



 



A224
584
VAYAAQGYKVLVLNP



 



A225
585
ITSTYGKFLADGGC



 



A226
586
TYSTYGKFLADGGCS



 



A227
587
YSTYGKFLADGCCSG



 



A228
588
STYGKFLADGGCSGG



 



A229
589
TYGKFLADGGCSGGA



 



A230
590
YGKFLADGGCSGGAY



 



A231
591
GKFLADGGCSGGAYD



 



A232
592
KFLADGGCSGGAYDI



 



A233
593
FLADGGCSGGAYDII



 



A234
594
LADGGCSGGAYDIII



 



C75
595
FPDLGVRVCEKMALY



 



C76
596
PDLGVRVCEKMALYD



 



C77
597
DLGVRVCEKMALYDV



 



C78
598
KGGKKAARLIVYPDL



 



C79
599
GGKKAARLIVYPDLG



 



C80
600
GKKAARLIVYPDLGV



 



C81
601
KKAARLIVYPDLGVR



 



C82
602
KAARLIVYPDLGVRV



 



C83
603
AARLIVYPDLGVRVC



 



C84
604
ARLIVYPDLGVRVCE



 



C85
605
RLIVYPDLGVRVCEK



 



C86
606
LIVYPDLGVRVCEKM



 



C87
607
IVPDLGVRVCEKMA



 



C88
608
VYPDLGVRVCEKMAL



 



C89
609
YPDLGVRVCEKMALY



 



C90
610
AQPGYPWPLYGNEGL



 



C91
611
GQPGYPWPLYGNEGL



 



C92
612
AFCSAMYVGDLCGSV



 



C93
613
AFCSALYVGDLCGSV



 



C94
614
ETVQDCNCSIYPGHV



 



C95
615
EFVQDCNCSIYPGHV



 



C96
616
GVLAGLAYYSMVGNW



 



C97
617
GVLFGLAYFSMVGNW



 



C98
618
DQRPYCWHYAPRPCG



 



C99
619
DQRPYCWYPPRPCG



 



C100
620
TCPTDCFRKHPEATY



 



C101
621
YTKCGSGPWLTPRCL



 



C102
622
LNAACNWTRGERCDL



 



C103
623
LNAACNFTRGERCDL



 



C104
624
IAQAEAALENLVVLN



 



C105
625
IAQAEAALEKLVVLH



 



C106
626
TRVPYFVRAQGLIRA



 



C107
627
TRVPYFVRAHGLIRA



 



C108
628
HAGLRDLAVAVEPVV



 



C109
629
AAGLRDLAVAVEPIV



 



C110
630
ITWGADTAACGDIIL



 



C111
631
ITWGAETAACGDIIL



 



C112
632
GQGWRLLAPITAYSQ



 



C113
633
TAYSQQTRGLLGCII



 



C114
634
TAYSQQTRGLLGCIV



 



C115
635
GCIITSLTGRDKNQV



 



C116
636
GCIVVSMTGRDKTQV



 



C117
637
VNGVCWTVYHGAGSK



 



C118
638
KGPITQMYTNVDQDL



 



C119
639
KGPITQMYSSAEQDL



 



C120
640
GDSRGSLLSPRPVSY



 



C121
641
GDSRGALLSPRPVSY



 



C122
642
SYLKGSSGGPLLCPS



 



C123
643
SYLKGSSGGPVLCPS



 



C124
644
GHAVGIFRAAVCTRG



 



C125
645
GVDPNIRTGVRTITT



 



C126
646
VPHPNIEEVALSNTG



 



C127
647
TGEIPFYGKAIPIEV



 



C128
648
TGEIPFYGKAIPLEV



 



C129
649
PTSGDVVVVATDALM



 



C130
650
QTVDFSLDPTFTIET



 



C131
651
TLHGPTPLLYRLGAV



 



C132
652
VQNEVTLTHPITKYI



 



C133
653
LYREFDEMEECASHL



 



C134
654
TTLLFNILGGWVAAQ



 



C135
655
TTLLNILGGWLAAQ



 



C136
656
PSAASAFVGAGIAGA



 



C137
657
PSAATGFVVSGLAGA



 



C138
658
TPCSGSWLRDVWDWI



 



C139
659
VAAEEYVEVTRVGDF



 



C140
660
VAAEEYAEVTRHGDF



 



C141
661
FFTEVDGVRLHRYAP



 



