Office europeen des brevets (fi) Publication number : 0 624 597 A1
@ EUROPEAN PATENT APPLICATION
@ Application number: 94830208.8 @ Int. CI.5: C07K 7/10, G01N 33/576
(22) Date of filing : 02.05.94
The applicant has subsequently filed a @ Representative : de Simone, Domenico et al sequence listing and declared, that it includes Ing. Barzano & Zanardo Roma S.p.A. no new matter. Via Piemonte 26 1-00187 Roma (IT) (§) Priority : 12.05.93 IT RM930309
@ Date of publication of application : 17.11.94 Bulletin 94/46
@ Designated Contracting States : AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE
(ft) Applicant : SORIN BIOMEDICA S.p.A. Via Crescentino 1-13040 Saluggia (Vercelli) (IT)
(72) Inventor : Rosa, Carlo Sorin Biomedica S.p.A., Via Crescentino-13040 Saluggia (IT) Inventor : Osborne, Stephen John Sorin Biomedica S.p.A., Via Crescentino-13040 Saluggia (IT) Inventor : Griva, Silvia Sorin Biomedica S.p.A., Via Crescentino-13040 Saluggia (IT) Inventor : Garetto, Fulvio Sorin Biomedica S.p.A., Via Crescentino-13040 Saluggia (IT) Inventor : Bonelli, Fabrizio Sorin Biomedica S.p.A., Via Crescentino-13040 Saluggia (IT)
(54) HCV peptides and uses thereof. < fx* (57) Peptides comprising at least one of the dominant epitopes of the NS4b or NS5a HCV proteins and OJ uses thereof for immunodiagnostics are described.
O
LU Jouve, 18, rue Saint-Denis, 75001 PARIS c EP 0 624 597 A1
The invention relates to HCV peptides and to HCV derived chimeric peptides, comprising the dominant epitopes of non-structural proteins coded by NS4b and NS5a HCV genes, a process to obtain such peptides and uses thereof in developing immunodiagnostic kits. HCV is known as being the main agent of non-A non-B hepatitis (1). The HCV genome consists of a RNA 5 positive helix of 9,400 nucleotides appr., which codifies three structural proteins, namely: the core (C), two envelopes (envl and env2) and six non-structural proteins (NS), i.e. NS2, NS3, NS4a, NS4b, NS5a and NS5b. Such RNA regions comprise non coding fragments both at 5'- and at 3'- terminus, thus showing a similar or- ganisation as detected in Flavivirus and Pestivirus (2). In order to prepare diagnostic kits to detect HCV infections, virus coding sequences both of structural and 10 of non structural regions were expressed in E. coli, yeast and Baculovirus (3). The resulting proteins were used both as single protein or as combination thereof, to develop immunological kits that are able to capture anti- HCV anti antibodies from samples. Data known in the art from these assays show that HCV is the main responsible agent for the post- transfusional sporadic hepatitis, and that the use of said proteins is very advantageous in analyzing blood sam- 15 pies from donors (6,7). However, with assays known in the art and available on the market, some samples escape from the screening and give false negative results. Serological studies either on chimpazees or on post-transfusion HCV infected patients showed that the most important epitopes reside into the nucleocapside protein, consisting of NS3 and NS4b products (8,9,11,12). 20 Recently it was reported (13) that also the gene for the NS5a product codifies a product able to efficiently capture anti-HCV antibodies. The authors expressed a recombinant protein in E. coli named (C)-NS3, that is able to bind both to anti-C and to anti-NS3 antibodies (13). The chimeric protein is suitable to develop a sen- sitive assay to detect anti-HCV antibodies. Following a number of studies of serum conversion, in order to make available a proper test for the pres- 25 ence of anti-HCV antibodies in the blood from donors, the diagnostic kit must also contain the NS4b gene prod- uct, preferably the antigenic determinants thereof. This is confirmed through assays based on a NS4b derived polypeptide, known as c100-3, which, however, did not show satisfactory specificity and sensitivity. It was recently found (7) that 90% appr. of the HCV induced hepatitis were detected by carrying out a test, based on an ELISA assay, comprising the nucleocapsid, the NS3 product and the c100-3 polypeptide. 