WO2002037115A1 - Detection of infectious agents using antigen mimics - Google Patents

Abstract

A method for making a diagnosis of an antigen, comprising identifying the binding specificity of the anti-antigen antibody molecules in the serum by the Antibody Detection by Antigen Mimics (ADAM) methodology, comprising screening phage libraries using sera from antigen-infected patients and non-infected individuals, identifying peptides binding antibodies (ligands) specifically associated with said antigen. Improvements of the method are given by in vitro maturation strategies; linking the ligands to a common core, such as MAP. In particular the method applies to HCV.

Description

DETECTION OF INFECTIOUS AGENTS USING ANTIGEN MIMICS

The present invention relates to a diagnostic assay for detecting infectious agents, in particular viral agents, more in particular human Hepatitis C Virus (hereinafter briefly named HCV), anti-infectious agent antibody binding peptides useful in said assay, to processes for preparing said peptides and to kits for performing said assay. The present invention relates in particular to HCV, anti- HCV antibody binding peptides useful in diagnostic assay, processes for the preparation thereof and to kits for performing said assays. Background of the invention

Hepatitis C virus (HCV) is the major etiological agent of parenterally transmitted, non-A, non-B hepatitis. This virus very often causes persistent infection and frequently leads to the development of chronic hepatitis and liver cirrhosis, constituting a major worldwide cause of chronic liver disease (Boyer N, Marcellin P, Pathogenesis, diagnosis and management of hepatitis C, J Hepatol 2000; 32:98-112).

Viral infection is diagnosed by detecting anti-HCV antibodies in the serum or by revealing viral RNA through nucleic acid amplification methods (Robbins DJ, Pasupuleti V, Cuan J, Chiang CS, Reverse transcriptase PCR quantitation of hepatitis C virus, Clin Lab Sci 2000 Winter; 13:23-30). These latter methods are very sensitive, but are expensive and prone to technical or laboratory error. In addition, reliability and specificity of the PCR technique are not standardized (Gretch DR, Diagnostic tests for hepatitis C, Hepatology 1997 Sep;26(3 Suppl 1):43S-47S)

The present invention relates to diagnosis of infectious diseases, such as viral infections, in particular to HCV infections, by means of detecting antibodies raised against the infectious agent, in particular anti-HCV antibodies.

Very recently, a system for the sensitive detection of HCV core protein has been reported (Komatsu F, Takasaki K, Liver 1999 Oct; 19(5):375-8O). An impressive number of patent and non patent literature is dedicated to diagnostic methods and kits therefore for the detection of HCV.

US 5,985,542, assigned to Sumitomo Chemical Company, provides a kit for liver diseases, such as hepatitis C and alcoholic cirrhσsis7 which contains an antibody capable of recognizing cytochrome P450.

US 5,972,347, assigned to Baxter Aktiengeselleschaft, provides a composition for the treatment of HCV infection comprising inactivated HCV bound to neutralizing human HCV antibodies, wherein the antibodies are against at least one protein selected from the group consisting of HCV-core protein and NS3-protein.

US 5,939,262, US 5,919,625, and US 5,677, 124, assigned to Ambion and Cenetron, provide a very wide method for determining the presence of a tested nucleic acid sequence in a sample, wherein the obtainment of a (ribo)nuclease resistant (ribo)nucleic acid segment is involved.

US 5,866, 139, assigned to Institut Pasteur, provides a kit for determining the presence of HCV E-l specific antibodies. See also US 5,854,001, to Abbott.

US 5,800,982, assigned to Tonen, discloses antigenic peptides capable of reacting specifically with antibodies directed against Group II of HCV. JP 10019897, to Tonen, provides a kit containing a peptide having amino acid sequence of at least five continuous amino acids constituting protein of a non-structure region 4A (NS4A) of HCV and peptide having amino acid sequence of at least five continuous amino acids constituting protein of a non- structure region 4B (NS4B) of HCV.

For a picture of the state of the art see also US 5,871,904, US 5,869,253, US 5,750,331, US 5,747,241, US 5,667,992, US 5,645,983, US 5,625,034, US 5,610,009, WO 9707400, WO 9637783, EP 593291, EP 593290, EP 586065, JP 4349885.

Detection of anti-HCV antibodies in the serum, remains by far the most widely used test to assess HCV infection. Since 1987, when the HCV genome was cloned, various improved versions of anti-HCV diagnostic assays have been developed. Currently available diagnostic kits reveal serum antibodies against a mixture of four recombinant antigens corresponding to part of the HCV core, nonstructural 3, nonstructural 4, and nonstructural 5 regions of the HCV polypeptide (Gretch DR, Diagnostic tests for hepatitis C, Hepatology 1997 Sep;26(3 Suppl 1):43S-47S). Samples that are scored positive by this screening ELISA then must be confirmed by an immunoblot assay where the presence of antibodies to the same four antigens is separately detected. A positive diagnosis requires the detection of antibodies against at least two recombinant antigens.

For a relevant part of the population with anti-HCV antibodies, reactivity towards only one antigen is revealed, thus making a conclusive diagnosis impossible to formulate. An additional problem derives from the use of recombinant antigens, which can be recognized by HCV-unrelated antibodies and generate a false positive diagnosis. Supplementary investigation and long term monitoring of the patient are then required to reach a conclusive diagnosis.

The contact of a foreign antigen with an organism activates a specific immune response. The image of the antigen that is detected by the humoral response is defined by the epitopes recognized by the host antibodies. Assuming that the antibody binding site is the negative image of the epitope, a molecule that specifically binds the paratope should represent a positive image of the epitope. This line of thought suggests that, as far as humoral response is concerned, an antigen could be faithfully described by specific ligands that bind to the antigen- specific antibodies. Identifying these ligands would offer a way to detect the specific humoral response against an antigen, independently from whether or not that same antigen is known and/ or available.

These concepts were applied for developing a diagnostic assay for the detection of antibodies associated with infection, comprising HCV infection, in humans. Screening phage libraries with HCV- positive sera identified peptide ligands that specifically bind anti- HCV antibodies. This was achieved by adopting ad hoc selection strategies, since sera from infected patients contained virus- specific antibodies interspersed among a very large population of other antibodies with different binding specificities.