C142
662
FFTELDGVRLHRYAP



 



C143
663
FFTWVDGVQIHRYAP



 



C144
664
FFTWLDGVQIHRYAP



 



C145
665
YLVGSQLPCEPEPDV



 



C146
666
YLVGSQLPCDPEPDV



 



C147
667
LPTWARPDYNPPLLE



 



C148
668
ASLRQKKVTFDRLQV



 



C149
669
ASLRAKKVTFDRLQV



 



C150
670
HIRSVWKDLLEDTET



 



C151
671
IDTTIMAKNEVFCVQ



 



C152
672
VMGSSYGFQYSPGQR



 



C153
673
DCTMLVCGDDLVVIC



 



A235
674
ADGGCSGGAYDIIIC



 



A236
675
DGGCSGGAYDIIICD



 



A237
676
GGCSGGAYDIIICDE



 



A238
677
GCSGGAYDIIICDEC



 



A239
678
CSGGAYDIIICDECH



 



A240
679
SGGAYDIIICDECHS



 



A241
680
TTILGIGTVLDQAET



 



A242
681
TILGIGTVLDQAETA



 



A243
682
ILGIGTVLDQAETAG



 



A244
683
LGIGTVLDQAETAGA



 



A245
684
GIGTVLDQAETAGAR



 



A246
685
IGTVLDQAETAGARL



 



A247
686
GTVLDQAETAGARLV



 



A248
687
TVLDQAETAGARLVV



 



A249
688
VLDQAETAGARLVVL



 



A250
689
LDQAETACARLVVLA



 



A251
690
DQAETAGARLVVLAT



 



A252
691
QAETAGARLVVLATA



 



A253
692
AETAGARLVVLATAT



 



A254
693
ETAGARLVVLATATP



 



A255
694
TAGARLVVLATATPP



 



A256
695
AGARLVVLATATPPG



 



A257
696
GARLVVLATATPPGS



 



A258
697
ARLVVLATATPPGSV



 



A259
698
RLVVLATATPPGSVT



 



A260
699
TSILGIGTVLDQAET



 



A261
700
SILGIGTVLDQAETA



 



A262
701
LGIGTVLDQAETAGV



 



A263
702
GIGTVLDQAETAGVR



 



A264
703
IGTVLDQAETAGVRL



 



A265
704
GTVLDQAETAGVRLT



 



A266
705
TVLDQAETAGVRLTV



 



A267
706
VLDQAETAGVRLTVL



 



A268
707
LDQAETAGVRLTVLA



 



A269
708
DQAETAGVRLTVLAT



 



A270
709
QAETAGVRLTVLATA



 



A271
710
AETAGVRLTVLATAT



 



A272
711
ETAGVRLTVLATATP



 



A273
712
TAGVRLTVLATATPP



 



A274
713
AGVRLTVLATATPPG



 



A275
714
GVRLTVLATATPPGS



 



A276
715
VRLTVLATATPPGSV



 



B5
716
SGMFDSSVLCECYDA



 



B6
717
GMFDSSVLCECYDAG



 



B7
718
MFDSSVLCECYDAGC



 



B8
719
FDSSVLCECDAGCA



 



B9
720
DSSVLCECYDAGCAW



 



B10
721
SSVLCECYDAGCAWY



 



B11
722
SVLCECYDAGCAWYE



 



B12
723
VLCECYDAGCAWYEL



 



B13
724
LCECYDAGCAWYELT



 



B14
725
CECYDAGCAWYELTP



 



B15
726
ECYDAGCAWYELTA



 



B16
727
CYDAGCAWYELTPAE



 



B17
728
YDAGCAWYELTPAET



 



B18
729
DAGCAWYELTPAETT



 



B19
730
AGCAWYELTPAETTV



 



B20
731
CCAWYELTPAETTVR



 



B21
732
CAWYELTPAETTVRL



 



B22
733
AWYELTPAETTVRLR



 



B23
734
WYELTPAETTVRLRA



 



B24
735
YELTPAETTVRLRAY



 



B25
736
DAGCAWYELTPAETS



 



B26
737
AGCAWYELTPAETSV



 



B27
738
GCAWYELTPAETSVR



 



B28
739
CAWYELTPAETSVRL



 



B29
740
AWYELTPAETSVRLR



 



B30
741
WYELTPAETSVRLRA



 



B31
742
GMFDSVVLCECYDAG



 



