CA2074370C - Synthetic antigens for the detection of antibodies to hepatitis c virus - Google Patents
Synthetic antigens for the detection of antibodies to hepatitis c virus Download PDFInfo
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- CA2074370C CA2074370C CA2074370A CA2074370A CA2074370C CA 2074370 C CA2074370 C CA 2074370C CA 2074370 A CA2074370 A CA 2074370A CA 2074370 A CA2074370 A CA 2074370A CA 2074370 C CA2074370 C CA 2074370C
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
Peptide sequences are provided which are capable of mimicking proteins encoded by HCV for use as reagents for screening of blood and blood products for prior exposure to HCV. The peptides are at least 5 amino acids long and can be used in various specific assays for the detection of antibodies to HCV, for the detection of HCV antigens, or as immunogens.
Description
2074370, SYNTHETIC ANTIGENS FOR THE DETECTION OF ANTIBODIES
TO HEPATITIS C VIRUS
The implementation of systematic testing for hepatitis B virus (HBV) has been instrumental in eliminating this virus from the blood supply.
Nevertheless, a significant number of post-transfusion hepatitis (PTH) cases still occur. These cases are generally attributable to non-A, non-B
io hepatitis (NANBH) virus(es), the diagnosis of which is usually made by exclusion of other viral markers.
The etiological agent responsible for a large proportion of these cases has recently been cloned (Choo, Q-L et al. Science (1988) 244:359-362) is and a first-generation antibody test developed (Kuo, G. et al. Science (1989) 244:362-364). The agent has been identified as a positive-stranded RNA virus, and the sequence of its genome has been partially determined. Studies suggest that this virus, referred to subsequently as hepatitis C virus (HCV), may be related to flaviviruses and pestiviruses.
zo A portion of the genome of an HCV isolated from a chimpanzee (HCVCDC/CHI ) is disclosed in EPO 88310922.5. The coding sequences disclosed in this document do not include sequences originating from the 5'-end of the viral genome which code for putative structural proteins.
Recently however, sequences derived from this region of the HCV
u genome have been published (Okamoto, H. et al., Japan L Ev-., Med.
60:167-177, 1990.). The amino acid sequences encoded by the Japanese clone HC-J1 were combined with the HCVCDC/CHI sequences in a region where the two sequences overlap to generate the composite sequence depicted in Figure 1. Specifically, the two sequences were 3o joined at glycine451 . It should be emphasized that the numbering $u rru1'S SHE
.2-system used for the HCV amino acid sequence is not intended to be absolute since the existence of variant HCV strains harboring deletions or insertions is highly probable. Sequences corresponding to the 5', end of the HCV genome have also recently been disclosed in EPO
$ 90302866Ø
In order to detect potential carriers of HCV, it is necessary to have access to large amounts of viral proteins. In the case of HCV, there is currently no known .method for culturing the virus, which precludes the 1o use of virus-infected cultures as a source of viral antigens. The current first-generation antibody test makes use of a fusion protein containing a sequence of 363 amino acids encoded by the HCV genome. It was found that antibodies to this protein could be detected in 75 to 85% of chronic NANBH patients. In contrast, only approximately 15% of those is patients who were in the acute phase of the disease, had antibodies which recognized this fusion protein (Kuo, G. et al. Science (1989) 244:362-364). The absence of suitable confirmatory tests, however, makes it difficult to verify these statistics. The seeming similarity between the HCV genome and that of flaviviruses makes it possible to predict the 20 location of epitopes which are likely to be of diagnostic value. An analysis of the HCV genome reveals the presence of a continuous long open reading frame. Viral RNA is presumably translated into a long polyprotein which is subsequently cleaved by cellular and/or viral proteases. By analogy with, for example, Dengue virus, the viral is structural proteins are presumed to be derived from the amino-terminal third. of the viral polyprotein. At the present time, the precise sites at which the polyprotein is cleaved can only be surmised. Nevertheless, the structural proteins are likely to contain epitopes which would be useful for diagnostic purposes, both for the detection of antibodies as well as 3o for raising antibodies which could subsequently be used for the detection ,USSYM-M
- 2a-of viral antigens. Similarly, domains of nonstructural proteins are also expected to contain epitopes of diagnostic value, even though these proteins are not found as structural components of virus particles.
Summary of the Invention In one aspect of the present invention there is provided a peptide selected from:
(a) the group of amino acid sequences consisting of.
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) - 2b -(61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, and (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (VII), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (VII) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
wherein the variant consisting of the sequence: Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, is excluded; and (c) fragments of peptides (I) to (VII) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80 and 73-92 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV; and provided that said peptides are different from the following list of peptides:
(1) Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Xi-Lys, wherein Xi represents Arg, (2) Pro-Lys-Pro-Gln-Xi-Lys-X2-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, wherein Xi represents Arg, and X2 represents Thr, (3) Gln-Asn-Val-Lvs-Phe-Pro.
- 2c-(4) Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly, (5) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg, (6) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (7) Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (8) Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile, (9) Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg, (10) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and (11) Gln-Arg-Lys-Thr-Lys-Arg.
In another aspect of the present invention there is provided a peptide composition comprising a peptide described herein, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-C ys-S er-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gin-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) -2d-(1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) (2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val- Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X, and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) - 2e -wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (VIII) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (VIII) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (VIII) to (XIX) having at least 6 amino acids of any of the peptide sequences 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743, 1730-1749, 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV.
In another aspect of the present invention there is provided use of a peptide or peptide composition described herein, for the incorporation into an immunoassay for detecting the presence of antibodies to Hepatitis C virus present in a body fluid.
In another aspect of the present invention there is provided a method for the in vitro detection of antibodies to Hepatitis C virus present in a body fluid comprising at least the steps of. (a) contacting body fluid of a person to be diagnosed with the peptide or peptide composition according to any one of claims 1 to 9; and, (b) detecting the immunological complex formed between said antibodies and the peptide(s) used.
In another aspect of the present invention there is provided a kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising at least the following components:
a nentide or nentide composition described herein -2f-the means for detecting an immunological complex formed between said peptides and said antibodies.
In another aspect of the present invention there is provided use of a peptide or peptide composition described herein, for incorporation into a vaccine composition against HCV.
In another aspect of the present invention there is provided a peptide or peptide composition described herein, for raising antibodies against HCV.
In another aspect of the present invention there is provided a peptide or peptide composition obtained by cyclizing any of the peptides described herein, or by coupling together two peptides described herein.
In another aspect of the present invention there is provided a peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-lle-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) -2g-(1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) to (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gin-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a process for preparing a peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) - 2h -(1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids.
In another aspect of the present invention there is provided a process for preparing a peptide (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-GIu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a -4.v nr -Nrl *%bnoa nmmriricinn at-net nn,- aminn an;rl and ,c many ne (.(1 amine - 2j -acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids and with said at least two peptides being mixed.
In another aspect of the present invention there is provided a process for the preparation of a kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising gathering at least the following components:
a peptide described herein, a means for detecting an immunological complex formed between said peptides and said antibodies.
In another aspect of the present invention there is provided a process for the preparation of a peptide composition described herein, wherein said peptides are such as obtained by synthesizing a peptides described herein directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides.
In another aspect of the present invention there is provided a peptide selected from:
(a) the group of amino acid sequences consisting of:
(2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) - 2k -(2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XV III)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-V al-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X, and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (XV) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (XV) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (XV) to (XIX) having at least 6 amino acids of any of the peptide sequences 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV, and provided that said peptides are different from the following list of peptides: Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg (2265-2280) and Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-m_.. A 1 _ __ /nnOfN r-nnm In another aspect of the present invention there is provided a peptide composition comprising a peptide described herein, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-S er-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys- V al-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, (80) - 2m-(73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) - 2n-wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (XIV), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (XIV) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
and (c) fragments of peptides (I) to (XIV) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80, 73-92, 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743 and 1730-1749, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV, provided that the combinations containing at least one peptide selected from the group of amino acid sequences consisting of I, II, IIA, III, IV, V, VI, VII and at least one peptide selected from the group of amino acid sequences consisting of VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX are excluded.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
-2o-In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVIII) Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVIII) Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro.
In another aspect of the present invention there is provided a peptide consisting of the amino - 2p -acid sequence: (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
In another aspect of the present invention there is provided a peptide selected from the group consisting of: (a) a peptide consisting of a combination of at least two of amino acid sequences: II, III, V, IX and XVIII; (b) a peptide consisting of a combination of at least two of amino acid sequences: I, II, V, IX, XI, XVI and XVIII; (c) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV, V, VIII, XI, XVI and XVIII; (d) a peptide consisting of a combination of at least two of amino acid sequences: II, IX and XVIII; (e) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV and V; (f) a peptide consisting of a combination of at least two of amino acid sequences: VIII, IX, XI, XIII and XIV; and (g) a peptide consisting of a combination of at least two of amino acid sequences: XV, XVI, XVII, XVIII and XIX;
wherein the amino acid sequences I, II, III, IV, V are as defined herein, and wherein the amino acid sequences VIII, IX, XI, XIII, XIV, XV, XVI, XVII, XVIII, and XIX
are as defined herein.
In another aspect of the present invention there is provided a peptide consisting of a combination of at least two peptides selected from the group consisting of:
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala;
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn;
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr;
(XV III)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-V al-His-Gly-Cys-Pro-Leu-Pro-Pro; and (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-- 2q -Arg-Glu-Phe-Asp-Glu, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-t0 Glu-Glu-Cys-Ser-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (X) Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XI) Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys -Gln-Lys .
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XII) Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
- 2r -In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala.
In another aspect of the present invention there is provided a peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV;
wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are as defined herein.
In another aspect of the present invention there is provided a peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are as defined herein.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-VaI-Lys-Phe-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (III)Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
-2s-In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly, and a linker arm which is I to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-V aI-Lys -Phe-Pro- Gl y.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly.
-2t-In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VII)Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly.
In another aspect of the present invention there is provided A peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; and wherein the amino acid sequences I, II, III, IV, V, VI and VII are as defined herein.
In another aspect of the present invention there is provided A peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences I, II, III, IV, V, VI, and VII are as defined herein.
s Brief Description of the Drawings Figure 1 shows the amino acid sequence of the composite HCVHC-io Figure 2 shows the antibody binding to individual peptides and various mixtures in an ELISA assay Description of the Specific Embodiments 15 It is known that RNA viruses frequently exhibit a high rate of spontaneous mutation and, as such, it is to be expected that no two HCV isolates will be completely identical, even when derived from the same individual. For the purpose of this disclosure, a virus is considered to be the same or equivalent to HCV if it exhibits a global homology 20 of 60 percent or more with the HCVHC-J1/CDC/CHI composite sequence at the nucleic acid level and 70 percent at the amino acid level.
Peptides are described which immunologically mimic proteins encoded by HCV. In order to accommodate strain-to-strain variations in sequence, 25 conservative as well as non-conservative amino acid substitutions may be made. These will generally account for less than 35 percent of a specific sequence. It may be desirable in cases where a peptide corresponds to a region in the HCV polypeptide which is highly polymorphic, to vary one or more of the amino acids so as to better mimic the different 30 epitopes of different viral strains.
!0074370 The peptides of interest will include at least five, sometimes six, sometimes eight, sometimes twelve, usually fewer than about fifty, more usually fewer than about thirty-five, and preferably fewer than about twenty-five amino acids included within the sequence encoded by the s HCV genome. In each instance, the peptide will preferably be as small as possible while still maintaining substantially all of the sensitivity of the larger peptide. It may also be desirable in certain instances to join two or more peptides together in one peptide structure.
to It should be understood that the peptides described need not be identical to any particular HCV sequence, so long as the subject compounds are capable of providing for immunological competition with at least one strain of HCV. The peptides may therefore be subject to insertions, deletions, and conservative or non-conservative amino acid substitutions is where such changes might provide for certain advantages in their use.
Substitutions which are considered conservative are those in which the chemical nature of the substitute is similar to that of the original amino acid. Combinations of amino acids which could be considered zo conservative are Gly, Ala; Asp, Glu; Asn, Gin; Val, Ile, Leu; Ser, Thr; Lys, Arg; and Phe, Tyr.
Furtheiinore, additional amino acids or chemical groups may be added to the amino- or carboxyl terminus for the purpose of creating a "linker is arm" by which the peptide can conveniently be attached to a carrier.
The linker arm will be at least one amino acid and may be as many as 60 amino acids but will most frequently be 1 to 10 amino acids. The nature of the attachment to a solid phase or carrier need not be covalent.
-s 2074370 Natural amino acids such as cysteine, lysine, tyrosine, glutamic acid, or aspartic acid may be added to either the amino- or carboxyl terminus to provide functional groups for coupling to a solid phase or a carrier.
However, other chemical groups such as, for example, biotin and s thioglycolic acid, may be added to the termini which will endow the peptides with desired chemical or physical properties. The termini of the peptides may also be modified, for example, by N-terminal acetylation or terminal carboxy-amidation. The peptides of interest are described in relation to the composite amino acid sequence shown in Figure 1. The 1o amino acid sequences are given in the conventional and universally accepted three-letter code. In addition to the amino acids shown, other groups are defined as follows: Y is, for example, NH2 , one or more N-terminal amino acids, or other moieties added to facilitate coupling. Y
may itself be modified by, for example, acetylation. Z is a bond, (an) is amino acid(s), or (a) chemical group(s) which may be used for linking.