Screening peptides with a large number of negative sera eliminated those ligands which detected antibodies in negative samples (false positive), leading to the selection of a set of highly specific peptides. The incidence of unspecific reactivities was also reduced by the use of short peptide sequences and outside the context of the natural antigen. This technique is disclosed in EP 0 698 091 Bl, published on 28.02.1996. See also J. Mol. Biol. (1991), 222, 301-310; Gene, 128 (1993), 51-57; Gene, 148 (1994), 7-13; The EMBO Journal, vol.13, no.9, 2236-2243, 1994; Bacterial Protein Toxins, Zb. Bakt.Suppl 24, 415-425 (1994); The Journal of Immunology (1996) 4504-4513; Methods in Molecular Biology, vol.87, Humana Press Inc. pp. 195-208; Combinatorial Libraries (R.Cortese ed.) Walter de Gruyter 1996, chapter 8; Methods in Enzymology, (1996) vol. 267, .116-129; Biol. Chem. Vol. 378, 495- 502, June 1997; The EMBO Journal Vol. 17, No. 13, 3521-3533, 1998; Nature Biotechnology Volume 16, November 1998, 1068-1073 for a review on the mimotope and phagotope technology disclosed in the above EP 0 698 091 and the methods for carrying out the same. These selection strategies identified those peptide structures which best bind immunodominant anti-HCV antibodies. In addition, the multivalent display of the ligand contributed a remarkable avidity effect to detection sensitivity. In spite of these considerations, testing the ADAM-HCV (ADAM = Antibody Detection by Antigen Mimics) mix on a panel of sera that has not been used for screening, resulted in a sensitivity lower than 100%, though quite high. The particular feature of anti-HCV humoral response can explain this result, as it does not involve a major immunodominant epitope but is rather directed towards a number of different viral determinants. The existence of different viral genotypes further complicates the issue.

Currently available diagnostic kits reveal serum antibodies against four recombinant antigens corresponding to large regions of the HCV polypeptide (Gretch DR, Diagnostic tests for hepatitis C, Hepatology 1997 Sep;26(3 Suppl 1):43S-47S). A positive diagnosis requires the detection of serum antibodies against at least two recombinant antigens: for this reason, it is impossible to formulate a conclusive diagnosis for a relevant part of the population with anti- HCV antibodies, since a unique reactivity with only one antigen can be revealed. ADAM-HCV EIA (Enzymatic Immuno Assay) employs mimics of several different immunodominant epitopes from various antigens, thus ensuring a greater power of resolution in the analysis. As an immediate result, the frequency .of indeterminate samples can be drastically reduced.

We devoted significant effort to the technical development of the ADAM-HCV assay (using a strategy basically disclosed in the above mentioned EP 0 698 091). Peptides selected from phage libraries are displayed as N-terminal fusions to the major capsid protein pVIII. Using the phage as a diagnostic reagent has many advantages: it is an extremely flexible reagent, which lends itself to different types of immuno assay (Dente et al., 1994, F.Felici, G. Galfre, A. Luzzago, P. Monaci, A. Nicosia and R. Cortese "Phage- displayed peptides as tools for the characterization of human sera" Methods in Enzymology 267, 116-129 - 1996, Bartoli et al. Nature Biotechnology Volume 16, November 1998, 1068-1073), and low- scale production is easy and cheap.

In spite of these advantages, the size of the phage particle limits the concentration of peptide molecules that can be achieved in the assay. Interference of serum antibodies against the phage capsid requires the addition of carrier phage in the assay mixture to sequester anti-phage antibodies. Finally, the large-scale production of this, as any, biological reagent faces problems of microbiological contamination, reproducibility, quality control, purification and production costs.

Simple, linear synthetic peptides deriving from the peptide sequences displayed on phage in most of the cases did not retain either sensitivity or specificity to achieve an effective antibody detection.

Abstract of the invention

It has now been found, and it is an object of the present invention, a method for making a diagnosis of infectious diseases, such as viral infections, in particular to hepatitis C, comprising identifying the binding specificity of the anti-antigen antibody molecules in the serum by the Antibody Detection by Antigen Mimics (ADAM) methodology, comprising screening phage libraries using sera from antigen-infected patients and non-infected individuals, identifying peptides binding antibodies (ligands) specifically associated with said antigen.

In a particularly preferred embodiment, the present invention relates to HCV infection.

Detailed description of the invention In a first preferred embodiment, in the method according to the present invention, said ligands are improved by in vitro maturation strategies.

In a second preferred embodiment, in the method according to the present invention, said ligands are synthetic peptides. In a third preferred embodiment, said ligands are linked to a common core. In a particularly preferred embodiment, said ligands, together with said common core is MAP, according to the definition given hereinafter. It is also another object of the present invention a collection of

HCV-specific ligands obtainable by the process comprising: a) first panning a phage library on n positive sera to generate a first series of n phage pools; b) preparing n pool mixtures containing n-1 pools; c) selection affinity of each of n mixtures against the serum that generated the excluded pool of phage, to give a second series of n phage pools, and optionally d) additional panning each of the second series of n phage pools on a mix composed of all the n original sera, except that used for the first panning; e) immunoscreening of the resulting second series of n phage pools using a mixture of all the n original sera to give positive clones; f) testing the individual reactivity of all the positive clones with a panel of positive and negative sera by using an ordered array of said clones as phage- secreting colonies; g) generating replicas of said phage-secreting colonies; h) screening each replica for their reactivity with positive and negative sera, revealing clones specifically reacting with positive sera; i) using each of said specifically reacting phage as ligate to affinity purify antibodies from a positive serum; j) testing said antibodies for their reactivity with previously identified HCV-peptides; k) singling out clones detecting serum antibodies. The single peptides coming out from the above process are also an object of the present invention.

In particular, in the preferred embodiment of the present invention applied to HCV, the following peptides are a further object of the present invention:

YSREQLNKLFGIDMT; YSREQLNKMFGIEIS; YSREQLSKLFGIEPM; - NSRWLSKAHGIEGM;

YSREQLNKLFGIEVM; YSREQLSKLFGIDTQ; KSREQLSKLHGVDTS; RSREQLSKLFGIDLT; - MWRTWLM THGIESW;

MLRTWLMKYQGIESW; YSRSWLMKAHGLELG; MMRSYLMKAHGIESL; MSRLWLMKAHGISSE; KHSEWLNKARGIESW; .

MSRTFLMKAHGIESW; . MSRTWLMKAHGIESW; ;

AEGEKKLRRSTNWGDPAK; - AEGEFKTRRQTNYQDPAK;

AEGEFKTLRNANRLDPAK;

AEGEFKTLRNSNRLDPAK;

AEGEFKKFPGSSTPKDPAKAAFDSL;

AEGEFPQDARFPGGGDPAKAAFDSL; - PQDARFPGGGDPAKAAFDSL;

AEGEFKGAGGAQTVDWALLVDPAK;

AEGEFMQKHFGGAQWIMGDPAK;

AEGEFLSLKGSGGGQLRALVDPAK;

AEGEFLSLKGSGGAQLRALVDPAK; - AEGEFYLLKRSSPPDPAKAAFDSL;

AEGEFPILVGPYLLPRRSREEAVDPAK;

AEGEFPILVGPYLLPRRSREEAVDPAKGK;

AEGEFRLGVRAPRKALDPAK;

AEGEFRLGVRALRKALDPAK; - AEGEFRLGVRALRKAPDPAK;

RLGVRALRKAPDPAK;

AEGEFTQPRGHSYQDPAK;

AEGEFLKERAEMSARKTLGADPAK;

AEGEFFYQIPRRMETKYGDPAK; AEGEFSREQLNKLFGIEGDPAK;

AEGEFNSREWLSKAHGIEGMDPAK;

AEGEFRSREQLSKLFGIDLTDPAK;

AEGEFYSREQLNKLFGIDMTDPAK; - AEGEFYSREQLNKMFGIETSDPAK;