B32
743
SGMFDSVVLCECYDA



 



B33
744
GMFDSVVLCECYDAG



 



B34
745
MFDSVVLCECYDAGA



 



B35
746
FDSVVLCECYDAGAA



 



B36
747
DSVVLCECYDAGAAW



 



B37
748
SVVLCECYDAGAAWY



 



B38
749
VVLCECYDAGAAWYE



 



B39
750
VLCECYDAGAAWYEL



 



B40
751
LCECYDAGAAWYELT



 



C154
752
DCTMLVNGDDLWIC



 



C155
753
DPTMLVCGDDLVVIC



 



C156
754
DPTMLVNGDDLWIC



 



C157
755
LWARMILMTHFFSIL



 



C158
756
LWVRMVLMTHFFSIL



 



C159
757
DLPQIIERLHGLSAF



 



C160
758
DLPQIIQRLHGLSAF



 



C161
759
AVRTKLKLTPIPAAS



 



C162
760
AVRTKLKLTPLPAAS



 



C163
761
SGGDIYHSLSRARPR



 



C164
762
SGGDIYHSVSRARPR



 



C165
763
WGENETDVLLLNNTR



 



C166
764
GENETDVLLLNNTRP



 



C167
765
WFGCTWMNSTGFTKT



 



C168
766
FGCTWMNSTGFTKTC



 



C169
767
GCTWMNSTGFTKTCG



 



C170
768
GLPVSARRGREILLG



 



C171
769
LPVSARRGREILLGP



 



C172
770
PVSARRGREILLGPA



 



C173
771
VSARRGREILLGPAD



 



C174
772
GLPVSALRGREILLG



 



C175
773
LPVSALRGREILLGP



 



C176
774
PVSALRGREILLGPA



 



C177
775
VSALRGREILLGPAD



 



C178
776
PDREVLYREFDEMEE



 



C179
777
DREVLYREFDEMEEC



 



C180
778
REVLYREFDEMEECA



 



C181
779
EVLYREFDEMEECAS



 



C182
780
VLYREFDEMEECASH



 



C183
781
LYREFDEMEECASHL



 



C184
782
YYLTRDPTTPLARAA



 



C185
783
YLTRDPTTPLARAAW



 



C186
784
LTRDPTTPLARAAWE



 



C187
785
TRDPTTPLARAAWET



 



C188
786
RDPTTPLARAAWETA



 



C189
787
DPTTPLARAAWETAR



 



C190
788
PTTPLARAWETARH



 



C191
789
YYLTRDPTTPLARAA



 



C192
790
YLTRDPTTPLARAAW



 



C193
791
LTRDPTTPLARAAWE



 



C194
792
TRDPTTPLARAAWET



 



C195
793
RDPTTPLARAAWETV



 



C196
794
DPTTPLARAAWETVR



 



C197
795
PTTPLARAAWETVRH













Peptide ID
SEQ ID



(Ipep)
NO:





1506
796
MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVY




LLPRRGPRLGVRATRKTSERSQPRGRRQPIPK


 


1526
797
VNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQ




WMNRLIAFASRGNHVSPTHYV


 


1545
798
IKGGRHLTFCHSKKKCDELA


 


1546
799
TVPQDAVSRSQRRGRTGRGR


 


1547
800
YLVAYQATVCARAQAPPPSWD


 


1551
801
HLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATL




GFGAY


 


1552
802
YLHAPTGSGKSTKVPVAYAAQGYKVLVLNPSVAATL




GFGAY


 


1553
803
GAAVGSICLGKVLVDILAGYGAGVAGAL




VAFKIMSGE


 


1554
804
GAAVGSIGLGKVLVDILAGYGAGVAGAL




VAFKVMSGE


 


1555
805
GAAVGSIGLGKVLVDILAGYGAGISGAL




VAFKIMSGE


 


1556
806
FTEAMTRYSAPPGDPP


 


1557
807
SSMPPLEGEPGDPDL


 


1558
808
CGYRRCRASGVLTTS


 


1559
809
PVNSWLGNIIMYAPT


 


1560
810
PVNSWLGNIIQYAPT


 


1561
811
SGMFDSSVLCECYDAGCAWYELTPAETTVRLRAY


 


1562
812
SGMFDSSVLCECYDAGCAWYELTPAETSVRLRAY


 


1563
813
SGMFDSVVLCECYDAGAAWYELTPAETTVRLRAY


 


