Multi-mer peptides derived from hepatitis C virus envelope proteins for diagnostic use and vaccination purposes

Abstract

Multimer peptides (e.g. 30- to 45-mer peptides) derived from hepatitis C virus envelope proteins reacting with the majority of anti-HCV antibodies present in patient sera are described. The usage of the latter peptides to diagnose, and to vaccinate against, an infection with hepatitis C virus is also disclosed.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to multi-mer peptides derived from hepatitis C virus envelope proteins which react with the majority of anti-HCV antibodies present in patient sera. Consequently, the present invention relates to the usage of the latter peptides to diagnose, and to vaccinate against, an infection with hepatitis C virus.

BACKGROUND OF THE INVENTION

[0002] Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus, amounting up to 175 million chronic infections worldwide. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, there is evidence implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic agents is high. There is also an urgent need to characterize new epitopes which can be used in the design of effective vaccines against hepatitis C.

[0003] HCV is a positive stranded RNA virus of about 9,8 kilobases which code for at least three structural and at least six non-structural proteins. The structural proteins have not yet been functionally assigned, but are thought to consist of a single core protein and two envelope proteins E1 and E2. The E1 protein consists of 192 amino acids and contains 5 to 6 N-glycosylation sites, depending on the HCV genotype, whereas the E2 protein consists of 363 to 370 amino acids and contains up to 11 N-glycosylation sites, depending on the HCV genotype (for review see Maertens and Stuyver, 1997).

[0004] The E1 and E2 proteins are currently not included in HCV antibody (Ab) assays, primarily because of their complex conformational structures which require expression in mammalian cells as well as non-denaturing purification techniques. Indeed, after expression of E2 in Escherichia coli, the reactivity of HCV sera with the recombinant protein ranged from 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992), whereas expression in eukaryotic systems yields reactivities of 13 to 97% (Inoue, 1992; Chien, 1993). Others demonstrated that the E1 protein expressed as a single protein from eukaryotic cells did not shown high reactivity with patient sera (from 6 to 60%; Kohara et al. (1992), Hsu et al. (1992), Chien et al. (1993)). We previously reported that high prevalences of Ab's to both of the purified recombinant E1 and E2 proteins, which were expressed in mammalian cells, could be found in sera from chronic hepatitis C patients (WO 96/04385 to Maertens et al.). In this regard, we also demonstrated that the majority of anti-E1 and anti-E2 antibodies in sera from HCV patients could not be mapped using 20-mer peptides (WO 96/04385 to Maertens et al.). Indeed, although all of the murine monoclonal Ab's against E1 could be mapped to reactivity with two 20-mer peptides, denoted as epitope A (amino acids (aa) 313-326) and epitope B (aa 208-224), at most 50% of patient sera reactive with recombinant proteins recognized epitope A and B. With regard to the E2 protein, only three out of twenty four murine monoclonal Ab's could be mapped using 20-mer peptides. These three Ab's were mapped to the hypervariable region I (HVR I) covered by peptide E2-67 (aa 394-413) and to a region covered by a peptide denoted E2-13B (aa 523-542). The remaining twenty-one Ab's could not be mapped using 20-mer peptides. The relative map positions of seven of these Ab's could be deduced from competition studies using recombinant E2 protein.

[0005] Taken together, it appears that anti-E1 and anti-E2 Ab's might be highly prevalent in sera of HCV patients. However, determining the presence of these Ab's is problematic due to the need to use eukaryotically expressed E1 and E2, which have to be purified using cumbersome non-denaturing techniques. As an alternative, chemically synthesized 20-mer peptides derived from the E1 and/or E2 proteins were produced. However, these synthesized 20-mer peptides were not able to recognize the anti-E1 and anti-E2 Ab's in sera from HCV patients.

[0006] There is thus a need to design alternative methods to screen for HCV envelope Ab's.

AIMS OF THE INVENTION

[0007] It is clear from the literature cited above that the E1 and E2 proteins probably have complex conformational structures which are essential for recognizing (and binding to) the anti-E1 and anti-E2 Ab's in sera from HCV patients. This could explain why prokaryotically expressed complete or near-complete E1 and E2 proteins, which might be malfolded due to the lack of glycosylations, relevant chaperones or correct cysteine bridles, and 20-mer peptides, which might be unable to mimic a complex conformational structure, are not able to recognize these Ab's.

[0008] The present invention relates to the surprising finding that multi-mer peptides (eg 30- to 45-mer peptides) are able to recognize the majority of anti-E1 and anti-E2 Ab's in sera from HCV patients. It should be clear that this is a surprising finding because there is no guidance which would suggest that 30- to 45-mer peptides derived from E1 and E2 would acquire proper folding and would efficiently recognize the majority of HCV envelope Ab's. In contrast, one would assume that the chance that multi-mer peptides malfold would be as great, or even greater, than the chance that prokaryotically expressed complete proteins malfold as is suggested above. In the case of the HCV NS3 protein for example, which reacts with more than 90% of patient samples as expressed from E. coli, 20-50 mer peptides only react very weakly.

[0009] Therefore, the present invention aims at providing a peptide of more than 20 contiguous amino acids derived from the envelope region of HCV-related viruses which binds and recognizes anti-HCV-related virus antibodies. HCV-related viruses, including HCV, GBV-B virus, GBV-A virus and GBV-C (HGV or hepatitis G virus), are a division of the Flaviviruses, which further comprise Dengue virus, Yellow fever virus, Pestiviruses such as Classical Swine Fever Virus and Bovine Viral Diarrhea Virus (Wengler, 1991).

[0010] More specifically, the present invention aims at providing a peptide which binds and recognizes an anti-HCV antibody or an anti-HGV antibody present in a sample of body fluid and which is chosen from the group consisting of the sequences as represented in SEQ ID NOs 1 to 38 (see Table 1) or a functionally equivalent variant or fragment thereof.

[0011] In this respect, the present invention aims specifically at providing a peptide as described above, wherein said anti-HCV antibody present in a sample of body fluid is an anti-HCV-E1 antibody or an anti-HCV-E2 antibody.

[0012] The present invention thus aims also at providing a peptide as described above, wherein said anti-HGV antibody present in a sample of body fluid is an anti-HGV-E1 antibody or an anti-HGV-E2 antibody.

[0013] Moreover, the present invention aims at providing a peptide as described above, wherein said peptide is synthesized chemically or is synthesized using recombinant DNA techniques.

[0014] The present invention also aims at providing a peptide as described above, wherein said peptide is biotinylated or contains cysteine bridges.

[0015] Furthermore, the present invention aims at providing any combination of peptides as described above, as well as compositions containing said combination of peptides or peptides as described above .

[0016] In addition, the present invention aims at providing a method for diagnosing exposure to or infection by HCV-related viruses comprising contacting anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above or with a combination of peptides as described above, determining the binding of anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above or with a combination of peptides as described above.

[0017] In addition, the present invention aims at providing an assay kit for detecting the presence of anti-HCV-related virus antibodies within a sample of body fluid comprising a solid support, a peptide as described above or a combination of peptides as described above, appropriate markers which allow to determine the complexes formed between anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above or a combination of peptides as described above.

[0018] In addition, the present invention aims at providing a bioassay for identifying compounds which modulate the interaction between a peptide and an anti-HCV-related virus antibody, said bioassay comprising contacting anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above, determining the binding of anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above, adding a modulator (ie a compound which is able to modulate the interaction between an envelope protein and an anti-HCV-related virus antibody) or a combination of modulators to the contacted anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above, determining the modulation of binding of anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above

[0019] In addition, the present invention aims at providing a bioassay for identifying compounds which modulate the interaction between a peptide and an anti-HCV-related virus antibody, said bioassay comprising determining the binding of anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above, contacting a modulator with a peptide as described above or a combination of peptides as described above, adding anti-HCV-related virus antibodies to the contacted modulator with the peptide as described above or a combination of peptides as described above, determining the modulation of binding between anti-HCV-related virus antibodies with a peptide as described above or a combination of peptides as described above.

[0020] Moreover, the present invention aims at providing a modulator, a composition containing a modulator, or a combination of modulators when produced by the bioassay as described above or when identified by the above-described bioassays.

[0021] Moreover, the present invention aims at providing a composition comprising a plasmid vector comprising a nucleotide sequence encoding a peptide as described above, or a modulator as described above, operably linked to transcription regulatory elements.

[0022] Moreover, the present invention aims at providing a composition as described above for use to vaccinate or therapeutically treat humans against infection with HCV-related virus or any mutated strain thereof

[0023] Moreover, it is an aim of the present invention to provide an antibody, more particularly a monoclonal antibody, characterized in that it specifically recognizes an HCV-related virus polypeptide as described above.

[0024] Finally, it is an aim of the present invention to provide a method to immunize humans against infection with HCV-related virus or any mutated strain thereof, comprising the use of a peptide as described above or a combination of peptides as described above.

[0025] All the aims of the present invention are considered to have been met by the embodiments as set out below. Other advantages and features of the instant invention will be evident from the following claims and detailed description.

BRIEF DESCRIPTION OF TABLES AND DRAWINGS

[0026] Table 1 provides information on the envelope protein and the HCV genotype from which the peptides of the present invention are derived. This table also provides the name, the amino acid sequence, the position within the envelope proteins and the sequence identity (SEQ ID) of the peptides of the present invention.

[0027] Table 2 shows ELISA results (in mOD) of reactivities of multimer peptides and recombinant E2 with 60 HCV positive samples and 4 control samples.

[0028] Table 3 shows the analysis for E1 antibodies of 23 sera from responders to interferon treatment.

[0029] Table 4 shows the analysis of E2 antibodies of 23 sera from responders to interferon treatment.