X is intended to represent OH, NH2 , or a linkage involving either of these two groups.
Peptide I corresponds to amino acids 1 to 20 and has the following 20 amino acid sequences:
(I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X.
2s Peptide II corresponds to amino acids 7 to 26 and has the amino acid sequence:
(II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X.
SHOT
20743'74 Of particular interest is the oligopeptide IIA:
(IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X.
s Peptide III corresponds to amino acids 13 to 32 and has the sequence:
(UI) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X.
Peptide IV corresponds to amino acid 37 to 56 and has the sequences:
(IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X.
Is Peptide V corresponds to amino acids 49 to 68 and has the sequence:
(V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X.
Peptide VI corresponds to amino acid 61 to 80 and has the following sequence:
(VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X.
Peptide VII corresponds to amino acids 73 to 92 and has the sequence:
(VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-3o Gly-Cys-Gly-Z-X.
svirvET
S
Peptide VIII corresponds to amino acids 1688 to 1707 and has the sequence:
O
Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-s Asp-Glu-Z-X.
Peptide IX corresponds to amino acids 1694 to 1713 and has the sequence:
(IX) io Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X.
Peptide X corresponds to amino acids 1706 to 1725 and has the sequence:
is (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ue-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X.
Peptide XI corresponds to amino acids 1712 to 1731 and has the 20 sequence:
(XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X.
is Peptide XII corresponds to amino acids 1718 to 1737 and has the sequence:
(XII) Y-Ile-Glu-Gin-Gly-Met-Met-Leu-Ala-Glu-Gin-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X.
Peptide 0'7 ~~ 3'7 0 XIII corresponds to amino acids 1724 to 1743 and has the sequence:
(X~) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-s Arg-Gln-Ala-Z-X.
Peptide XIV corresponds to amino acids 1730 to 1749 and has the sequence:
(X.V) to Y-GIn-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X.
Peptide XV corresponds to amino acids 2263 to 2282 and has the sequence:
1s (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X.
Peptide XVI corresponds to amino acids 2275 to 2294 and has the 20 sequence:
(XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X.
u Peptide XVII corresponds to amino acids 2287 to 2306 and has the sequence:
(XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys 3o Pro-Asp-Tyr-Z-X.
U sHr~
s ...... .....
Peptide XVIII corresponds to amino acids 2299 to 2318 and has the sequence:
(XVIII) Y-G lu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al- V al-His-Gly-Cys-Pro-s Leu-Pro-Pro-Z-X.
Peptide XIX corresponds to amino acids 2311 to 2330 and has the sequence:
(XIX) io Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X.
Of particular interest is the use of the mercapto-group of cysteines or thioglycolic acids used for acylating terminal amino groups for cyclizing is the peptides or coupling two peptides together. The cyclization or coupling may occur via a single bond or may be accomplished using thiol-specific reagents to form a molecular bridge.
The peptides may be coupled to a soluble carrier for the purpose of 20 either raising antibodies or facilitating the adsorption of the peptides to a solid phase. The nature of the carrier should be such that it has a -molecular weight greater than 5000 and should not be recognized by antibodies in human serum. Generally, the carrier will be a protein.
Proteins which are frequently used as carriers are keyhole limpet 2s hemocyanin, bovine gamma globulin, bovine serum albumin, and poly-L-lysine.
There are many well described techniques for coupling peptides to carriers. The linkage may occur at the N-terminus, C-terminus or at an 30 internal site in the peptide. The peptide may also be derivatized for UBS. r M SHE
coupling. Detailed descriptions of a wide variety of coupling procedures are given, for example, in Van Regenmortel, M.H.V., Briand, J.P., Muller, S., and Plaue, S., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19, Synthetic Polypeptides as Antigens, Elsevier Press, Amsterdam, New York, Oxford, 1988.
The peptides may also be synthesized directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides. The number of lysines io comprising the core is preferably 3 or 7. Additionally, a cysteine may be included near or at the C-terminus of the complex to facilitate the formation of homo- or heterodimers. The use of this technique has been amply illustrated for hepatitis B antigens (Tam, J.P., and Lu, Y-A., Proc.
Natl. Acad. Sci. USA (1989) 86:9084-9088) as well as for a variety of is other antigens (see Tam, J.P., Multiple Antigen Peptide System: A Novel Design for Synthetic Peptide Vaccine and Immunoassay, in Synthetic Peptides, Approaches to Biological Problems, Tam, J.P., and Kaiser, E.T., ed. Alan R. Liss Inc., New York, 1989).
20 Depending on their intended use, the peptides may be either labeled or unlabeled. Labels which may be employed may be of any type, such as enzymatic, chemical, fluorescent, luminescent, or radioactive. In addition, the peptides may be modified for binding to surfaces or solid phases, such as, for example, microtiter plates, nylon membranes, glass or plastic is beads, and chromatographic supports such as cellulose, silica, or agarose.
The methods by which peptides can be attached or bound to solid support or surface are well known to those versed in the art.
Of particular interest is the use of mixtures of peptides for the detection so of antibodies specific for hepatitis C virus. Mixtures of peptides which UBS T1~~ SFigBT
S
li 2074370 are considered particularly advantageous are:
A. II, III, V, IX, and XVIII
B. I, II, V, IX, XI, XVI, and XVIII
s C. II, III, IV, V, VIII, XI, XVI, and XVIII
D. II, IX, and XVIII
E. II, III, IV, and V
F. VIII, IX, XI, XIII, and XIV
G. XV, XVI, XVII, XVIII, and XIX
Antibodies which recognize the peptides can be detected in a variety of ways. A preferred method of detection is the enzyme-linked immunosorbant assay (ELISA) in which a peptide or mixture of peptides is bound to a solid support. In most cases, this will be a microtiter.
is plate but may in principle be any sort of insoluble solid phase. A
suitable dilution or dilutions of serum or other body fluid to be tested is brought into contact with the solid phase to which the peptide is bound. The incubation is carried out for a time necessary to allow the binding reaction to occur. Subsequently, unbound components are removed by washing the solid phase. The detection of immune complexes is- achieved using antibodies which specifically bind to human immunoglobulins, and which have been labeled with an enzyme, preferably but not limited to either horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a 2s colorless or nearly colorless substrate or co-substrate into a highly colored product or a product capable of forming a colored complex with a chromogen. Alternatively, the detection system may employ an enzyme which, in the presence of the proper substrate(s), emits light. The amount of product formed is detected either visually, 3o spectrophotometrically, electrochemically, or luminometrically, and is u TS SH~'E't S
TO HEPATITIS C VIRUS
The implementation of systematic testing for hepatitis B virus (HBV) has been instrumental in eliminating this virus from the blood supply.
Nevertheless, a significant number of post-transfusion hepatitis (PTH) cases still occur. These cases are generally attributable to non-A, non-B
io hepatitis (NANBH) virus(es), the diagnosis of which is usually made by exclusion of other viral markers.
The etiological agent responsible for a large proportion of these cases has recently been cloned (Choo, Q-L et al. Science (1988) 244:359-362) is and a first-generation antibody test developed (Kuo, G. et al. Science (1989) 244:362-364). The agent has been identified as a positive-stranded RNA virus, and the sequence of its genome has been partially determined. Studies suggest that this virus, referred to subsequently as hepatitis C virus (HCV), may be related to flaviviruses and pestiviruses.
zo A portion of the genome of an HCV isolated from a chimpanzee (HCVCDC/CHI ) is disclosed in EPO 88310922.5. The coding sequences disclosed in this document do not include sequences originating from the 5'-end of the viral genome which code for putative structural proteins.
Recently however, sequences derived from this region of the HCV
u genome have been published (Okamoto, H. et al., Japan L Ev-., Med.
60:167-177, 1990.). The amino acid sequences encoded by the Japanese clone HC-J1 were combined with the HCVCDC/CHI sequences in a region where the two sequences overlap to generate the composite sequence depicted in Figure 1. Specifically, the two sequences were 3o joined at glycine451 . It should be emphasized that the numbering $u rru1'S SHE
.2-system used for the HCV amino acid sequence is not intended to be absolute since the existence of variant HCV strains harboring deletions or insertions is highly probable. Sequences corresponding to the 5', end of the HCV genome have also recently been disclosed in EPO
$ 90302866Ø
In order to detect potential carriers of HCV, it is necessary to have access to large amounts of viral proteins. In the case of HCV, there is currently no known .method for culturing the virus, which precludes the 1o use of virus-infected cultures as a source of viral antigens. The current first-generation antibody test makes use of a fusion protein containing a sequence of 363 amino acids encoded by the HCV genome. It was found that antibodies to this protein could be detected in 75 to 85% of chronic NANBH patients. In contrast, only approximately 15% of those is patients who were in the acute phase of the disease, had antibodies which recognized this fusion protein (Kuo, G. et al. Science (1989) 244:362-364). The absence of suitable confirmatory tests, however, makes it difficult to verify these statistics. The seeming similarity between the HCV genome and that of flaviviruses makes it possible to predict the 20 location of epitopes which are likely to be of diagnostic value. An analysis of the HCV genome reveals the presence of a continuous long open reading frame. Viral RNA is presumably translated into a long polyprotein which is subsequently cleaved by cellular and/or viral proteases. By analogy with, for example, Dengue virus, the viral is structural proteins are presumed to be derived from the amino-terminal third. of the viral polyprotein. At the present time, the precise sites at which the polyprotein is cleaved can only be surmised. Nevertheless, the structural proteins are likely to contain epitopes which would be useful for diagnostic purposes, both for the detection of antibodies as well as 3o for raising antibodies which could subsequently be used for the detection ,USSYM-M
- 2a-of viral antigens. Similarly, domains of nonstructural proteins are also expected to contain epitopes of diagnostic value, even though these proteins are not found as structural components of virus particles.
Summary of the Invention In one aspect of the present invention there is provided a peptide selected from:
(a) the group of amino acid sequences consisting of.
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) - 2b -(61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, and (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (VII), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (VII) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
wherein the variant consisting of the sequence: Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, is excluded; and (c) fragments of peptides (I) to (VII) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80 and 73-92 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV; and provided that said peptides are different from the following list of peptides:
(1) Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Xi-Lys, wherein Xi represents Arg, (2) Pro-Lys-Pro-Gln-Xi-Lys-X2-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, wherein Xi represents Arg, and X2 represents Thr, (3) Gln-Asn-Val-Lvs-Phe-Pro.
- 2c-(4) Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly, (5) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg, (6) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (7) Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (8) Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile, (9) Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg, (10) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and (11) Gln-Arg-Lys-Thr-Lys-Arg.
In another aspect of the present invention there is provided a peptide composition comprising a peptide described herein, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-C ys-S er-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gin-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) -2d-(1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) (2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val- Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X, and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) - 2e -wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (VIII) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (VIII) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (VIII) to (XIX) having at least 6 amino acids of any of the peptide sequences 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743, 1730-1749, 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV.
In another aspect of the present invention there is provided use of a peptide or peptide composition described herein, for the incorporation into an immunoassay for detecting the presence of antibodies to Hepatitis C virus present in a body fluid.
In another aspect of the present invention there is provided a method for the in vitro detection of antibodies to Hepatitis C virus present in a body fluid comprising at least the steps of. (a) contacting body fluid of a person to be diagnosed with the peptide or peptide composition according to any one of claims 1 to 9; and, (b) detecting the immunological complex formed between said antibodies and the peptide(s) used.
In another aspect of the present invention there is provided a kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising at least the following components:
a nentide or nentide composition described herein -2f-the means for detecting an immunological complex formed between said peptides and said antibodies.
In another aspect of the present invention there is provided use of a peptide or peptide composition described herein, for incorporation into a vaccine composition against HCV.
In another aspect of the present invention there is provided a peptide or peptide composition described herein, for raising antibodies against HCV.
In another aspect of the present invention there is provided a peptide or peptide composition obtained by cyclizing any of the peptides described herein, or by coupling together two peptides described herein.
In another aspect of the present invention there is provided a peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-lle-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) -2g-(1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) to (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gin-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a process for preparing a peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) - 2h -(1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids.
In another aspect of the present invention there is provided a process for preparing a peptide (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-GIu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a -4.v nr -Nrl *%bnoa nmmriricinn at-net nn,- aminn an;rl and ,c many ne (.(1 amine - 2j -acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids and with said at least two peptides being mixed.
In another aspect of the present invention there is provided a process for the preparation of a kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising gathering at least the following components:
a peptide described herein, a means for detecting an immunological complex formed between said peptides and said antibodies.
In another aspect of the present invention there is provided a process for the preparation of a peptide composition described herein, wherein said peptides are such as obtained by synthesizing a peptides described herein directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides.