1564
814
SGMFDSVVLCECYDAGAAWYELTPAETSVRLRAY


 


1565
815
FWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAF


 


1577
816
GEVQVVSTATQSFLAT


 


1578
817
GEVQVLSTVTQSFLGT


 


1579
818
FTDNSSPPAVPQTFQV


 


1580
819
FSDNSTPPAVPQTYQV


 


1581
820
NAVAYYRGLDVSVIPT


 


1587
821
VNLLPGILSPGALVVGVICAAILRRHVGPGEGAVQ




WMNRLIAFASRGNHVAPTHYV


 


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
ARALAHGVRVLEDGVNYATGNLPGCSF




SIFLLALLSC


 


1648
854
DPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGG


 


1649
855
DPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLGG


 


1650
856
VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL


 


1651
857
VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL


 


1652
858
XGGRKPARLIVFPDLGVRVCEKMALYDV


 


1653
859
KGGKKAARLIVYPDLGVRVCEKMALYDV


 


1654
860
IQLINTNGSWHINRTALNCNDSL


 


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 DRB1*0701 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




 


A122
864
NGSWHINRTALNCND
*

*
*
new DRB1*0101,


 


A124
865
SWHINRTALNCNDSL
*












*0404, *0701: 45-









55%


 


B25
866
DAGCAWYELTPAETS
***
*
***


 


B26
867
AGCAWYELTPAETSV
***
***
***
nb
new DRB1*0101,


 


B28
868
CAWYELTPAETSVRL
***
**
***
*
*0404, *0701: 45-


 


B30
869
WYELTPAETSVRLRA
**
*
**
nb
55%


 


B46
870
AGAAWYELTPAETTV
***

***
nb
new DRB1*0101,


 


B48
871
AAWYELTPAETTVRL
***

***
**
*0404, *0701: 45-









55%


 


B84
872
GSIGLGKVLVDILAG
*
*
*


 


B86
873
IGLGKVLVDILAGYA
*
**
*
**
new DRB1*0101,


 


B88
874
LGKVLVDILAGYGAG
*
**
*

*0404, *0701: 45-


 


B92
875
LVDILAGYGAGVAGA
*
nb


55%


 


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


 


B95
887
ILAGYGAGVAGALVA
*
nb
nb

new DRB1*0101,


 


B96
888
LAGYCAGVAGALVAF
**
**
nb
*
*0404, *0701: 40-


 


B97
889
AGYGAGVAGALVAFK
*
nb
nb

50%


 


B98
890
GYGAGVACALVAKFI
*
nb
nb
nb


 


A272
891
ETAGVRLTVLATATT
*
*
nb


 


A274
892
AGVRLTVLATATPPG
*
nb

nb
new DRB1*0101,


 


A276
893
VRLTVLATATPPGSV

nb

*
*0404, *0701: 40-









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
AGAAWYELTPAETSV
***
***
***

new DRB1*0101,


 


B52
904
AAWYELTPAETSVRL
***
***
***
nb
*0401, *0404: ~40


 


1623
905
YLVAYQATVCARAKAPPPSWD
*

**

new DRB1*0101,









*0401, *0404: ~40


 


C130
906
QTVDFSLDPTFTIET
**
***
**
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
YYLTRDPTTPLARAA
nb
***
nb

new DRB1*0401,









*0701: ~40


 


A216
910
SGKSTKVPVAYAAQG
nb
*
nb
nb


 


A218
911
KSTKVPVAYAAQGYK
nb
*
nb

new DRB1*0101,


 


A220
912
TKVPVAYAAQGYKVL
*
**
nb

*0401 ~35


 


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
AFCSAMYVGDLCGSV
*
**
nb
nb
new DRB1*0101,


 


C93
918
AFCSALYVGDLCGSV
*
*
nb

*0401: ~35


 


A174
919
PALSTGLLHLHQNIV

nb
*
*
new DRB1*0404,









*0701: ~25-30


 


B32
920
SGMFDSVVLCECYDA
*
nb
***


 


B34
921
MFDSVVLCECYDAGA
*
nb
***
nb
new DRB1*0101,


 


B36
922
DSVVLCECYDAGAAW
*
nb
***

*0404: ~20-25


 


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
MWNFISGIQYLAGLS
*
nb
nb

new DRB1*0101: ~20


 


B112
935
VDILAGYGAGISGAL
*


 