[0030] Table 5 shows the monitoring of disease over time by measuring antibodies to the HCV E1 and E2 regions in 18 patients.

[0031] Table 6 indicates the reactivity of HGV (Hepatitis G virus) RNA positive sera with the HGV E1 peptide V1V2.

[0032] FIG. 1 demonstrates the positions of the multi-mer peptides of the present invention relative to the conserved and variable regions of the E1 envelope protein of HCV (HVR=hypervariable regions; V=variable regions; C=conserved regions; HR=hydrophobic region; SA=signal anchor domain; Y=glycosylation; I=cysteine).

[0033] FIG. 2 demonstrates the positions of the multi-mer peptides of the present invention relative to the conserved and variable regions of the E2 envelope protein of HCV (HVR=hypervariable regions; V=variable regions; C=conserved regions; SA=signal anchor domain; Y=glycosylation; I=cysteine).

[0034] FIG. 3 shows the reactivity of 20-mer E2 peptides. The OD values of serum samples from patients with chronic active hepatitis C were added and plotted against the different peptides.

[0035] FIG. 4 shows the reactivity of mulit-mer E2 peptides. The OD values of the samples were added and plotted against the different peptides. The samples were identical as used for FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All these publications and applications, cited previously or below are hereby incorporated by reference.

[0037] The present invention is based on the finding that multimer peptides, as of a certain length, derived from the envelope proteins of HCV-related viruses, eg HCV and HGV, recognize and bind anti-HCV-related virus antibodies, eg anti-HCV antibodies and anti-HGV antibodies, respectively. Therefore, the present invention provides a peptide of more than 20 contiguous amino acids derived from the envelope region of HCV-related viruses which binds and recognizes anti-HCV-related virus antibodies.

[0038] HCV-related viruses include, but are not limited to HCV, GBV-B virus, GBV-A virus and GBV-C virus (HGV or hepatitis G virus)(Linnen et al., 1996). HCV constitutes a genus within the Flaviviridae, and is closely related to hepatitis G virus (26.8% at the amino acid level). The term "envelope region" of HCV-related viruses is a well-known region by a person skilled in the art (Wengler, 1991), and comprises the E1 protein as well as the E2 protein, which was previously called non-structural protein 1 (NS 1) or E2/NS1.

[0039] Furthermore, the present invention relates to a peptide, which binds and recognizes an anti-HCV antibody or an anti-HGV antibody present in a sample of body fluid, and which is chosen from the group consisting of the sequences as represented in SEQ ID 1 to 38 (see Table 1) or a functionally equivalent variant or fragment thereof.

[0040] The present invention relates also to a peptide as described above, wherein said anti-HCV antibody or said anti-HGV antibody present in a sample of body fluid is an anti-HCV-E1 or anti-HCV-E2 antibody, or an anti-HGV-E1 or anti-HGV-E2 antibody, respectively.

[0041] The term "a peptide" refers to a polymer of amino acids (aa's) derived (i.e. containing less aa's) from the well-known HCV-related virus envelope proteins E1 and E2 (Linnen et al., 1996, Maertens and Stuyver, 1997), which binds anti-HCV-related virus antibodies. The term "a peptide" refers in particular to a polymer of aa's derived from HCV envelope proteins E1 and E2, which binds anti-HCV antibodies, or from HGV envelope proteins E1 and E2, which binds anti-HGV antibodies.

[0042] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein.

[0043] The term "an anti-HCV-related virus antibody" refers to any polyclonal or monoclonal antibody binding to a HCV-related virus particle or any molecule derived from said viral particle. More particularly, the term "an anti-HCV-related virus antibody" refers to antibodies binding to the natural, recombinant or synthetic E1 and/or E2 proteins derived from HCV or HGV proteins (anti-HCV-E1 or anti-HCV-E2 antibody, or anti-HGV-E1 or anti-HGV-E2 antibody, respectively).

[0044] The term "monoclonal antibody" used herein refers to an antibody composition having a homogeneous antibody population. The term is not limiting regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.

[0045] In addition, the term "antibody" also refers to humanized antibodies in which at least a portion of the framework regions of an immunoglobulin are derived from human immunoglobulin sequences and single chain antibodies as described in U.S. Pat. No. 4,946,778 and to fragments of antibodies such as F.sub.ab, F.sub.'(ab))2, F.sub.v, and other fragments which retain the antigen binding function and specificity of the parent antibody.

[0046] The term "a sample of body fluid" as used herein refers to a fluid obtained from an organism, such as serum, plasma, saliva, gastric secretions, mucus, spinal cord fluid, and the like. The term "the group consisting of the sequences as represented in SEQ ID NOs 1 to 33" as used herein refers to the thirty-eight peptides shown in Table 1 of the present application. In this table, it is indicated:

[0047] in the column named "protein" from which HCV envelope protein the peptide is derived, but for the envelope protein of HGV, which is denoted E1(HGV),

[0048] in the column named "genotype" the HCV genotype from which the envelope protein is derived, and thus the peptide is derived, except for HGV which was not determined (ND),

[0049] in the column named "peptide" the assignment of the peptide region.

[0050] the aa sequence of the peptide and,

[0051] in the column named "position", the well-known (Maertens and Stuyver, 1997) aa position of the peptides within the HCV envelope proteins. Note that the position for the E1 envelope protein is not determined, which is denoted as "ND".

[0052] The term "functionally equivalent" as used in "functionally equivalent variant or fragment thereof" refers to variants and fragments of the peptides represented by SEQ ID 1 to 38, which bind anti-HCV-related virus antibodies. The term "variant or fragment" as used in "functionally equivalent variant or fragment thereof" refers to any variant or any fragment of the peptides represented by SEQ ID 1 to 38. Furthermore, the latter terms do not refer to, nor do they exclude, post-translational modifications of the peptides represented by SEQ ID 1 to 38 such as glycosylation, acetylation, phosphorylation, modifications with fatty acids and the like. Included within the definition are, for example, peptides containing one or more analogues of an aa (including unnatural aa's), peptides with substituted linkages, mutated versions or natural sequence variations of the peptides (for examples corresponding to the genotypes HCV, as described in WO 94/12670 to Maertens et al.), peptides containing disulfide bounds between cysteine residues, or other cysteine modifications, biotinylated peptides, as well as other modifications known in the art. Modification of the structure of the polypeptides can be for such objectives as increasing therapeutic or prophylactic efficacy, stability (e.g. ex vivo shelf life and in vivo resistance to proteolytic degradation), or post-translational modifications (e.g. to alter the phosphorylation pattern of protein). Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein are considered functional equivalents of the polypeptides described in more detail herein. Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic: aspartate, glutamate; (2) basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similar fashion, the amino acid repertoire can be grouped as (1) acidic: aspartate, glutamate; (2) basic: lysin, arginine histidine, (3) aliphatic: glycine, alanine, valine, leucine, isoleucine, serine, threoaine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic: phenylalanine, tyrosine, tryptophan, (5) amide: asparagine, glutamine; and (6) sulfur-containing: cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acid sequence of a peptide results in a functional homologue (e.g. functional in the sense that the resulting polypeptide mimics the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in e.g. ELISAs in a fashion similar to the wild-type protein, or to competitively inhibit such a response. Polypeptides in which more than one replacement has been introduced can be readily tested in the same manner. It should also be clear that the region of a peptide represented by SEQ ID 1 to 38 which bind to an antibody (the so-called epitope) need not to be composed of a contiguous aa sequence. In this regard, the term "fragment" includes any fragment which comprises these non-contiguous binding regions or parts thereof. In other words, fragments which include these binding regions may be separated by a linker, which is not a functional part of the epitope. This linker does not need to be an amino acid sequence, but can be any molecule, eg organic or inorganic, that allows the formation of the desired epitope by two or more fragments.

[0053] Moreover, it should be clear that the variants and fragments of SEQ ID NOs 1 to 5, 7 to 9, and 18 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's or 33 aa's, or 34 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 6 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 10 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NOs 11, 15, 21, 34 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NOs 12, 24 or 32 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's. or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, or 35 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NOs 13, 22, or 34 used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's. Moreover, it should be that clear the variants and fragments of SEQ ID NO 16 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39 aa's, or 40 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 17 as used herein refers to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 19 as used herein refers to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NOs 20 and 30 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 23 as used herein include to peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39 aa's, or 40 aa's, or 41 aa's, or 42 aa's, or 43 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NOs 25 or 29 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 26 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's or 29 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 27 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39 aa's, or 40 aa's, or 41 aa's, or42 aa's, or 43 aa's, or 44 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 28 or 31 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's. Moreover, it should be clear that the variants and fragments of SEQ ID NO 33 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39 aa's, or 40 aa's, or 41 aa's. Moreover it should be clear that the variants and fragments of SEQ ID NOs 14 or 37 as used herein include peptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39 aa's.

[0054] In addition, it shall be appreciated by the person skilled in the art that the amino acid regions of the peptides, which are disclosed in the present invention and that bind anti-HCV antibodies, can be delineated in more detail by experimentation.

[0055] In addition, it should be clear that the variants and fragments of the peptides represented by SEQ ID 1 to 38, as herein described, can be prepared by any method known in the art such as classical chemical synthesis, as described by Houbenweyl (1974) and Atherton & Shepard (1989), or by means of recombinant DNA techniques as described by eg Maniatis et al. (1982), or Sambrook et al. (1989).

[0056] Similarly, it should be clear that also the peptides represented by SEQ ID 1 to 38 of the present invention can be prepared by any method known in the art and more particularly by means of classical chemical synthesis, as described by Houbenweyl (1974) and Atherton & Shepard (1989), or by means of recombinant DNA techniques such as described by eg Maniatis et al. (1982), or Sambrook et al. (1989).