AEGEFYSREQLNKLFGIEVMDPAK;

AEGEFKSREQLRKLHGFDTSDPAK;

AEGEFKMRNYLNKAFGIEGMDPAK;

AEGEFRSREQLSKLFGIELTDPAK; - AEGEFSRREYSNKAFGIETQDPAK;

AEGEFRRREYLNKAFGIEGGDPAK;

AEGEFSRREWLNKRFGIEYLDPAK;

AEGEFMSRTWLMKAHGIESWDPAK;

AEGEFYSPEWLNKARGIDRSDPAK; - AEGEFKSREQLSKLHGVDTSDPAK;

AEGEFYSREQLNKMFGIEISDPAK;

AEGEFYSRSWLMKAHGLELGDPAK;

AEGEFMMRSYLMKAHGIESLDPAK;

AEGEFMSRLWLMKAHGISSEDPAK; - AEGEFPQPQEVHVYREQLGLDPAKAAFDSL;

AEGEFGEVLYRGFDEVGGDPAKAAFDSL;

AGEPYVIERGMQDPAK;

AEGEFTTASPRHFLVPLDPAKAAFD SL;

AEGEFTTASPAHFLVPLDPAKAAFD SL; AEGEFTTASPSHFLVPLDPAKAAFDSL; AEGEFATAPPRHYSWDPAK; AEGEFATAPPAHYSWDPAK; AEGEFATAPPSHYSWDPAK; - AEGEFRFWKVPDYDPPAAGGDPAK;

AEGEFTESSVSSTLADLASKTFGSADPAK; AEGEFTLADLATMTFGSTDPAK; AEGEFGLADLATLTFGSPDPAK; For the collection of the present invention, a preferred phage library of step a) is pVIII- 12aa.

For the collection of the present invention, the inserts of the preferred clones singled out from step k), have the sequences

1. SREQLNKLFGIEG;

2. RATLSNEHGITIG; 3. DQRENWFKYHGFG;

4. EWRRYMSDIHGYG;

5. DSLRYMYVMPGFG.

In the collection of the present invention, preferably a phage library is generated in which the reacting clones, more preferably the best reacting clones, are partially mutagenized so that each amino acid of the clone sequence is independently substituted by any other amino acid.

In the collection of the present invention, preferably said clones have the preferred insert sequence: SREQLNKLFGIEG. In the collection of the present invention, in the phage library, the random sequence may be flanked by two cysteine residue. This aspect may apply in the general exploitation of the present invention and may not be limited to the case of HCV. It is an object of the present invention the use of the above collection for the preparation of a diagnostic assay for detecting infectious agents, such as viruses, in particular HCV in a subject suspected to be affected by said infectious agents, such as viruses, in particular HCV. It is an object of the present invention a kit for diagnostic purposes, comprising the above collection.

It is an object of the present invention the immunogenic peptides obtainable, either in the form of a collection or as single peptide, from, the process herein disclosed. Said peptides are useful as immunogenic, therefore are useful for the preparation of vaccines, in particular against HCV. Conveniently, the peptides are in the form of the kit above mentioned.

Advantageously, in contrast to antigen-based systems, the diagnostic assay we tested has an in-built upgrading capacity: ad hoc selections can be performed with those sera for which no response was detected. A similar methodology might be used to identify a peptide panel which discriminates among different HCV genotypes, thus replacing expensive and labour-intensive PCR methods with a cheaper and faster EIA. The present invention shall be described in further details also by means of examples and figures, wherein in the latter:

Figure 1 represents the characterization of clones derived from screening. The amino acid sequences of selected clones are reported in single letter code. Top and lower panels display sequences derived from the original or the secondary library, respectively. pVIII sequences flanking the foreign epitope are (NH2)AEGEF[foreign epitope] DPAK. Gray boxes indicate residues more frequently present at any given position in clones from original library. Residues contributing to the consensus sequence of the peptides from the secondary library are indicated in bold characters. Data reported are average values from two independent assays and refer to the difference between the absorbance (A = A_450nm - A_620nm) measured on the indicated phage and that measured on wt phage pC89 (Felici et al., 1991). * indicates sera not tested.

Figure 2 represents the identification of phage mimicking the same antigen determinant. Antibodies affinity purified by positive serum C65 by clones PAl, PA3, PA8, PA 12 or P18 (listed in the first column) were tested in ELISA for their reactivity against the same phage (listed in the first row) .

Data reported are average values from two independent assays and refer to the difference between the absorbance (A = A_450nm - A_620nm) measured on the indicated phage and that measured on wt phage ρC89 (Felici et al., 1991). Figure 3 represents the ELISA reactivity of pool derived from selection of library pVIIIA12. Phage pool derived from library panning library pVIIIA12 on positive sera C76 was tested in ELISA for its reactivity with positive sera C12, C13, C29, c40, C47, C65, C73, C74, C76, C83 and C85. White, grey and black bars indicate reactivity of wt phage, pVIIIA12 library and pool p76^π, respectively. The ELISA results are expressed as A = A405nm - A620nm- Data reported are average values from two independent assays.

Figure 4 represents A. Reactivity of ADAM-HCV mix with sera. Cut-off value (CO = 0.232) was calculated as CO = N + 5σ where N and σ are the average and the standard deviations, respectively, of the data obtained with negative sera. B. ADAM-HCV EIA on a panel of sera obtained from Italian Red Cross. Cut-off value (CO = 0.252) was calculated as CO = N + 5σ where N and σ are the average and the standard deviations, respectively, of the data obtained from five negative control sera. ELISA as described in Materials and Methods detected binding of peptides to antibodies present in human sera. Average values from two independent experiments were collected. Results are expressed as the ratio between the measure signal and the cut-off value (S/CO). Number of sera tested for each group of sera is indicated.

Figure 5 ADAM /HCV EIA on a panel of indeterminate sera. The left-most column indicates the name of the HCV peptides tested, grouped according to their binding specificity. The next four columns report the reactivities of the listed peptides with positive (c25 and rl5) and negative (r6 and rl3) control sera. Each additional column reports the reactivities of the listed peptides with indeterminate sera. Binding of antibodies to HCV peptides present in human sera was detected by ELISA as described in Material and Methods. Average values from two independent experiments have been determined. For each peptide, cut-off value (CO) was calculated as CO = N + 5σ where N and σ are the average and the standard deviation, respectively, of the data obtained from 31 negative control sera. Results are expressed as the ratio between the measure signal and the cut-off value (S/CO).