30 However it is advantageous to include also the NS5a gene product as a sensitive marker for the HCV in- duced infection in order to make available a more sensitive assay. Accordingly, in order to develop a suitable immunodiagnostic kit to detect HCV induced infections, having highest specif ity and sensitivity levels, an obvious need exists to have available various HCV proteins, orfrac- tions thereof. In order to prepare kits comprising more specific combined ligands, a further need exists to iden- 35 tify antigenic determinants of said proteins. Among the many theoretical sequences, the authors identified the main antigenic determinants of NS4b and NS5a, and analyzed the immunoreactivity thereof in a HCV detection test, based on the identification of such epitopes. The authors also synthesized chemically such epitopes into one chimeric peptide, comprising both the epitopes, and demonstrated how advantageously this peptide could be used to detect anti-HCV an- 40 tibodies, as having a specif ity and a sensitivity, which are comparable to those of peptides containing the epit- opes separately; however being more easily and economically used to develop immunodiagnostic kits on a large scale. Accordingly, it is an object of the invention a peptide comprising at least one of the dominant epitopes of either Hepatitis C Virus (HCV) NS4b or NS5a protein, preferably comprising at least one of the epitopes having 45 the aminoacid sequence, as shown in SEQ ID No. 1 or SEQ ID No. 2. In a preferred embodiment of the invention said peptide comprises at least one of said NS4b and one of said NS5a protein dominant epitopes, more pre- ferably the epitopes having the amino acid sequences of SEQ ID No. 1 and SEQ ID No. 2; most preferably said peptide comprises the amino acid sequence of SEQ ID No. 3. It is a further object of the invention a method to detect in vitro anti-HCV antibodies from biological fluids 50 through an immunologic assay, comprising the following steps: - to pick up a biologic fluid sample from a subject; - to incubate said sample together with an excess of the peptide according to this invention; - to remove the sample not bound to said peptide; and - to put in evidence the amount of bound antibodies to said peptide by using a detection system. 55 it is a further object of the invention a diagnostic kit to detect in vitro anti-HCV antibodies from biologic fluids, comprising: - an inert solid carrier, to which the peptide according to the invention is bound; - a container with a buffer to dilute the biologic fluid sample; 3 EP 0 624 597 A1
- a container with a washing buffer; - a container with anti-human IgG antibodies that are conjugated with a detecting system; - a container with a positive control sample; and - a container with a negative control sample. 5 The invention will be now described with reference to some clarifying, but not limiting Examples as well as to the annexed figures, wherein: - fig.1 is a bar diagram showing different reactivities of 14 peptides from NS4b against the BBI serum panel; - fig.2 is a table summarizing the NS4b-13 peptide (SEQ ID No. 1) reactivity against the BBI serum panel; 10 - f ig.3 is a map of the NS5a peptide sequences; - fig.4 shows the reactivity of peptides of fig. 3 when tested by an ELISA assay with no. 2 (a), no. 11 (b), no. 24 (c) samples of the BBI serum panel, and with a HCV negative serum (d); - f ig.5 is a table summarizing the NS5a-44 peptide (SEQ ID No. 2) reactivity against the BBI serum panel; - f ig.6 is a table summarizing the NS4b-NS5a chimeric peptide (SEQ ID No. 3) reactivity against the BBI 15 serum panel; - fig.7 is a neutralisation test of the serum sample n.3, previously treated with the NS4b-1 3 peptide before the test with the NS4b-NS5b chimeric peptide; - fig.8 is a neutralisation test of the serum sample n.11, previously treated with the NS5a-44 peptide before the test with the NS4b-NS5a chimeric peptide. 20 Market available sera are supplied by Boston Biomedica, Inc. (Massachussets, U.S.) (BBI serum panel). Donor blood samples are supplied by "Ospedale Santo Spirito" at Casale Monferrato, Italy. All the serum sam- ples are maintained at - 20°C until the tests were carried out.