B114
936
ILAGYGAGISGALVA
***



new DRB1*0101: ~20


 


B116
937
AGYGAGISGALVAFK
***

nb
nb


 


B118
938
YGAGISGALVAFKIM
***

nb


 


B18
939
DAGCAWYELTPAETT
***

nb
nb


 


B20
940
GCAWYELTPAETTVR
***
nb


new DRB1*0101: ~20


 


B22
941
AWYELTPAETTVRLR
**

nb
nb


 


C112
942
GQGWRLLAPITAYSQ
**
nb


new DRB1*0101: ~20


 


C116
943
GCIVVSMTGRDKTQV
*
nb
nb

new DRB1*0101: ~20


 


C122
944
SYLKGSSGGPLLCPS
*
nb
nb

new DRB1*0101: ~20


 


C127
945
TGEIPFYGKAIPIEV
*
nb


new DRB1*0101: ~20


 


C144
946
FFTWLDGVQIHRYAP
**
nb
nb

new DRB1*0101: ~20


 


C159
947
DLPQIIERLHGLSAF
*
nb
nb
nb
new DRB1*0101: ~20


 


C160
948
DLPQIIQRLHGLSAF
*
nb


 


C174
949
GLPVSALRGREILLG
*
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
LWARMILMTHFESIL
nb
nb
*
nb
new DRB1*0404: ~5


 


C158
955
LWVRMVLMTHFFSIL

nb
*


 


A254
956
ETAGARLVVLATATP
nb
nb
*


 


A256
957
AGARLVVLATATPPG
nb
nb
*

new DRB1*0404: ~5


 


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


 


A72
965
GFADLMGYIPLVGAP
***
*
**

known class II


 


A74
966
DLMGYIPVVGAPLGG
***

***
**
DR*0101, 0404,









0701


 


A88
967
CGFADLMGYIPVVGA
**

**
*


 


A90
968
FADLMGYIPVVGAPL
***

***

known class II


 


A92
969
DLMGYIPVVGAPLGG
***

***
***
DR*0101, 0404,









0701


 


A96
970
LAHGVRVLEDGVNYA
nb
***
nb


 


A98
971
HGVRVLEDGVNYATG
nb
***
***
**
known DR11


 


A100
972
VRVLEDGVNYATGNL
nb
***
***
*
new DR*0401,


 


A102
973
VLEDGVNYATGNLPG
nb

*

0404, 0701


 


A104
974
EDGVNYATGNLPGCS
nb
*
nb
nb


 


A200
975
AAQGYKVLVLNPSVA

nb
***
**


 


A202
976
QGYKVLVLNPSVAAT
***
***
***
***
known DRB1*0401,


 


A204
977
YKVLVLNPSVAATLG
***
***
***
***
0701, DR11, DR15


 


A206
978
VLVLNPSVAATLGFG
***
***
***
***
new DR*0101


 


C30
979
AVQWMNRLIAFASRG
*

nb

known DR11, DQ5,









also DR*0101


 


B60
980
NFISGIQYLAGLSTL
**

nb
8


 


B62
981
ISGIQYLAGLSTLPG
***

***
**


 


B64
982
GIQYLAGLSTLPGNP
***

***
**
know DR#0401,


 


B66
983
QYLAGLSTLPGNPAI
*

***
**
1101


 


B68
984
LAGLSTLPGNPAIAS
*

***
**
new 0101, 0404,









0701


 


C124
985
GHAVGIFRAAVCTRG
**
*
nb
**
known DR*0101,









0401, 0701


 


1620
986
TGDFDSVIDCNTCVTQ
nb
nb
*

new DR*0404


 


1621
987
TGDFDSVIDCNVAVTQ
*

nb
nb
known DR13, llso









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 HLA-ligands with confirmed immunogenicity in HLA-transgenic mice


Boldface peptide sequences indicate putative core binding regions based on prediction algorithms as described 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 *07.01, 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









SEQ ID




Ipep
DRB1*0401
A*0201
B*0702
NO
Sequence





















1506


+
990
MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQ








PRGRRQPIPK


 


1526
+
+
+
991
VNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYV


 


1545

+

992
IKGGRHLIFCHSKKKCDELA


 


1547
++

+++
993
YLVAYQATVCARAQAPPPSWD


 


1552

+
+
994
YLHAPTGSGKSTKVPVAYAAQGYKVLVLNPSVAATLGFGAY


 


