SK5362003A3 - 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

Technical field

The present invention relates to the diagnostic determination of infectious agents, in particular viruses, in particular human hepatitis C virus (abbreviated herein as HCV), peptide antibodies to an infectious agent for such determination, the preparation of said peptides and kits for carrying out such determinations. In particular, the present invention relates to HCV, anti-HCV antibody binding peptides for diagnostic assays, methods for their preparation and kits for carrying out such assays.

BACKGROUND OF THE INVENTION

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

Viral infection is diagnosed by detection of anti-HCV antibodies in serum, or by detection of viral RNA by nucleic acid amplification methods (Robbins DJ, Pasupuleti V., Cuan J., Chiang CS: Reverse transcriptase PCR quantification of hepatitis C virus. Clin Lab Sci 2000, Winter; 13: 23-30). These methods are very sensitive, but they are expensive and not very reliable and reproducible. In addition, the reliability and specificity of PCR techniques are not standardized (Gretch, D.R .: Diagnostic tests for hepatitis C. Hepatology 1997, 26 (3 Suppl 1): 43S-47S.

The present invention relates to the diagnosis of infectious diseases such as viral infection, particularly HCV infection, ca Ce, - r ty r Γ erceff <fc * nocrrrf ^rr f Γ CR RC, CC · by measurement of antibodies to infectious agents, particularly anti -HCV.

Recently, a system for sensitive detection of HCV protein has been published (Komatsu F., Takasaki K .: Liver 1999, 19 (5): 375-80).

A very large amount of patented and non-patented literature data is focused on diagnostic methods and kits for HCV detection.

US 5,985,542 (Sumitomo Chemical Company), provides a kit for liver diseases such as hepatitis C and alcoholic cirrhosis, which comprises an antibody capable of recognizing cytochrome P450.

US 5,972,347 (Baxter Aktiengeselleschaft) discloses a composition for treating an HCV infection comprising inactivated bound HCV for neutralizing human antibodies to HCV, wherein the antibodies are against at least one protein selected from the group consisting of a major HCV protein and an NS3 protein.

US 5,939,262, US 5,919,625 and US 5,677,124 (Ambion and Cenetron) provide a very broad method for determining the presence of a test nucleic acid sequence in a sample, including obtaining a (ribo) nuclease resistant nucleic acid.

US 5,866,139 (Pasteur Institute) provides a kit for determining the presence of specific antibodies to HCV E-1. See also US 5,854,001 (Abbot).

US 5,800,982 (Tonen) describes antigenic peptides capable of specifically reacting with HCV Group II antibodies. JP 10019897 (Tonen) provides a kit comprising a peptide having an amino acid sequence, wherein at least five protein-forming amino acids are located in the non-structural region of the 4A protein (NS4A) in HCV and in a peptide having an amino acid sequence. 4B (NS4B) in HCV.

US

US

WO

JP efrccr • ccfr fo ^¾ fo * rc '

They also complement the overview of the prior art

5,871,904, 5,869,253, 5,667,992, 5,645,983, 9707400, WO 9637783, EP 4349885.

US 5,750,331; US 5,625,034;

593291, EP 593290

5,747,241, 5,610,009, EP 586065,

The most widely used assay for HCV infection is the determination of anti-HCV antibodies in serum. Since 1987, when the HCV genome was cloned, a number of anti-HCV diagnostic assays have been developed. Commercially available diagnostic kits determine antibodies in serum for a mixture of four recombinant antigens corresponding to the HCV core, non-structural region 3, non-structural region 4, and non-structural region 5 of the HCV polypeptide (Gretch DR: Diagnostic tests for hepatitis C. Hepatology 1997,26 (3 Suppl 1): 43S-47S). Samples that are positive by this ELISA assay must be confirmed by immunoblot method when the presence of antibodies against the same antigens is separately determined. A positive result requires the determination of antibodies to at least two recombinant antigens.

For some of the population with anti-HCV antibodies, reactivity to only one antigen was found, making it impossible to make a clear diagnosis. A further problem lies in the use of recombinant antigens which can be recognized by non-HCV-related antibodies and which constitute a false positive diagnosis. Further examination and long-term patient monitoring are required for a final diagnosis.

Contact with a foreign antigen in the body activates a specific immune response. The properties of the antigen that is detected by a humoral response are defined by epitopes recognized by the host antibodies. Assuming that the antibody binding site is a negative image of the epitope, the molecule that specifically binds the paratope represents a positive

C <rec <: r re crcccprrrrc ^r r Γ C r C r epitope image. This idea suggests, as regards the humoral response, that the antigen can be reliably described by specific ligands that bind to antigen-specific antibodies. Identifying these ligands would provide a method of detecting a specific humoral response to an antigen, regardless of whether the same antigen is known and / or available.

This concept was used to develop a diagnostic assay for the detection of antibodies causing infection, HCV infection in humans. Screening of phage libraries of HCV positive sera identified peptide ligands that specifically bind anti-HCV antibodies. This was achieved by an ad hoc strategy, although the sera of infected patients contained virus-specific antibodies in a large population of other antibodies with different binding specificities.

Screening of peptides with a large number of negative sera excluded those ligands that detected antibodies in negative samples (false positive), resulting in the selection of a set of highly specific peptides. The incidence of non-specific reactions was reduced by using a short peptide sequence outside the natural antigen. This procedure is described in EP 0 698 091 B1, published February 28, 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. No. 87, Humana Press Inc. p. 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, Vol. 16, November 1998, 1068-1073.

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These selection strategies identify those peptide structures that best bind immunodominant anti-HCV antibodies. In addition, ligand binding contributes to remarkable avidity in determining susceptibility. In doing so, testing with ADAM-HCV (ADAM = antibody mimicking antigen detection) of multiple sera that were not used in screening resulted in a sensitivity of less than 100%, i.e. quite high. The appropriate anti-HCV humoral response may explain this outcome as it does not include a major immunodominant epitope that is rather directed towards many viral determinants. This is further complicated by the existence of different viral genotypes.

Commercially available diagnostic kits determine antibodies in serum for four recombinant antigens corresponding to large regions of the HCV polypeptide (Gretch D.R: Diagnostic tests for hepatitis C. Hepatology 1997, 26 (3 Suppl 1): 43S-47S). A positive diagnosis requires the determination of antibodies in serum against at least two recombinant antigens; therefore, it is impossible to make a definitive diagnosis for a certain population with antibodies to HCV, although exceptionally, reactivity with only one antigen can be detected. ADAM-HCV EIA (Enzyme Immunoassay) employs multiple mimicking immunodominant epitopes from different antigens, ensuring assurance of analysis. The incidence of positive samples is greatly reduced.