EXAMPLE 1 - MAPPING OF EPITOPES OF NS4b AND NS5a GENE PRODUCTS AND CHIMERIC PEP- 25 TIDE SYNTHESIS
The NS4b and NS5a proteic sequences were analyzed using combined algorithms to predict potentially useful epitopes as capturing antigens. Depending upon hydrophilic and hydrophobic profiles of the HCV poly- protein sequence (1 8, 1 9) as well as the predicted secondary structure thereof (20), 1 5 peptides of various sizes 30 from NS4b sequence and 52 octapeptides from NS5a sequence are synthesized.
IMMUNOASSAYS: PROCEDURES
The specificity of synthetic peptides is tested using 300 serum samples from blood donors, as well as 35 through a further neutralisation assay, using separately both NS4b and NS5a epitopes as soluble antigens. The neutralisation assay is carried out as follows: samples no.3 and no.11 of the BBI serum panel, which react respectively only against C-100-3 and C 22C and against 5-1-1, C 33C, C 22C, according to the Ortho Riba II assay, are incubated with the NS4b and NS5a epitopes, or with a non correlated epitope, before being trans- ferred to tray wells, coated with the peptide to be analyzed; sample amounts (20 ^l) are mixed with PBS buffer 40 (20 nl), containing decreasing amounts (from 5 p.g up to 0.05 ng) of synthesized peptides. After having been incubated for 18 hours at room temperature, mixtures are transferred on ELISA assay trays and the procedure, as described hereinafter, is carried out. Results are reported as binding activity per- centages of peptides as compared to immunoreactivity when no soluble antigens are used.
45 ANTI-HCV ELISA ASSAY
The antigenic activity is tested through indirect, solid phase ELISA assays, consisting of microtitration trays, that are prepared as follows: 1) synthetic peptides are solubilized with the smallest volume of 1 00% tri- f luoroacetic acid and then diluted to a final concentration of 5 ng/ml in a 0.05 M carbonate buffer, pH 9.6. Cys- 50 teine containing peptides, which must be maintained as linear monomers, are resuspended in citrate buffer, pH 4, to be used to coat the tray wells. The synthetic peptide is dissolved in 100% trif luoroacetic acid to 2 mg/ml. Then, the solution is diluted to 5 ng/ml with carbonate buffer, pH 9.6. 200 ^l are added to each well and an incubation for 1 hour at 37°C is carried out; 2) wells are then washed out and blocked using a solution containing proteins (1 00 mM Tris-HCI, 0.2% BSA); 3) after having removed the fluid, wells are dried for 1 8 hours 55 at room temperature and then sealed into polyethylene bags and maintained at 4°C until its use. The assay is carried out as follows: peptide coated wells are incubated for 30 minutes at 37°C with 200 ^l of 1:21 diluted serum, into the same solid phase blocking solution; then wells are washed 5 times with a 10 mM PBS, pH 7.4 buffer with 0.25% Triton x-100 . The enzyme tracer, consisting of 100 ^l of goat anti-human 4 EP 0 624 597 A1
IgG serum, is then added to each well after having been diluted with the diluent as above. After having been incubated for 30 minutes at 37°C, during which the enzyme tracer reacts with solid phase bound proteins, wells are washed with washing buffer; then, 100 ^l of a solution containing a chromogen substrate (0.01% tetrame- thylbenzidine in 0.005% of H202) is added; after having been incubated for 30 minutes at room temperature, 5 the reaction is blocked with 100 ^l of a 2N H2S04 blocking solution. Differential optical densities (at 450 nm and 630 nm) of the solution in each well are measured on a colorimeter (BIOTEK EL 311) and related to the presence of the antibody, which reacts with the peptide in solid phase. Oligopeptides, which are synthesized directly in solid phase as previously described, are blocked using the same buffer, as described in the conventional ELISA assay. 10 RESULTS
MAPPING OF NS4b EPITOPES WITH SYNTHETIC PEPTIDES NS4b PEPTIDE SYNTHESIS