Figure 6 ADAM-HCV/ SIA on positive, negative and indeterminate sera. The following ADAM-HCV peptides were grouped according to their binding specificity and immobilized onto nylon membrane to obtain 10 bands: ml909.2 and ml913.2 (A); ml901.31, m3322.3, m3362.3 (B); ml977.1 (C); m3551.3 (D); m3566.3 (E); m858, mF78 and mHl (F); mA12.1, mA12.2 and mA12.12 (G); mB11.17 (H); mG21.2 (I); ml929A3.1, ml929C3.4 and ml 929.21 (J). An additional line containing purified human IgG was included as internal positive control. Binding of antibodies to HCV peptides present in human sera was detected as described in Material and Methods

In the most preferred embodiment of the present invention, multiple antigen peptides (MAP) in which the C-terminus of eight identical peptide sequences is linked to a common core ( Tarn, J.P.: Synthetic peptide vaccine design: synthesis and properties of a high- density multiple antigenic peptide system. Proc. Natl. Acad. Sci. 85 (1988) 5409-5413), were synthesised, providing reagents which maintain a level of specificity comparable to that of phage-borne peptides. The removal of the phage scaffold allows a higher peptide concentration of immobilized ligand, which results in a higher sensitivity.

Preferably, the panel of HCV- specific ligands obtainable according to the present invention, includes peptides mimicking HCV NS3, an immunodominant HCV antigen. In the case of the preferred embodiment, to our knowledge, this is the first time that a short peptide which mimics an immunodominant NS3 determinant is described. Pepscan analysis of the NS3 protein revealed longer sequences which rarely reacted with positive sera (Khudyakov, Y., et al. 1995. Virology, 206:666-672).

In contrast, other approaches (Santini C, Brennan D, Mennuni C, Hoess RH, Nicosia A, Cortese R, Luzzago A, Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda. J Mol Biol 1998 Sep 11;282(1): 125-35; Pereboeva J. Med. Virol 1998 Oct; 56 (2): 105-11) have isolated large protein domains which retain antigenic properties. The approach we have developed thus contain the intrinsic properties of not relying on the use of or information on the HCV antigens, and might even be applied to systems where the antigen is unknown, thus creating a process that leads to antigen discovery.

Phage displayed random peptide libraries (phage libraries) represent a powerful tool to identify ligands which are specifically recognized by anti-HCV serum antibodies. Phage-borne peptides have a broad mimicking potential, as they are able to mimic linear, conformational and even non proteinaceous epitopes (for a review see Felici F, Luzzago A, Monaci P, Nicosia A, Sollazzo M, Traboni C. Peptide and protein display on the surface of filamentous bacteriophage. Biotechnol Annu Rev. 1995; 1 : 149-83; Zwick MB, Shen J, Scott JK. Phage-displayed peptide libraries. Curr Opin Biotechnol.-.1998 Aug;9(4):427-36).

In the present invention, there is reported the identification of a wider collection of efficient HCV- specific ligands and the development of a novel type of diagnostic kit for the detection of anti- HCV antibodies in the serum.

The peptides provided in the present invention are useful as diagnostic or as immunogens or vaccines. Pharmaceutical compositions comprising immunogens and vaccines according to the present invention are prepared conventionally as normally understood by those having ordinary experience in the art. For example, they may be prepared as described in EP 0 698 091. The following example further illustrate the invention.

EXAMPLE

Identification of HCV-specific ligands with novel binding specificities Phage library pVIII-12aa was panned on 8 positive sera (C13,

C14, C27, C29, C40, C47, C62 and C65) to generate 8 phage pools

(indicated as pl3ⁱ, ρl4ⁱ, ρ27*, p29*, p40ⁱ, p47*, ρ62* and pόδ¹, respectively). Eight mixtures were prepared, each containing a different combination of seven out of these eight phage pools. Each mix was affinity selected using the serum that generated the excluded pool of phage: e.g., the mixture mixΔp65, composed of pools pl3^!, ρl4^∑, p27^!, p29^!, p40^:, p47^: -and p62^!, was panned on serum C65. Each of the resulting eight phage pools (indicated as pl3^π, pl4^π, etc.) were individually panned again on a mix composed of all the original sera except that used for the previous selection.

For example, pool pl3^π was panned on a mixture of sera C14, C27,

C29, C40, C47, C62 and C65.

Immuno screening the resulting eight phage pools using a mixture of all the eight original sera revealed a large number of positive clones. We tested the individual reactivity of all these clones with a panel of positive and negative sera by generating an ordered array of the clones as phage- secreting colonies. Following their growth, the entire set of clones was spotted as an ordered array on a nitrocellulose filter using a multipin device. This same procedure was repeated to generate many replicas, which were then individually and simultaneously screened for their reactivity with positive and negative sera, revealing many clones which specifically reacted with positive sera. Each of these phage was used as ligate to affinity purify antibodies from a positive serum. These antibodies were then tested in ELISA for their reactivity with any of the 4 groups of HCV-peptides previously identified (ref. Prezzi, C, Nuzzo, M., Meola, A., Delmastro, ^ΛR, Galfre', C, Cortese, R., Nicosia, A. and Monaci, P. 1996. 1. Immunology. 156: 4504-4513. , Bartoli et al. Nature Biotechnology Volume 16, November

1998, 1068- 1073). This analysis singled out 12 clones that detected serum antibodies with novel binding specificity, thus indicating they mimic antigenic determinants different from those already identified.

Sequence analysis revealed 5 different sequences. Culture supernatants from each of these 5 clones (PAl, PA3, PA8, PA12 and PA18) were prepared and their ELISA reactivity tested with 30 different positive and 24 negative sera (Figure 1). Only clones PA8 and PA 12 displayed statistically significant reactivity with positive sera (ρ<0.2). Phage PAl was then used to immunopurify antibodies from serum C65. These purified antibodies specifically reacted with clones PA3, PA8, PA 12, PA 18, as well as with clone PAl itself, indicating that all these phage can be grouped into a unique class recognized by antibodies of the same specificity (Figure 2). No reactivity with any of the above clones was detected when wild type was used as ligate, or when antibodies were affinity-purified from a negative serum.

When a different clone from the same group was used to immuno-purify the serum antibodies or when a different positive serum was used, we obtained results consistent with the reactivities reported in Figure 1. For example, when phage PA3 was used to immuno-purify antibodies from the same serum, these antibodies reacted with clones PA8 and PA 12, in addition to the same PA3. These results indicate that clones PAl, PA3, PA8, PA12 and PA18 detect antibodies reacting with the same B-cell epitope, as suggested by the weak similarity among the peptide sequences (Figure 1).

Affinity maturation of HCV peptide

A phage library was generated in which the sequence of clone PA12 was partially mutagenized. In this "secondary" library, named pVIIIA12, oligonucleotides were synthesized so that each amino acid of the SREQLNKLFGIEG sequence was independently substituted by any other amino acid: in theory, a substitution at each position would occur at a frequency of 20 percent. In addition, a random residue was included at both sites of the foreign peptide sequence. The pVHIA12 library was panned twice on 12 positive sera (C8, CIO, C12, C13, C22, C58, C60, C76, C83, C85, C141 arid C177).