In another aspect of the present invention there is provided a peptide selected from:
(a) the group of amino acid sequences consisting of:
(2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) - 2k -(2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XV III)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-V al-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X, and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (XV) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (XV) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (XV) to (XIX) having at least 6 amino acids of any of the peptide sequences 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV, and provided that said peptides are different from the following list of peptides: Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg (2265-2280) and Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-m_.. A 1 _ __ /nnOfN r-nnm In another aspect of the present invention there is provided a peptide composition comprising a peptide described herein, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-S er-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys- V al-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, (80) - 2m-(73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) - 2n-wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated; Z
is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids; and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (XIV), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (XIV) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
and (c) fragments of peptides (I) to (XIV) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80, 73-92, 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743 and 1730-1749, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV, provided that the combinations containing at least one peptide selected from the group of amino acid sequences consisting of I, II, IIA, III, IV, V, VI, VII and at least one peptide selected from the group of amino acid sequences consisting of VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX are excluded.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
-2o-In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XVIII) Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XVIII) Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro.
In another aspect of the present invention there is provided a peptide consisting of the amino - 2p -acid sequence: (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
In another aspect of the present invention there is provided a peptide selected from the group consisting of: (a) a peptide consisting of a combination of at least two of amino acid sequences: II, III, V, IX and XVIII; (b) a peptide consisting of a combination of at least two of amino acid sequences: I, II, V, IX, XI, XVI and XVIII; (c) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV, V, VIII, XI, XVI and XVIII; (d) a peptide consisting of a combination of at least two of amino acid sequences: II, IX and XVIII; (e) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV and V; (f) a peptide consisting of a combination of at least two of amino acid sequences: VIII, IX, XI, XIII and XIV; and (g) a peptide consisting of a combination of at least two of amino acid sequences: XV, XVI, XVII, XVIII and XIX;
wherein the amino acid sequences I, II, III, IV, V are as defined herein, and wherein the amino acid sequences VIII, IX, XI, XIII, XIV, XV, XVI, XVII, XVIII, and XIX
are as defined herein.
In another aspect of the present invention there is provided a peptide consisting of a combination of at least two peptides selected from the group consisting of:
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala;
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn;
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr;
(XV III)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-V al-His-Gly-Cys-Pro-Leu-Pro-Pro; and (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-- 2q -Arg-Glu-Phe-Asp-Glu, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-t0 Glu-Glu-Cys-Ser-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (X) Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XI) Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys -Gln-Lys .
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XII) Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
- 2r -In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala.
In another aspect of the present invention there is provided a peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV;
wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are as defined herein.
In another aspect of the present invention there is provided a peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are as defined herein.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-VaI-Lys-Phe-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (III)Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
-2s-In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide comprising the amino acid sequence: (VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly, and a linker arm which is I to 60 amino acids and are different from any naturally occurring HCV flanking regions.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-V aI-Lys -Phe-Pro- Gl y.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly.
-2t-In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly.
In another aspect of the present invention there is provided a peptide consisting of the amino acid sequence: (VII)Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly.
In another aspect of the present invention there is provided A peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; and wherein the amino acid sequences I, II, III, IV, V, VI and VII are as defined herein.
In another aspect of the present invention there is provided A peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences I, II, III, IV, V, VI, and VII are as defined herein.
s Brief Description of the Drawings Figure 1 shows the amino acid sequence of the composite HCVHC-io Figure 2 shows the antibody binding to individual peptides and various mixtures in an ELISA assay Description of the Specific Embodiments 15 It is known that RNA viruses frequently exhibit a high rate of spontaneous mutation and, as such, it is to be expected that no two HCV isolates will be completely identical, even when derived from the same individual. For the purpose of this disclosure, a virus is considered to be the same or equivalent to HCV if it exhibits a global homology 20 of 60 percent or more with the HCVHC-J1/CDC/CHI composite sequence at the nucleic acid level and 70 percent at the amino acid level.
Peptides are described which immunologically mimic proteins encoded by HCV. In order to accommodate strain-to-strain variations in sequence, 25 conservative as well as non-conservative amino acid substitutions may be made. These will generally account for less than 35 percent of a specific sequence. It may be desirable in cases where a peptide corresponds to a region in the HCV polypeptide which is highly polymorphic, to vary one or more of the amino acids so as to better mimic the different 30 epitopes of different viral strains.
!0074370 The peptides of interest will include at least five, sometimes six, sometimes eight, sometimes twelve, usually fewer than about fifty, more usually fewer than about thirty-five, and preferably fewer than about twenty-five amino acids included within the sequence encoded by the s HCV genome. In each instance, the peptide will preferably be as small as possible while still maintaining substantially all of the sensitivity of the larger peptide. It may also be desirable in certain instances to join two or more peptides together in one peptide structure.
to It should be understood that the peptides described need not be identical to any particular HCV sequence, so long as the subject compounds are capable of providing for immunological competition with at least one strain of HCV. The peptides may therefore be subject to insertions, deletions, and conservative or non-conservative amino acid substitutions is where such changes might provide for certain advantages in their use.
Substitutions which are considered conservative are those in which the chemical nature of the substitute is similar to that of the original amino acid. Combinations of amino acids which could be considered zo conservative are Gly, Ala; Asp, Glu; Asn, Gin; Val, Ile, Leu; Ser, Thr; Lys, Arg; and Phe, Tyr.
Furtheiinore, additional amino acids or chemical groups may be added to the amino- or carboxyl terminus for the purpose of creating a "linker is arm" by which the peptide can conveniently be attached to a carrier.
The linker arm will be at least one amino acid and may be as many as 60 amino acids but will most frequently be 1 to 10 amino acids. The nature of the attachment to a solid phase or carrier need not be covalent.
-s 2074370 Natural amino acids such as cysteine, lysine, tyrosine, glutamic acid, or aspartic acid may be added to either the amino- or carboxyl terminus to provide functional groups for coupling to a solid phase or a carrier.
However, other chemical groups such as, for example, biotin and s thioglycolic acid, may be added to the termini which will endow the peptides with desired chemical or physical properties. The termini of the peptides may also be modified, for example, by N-terminal acetylation or terminal carboxy-amidation. The peptides of interest are described in relation to the composite amino acid sequence shown in Figure 1. The 1o amino acid sequences are given in the conventional and universally accepted three-letter code. In addition to the amino acids shown, other groups are defined as follows: Y is, for example, NH2 , one or more N-terminal amino acids, or other moieties added to facilitate coupling. Y
may itself be modified by, for example, acetylation. Z is a bond, (an) is amino acid(s), or (a) chemical group(s) which may be used for linking.
X is intended to represent OH, NH2 , or a linkage involving either of these two groups.
Peptide I corresponds to amino acids 1 to 20 and has the following 20 amino acid sequences:
(I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X.
2s Peptide II corresponds to amino acids 7 to 26 and has the amino acid sequence:
(II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X.
SHOT
20743'74 Of particular interest is the oligopeptide IIA:
(IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X.
s Peptide III corresponds to amino acids 13 to 32 and has the sequence:
(UI) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X.
Peptide IV corresponds to amino acid 37 to 56 and has the sequences:
(IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X.
Is Peptide V corresponds to amino acids 49 to 68 and has the sequence:
(V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X.
Peptide VI corresponds to amino acid 61 to 80 and has the following sequence:
(VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X.
Peptide VII corresponds to amino acids 73 to 92 and has the sequence:
(VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-3o Gly-Cys-Gly-Z-X.
svirvET
S
Peptide VIII corresponds to amino acids 1688 to 1707 and has the sequence:
O
Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-s Asp-Glu-Z-X.
Peptide IX corresponds to amino acids 1694 to 1713 and has the sequence:
(IX) io Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X.
Peptide X corresponds to amino acids 1706 to 1725 and has the sequence:
is (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ue-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X.
Peptide XI corresponds to amino acids 1712 to 1731 and has the 20 sequence:
(XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X.
is Peptide XII corresponds to amino acids 1718 to 1737 and has the sequence:
(XII) Y-Ile-Glu-Gin-Gly-Met-Met-Leu-Ala-Glu-Gin-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X.
Peptide 0'7 ~~ 3'7 0 XIII corresponds to amino acids 1724 to 1743 and has the sequence:
(X~) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-s Arg-Gln-Ala-Z-X.
Peptide XIV corresponds to amino acids 1730 to 1749 and has the sequence:
(X.V) to Y-GIn-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X.
Peptide XV corresponds to amino acids 2263 to 2282 and has the sequence:
1s (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X.
Peptide XVI corresponds to amino acids 2275 to 2294 and has the 20 sequence:
(XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X.
u Peptide XVII corresponds to amino acids 2287 to 2306 and has the sequence:
(XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys 3o Pro-Asp-Tyr-Z-X.
U sHr~
s ...... .....
Peptide XVIII corresponds to amino acids 2299 to 2318 and has the sequence:
(XVIII) Y-G lu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al- V al-His-Gly-Cys-Pro-s Leu-Pro-Pro-Z-X.
Peptide XIX corresponds to amino acids 2311 to 2330 and has the sequence:
(XIX) io Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X.
Of particular interest is the use of the mercapto-group of cysteines or thioglycolic acids used for acylating terminal amino groups for cyclizing is the peptides or coupling two peptides together. The cyclization or coupling may occur via a single bond or may be accomplished using thiol-specific reagents to form a molecular bridge.
The peptides may be coupled to a soluble carrier for the purpose of 20 either raising antibodies or facilitating the adsorption of the peptides to a solid phase. The nature of the carrier should be such that it has a -molecular weight greater than 5000 and should not be recognized by antibodies in human serum. Generally, the carrier will be a protein.
Proteins which are frequently used as carriers are keyhole limpet 2s hemocyanin, bovine gamma globulin, bovine serum albumin, and poly-L-lysine.
There are many well described techniques for coupling peptides to carriers. The linkage may occur at the N-terminus, C-terminus or at an 30 internal site in the peptide. The peptide may also be derivatized for UBS. r M SHE
coupling. Detailed descriptions of a wide variety of coupling procedures are given, for example, in Van Regenmortel, M.H.V., Briand, J.P., Muller, S., and Plaue, S., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19, Synthetic Polypeptides as Antigens, Elsevier Press, Amsterdam, New York, Oxford, 1988.
The peptides may also be synthesized directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides. The number of lysines io comprising the core is preferably 3 or 7. Additionally, a cysteine may be included near or at the C-terminus of the complex to facilitate the formation of homo- or heterodimers. The use of this technique has been amply illustrated for hepatitis B antigens (Tam, J.P., and Lu, Y-A., Proc.
Natl. Acad. Sci. USA (1989) 86:9084-9088) as well as for a variety of is other antigens (see Tam, J.P., Multiple Antigen Peptide System: A Novel Design for Synthetic Peptide Vaccine and Immunoassay, in Synthetic Peptides, Approaches to Biological Problems, Tam, J.P., and Kaiser, E.T., ed. Alan R. Liss Inc., New York, 1989).
20 Depending on their intended use, the peptides may be either labeled or unlabeled. Labels which may be employed may be of any type, such as enzymatic, chemical, fluorescent, luminescent, or radioactive. In addition, the peptides may be modified for binding to surfaces or solid phases, such as, for example, microtiter plates, nylon membranes, glass or plastic is beads, and chromatographic supports such as cellulose, silica, or agarose.
The methods by which peptides can be attached or bound to solid support or surface are well known to those versed in the art.
Of particular interest is the use of mixtures of peptides for the detection so of antibodies specific for hepatitis C virus. Mixtures of peptides which UBS T1~~ SFigBT
S
li 2074370 are considered particularly advantageous are:
A. II, III, V, IX, and XVIII
B. I, II, V, IX, XI, XVI, and XVIII
s C. II, III, IV, V, VIII, XI, XVI, and XVIII
D. II, IX, and XVIII
E. II, III, IV, and V
F. VIII, IX, XI, XIII, and XIV
G. XV, XVI, XVII, XVIII, and XIX
Antibodies which recognize the peptides can be detected in a variety of ways. A preferred method of detection is the enzyme-linked immunosorbant assay (ELISA) in which a peptide or mixture of peptides is bound to a solid support. In most cases, this will be a microtiter.
is plate but may in principle be any sort of insoluble solid phase. A
suitable dilution or dilutions of serum or other body fluid to be tested is brought into contact with the solid phase to which the peptide is bound. The incubation is carried out for a time necessary to allow the binding reaction to occur. Subsequently, unbound components are removed by washing the solid phase. The detection of immune complexes is- achieved using antibodies which specifically bind to human immunoglobulins, and which have been labeled with an enzyme, preferably but not limited to either horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a 2s colorless or nearly colorless substrate or co-substrate into a highly colored product or a product capable of forming a colored complex with a chromogen. Alternatively, the detection system may employ an enzyme which, in the presence of the proper substrate(s), emits light. The amount of product formed is detected either visually, 3o spectrophotometrically, electrochemically, or luminometrically, and is u TS SH~'E't S
2 0'7 43r 0 compared to a similarly treated control. The detection system may also employ radioactively labeled antibodies, in which case the amount of immune complex is quantified by scintillation counting or gamma counting.
s Other detection systems which may be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Staphylococcus sp. (strain 26RP66), or systems which make use of the high affinity biotin-avidin or streptavidin binding io reaction.
Antibodies raised to carrier-bound peptides can also be used' in conjunction with labeled peptides for the detection of antibodies present in serum or other body fluids by competition assay. In this case, is antibodies raised to carrier-bound peptides are attached to a solid support which may be, for example, a plastic bead or a plastic tube. Labeled peptide is then mixed with suitable dilutions of the fluid to be tested and this mixture is subsequently brought into contact with the antibody bound to the solid support. After a suitable incubation period, the solid 20 support is washed and the amount of labeled peptide is quantified. A
reduction in the amount of label bound to the solid support is indicative of the presence of antibodies in the original sample. By the same token, the peptide may also be bound to the solid support. Labeled antibody may then be allowed to compete with antibody present in the sample 2s under conditions in which the amount of peptide is limiting. As in the previous example, a reduction in the measured signal is indicative of the presence of antibodies in the sample tested.