15 Peptide synthesis is carried out using an automatic Milligen 9050 synthesizer (Mi llipore Co., Ma, U.S.) us- ing a Fmoc chemical technique. Peptide purity levels are checked through reverse phase HPLC and result in the range 85-90%. Amino acid sequences are checked by a PI2090 E microsequencer. The amino acid num- bered positions refer to those used in the PCT Application No. W092/03458. Sequences of the 14 synthesized peptides are shown in Table 1 herebelow. 20 Table 1 NS4b PEPTIDES Peptide Sequence 25 NS4b-l 1688-1720 NS4b-2 1710-1721 NS4b-3 1725-1734 30 NS4b-4 1734-1753 NS4b-5 1742-1758 NS4b-6 1781-1796 1794-1805 35 NS4b-7
NS4b-8 1861-1881 NS4b-9 1867-1881 40 NS4b-10 1902-1918 NS4b-ll 1911-1926 NS4b-12 1918-1935 45 NS4b-15 1924-1935 NS4b-13 1926-1943 NS4b-14 1935-1952
50 The results of an assay on 25 samples, which are positive in respect of anti-HCV antibodies of BBI serum panel, with the 1 5 synthesized peptides from NS4b are shown in f ig.1 . Numbers of amino acid positions refer to those shown in the PCT No. WO92/03458. Values are calculated as cumulated absorbance (i.e. 450 nm optical density less 630 nm optical density, 55 divided by the number of the samples, less the optical density average from the donor blood samples). Major antigenic domains map in the following peptides: NS4b-1 (1688-1720), NS4b-12 (1918-1935) and NS4b-13 (1926-1943). However the 1688-1720 peptide, when tested with some sample from blood donors, showa too high percentage (4%) of false positive reactions, while the 1918-1935 and 1926-1943 peptides show a high 5 EP 0 624 597 A1
specificity level. In order to better define the minimum epitope into the two peptides, the NS4b-1 5 (1924-1935) is analyzed. This peptide reacts more than the 1918-1935 peptide, but less than the 1926-1943 peptide. Accordingly, the 1926-1943 region is critical in order to define the highest sensitivity. 5 As shown in fig.2, the NS4b-13 peptide is used in an ELISA assay with different sera of the BBI panel and found to be immunoreactive for 19 samples out of 25. NS4b sequence is shown in SEQ ID No. 1.
NS5a EPITOPE MAPPING WITH SUPERIMPOSING OCTAPEPTIDES NS5a SYNTHESIS
10 Octapeptides superimposing for only one amino acid, comprised into the highest hydrophilic region of the NS5a gene product, are synthesized on polyethylene substrates using materials supplied by Cambridge Re- search Biochemicals, Inc., Cambridge, U.K., according to the Manufacturer's instructions (Epitope Scanning Pin Kit). The process is carried out by combining activated esters for Fmoc with an HOBT (t-butyloxycarboxyl) for protection purposes. This method is described by Geysen and al. (14, 16) and is carried out as modified, 15 as specified in (21). In such a way 52 octapeptides are synthesized throughout the more hydrophobic domain of the NS5a polypeptide, having the sequences as shown in fig. 3. Preliminary ELISA assays show that there is a no specific binding among human IgG from donor blood and substrates bearing the octapeptides, when a serum dilution less than 1:1000 are used, thus allowing to define the useful serum dilution to get a positive sample. 20 The substrate immunoreactivity is evaluated using three samples of the BBI serum panel (namely nos. 2, 11 and 24 samples), already previously classified as having an high anti-NS5a titer, according to the commer- cially available Inno-Lia assay (Innogenetics, Belgium). The ELISA assay results, based on the octapeptides with the three samples of sera of the BBI panel and the donor's blood sample, are shown in the fig. 4a, 4b, 4c and 4d. Data show a significant reactivity for most 25 of the analyzed samples; anywhere the most reactive region comprises the sequence from aa. 1 to aa. 8 of SEQ ID No. 2. The results of the ELISA assays with the octapeptides are highly reproducible and control samples, incu- bated with positive and negative sera, show that this procedure is effective in fully removing the substrate bound IgGs. 30 After having the epitopes been defined, a peptide, having the SEQ ID No. 2 sequence (NS5a-44) is syn- thesized. As shown in fig. 5, this peptide is subjected to an ELISA assay, showing that the peptide is immunoreactive against the same three samples that were used in the epitope mapping; moreover, this peptide shows the ability to detect furthermore four other samples from the BBI serum panel. 35 This peptide does not show any false positive reaction when used with donor's blood samples.