[0057] The present invention further relates to the peptides represented by SEQ ID 1 to 38 and functionally equivalent variants or fragments thereof, all as defined above, which are biotinylated or contain cysteine bridges. Biotinylated peptides can be obtained by any method known in the art, such as the one described in WO93/18054 to De Leys. Methods for obtaining peptides containing inter- and/or intramolecular cysteine bridges are extensively described in WO 96/13590 to Maertens & Stuyver.

[0058] The present invention also relates to any combination of peptides represented by SEQ ID 1 to 38 and functionally equivalent variants or fragments thereof as defined above. The terms "any combination" refers to any possible mixture of above-described peptides or any possible linkage (covalently or otherwise) between above-described peptides. Examples of the latter peptide combinations are simple mixtures, homo- or hetero-branched peptides, combinations of biotinylated peptides presented on streptavidin, avidin or neutravidin, chemically cross-linked peptides with or without spacer, condensed peptides and recombinantly produced peptides.

[0059] The present invention relates also an antibody, more particularly a monoclonal antibody, characterized in that it specifically recognizes an HCV-related virus polypeptide as described above.

[0060] The present invention also relates to a method for diagnosing exposure to or infection by HCV-related viruses comprising contacting anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above or with a combination of peptides as described above, and, determining the binding of anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above or with a combination of peptides as described above. As used herein, the term "a method for diagnosing" refers to any immunoassay known in the art such as assays which utilize biotin and avidin or streptavidin, ELISAs and immunoprecipitation and agglutination assays. A detailed description of these assays is given in WO 96/13590 to Maertens & Stuyver.

[0061] In this regard, the present invention also relates to an assay kit for detecting the presence of anti-HCV-related virus antibodies comprising a solid support, a peptide as described above or a functionally equivalent variant or fragment thereof, or combination of peptides as described above, and appropriate markers which allow to determine the complexes formed between anti-HCV-related virus antibodies within a sample of body fluid with a peptide as described above, or a functionally equivalent variant or fragment thereof, or combination of peptides as described above.

[0062] The term "a solid support" refers to any solid support known in the art.

[0063] Similarly, the term "appropriate markers" refers to any marker known in the art.

[0064] It should also be clear that the term "a method for diagnosing" encompasses screening, detection, confirmation, monitoring and serotyping methods.

[0065] The present invention further pertains to a bioassay for identifying compounds which modulate the binding between a peptide and an anti-HCV-related virus antibody, comprising contacting anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above, and determining the binding of anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above, adding a modulator or a combination of modulators to the contacted anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above, and finally determining the modulation of binding of anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above.

[0066] In another embodiment the present invention features a bioassay for identifying compounds which modulate the binding between a peptide and an anti-HCV-related virus antibody, comprising determining the binding of anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above, contacting a modulator with a peptide as described above, or a combination of peptides as described above, adding anti-HCV-related virus antibodies to the contacted modulator with a peptide as described above, or a combination of peptides as described above, determining the modulation of binding of anti-HCV-related virus antibodies with a peptide as described above, or a combination of peptides as described above.

[0067] The term "compound" as used herein refers to a composition, which has a molecular weight of less than about 25 KDa, preferably less than 10 KDa, and most preferably less than 5 KDa. Compounds can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules, or antibodies which may be generated by the host itself upon vaccination.

[0068] The term "binding" as used herein indicates that a peptide as described above is physically connected to, and interacts with antibodies. Binding of the peptide to the antibody can be demonstrated by any method or assay known in the art such as binding-, ELISA, and RIA-type of assays or competition assays (eg see Examples section and Current protocols in immunology).

[0069] The terms "modulation" or "modulate" as used herein refer to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating)) and downregulation (i.e. inhibition or suppression (e.g. by antagonizing, decreasing or inhibiting) of the binding between a peptide and an anti-HCV antibody.

[0070] The term "modulator" as used herein refer to the ability of a compound as described above to modulate as described above.

[0071] The term "peptidomimetics" as used herein refers to molecules which can be manufactured and which mimic those residues of peptides which modulate the interaction of the antibody with the peptide as described above. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), PNA, substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), ketomethylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295: and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), .beta.-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett

[0072] 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and .beta.-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun, 126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).

[0073] The present invention pertains to a modulator produced by a bioassay as described above.

[0074] The present invention pertains also to a modulator for the interaction between a peptide and an anti-HCV-related virus antibody, when said modulators are identified by a bioassay as described above.

[0075] The present invention features a composition comprising as an active substance a peptide as described above or a combination of peptides as described above.

[0076] The present invention features also a composition comprising as an active substance a modulator as described above or a combination of modulators as described above.

[0077] In another embodiment, the present invention relates to a composition comprising a plasmid vector comprising a nucleotide sequence encoding a peptide as described above, operably linked to transcription regulatory elements. Upon introduction in a human tissue said plasmid vector induces the expression in vivo, of the nucleotide sequence thereby producing the encoded protein. If said protein elicits an immunogenic response it is referred to as a DNA vaccine. It is readily apparent to those skilled in the art that variations or derivatives of the nucleotide sequence can be produced which alter the nucleotide sequence. The altered polynucleotide may have an altered nucleic sequence, yet still encodes a protein as described above, and which reacts with anti-HCV-related virus antibodies, and is considered a to be functional equivalent

[0078] In a preferred embodiment, the present invention relates to a composition as described herein for use as to vaccinate humans against infection with HCV-related virus or any mutated strain thereof.

[0079] In another preferred embodiment, the present invention relates to a composition as described herein for use as to therapeutically treat humans against infection with HCV-related virus or any mutated strain thereof.

[0080] A composition of the present invention can be, as appropiate, any of the preparations described herein, including peptides, functionally equivalent variants or fragments thereof, a combination of peptides, or modulators (e.g. as identified in the bioassay provided herein). Specifically, the term "a composition" relates to an immunogenic composition capable of eliciting protection against HCV-related virus, in particular against HCV and/or HGV, whether partial or complete. The term "as an active substance" relates to the component of the vaccine composition which elicits protection against HCV-related viruses, in particular against HCV and/or HGV. An active substance (e.g. the peptides or the modulators of the present invention) can be used as such, in a biotinylated form (as explained in WO 93/18054) and/or complexed to Neutralite Avidin according to the manufacturer's instruction sheet (Molecular Probes Inc., Eugene, Oreg.).

[0081] It should also be noted that "a composition" comprises, in addition to an active substance, a suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Suitable carriers are typically large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric aa's, aa copolymers and inactive virus particles. Such carriers are well known to those skilled in the art. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: aluminium hydroxide, aluminium in combination with 3-0-deacylated monophosphoryl lipid A as described in WO 93/19780, aluminium phosphate as described in WO 93/24148, N-acetyl-muramyl-L-threo- nyl-D-isoglutamine as described in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-is- oglutamyl-L-alanine2(1'2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine and RIBI (ImmunoChem Research Inc., Hamilton, Mont.), which may contain one or all of the following elements: monophosphoryl lipid A (detoxified endotoxin), trehalose-6,6-dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the three components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass.), MF 57 (Chiron) or SAF-1 (Syntex) may be used, as well as adjuvants such as combinations between QS21 and 3-de-O-acetylated monophosphoryl lipid A (WO94/00153), or MF-59 (Chiron), or poly[di(carboxylatophenoxy) phosphazene] based adjuvants (Virus Research Institute), or blockcopolymer based adjuvants such as Optivax (Vaxcel) or GammaInulin (Anutech), or Gerbu (Gerbu Biotechnik). Furthermore, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be used for non-human applications and research purposes. "A composition" will further contain excipients and diluents, which are inherently non-toxic and non-therapeutic, such as water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, preservatives, and the like. Typically, a vaccine composition is prepared as an injectable, either as a liquid solution or suspension. Solid forms, suitable for solution on, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect. The polypeptides may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS). Compositions, which can be used as a vaccine, comprise an immunologically effective amount of the polypeptides of the present invention and/or modulators, as well as any other of the above-mentioned components. "Immunologically effective amount" means that the administration of that amount to an individual, either in a single dosis or as part of a series, is effective for prevention or treatment This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to mount an effective immune response, the degree of protection desired, the formulation of the vaccine, the treating's doctor assessment, the strain of the infecting HCV and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 .mu.g/dose, more particularly from 0.1 to 100 .mu.g/dose. Compositions, which can be used as a vaccine are conventionally administered parenterally, typically by injection, for example, subcutaneously or intramuscularly.

[0082] In the case of DNA vaccines, particular useful methods for eliciting an immune response include the coating of gold particles with the plasmid vector encoding the desired peptide, and injecting them under high pressure into the epidermis and/or dermis, eg by means of a device called gene gun (eg as produced by Powderject Vaccines, Madison, Wis., USA).

[0083] Additional formulations suitable for other methods of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents. It should be noted that a vaccine may also be useful for treatment of an individual, in which case it is used as a to "therapeutically treat humans".

[0084] As used herein, a "plasmid vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they have been linked. In general, but not limited to those, plasmid vectors are circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. For expression purposes, promoters are required. For DNA vaccination, a very suitable promoter is the Major Immediate Early (MIE) of human cytomegalovirus.

[0085] As used herein, a "nucleotide sequence" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and single (sense or antisense) and double-stranded polynucleotides.

[0086] As used herein, the term "transcription regulatory elements" refers to a nucleotide sequence which contains essential regulatory elements, ie such that upon introduction into a living vertebrate cell it is able to direct the cellular machinery to produce translation products encoded by the polynucleotide.

[0087] The term "operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, transcription regulatory elements operably linked to a nucleotide sequence are capable of effecting the expression of said nucleotide sequence. Those skilled in the art can appreciate that different transcriptional promoters, terminators, carrier vectors or specific gene sequences may be used successfully.