When tested in ELISA, phage pools ρ76^π, pl41 and ρl77^π (derived from selection using sera C76, C141 and C177, respectively) showed the highest and broadest reactivity with a panel of positive sera and were further analyzed (Figure 3). On the basis of this reactivity, phage pools p76^π, p l41^π and pl77^π were immunoscreened by using sera C40, C141 and C177, respectively. For each pool, this analysis picked out several clones that were then individually tested for their reactivity with many different positive and negative sera by a filter-replica protocol, as detailed above. This final screening' singled out 51 clones specifically reacting with positive sera. Sequence analysis revealed 16 different sequences (8, 5 and 3 derived from pool p76^π, pl41^π and pl77^π, respectively). Culture supernatants from each of these clones were prepared and their ELISA reactivity tested with 33 different positive and 24 negative sera (Figure 1). Clones P40.17 and P40.7 reacted with the more positive sera tested (42% each). Their combination with clones PI 41.7 and P177.22 scored 70% of the positive sera of the panel. Alignment of the selected peptides defines a consensus sequence (M/Y)SRE(W/Q)L(M/N)K(A/L)(H/F)GIES(W/M). Identification of additional HCV-specific ligands

Similar selection strategies were carried out aiming at the identification of ligands with novel binding specificities different from the ones previously identified. Phage libraries of various lengths were screened in which the random sequence was either completely random or flanked by two cysteine residues that constrain the conformation of the displayed peptide.

Various combinations of sera and phage pools were employed to select phage libraries. Phage pools exhibiting interesting reactivity profiles with positive sera were further analyzed. Assessing the reactivity of a large number of individual clones by replica screening singled out phage displaying a specific reactivity with positive sera.

We focused our attention on clones with interesting reactivity patterns. Probing these clones with antibodies affinity purified from HCV peptides eliminated those clones which mimicked antigenic determinants of the previously identified peptides. Clones that survived these selection steps were tested in ELISA with a large number of positive and negative sera to statistically define their HCV specificity.

These selected clones were further improved by creating and screening secondary libraries in which the sequence of the original clone or a population of clones was partially mutagenized and screened again to pick out variants with improved binding properties. This step was carried out adopting different strategies that have been already described (ref. paper Urbanelli, Zhu). This extensive and iterative effort identified 7 novel groups of ligands which specifically bind HCV-specific serum antibodies with different binding specificities. Screening a phage- displayed repertoire of HVR1 variants using sera isolated peptides specifically reacting with a large number of positive sera (Puntoriero et al.; Nicosia unpublished). A set of HVR1 phage-borne peptides derived from this screening was analyzed for their reactivity with our panel of sera. Three peptides were identified with the highest and specific frequency of reactivity with positive sera (mF78, mHl and m858).

In summai , 12 groups of ligands were identified, including the 4 groups previously identified (Bartoli et al. Nature Biotechnology Volume 16, November 1998, 1068- 1073, Prezzi, C, Nuzzo, M., Meola, A., Delmastro, ^ΛR, Galfre', C, Cortese, R., Nicosia, A. and Monaci, P. 1996. 1. Immunology. 156: 4504-4513.); see figure 5, groups A to L.

From phage-borne to synthetic peptide Twenty- two peptide sequences derived from the 12 groups of

HCV-ligands were synthesized as octa-branching multiple antigen peptides (ADAM-HCV peptides). In this molecule, eight identical peptide sequences are linked through a lysine fork to a common core to form a multiple display similar to that of pVIII-fused peptides on the phage capsid. Sequences of ADAM-HCV peptides are reported hereinbelow, m858 ETYTTGGAAARTTSGLTSLFSPGPSQN ml901.31 AEGEFKKFPGSSTPKDPAKAAFDSL ml901.34 AEGEFPEDTFPGSKLILSGDPAKAAFDSL ml 909.2 AEGEFKTRRNTNYQDPAK m 1913.2 AEGEFKTLRNTNRLDPAK m 1929.21 AEGEFATASPTHYTSELDPAK m 1929A3.1 AEGEFTTASPTHFLVPLDPAK ml929C3.4 AEGEFATAPPSHYSWDPAK m 1977.1 AEGEFPYLLPRRSREEAVDPAK m3322.3 AEGEFPQDARFPGGGDPAK m3362.3 AEGEFLSLKGSGGGQLRALVDPAK m3551.3 AEGEFRLGVRALRKAPDPAK m3566.3 AEGEFKTSVRSVPRARPPINGDPAK mA12.1 AEGEFNSREWLSKAHGIEGMDPAK mA12.2 AEGEFRSREQLSKLFGIDLTDPAK mAl 2.13 AEGEFMSRTWLMKAHGIESWDPAK mB 11.17 AEGEFRELLYEAFDDMEGDPAK mF78 QTHTTGGQAGHQAHSLTGLFSPGAKQN mG21.2 AGEPYVIEQGMMDPAK mHl QTHTTGGWGHATSGLTSLFSPGPSQN mN15.3 AEGEFGLADLATLTFGSTDPAK mS48.5 AEGEFRFWKVPDYDPPAAGGDPAK while data on their analytical performance are summarized in

Table 1, herein appended with the Figures.

In many cases, pVIII sequences flanking the foreign epitope (NH2AEGEF and/ or DPAK-COOH) were shown to be relevant for the binding specificity of the corresponding peptide. A mixture containing these 22 ADAM-HCV peptides (ADAM- HCV mix) was used to detect the presence of anti-HCV antibodies by an EIA (ADAM/ HCV EIA). The ADAM-HCV mix of peptides was immobilized by passive coating at the bottom of a multi-well ELISA plate and incubated with 1 :40 diluted sera samples for 40 min. Human antibodies bound to the peptides were detected by incubating for 20 min with anti-human conjugate and measured through a chromogenic enzymatic reaction.

As reported in Figure 4A, ADAM-HCV EIA efficiently discriminates between positive and negative sera.

ADAM-HCV /EIA

A panel of HCV-positive and HCV-negative sera was tested for the presence of anti-HCV antibodies by ADAM/HCV-EIA (Figure 4B)The test identified all positive sera included in thepanel and also demonstrated 100% specificity by identifying all negative samples.

Indeterminate samples

A collection of sera diagnosed as indeterminate according to a commercially available HCV-confirmatory assay was obtained from various sources. The 23 ADAM-HCV peptides were individually tested in ELISA for their reactivity with 31 samples (Figure 5). 6 samples did not react with any of the tested MAP and were thus scored as negative. 8 samples recognized only one antigen, thus confirming the indeterminate analysis. Finally, 17 samples showed two or more reactions against different groups of peptides, thus being identified as positive.

ADAM-HCV strip immunoblot assay (ADAM-HCV /SIA). ADAM-HCV peptides were covalently immobilized onto an activated nylon membrane to obtain a strip with ten bands. Each line included different peptides with the same binding specificity, as detailed in the legend of Figure 6. A control line containing purified human IgG was also included as internal positive control. A selected number of samples from the panel of indeterminate sera were tested for their reactivity with immobilized antigens by incubating serum samples with the strip. Anti-HCV antibodies captured by individual antigens were visualized by incubating the strips with anti-human enzyme-conjugate followed by a colorimetric enzymatic reaction. The reactivity of specimens with peptide bands was determined by visually comparing the intensity of each band with that of the internal positive control.