CHIMERIC PEPTIDE SYNTHESIS
After having been defined the minimum immunodominant sequences for both NS4b and NS5a peptides, 40 a chimeric peptide, comprising the NS4b and NS5a peptides, spaced by two glycine residues, is synthesized, having the sequence as follows (SEQ ID No. 3):
Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Gly Gly Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly.
The chimeric peptide is analyzed through an indirect ELISA assay with the BBI serum panel, and the results so are shown in fig.6. 21 sample of anti-HCV positive sera are captured by the immobilized chimeric peptide, with the ELISA assay. Also the specificity in respect of 300 blood donor samples is tested and no false positive reaction detected (average value: 0.085; standard deviation: 0.028). To carry out the neutralisation assay, no. 3 and 11 samples of the BBI serum panel are used. As shown 55 in fig. 7, a pre-incubation of the no.3 sample with the soluble NS4b-14 peptide before adding the NS4b-NS5a chimeric peptide to the coated wells, causes an immunoinhibition of the binding up to 80%; while no significant inhibition occurs when a non correlated peptide is used. In a similar way, the no. 11 sample binding shows to be inhibited up to 60%, after having been incubated with the NS5a-44 peptide, while no significant inhibition 6 EP 0 624 597 A1
occurs when a non correlated peptide is used, as shown in fig.8. The invention has been described with reference to specific peptides, but it us to be understood that the method and the diagnostic kit may comprise further HCV peptides not specifically disclosed in the instant spec- if ication but having reactivities comparable. 5 REFERENCES
1. Choo, Q.Land al.: Science 244, 359-362, 1989. 2. Grakoni, A. and al.: Journ. of Virology 67, 1385-1395, 1993. w 3. Nasoff, M.S. and al.: Proc. Natl. Acad. Sci. USA 88, 5462-5466, 1 991 . 4. Kuo, G. and al.: Science 244, 362-364, 1989. 5. Chiba, J. and al.: Proc. Natl. Acad. Sci. 88, 4641-4645, 1991. 6. Alter, M.G. and al.: JAMA 264, 2231-2235, 1990. 7. Lelie, P.N. and al.: Journ. of Medical Virology, vol.37, 203-209. 15 8. Beach, M.J. and al.: Journ. of Medical Virology, 36, 226-237, 1992. 9. Vallari, D.S. and al.: Journ. Clin. Microbiol., 30, 552-556, 1992. 11. Desai, S.M. and al.: 1st annual meeting, July 6-9 1992,Fondazione Cini, Venice, Italy 12. Lesniewki R.R. and al.: 1st annual meeting, July 6-9, 1992, Fondazione Cini, Venice, Italy 13. Chien, D.Y. and al.: Proc. Natl. Acad. Sci. USA, 89, 10011-10015, 1992. 20 14.0sborne S.E. and al.: Proceedings of the 1993 Miami Biotechnology Winter Symposium; edited by Brew, K., Petsko G.A., Ahmad, F., Bialy, H., Black, S., Fernandez, A, Fenna, R.E., Lee, E.Y.C. and Whelan, W.J., pag.68. 15. Dawson, G.J. and al.: J. Clin. Microbiol., 29, 551-556, 1991. 16. Nicholson, S. and al.: J. of Virological Methods, 33, 311-317. 25 17. Alter, M.G. and al.: The New England Journal of Medicine, 27, 1899-1905, 1993. 18. Hopp, T.P. and Woods, K.R.: Proc. Natl. Acad. Sci. USA, 78, 3824, 1981. 19. Kyte, J. and Doolittle, R.F.: J. Mol. Biol., 157, 105, 1982. 20. Chou, P.Y and Fasman, G.P., Biochemistry, 13, 22, 1974. 21. Cambridge Research Biochemicals Epitope scanning Pin recorder Kit.