We have devoted efforts to technical development of the ADAM-HCV assay (strategy published in the above-mentioned EP 0 698 091). Peptides from phage libraries are selected based on the N-terminal fusion of the major capsid protein pVIII. The use of phage display as a diagnostic reagent has many advantages: it is exceptionally flexible reagent, which is directed at different types of immunoassays (Dente et al., 1994; Felici F., t · Γ β RFC rrr ^r er * ♦ t e ^{r R.} rrr ·

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G. Galfre, A. Luzzago, P. Monaci, A. Nicosia, and R. Cortese “Phage-displayed peptides as tools for characterization of human serum. Methods in Enzymology 267: 116-129 (1996); Bartoli et al., Nature Biotechnology, Vol. 16, November 1998, 1068-1073), and a low range of production is light and cheap.

Despite these advantages, the concentration of the peptide molecule limits the size of the phage particle in the assay. Serum antibody interference against the phage capsid requires the addition of carrier phage to the mixture, which sequesteres the anti-phage antibodies. Finally, the generation of a wide range of antibodies raises problems for biological reagents of microbiological contamination, repetition, quality control, purification and cost.

Simple, linear synthetic peptides derived from the phage peptide sequence do not retain sensitivity and specificity in most of these cases for detection of an effective antibody.

annotation

It has been found, and object of the present invention, that the method for diagnosing infectious diseases, such as infectious diseases, in particular hepatitis C, is based on identifying the binding specificity of anti-antigen antibody molecules in the serum by an antibody mimicking antigen (ADAM) method. It comprises screening a library of phages from the sera of patients infected with antigen and uninfected individuals to identify antibodies (ligands) binding specific peptides to said antigen. In a preferred embodiment, the present invention relates to HCV infection.

DETAILED DESCRIPTION OF THE INVENTION

A first, preferred embodiment of the invention relates to a method wherein the ligands are treated by an in vitro maturation process.

A second embodiment of the present invention is a method wherein the ligands are synthetic peptides.

In a third embodiment of the invention said ligands are attached to a common core. In a particularly preferred embodiment, said ligands are together with said common MAP, as defined below.

Another cell of the present invention is a collection of HCV specific ligands obtained by a process comprising:

(a) first selective selection of n positive serum phage library to form first series of n phage pools;

(b) preparing a mixture of n funds containing n-1 funds;

(c) selecting the affinity of each of the n mixtures for the serum that exclusively constitutes the phage pool to produce second sera of the n phage pools, and optionally

d) further selective selection of each of the second series of n phage pools on mixtures containing all n original sera, except for those used in the first selective selection;

e) immunoscreening the resulting second series of n phage pools using a mixture of all n parent sera to form positive clones;

f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an ordered array of said clones as phage-secreting colonies;

g) creating replicates of said phage secreting colonies;

h) screening each replica for reactivity with positive and negative sera, obtaining clones reacting with positive sera;

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i) use of each of said specifically reacting phage as a ligate for affinity purification of antibodies from positive serum;

j) testing said antibodies for their reactivity with the antigen-specific peptides already identified;

k) separating clones that detect antibodies.

The individual peptides derived from the above process are also within the scope of the present invention.

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

YSREQLNKLFGIDMT;

YSREQLNKMFGIEIS;

YSREQLSKLFGIEPM;

NSRWLSKAHGIEGM;

YSREQLNKLFGIEVM;

YSREQLSKLFGIDTQ;

KSREQLSKLHGVDTS;

RSREQLSKLFGIDLT;

MWRTWLMKTHGIESW;

MLRTWLMKYQGIESW;

YSRSWLMKAHGLELG;

MMRSYLMKAHGIESL;

MSRLWLMKAHGIS SE;

KHS EWLNKARGIE SW;

MSRTFLMKAHGIESW;

MSRTWLMKAHGIESW;

AEGEKKLRRSTNWGDPAK;

AEGEFKTRRQTNYQPAK;

AEGEFKTLRNANRLDPAK;

AEGEFKTLRNSNRLDPAK;

AEGEFKKFPGSSTPKDPAKAAFDSL;

e t rf r c Γ r.c c

AEGEFPQDDARFPGGGDPAKAAFDSL; PQDARFPGGGDPAKAAFDSL; AEGEFKGAGGAQTVDWALLVDPAK; AEGEFMQKHFGGAQWIMGDPAK; AEGEFLSLKGSGGGQLRALVDPAK; , AEGEFLSLKGSGGAQLRALVDPAK; AEGEFYLLKRSSPPDPAKAAFDSL; AEGEFPILVGPYLLPRRSREEAVDPAK; AEGEFPILVGYLLPRRSREEAVDPAKGK; AEGEFRLGVRAPRKALDPAK; AEGEFRLGVRALRKALDPAK; AEGEFRLGVRALRKAPDPAK; RLGVRALRKAPDPAK;

AEGEFTQPRGHSYQDPAK; AEGEFLKERAEMSARKTLGADPAK; AEGEFFYQIPRRMETKYGDPAK; AEGEFSREQLNKLFGIEGDPAK; AEGEFNSREWLSKAHGIEGMDPAK; AEGEFRSREQLSKLFGIDLTDPAK; AEGEFYSREQLNKLFGIDMTDPAK; AEGEFYSREQLNKMFGIETSDPAK; AEGEFYSREQLNKLFGIEVMDPAK; AEGEFKSREQLRKLHGFDTSDPAK; AEGEFKMRNYLNKAFGIEGMDPAK; AEGEFRSREQLSKLFGIELTDPAK; AEGEFSRREYSNKAFGIETQDPAK; AEGEFRRREYLNKAFGIEGGDPAK; AEGEFSRREWLNKRFGIEYLDPAK; AEGEFMSRTWLMKAHGIESWDPAK; AEGEFYSPEWLNKARGIDRSDPAK; AEGEFKSREQLSKLHGVDTSDPAK; AEGEFYSREQLNKMFGIEISDPAK;

r r f re (r r e e e f r cc

AEGEFYSRSWLMKAHGLELGDPAK;

AEGEFMMRSYLMKAHGIESLDPAK;

AEGEFMSRLWLMKAHGISSEDPAK; AEGEFPQPQEVHVYREQLGLDPAKAAFDSL; AEGEFGEVLYRGFDEVGGDPAKAAFDSL;

AGEPPYVIERGMQDPAK;

AEGEFTTASPAHFLVPLDPAKAAFDSL; AEGEFTTASPAHFLVPLDPAKAAFDSL; AEGEFTTASPSHFLVPLDPAKAAFDSL;

AEGEFATAPPRHYSWDPAK;

AEGEFATAPPAHYSWDPAK;

AEGEFATAPPSHYSWDPAK;

AEGEFRFWKVPDYDPPAAGGDPAK;

AEGEFTE S SVS STLADLASKTFGSADPAK; AEGEFTLADLATMTFGSTDPAK;

AEGEFGLADLATLTFGSPDPAK;

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

For the collection of the present invention, the inserts of the preferred clones separated in step k) have sequences

1. SREQLNKLFGIEG;

2. RATLSNEHGITIG;

3. DQRENWFKYHGFG;

4. EWRRYMSDIHGYG;

5. DSLRYMYVMPGFG.

In the collection of the present invention, a phage library of reactive clones, more preferably the best reacting clones, is preferably generated that are partially mutated such that each amino acid in the clone sequence is independently substituted with any other amino acid.