ADAM-HCV/ SIA revealed the reactivity of all the 8 positive sera tested to several different viral determinants mimicked by ADAM- HCV peptides. No reactivity was detected when 8 negative sera were tested (Figure 6). We also analyzed by ADAM- SIA a selected number of indeterminate samples. As shown in Figure 6, analysis of 8 indeterminated sera confirmed the results obtained by ADAM- HCV/ EIA using individual peptides, indicating a comparable sensitivity of the two assays. Materials and methods

Phage libraries

Four different phage displayed random peptide libraries were used as a source of ligands: pVIII9aa, pVIII9aa_cys, pVIII12aa, pVIII15aa and pVIIIA12. pVIII9aa (Felici et al., 1991), pVIII12aa and pVIII15aa are three different libraries composed of random 9-mers,

12-mers and 15-mers, respectively, which are displayed on filamentous phage as fusion to the NH2-terminus of the major coat protein pVIII. pVIII9aa_cys is a library in which the random nonapeptide is flanked by two cysteine residues (Luzzago et al., 1993). In this latter library, cysteines promote the formation of a disulfide bridge that constrains to some extent the conformation of the displayed peptide. The pVIIIA12 library was constructed by synthesizing an oligonucleotide encoding the amino acid sequence SREQLNKLFGIEG. By adopting a resin-splitting synthesis methodology (Glaser et al., 1992), each amino acid position was substituted with a NNS triplet with a frequency of 20 percent. In addition, a random residue was included at both sites of the foreign peptide sequence. All five libraries have been generated as described (Folgori et al., 1998). Library complexity as derived from the number of individual clones obtained upon bacterial transformation was about lxl 0⁸ for each of the five libraries.

Human sera

Human sera used in this study were randomly collected from rejected units of donors' blood from transfusional centres and hospital departments and units from healthy volunteers. Namely, many of the indeterminate samples used in this study were obtained from the Laboratory of Virology, Istituto Superiore di Sanita, Roma (Italy) and from the Centro Nazionale Trasfusione Sangue della Croce Rossa Italiana, Roma (Italy). Sera have been tested for the presence of antibodies to HCV by the second generation HCV ELISA test system (Ortho Diagnostic Systems, Bersee, Belgium) and confirmed with a first generation dot blot immuno-assay RIBA HCV test system, (Chiron Co., Emeryville, CA). Sera were also tested for the absence of antibodies to HBsAg and to HIV-l/HIV-2 by AUSAB EIA test (Abbott Labs, Chicago, IL) and by the third generation HIV- l/HIV-2 EIA test (Abbott Labs, South Pasadena, CA). Samples positive for the presence of anti-HCV antibodies, but negative for anti-HBsAg and anti-HIV antibodies, were included in this study as HCV-positive sera. Sera negative for the presence of antibodies against all the three antigens were included in this study as HCV- negative sera.

Affinity selection and immuno screening HCV-positive sera were used to affinity select phage- displayed random peptide libraries as described (Folgori et al. 1998; Felici et al., 1996; Prezzi et al., 1996). Clones derived from the affinity selection were immuno-screened using sera as described (Prezzi et al. 1996, Minenkova et al). ELISA using phage clones

ELISA using phage supernatant and human sera was performed as follows. Phage supernatants were prepared from DH5α-F' infected cells as previously described (Felici et al., 1991). Multi-well plates (Immunoplate Maxisorp, Nunc, Roskilde, Denmark) were coated overnight at 4°C with 200μl of the anti-pill monoclonal antibody 57D1 (Dente et al., 1994) at a concentration of 1 μg of antibody/ml in 50 mM NaHC0₃ pH 9.6. After discarding coating solution, plates were incubated at 37°C for 60 min with ELISA blocking buffer (0.1% casein, 1% Triton-XlOO in PBS). Plates were washed several times with PBS/0.05% Tween-20 (washing buffer). A 1 : 1 mixture of ELISA blocking buffer and cleared phage supernatant was added to each well and allowed to bind for 1 hr at 37°C. A 1 :40 diluted human serum was incubated for 30 min at room temperature with 5x1010 plaque forming units (pfu)/ml of phage fl l . l (Dente et al., 1996), 25 μl/ml of a protein extract from DH5 - F' cells infected with phage fl l . l (dente et al.) and 25 μl/ml of supernatant from unrelated rat hybridoma cells in ELISA blocking buffer. After having discarded the mix containing phage supernatant, plates were washed with washing buffer and 200μl of the pre-incubated serum mixture were added to each well and incubated 60 min at 37°C. Plates were then washed with washing buffer, and a 1 :20.000 dilution of goat anti-human IgG HRP-conj. (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in secondary antibody blocking buffer (1% Triton, 1% horse serum, 50% fetal calf serum in PBS, Mab supernatant 5μl/well) was added to each well. After a 30-min incubation at 37°C, plates were washed and peroxidase activity was detected by incubation with 200μl of TMB liquid substrate system (SIGMA, St. Louis, MO). After 15 minutes of developing, reaction was stopped by adding 25μl 2M H2S04. The plates were read by an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and the

results were expressed as A = A450nm ~ -^620nm- ELISA data reported in display items are average values from two independent assays. They were considered statistically significant if they were greater than the 0.3 cut-off and differed more than 3σ (σ = {l/2[σ p+ σ 2 w]} 1 /2 ) from the background signal observed for the wt phage.

The p value referred to clones PA8 and PA 12 is the probability that the observed frequency distributions of reactivities of the positive and negative sera are statistically the same according to the χ test.

Affinity-purification of phagotope-specific antibodies from serum A 60 mm diameter polystyrene Petri dish (Becton Dickinson

Labware, NJ) was coated overnight at 4°C with a solution of lxl 0¹¹

CsCl-purified phage particles/ml in 50 mM NaHCθ3 pH 9.6. After washing with PBS/Tween, dish was incubated for 60 min at 37°C with ELISA blocking buffer. A mixture of human serum (dilution 1/ 100 in ELISA blocking buffer), 1x10 *2 fn . i _pf_u/_mi and 25 μl/ml of XLl-blue cell protein extract was incubated for 60 min at room temperature. After having discarded the blocking solution, pre- incubated mix was added to the plate and incubated overnight at 4°C. The serum dilution was discarded and the dish washed with washing buffer. Bound antibodies were eluted by 0.1 M glycine-HCl pH 2.7 added with 10 μg/ml BSA and neutralized.

Characterization of phage clones

Affinity-purified antibodies were tested in standard ELISA for their reactivity against phagotopes. Typically, multi-well plates were coated with 100 μl/well of a solution of 1x10 H TU/ml CsCl-purified phage in 50 mM NaHCθ3 pH 9.6, overnight at 4°C. After washing with PBS/Tween, plates were incubated for 60 min at 37°C with blocking buffer. Afterwards, 100 μl of affinity-purified antibodies were added to each well and allowed to bind overnight at 4°C.