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5 SEQUENCE LISTING
(1) GENERAL INFORMATION: 70 (i) APPLICANT: (A) NAME: Sorin Biomedica S.p.A. (B ) STREET: Via Crescentino (C) CITY: Saluggia (D) STATE: VC 7S (E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 13040
(ii) TITLE OF INVENTION: HCV peptides and uses thereof
(iii) NUMBER OF SEQUENCES: 55 20 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS /MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) 25 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids 30 (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO 35
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala 40 1 5 10 15
Arg
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50 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
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(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
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Arg Gly Gly Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val. 20 25 30
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(D) TOPOLOGY: linear
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{XXX) HYPOTHETICAL: NO
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40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: lie Ser Val Pro Ala Glu lie Leu 1 5
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JO
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(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Lys Ser Arg Arg Phe Ala Gin Ala 1 5
INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
16 EP 0 624 597 A1
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO : 2 6 : Ser Arg Arg Phe Ala Gin Ala Leu ■1 i-
l^J XJN c UKMAT1UN tOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 15 (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
iXl) ityULNUE DESCRIPTION: SEQ ID NO : 2 / : Arg Arg Phe Ala Gin Ala Leu Pro 25 1 5
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids 30 (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO 35
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Arg Phe Ala Gin Ala Leu Pro Val to 1 5
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids '5(5 (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
7 EP 0 624 597 A1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Phe Ala Gin Ala Leu Pro Val Trp 1 5
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Ala Gin Ala Leu Pro Val Trp Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B ) TYPE: amino acid 30 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Gin Ala Leu Pro Val Trp Ala Arg 1 5 40 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid 45 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO 50
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
55
18 EP 0 624 597 A1
Ala Leu Pro Val Trp Ala Arg Pro 1 5
INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Leu Pro Val Trp Ala Arg Pro Asp 1 5
INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Pro Val Trp Ala Arg Pro Asp Tyr 1 5
INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
19 EP 0 624 597 A1
Val Trp Ala Arg Pro Asp Tyr Asn 1 5
(2) INFORMATION FOR SEQ ID NO: 36:
10 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 15 (iii) HYPOTHETICAL: NO
DESCRIPTION: SEQ ID NO:36: 20 (xi) SEQUENCE Trp Ala Arg Pro Asp Tyr Asn Pro 1 5
(2) INFORMATION FOR SEQ ID NO: 37: 25 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
30 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Ala Arg Pro Asp Tyr Asn Pro Pro 1 5
(2) INFORMATION FOR SEQ ID NO: 38: 40 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
45 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Arg Pro Asp Tyr Asn Pro Pro Leu
55
20 P 0 624 597 Al
1 5
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS: 100 (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
15'5 (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 20 Pro Asp Tyr Asn Pro Pro Leu Val 1 5
(2) INFORMATION FOR SEQ ID NO: 40: CHARACTERISTICS: 25 (i) SEQUENCE (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide 30 (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 35 Asp Tyr Asn Pro Pro Leu Val Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 41:
40 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide 45 (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: 50 Tyr Asn Pro Pro Leu Val Glu Thr 1 5
55
21 EP 0 624 597 A1
5
2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Asn Pro Pro Leu Val Glu Thr Trp 1 5
(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
35 Pro Pro Leu Val Glu Thr Trp Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS : 40 (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
45 (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
50 Pro Leu Val Glu Thr Trp Lys Lys 1 5
55
22 EP 0 624 597 A1
J.JNEUKMATXUN t'OK SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid 10 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO IO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Leu Val Glu Thr Trp Lys Lys Pro 1 5
2) INFORMATION FOR SEQ ID NO : 4 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid CO (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 6 :
Val Glu Thr Trp Lys Lys Pro Asp 1 5 JO (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO to
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Glu Thr Trp Lys Lys Pro Asp Tyr 50 1 5 (2) INFORMATION FOR SEQ ID NO: 48:
?o
!3 EP 0 624 597 A1
(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 10 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Thr Trp Lys Lys Pro Asp Tyr Glu 1 5
20 (2) INFORMATION FOR SEQ ID NO : 4 9 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid TOPOLOGY: linear 25 (D) (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 9 :
Trp Lys Lys Pro Asp Tyr Glu Pro 1 5 35 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid 40 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50;
Lys Lys Pro Asp Tyr Glu Pro Pro 1 5 50 (2) INFORMATION FOR SEQ ID NO : 5 1 :
55
24 EP 0 624 597 A1
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Lys Pro Asp Tyr Glu Pro Pro Val 1 5
INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Pro Asp Tyr Glu Pro Pro Val Val 1 5
INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO
Asp Tyr Glu Pro Pro Val Val His 1 5
INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
25 EP 0 624 597 A1
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Tyr Glu Pro Pro Val Val His Gly 1 5
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Glu Pro Pro Val Val His Gly Cys 1 5
Claims
1 . A peptide comprising at least one of the dominant epitopes of either Hepatitis C Virus (HCV) NS4b or NS5a protein.