In the collection of the present invention, said preferred clones have a preferred insertion sequence: SREQLNKLFGIEG.

er · e · efpeec * - <. _e eec r. · Rprree ^{fP Γ r} '

In the collection of the present invention, in the phage library, the random sequence may be flanked by two cysteine residues. This aspect can generally be used in the dissemination of the present invention and is not limited to the case of HCV.

It is an object of the present invention to use the above collection for the preparation of diagnostic assays for the determination of infectious agents, such as viruses, particularly HCV, in individuals suspected of having infectious agents, such as viruses, especially HCV.

An object of the present invention is a diagnostic kit comprising the above collection.

It is an object of the present invention to provide immunogenic peptides obtained, either as a collection or as a single peptide, as described herein. Said peptides are useful as immunogens and are therefore suitable for the preparation of vaccines, especially against HCV. Typically, the peptides are in the form of the above kit.

Advantageously, in contrast to the antigen-based system, the diagnostic assay that we have tested has an inbuilt increasing capacity: an ad hoc selection of those sera for which no response was found can be made. A similar procedure can be used to identify a peptide panel that differentiates different HCV genotypes, replacing expensive and laborious PCR techniques with cheaper and faster EIAs.

The present invention will be described in more detail by way of examples and figures.

Figure 1 shows the characterization of clones derived from screening. The amino acid sequences of the selected clones are indicated by single letter symbols. The top and bottom panels record sequences derived from the original or secondary library. The pVIII sequences flanking the foreign epitope are (NH2) AEGEF (foreign epitope) DPAK. Gray squares are residues that are more often present at any position in the clones

Γ C ree »« · _r · ee crror rreec _e rr <r. ;

^XZ e, yy r. rr ^r _r /.

r cer r. r ^crr 'of the original library. Residues that contribute to the conventional sequence of peptides from the secondary library are marked in black. The _reported values are the average of two independent assays and refer to the difference between the absorbance ( _λ = 0 _45nm - As 2nm) of the phage and wt phage of pC89 (Felici et al., 1991). * indicates untested sera.

Figure 2 shows the identification of a phage mimicking the same antigen determinant. Affinity purified antibodies from positive serum C65 using clones PA1, PA3, PA8, and PA12 or PA18 (shown in the first column) were tested for their reactivity against the same phage (shown in the first row).

The results presented are average values from two independent assays and relate to the difference between the absorbance (λ = _{5 5} µm - _{6 6} 20nm) of the phage and wt phage of pC89 (Felici et al., 1991).

Figure 3 shows the ELISA reactivity of the mixture derived from the pVIIIA12 secondary library. Phage mixture after selective selection of the pVIIIA12 library from C76 positive serum was tested by ELISA with C12, C13, C29, C40, C47, C65, C73, C74, C76, C83 and C85 positive sera. The white, gray and black bars indicate the reactivity of wt phage, the pVIII12 library, and the p76 ^XI mixture. ELISA results are reported as A = A4 ₅ onm - A ₆ 20wn · The values given are the average of two independent determinations.

Figure 4 shows the A: reactivity of the ADAM-HCV mixture with serum. The cut-off value (00 = 0.232) was calculated as CO = N + 5σ, where N is the mean and σ is the standard deviation of the values obtained using negative serum. B: ADAM-HCV EIA sera obtained from the Italian Red Cross. The cut-off value (CO = 0.252) was calculated as CO = N + 5σ, where N is the mean and σ is the standard deviation of the values obtained from the five negative sera. ELISA as described in Materials and Methods, revealed the binding of peptides to antibodies present in human Sri e π ^r errrerrrcrccr levels. Mean values of two independent experiments are shown.

The results are expressed as the ratio between the measured signal and the cut-off value (S / CO). The number of sera tested is shown in each group.

Figure 5: ADAM / HCV EIA on uncertain serum panel. The left column shows the designations of the HCV peptides grouped according to their binding capacity. The next four bars record the reactivity of the peptides with the positive (c25 and r15) and negative (r6 and r13) control sera. Each additional column records the reactivity of said peptides by indeterminate sera. Binding of antibodies to HCV peptides present in human serum was tested by ELISA as described in the Materials and Methods chapter. Mean values from two independent experiments were determined. For each peptide, the cut-off value (CO) was calculated as CO = N + 5σ, where N is the mean value and σ means the standard error of the mean, from 31 negative control sera. The results are expressed as the ratio between the measured signal and the cut-off value (S / CO).

Figure 6: ADAM-HCV / SIA positive, negative and indeterminate sera. The following ADAM-HCV peptides were grouped according to their binding specificity and were immobilized on a nylon membrane to obtain 10 bands: ml909.2 and ml913.2 (A); m1,901.31, m 3 332.2, m 3362.3 (B); ml977.1 (C); m3551.3 (D); m3566.3 (E); m858, mF78 and mH1 (F); mA12, 1, mA12,2 and mA12,12 (G); m B1, 17 (H); mG21.2 (I); m1929A3.1, m1929C3.4 and m1929.21 (J). Purified human IgG (pos. Ctrl.) Was used as an internal positive control. Binding of antibodies to HCV peptides present in human serum was detected as described in the Materials and Methods chapter.

In a most preferred embodiment of the present invention, multiple antigen peptides (MAPs) have been synthesized in which the C-terminus of the eight identical peptide sequences is ee * er cr cr attached to a common nucleus (Tam, JP: Synthetic peptide vaccine design Synthesis and properties of a high-density multiple antigenic peptide system (Proc. Natl. Acad. Sci. 85 (1988), 5409-5413).