Another preferred method of antibody detection is the homogeneous so immunoassay. There are many possible variations in the design of such $H ST
USST
WO 92/10514 PCf/EP91 /02409 _13-assays. By way of example, numerous possible configurations for homogeneous enzyme immunoassays and methods by which they may be performed are given in Tijssen, P., Practice and Theory of Enzyme Immunoassays, Elsevier Press, Amersham, Oxford, New York, 1985.
s Detection systems which may be employed include those based on enzyme channeling, bioluminescence, allosteric activation and allosteric inhibition. Methods employing liposome-entrapped enzymes or coenzymes may also be used (see Pinnaduwage, P. and Huang, L., Clin, Chem.
(1988) 34/2: 268-272, and Ullman, E.F. et al., Clin. Chem, (1987) 33/9:
1579-1584 for examples).
The synthesis of the peptides can be achieved in solution or on a solid support. Synthesis protocols generally employ the use t-butyloxycarbonyl-or 9-fluorenylmethoxy-carbonyl-protected activated amino acids. The .
is procedures for carrying out the syntheses, the types of side-chain protection, and the cleavage methods are amply described in, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, 1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IRL Press, 1989.
Experimental 1. Peptide Synthesis All of the peptides described were synthesized on Pepsyn K polyamide-Kieselguhr resin (Milligen, Novato, California) which had been functionalized with ethylenediamine and onto which the acid-labile linker 4-(alpha-Fmoc-amino-2',4'-dimethoxybenzyl) phenoxyacetic acid had been coupled (Rink, Tetrahedron Lett. (1987) 28:3787). t-Butyl-based side-chain protection and Fmoc alpha-amino-protection was used. The 1 8H~~
2074.370 -14_ guanidino-group of arginine was protected by the 2,2,5,7,8-pentamethylchroman-6-sulfonyl moiety. The imidazole group of histidine was protected by either t-Boc or trityl and the sulfhydryl group of cysteine was protected by a trityl group. Couplings were carried out s using performed 0-pentafluorophenyl esters except in the case of arginine where diisopropylcarbodiimide-mediated hydroxybenzotriazole ester formation was employed. Except for peptide I, all peptides were N-acetylated using acetic anhydride. All syntheses were carried out on a Milligen 9050 PepSynthesizer (Novato, California) using continuous flow io procedures. Following cleavage with trifluoroacetic acid in the presence of scavengers and extraction with diethylether, all peptides were analyzed by C18 -reverse phase chromatography..
is II. Detection of Antibodies to Hepatitis C Virus A. Use of peptides bound to a nylon membrane.
Peptides were dissolved in a suitable buffer to make a concentrated 20 stock solution which was then further diluted in phosphate-buffered saline (PBS) or sodium carbonate buffer, pH 9.6 to make working solutions. The peptides were applied as lines on a nylon membrane (Pall, Portsmouth, United Kingdom), after which the membrane was treated with casein to block unoccupied binding sites. The 25 membrane was subsequently cut into strips perpendicular to the direction of the peptide lines. Each strip was then incubated with a serum sample diluted 1 to 100, obtained from an HCV-infected individual. Antibody binding was detected by incubating the strips with goat anti-human immunoglobulin antibodies conjugated to the 30 enzyme alkaline phosphatase. After removing unbound conjugate by s USSU" H
S
.2074370 washing, a substrate solution containing 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium was added.
Positive reactions are visible as colored lines corresponding to the' positions of the peptides which are specifically recognized. The reaction patterns of thirty-six different sera are tabulated in Table 1. The results shown in Table 1 are further summarized in Table 2.
B. Use of peptides in an enzyme-linked im_Tnunosorbent assay (ELISA).
Peptide stock solutions were diluted in sodium carbonate buffer, pH
9.6 and used to coat microtiter plates at a peptide concentration of.
1s 2 micrograms per milliliter. A mixture consisting of peptides II, III, V, IX, and XVIII was also used to coat plates. Following coating, the plates were blocked with casein. Fifteen HCV-antibody-positive sera and control sera from seven uninfected blood donors were diluted 1 to 20 and incubated in wells of the peptide-coated plates.
Antibody binding was detected by incubating the plates with goat anti-human' immunoglobulin antibodies conjugated to the enzyme horseradish peroxidase. Following removal of unbound conjugate by washing, a solution containing H202 and 3,3',5,5'-tetramethylbenzidine was added. Reactions were stopped after a u suitable interval by addition of sulfuric acid. Positive reactions gave rise to a yellow color which was quantified using a conventional microtiter plate reader. The results of these determinations are tabulated in Table 3. To correct for any aspecific binding which could be attributable to the physical or chemical properties of the peptides themselves, a cut-off value was determined for each peptide 2O'7437U -16-individually. This cut-off absorbance value was calculated as the average optical density of the negative samples plus 0.200. Samples giving absorbance values higher than the cut-off values are considered positive. The results for the fifteen positive serum s samples are further summarized in Table 4.
While it is evident that some of the peptides are recognized by a large percentage of sera from HCV-infected individuals, it is also clear that no single peptide is recognized by all sera. In contrast, the peptide mixture was recognized by all fifteen sera and, for six of the fifteen sera, the optical densities obtained were, equal to or higher than those obtained for any of the peptides individually.
These results serve to illustrate the advantages of using mixtures of peptides for the detection of anti-HCV antibodies.
is C. Binding of antibodies in sera from HCV-infected patients to various {
individual peptides and peptide mixtures in an ELISA.
Five peptides were used individually and in seven different combinations to coat microliter plates. The plates were subsequently incubated with dilutions of fifteen HCV antibody-positive sera in order to evaluate the relative merits of using mixtures as compared to individual peptides for antibody detection. The mixtures used and the results obtained are shown in Figure 2. -In general, the mixtures functioned better than individual peptides.
This was particularly evident for mixture 12 (peptides I, III, V, IX, and XVIII) which was recognized by all twelve of the sera tested.
These results underscore the advantages of using mixtures of peptides SHOT
SUSS. ls in diagnostic tests for the detection of antibodies to HCV.
D. Use of a mixture of peptides in an ELISA assay for the detection of anti-HCV antibodies.
A mixture of peptides II, III, V, IX, and XVIII was prepared and used to coat microtiter plates according to the same procedure used to test the individual peptides. A total of forty-nine sera were tested from patients with clinically diagnosed but undifferentiated chronic non A non B hepatitis as well as forty-nine sera from healthy blood donors. Detection of antibody binding was accomplished -using goat anti-human immunoglobulin antibodies conjugated to horseradish peroxidase. The resulting optical density.
is values are given in Table 5. These results indicate that the mixture of peptides is not recognized by antibodies in sera from healthy donors (0/49 reactives) but is recognized by a large proportion (41/49, or 84%) of the sera from patients with chronic NANBH.
These results demonstrate that the peptides described can be used effectively as mixtures for the diagnosis of HCV infection.
E. Detection of anti-HCV antibodies in sera from patients with acute NANB infection using individual peptides bound to nylon membranes and a mixture of peptides in an ELISA assay, and comparison with zs commercially available kit.
Peptides were applied to nylon membranes or mixed and used to coat microtiter plates as previously described. The peptide mixture consisted of peptides II, III, V, IX, and XVIII. Sera obtained from twenty-nine patients with acute non-A, non-B hepatitis were then tested for the presence of antibodies to hepatitis C virus. These same sera were also evaluated using a commercially available kit (Ortho, Emeryville, CA, USA).
s The results of this comparative study are given in Table 6. In order to be able to compare the peptide-based ELISA with the commercially available kit, the results for both tests are also expressed aS signal to noise ratios (S/N) which were calculated by dividing he measured optical density obtained for each sample by 0 io the 'cut-off value. A signal-to-noise ratio greater or equal to 1.0 is taken to represent a positive reaction. For the commercially available kit, the cut-off value was calculated according to the manufacturer's instructions. The cut-off value for the peptide-based ELISA was calculated as the average optical density of five negative is samples plus 0.200.
The scale used to evaluate antibody recognition of nylon-bound peptides was the same as that given in Table 1. Of the twenty-nine samples tested, twenty-five (86%) were positive in the peptide-based 20 ELISA and recognized one or more nylon-bound peptides. In contrast, only fourteen of the twenty-nine sera scored positive in the commercially available ELISA. These results serve to illustrate the advantages of using peptide mixtures for the detection of anti-HIV
antibodies as well as the need to include in the mixtures peptides is which contain amino acid sequences derived from different regions of the HCV polyprotein.
S
Table 1. Recognition of peptides bound to nylon membranes by sera from persons infected by HCV.
PEPTIDE
Serom et. I ^ N Y N V11 VI IX
3 1 0.5 2 1 0.5 2 0.5 e 2 1 0.5 7 0.5 1 2 1 05 3 2 e 0.5 1 3 1 1 1 1 1 0.5 3 1 0.5 2 1 is 2 1 0.5 0.5 1 0.5 2 0.5 1e 1 1 3 0.5 2 05 24 1 0.5 2 1 0.5 OS 03 2 1 05 2 0.5 27 03 0.5 1 3 2 29 0.5 3 2 1 1 05 OS 0.5 1 1 0.51 31 1 0.5 44 1 2 1 0.5 4e 0.5 2 0.5 0.5 05 2 47 0.5 0.5 0.5 1 4e 1 2 2 0.5 2 49 1 1 05 0.5 0.5 50 1 2 1 2 0.5 51 2 0.5 0.5 10.5 52 2 0.5 05 54 2 0.5 0.5 1 05 5e 10 FD !0 :t0 !0 2 Blank: no reaction; 0.5: weakly positive; 1: clearly positive;
2: strong reaction; 3: intense reaction; ND: not determined Sue8'TRU t BHS
s Other detection systems which may be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Staphylococcus sp. (strain 26RP66), or systems which make use of the high affinity biotin-avidin or streptavidin binding io reaction.
Antibodies raised to carrier-bound peptides can also be used' in conjunction with labeled peptides for the detection of antibodies present in serum or other body fluids by competition assay. In this case, is antibodies raised to carrier-bound peptides are attached to a solid support which may be, for example, a plastic bead or a plastic tube. Labeled peptide is then mixed with suitable dilutions of the fluid to be tested and this mixture is subsequently brought into contact with the antibody bound to the solid support. After a suitable incubation period, the solid 20 support is washed and the amount of labeled peptide is quantified. A
reduction in the amount of label bound to the solid support is indicative of the presence of antibodies in the original sample. By the same token, the peptide may also be bound to the solid support. Labeled antibody may then be allowed to compete with antibody present in the sample 2s under conditions in which the amount of peptide is limiting. As in the previous example, a reduction in the measured signal is indicative of the presence of antibodies in the sample tested.
Another preferred method of antibody detection is the homogeneous so immunoassay. There are many possible variations in the design of such $H ST
USST
WO 92/10514 PCf/EP91 /02409 _13-assays. By way of example, numerous possible configurations for homogeneous enzyme immunoassays and methods by which they may be performed are given in Tijssen, P., Practice and Theory of Enzyme Immunoassays, Elsevier Press, Amersham, Oxford, New York, 1985.
s Detection systems which may be employed include those based on enzyme channeling, bioluminescence, allosteric activation and allosteric inhibition. Methods employing liposome-entrapped enzymes or coenzymes may also be used (see Pinnaduwage, P. and Huang, L., Clin, Chem.
(1988) 34/2: 268-272, and Ullman, E.F. et al., Clin. Chem, (1987) 33/9:
1579-1584 for examples).
The synthesis of the peptides can be achieved in solution or on a solid support. Synthesis protocols generally employ the use t-butyloxycarbonyl-or 9-fluorenylmethoxy-carbonyl-protected activated amino acids. The .
is procedures for carrying out the syntheses, the types of side-chain protection, and the cleavage methods are amply described in, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, 1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IRL Press, 1989.
Experimental 1. Peptide Synthesis All of the peptides described were synthesized on Pepsyn K polyamide-Kieselguhr resin (Milligen, Novato, California) which had been functionalized with ethylenediamine and onto which the acid-labile linker 4-(alpha-Fmoc-amino-2',4'-dimethoxybenzyl) phenoxyacetic acid had been coupled (Rink, Tetrahedron Lett. (1987) 28:3787). t-Butyl-based side-chain protection and Fmoc alpha-amino-protection was used. The 1 8H~~
2074.370 -14_ guanidino-group of arginine was protected by the 2,2,5,7,8-pentamethylchroman-6-sulfonyl moiety. The imidazole group of histidine was protected by either t-Boc or trityl and the sulfhydryl group of cysteine was protected by a trityl group. Couplings were carried out s using performed 0-pentafluorophenyl esters except in the case of arginine where diisopropylcarbodiimide-mediated hydroxybenzotriazole ester formation was employed. Except for peptide I, all peptides were N-acetylated using acetic anhydride. All syntheses were carried out on a Milligen 9050 PepSynthesizer (Novato, California) using continuous flow io procedures. Following cleavage with trifluoroacetic acid in the presence of scavengers and extraction with diethylether, all peptides were analyzed by C18 -reverse phase chromatography..
is II. Detection of Antibodies to Hepatitis C Virus A. Use of peptides bound to a nylon membrane.