2. A peptide according to claim 1 comprising at least one of the epitopes having the amino acid sequenceas shown in SEQ ID No. 1 or SEQ ID No. 2.
3. A peptide according to claim 1 comprising at least one of said NS4band one of said NS5a protein dominant epitopes
4. A peptide according to claim 3 comprising the amino acid sequences of SEQ ID No. 1 and of SEQ ID No. 2.
5. A peptide according to claim 4 comprising the amino acid sequence of SEQ ID No. 3.
6. Amethod to detect in vitro anti-HCV antibodies from biological fluids through an immunologic assay, com- prising at least the following steps: - to pick up a biologic fluid sample from a subject; - to incubate said sample together with an excess of a peptide according to any of previous claims; - to remove the sample not bound to said peptide; and
26 EP 0 624 597 A1
- to put in evidence the amount of bound antibodies to said peptide by using a detection system.
Diagnostic kit to detect in vitro anti-HCV antibodies from biologic fluids, comprising at least: - an inert solid carrier, to which a peptide according to any of claims from 1 to 5 is bound; - a container with a buffer to dilute the biologic fluid sample; - a container with a washing buffer; - a container with anti-human IgG antibodies that are conjugated with a detecting system; - a container with a positive control sample; and - a container with a negative control sample.
27 c
- EP 0 624 597 A1
HCV BBI SERA NSLh-13 fflBA ll QtW,
5-1-1 C-100-3 C33C CZZC
I 0.600 +/- - 2 4 Z 0.015 12 4 4 3 2.403 - 1 -4 4 0.012 - 5 0.754 2 +/- +/- 4 B 3,429 2 1 4 4 7 0.011 - - " - B 0.580 - +/- 3 9 1.881 1-44 10 0.166 +/- +/- 2 II 3.551 2 +/- 3 3 12 2.390 2 2 2 3 13 0.467 4 4 4 4 14 0.013 2 +/. 4 4 15 3.473 2 +/- 4 4 16 3.540 4 4 4 4 17 3.542 4 4 4 4 18 3.585 +/- 2 4 3 19 0.017 - +/- 20 0.266 1 1 1 +/- 21 2.996 1 +/- 3 4 22 3.350 +/- - 4 4 23 3.410 4 3 4 4 24 3.618 2 4 4 4 25 3.636 4 1 4 4
FIG. 2
29 r* U t>24 Al
(OCTA PEPTIDE SEQUENCES) 256-FDPLVAEE BPLYAEED PLVAEEDE LVAEEDER iAEEDERE AEEDERE1 EEDEREiS EDEREISV QEREISVP EREISVPA BEISVPAE EISVPAEI 1SVPAEIL fiVPAEILR YPAEILRK EAEILRKS AEILRKSR EILRKSRR 1LRKSRRF LRKSRRFA BKSRRFAQ KSRRFAQA SRRFAQAL ERFAQALP BfAQALPV FAQALPYW AQALPVWA QALPVWAR ALPVWARP LPVWARPD PVWARPDY VWARPDYN SffARPDYNP ARPDYNPP BPDYNPPL PDYNPPLV DYNPPLVE YNPPLVET NPPLVETW PPLVETWK ELVETWKK LVETWKKP XETWKKPD - - ETWKKPDY \- \ln < IWKKPDYE ' HKKPDYEP KKPDYEPP KPDYEPPV EDYEPPVV fiYEPPVVH YJPPVVHG EPPVVHGC-2307 k
i 1