[0088] Finally, the present invention provides a method to immunize humans against infection with HCV-related virus or any mutated strain thereof, comprising the use of a peptide as described above or a combination of peptides as described above.

[0089] The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and can not be construed as to restrict the invention in any way.

EXAMPLES

Example 1

[0090] Synthesis of Multimer E1 and E2 Peptides

[0091] We aimed at synthesizing peptides which would display epitopes, similar to the ones present on E1 and E2 peptides expressed in mammalian cells. Since such epitopes do not seem to be present in E1 and E2 proteins expressed in E. coli, the design of such peptides was not an easy task. We first aligned E1 and E2 primary amino acid sequences of different HCV genotypes and delineated variable and constant domains. It was reasoned that these domains, or a combination of two or more of these domains might represent conformational domains, ie form or constitute independent conformational units. If displayed as 3D structure, these conformational domains may also contain conformational epitopes. The latter domains may therefore be able to adopt a native-like structure as is present in the envelope proteins when these envelope proteins are expressed in mammalian cells. In contrast, such structures are absent when the envelope proteins are expressed in prokaryotic cells, like E. coli.

[0092] The following domains were assigned:

[0093] V1, V2, V3, V4, V5, V6=variable regions; C1, C2, C3, C4 conserved domains; HR=hydrophobic region; SA=signal anchor sequence; HVRI, HVRII=hypervariable regions of E2.

1 Protein Region Amino acid position E1 V1 192-203 C1 204-217 V2 218-223 C2 224-229 V3 230-242 C3 243-247 V4 248-257 HR 258-293 V5 294-303 C4 304-329 V6 330-342 SA 343-383 E2 HVRI 384-411 C1 412-470 HVRII 471-482 C2 483-521 V3 522-548 C3 549-569 V4 570-580 C4 581-704 SA 705-746

[0094] Based on these domains of the BE11 subtype 1b isolate (SEQ ID 50 in PCT/EP 95/03031), we designed a series long peptides of 24 to 45 amino acids. For some extended domains of the envelope proteins more than one multimer peptide was synthesized in order to encompass the domain of interest. Table 1 gives an overview of the peptides with their respective amino acid positions; numbering starts from the first initiation codon of the HCV polyprotein. Peptides were synthesized using t-Boc technology as explained in detail in WO 93/18054.

Example 2

[0095] Reactivity of Multimer Peptides with E1 and E2 Antibodies in Patient Sera

[0096] A series of 60 randomly chosen samples from patients with chronic active hepatitis C were tested for reactivity with the multimer peptides. These samples did not show any notable reactivity with 20-mer peptides except for some 20-mer peptides derived from the HVRI. For comparison, reactivity with the hydrophylic ectodomain of E2, the recombinant E2h protein, was assayed (E2h extends from aa 384-708 and was cloned from SEQ ID NO 45, and expressed and purified as described in PCT/EP 95/03031). Peptides were coated onto streptavidin-coated plates (5 .mu.g/ml) and antibodies in serum samples were left to react and detected using the reagents and procedures as described in the package insert of the INNOTEST HCV Ab III kit (Innogenetics, Gent, Belgium). Table 2 shows the results of the ELISA tests, in which a cutoff of 150 mOD was used. In this series, 5 sera did not show reactivity with the E2h protein, only one of these reacted with the HVRI peptide. Five out of 60 sera (8%; e.g. sample 17758) only reacted with the E2h protein,

[0097] 34 (57%) recognized HVRI 24(40%) reacted with C1-a, 18 (30%) with C1-b, 21 (35%) with HVRII, 17 (28%) C2-a, 22 (37%) with C2-b, 18 (30%) with C3, 18 (30%) with C3', 17 (28%) with C3", 18 (30%) with V4, 22 (37%) with C-4, 21 (35%) with C4-a, 35 (58%) with C4-b, and 24 (40%) with C4-c. This experiment surprisingly learned that, while none of the samples recognized any of the 20-mer peptides, except for those derived from the HVRI, 50 out of 55 (91%) E2h reactive sera could be detected using the peptides of the present invention.

[0098] In a second series of 23 sera derived from chronic hepatitis C patients who were long-term responders to interferon-alpha treatment and 3 HCV infected chimpanzees, E1 and E2 antibodies were tested. Eighteen out of 23 samples (78%) reacted with recombinant E1s protein, expressed and purified from mammalian cells as described in PCT/EP 95/0303 1. Nine samples (39%) reacted with the C4V6 region, another 9 (39%) with the V1V2 region, and 3 with V2V3 (Table 4). For comparative purposes peptide V5, ie SQLFTISPRRHETVQD, is shown.

[0099] Different reactivities to E2 were observed (Table 4) as compared with the first series of samples. 21 samples (91%) reacted with E2h, with 13 (57%) reactive on HVRI, 9 (39%) with C1-a, 11 (48%) with C1-b, 1 with HVRII, C2-a, and C2-b each, 2 with C3, 3 with C4-a, 4 (17%) with C4-b, and 4 (17%) with C4-c. In this series of patients with a benign evolution of disease, the C1 region was more frequently recognized and fewer antibodies to the C4 region were detected as compared to the series of samples obtained from patients with chronic active hepatitis. These results indicate that peptides from the C1, C2, and C4 regions may be particularly useful in monitoring the course on HCV-related virus disease. More specifically, antibodies to the Cl region may better neutralize HCV as compared to anti-C4 antibodies. The C1 domain may therefore be functionally important, eg exhibit receptor-binding activity. Neutralization of this region may therefore result in lesser activity of the disease and may lead to resolvement The E2-C1 region may therefore be particularly useful in therapeutic interventions. It should also be noted that, once reactivity to a given domain is established, it can be further mapped to smaller peptides, e.g. reactivities of 1 chimpanzee serum to C3 could be mapped to smaller region of 25 amino acids (peptide C3").

Example 3

[0100] Monitoring of E1 and E2 Antibodies in Patients with Response to Interferon-alpha Therapy

[0101] In Table 5, results of E2 antibody tests as described in example 2 are given for consecutive samples obtained from patients with response to interferon therapy. A decline in E2Ab, and to a larger extend E1Ab, has been described in PCT/EP 95/03031 in case of a long-term response to interferon treatment. Reactivities to several peptides of the present invention also show similar declining levels. Peculiar reactivities could sometimes be detected as exemplified in patient 2: upon the detection of reappearing virus, antibody responses to the (E1)V4V5 region and the (E2)HVRII region could be detected; these quickly disappeared simultaneously with viral clearance. (E1)V4V5 and (2)HVRII may therefore be particularly useful peptides for disease monitoring, especially in treatment of disease. Other peptides such as (E2)C1 (example 2) and those shown in bold in Table 5 also seem to be useful for purposes such as monitoring. Table 2 also shows the presence of reactivity in patient 2 to a new peptide HVRI-C1, which overlaps the junction between HVRI and C1 (Table 2), in the absence of detectable reactivity to the HVRI or C1 peptides. Similarly, peptide C4-bc encompassing the region between C4-b and C4-c (Table 2), was tested in this series, and showed almost identical reactivities as compared to peptide C4-b. Therefore, it is possible that the C4-b epitope lies between aa 658 and 673, but surprisingly, the epitope does not seem to be presented in peptide SEQ ID 92 of PCT/EP 95/03031 (aa 655-674). The C4-c epitope is not present in C4-bc and therefore can be localized between aa 683 and 706.

Example 4

[0102] Application to Other Flaviviruses

[0103] To examine the applicability of the invention to envelope proteins of other HCV-related viruses, a peptide spanning the V1V2 region of the hepatitis G virus (GBV-C; Linnen et al., 1996; Simons et al., 1996) E1 region was synthesized, see also SEQ ID NO 38 (Table 1): NH2-THACRANGQYFLTNCCAPEDIGFCLEGGCLVALGGK-biotin.

[0104] So far, only reactivity to the complete HGV E2 protein seemed to be useful in the diagnosis of HGV. Peptide epitopes have not yet been described for GBV envelope proteins E1 or E2. Sixteen HGV RNA-positive sera were tested and 1 of these was reactive with the E1 peptide as shown in Table 6. Antibody reactivity to the recombinant HGV E2 protein (but not to HGV E2 peptides) is found in up to 15% of the European population, but cases with both HGV RNA and E2Ab are rare as they probably represent cases in which seroconversion and elimination of the virus is ongoing. Antibody reactivity to the HGV E1 protein has not yet been reported. Therefore, the HGV E1 peptide V1V2 is new and it may display higher reactivities in a series of HGV anti-E2 reactive sera. Using similar approaches as described in the present invention, HGV E2 peptides may also be synthesized. Multimer peptides from GBV-A or GBV-B can be synthesized in a similar approach as described for HCV and HGV.

Example 5

[0105] Reactivity of 20-mer E2 Peptides Compared to Multimer E2 Peptides.

[0106] E2 peptides listed in Table 1 were analyzed for their reactivity with 32 serum samples from patients with chronic active hepatitis C. In addition, a series of overlapping 20-mer peptides were synthesized with exactly the same HCV subtype 1b sequence as used for the longer peptides and as shown in Table 1. The ELISA test used was the same as described in Example 2. FIGS. 3 and 4 show the reactivities of the series of 20-mer and longer peptides, respectively. Peptides with a sum of>5 (HVR I HVR I/C1, C1a, C1b, C4a, C4b, C4c, C4b-c) were considered to be very useful for the detection of antibodies directed against E2. A total of six of these peptides (peptides C4b-c and C1a were not included as these peptides are almost entirely represented by other peptides) were combined together with 20-mer peptide 1350 (Table 1), which occasionally reacted with some patient sera. The combination of these peptides was tested on a panel of 128 sera from chronic active HCV carriers. Hundred and twenty six of these sera tested positive on recombinant E2s protein. Of these 126 sera, 33 sera showed at least two times higher OD values with the peptide mixture as compared to the recombinant E2 protein, 64 sera showed a similar reactivity, 16 sera showed reactivities which were 2- to 4-fold higher with the recombinant protein than with the peptide mixture, and 13 sera only reacted with the recombinant protein.