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

Other approaches (Santini, C., Brennan, D., Mennuni, C., Hoess, RH, Nicosia, A., Cortese, R., Luzzago, A .: Efficient display of HCV cDNA expression library lambda, J. Mol Biol 1998, 282 (1): 125-135, Pereboeva J .: Med Virol 1998, 56 (2): 105-111) isolated large protein domains that retain antigenic properties. The approach we have developed, therefore, contains intrinsic properties that are not based on the use or information of HCV antigens, can even be applied to systems with unknown antigen, thus creating a process leading to antigen detection. Phages of random peptide libraries (phage libraries) represent an effective tool for identifying ligands that are specifically recognized by anti-HCV serum antibodies. Phage peptides have a broad potential for mimicking, as they are capable of mimicking linear, conformational and even non-protein epitopes (see review Felici F., Luzzago A., Monaci P., Nicosia A., Sollazzo M., Traboni C .: Peptide and protein display on the sufrace of filamentous bacteriophage.

y y y y y

í (> f * r * (() ((r!! (r)

1995, 1: 149-183; Zwick M.B., Shen J., Scott J.K .: Phagedisplayed peptide libraries. Curr. Opin. Biotechnol. 1998, 9 (4): 427-436.

The present invention identifies a broader collection of active HCV-specific ligands and develops a new type of diagnostic kit for the determination of anti-HCV antibodies in serum.

The peptides of the present invention may be used for diagnosis or as immunogens or as vaccines.

Pharmaceutical compositions containing the immunogens and vaccines of the present invention are prepared by methods commonly known to those skilled in the art. For example, they can be prepared as disclosed in EP 0 698 091.

Example for

Identification of HCV-specific ligands of novel binding properties

Selective selection of the pVIII-12aa phage library was performed from 8 positive sera (C13, C14, C27, C29, C40, C47, C62 and C65). Thus, 8 phage pools (designated as p13 ^x , ρ14 ^χ , p27 ^x , p29 ^x , P40 ¹ , p47 ^x , p62 ^T and p65 ^x ) were created. Eight mixtures were prepared, each containing a combination of seven of the eight phage pools. Each mixture was affinity selected against using serum which formed the pool of phage exclusive: for example, a mixture of mixAp65, comprised of funds ^x PL3, PL4 ^{x, ρ27} τ, ^x p29, p40, ^{x, x} p47 and p62 ^x be selectively selected from the serum-C65. Each of the remaining eight phage pools (designated p13 ⁿ , p14 ^T1 , etc.) was individually selectively selected from a mixture of all original sera except that used in the previous selection. For example, the pool p13 ^XI was selectively selected from a mixture of sera C14, C27, C29, C40, C47, C62 and C65.

r r r f r c e e β rt r r. t y c y y y y y y y y y y y y y y * *

Immunoscopy of the resulting eight phage pools using a mixture of all eight original sera yielded a large number of positive clones. We tested the individual reactivity of all these clones in a panel of positive and negative sera to create an ordered list of clones as phage-secreting colonies. According to their growth, a whole set of clones were applied to a nitrocellulose filter. This procedure was repeated to generate multiple replicas, which were then individually and simultaneously tested for their reactivity with positive and negative sera. Thus, many clones were obtained that specifically reacted with the positive serum. Each of these phages was used as a ligate for affinity purification of antibodies from positive serum. These antibodies were then tested by ELISA for their reactivity with any of the 4 HCV-peptide groups already identified (Prezzi C., Nuzzo M., Meola A., Delmastro R., Galfre C., Cortese R., Nicosia A., Monaci P. 1996, 1. Immunology, 156: 4504-4513; Bartoli et al Nature Biotechnology, Vol. 16, 1998, 1068-1073). This analysis separated 12 clones that detected serum antibodies with a new binding specificity, indicating their antigenic properties that are different from those already known.

Sequence analysis identified 5 different sequences. Culture supernatants of each of these 5 clones (PA1, PA3, PA8, PA12 and PA18) were prepared and their ELISA reactivity was tested with 30 different positive and 24 negative sera (Figure 1). Only clones PA8 and PA12 showed statistically significant reactivity with positive sera (p <0.2).

Phage PA1 was then used to immunopurify antibodies from serum C65. These purified antibodies specifically reacted with clones PA3, PA8, PA12, PA18, as well as the clone PA1 itself. This means that these phages can be grouped into an exceptional class recognized by antibodies of the same specificity cfrrc * erfr r. rfr ^e «rer r> rrrr Γ r C r.

(Figure 2). When using wild-type ligate, or when the antibodies were affinity purified from negative serum, no reactivity was noted with any of the above clones.

When a different clone from the same group was used for immunopurification of serum antibodies, or when another positive serum was used, we obtained results consistent with the reactivity shown in Figure 1. For example, when phage PA3 was used to immunopurify antibodies from the same serum, these antibodies reacted with clones PA8 and PA12, in addition with the same PA3. These results indicate that clones PA1, PA3, PA8, PA12, and PA18 detect antibodies by reacting with the same B cell epitope, which is assumed based on the low peptide sequence similarity (Figure 1).

Affinity maturation of HCV peptide

A phage library was created in which the sequence of the PA12 clone was partially mutated. In this "secondary library, called pVIIIA12, oligonucleotides were synthesized such that each amino acid of the SREQLNKLFGIEG sequence was independently substituted with any other amino acid: in theory, the substitution at each position may occur at a frequency of 20%. In addition, a random residue was included on both sides of the foreign peptide sequence. The pVIIIA12 library was selected selectively from 12 positive sera (C8, C10, C12, C13, C22, C58, C60, C76, C83, C85, C141 and C177).

ELISA testing showed that the p76 ^IT , ρ141 ^ΙΣ and ρ177 ^ΣΙ phage ^pools (derived from selection C76, C141 and C177) have the highest and broadest reactivity with positive sera and were therefore further analyzed (Figure 3). Based on the reactivity were phage funds p76 ^IT, pl41 and pl77 ^{JI ZI} subjected to immunoscreening using sera C40, C141 and C177. The analysis selected a few clones from each pool that were then individually tested for their reactivity with many different positive and negative sera according to the filter-replica protocol as described in detail above. This final screening separated 51 clones specifically reacting with positive sera.

Sequence analysis revealed 16 different sequences (8, 5 and 3 derived from pool p76 ^IX , p141 ^IX and p177 ^IT . Supernatants were prepared from cultures of these clones and their ELISA reactivity was tested with 33 different positive and 24 negative sera (Figure 1).

Clones P40.17 and P40.7 reacted with positive sulfur (42%). Their combination with clones P141.7 and P177.22 gave a 70% reaction with positive sera. The alignment of selected peptides defines the consensus sequence (M / Y) of SRE (W / Q) L (M / N) K (A / L) (H / F) GIES (W / M).