Plates were then washed with cold PBS/Tween and 100 μl/well of goat anti-human IgG (Fc specific) alkaline-phosphatase conjugated antibodies (Sigma, St. Louis, MO), diluted 1/5000 in blocking buffer, were added. After incubation at room temperature for 2 hrs, plates were washed and alkaline phosphatase revealed as described above

Synthetic peptides

We have used synthetic octabranching multiple antigen peptides (MAPs): (Tarn, 199X). The synthesis was performed by the Flow-poly amide method (Pessi et al., 1990). Peptides were dissolved in dimethyl sulf oxide.

ELISA using synthetic peptides

Multi-well plates (Immuno plate Maxisorp, Nunc, Roskilde, Denmark) were coated overnight at 4°C with a MAP solution at a concentration of 10 μg /ml in 50 m^'M NaHCθ3 pH 9.6. After discarding coating solution, plates were incubated at 37°C for 60 min with ELISA blocking buffer (0.1% casein, 1% Triton-XlOO in PBS). Plates were washed several times with PBS/0.05% Tween-20 (washing buffer). A 1:40 diluted human serum was added to each well and incubated 40 min 37°C. Plates were then washed with washing buffer, and a 1 :20.000 dilution of goat anti-human IgG HRP-conj. (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in ELISA blocking buffer was added to each well. After a 20 min incubation at 37°C, plates were washed and peroxidase activity was detected by incubation with TMB liquid substrate ((SIGMA, St. Louis, MO). After 15 minutes of developing, reaction was stopped by adding 2M H2SO4. The plates were read by an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and the results were expressed as A = A450nm " A520nm- ELISA data reported in display items are average values from two independent assays.

References

Smith, C. P. and Petrenko, V.A. 1997. Phage display. Chem. Rev. 97:391-410 Folgori, A., Tafi, R., Meola, A., Felici, F., Galfre, G., Cortese, R., Monaci, P. and Nicosia, A. 1994. A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO 1. 13:2236-"43. Prezzi, C, Nuzzo, M., Meola, A., Delmastro, ^ΛR, Galfre¹, C,

Cortese, R., Nicosia, A. and Monaci, P. 1996. 1. Immunology. 156: 4504-4513.

Alter, H. j. 1995. To C or not to C: these are the questions'. Blood. 85: 1681. Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R. and

Cesareni, C. 1991. Selection of antibodies ligands from a large library of oligopeptides expressed on a multivalent exposition vector, f. Mol. Biol. 222:301-310.

Luzzago, A., _Felici, F., Tramontano, A., Pessi, A. and Cortese, R. 1993. Mimicking of discontinuous epitopes by phage displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Cene. 128:51-57.

Dente, L., Cesareni, G., Micheli, C, Felici, F., Folgori, A., Luzzago, A., Monaci, P., Nicosia, A. and Delmastro, P. 1994. Monoclonal antibodies that recognise filamentous phage. Useful tools for phage display technology. Gene. 148:7

Sambrook, J., Fritsch, T. and Maniatis, T. 1989. Molecular Cloning: a laboratory manual (second edition)

Takamizawa, A., Mori, C, Fuke, I., Manabe, S., Murakami, S., Fujita, J., Onoshi, E., Andoh, T., Yoshida, I. and Okayama, H. 1991. Structure and organisation of the Hepatitis C virus genome isolated from human carriers. I. Virol. 65: 1105- 1113.

Smith, D. B. 1993. Purification of glutathione S-Transf erase fusion proteins. Methods in molecular and cellular biology. 4:220- 229.

Frangioni, f.V. and Neel, B.J. 1993. Solubilization and purification of enzymatically active glutathione S-transferase (pGex) fusion proteins. Analyt. Biochem. 210: 179-187. Komatsu F, Takasaki K 1999

Determination of serum hepatitis C virus (HCV) core protein using a novel approach for quantitative evaluation of HCV viraemia in anti-HCV-positive patients. Liver 19:375-80

Claims

1. A method for making a diagnosis of an antigen, comprising identifying the binding specificity of the anti- antigen antibody molecules in the serum by the Antibody Detection by Antigen Mimics (ADAM) methodology, comprising screening phage libraries using sera from antigen-infected patients and non- infected individuals, identifying peptides binding antibodies (ligands) specifically associated with said antigen.

2. A method according to claim 1, wherein said ligands are improved by in vitro maturation strategies.

3. A method according to claim 1 or 2, wherein said ligands are synthetic peptides.

4. A method according to claim 1 or 2 or 3, wherein said ligands are linked to a common core.

5. A method according to claim 4, wherein said ligands together with said common core is MAP.

6. A collection of antigen-specific ligands obtainable by the process comprising: a) first panning a phage library on n positive sera to generate a first series of n phage pools; b) preparing n pool mixtures containing n-1 pools; c) selection affinity of each of n mixtures against the serum that generated the excluded pool of phage, to give a second series of n phage pools, and optionally d) additional panning each of the second series of n phage pools on a mix composed of all the n original sera, except that used for the first panning; e) immunoscreening the resulting second series of n phage pools using a mixture of all the n original sera to give positive clones; f) testing the individual reactivity of all the positive clones with a panel of positive and negative sera by using an ordered array of said clones as phage-secreting colonies; g) generating replicas of said phage-secreting colonies; h) screening each replica for their reactivity with positive and negative sera, revealing clones specifically reacting with positive sera; i) using each of said specifically reacting phage as ligate to affinity purify antibodies from a positive serum; j) testing said antibodies for their reactivity with previously identified antigen-specificpeptides; k) singling out clones detecting serum antibodies.

7. A method for making a diagnosis of hepatitis C comprising identifying the binding specificity of the anti-HCV antibody molecules in the serum by the Antibody Detection by Antigen

Mimics (ADAM) methodology, comprising screening phage libraries using sera from HCV patients and non-infected individuals, identifying peptides binding antibodies (ligands) specifically associated with HCV infection.

8. A method according to claim 7, wherein said ligands are improved by in vitro maturation strategies.

9. A method according to claim 7 or 8, wherein said ligands are synthetic peptides.

10. A method according to claim 6 or 7 or 8 wherein said ligands are linked to a common core.

11. A method according to claim 8, wherein said ligands together with said common core is a MAP.

12. A collection of HCV-specific ligands obtainable by the process comprising: a) first panning a phage library on n positive sera to generate a first series of n phage pools; b) preparing pool mixtures containing n-1 pools; c) selection affinity of each of n mixtures against the serum that generated the excluded pool of phage, to give a second series of n phage pools, and optionally d) additional panning each of the second series of n phage pools on a mix composed of all the n original sera, except that used for the first panning; e) immunoscreening the resulting second series of n phage pools using a mixture of all the n original sera to give positive clones; f) testing the individual reactivity of all the positive clones with a panel of positive and negative sera by using an ordered array of said clones as phage-secreting colonies; g) generating replicas of said phage-secreting colonies; h) screening each replica for their reactivity with positive and negative sera, revealing clones specifically reacting with positive sera; i) using each of said specifically reacting phage as ligate to affinity purify antibodies from a positive serum; j) testing said antibodies for their reactivity with previously identified HCV-peptides; k) singling out clones detecting serum antibodies.