[0107] In summary, almost 90% of the sera containing antibodies against recombinant E2 protein could be detected using the above peptide mixture. For 26% of the sera, detection was even better using the peptides of the invention, than using recombinant E2 protein. A sum of OD values of>5, ie exhibited by peptides HVR I, HVR I/C1, C1a, C1b, C4a, C4b, C4c, and C4b-c (FIG. 4) is therefore considered a surprisingly high value for the serodiagnosis of antibodies directed against the E2 protein of HCV. From the experiment described above, it is also clear that a combination of recombinant E2 with the peptides of the invention is a particularly useful composition.

[0108] Given the variability of the E2 protein in different HCV genotypes, the addition of genotype-specific peptides to recombinant E2 proteins may be a desired way of improving sensitivity of E2 antibody assays. For example, a variant of peptide C1a based on a reported HCV type 2a sequence HC-J6 could be LINTNGSWHINRTALNCNDSLHTGFLASLFYTHS, and similar useful variants e.g. based on a genotype 3a sequence, could be synthesized and tested for reactivity. It should be noted that the HCV E2 protein may contain insertions or deletions in any given HCV genotype. For example, while subtype 1a and 1b sequences show contiguous sequences which can be aligned without having to insert gaps, HCV type 2a isolates encode E2 proteins which are 4 aa's longer as compared to type 1 sequences. For example, 2 additional amino acids are inserted in HCV type 2a and 2b sequences around hypervariable region II (HVR II). Therefore, a potentially useful variant of peptide HVRII, based on the HC-J6 prototype 2a sequence, would be RSIEAFRVGWGALQYEDNVTNPEDMRPYCW, which is a 30-mer peptide while the subtype 1b sequence-based peptide depicted in Table 1 (SEQ ID 20) is only 28 aa's long. The two glutamates (symbol E) which are inserted in the subtype 2a sequence are shown underlined. Similar peptides can be easily constructed based on sequences and alignments previously published (e.g. Maertens and Stuyver, 1997).

List of References

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[0115] Kohara M, Tsukiyama-Kohara K, Maki N, et al. Expression and characterization of glycoprotein gp35 of hepatitis C virus using recombinant vaccinia virus. J. Gen. Virol. 1992; 73: 2313-8.

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2TABLE 1 GENO SEQ ID PROTEIN TYPE PEPTIDE AMINO ACID SEQUENCE POSITION NUMBER E1 1a V1V2T1a YQVRNSTGLYHVTNDCPNSSIVYEAADAILHTPGC 192-226 Seq ID 1 1b V1V2T1b YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGC 192-226 Seq ID 2 2c V1V2T2c VEVKNNSNSYMATNDCSNSSIIWQLEGAVLHTPGC 192-226 Seq ID 3 2c V1V2T2c' VEVKNTSTSYMVTNDCSNSSIVWQLEGAVLHTPGC 192-226 Seq ID 4 3a V1V2T3a LEWRNTSGLYVLTNDCSNSSIVYEADDVILHTPGC 192-226 Seq ID 5 3a V2T3a LTNDCSNSSIVYEADDVILHTPGC 203-226 Seq ID 6 4c/4k V1V2T4a INYRNVSGIYHVTNDCPNSSIVYEADHHILHLPGC 192-226 Seq ID 7 5a V1V2T5a VPYRNASGIYHITNDCPNSSIVYEADNL- ILHAPGC 192-226 Seq ID 8 6a V1V2T6a LTYGNSSGLYHLTNDCSNSSIVLEADAMILHLPGC 192-226 Seq ID 9 1b V2V3 IVYEAADMIMHTPGCVPCVRENNSSRCWV 212-240 Seq ID 10 1b V3V4 VRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 230-263 Seq ID 11 1b PC-V3V4 PCVRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 228-263 Seq ID 12 1b HR HVDLLVGAAAFCSAMYVGDLCGSVFLVSQL 260-290 Seq ID 13 1b V5C4 SQLFTISPRRHETVQDCNCSIYPGHITGHRMAWDMMMNWS 288-327 Seq ID 14 1b C4V6 SIYPGHITGHRMAWDMMMNWSPTTALVVSQLLRI 307-340 Seq ID 15 1b SA PQAVVDMVAGAHWGVLAGLAYYSMVGNWAKVLVVMLLFAG- V 341-381 Seq ID 16 1b V4V5 VALTPTLAARNASVPTTTIRRHVDSQLFT- ISPRRHETVQD 240-303 Seq ID 37 E1(HGV) ND V1V2 THACRANGQYFLTNCCAPEDIGFCLEGGCLVALGGK ND Seq ID 38 E2 1b HVR I HTRVSGGAAASNTRGLVSLFSPGSAQKIQLVN 384-415 Seq ID 17 1b C1a LVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKF 413-447 Seq ID 18 1b C1b NDSLQTGFFAALFYKHKFNSSGCPERLASCRSIDKFAQ 430-467 Seq ID 19 1b HVR II RSIDKFAQGWGPLTYTEPNSSDQRPYCW 460-487 Seq ID 20 1b C2a SDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSP 480-513 Seq ID 21 1b C2b SQVCGPVYCFTPSPVVVGTTDRFGVPTYNWG 500-530 Seq ID 22 1b V3C3 GVPTYNWGANDSDVLILNNTRPPRGNWFGCTWMNGTGFTKTCGG 523-566 Seq ID 23 1b V3C3' ANDSDVLILNNTRPPRGNWFGCTWMNGTG- FTKTCGG 531-566 Seq ID 24 1b C3" TRPPRGNWFGCTWMNGTGFTKTCG- G 542-566 Seq ID 25 1b V4 TKTCGGPPCNIGGAGNNTLTCPTDCFRKHP 561-590 Seq ID 26 1b C4 TDCFRKHPEATYARCGSGPWLTPRCMVHYPYR- LWHYPCTVNFTIF 583-627 Seq ID 27 1b C4' ARCGSGPWLTPRCMVHYPYRLWHYPCTVNFTIF 595-627 Seq ID 28 1b C4" LTPRCMVHYPYRLWHYPCTVNFTIF 603-627 Seq ID 29 1b C4a TVNFTIFKVRMYVGGVEHRFEAACNWTR 621-648 Seq ID 30 1b C4b EAACNWTRGERCDLEDRDRSELSPLLLSTTEWQ 641-673 Seq ID 31 1b C4c QWQILPCSFTTLPALSTGLIHLHQNIVDVQYLYGVG 671-706 Seq ID 32 E2 1b SA GVGSAVVSLVIKWEYVLLLFLLLADARICACLWMMLLIAQAE 704-745 Seq ID 33 1b HVR I/C1 NTRGLVSLFSPGSAQKIQLVNTNGSWHINRTALN 395-428 Seq ID 34 1b C4b-c DRSELSPLLLSTTEWQILPCSFTTLPALSTG 658-688 Seq ID 35 1b 1350 VGTTDRFGVPTYNWGANDSD 516-535 Seq ID 36

[0126]