Identification of additional HCV-specific ligands

A similar selection strategy was carried out to identify ligands of new binding properties that differ from the known ligands. Phage libraries of varying length were screened in which the random sequence was either completely or randomly flanked by two cysteine residues, creating a conformation of the peptide.

Various combinations of sera and phage pools were used to select phage libraries. Phage pools that showed interesting reactivity profiles with positive sera were further analyzed. Determination of the reactivity of a large number of individual clones by repeated screening separated phages showing specific reactivity with positive sera.

We focused on clones with interesting reactive properties.

• f * β e e r r r <r

Testing these clones with antibodies that were affinity purified from HCV peptides excluded those clones that mimic the antigenic properties of the peptides already identified. Clones remaining after this selection were tested by ELISA with a large number of positive and negative sera, thus statistically defining their HCV specificity.

These selected clones were further enhanced by creating and screening secondary libraries where the sequence of the original clone or population of clones was partially mutated and subjected to screening again. Thus, variants with improved binding properties were selected. This step was accomplished by adopting various strategies that have already been described (for example, Urbanelli, Zhu). This extensive and repeated effort has identified 7 new groups of ligands that specifically bind HCV-specific serum antibodies with different binding specificity.

Screening the phage range of HVR1 variants using serum-isolated peptides specifically reacting with a plurality of positive sera (Puntoriero et al .; Nicosia, unpublished). A set of HVR1 phage peptides derived from this screening was analyzed for their reactivity with a panel of our sera. Three peptides with high reactive specificity were identified using positive sera mF78, mH1 and m858.

In conclusion, 12 groups of ligands were identified, including 4 groups identified in the past (Bartoli et al., Nature Biotechnology, 1998 vol. 16, 1068-1043; Prezzi C., Nuzzo M., Meola A., Delmastro R., Galfre C., Cortese R., Nicosia A., Monaci P. 1996, 1. Immunology, 156: 4504-4513). See Figure 5, Groups A to L.

From phage to synthetic peptide

Twenty-two peptide sequences derived from 12 groups of HCV-ligands were synthesized as octa-branched multiple 20-fold antigenic peptides (ADAM-HCV peptides). In this molecule, eight identical peptide sequences are linked via a forked lysine to a common nucleus to form a disjointed image similar to the pVIII-fusion peptide on the phage kapside. The sequences of ADAM-HCV peptides are as follows:

m858 ml901.31 ml901.34 ml909.2 ml913.2 ml929.21 ml929A3, 1 ml929C3.4 ml977, 1 m3322.3 m3362.3 m3551.3 m3566.3 mAl2.1 mA12.2 mA12.13 mBll, 17 mF78 mG21.2 mHl mN15.3 mS48.5 ethyttggaaarttsgltslfspgpsqn

AEGEFKKFPGSSTPKDPAKAAFDSL

AEGEFPEDTFPGSKLILSGDPAKAAFDSL

AEGEFKTRRNTNYQDPAK

AEGEFKTLRNTNRLDPAK

AEGEFATASPTHYTSELDPAK

AEGEFTTASPTHFLVPLDPAK

AEGEFATAPPSHYSWDPAK

AEGEFPYLLPRRSREEAVDPAK

AEGEFPQDARFPGGGDPAK

AEGEFLSLKGSGGGQLRALVDPAK

AEGEFRLGVRALRKAPDPAK

AEGEFKTSVRSVPRARPPINGDPAK

AEGEFNSREWLSKAHGIEGMDPAK

AEGEFRSREQLSKLFGIDLTDPAK

AEGEFMSRTWLMKAHGIESWDPAK

AEGEFRELLYEAFDDMEGDPAK

QTHTTGGQAGHQAHSLTGLFSPGAKQN

AGEPYVIEQGMMDPAK

QTHTTGGWGHATSGLTSLFSPGPSQN

AEGEFGLADLATLTFGSTDPAK

AEGEFRFWKVPDYDPPAAGGDPAK their analytical properties are summarized in Table 1, which is incorporated into the figures.

In many cases, the pVIII sequences flanking a foreign epitope (NH2AEGEF and / or DPAK-COOH) have been shown to be relevant for their binding specificity to the respective peptide.

~ r Γ C O Γ

- yy yy yy - yy yy

A mixture containing these 22 ADAM-HCV peptides (ADAM-HCV mixture) was used to detect the presence of anti-HCV antibodies by EIA (ADAM / HCV EIA). The ADAM-HCV peptide mixture was immobilized by passive coating to the bottom of a multiwell ELISA plate and incubated with 1:40 diluted serum for 40 minutes. Peptide-bound human antibodies were detected by incubating for 20 minutes with an anti-human conjugate and measured by a chromogenic enzymatic reaction.

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

ADAM-HCV EIA /

HCV-positive and HCV-negative sera were tested for the presence of anti-HCV antibodies using ADAM / HCV-EIA (Figure 4B). The assay recognized all positive sera and also showed 100% specificity in identifying negative samples.

Indeterminate samples

A collection of sera diagnosed as indeterminate samples according to commercially available HCV-confirmatory assays was obtained from various sources. 23 ADAM-HCV peptides were separately tested by ELISA for their reactivity with 31 samples (Figure 5). 6 samples did not react with any MAP tested and were therefore considered negative. 8 samples recognized only one antigen, confirming indeterminate analysis. Finally, 17 samples showed two or more responses to different groups of peptides, which means they are positive.

ADAM-HCV immunoblot assay (ADAM-HCV / SIA)

ADAM-HCV peptides were covalently immobilized on an activated nylon membrane to give a band of ten bands. Each line represents different peptides of the same binding specificity as shown in detail in Figure 6. As a control, a purified human IgG sample was used and represented an internal positive control. A select number of samples from uncertain sera were tested for their reactivity with immobilized antigens by incubating the serum sample with the strip. Anti-HCV antibodies captured by individual antigens were visualized by incubating the strips with the anti-human enzyme conjugate, followed by a colorimetric enzymatic reaction. The reactivity of the samples with peptide bands was determined visually by comparing the intensity of each band with the corresponding internal positive control band.

ADAM-HCV / SIA showed the reactivity of all 8 positive sera tested to many different viral determinants mimicking ADAM-HCV peptides. No reactivity was found when testing 8 negative sera (Figure 6).

We also analyzed a selected number of indeterminate samples using ADAM-SIA. As shown in Figure 6, analyzes of 8 indeterminate sera confirmed the results obtained with ADAM-HCV / EIA using individual sera. This means a comparable sensitivity of the two assays.