Peptides were dissolved in a suitable buffer to make a concentrated 20 stock solution which was then further diluted in phosphate-buffered saline (PBS) or sodium carbonate buffer, pH 9.6 to make working solutions. The peptides were applied as lines on a nylon membrane (Pall, Portsmouth, United Kingdom), after which the membrane was treated with casein to block unoccupied binding sites. The 25 membrane was subsequently cut into strips perpendicular to the direction of the peptide lines. Each strip was then incubated with a serum sample diluted 1 to 100, obtained from an HCV-infected individual. Antibody binding was detected by incubating the strips with goat anti-human immunoglobulin antibodies conjugated to the 30 enzyme alkaline phosphatase. After removing unbound conjugate by s USSU" H
S
.2074370 washing, a substrate solution containing 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium was added.
Positive reactions are visible as colored lines corresponding to the' positions of the peptides which are specifically recognized. The reaction patterns of thirty-six different sera are tabulated in Table 1. The results shown in Table 1 are further summarized in Table 2.
B. Use of peptides in an enzyme-linked im_Tnunosorbent assay (ELISA).
Peptide stock solutions were diluted in sodium carbonate buffer, pH
9.6 and used to coat microtiter plates at a peptide concentration of.
1s 2 micrograms per milliliter. A mixture consisting of peptides II, III, V, IX, and XVIII was also used to coat plates. Following coating, the plates were blocked with casein. Fifteen HCV-antibody-positive sera and control sera from seven uninfected blood donors were diluted 1 to 20 and incubated in wells of the peptide-coated plates.
Antibody binding was detected by incubating the plates with goat anti-human' immunoglobulin antibodies conjugated to the enzyme horseradish peroxidase. Following removal of unbound conjugate by washing, a solution containing H202 and 3,3',5,5'-tetramethylbenzidine was added. Reactions were stopped after a u suitable interval by addition of sulfuric acid. Positive reactions gave rise to a yellow color which was quantified using a conventional microtiter plate reader. The results of these determinations are tabulated in Table 3. To correct for any aspecific binding which could be attributable to the physical or chemical properties of the peptides themselves, a cut-off value was determined for each peptide 2O'7437U -16-individually. This cut-off absorbance value was calculated as the average optical density of the negative samples plus 0.200. Samples giving absorbance values higher than the cut-off values are considered positive. The results for the fifteen positive serum s samples are further summarized in Table 4.
While it is evident that some of the peptides are recognized by a large percentage of sera from HCV-infected individuals, it is also clear that no single peptide is recognized by all sera. In contrast, the peptide mixture was recognized by all fifteen sera and, for six of the fifteen sera, the optical densities obtained were, equal to or higher than those obtained for any of the peptides individually.
These results serve to illustrate the advantages of using mixtures of peptides for the detection of anti-HCV antibodies.
is C. Binding of antibodies in sera from HCV-infected patients to various {
individual peptides and peptide mixtures in an ELISA.
Five peptides were used individually and in seven different combinations to coat microliter plates. The plates were subsequently incubated with dilutions of fifteen HCV antibody-positive sera in order to evaluate the relative merits of using mixtures as compared to individual peptides for antibody detection. The mixtures used and the results obtained are shown in Figure 2. -In general, the mixtures functioned better than individual peptides.
This was particularly evident for mixture 12 (peptides I, III, V, IX, and XVIII) which was recognized by all twelve of the sera tested.
These results underscore the advantages of using mixtures of peptides SHOT
SUSS. ls in diagnostic tests for the detection of antibodies to HCV.
D. Use of a mixture of peptides in an ELISA assay for the detection of anti-HCV antibodies.
A mixture of peptides II, III, V, IX, and XVIII was prepared and used to coat microtiter plates according to the same procedure used to test the individual peptides. A total of forty-nine sera were tested from patients with clinically diagnosed but undifferentiated chronic non A non B hepatitis as well as forty-nine sera from healthy blood donors. Detection of antibody binding was accomplished -using goat anti-human immunoglobulin antibodies conjugated to horseradish peroxidase. The resulting optical density.
is values are given in Table 5. These results indicate that the mixture of peptides is not recognized by antibodies in sera from healthy donors (0/49 reactives) but is recognized by a large proportion (41/49, or 84%) of the sera from patients with chronic NANBH.
These results demonstrate that the peptides described can be used effectively as mixtures for the diagnosis of HCV infection.
E. Detection of anti-HCV antibodies in sera from patients with acute NANB infection using individual peptides bound to nylon membranes and a mixture of peptides in an ELISA assay, and comparison with zs commercially available kit.
Peptides were applied to nylon membranes or mixed and used to coat microtiter plates as previously described. The peptide mixture consisted of peptides II, III, V, IX, and XVIII. Sera obtained from twenty-nine patients with acute non-A, non-B hepatitis were then tested for the presence of antibodies to hepatitis C virus. These same sera were also evaluated using a commercially available kit (Ortho, Emeryville, CA, USA).
s The results of this comparative study are given in Table 6. In order to be able to compare the peptide-based ELISA with the commercially available kit, the results for both tests are also expressed aS signal to noise ratios (S/N) which were calculated by dividing he measured optical density obtained for each sample by 0 io the 'cut-off value. A signal-to-noise ratio greater or equal to 1.0 is taken to represent a positive reaction. For the commercially available kit, the cut-off value was calculated according to the manufacturer's instructions. The cut-off value for the peptide-based ELISA was calculated as the average optical density of five negative is samples plus 0.200.
The scale used to evaluate antibody recognition of nylon-bound peptides was the same as that given in Table 1. Of the twenty-nine samples tested, twenty-five (86%) were positive in the peptide-based 20 ELISA and recognized one or more nylon-bound peptides. In contrast, only fourteen of the twenty-nine sera scored positive in the commercially available ELISA. These results serve to illustrate the advantages of using peptide mixtures for the detection of anti-HIV
antibodies as well as the need to include in the mixtures peptides is which contain amino acid sequences derived from different regions of the HCV polyprotein.
S
Table 1. Recognition of peptides bound to nylon membranes by sera from persons infected by HCV.
PEPTIDE
Serom et. I ^ N Y N V11 VI IX
3 1 0.5 2 1 0.5 2 0.5 e 2 1 0.5 7 0.5 1 2 1 05 3 2 e 0.5 1 3 1 1 1 1 1 0.5 3 1 0.5 2 1 is 2 1 0.5 0.5 1 0.5 2 0.5 1e 1 1 3 0.5 2 05 24 1 0.5 2 1 0.5 OS 03 2 1 05 2 0.5 27 03 0.5 1 3 2 29 0.5 3 2 1 1 05 OS 0.5 1 1 0.51 31 1 0.5 44 1 2 1 0.5 4e 0.5 2 0.5 0.5 05 2 47 0.5 0.5 0.5 1 4e 1 2 2 0.5 2 49 1 1 05 0.5 0.5 50 1 2 1 2 0.5 51 2 0.5 0.5 10.5 52 2 0.5 05 54 2 0.5 0.5 1 05 5e 10 FD !0 :t0 !0 2 Blank: no reaction; 0.5: weakly positive; 1: clearly positive;
2: strong reaction; 3: intense reaction; ND: not determined Sue8'TRU t BHS
TABLE 1 continued x X! X/ xv x111 xv1 VA Xlx 0.5 2 2 1 1 0.5 1 2 2 1 0.5 2 1 1 2 0.5 1 1 1 0.5 2 2 2 2 1 1 0.5 0.5 0.S 1 0.5 % 1 0.5 1 0.5 0.5 0.5 0.5 2 1 1 21 1 1 21 1 0.5 0.5 0.5 0.5 1 0.S 1 1 1 0.5 Os =
i 1 0.5 2 0.5 0.5 1 1 I 1 0.5 0.5 0.5 0s 1 1 1 1 1 1 1 0.5 0.5 1 1 0.5 0s I
0.5 1 2 1 $V$S SHRET
2074370.
Table 2. Summary of antibody binding to nylon-bound HCV peptides by sera from infected patients.
s Peptide No. reactive sera % reactive sera is VII 11/35 31 u XVII 14/36 39 SHOST
SUSST"Is almIANAQI=-N=-r~~pCltAOfi'f tAt~V9tpn~{pp^ ~p^Q) Vf1~tO NN tD CO ^pp0p0 00-YC tDpa1 1,-^c'1 trJrN r 1,7 t 0 0 0-0-000 0- O coo 606 tiOaf-(p D000~~N 00000 0-~
to '~P f~ 0 ca t/f Q IAmNl.nM-P~-0n W 0Nn~p ^^ =IA^ N NNco Or-e=fONf-pr<DtAOr~P- o ~r0-W =-W IV
,.000-x{00 IONp000GO 000000 OGC
O 000 000 000=- 00 000 000 .rao p.- c' 'Matt ,au ^t?flANtqr IAN =%n ~..~ Ot'9aDa~c'fwe'ff.-W pMa-rgo -W 00=~tA 00 00r~-r000rt'f OOOtOp Opp000 OC.-u s O O O O O O O O O O O 0 O 0 0 0 0 0 0 0 C -x N lr Ir W N Qf Cfl Q1 iD N N to r U~ to tp f- u, rf r- ~
r P7 to N ti ^ tD y ^ 1- N O ti r) r1 e~ r! rl N e7 ^
to 00{DtONN~-Of~Cft'9~ 00c~ -0 coo 00N
~=^ y 0 0 0 0^ O C O 0 0 0 0 0 ^ 0 0 0 0 0 0 0 0 0 N^QI~WNppOprNlQp9~~1~11pD1Q~~'Iee~ppN^al tArrrf rl r1N
NO!'ftO~f000v~m-0. t-N 00 p'000 OSO
r7 4..t G ^NIDNn~e'f=rtD~usNm=t= a~=rtim~r) - =r O O~=N~p ~Otr.~NNaD ^rn0 rlr)^'rNN ^
C) N0tD1A OrR~OIO~COrC fl ^~=~=~=== == ON
O ^ 00 -X000^O0O 000000 000 O
=~ ^ QfaltrlOltD^Npp~N~lf~~r1^pp rf Nt0wfo tfN^
p ~OQ1CY^iVOtDO~~ay0^NCf 000000 O0 0~ 000000 000 V w ---- --- -- - -- - -- -----tD to N to Qf t~ r N N Q tD r rl a1 r =- r ^ 1- ^ tppA Q~
COr - Nya~ppw r N1-000tO 000000 000 I-.OaO~r SCN001D6OC9 - 0 ^
y - 000----00 -0 ^- 00 000 000 L ----- -- - - - --- ---------ot0_ ^tcotn tD
NtD th V p ^Ntft~a00 V)mw fnt7 ^N{p Q ^ Q ~_ N N Y^ r to Q Q Q Q Q
see --=ss.=asma~~._s-'-sera AM ton rn'n SHED
WO 92/10514 2 3 PCT/EP9i/02409 207.4370 IV W: o0Npao o N ' 'n0T ~j oo~
0000 NNtO
rf00 On O 0 0^0,-00 00 0-6 000 00 000 t'Y la N f.- Vf Vf N N W w 1.- N V) ID h IC -8 Dow f a^0 -OCd O 00Coo0 00^
,~ ~oooo~-occ oo0-o ood o 000 {OQI~~- -frlQ9~lQr~P e'lg7i~CNIQ NIDfbP-O ' CO Nr Wt90000000000000 000000 08.0 x o0000000000oo0d dd0000 000 {'yrNNN rN=OQ1 ry t-0N Nn N mw (cpp to TT vv~~ N C1 e0 N
s 0 N O N N M M IV N N N N N N NON N o r o 0 0 0 0 0 0 0 0 0 0 0 0 O O O o 0 0 0 0 0 lrtt= QQi^8 rqsl40 0A rrgrrf0"f V Am ~OOVf tr~0 Vf 000O'-p0 00-000 CON
>K 0000000000000 O 000000 000 00 ~~ oo o~~0 4ooooc o8g Odddddoddddodoo ood cc coo OagNoa~a^^aaa 000000 0Ns.
U ~( Oo00 0000000 00. O Oo 0 000 a ~~~..t0 NTOT~u~f=ri MrIN C-W
ooao00.-00000000 00000 00 F it dooo0oo0o00co00 00 000 000 o ~a^ ~~a - CD O00aoo asg 4i 000~0000000 O=- 00000 000 mO O OC4 gVNao 0 a 0 x odoooo000000000 00 occ 000 uBSTT iTV SHED
S
Table 4 Summary of antibody-binding to individual peptides in an ELISA assay.
Peptide No. reactive sera % reactive sera is VI 7 47 s XVI 5 33 SHOT
20.7.4370.