3TABLE 2 Sample HVR R c # HVR I C1-a C1-b II C2-a C2-b E2-13 B C3 C3' C3" V4 C4 C4-a C4-b C4-c SA E2 17758 69 48 47 52 49 48 47 49 38 44 43 52 44 55 48 46 1355 17763 88 54 44 49 52 48 51 51 46 45 48 49 45 133 104 50 361 17764 100 148 138 134 128 136 141 136 136 65 130 145 144 242 128 127 371 17766 91 97 145 96 80 87 90 90 95 47 75 89 163 139 99 86 173 17771 307 79 54 65 51 50 65 68 50 45 60 65 59 96 132 58 393 17775 49 50 46 39 50 271 43 51 48 45 50 55 52 54 47 50 228 17777 60 133 105 130 129 123 118 118 130 95 119 133 129 357 177 113 850 17779 373 328 285 330 284 343 281 323 316 283 297 318 343 341 309 282 720 17785 81 80 73 71 76 66 81 70 74 70 69 79 79 87 119 73 146 17786 341 863 693 152 164 179 148 139 146 136 137 158 160 163 148 157 720 17788 111 553 120 137 69 121 121 119 111 110 103 140 132 131 48 47 934 17789 1316 49 47 46 49 45 53 51 48 43 42 50 49 52 48 48 1178 17790 234 233 182 223 130 224 185 185 186 184 179 216 218 1347 853 207 1534 17791 269 194 177 192 123 203 172 192 157 184 184 200 195 211 187 190 287 17797 260 264 248 257 240 281 249 237 246 221 223 283 261 272 231 243 1357 17798 52 53 50 47 52 54 50 53 49 51 50 51 50 1036 51 51 1161 17799 225 89 81 86 85 100 76 85 87 82 84 86 92 115 86 76 362 17802 42 51 44 47 50 133 48 52 51 48 51 56 76 773 157 56 882 17807 49 133 60 59 66 62 62 59 57 56 57 63 65 62 57 52 605 17808 89 121 117 109 106 1051 118 875 133 116 123 126 393 228 109 126 1354 17810 327 220 199 222 195 200 221 182 197 182 196 209 266 222 195 199 422 17818 224 134 115 126 118 115 128 108 109 98 111 113 112 117 109 108 230 17821 671 243 214 282 238 232 228 217 234 197 216 222 218 557 810 205 1046 17825 397 320 264 284 282 286 289 277 276 274 276 306 273 391 399 277 514 17826 92 109 111 99 114 126 113 98 104 84 105 121 122 126 145 113 695 17827 45 47 46 47 48 49 48 49 49 47 49 50 50 261 113 47 320 17832 151 65 55 70 78 63 77 72 68 59 64 70 62 54 57 49 288 17838 212 167 166 164 156 165 164 146 160 154 150 165 165 161 272 157 305 17839 48 94 117 61 61 51 58 51 46 52 58 55 87 60 95 66 182 17840 318 323 347 317 329 338 320 305 326 302 312 343 355 322 318 337 417 17842 161 174 185 176 168 163 159 157 163 156 150 168 151 154 138 153 195 17844 122 94 90 88 98 78 92 88 84 77 85 94 61 214 51 73 166 17849 1469 68 75 49 54 629 52 53 46 46 51 54 119 1102 55 47 1393 17870 125 236 148 114 128 133 135 116 132 109 135 151 118 293 120 45 197 17879 209 195 201 222 195 215 225 191 194 181 218 209 209 255 253 199 325 17983 438 54 50 48 52 46 50 54 46 46 51 52 46 55 53 48 216 17999 276 201 200 202 190 187 191 169 176 150 190 205 186 321 535 198 697 8242 162 114 114 127 140 114 120 117 103 120 117 107 112 161 152 128 340 8243 188 191 171 175 204 172 189 174 186 174 176 205 200 206 177 178 225 8247 248 169 137 127 120 110 122 96 111 104 114 128 104 130 150 118 215 8250 129 161 127 150 164 144 154 125 134 122 142 151 125 146 137 140 165 8317 112 131 115 123 113 111 144 95 103 95 108 118 108 158 126 111 198 8320 463 433 337 473 435 445 363 345 503 384 362 369 405 446 432 378 474 8329 119 126 123 160 143 145 142 117 135 121 122 126 131 152 148 132 163 8330 198 271 210 210 207 196 216 178 194 206 209 215 186 356 45 51 536 8332 154 141 128 141 132 116 129 110 123 112 135 140 123 147 312 144 290 8333 57 67 50 51 52 52 50 54 50 50 50 56 48 480 65 52 1108 8334 283 66 64 80 68 69 84 79 65 52 67 74 72 180 191 90 348 8337 162 105 99 108 103 92 104 86 93 80 101 107 108 124 118 110 142 8339 50 49 52 62 54 46 54 51 47 41 51 55 53 413 49 50 247 8344 59 52 50 51 58 48 54 52 47 48 55 53 58 63 63 60 59 8351 163 114 105 111 101 91 98 97 92 78 110 111 115 141 179 112 154 8362 211 54 50 47 55 119 53 53 44 45 51 54 59 60 58 55 165 8364 110 308 106 112 112 107 98 102 108 92 116 152 133 208 169 132 671 8365 69 84 94 67 77 74 55 73 70 69 70 79 73 69 88 66 86 8367 218 189 171 201 204 174 191 156 158 140 183 186 294 197 186 171 303 8374 575 113 95 114 110 93 100 92 106 88 103 125 118 112 111 106 143 8377 364 232 229 225 211 202 233 189 207 170 209 205 230 234 218 221 293 8382 314 211 187 196 207 173 208 181 158 150 181 187 201 223 189 211 265 8383 51 100 102 55 58 48 57 53 53 50 52 57 66 94 63 56 285 V1200 52 55 52 56 55 53 50 54 50 52 51 50 50 52 53 54 50 V1201 118 147 138 136 224 144 123 137 140 111 135 154 166 171 137 155 162 V1202 274 308 284 170 290 286 282 248 277 229 271 306 287 330 268 295 329 V1204 130 134 135 127 141 128 79 113 119 106 131 144 145 144 130 144 159

[0127]

4 TABLE 3 E1 antigens Sample# No peptide V1V2 V2V3 V3V4 HR/SA V5 C4V6 rec E1s No sample 0.011 0.007 0.011 0.014 0.009 0.007 0.009 0.056 30108 0.03 0.035 0.04 0.034 0.032 0.03 0.234 0.378 30109 0.032 0.033 0.035 0.028 0.024 0.026 0.227 0.368 30110 0.021 0.545 0.02 0.019 0.016 0.017 0.047 0.669 30111 0.017 0.614 0.019 0.018 0.017 0.015 0.064 0.796 30112 0.037 0.069 0.035 0.034 0.031 0.031 0.048 0.187 30113 0.042 0.083 0.136 0.039 0.034 0.035 0.063 0.226 30114 0.042 0.099 0.036 0.035 0.035 0.037 0.058 0.267 30115 0.021 0.114 0.023 0.021 0.02 0.02 0.189 0.339 30116 0.019 0.442 0.025 0.022 0.022 0.018 0.056 0.645 30117 0.027 0.062 0.047 0.043 0.041 0.038 0.066 0.164 30118 0.122 0.216 0.126 0.12 0.11 0.125 0.696 0.923 30119 0.023 0.028 0.031 0.028 0.023 0.024 0.23 0.426 30120 0.025 0.024 0.027 0.025 0.039 0.027 0.03 0.024 30121 0.03 0.033 0.033 0.029 0.052 0.034 0.037 0.032 30122 0.029 0.031 0.056 0.03 0.052 0.033 0.035 0.03 30123 0.085 0.081 0.076 0.075 0.087 0.071 0.094 0.137 30124 0.022 0.084 0.022 0.022 0.023 0.022 0.193 0.391 30125 0.095 0.128 0.091 0.089 0.172 0.159 0.47 0.708 17805 0.038 0.051 0.039 0.033 0.09 0.154 0.738 1.169 13059 0.011 0.011 0.012 0.012 0.014 0.012 0.229 0.681 Chimp1 0.095 0.38 0.276 0.126 0.098 0.095 0.099 0.805 Chimp2 0.026 0.234 0.143 0.035 0.036 0.038 0.354 0.822 Chimp3 0.018 0.017 0.02 0.022 0.023 0.019 0.141 0.353

[0128]

5 TABLE 4 E2 antigens Sample peptide HVR I C1-a C1-b HVR II C2-a C2-b C3 C3' C3" V4 C4 C4-a C4-b C4-c recE2h No sample 0.006 0.009 0.011 0.015 0.007 0.006 0.01 0.01 0.01 0.01 0.01 0.01 0.007 0.007 0.009 0.032 30108 0.036 0.747 0.848 0.969 0.032 0.033 0.03 0.04 0.02 0.02 0.03 0.03 0.041 0.026 0.031 0.988 30109 0.027 0.849 0.93 1.053 0.027 0.032 0.03 0.03 0.02 0.02 0.03 0.02 0.038 0.023 0.026 1.079 30110 0.018 0.026 0.021 0.044 0.019 0.024 0.02 0.02 0.02 0.02 0.02 0.03 0.023 0.026 0.056 0.11 30111 0.017 0.02 0.021 0.088 0.018 0.02 0.02 0.02 0.01 0.02 0.02 0.03 0.022 0.028 0.07 0.137 30112 0.037 0.092 0.052 0.177 0.044 0.048 0.04 0.04 0.04 0.04 0.04 0.05 0.043 0.562 0.053 0.947 30113 0.045 0.104 0.054 0.276 0.051 0.047 0.05 0.03 0.04 0.04 0.04 0.05 0.054 0.633 0.07 1.003 30114 0.045 0.112 0.075 0.726 0.046 0.041 0.05 0.05 0.03 0.04 0.04 0.06 0.054 0.646 0.067 1.065 30115 0.022 0.982 0.034 0.064 0.025 0.025 0.02 0.03 0.02 0.03 0.03 0.02 0.03 0.097 0.031 0.413 30116 0.015 0.023 0.02 0.04 0.017 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.023 0.022 0.046 0.084 30117 0.04 0.087 0.048 0.119 0.037 0.044 0.05 0.05 0.03 0.04 0.04 0.04 0.041 0.547 0.049 0.935 30118 0.112 0.213 0.122 0.119 0.119 0.121 0.12 0.12 0.11 0.05 0.11 0.1 0.117 0.105 0.2 0.289 30119 0.03 0.954 1.012 1.128 0.026 0.029 0.03 0.03 0.02 0.02 0.03 0.03 0.035 0.026 0.03 1.123 30120 0.031 0.427 0.208 0.208 0.03 0.033 0.03 0.04 0.03 0.03 0.03 0.03 0.033 0.032 0.032 0.577 30121 0.033 0.734 0.463 0.398 0.037 0.042 0.04 0.05 0.04 0.03 0.04 0.03 0.04 0.034 0.037 0.963 30122 0.03 0.661 0.413 0.365 0.043 0.034 0.03 0.04 0.04 0.03 0.03 0.03 0.038 0.03 0.034 0.907 30123 0.079 0.11 0.576 0.789 0.09 0.108 0.09 0.08 0.08 0.06 0.07 0.06 0.091 0.078 0.077 0.916 30124 0.02 0.939 0.041 0.065 0.028 0.237 0.04 0.04 0.02 0.03 0.02 0.02 0.038 0.108 0.049 0.4 30125 0.096 0.133 0.103 0.096 0.097 0.115 0.15 0.14 0.09 0.09 0.09 0.1 0.1 0.092 0.183 0.227 17805 0.042 0.255 0.074 0.078 0.071 0.045 0.06 0.06 0.05 0.04 0.06 0.04 0.163 0.043 0.831 0.881 13059 0.013 0.47 0.02 0.019 0.018 0.022 0.02 0.03 0.02 0.01 0.01 0.02 0.36 0.052 0.904 0.944 Chimp1 0.102 0.103 0.116 0.118 0.23 0.109 0.12 0.19 0.17 0.19 0.1 0.1 0.087 0.098 0.095 0.581 Chimp2 0.028 0.181 0.267 0.261 0.056 0.032 0.03 0.04 0.04 0.04 0.04 0.04 0.188 0.035 0.033 1.008 Chimp3 0.058 0.035 0.162 0.086 0.026 0.062 0.02 0.03 0.04 0.02 0.03 0.03 0.023 0.02 0.026 1.327