MATERIAL AND METHODS

Phage libraries

Four random peptide phage libraries were used as ligand source: pVIII9aa, pVIII9aa_cys, pVIII12aa, pVIII15aa and pVIIIA12. pVIH9aa (Felici et al., 1991), pVIII12aa and pVIII15aa are three different libraries consisting of random 9-mer, 12-mer and 15-mer, which appear on filamentous phage as a fusion with the NH ₂ terminus of the major pVIII protein. pVIH9aa_cys is a library in which a random nonapeptide is flanked by two cysteine residues (Luzzago et al., r.

f Cf

1993). In this library, cysteines condition the formation of a disulfide bridge which contributes to some extent to the conformation of the peptide. The pVIIIA12 library was generated by synthesis of an oligonucleotide encoding the amino acid sequence SREQLNKLFGIEG. By adjusting the resin-cleavage synthesis method (Glaser et al., 1992), each amino acid was substituted with an NNS triplet at a frequency of 20%. In addition, random residues at both ends of the foreign peptide sequence were included. All five libraries were constructed as described (Folgori et al., 1998). For each of the five libraries, the complexity of the library based on the individual clones obtained after transformation of the bacteria was about 1x10®.

Human sera

The human sera used in this study were randomly collected from healthy volunteers, blood donors for transfusions, and clinical departments. Namely, many indeterminate samples used in this study were obtained from the Laboratory of Virology, the Instituto Superiore di Sanita, Roma (Italy) and the Centro Nazionale Transfusione Sangue delia Croce Rossa Italiana, Roma (Italy). Sera were tested for HCV antibodies by second generation HCV ELISA kits (Ortho Diagnostic Systems, Bersee, Belgium) and were confirmed by first generation dot blot immunoanalytic RIBA HCV assay (Chiron Co., Emeryville, CA). Sera were also tested for the absence of antibodies to HBsAg and HIV-1 / HIV-2 viruses using the AUSAB EIA test (Abbot Labs, Chicago, IL) and the third generation HIV-1 / HIV-2 EIA tests (Abbot Labs, South Pasadena, CA). Positive samples 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 to all three antigens as HCV negative sera were included in the study.

Affinity Selection and Immunoscreening

HCV-positive sera were used for affinity selection of phage random peptide libraries as described by Folgori et al., 1998; Felici et al., 1996; Prezzi et al., 1996. Clones after affinity selection were analyzed by immuno-screening using sera as described by Prezzi et al., 1996 and Minenkova et al.

ELISA using phage clones

The ELISA assay using phage and human serum clones was as follows. Phage supernatants were prepared from DH5α-F 'infected cells as described (Felici et al., 1991). Multwell plates (Immunoplate Maxisorp, Nunc, Roskilde, Denmark) were coated overnight at 4 ° C with 200 μΐ anti-pIII monoclonal antibody 57D1 (Dente et al., 1994) at a concentration of 1 μg antibody / ml in 50 mM NAHCO3, pH 9.6. After removal of the solution, the plates were incubated at 37 ° C for 60 minutes with ELISA blocking buffer (0.1% casein, 1% TRITON-X100 in PBS). Plates were washed several times with PBS / 0.05% Tween-20 (wash buffer). Then, a 1: 1 mixture of ELISA blocking buffer and clear phage supernatant was added to each well and the reaction lasted for 1 hour at 37 ° C. 1:40 diluted human serum was incubated for 30 minutes at room temperature with 5χ10 ^1θ plaque forming units (pfu) of phage f11, 1 (Dente et al., 1996), 25 μΙ / ml protein extract from DH5-F 'cells infected with phage f11 , l (Dente et al.) and 25 μΐ of rat hybridoma cell supernatant in ELISA blocking buffer. After discarding the supernatant, the plates were washed with wash buffer and 200 µL of pre-incubated serum was added to each well. Following a 60 minute incubation at 37 ° C, the plates were then washed with wash buffer. Then, 1:20,000 diluted goat anti-human IgG HRP P rrrrrr 0 ctrr r was added to each well. 'ffe''' y - ecnr '· t »rrr CC Λ cc <

konj. (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in second antibody blocking buffer (1% Triton, 1% horse serum, 50% calf serum in PBS, Mab supernatant 5 μΙ / well). After incubation for 30 minutes at 37 ° C, the plates were washed and peroxidase activity was evaluated after incubation with a 200 μΐ TMB substrate system (SIGMA, St. Louis, Mo). After 15 minutes, the reaction was stopped by adding 25 μΐ of 2M H2SO4. Plates were evaluated on an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and the results were expressed as A = A _450n m - Aa ₆ 20nm. ELISA values are the average of two independent determinations. Statistically significant differences are when they were measured more than 3σ (σ = {1/2 [a ² p ⁺ and ² w]} ^1/2 ) from the background signal for wt phage.

The p value for PA8 and PA12 clones is the probability that the observed frequency of reactivity of positive and negative sera is statistically the same according to the χ ² assay.

Affinity purification of phagotope-specific antibodies from serum

60 mm diameter Petri dishes (Becton Dickinson Labware, NJ) were coated overnight at 4 ° C with a solution of 1x10 ¹¹ CsCl-purified phage particles / ml in 50 mM NaHCO 3, pH 9.6. After washing with PBS / Tween, the plates were incubated for 60 minutes at 37 ° C with ELISA blocking buffer. A mixture of human serum (diluted 1/100 with ELISA blocking buffer), 1x10 ¹² f11.1 pfu / ml and 25 μ ml / ml XL1-blue protein extract of cells was incubated for 60 minutes at room temperature. After removing the blocking buffer, the preincubated mixture was added to the plate. Incubation was performed at 4 ° C and continued overnight. The diluted serum was removed and the plate was washed with wash buffer. Bound antibodies were eluted with 0.1 M glycine-HCl buffer pH 2.7 with 10 μΐ BSA for neutralization.

n r rr '

Characterization of phage clones

Affinity purified clones were tested by standard ELISA for their reactivity to phagotopes. Typically, the multiwell plate was coated (100 μΙ / well) with a solution of 1x10 ¹¹ TU / ml CsCl-purified phage in 50 mM NaHCO ₃ , pH 9.6 at 4 ° C overnight. After washing with PBS / Tween, the plates were incubated for 60 minutes at 37 ° C with blocking buffer. Then, 100 μΐ of affinity purified antibodies was added to each well and incubated at 4 ° C overnight. Plates were then washed with cold PBS / Tween and 100 µl / well goat anti-human IgG (Fc specific) antibody-conjugated alkaline phosphatase (Sigma, St. Louis, MO) was added, diluted 1/5,000 in blocking buffer. After a 2 hour incubation at room temperature, the plates were washed and alkaline phosphatase was evaluated as described above. Synthetic peptides

Synthetic octavained peptide peptides (MAPs) were used: (Tam, 199X). Synthesis was performed by the flow-polyamide method (Pessi et al., 1990). The peptides were dissolved in dimethyl sulfoxide.