Table 5 Use of a peptide mixture for the detection of antibodies to HCV in sera from chronic NANBH patients and comparison to sera from healthy blood donors.
s Chronic NANB Sera Control Sera Serum nr. Optical Density Serum nr. Optical Density 101 0.041 1 0.049 102 1.387 2 0.047 is 103 1.578 3 0.049 104 1.804 4 0.046 105 1.393 5 0.049 107 1.604 6 0.045 108 1.148 7 0.043 109 1.714 8 0.053 110 1.692 9 0.049 112 0.919 10 0.047 113 1.454 11 0.060 114 0.936 12 0.044 115 0.041 13 0.049 116 1.636 14 0.051 118 1.242 15 0.056 119 1.568 16 0.050 120 1.290 17 0.049 121 1.541 18 0.055 122 1.422 19 0.054 123 1.493 20 0.058 vT 544E
124 1.666 21 0.050 125 1.644 22 0.044 126 1.409 23 0.043 127 1.625 24 0.045 128 1.061 25 0.046 129 1.553 26 0.049 130 .1.709 27 0.050 131 0.041 28 0.047 132 0.044 29 0.050 133 1.648 30 0.053 134 0.043 31 0.051 135 1.268 32 0.053 136 1.480 33 0.055 138 0.628 34 0.064 is 139 0.042 35 0.063 140 0.040 36 0.057 141 0.039 38 0.048 142 1.659 39 0.045 143 1.457 40 0.046 144 0.722 41 0.046 145 1.256 42 0.051 146 0.373 43 0.057 147 1.732 44 0.050 148 1.089 45 0.050 2s 149 - 1.606 46 0.045 150 1.725 47 0.041 151 1.449 48 0.064 154 1.639 49 0.040 155 1.775 50 0.036 UBSThT' SHED
.y,~s...,.......J
St 0 0 0 0 ~- O O O X 0 0 0 O O ~- X 0 0 O O ~~ rJ J`1 N n 0 0 0 O
0 00 con N ""M 000 00" 00 0 0 0 D O -- 0 0 0 0 0000 N --NOOOOO 00C Gl0C=00 o~OPI0000 q C1 O C G
==o O llQ O cc c o o 00 0 0 0 0 0 0 0 O N .- o 0 0 0 G K 0 0 0 0 0 0 0 0 N 0 0 0 0 0 ~ ~ O f` S n n n N N p n 0 0 0~-8 00000660N 00000 nQ000 NNN0000 -0 S 0 o0006100000000 ---- 0000000000000 O
=V.U G
E > 0000nNNN0~~~c;~NN~~.-.Od000GG^..
U O CD OOoN--NQCoco= .. ..Or= .-N00000o 000 2 N ' y t0 ,D h C
~, a o o a o N N N N o 0 0 0 0- o .= 0 d o 0 0 0 0 0 O vVi e~'f A C, O
in cj~
=g G 0 0 0 0 -- --40 0 0 co C) 0 0 0 0 0 C =~
Q u =~ lu U G ti O ~ tC
C
-- N n = Y1 b n O -- n = Y'1 ~D n Of O N n =1 n 4D n QI -r in tD f. 93 0 f= PI C .v tJ h1 N N .==~ n al in n r) C .. -=. ~. r r -- ~- .- N N N C-4 C-4 N N N N N N N N N CJ N N N C4 N N N
E
caa 2 c ci E' JX .
$u ASS T
vvnry~vnlIf ('4~^~~~ar'iaNO`~ae~o~womoy11WI
a 00004000.;0000=-:va~.:.:nv~.i~aod-N
.z NA^P1CbnY p^ 0 PI in OJQOi OO.O On U1-=y+ ~' p .
O .Y A W A e m Y +A. p N 0 0 O~ ~~ O O co, O O 01 O 1 0 0 fn N C ~fl C. Q V.
=C ..
,a NNr= O/1Y r1p r =- =OaD00"~000~ OOOON' r, .
e000 000 0AM 0C:JC1.jpj Cpjpi rjNpjpjAn An 000- /C` 7 =
~t1=
U u = ey.lsor.ea. NoolnlnoA.r r M11YN~ r Y ONOY O 9 u ~ . ~ . - ~ANOMY r^h(+N r N V NN(~ A aO'. O u on vn eo004lol WW,,j errilriw owilrir~ Irihhri~ +: O u `
= ur,aey o c u NNAYNAMOIOAY/% NOI'-IOeN10Y Ob1AC7 r.. Y1V 10 V =
Y hr A~~ppNN pp0" 21~a"4 "N 10 .= 0 Vf 1~1O 43NN 1.1171 ,a 0 0 0 0 ID Ifl 1/1 1r1 10 on M 1p ID $I All in V 001 ^ h r= co ^ V V on Y r O
cciCiOr'~ ~~. GOCO~ .: .%. .. ~%.: r r 00 O a' -.~ N A Y h 10 N O .h Y 1A 40 n 01, c3 r N on Y h 1D P- An Y in 10 N O A C. C.
C. CA 14 C-4 C4 10 All r ~'FA. p N N N N CIA N N N N N C C=1 C( C= Cl N N N C
i N (+
Ii v ~ SHEET
S
i 1 0.5 2 0.5 0.5 1 1 I 1 0.5 0.5 0.5 0s 1 1 1 1 1 1 1 0.5 0.5 1 1 0.5 0s I
0.5 1 2 1 $V$S SHRET
2074370.
Table 2. Summary of antibody binding to nylon-bound HCV peptides by sera from infected patients.
s Peptide No. reactive sera % reactive sera is VII 11/35 31 u XVII 14/36 39 SHOST
SUSST"Is almIANAQI=-N=-r~~pCltAOfi'f tAt~V9tpn~{pp^ ~p^Q) Vf1~tO NN tD CO ^pp0p0 00-YC tDpa1 1,-^c'1 trJrN r 1,7 t 0 0 0-0-000 0- O coo 606 tiOaf-(p D000~~N 00000 0-~
to '~P f~ 0 ca t/f Q IAmNl.nM-P~-0n W 0Nn~p ^^ =IA^ N NNco Or-e=fONf-pr<DtAOr~P- o ~r0-W =-W IV
,.000-x{00 IONp000GO 000000 OGC
O 000 000 000=- 00 000 000 .rao p.- c' 'Matt ,au ^t?flANtqr IAN =%n ~..~ Ot'9aDa~c'fwe'ff.-W pMa-rgo -W 00=~tA 00 00r~-r000rt'f OOOtOp Opp000 OC.-u s O O O O O O O O O O O 0 O 0 0 0 0 0 0 0 C -x N lr Ir W N Qf Cfl Q1 iD N N to r U~ to tp f- u, rf r- ~
r P7 to N ti ^ tD y ^ 1- N O ti r) r1 e~ r! rl N e7 ^
to 00{DtONN~-Of~Cft'9~ 00c~ -0 coo 00N
~=^ y 0 0 0 0^ O C O 0 0 0 0 0 ^ 0 0 0 0 0 0 0 0 0 N^QI~WNppOprNlQp9~~1~11pD1Q~~'Iee~ppN^al tArrrf rl r1N
NO!'ftO~f000v~m-0. t-N 00 p'000 OSO
r7 4..t G ^NIDNn~e'f=rtD~usNm=t= a~=rtim~r) - =r O O~=N~p ~Otr.~NNaD ^rn0 rlr)^'rNN ^
C) N0tD1A OrR~OIO~COrC fl ^~=~=~=== == ON
O ^ 00 -X000^O0O 000000 000 O
=~ ^ QfaltrlOltD^Npp~N~lf~~r1^pp rf Nt0wfo tfN^
p ~OQ1CY^iVOtDO~~ay0^NCf 000000 O0 0~ 000000 000 V w ---- --- -- - -- - -- -----tD to N to Qf t~ r N N Q tD r rl a1 r =- r ^ 1- ^ tppA Q~
COr - Nya~ppw r N1-000tO 000000 000 I-.OaO~r SCN001D6OC9 - 0 ^
y - 000----00 -0 ^- 00 000 000 L ----- -- - - - --- ---------ot0_ ^tcotn tD
NtD th V p ^Ntft~a00 V)mw fnt7 ^N{p Q ^ Q ~_ N N Y^ r to Q Q Q Q Q
see --=ss.=asma~~._s-'-sera AM ton rn'n SHED
WO 92/10514 2 3 PCT/EP9i/02409 207.4370 IV W: o0Npao o N ' 'n0T ~j oo~
0000 NNtO
rf00 On O 0 0^0,-00 00 0-6 000 00 000 t'Y la N f.- Vf Vf N N W w 1.- N V) ID h IC -8 Dow f a^0 -OCd O 00Coo0 00^
,~ ~oooo~-occ oo0-o ood o 000 {OQI~~- -frlQ9~lQr~P e'lg7i~CNIQ NIDfbP-O ' CO Nr Wt90000000000000 000000 08.0 x o0000000000oo0d dd0000 000 {'yrNNN rN=OQ1 ry t-0N Nn N mw (cpp to TT vv~~ N C1 e0 N
s 0 N O N N M M IV N N N N N N NON N o r o 0 0 0 0 0 0 0 0 0 0 0 0 O O O o 0 0 0 0 0 lrtt= QQi^8 rqsl40 0A rrgrrf0"f V Am ~OOVf tr~0 Vf 000O'-p0 00-000 CON
>K 0000000000000 O 000000 000 00 ~~ oo o~~0 4ooooc o8g Odddddoddddodoo ood cc coo OagNoa~a^^aaa 000000 0Ns.
U ~( Oo00 0000000 00. O Oo 0 000 a ~~~..t0 NTOT~u~f=ri MrIN C-W
ooao00.-00000000 00000 00 F it dooo0oo0o00co00 00 000 000 o ~a^ ~~a - CD O00aoo asg 4i 000~0000000 O=- 00000 000 mO O OC4 gVNao 0 a 0 x odoooo000000000 00 occ 000 uBSTT iTV SHED
S
Table 4 Summary of antibody-binding to individual peptides in an ELISA assay.
Peptide No. reactive sera % reactive sera is VI 7 47 s XVI 5 33 SHOT
20.7.4370.
Table 5 Use of a peptide mixture for the detection of antibodies to HCV in sera from chronic NANBH patients and comparison to sera from healthy blood donors.
s Chronic NANB Sera Control Sera Serum nr. Optical Density Serum nr. Optical Density 101 0.041 1 0.049 102 1.387 2 0.047 is 103 1.578 3 0.049 104 1.804 4 0.046 105 1.393 5 0.049 107 1.604 6 0.045 108 1.148 7 0.043 109 1.714 8 0.053 110 1.692 9 0.049 112 0.919 10 0.047 113 1.454 11 0.060 114 0.936 12 0.044 115 0.041 13 0.049 116 1.636 14 0.051 118 1.242 15 0.056 119 1.568 16 0.050 120 1.290 17 0.049 121 1.541 18 0.055 122 1.422 19 0.054 123 1.493 20 0.058 vT 544E
124 1.666 21 0.050 125 1.644 22 0.044 126 1.409 23 0.043 127 1.625 24 0.045 128 1.061 25 0.046 129 1.553 26 0.049 130 .1.709 27 0.050 131 0.041 28 0.047 132 0.044 29 0.050 133 1.648 30 0.053 134 0.043 31 0.051 135 1.268 32 0.053 136 1.480 33 0.055 138 0.628 34 0.064 is 139 0.042 35 0.063 140 0.040 36 0.057 141 0.039 38 0.048 142 1.659 39 0.045 143 1.457 40 0.046 144 0.722 41 0.046 145 1.256 42 0.051 146 0.373 43 0.057 147 1.732 44 0.050 148 1.089 45 0.050 2s 149 - 1.606 46 0.045 150 1.725 47 0.041 151 1.449 48 0.064 154 1.639 49 0.040 155 1.775 50 0.036 UBSThT' SHED
.y,~s...,.......J
St 0 0 0 0 ~- O O O X 0 0 0 O O ~- X 0 0 O O ~~ rJ J`1 N n 0 0 0 O
0 00 con N ""M 000 00" 00 0 0 0 D O -- 0 0 0 0 0000 N --NOOOOO 00C Gl0C=00 o~OPI0000 q C1 O C G
==o O llQ O cc c o o 00 0 0 0 0 0 0 0 O N .- o 0 0 0 G K 0 0 0 0 0 0 0 0 N 0 0 0 0 0 ~ ~ O f` S n n n N N p n 0 0 0~-8 00000660N 00000 nQ000 NNN0000 -0 S 0 o0006100000000 ---- 0000000000000 O
=V.U G
E > 0000nNNN0~~~c;~NN~~.-.Od000GG^..
U O CD OOoN--NQCoco= .. ..Or= .-N00000o 000 2 N ' y t0 ,D h C
~, a o o a o N N N N o 0 0 0 0- o .= 0 d o 0 0 0 0 0 O vVi e~'f A C, O
in cj~
=g G 0 0 0 0 -- --40 0 0 co C) 0 0 0 0 0 C =~
Q u =~ lu U G ti O ~ tC
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-- N n = Y1 b n O -- n = Y'1 ~D n Of O N n =1 n 4D n QI -r in tD f. 93 0 f= PI C .v tJ h1 N N .==~ n al in n r) C .. -=. ~. r r -- ~- .- N N N C-4 C-4 N N N N N N N N N CJ N N N C4 N N N
E
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.z NA^P1CbnY p^ 0 PI in OJQOi OO.O On U1-=y+ ~' p .
O .Y A W A e m Y +A. p N 0 0 O~ ~~ O O co, O O 01 O 1 0 0 fn N C ~fl C. Q V.