1553

+
+
995
GAAVGSIGLGKVLVDILAGYGAGVAGALVAFKIMSGE


 


1555
+
+
+
996
GAAVGSIGLGKVLVDILAGYGAGISGALVAFKIMSGE


 


1558
++
+
++
997
CGYRRCRASGVLTTS


 


1559
+
++

998
PVNSWLGNIIMYAPT


 


1560
+
++

999
PVNSWLGNIIQYAPT


 


1562


+
1000
SGMFDSSVLCECYDAGCAWYELTPAETSVRLRAY


 


1563
+

+
1001
SGMFDSVVLCECYDAGAAWYELTPAETTVRLRAY


 


1565
++
++
+
1002
FWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAF


 


1577

+
+
1003
GEVQVVSTATQSFLAT


 


1578


+
1004
GEVQVLSTVTQSFLGT


 


1580


+
1005
FSDNSTPPAVPQTYQV


 


1587

+
+
1006
VNLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIAFASRGNHVAPTHYV


 


1592
++
++
+
1007
FWAKHMWNFISGIQYLAGLSTLPGNPAVASMMAF


 


1604
++
++
++
1008
VVCCSMSYTWTGALITPC


 


1605
+
+
+
1009
VVCCSMSYSWTGALITPC


 


1615

+

1010
LTPPHSAKSKFGYGAKDVR


 


1616
+


1011
LTPPHSAKSKYGYGAKEVR


 


1617

+

1012
LTPPHSARSKYGYGAKEVR


 


1621
+
+
+
1013
TGDFDSVIDCNVAVTQ


 


1623
+
+
+
1014
YLVAYQATVCARAKAPPPSWD


 


1624


+
1015
LEDRDRSELSPLLLSTTEW


 


1625
+


1016
LEDRDRSQLSPLLHSTTEW


 


1627
+
+
++
1017
PEYDLELITSCSSNVSVA


 


1628


++
1018
VCGPVYCFTPSPVVVGTTDR


 


1630
++
++

1019
LLFLLLADARVCACLWM


 


1631
+
+

1020
SGHRMAWDMMMNWSPT


 


1632

+

1021
TGHRMAWDMMMNWSPT


 


1641

+

1022
ITYSTYGKFLADGGCSGGAYDIIICDECHS


 


1647

+
+
1023
ARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSC


 


1649
+


1024
DPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLGG


 


1650
++
++
+
1025
VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL


 


1651
++
++
+
1026
VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL


 


1652
++
+

1027
KGGRKPARLIVFPDLGVRVCEKMALYDV


 


1653

+

1028
KGGKKAARLIVYPDLGVRVCEKMALYDV


 


1654
++
+

1029
IQLINTNGSWHINRTALNCNDSL


 


1655
++
+

1030
IQLVNTNGSWHINRTALNCNDSL


 


1656
+


1031
LPALSTGLIHLHQNIVDYQYLYG









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 A2epitopes.
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 tg mice (CD8+ fraction for B7 mice containing 97% of CD8 and 1.5% of CD4 cells and for A2 tg 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.
FIG. 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 Ipep14835+IC31, significant interferon-gamma induction against Ipep14843 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 Ipep14587 a different approach was taken: the sequence of Ipep14587 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 Ipep14838 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
Peptide sequence
SEQ ID



ID
(1 amino acid code)
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 ore 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.


Numbers represent peptide-specific IFN-gamma secreting T-


cells/106 PBMC calculated from ELIspot results (duplicate


determinations); values >8 (>3x over background) were regarded


statistically significant. Donors H32 and H33 are spontaneously


recovered patients.


















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














. . . reactive after 2



Donors reactive directly ex vivo in IFN-gamma ELIspot
rounds of in vitro



with HCV-derived peptides
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 









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 (“+++” strong


affinity, “++” intermediate affinity, “+” weak affinity, “−” no


affinity, “nd” not done; core binding motifs are underlined)










Peptide ID





HLA-DRB1*


SEQ ID

Binding to soluble













NO:
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
AGAAWYELTPAETTV
+++
+++
+++
−
+/−


 


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









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.



Hotspots (incl. some variations) are shown in bold, epitopes


contained within the hotspots in normal font. Peptide number and


sequence, as well as HLA-class I and class II coverage are


given. Source data refers to Examples and Tables within this


specification, or literature references.