[0129]

6TABLE 5 HCV E1 peptides Sample PCR Genotype V1V2 V2V3 V3V4 V4V5 HR/SA V5C4 C4V6 E1s Patient 1 14/8/90 pos 3a 0.014 0.03 0.06 0.034 0.037 0.048 0.045 0.051 01/06/91 0.03 0.032 0.064 0.041 0.041 0.051 0.048 0.045 20/9/91 neg 0.06 0.064 0.064 0.037 0.039 0.05 0.398 0.045 13/3/92 0.034 0.041 0.037 0.034 0.037 0.046 0.044 0.04 04/09/92 neg 0.037 0.041 0.039 0.037 0.037 0.052 0.048 0.043 24/9/93 0.048 0.051 0.05 0.046 0.052 0.048 0.047 0.042 20/10/94 neg 0.045 0.048 0.398 0.044 0.048 0.047 0.045 0.041 23/10/95 0.051 0.045 0.045 0.04 0.043 0.042 0.041 0.051 10/12/96 pos? 0.037 0.041 0.033 0.034 0.035 0.039 0.038 0.045 Patient 2 15/2/90 0.106 0.103 0.104 0.108 0.104 0.949 0.872 1.03 03/05/90 pos 1a 0.103 0.109 0.106 0.104 0.108 0.828 0.859 1.04 04/12/90 0.096 0.103 0.105 0.103 0.095 0.737 0.848 1.218 23/9/91 0.063 0.078 0.078 0.067 0.072 0.318 0.354 0.66 14/4/92 0.099 0.106 0.099 0.1 0.096 0.219 0.255 0.491 18/12/92 0.104 0.106 0.102 0.105 0.101 0.222 0.249 0.448 26/3/93 0.089 0.095 0.09 0.085 0.082 0.168 0.194 0.357 30/9/93 neg 0.092 0.081 0.089 0.09 0.088 0.17 0.18 0.35 17/6/94 pos 1a 0.084 0.09 0.096 0.599 0.095 0.154 0.166 0.32 18/12/95 0.072 0.077 0.077 0.077 0.081 0.111 0.121 0.206 23/12/96 neg 0.065 0.078 0.074 0.073 0.078 0.106 0.108 0.199 Patient 3 15/04/93 0.005 0.006 0.005 0.004 0.006 0.005 0.006 0.007 06/09/94 pos 3a 0.007 0.008 0.007 0.008 0.007 0.006 0.006 0.009 30/10/95 neg 0.007 0.01 0.009 0.009 0.009 0.008 0.007 0.011 18/11/96 pos? 1b 0.012 0.012 0.012 0.011 0.01 0.009 0.009 0.012 Patient 4 12/04/91 pos 1a 0.006 0.007 0.006 0.006 0.007 0.006 0.006 0.01 23/09/91 neg 0.01 0.01 0.008 0.009 0.009 0.006 0.008 0.013 27/07/92 neg 0.007 0.009 0.007 0.008 0.007 0.006 0.007 0.01 11/06/93 neg 0.009 0.011 0.009 0.01 0.009 0.007 0.006 0.011 29/11/96 pos 1a 0.007 0.01 0.008 0.007 0.007 0.005 0.006 0.008 Patient 5 18/09/92 pos 0.017 0.01 0.008 0.007 0.008 0.178 0.196 0.537 17/12/93 neg 0.012 0.014 0.011 0.01 0.011 0.039 0.04 0.231 15/11/96 neg 0.012 0.014 0.012 0.01 0.01 0.026 0.017 0.116 Patient 6 10/05/90 pos 0.311 0.006 0.007 0.005 0.006 0.004 0.01 0.544 11/10/91 neg 0.284 0.007 0.007 0.006 0.007 0.006 0.013 0.605 Patient 7 10/10/91 pos 1b 0.009 0.01 0.009 0.008 0.008 0.008 0.01 0.043 18/12/92 neg 0.01 0.011 0.011 0.009 0.009 0.008 0.011 0.043 28/06/93 neg 0.006 0.006 0.007 0.006 0.007 0.005 0.008 0.021 10/03/97 pos 1b 0.008 0.008 0.007 0.008 0.007 0.006 0.008 0.012 Patient 8 19/08/91 neg 0.008 0.009 0.008 0.008 0.008 0.006 0.008 0.009 17/07/95 pos 1b 0.01 0.009 0.009 0.009 0.006 0.007 0.007 0.018 09/10/95 pos 1b 0.007 0.007 0.008 0.005 0.006 0.007 0.007 0.009 15/12/95 neg 0.008 0.009 0.008 0.009 0.008 0.007 0.007 0.011 04/03/96 neg 0.009 0.011 0.01 0.011 0.009 0.008 0.007 0.01 02/09/96 neg 0.01 0.011 0.011 0.01 0.01 0.008 0.008 0.013 Patient 9 26/08/91 pos 1b/2ac 0.044 0.015 0.022 0.023 0.028 0.031 0.034 0.115 21/12/93 neg 0.033 0.017 0.021 0.027 0.022 0.025 0.023 0.048 20/12/94 pos 1b 0.023 0.016 0.015 0.028 0.019 0.028 0.034 0.077 21/12/95 pos 1b 0.019 0.029 0.024 0.027 0.027 0.031 0.034 0.048 Patient 10 27/04/92 pos 1b 0.128 0.024 0.02 0.023 0.026 0.118 0.449 0.68 01/06/93 neg 0.107 0.03 0.029 0.027 0.026 0.098 0.385 0.667 Patient 11 09/11/90 neg 0.018 0.019 0.012 0.013 0.015 0.087 0.141 0.591 12/07/91 pos 1b 0.023 0.023 0.016 0.02 0.018 0.073 0.1 0.466 28/05/93 pos 0.008 0.009 0.009 0.005 0.008 0.123 0.173 0.495 20/01/95 neg 0.011 0.009 0.008 0.007 0.007 0.026 0.047 0.187 08/01/96 neg 0.012 0.013 0.01 0.009 0.009 0.025 0.031 0.21 07/02/97 neg 0.019 0.019 0.014 0.014 0.013 0.027 0.051 0.203 Patient 12 11/05/92 pos 1b 0.017 0.013 0.011 0.014 0.015 0.227 0.173 0.425 26/02/93 neg 0.022 0.014 0.013 0.013 0.014 0.178 0.264 0.417 12/08/93 pos 1b 0.016 0.016 0.016 0.014 0.015 0.29 0.387 0.63 Patient 13 07/01/91 pos 1b 0.027 0.017 0.021 0.026 0.026 0.04 0.074 0.062 19/08/91 neg 0.018 0.018 0.015 0.013 0.012 0.021 0.009 0.043 21/08/92 pos 0.015 0.012 0.015 0.014 0.017 0.015 0.021 0.023 06/08/93 neg 0.019 0.018 0.016 0.021 0.016 0.01 0.011 0.02 06/03/95 pos 1b 0.027 0.026 0.018 0.015 0.018 0.02 0.023 0.028 12/04/96 neg 0.03 0.017 0.018 0.036 0.021 0.027 0.027 0.022 Patient 14 22/11/94 pos 1b 0.016 0.011 0.013 0.013 0.026 0.318 0.437 0.461 11/10/95 pos 0.024 0.014 0.014 0.018 0.019 0.039 0.061 0.059 15/02/96 neg 0.032 0.022 0.021 0.023 0.016 0.031 0.041 0.102 Patient 15 04/12/90 pos 1b 0.003 0.005 0.005 0.004 0.005 0.005 0.005 0.019 29/11/90 neg 0.005 0.005 0.005 0.006 0.005 0.008 0.006 0.011 09/10/92 pos 1b 0.006 0.008 0.007 0.007 0.007 0.006 0.005 0.012 25/03/96 neg 0.006 0.008 0.007 0.006 0.006 0.004 0.007 0.012 Patient 16 16/12/91 pos 3a 0.003 0.004 0.006 0.004 0.004 0.08 0.102 0.435 04/10/93 neg 0.006 0.007 0.007 0.006 0.008 0.028 0.033 0.253 12/09/94 neg 0.004 0.008 0.006 0.005 0.005 0.034 0.038 0.197 09/09/96 neg 0.004 0.008 0.007 0.006 0.005 0.008 0.013 0.08 Patient 17 24/04/97 pos 1b 0.076 0.006 0.008 0.004 0.009 0.203 0.327 1.196 Patient 18 08/01/97 neg 0.006 0.007 0.007 0.007 0.006 0.006 0.008 0.009 Blank 0.006 0.009 0.009 0.006 0.006 0.007 0.006 0.009

[0130]

7TABLE 6 Sample# Blank E1 V1V2 20188 68 74 20189 77 73 20251 170 150 20252 490 1319 20253 92 70 20254 50 55 20255 81 88 20256 56 62 20266 119 134 20271 77 78 20272 61 69 21010 129 135 21011 159 161 21012 120 93 21286 108 105

[0131]