=C ..
,a NNr= O/1Y r1p r =- =OaD00"~000~ OOOON' r, .
e000 000 0AM 0C:JC1.jpj Cpjpi rjNpjpjAn An 000- /C` 7 =
~t1=
U u = ey.lsor.ea. NoolnlnoA.r r M11YN~ r Y ONOY O 9 u ~ . ~ . - ~ANOMY r^h(+N r N V NN(~ A aO'. O u on vn eo004lol WW,,j errilriw owilrir~ Irihhri~ +: O u `
= ur,aey o c u NNAYNAMOIOAY/% NOI'-IOeN10Y Ob1AC7 r.. Y1V 10 V =
Y hr A~~ppNN pp0" 21~a"4 "N 10 .= 0 Vf 1~1O 43NN 1.1171 ,a 0 0 0 0 ID Ifl 1/1 1r1 10 on M 1p ID $I All in V 001 ^ h r= co ^ V V on Y r O
cciCiOr'~ ~~. GOCO~ .: .%. .. ~%.: r r 00 O a' -.~ N A Y h 10 N O .h Y 1A 40 n 01, c3 r N on Y h 1D P- An Y in 10 N O A C. C.
C. CA 14 C-4 C4 10 All r ~'FA. p N N N N CIA N N N N N C C=1 C( C= Cl N N N C
i N (+
Ii v ~ SHEET
S
Claims (123)
1. A peptide selected from:
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, and (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (VII), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (VII) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
wherein the variant consisting of the sequence:
Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, is excluded; and (c) fragments of peptides (I) to (VII) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80 and 73-92 as defined in part (a);
and said fragments being capable of providing for immunological competition with at least one strain of HCV;
and provided that said peptides are different from any of those in the following list of peptides:
(1) Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-X1-Lys, wherein X1 represents Arg, (2) Pro-Lys-Pro-Gln-X1-Lys-X2-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, wherein X1 represents Arg, and X2 represents Thr, (3) Gln-Asp-Val-Lys-Phe-Pro, (4) Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly, (5) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg, (6) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (7) Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (8) Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile, (9) Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg, (10) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and (11) Gln-Arg-Lys-Thr-Lys-Arg.
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, and (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (VII), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (VII) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV;
wherein the variant consisting of the sequence:
Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, is excluded; and (c) fragments of peptides (I) to (VII) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80 and 73-92 as defined in part (a);
and said fragments being capable of providing for immunological competition with at least one strain of HCV;
and provided that said peptides are different from any of those in the following list of peptides:
(1) Met-Ser-Thr-Asn-Pro-Lys-Pro-Gln-X1-Lys, wherein X1 represents Arg, (2) Pro-Lys-Pro-Gln-X1-Lys-X2-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, wherein X1 represents Arg, and X2 represents Thr, (3) Gln-Asp-Val-Lys-Phe-Pro, (4) Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly, (5) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg, (6) Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (7) Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser, (8) Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile, (9) Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg, (10) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and (11) Gln-Arg-Lys-Thr-Lys-Arg.
2. A peptide composition comprising the peptide according to claim 1, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) (2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X,and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (VIII) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (VIII) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (VIII) to (XIX) having at least 6 amino acids of any of the peptide sequences 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743, 1730-1749, 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV.
(a) the group of amino acid sequences consisting of:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) (2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X,and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (VIII) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (VIII) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (VIII) to (XIX) having at least 6 amino acids of any of the peptide sequences 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743, 1730-1749, 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV.
3. A peptide composition containing at least one of the following mixtures of peptides as defined in claim 1 or 2:
(a) peptides II, III, V, IX, and XVIII;
(b) peptides I, II, V, IX, XI, XVI, and XVIII;
(c) peptides II, III, IV, V, VIII, XI, XVI, and XVIII;
(d) peptides II, IX and XVIII; and (e) peptides II, III, IV, and V.
(a) peptides II, III, V, IX, and XVIII;
(b) peptides I, II, V, IX, XI, XVI, and XVIII;
(c) peptides II, III, IV, V, VIII, XI, XVI, and XVIII;
(d) peptides II, IX and XVIII; and (e) peptides II, III, IV, and V.
4. The peptide or peptide composition according to any one of claims 1 to 3, wherein said peptides are coupled N-terminally, C-terminally or internally to a carrier molecule for the purpose of raising antibodies or facilitating the adsorption of said peptides to a solid phase.
5. The peptide or peptide composition according to any one of claims 1 to 4, wherein said peptides contain a detectable label.
6. The peptide or peptide composition according to any one of claims 1 to 5, wherein Y
is H, or 1 to 10 amino acids and Z is a bond or 1 to 10 amino acids.
is H, or 1 to 10 amino acids and Z is a bond or 1 to 10 amino acids.
7. The peptide or peptide composition according to any one of claims 1 to 6, wherein Y
is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
8. The peptide or peptide composition according to any one of claims 1 to 7, wherein Z
is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
9. The peptide or peptide composition according to any one of claims 1 to 5, wherein the group for coupling the peptide to a solid phase or carrier is selected from the group consisting of biotin and thioglycolic acid.
10. Use of the peptide or peptide composition according to any one of claims 1 to 9, for the incorporation into an immunoassay for detecting the presence of antibodies to Hepatitis C virus present in a body fluid.
11. A method for the in vitro detection of antibodies to Hepatitis C virus present in a body fluid comprising at least the steps of:
(a) contacting body fluid of a person to be diagnosed with the peptide or peptide composition according to any one of claims 1 to 9, and, (b) detecting the immunological complex formed between said antibodies and the peptide(s) used.
(a) contacting body fluid of a person to be diagnosed with the peptide or peptide composition according to any one of claims 1 to 9, and, (b) detecting the immunological complex formed between said antibodies and the peptide(s) used.
12. A kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising at least the following components:
- the peptide or peptide composition according to any one of claims 1 to 9, - means for detecting an immunological complex formed between said peptides and said antibodies.
- the peptide or peptide composition according to any one of claims 1 to 9, - means for detecting an immunological complex formed between said peptides and said antibodies.
13. The method according to claim 11, wherein said peptides are applied as lines on a nylon membrane.
14. The kit according to claim 12, wherein said peptides are applied as lines on a nylon membrane.
15. The kit according to claim 12, wherein said peptides, separately or in combination, are used to coat the wells of microtiter plates.
16. Use of the peptide or peptide composition according to any one of claims 1 to 9, for incorporation into a vaccine composition against HCV.
17. The peptide or peptide composition according to any one of claims 1 to 9, for raising antibodies against HCV.
18. A peptide or peptide composition obtained by cyclizing any of the peptides of claims 1 to 2, or by coupling together two peptides of claims 1 to 2.
19. The peptide or peptide composition according to any one of claims 1 to 9, wherein said peptides are such as obtained by synthesizing the peptides of claims 1 to 2 directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides.
20. A peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions.
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions.
21. The peptide according to claim 20, wherein said peptides are coupled N-terminally, C-terminally or internally to a carrier molecule for the purpose of raising antibodies or facilitating the adsorption of said peptides to a solid phase.
22. The peptide according to claim 20 or 21, wherein said peptides contain a detectable label.
23. The peptide according to any one of claims 20 to 22, wherein Y is H, or 1 to 10 amino acids and Z is a bond or 1 to 10 amino acids.
24. The peptide according to any one of claims 20 to 23, wherein Y is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
25. The peptide according to any one of claims 20 to 24, wherein Z is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
26. The peptide according to any one of claims 20 to 22, wherein the group for coupling the peptide to a solid phase or carrier is selected from the group consisting of biotin and thioglycolic acid.
27. Use of the peptide according to any one of claims 20 to 26, for the incorporation into an immunoassay for detecting the presence of antibodies to Hepatitis C virus present in a body fluid.
28. A method for the in vitro detection of antibodies to Hepatitis C virus present in a body fluid comprising at least the steps of:
(a) contacting body fluid of a person to be diagnosed with the peptide according to any one of claims 20 to 26, and, (b) detecting an immunological complex formed between said antibodies and the peptide(s) used.
(a) contacting body fluid of a person to be diagnosed with the peptide according to any one of claims 20 to 26, and, (b) detecting an immunological complex formed between said antibodies and the peptide(s) used.
29. A kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising at least the following components:
- the peptide according to any of claims 20 to 26, - a means for detecting an immunological complex formed between said peptides and said antibodies.
- the peptide according to any of claims 20 to 26, - a means for detecting an immunological complex formed between said peptides and said antibodies.
30. The method according to claim 28, wherein said peptides are applied as lines on a nylon membrane.
31. The kit according to claim 29, wherein said peptides are applied as lines on a nylon membrane.
32. The kit according to claim 29, wherein said peptides, separately or in combination, are used to coat the wells of microtiter plates.
33. Use of the peptide according to any one of claims 20 to 26, for incorporation into a vaccine composition against HCV.
34. The peptide according to any one of claims 20 to 26, for raising antibodies against HCV.
35. A peptide or peptide composition obtained by cyclizing any of the peptides of claim 20 or by coupling together two peptides of claim 20.
36. The peptide according to any one of claims 20 to 26, wherein said peptides are such as obtained by synthesizing the peptides of claim 20 directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth joints for the peptides.
37. A process for preparing a peptide selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids.
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids.
38. A process for preparing a peptide composition comprising at least two peptides selected from:
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-IIe-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids and with said at least two peptides being mixed.
(1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-IIe-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions, with said peptides being synthesized in solution or on a solid support employing t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids and with said at least two peptides being mixed.
39. The process according to claim 37 or 38, further characterized in that said peptides are coupled N-terminally, C-terminally or internally to a carrier molecule for the purpose of raising antibodies or facilitating the adsorption of said peptides to a solid phase.
40. The process according to any one of claims 37 to 39, wherein said peptides contain a detectable label added to said peptide.
41. The process according to any one of claims 37 to 40, wherein Y is H, or 1 to 10 amino acids and Z is a bond or 1 to 10 amino acids.
42. The process according to any one of claims 37 to 41, wherein Y is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
43. The process according to any one of claims 37 to 42, wherein Z is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
44. The process according to any one of claims 37 to 40, wherein the group for coupling the peptide to a solid phase or carrier is selected from the group consisting of biotin and thioglycolic acid.
45. A process for the preparation of a kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising gathering at least the following components:
- the peptide according to claim 20, - a means for detecting an immunological complex formed between said peptides and said antibodies.
- the peptide according to claim 20, - a means for detecting an immunological complex formed between said peptides and said antibodies.
46. The process for the preparation of a kit according to claim 45, wherein said peptides are applied as lines on a nylon membrane.
47. The process for the preparation of a kit according to claim 45, wherein said peptides, separately or in combination, are used to coat the wells of microtiter plates.
48. The process according to any one of claims 37 to 44, for incorporation into a vaccine composition against HCV.
49. The process according to any one of claims 37 to 44, for raising antibodies against HCV.
50. A process according to any one of claims 37 to 44, wherein said peptides are such as obtained by synthesizing the peptides of claim 20 directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides.
51. A peptide selected from:
(a) the group of amino acid sequences consisting of:
(2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X,and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (XV) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (XV) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (XV) to (XIX) having at least 6 amino acids of any of the peptide sequences 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV, and provided that said peptides are different from the following list of peptides:
Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg (2265-2280), and Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg (2280-2290).
(a) the group of amino acid sequences consisting of:
(2263) (XV) Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X, (2282) (2275) (XVI) Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X, (2294) (2287) (XVII) Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X, (2306) (2299) (XVIII)Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X,and (2318) (2311) (XIX) Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X, (2330) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (XV) to (XIX), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (XV) to (XIX) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (XV) to (XIX) having at least 6 amino acids of any of the peptide sequences 2263-2282, 2275-2294, 2287-2306, 2299-2318 and 2311-2330 as defined in part (a); and said fragments being capable of providing for immunological competition with at least one strain of HCV, and provided that said peptides are different from the following list of peptides:
Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg (2265-2280), and Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg (2280-2290).
52. A peptide composition comprising the peptide according to claim 51, and further comprising at least one peptide selected from:
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-V al-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (XIV), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (XIV) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (I) to (XIV) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80, 73-92, 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743 and 1730-1749, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV, provided that the combinations containing at least one peptide selected from the group of amino acid sequences consisting of I, II, IIA, III, IV, V, VI, VII and at least one peptide selected from the group of amino acid sequences consisting of VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX are excluded.