i " I- - • ~
Q
0 r
4j EP 0 624 597 A1
HCV BBI SERA NS5a-M RJBA ll QEH
5-M C-100-3 C33C C22C
1 0.400 +/- 2 4 2 2.194 1 2 4 4 3 0.083 - 1 - 4 4 0.034 5 0.020 2 +/- +/- 4 6 0.140 2 1 4 4 7 0.005 .... 8 0.076 - +/- 3 - 9 0.015 1-44 10 0.009 +/- +/- 2 11 2.301 2 +/- 3 3 12 0.167 2 2 2 3 13 0.043 4 4 4 4 14 0.055 2 +/- 4 4 15 2.114 2 +/- 4 4 16 0.065 4 4 4 4 17 0.093 4 4 4 4 18 1.523 +/- 2 4 3 19 0.012 - +/- 20 0.342 1 1 1 +/■ 21 0.054 1 +/- 3 4 22 0.053 +/- - 4 4 23 2.397 4 3 4 4 24 2.374 2 4 4 4 25 0.115 4 1 4 4
FIG. 5
35 EP 0 624 597 A1
HCV BBI SERA NSl*b-NS5a. RIBA II GEN.
5-1-1 C-100-3 C33C C22C
1 1.305 +/- - 2 4 2 3.179 1 2 4 4 3 2.231 1 - 4 4 0.068 .... 5 0.866 2 +/- +/- 4 6 3.210 2 1 4 4 7 0.090 .... 8 0.914 - +/- 3 9 1.907 1-44 10 0.463 +/- +/- 2 11 2.949 2 +/- 3 3 12 2.130 2 2 2 3 13 0.309 4444 14 0.076 2 +/- 4 4 15 3.059 2 +/- 4 4 16 3.199 4 4 4 4 17 3.102 4 4 4 4 18 3.150 +/- 2 4 3 19 0.073 - +/- - 20 0.302 1 1 1 +/'- 21 2.915 1 +/- 3 4 22 1.349 +/- 4 4 23 3.260 4 3 4 4 24 3.237 2 4 4 4 25 3.288 4 1 4 4
FIG. 6
36 T
ff\ 1
S%
I 1 1 1 1 1 1 1 300000000
NOIllQlHNIONnMUl %
37 T _ EP 0 624 597 A1
Patent i) European EUROPEAN SEARCH REPORT vppiicauon numoer Dfficc
Ef y4BJU/UB.B DOCUMENTS CONSIDERED TO BE RELEYAN I Citation of document with indication, where appropriate, Kctcvam ^LAaoirivAiivsi'* ur inc. Category of relevant passages to daim APPLICATION (Int. CI.S) wo ■ 33/06 488 1-J , b L- U / Is. //1U [GENELABS TECHNOLOGIES) 7 G 01 N 33/576 * Page 124, SEQ. ID. NO. 7, AA 6-22; claims 1,5-8,14
P.X WO 93/18 054 (N.V. INNOGENETICS ) 7 * Claim 1; claim 4, page 94, SEQ. ID. NO. 18, AA 1-15 *
EP 0 489 968 1- J , b INNOGENETICS N.V.) 7 * Claims 18,22-25
EP - A 0 468 527 (UNITED BIOMEDICAL INC.) * Page 81, SEQ. ID. NO. 309B, AA 2-16; page 81, SEQ. ID. No. 309C, AA 11-25
SEARCHED (lot CIS)
C UV K //UU G 01 N 33/00
The present search report has been drawn up for all claims ^TlSRNA lgKtfffB'TW4~" SCftKKF
CATEGORY OF CITED DOCUMfcNl b I : theory or principle underlying tne invention E : earlier patent document, but published on, or X : particularly relevant if liken alone after the Filing date Y : particularly relevant if combined with another D : document cited in the application tfoenment of the same category L : document died for other reasons A : technological background O : tio*- written disclosure tt : member of the same patent family, corresponding P : ix-termediate document document
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