13. The collection according to claim 12, wherein the phage library of step a) is pVIII- 12aa

14. The collection according to claim 12 and 13, wherein the inserts of the clones singled out from step k), in claim 12 have the following- sequences: SREQLNKLFGIEG; RATLSNEHGITIG, DQRENWFKYHGFG, EWRRYMSDIHGYG, DSLRYMYVMPGFG.

15. The collection according to claim 12, wherein a phage library is generated in which the best reacting clones are partially mutagenized so that each amino acid of the clone sequence is independently substituted by any other amino acid.

16. The collection according to claim 12, wherein a phage library is generated in which the reacting clones are partially mutagenized so that each amino acid of the clone sequence is independently substituted by any other amino acid.

17. The collection according to claim 15 or 16, wherein said clones have the following insert sequence SREQLNKLFGIEG.

18. The collection according to any one of claims 12-17, wherein in said phage library, the random sequence is flanked by two cysteine residues.

19. The collection according to any one of claims 12-18, wherein the peptide sequence is linked on a common core.

20. The collection according to claim 17, wherein said peptide together with said common core is MAP .

21. Use of the collection of any one of claims 12-20 for the preparation of a diagnostic assay for detecting HCV in a subject suspected to be affected by said HCV.

22. A kit for diagnostic purposes, comprising a collection of claim 6.

23. A kit for the diagnosis of HCV, comprising a collection of any one of claims 12-20.

24. A kit according to claim 22 or 23, comprising strips wherein said collection is immobilised thereto.

25. A kit according to claim 24, wherein said strip further contains an internal standard. 26. The peptide selected from the group consisting of: i) SREQLNKLFGIEG; ii) RATLSNEHGITIG; iii) DQRENWFKYHGFG; iv) EWRRYMSDIHGYG; v) DSLRYMYVMPGFG; vi) YSREQLNKLFGIDMT; vii) YSREQLNKMFGIEIS; viii) YSREQLSKLFGIEPM; ix) NSRWLSKAHGIEGM; x) YSREQLNKLFGIEVM; xi) YSREQLSKLFGIDTQ; xii) KSREQLSKLHGVDTS; xiii) RSREQLSKLFGIDLT; xiv) MWRTWLMKTHGIESW; xv) MLRTWLMKYQGIESW; xvi) YSRSWLMKAHGLELG; xvii) MMRSYLMKAHGIESL; xviii) MSRLWLMKAHGISSE; xix) KHSEWLNKARGIESW; xx) MSRTFLMKAHGIESW; xxi) MSRTWLMKAHGIESW; ; xxii) AEGEKKLRRSTNWGDPAK; xxiii) AEGEFKTRRQTNYQDPAK; xxiv) AEGEFKTLRNANRLDPAK; xxv) AEGEFKTLRNSNRLDPAK; xxvi) AEGEFKKFPGSSTPKDPAKAAFDSL; xxvii) AEGEFPQDARFPGGGDPAKAAFDSL; xxviii) PQDARFPGGGDPAKAAFDSL; xxix) AEGEFKGAGGAQTVDWALLVDPAK; xxx) AEGEFMQKHFGGAQWIMGDPAK; xxxi) AEGEFLSLKGSGGGQLRALVDPAK; xxxii) AEGEFLSLKGSGGAQLRALVDPAK; xxxiii) AEGEFYLLKRSSPPDPAKAAFDSL; xxxiv) AEGEFPILVGPYLLPRRSREEAVDPAK; xxxv) AEGEFPILVGPYLLPRRSREEAVDPAKGK; xxxvi) AEGEFRLGVRAPRKALDPAK; xxxvii) AEGEFRLGVRALRKALDPAK; xxxviii) AEGEFRLGVRALRKAPDPAK; xxxix) RLGVRALRKAPDPAK; xl) AEGEFTQPRGHSYQDPAK; xli) AEGEFLKERAEMSARKTLGADPAK; xlii) AEGEFFYQIPRRMETKYGDPAK; xliii) AEGEFSREQLNKLFGIEGDPAK; xliv) AEGEFNSREWLSKAHGIEGMDPAK; xlv) AEGEFRSREQLSKLFGIDLTDPAK; xlvi) AEGEFYSREQLNKLFGIDMTDPAK; xlvii) AEGEFYSREQLNKMFGIETSDPAK; xlviii) AEGEFYSREQLNKLFGIEVMDPAK; xlix) AEGEFKSREQLRKLHGFDTSDPAK;

1) AEGEFKMRNYLNKAFGIEGMDPAK; li) AEGEFRSREQLSKLFGIELTDPAK; lii) AEGEFSRREYSNKAFGIETQDPAK; liii) AEGEFRRREYLNKAFGIEGGDPAK; liv) AEGEFSRREWLNKRFGIEYLDPAK; lv) AEGEFMSRTWLMKAHGIESWDPAK; lvi) AEGEFYSPEWLNKARGIDRSDPAK; i) AEGEFKSREQLSKLHGVDTSDPAK; lviii) AEGEFYSREQLNKMFGIEISDPAK; lix) AEGEFYSRSWLMKAHGLELGDPAK; lx) AEGEFMMRSYLMKAHGIESLDPAK; lxi) AEGEFMSRLWLMKAHGISSEDPAK; lxii) AEGEFPQPQEVHVYREQLGLDPAKAAFDSL; lxiii) AEGEFGEVLYRGFDEVGGDPAKAAFDSL; lxiv) AGEPYVIERGMQDPAK; lxv) AEGEFTTASPRHFLVPLDPAKAAFDSL; lxvi) AEGEFTTASPAHFLVPLDPAKAAFDSL; lxvii) AEGEFTTASPSHFLVPLDPAKAAFDSL; lxviii) AEGEFATAPPRHYSWDPAK; lxix) AEGEFATAPPAHYSWDPAK; lxx) AEGEFATAPPSHYSWDPAK; lxxi) AEGEFRFWKVPDYDPPAAGGDPAK; lxxii) AEGEFTESSVSSTLADLASKTFGSADPAK;

Ixxiii) AEGEFTLADLATMTFGSTDPAK; lxxiv) AEGEFGLADLATLTFGSPDPAK; 27. The use of the peptides of claim 26 in the method of. claims 7- 11.

28. A collection according to any one of claims 12-20 comprising at least one of the peptides of claim 26.

29. A kit for detection of an infection, comprising a collection of claim 6.

30. A._ ki . according to claim 29, comprising strips wherein said collection is immobilized thereto.

31. A kit according to claim 30, wherein said strip further contains an internal standard.

32. A kit for detection of a HCV infection, comprising a collection of any one of claims 12-20.

33. A kit for detection of HCV infection, comprising at least a peptide from the collection of claim 25.

34. A kit according to claim 32 or 33, comprising strips wherein said collection is. immobilized thereto.

35. A kit according to claim 34, wherein said strip further contains an internal standard.

36. Use of the collection of claim 6 for the preparation of vaccines.

37. Use of the collection of anyone of claims 12-20 or of at least one peptide of claim 26 for the preparation of vaccines against HCV.

38. A vaccine against HCV comprising at least a collection of anyone of claims 12-20 or at least a peptide of claim 26.