(a) the group of amino acid sequences consisting of:
(1) (I) Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X, (20) (7) (II) Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X, (26) (8) (18) (IIA) Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X, (13) (III) Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-V al-Gly-Z-X, (32) (37) (IV) Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X, (56) (49) (V) Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X, (68) (61) (VI) Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X, (80) (73) (VII) Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X, (92) (1688) (VIII) Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X, (1707) (1694) (IX) Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X, (1713) (1706) (X) Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X, (1725) (1712) (XI) Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X, (1731) (1718) (XII) Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X, (1737) (1724) (XIII) Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Z-X, and (1743) (1730) (XIV) Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X, (1749) wherein Y is H, a group for coupling the peptide to a solid phase or a carrier, 1 to 10 amino acids, a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 or is acetylated;
Z is a bond, 1 to 10 amino acids, or a linker arm by which the peptide can be attached to a carrier or solid phase comprising at least one amino acid and as many as 60 amino acids;
and X is NH2, OH or a linkage involving either of these two groups; and provided that when Y or Z-X are (an) amino acid(s), they are different from any naturally occurring HCV flanking regions;
(b) the variants of each of the above peptides (I) to (XIV), with said variants presenting conservative as well as non-conservative amino acid substitutions accommodating for less than 35% strain-to-strain variation in HCV sequences with respect to each of the amino acid sequences (I) to (XIV) provided that said variant peptides are capable of providing for immunological competition with at least one strain of HCV; and (c) fragments of peptides (I) to (XIV) having at least 6 amino acids of any of the peptide sequences 1-20, 7-26, 8-18, 13-32, 37-56, 49-68, 61-80, 73-92, 1688-1707, 1694-1713, 1706-1725, 1712-1731, 1718-1737, 1724-1743 and 1730-1749, as defined in part (a), and said fragments being capable of providing for immunological competition with at least one strain of HCV, provided that the combinations containing at least one peptide selected from the group of amino acid sequences consisting of I, II, IIA, III, IV, V, VI, VII and at least one peptide selected from the group of amino acid sequences consisting of VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX are excluded.
53. A peptide composition containing the following mixture of peptides as defined in claim 51 or 52:
(f) VIII, IX, XI, XIII, and XIX; and (g) XV, XVI, XVII, XVIII, and XIX.
(f) VIII, IX, XI, XIII, and XIX; and (g) XV, XVI, XVII, XVIII, and XIX.
54. The peptide composition or peptide according to any one of claims 51 to 53, wherein said peptides are coupled N-terminally, C-terminally or internally to a carrier molecule for the purpose of raising antibodies or facilitating the adsorption of said peptides to a solid phase.
55. The peptide composition or peptide according to any one of claims 51 to 54, wherein said peptides contain a detectable label.
56. The peptide or peptide composition according to any one of claims 51 to 55, wherein Y is H, or 1 to 10 amino acids and Z is a bond or 1 to 10 amino acids.
57. The peptide or peptide composition according to any one of claims 51 to 56, wherein Y is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
58. The peptide or peptide composition according to any one of claims 51 to 57, wherein Z is an amino acid selected from the group consisting of cysteine, lysine, tyrosine, glutamic acid and aspartic acid.
59. The peptide or peptide composition according to any one of claims 51 to 55, wherein the group for coupling the peptide to a solid phase or carrier is selected from the group consisting of biotin and thioglycolic acid.
60. Use of the peptide or peptide composition according to any one of claims 51 to 59, for the incorporation into an immunoassay for detecting the presence of antibodies to Hepatitis C virus present in a body fluid.
61. A method for the in vitro detection of antibodies to Hepatitis C virus present in a body fluid comprising at least the steps of:
(a) contacting said body fluid of a person to be diagnosed with the peptide composition or peptide according to any one of claims 51 to 59, and, (b) detecting an immunological complex formed between said antibodies and the peptide(s) used.
(a) contacting said body fluid of a person to be diagnosed with the peptide composition or peptide according to any one of claims 51 to 59, and, (b) detecting an immunological complex formed between said antibodies and the peptide(s) used.
62. A kit for the detection of anti-hepatitis C virus antibodies in a body fluid, comprising at least the following components:
- the peptide composition or peptide according to any one of claims 51 to 59, - a means for detecting an immunological complex formed between said peptides and said antibodies.
- the peptide composition or peptide according to any one of claims 51 to 59, - a means for detecting an immunological complex formed between said peptides and said antibodies.
63. The method according to claim 61, wherein said peptides are applied as lines on a nylon membrane.
64. The kit according to claim 62, wherein said peptides are applied as lines on a nylon membrane.
65. The kit according to claim 62 , wherein said peptides, separately or in combination, are used to coat the wells of microtiter plates.
66. Use of the peptide or peptide composition according to any one of claims 51 to 59, for incorporation into a vaccine composition against HCV.
67. The peptide or peptide composition according to any one of claims 51 to 59, for raising antibodies against HCV.
68. A peptide or peptide composition obtained by cyclizing any of the peptides of claims 51 to 52 or by coupling together two peptides of claims 51 to 52.
69. The peptide or peptide composition according to any one of claims 51 to 59, wherein said peptides are such as obtained by synthesizing the peptides of claims 51 or 52 directly on an oligo-lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the peptides.
70. The method according to any one of claims 11, 28 or 61, wherein said body fluid is serum or plasma.
71. A peptide comprising the amino acid sequence:
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
72. A peptide comprising the amino acid sequence:
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
73. A peptide comprising the amino acid sequence:
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
74. A peptide comprising the amino acid sequence:
(XVIII)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XVIII)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
75. A peptide comprising the amino acid sequence:
(XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
76. A peptide consisting of the amino acid sequence:
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala.
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala.
77. A peptide consisting of the amino acid sequence:
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn.
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn.
78. A peptide consisting of the amino acid sequence:
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr.
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr.
79. A peptide consisting of the amino acid sequence:
(XVIII)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro.
(XVIII)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-Gly-Cys-Pro-Leu-Pro-Pro.
80. A peptide consisting of the amino acid sequence:
(XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
(XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
81. The peptide according to any one of claims 71 to 80 wherein said peptide is cyclic.
82. A peptide selected from the group consisting of:
(a) a peptide consisting of a combination of at least two of amino acid sequences: II, III, V, IX and XVIII;
(b) a peptide consisting of a combination of at least two of amino acid sequences: I, II, V, IX, XI, XVI and XVIII;
(c) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV, V, VIII, XI, XVI and XVIII;
(d) a peptide consisting of a combination of at least two of amino acid sequences: II, IX
and XVIII;
(e) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV and V;
(f) a peptide consisting of a combination of at least two of amino acid sequences: VIII, IX, XI, XIII and XIV; and (g) a peptide consisting of a combination of at least two of amino acid sequences: XV, XVI, XVII, XVIII and XIX;
wherein the amino acid sequences I, II, III, IV, V are defined as in claim 1, and wherein the amino acid sequences VIII, IX, XI, XIII, XIV, XV, XVI, XVII, XVIII, and XIX are defined as in claim 2.
(a) a peptide consisting of a combination of at least two of amino acid sequences: II, III, V, IX and XVIII;
(b) a peptide consisting of a combination of at least two of amino acid sequences: I, II, V, IX, XI, XVI and XVIII;
(c) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV, V, VIII, XI, XVI and XVIII;
(d) a peptide consisting of a combination of at least two of amino acid sequences: II, IX
and XVIII;
(e) a peptide consisting of a combination of at least two of amino acid sequences: II, III, IV and V;
(f) a peptide consisting of a combination of at least two of amino acid sequences: VIII, IX, XI, XIII and XIV; and (g) a peptide consisting of a combination of at least two of amino acid sequences: XV, XVI, XVII, XVIII and XIX;
wherein the amino acid sequences I, II, III, IV, V are defined as in claim 1, and wherein the amino acid sequences VIII, IX, XI, XIII, XIV, XV, XVI, XVII, XVIII, and XIX are defined as in claim 2.
83. A peptide consisting of a combination of at least two peptides selected from the group consisting of:
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala;
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn;
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr;
(XV III)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-Val-His-Gly-Cys-Pro-Leu-Pro-Pro;and (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
(XV) Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala;
(XVI) Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn;
(XVII) Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr;
(XV III)Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-V al-Val-His-Gly-Cys-Pro-Leu-Pro-Pro;and (XIX) Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys.
84. A peptide comprising the amino acid sequence:
(VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
85. A peptide consisting of the amino acid sequence:
(VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu.
(VIII) Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu.
86. A peptide comprising the amino acid sequence:
(IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln, and a linker arm which is I to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln, and a linker arm which is I to 60 amino acids and are different from any naturally occurring HCV flanking regions.
87. A peptide consisting of the amino acid sequence:
(IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln.
(IX) Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln.
88. A peptide consisting of the amino acid sequence:
(X) Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala.
(X) Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala.
89. A peptide consisting of the amino acid sequence:
(XI) Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys.
(XI) Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys.
90. A peptide consisting of the amino acid sequence:
(XII) Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln.
(XII) Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln.
91. A peptide comprising the amino acid sequence:
(XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
92. A peptide consisting of the amino acid sequence:
(XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala.
(XIII) Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala.
93. The peptide according to any one of claims 84 to 92, wherein said peptide is coupled N-terminally, C-terminally or internally to a carrier molecule.
94. The peptide according to any one of claims 84 to 92, wherein said peptide contains a detectable label.
95. The peptide according to any one of claims 84 to 92, wherein said peptide has on its amino terminus an H, or one or more chemical linking groups, and has on its carboxy terminus an NH2, or one or more chemical linking groups.
96. The peptide according to any one of claims 84 to 92, wherein said peptide is cyclic and is immunoreactive with HCV antibodies.
97. The peptide of any one of claims 84 to 92 wherein said peptide is cyclic.
98. A peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are defined as in claim 2.
99. A peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences VIII, IX, X, XI, XII, XIII, and XIV, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV;
wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are defined as in claim 2.
wherein the amino acid sequences VIII, IX, X, XI, XII, XIII and XIV are defined as in claim 2.
100. A peptide comprising the amino acid sequence:
(I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
101. A peptide comprising the amino acid sequence:
(II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-VaI-Lys-Phe-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-VaI-Lys-Phe-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
102. A peptide comprising the amino acid sequence:
(III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
103. A peptide comprising the amino acid sequence:
(IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser; and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
104. A peptide comprising the amino acid sequence:
(V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
105. A peptide comprising the amino acid sequence:
(VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
106. A peptide comprising the amino acid sequence:
(VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
(VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly, and a linker arm which is 1 to 60 amino acids and are different from any naturally occurring HCV flanking regions.
107. A peptide consisting of the amino acid sequence:
(I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln.
(I) Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln.
108. A peptide consisting of the amino acid sequence:
(II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly.
(II) Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly.
109. A peptide consisting of the amino acid sequence:
(III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly.
(III) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly.
110. A peptide consisting of the amino acid sequence:
(IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser.
(IV) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser.
111. A peptide consisting of the amino acid sequence:
(V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val.
(V) Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val.
112. A peptide consisting of the amino acid sequence:
(VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly.
(VI) Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly.
113. A peptide consisting of the amino acid sequence:
(VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly.
(VII) Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly.
114. The peptide according to any one of claims 100 to 113, wherein said peptide is coupled N-terminally, C-terminally or internally to a carrier molecule.
115. The peptide according to any one of claims 91 to 97, wherein said peptide contains a detectable label.
116. The peptide of any one of claims 100 to 113, wherein said peptide is cyclic.
117. The peptide of any one of claims 100 to 113, wherein said peptide is cyclic and is immunoreactive with HCV antibodies.
118. The peptide of any one of claims 100 to 113, wherein said peptide has on its amino terminus an H, or one or more chemical linking groups, and has on its carboxy terminus an NH2, or one or more chemical linking groups.
119. A peptide comprising a linker arm which is 1 to 60 amino acids, and two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides having amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; and wherein the amino acid sequences I, II, III, IV, V, VI and VII
are defined as in claim 1.
are defined as in claim 1.
120. A peptide comprising two or more peptides joined together, said two or more peptides being selected from the group consisting of peptides consisting of amino acid sequences I, II, III, IV, V, VI, and VII, wherein said two or more peptides joined together is capable of providing immunological competition with at least one strain of HCV; wherein the amino acid sequences I, II, III, IV, V, VI, and VII are defined as in claim 1.
121. The method according to any one of claims 13, 30 and 63 wherein said nylon membrane is cut into strips perpendicular to the direction of the peptide lines, thus allowing each strip to be incubated with an appropriately diluted serum sample from an individual.
122. The kit according to any one of claims 14, 31 and 64 wherein said nylon membrane is cut into strips perpendicular to the direction of the peptide lines, thus allowing each strip to be incubated with an appropriately diluted serum sample from an individual.
123. The process according to claim 45 wherein said nylon membrane is cut into strips perpendicular to the direction of the peptide lines, thus allowing each strip to be incubated with an appropriately diluted serum sample from an individual.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SG1996005024A SG47062A1 (en) | 1990-12-14 | 1990-12-14 | Synthetic antigens for the detection of antibodies to hepatitis virus |
| EP90124241A EP0489968B1 (en) | 1990-12-14 | 1990-12-14 | Synthetic antigens for the detection of antibodies to hepatitis C virus |
| EP90124241.2 | 1990-12-14 | ||
| PCT/EP1991/002409 WO1992010514A2 (en) | 1990-12-14 | 1991-12-13 | Synthetic antigens for the detection of antibodies to hepatitis c virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2074370A1 CA2074370A1 (en) | 1992-06-15 |
| CA2074370C true CA2074370C (en) | 2011-02-15 |
Family
ID=43606393
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2074370A Expired - Lifetime CA2074370C (en) | 1990-12-14 | 1991-12-13 | Synthetic antigens for the detection of antibodies to hepatitis c virus |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2074370C (en) |
-
1991
- 1991-12-13 CA CA2074370A patent/CA2074370C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CA2074370A1 (en) | 1992-06-15 |
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| EEER | Examination request | ||
| MKEX | Expiry |