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
GEVQVVSTATQSFLAT
(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





B8,27,4402,60


 



     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

Wong 2001


 


1547
YLVAYQATVCARAQAPPPSWD
(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)
B08,527

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: 11670)
A3

Wong 1998


 


C92
AFCSAMYVGDLCGSV
(SEQ ID NO: 1168)
A2,B51
DR1,4
Tab.2,5


 


C97
GVLFGLAYFSMVGNW
(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









Example VII
HCV Epitope Hotspot Ipep 1426 Contains at Least HLA-A*201 and Several Promiscuous Class II T-Cell Epitopes
The major objective of this experiment was to compare the immunogenicity of the “hotspot” 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 426, 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. 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).









Donor HLA











A*0206, A*01,



A*0201, A*03, B7, B60;
B27, B50;



DRB1*1501, −B1*1302
DRB1*0401, −B1*1402












line


line raised



raised
line raised
line raised
against



against
against 1006 +
against
1006 +


Peptide ID
1426
1425 + 1334
1426
1425 + 1334





1006
++
++
++
++


1425
+++
+++
+++
++


1334
+
+
−
−


1006 + 1425 + 1334
++
++
++
++


1426
+++
+++
+++
++


84 (HCV
−
−
−
−


derived


negative


control)









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## Claims

1. A method for isolating Hepatitis C Virus peptides (HPs) or isolating HCV T cell epitopes which have a binding capacity to a purified, recombinant MHC/HLA molecule or a complex comprising the HCV-peptide or HCV T cell epitope and the purified, recombinant MHC/HLA molecule, the method comprising:
providing a pool comprising at least 10 HCV peptides, the pool containing HCV-peptides which bind to the purified, recombinant MHC/HLA molecule (binding HCV peptides) and HCV-peptides which do not bind to the purified, recombinant MHC/HLA molecule (non-binding HCV peptides);
contacting the purified, recombinant MHC/HLA molecule with the pool of HCV-peptides whereby a binding HCV-peptide binds to the purified, recombinant MHC/HLA molecule and a complex comprising the HCV-peptide and the purified, recombinant MHC/HLA molecule is formed;
detecting the complex and, optionally, separating the complex from non-binding HCV peptides;
optionally isolating and characterizing the binding HCV peptides from the complex;
assaying the complex or the optionally isolated binding HCV peptides in a T cell assay for T cell activation capacity; and
identifying optionally isolated binding HCV peptides with T cell activation capacity as a T cell epitope or the complex.

2. The method of claim 1, further comprising separating the complex from the HCV-peptides which do not bind to the purified, recombinant MHC/HLA molecule.

3. The method of claim 1, further comprising isolating and characterizing the HCV-peptide from the complex.

4. The method of claim 1, further defined as a method for isolating HPs.

5. The method of claim 1, further comprising isolating and characterizing the HCV-peptide from the complex before assaying the HCV-peptide.

6. The method of claim 1, wherein the pool of peptides is further defined as a pool of overlapping peptides, a pool of protein fragments, a pool of modified peptides, a pool obtained from antigen-presenting cells, a pool comprised of fragments of cells, a pool comprised of peptide libraries, a pool of (poly)-peptides generated from a recombinant DNA library, a pool of proteins and/or protein fragments from HCV, or a combination of any of these.

7. The method of claim 6, wherein the pool is obtained from a total lysate or fraction of antigen-presenting cells.

8. The method of claim 7, wherein the pool is obtained from a fraction eluted from surface or MHC/HLA molecules of the antigen-presenting cells.

9. The method of claim 6, wherein the pool is comprised of fragments of HCV-containing cells, tumor cells, or tissue cells.

10. The method of claim 9, wherein the pool is comprised of fragments from liver cells.

11. The method of claim 6, wherein the pool is generated from a recombinant DNA library derived from pathogens or tumor cells.

12. The method of claim 1, wherein the purified, recombinant MHC/HLA molecules are HLA class I molecules, HLA class II molecules, non classical MIHC/HLA molecules, MHC/HLA-like molecules or a mixture thereof.

13. The method of claim 1, wherein the characterizing of the HCV-peptides of the complex comprises at least one of mass spectroscopy, polypeptide sequencing, a binding assay, identification of HCV-peptides by determination of their retention factors by chromatography, or a spectroscopic technique.

14. The method of claim 13, wherein the characterizing of the HCV-peptides of the complex comprises a binding assay, further defined as an SDS-stability assay.