ELISA using synthetic peptides

Multiwell plates (Immuno plate Maxisorp, Nunc, Roskilde, Denmark) were coated overnight with a 10 µg / ml MAP solution in 50 mM HaHCO ₃ , pH 9.6. After liquid removal, the plates were incubated for 60 minutes at 37 ° C with ELISA blocking buffer (0.1% casein, 1% Triton-X100 in PBS). Plates were washed several times with PBS / 0.05% Tween-20 (wash buffer). Human serum was then added to each well diluted 1:40 and incubated for 40 minutes at 37 ° C. Plates were then washed with wash buffer and then added to each ELISA blocking buffer with 1:20,000 diluted goat anti-human IgG conjugated to HRP »r>

(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After incubation for 20 minutes at 37 ° C, the plates were washed and peroxidase activity was determined after addition of TMB substrate (SIGMA, St. Louis, MO). After 15 minutes, the reaction was quenched by the addition of 2M H 2 SO 4. Plates were evaluated by an automatic ELISA instrument (Labsystems Multiscan Bichromatic, Helsinki, Finland) and the results were expressed as A = A ₄ 50nm A620nm. The ELISA values are the result of two independent determinations.

LITERARY LINKS

Smith C.P., 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., Nicosia A .: 1994, A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human serum. EMBO 1. 13: 22362243.

Prezzi C., Nuzzo M., Meola A., Delmastro R., Galfre C., Cortese R., Nicosia A., 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., Cesareni C.: 1991, Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 222: 301-310.

Luzzago A., Felici F., Tramontano A., Pessi A., Cortese, R .: 1993, Mimicking of discontinuous epitopes by phage displayed peptides. I. Epitope mapping of human H ferritin using phage library of constrained peptides. Gene 128: 5057.

f 0 frr c. yy · ^y · yyyy

Dente L., Cesareni G., Micheli C., Felici F., Folgori

A., Luzzago, A., Monaci, P., Nicosia, A., Delmastro, P .: 1994,

Monoclonal antobodies that recognize filamentous phage.

Phage display technology. Gene 148: 7.

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

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

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

Frangioni F.V., Neel B.J .: 1993, Solubilization and purification of enzymatically active glutathione Stransferase (pGex) fusion proteins. Analyte. Biochem. 210: 179,187th

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Claims (36)