Sequence CWU 1

1

41 1 35 PRT Hepatitis C virus 1 Tyr Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr 20 25 30 Pro Gly Cys 35 2 35 PRT Hepatitis C virus 2 Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr 20 25 30 Pro Gly Cys 35 3 35 PRT Hepatitis C virus 3 Val Glu Val Lys Asn Asn Ser Asn Ser Tyr Met Ala Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Ile Trp Gln Leu Glu Gly Ala Val Leu His Thr 20 25 30 Pro Gly Cys 35 4 35 PRT Hepatitis C virus 4 Val Glu Val Lys Asn Thr Ser Thr Ser Tyr Met Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Trp Gln Leu Glu Gly Ala Val Leu His Thr 20 25 30 Pro Gly Cys 35 5 35 PRT Hepatitis C virus 5 Leu Glu Trp Arg Asn Thr Ser Gly Leu Tyr Val Leu Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Tyr Glu Ala Asp Asp Val Ile Leu His Thr 20 25 30 Pro Gly Cys 35 6 24 PRT Hepatitis C virus 6 Leu Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Asp Asp 1 5 10 15 Val Ile Leu His Thr Pro Gly Cys 20 7 35 PRT Hepatitis C virus 7 Ile Asn Tyr Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala Asp His His Ile Leu His Leu 20 25 30 Pro Gly Cys 35 8 35 PRT Hepatitis C virus 8 Val Pro Tyr Arg Asn Ala Ser Gly Ile Tyr His Ile Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala Asp Asn Leu Ile Leu His Ala 20 25 30 Pro Gly Cys 35 9 35 PRT Hepatitis C virus 9 Leu Thr Tyr Gly Asn Ser Ser Gly Leu Tyr His Leu Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Leu Glu Ala Asp Ala Met Ile Leu His Leu 20 25 30 Pro Gly Cys 35 10 29 PRT Hepatitis C virus 10 Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val 1 5 10 15 Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 20 25 11 34 PRT Hepatitis C virus 11 Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 1 5 10 15 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His 20 25 30 Val Asp 12 36 PRT Hepatitis C virus 12 Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr 1 5 10 15 Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg 20 25 30 Arg His Val Asp 35 13 30 PRT Hepatitis C virus 13 His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr 1 5 10 15 Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu 20 25 30 14 40 PRT Hepatitis C virus 14 Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp 1 5 10 15 Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala 20 25 30 Trp Asp Met Met Met Asn Trp Ser 35 40 15 34 PRT Hepatitis C virus 15 Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met 1 5 10 15 Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu 20 25 30 Arg Ile 16 41 PRT Hepatitis C virus 16 Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu 1 5 10 15 Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu 20 25 30 Val Val Met Leu Leu Phe Ala Gly Val 35 40 17 32 PRT Hepatitis C virus 17 His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asn Thr Arg Gly Leu 1 5 10 15 Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn 20 25 30 18 35 PRT Hepatitis C virus 18 Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn 1 5 10 15 Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys 20 25 30 His Lys Phe 35 19 38 PRT Hepatitis C virus 19 Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His 1 5 10 15 Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser 20 25 30 Ile Asp Lys Phe Ala Gln 35 20 28 PRT Hepatitis C virus 20 Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr 1 5 10 15 Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp 20 25 21 34 PRT Hepatitis C virus 21 Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 1 5 10 15 Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 20 25 30 Ser Pro 22 31 PRT Hepatitis C virus 22 Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val 1 5 10 15 Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly 20 25 30 23 44 PRT Hepatitis C virus 23 Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile 1 5 10 15 Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp 20 25 30 Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly 35 40 24 36 PRT Hepatitis C virus 24 Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg 1 5 10 15 Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys 20 25 30 Thr Cys Gly Gly 35 25 25 PRT Hepatitis C virus 25 Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly 1 5 10 15 Thr Gly Phe Thr Lys Thr Cys Gly Gly 20 25 26 30 PRT Hepatitis C virus 26 Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn 1 5 10 15 Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro 20 25 30 27 45 PRT Hepatitis C virus 27 Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly 1 5 10 15 Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg 20 25 30 Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe 35 40 45 28 33 PRT Hepatitis C virus 28 Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His 1 5 10 15 Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile 20 25 30 Phe 29 25 PRT Hepatitis C virus 29 Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr 1 5 10 15 Pro Cys Thr Val Asn Phe Thr Ile Phe 20 25 30 28 PRT Hepatitis C virus 30 Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val 1 5 10 15 Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg 20 25 31 33 PRT Hepatitis C virus 31 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp 1 5 10 15 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp 20 25 30 Gln 32 36 PRT Hepatitis C virus 32 Gln Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser 1 5 10 15 Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu 20 25 30 Tyr Gly Val Gly 35 33 42 PRT Hepatitis C virus 33 Gly Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp Glu Tyr Val 1 5 10 15 Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu 20 25 30 Trp Met Met Leu Leu Ile Ala Gln Ala Glu 35 40 34 34 PRT Hepatitis C virus 34 Asn Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys 1 5 10 15 Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala 20 25 30 Leu Asn 35 31 PRT Hepatitis C virus 35 Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln 1 5 10 15 Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly 20 25 30 36 20 PRT Hepatitis C virus 36 Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala 1 5 10 15 Asn Asp Ser Asp 20 37 40 PRT Hepatitis C virus 37 Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr 1 5 10 15 Thr Thr Ile Arg Arg His Val Asp Ser Gln Leu Phe Thr Ile Ser Pro 20 25 30 Arg Arg His Glu Thr Val Gln Asp 35 40 38 36 PRT Hepatitis C virus 38 Thr His Ala Cys Arg Ala Asn Gly Gln Tyr Phe Leu Thr Asn Cys Cys 1 5 10 15 Ala Pro Glu Asp Ile Gly Phe Cys Leu Glu Gly Gly Cys Leu Val Ala 20 25 30 Leu Gly Gly Lys 35 39 16 PRT Hepatitis C virus 39 Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp 1 5 10 15 40 35 PRT Hepatitis C virus 40 Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn 1 5 10 15 Cys Asn Asp Ser Leu His Thr Gly Phe Leu Ala Ser Leu Phe Tyr Thr 20 25 30 His Ser Phe 35 41 30 PRT Hepatitis C virus 41 Arg Ser Ile Glu Ala Phe Arg Val Gly Trp Gly Ala Leu Gln Tyr Glu 1 5 10 15 Asp Asn Val Thr Asn Pro Glu Asp Met Arg Pro Tyr Cys Trp 20 25 30

Claims

1. A peptide of more than 20 contiguous amino acids derived from the envelope region of HCV-related viruses which binds and recognizes anti-HCV-related virus antibodies.

2. A peptide which binds and recognizes an anti-HCV antibody or an anti-HGV antibody present in a sample of body fluid and which is chosen from the group consisting of the sequences as represented in SEQ D NOs 1 to 38.

3. A functionally equivalent variant or fragment of a peptide according to claim 2.

4. A peptide according to claims 2 or 3, wherein said anti-HCV antibody present in a sample of body fluid is an anti-HCV-E1 or anti-HCV-E2 antibody.

5. A peptide according to of claims 2 or 3, wherein said anti-HGV antibody present in a sample of body fluid is an anti-HGV-E1 or anti-HGV-E2 antibody.

6. A peptide according to any of claims 1 to 5, wherein said peptide is synthesized chemically.

7. A peptide according to any of claims 1 to 5, wherein said peptide is synthesized using recombinant DNA techniques.

8. A peptide according to any of claims 1 to 7, wherein said peptide is biotinylated or contains cysteine bridges.

9. A combination of peptides according to any of claims 1 to 8.

10. A method for diagnosing exposure to or infection by HCV-related viruses comprising: contacting anti-HCV-related virus antibodies within a sample of body fluid with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, determining the binding of anti-HCV-related virus antibodies within a sample of body fluid with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9.

11. An assay kit for detecting the presence of anti-HCV-related virus antibodies within a sample of body fluid comprising: possibly a solid support, a peptide according to any of claims 1 to 8 or a combination of peptides according to claim 9, appropriate markers which allow to determine the complexes formed between anti-HCV-related virus antibodies within a sample of body fluid with a peptide according to any of claims 1 to 8 or a combination of peptides according to claim 9.

12. A bioassay for identifying compounds which modulate the interaction between a peptide and an anti-HCV-related virus antibody, said bioassay comprising: contacting anti-HCV-related virus antibodies with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, determining the binding of anti-HCV-related virus antibodies with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, adding a modulator or a combination of modulators to the contacted anti-HCV-related virus antibodies and a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, determining the modulation of binding of anti-HCV-related virus antibodies with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9.

13. A bioassay for identifying compounds which modulate the interaction between a peptide and an anti-HCV-related virus antibody, said bioassay comprising: determining the binding of anti-HCV-related virus antibodies with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, contacting a modulator with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, adding anti-HCV-related virus antibodies to the contacted modulator with the peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9, determining the modulation of binding between anti-HCV-related virus antibodies with a peptide according to any of claims 1 to 8 or with a combination of peptides according to claim 9.

14. A method for producing a modulator as defined by claims 12 or 13.

15. A modulator for the interaction between a peptide and an anti-HCV-related virus antibody, wherein said modulator was identified by the method according to claims 12 or 13.

16. A composition containing a modulator or a combination of modulators wherein said modulator or combination of modulators was identified by the method according to claims 12 or 13.

17. A composition comprising a peptide according to any of claims 1 to 8 or a combination of peptides according to claim 9.

18. A plasmid vector comprising a nucleotide sequence encoding a polypeptide according to any of claims 1 to 5 or a modulator according to any of claims 12 to 16, operably linked to transcription regulatory elements.

19. A composition according to any of claims 16 to 18 for vaccinating humans against infection with HCV-related virus or any mutated strain thereof.

20. A composition according to any of claims 16 to 18 for therapeutically treating humans against infection with HCV-related virus or any mutated strain thereof.

21. An antibody, more particularly a monoclonal antibody, characterized in that it specifically recognizes an HCV-related virus peptide according to any of claims 1 to 9.

22. A method to immunize humans against infection with HCV-related virus or any mutated stain thereof, comprising the use of a peptide according to any of claims 1 to 8 or a combination of peptides according to claim 9.