15. The method of claim 13, wherein the characterizing of the HCV-peptides of the complex comprises identification of HCV-peptides by determination of their retention factors by HPLC.

16. The method of claim 13, wherein the characterizing of the HCV-peptides of the complex comprises violet (UV) spectroscopy, infra-red (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, or electron spin resonance (ESR) spectroscopy.

17. The method of claim 1, further comprising performing a cytokine secretion assay.

18. The method of claim 17, wherein the cytokine secretion assay is an Elispot assay, intracellular cytokine staining, FACS, or an ELISA.

19. The method of claim 1, wherein the T cell assay comprises the mixing and incubation of the complex with isolated T cells and subsequent measuring cytokine secretion or proliferation of the isolated T cells.

20. The method of claim 1, wherein the T cell assay comprises measuring up-regulation of an activation marker.

21. The method of claim 20, wherein the activation marker is CD69 or CD38.

22. The method of claim 20, wherein the T cell assay comprises measuring down-regulation of a surface marker.

23. The method of claim 22, wherein the surface marker is CD3, CD8 or TCR.

24. The method of claim 1, wherein the T cell assay comprises measuring up-/down-regulation of mRNAs involved in T cell activation.

25. The method of claim 24, further defined as comprising real-time RT-PCR.

26. The method of claim 1, wherein the T cell assay is a T cell assay measuring phosphorylation/de-phosphorylation of components downstream of the T cell receptor, T cell assay measuring intracellular Ca++ concentration or activation of Ca++-dependent proteins, T cell assay measuring formation of immunological synapses, or T cell assay measuring release of effector molecules.

27. The method of claim 26, wherein the T cell assay measures phosphorylation/de-phosphorylation of p56, lck, ITAMS of the TCR and the zeta chain, ZAP70, LAT, SLP-76, fyn, or lyn.

28. The method of claim 26, wherein the T cell assay measures release of perforin, a granzyme, or granulolysin.

29. A method for preparing an immunogenic composition comprising Hepatitis C Virus peptides (HPs) or isolated HCV T cell epitopes which have a binding capacity to a MHC/HLA molecule or a complex comprising the HCV-peptide or HCV T cell epitope and the MHC/HLA molecule, comprising:
providing a pool comprising at least 10 HCV peptides, the pool containing HCV-peptides which bind to the MHC/HLA molecule (binding HCV peptides) and HCV- peptides which do not bind to the MHC/HLA molecule (non-binding HCV peptides);
contacting the MHC/HLA molecule with the pool of HCV-peptides whereby a binding HCV-peptide binds to the MHC/HLA molecule and a complex comprising the HCV-peptide and the MHC/HLA molecule is formed;
detecting the complex and, optionally, separating the complex from non-binding HCV peptides;
optionally isolating and characterizing the binding HCV peptides from the complex; assaying the complex or the optionally isolated binding HCV peptides in a T cell assay for T cell activation capacity;
identifying optionally isolated binding HCV peptides with T cell activation capacity as a T cell epitope or the complex; and
formulating the peptide or epitope in an immunogenic composition.

30. A method for inducing an immune response directed against Hepatitis C Virus with an immunogenic composition comprising Hepatitis C Virus peptides (HPs) or isolated HCV T cell epitopes which have a binding capacity to a MHC/HLA molecule or a complex comprising the HCV-peptide or HCV T cell epitope and the MHC/HLA molecule, comprising:
providing a pool comprising at least 10 HCV peptides, the pool containing HCV-peptides which bind to the MHC/HLA molecule (binding HCV peptides) and HCV- peptides which do not bind to the MHC/HLA molecule (non-binding HCV peptides);
contacting the MHC/HLA molecule with the pool of HCV-peptides whereby a binding HCV-peptide binds to the MHC/HLA molecule and a complex comprising the HCV-peptide and the MHC/HLA molecule is formed;
detecting the complex and, optionally, separating the complex from non-binding HCV peptides;
optionally isolating and characterizing the binding HCV peptides from the complex; assaying the complex or the optionally isolated binding HCV peptides in a T cell assay for T cell activation capacity;
identifying optionally isolated binding HCV peptides with T cell activation capacity as a T cell epitope or the complex;
formulating the peptide or epitope in an immunogenic composition; and
administering the immunogenic composition to a subject, wherein an immune response specific for Hepatitis C Virus is induced.

31. The method of claim 30, wherein the subject is a human.