PATENT CLAIMS An antigen diagnosis method, characterized by identifying the binding specificity of antiantigen antibody molecules, in a method of detecting an antigen mimicking antibody (ADAM), which comprises screening phage libraries using sera of patients infected with antigen and uninfected individuals, by identifying the binding antibodies (ligands) specifically associated with said antigen. Method according to claim 1, characterized in that the better ligand properties are achieved by a maturation strategy. The method of claim 1 or 2, wherein said ligands are synthetic peptides. The method according to claim 1 or 2 or 3, characterized in that said ligands are attached to a common core. 5. The method of claim 4, wherein said ligands together with said common core are MAP. 6. Collection of antigen-specific ligands obtained by the procedure: (a) first selective selection of n positive serum phage library to form first series of n phage pools; (b) preparing a mixture of n funds containing n-1 funds; (c) selecting the affinity of each of the n mixtures for the serum that exclusively constitutes the phage pool to produce second sera of the n phage pools, and optionally d) further selective selection of each of the second series of n phage pools on mixtures containing all n original sera, except for those used in the first selective selection; e) immunoscreening the resulting second series of n phage pools using a mixture of all n parent sera to form positive clones; f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an ordered array of said clones as phage-secreting colonies; g) generating replications of said phage secreting colonies; h) screening each replica for reactivity with positive and negative sera, obtaining clones reacting with positive sera; i) use of each of said specifically reacting phage as a ligate for affinity purification of antibodies from positive serum; j) testing said antibodies for their reactivity with the antigen-specific peptides already identified; k) separating clones that detect antibodies. 7. A method for diagnosing hepatitis C comprises identifying the binding specificity of anti-HCV antibody molecules in serum by an antigen mimicking antibody detection method (ADAM), comprising screening phage libraries using sera from HCV patients and uninfected individuals, identifying peptide binding antibodies (ligands) specifically associated with HCV infection. 8. The method of claim 7, wherein said ligands have superior in vitro maturation properties. r r. r. f tf t t r C * t- C r r e e e r r < e e r r. r. r r r t- r f r y r - r 'y r c f y r * The method of claim 7 or 8, wherein said ligands are synthetic peptides. The method of claim 6 or 7 or 8, wherein said ligands are attached to a common core. 11. The method of claim 8, wherein said ligands together with a common core form MAP. 12. A collection of HCV-specific ligands obtained by a method comprising: (a) first selective selection of n positive serum phage library to form first series of n phage pools; b) preparing a mixture of n funds containing n-1 funds; (c) selecting the affinity of each of the n mixtures for serum, which exclusively forms a phage pool to form second serum n phage pools, and optionally d) further selective selection of each of the second series of n phage pools on mixtures containing all n original sera, except for those used in the first selective selection; e) immunoscreening the resulting second series of n phage pools using a mixture of all n parent sera to form positive clones; f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an ordered array of said clones as phage-secreting colonies; g) generating replications of said phage secreting colonies; C ecrff Ctrr r ^r tfrrcerecrr 'r Cer c r ·. r r- r; r r r < r c r r / .- «- f h) screening each replica for reactivity with positive and negative sera, obtaining positive sera-reactive clones; i) use of each of said specifically reacting phage as a ligate for affinity purification of antibodies from positive serum; j) testing said antibodies for their reactivity with the antigen-specific peptides already identified; k) separating clones that detect antibodies. 13. The collection of claim 12, wherein the phage library of step a) is pVIII-12aa. 14. The collection of claims 12 and 13, wherein the inserts of the clones separated in step k) of claim 12 have the following sequences; SREQLNKLFGIEG, RATLSNEHGITIG, DQRENWFKYHGFG, EWRRYMSDIHGYG, DSLRYMYVMPGFG. 15. The collection of claim 12, wherein a phage library is generated wherein the best reacting clones are partially mutated such that each amino acid of the clone sequence is independently substituted with any other amino acid. 16. The collection of claim 12, wherein a phage library is generated wherein the reactive clones are partially mutated such that each amino acid of the clone sequence is independently substituted with any other amino acid. 17. The collection of claim 15 or 16, wherein said clones have the following insert sequence: SREQLNKLFGIEG. r r r, O f C f C yy yy <· yy yy. r rt r r A collection according to any one of claims 12 to 17, characterized in that in said phage library the random sequence is flanked by two cysteine residues. A collection according to any one of claims 12 to 18, wherein the peptide sequence is linked to a common core. 20. The collection of claim 17, wherein said peptide together with said common core is MAP. Use of a collection of any one of claims 12 to 20 for the preparation of a diagnostic assay for the determination of HCV in an individual suspected of having said HCV infection. A kit for diagnostic purposes, comprising the collection of claim 6. An HCV diagnosis kit comprising a collection of any one of claims 12 to 20. Kit according to claim 22 or 23, comprising strips on which said collection is immobilized. The kit of claim 24, wherein said strip further comprises an internal standard. 26. The peptide is selected from: (i) SRQLNKLFGIEG; (ii) RATLSNEHGITIG; iii) DQRENWFKYHGFG; o r * * f f f f f f f f r. e r r r r r r c r c r r c c. j 4 C yy yy yy yy iv) (v) (vi) (vii) (viii) (ix) (x) (xi) (xii) (xiii) (xiv) XV) xvi) xvii) xviii) xix) XX) xxi) xxii) xxiii) xxiv) XXV) xxvi) xxvii) xxviii) xxix) XXX) xxxi) xxxii) xxxiii) xxxiv) XXXV) EWRRYMSDIHGYG; DSLRYMYVMPGFG; YSREQLNKLFGIDTM; YSREQLNKMFGIEIS; YSREQLSKLFGIEPM; NSRWLSKAHGESGM; YSREQLNKLFGIEVM; YSRWQLSKLFGIDTQ; KSREQLSKLHGVDTS; RSREQLSKLFGIDLT; MWRTLMKTHGIESW; MLRTWLMKYQGIESW; YSRSWLMKAHGLELG; MMRSYLMKAHGIESL; MSRLWLMKAHGISSE; KHSEWLNKARGIESW; MSRTFLMKAHGIESW; MSRTWLMKAHGIESW; AEGEKKLRRSTNWGDPAK; AEGRFKTRRQTNYQDPAK; AEGEFKTLRNARLDPAK; AEGEFKTLRNSNRLDPAK; AEGEFKKFPGSSTPKDPAKAAFDSL; AEGEFPQDARFPGGGDPAKAAFDSL; PQDARFPGGGDPAKAAFDSL; AEGEFKGAGGAQTVDWALLVDPAK; AEGEFMQKHFGGAQWIMGDPAK; AEGEFLSLKGSGGGQLRALVDPAK; AEGEFLSLKGSGGAQLRALVDPAK; AEGEFYLLKRSSPPDPAKAAFDSL; AEGEFPILVGPYLLPRRSREEAVDPAK; AEGEFPILVGPYLLPRRSREEAVDPAKGK; ecef · p po r ec r rr t · eec ·; <* ap P r 're er J 0 eeec P rrcc prerrcr · ^r rrr - * r' rrrrr XXXVI) AEGEFRLGVRAPRKALDPAK; XXXVII AEGEFRLGVRALRKALDPAK; XXXVIII) AEGEFRLGVRALRKAPDPAK; xxxix) RLGVRALRKAPDPAK; xl) AEGEFTQPRGHSYQDPAK; XLI) AEGEFLKERAEMSARKTLGADPAK; XLII) AEGEFFYQIPRRMETKYGDPAK; XLIII) AEGEF SREQLNKLFGIEGDPAK; xliv) AEGEFNSREWLSKAHGIEGMDAK; XLV) AEGEFRSREQLSLFGIDLTDPAK; xlvi) AEGEFYSREQLNKLFGIDMTDPAK; XLVII) AEGEFYSREQLNKMFGIETSDPAK; XLVIII) AEGEFYSREQLNKLFGIEVMDPAK; xlix) AEGEFKSREQLRKLHGFDTSDPAK; 1) AEGEFKMRNYLNKAFGIEGMDPAK; If) AEGEFRSREQLSKLFGIELTDPAK; iii) AEGEFSRREYSNKAFGIETQDPAK; liii) AEGEFRRRWYLNKAFGIEGGDPAK; liv) AEGEFSRREWLNKRFGIEYLDPAK; lv) AEGEFMSRTWLMKAHGIESWDPAK; lions AEGEFYS PEWLNKARGIDRSDPAK; LVII) AEGEFKSREQLSKLHGVDTSDPAK; LVIII) AEGEFYSREQLNKMFGIEISDPAK; lix) AGEFYSRSWLMKAHGLELGDPAK; lx) AEGEFMMRSYLMKAHGIESLDPAK; LXI) AEGEFMSRLWLMKAHGISSEDPAK; LXII) AEGEFPQPQEVHVYREQLGLDPAKAAFDSL; LXIII) AEGEFGEVLYRGFDEVGGDPAKAAFDSL; LXIV) AGEPYWIERGMQDPAK; LXV) AEGEFTTASPRHFLVPLDPAKAAFDSL; XXXIX) AEGEFTTASPAHFLVPLDPAKAAFDSL; e f f f R R R R R R f r f f f f f f f f f f · - LXVII) AEGEFTTASPSHFLVPLDPAKAAFDSL; LXVIII) AEGEFATAPPRHYSWDPAK; LXIX) AEGEFATAPPAHYSWDPAK; LXX) AEGEFATAPPSHYSWDPAK; LXXI) AEGEFRFWKVPDYDPPAAGGDPAK; LXXII) AEGEFTESSVSSTLADLASKTFGSADPAK; LXXIII) AEGEFTLADLATMTFGSTDPAK; LXXIV) AEGEFGLADLATLTFGSPDPAK; 27th The use The peptide of claim 26 in the method 11th 28th collection according to any one of the claims az comprising at least one peptide of claim 26. An infection detection kit comprising the collection of claim 6. Kit according to claim 29 comprising strips on which said collection is immobilized. The kit of claim 30, wherein said strip further comprises an internal standard. A kit for detecting HCV infection comprising a collection of any one of claims 12 to 20. A kit for detecting HCV infection comprising at least one peptide from the collection of claim 25. A kit according to claim 32 or 33 comprising strips on which said collection is immobilized. f ť C Γ C Γ r yy r The kit of claim 34, wherein said strip further comprises an internal standard. Use of the collection of claim 6 for the preparation of vaccines. Use of the collection of any one of claims 12 to 20, or at least one peptide of claim 26, for the preparation of vaccines against HCV. An HCV vaccine comprising at least a collection of any one of claims 12 to 20, or at least a peptide of claim 26.