CA2662090A1 - New sequences of hepatitis c virus genotypes and their use as therapeutic and diagnostic agents - Google Patents
New sequences of hepatitis c virus genotypes and their use as therapeutic and diagnostic agents Download PDFInfo
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- CA2662090A1 CA2662090A1 CA002662090A CA2662090A CA2662090A1 CA 2662090 A1 CA2662090 A1 CA 2662090A1 CA 002662090 A CA002662090 A CA 002662090A CA 2662090 A CA2662090 A CA 2662090A CA 2662090 A1 CA2662090 A1 CA 2662090A1
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
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- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to a polynucleic acid composition comprising or consisting of at least one polynucleic acid containing 8 or more contiguous nucleotides corresponding to a nucleotide sequence from the region spanning positions 417 to 957 of the Core/E1 region of HCV type 3; and/or the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3; and/or the region spanning positions 4892 to 5292 of the NS3/4 region of HCV type 3; and/or the region spanning positions 8 023 to 8 235 of the NS5 region of the BR36 subgroup of HCV type 3a; and/or the coding region of HCV
type 4a starting at nucleotide 379 in the core region; and/or the coding region of HCV
type 4; and/or the coding region of HCV type 5, with said nucleotide numbering being with respect to the numbering of HCV nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV
type 1, and/or HCV type 2 genomes in the above-indicated regions, or the complement thereof.
type 4a starting at nucleotide 379 in the core region; and/or the coding region of HCV
type 4; and/or the coding region of HCV type 5, with said nucleotide numbering being with respect to the numbering of HCV nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV
type 1, and/or HCV type 2 genomes in the above-indicated regions, or the complement thereof.
Description
WO 94/25601 . PCT/EP94/01323 NEW SEQUENCES OF HEPATITIS C VIRUS GENOTYPES AND THEIR USE AS
THERAPEUTIC AND DIAGNOSTIC AGENTS
---------------------The invention relates to new sequences of hepatitis C virus (HCV) genotypes and their use as therapeutic and diagnostic agents.
' The present invention relates to new nucleotide and amino acid sequences corresponding to the coding region of a new type 2 subtype 2d, type-specific sequences corresponding to HCV type 3a, to new sequences corresponding to the coding region of a new subtype 3c, and to new sequences corresponding to the coding region of HCV type 4 and type 5 subtype 5a;
a process for preparing them, and their use for diagnosis, prophylaxis and therapy.
The technical problem underlying the present invention is to provide new type-specific sequences of the Core, the El, the E2, the NS3, the NS4 and the NS5 regions of HCV type 4. and type 5, as well as of new variants of HCV types 2 and 3. These new HCV
sequences are useful to diagnose the presence of type 2 and/or type 3 and/or type 4 and/or type 5 HCV
genotypes in a biological sample. Moreover, the availability of these new type-specific sequences can increase the overall sensitivity of HCV detection and should also prove to be useful for therapeutic purposes. -Hepatitis C viruses (HCV) have been found to be the major cause of non-A, non-B
hepatitis. The sequences of cDNA clones covering the complete genome of several prototype isolates have been determined (Kato et al., 1990; Choo et al., 1991; Okamoto et al., 1991;
Okamoto et al., 1992). Comparison of these isolates shows that the variability in nucleotide sequences can be used to distinguish at least 2 different genotypes, type 1(HCV-1 and HCV-J) and type 2(HC-J6 and HC-J8), with an average homology of about 68%. Within each type, at least two subtypes exist (e.g. represented by HCV-1 and HCV-J), having an average homology of about 79%. HCV genomes belonging to the same subtype show average homologies of more than 90% (Okamoto et al., 1992). However, the partial nucleotide sequence of the NS5 region of the HCV-T isolates showed at most 67 % homology with the ~= previously published sequences, indicating the existence of a yet another HCV type (Mori et al., 1992). Parts of the 5' untranslated region (UR), core, NS3, and NS5 regions of this type 3 have been published, further establishing the similar evolutionary distances between the 3 major genotypes and their subtypes (Chan et al., 1992).
The identification of type 3 genotypes in clinical samples can be achieved by means of PCR with type-specific primers for the NS5 region. However, the degree to which this will SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCT/EP941t1323 be successful is largely dependent on sequence variability and on the virus titer present in the serum. Therefore, routine PCR in the open reading frame, especially for type 3'and the new type 4 and 5 described in the present invention and/or group V (Cha et al., 1992) genotypes can be predicted to be unsuccessful. A new typing system (LiPA), based on variation in the highly conserved 5' UR, proved to be more useful because the 5 major HCV
genotypes and their subtypes can be determined (Stuyver et al., 1993). The selection of high-titer isolates enables to obtain PCR fragments for cloning with only 2 primers, while nested PCR requires that 4 primers match the unknown sequences of the new type 3, 4 and 5 genotypes.
New sequences of the 5' untranslated region (5'UR) have been listed by Bukh et al.
(1992). For some of these, the El region has recently been described (Bukh et al., 1993).
Isolates with similar sequences in the 5'UR to a group of isolates including DK12and HK10 described by Bukh et al. (1992) and E-b 1 to E-b8 described and classified as type 3 by Chan et al. (1991), have been reported and described in the 5'UR, the carboxyterminal part of El, and in the NS5 region as group IV by Cha et al. (1992; WO 92/19743), and have also been described in the 5'UR for isolate BR56 and classified as type 3 by the inventors of this application (Stuyver et al., 1993).
The aim of the present invention is to provide new HCV nucleotide and amino acid sequences enabling the detection of HCV infection.
Another aim of the present infection is to provide new nucleotide and amino acid HCV
sequences enabling the classification of infected biological fluids into different serological groups unambiguously linked to types and subtypes at the genome level.
Another aim of the present invention is to provide new nucleotide and amino acid HCV
sequences ameliorating the overall HCV detection rate.
Another aim of the present invention is to provide new HCV sequences, useful for the design of HCV vaccine compositions.
Another aim of the present invention is to provide a pharmaceutical composition consisting of antibodies raised against the polypeptides encoded by these new HCV
sequences, for therapy or diagnosis.
The present invention relates more particularly to a composition comprising or consisting of at least one polynucleic acid containing at least 5, and preferably 8 or more contiguous nucleotides selected from at least one of the following HCV sequences:
- an HCV type 3 genomic sequence, more particularly in any of the following regions:
SUBSTITUTE SHEET (RULE 26) - the region spanning positions 417 to 957 of the Core/El region of HCV
subtype 3a, - the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3, - the region spanning positions 4892 to 5292 of the NS3/4 region of HCV
type 3, - the region spanning positions 8023 to 8235 of the NS5 region of the BR36 subgroup of HCV subtype 3a, - an HCV subtype 3c genomic sequence, more particularly the coding regions of the above-specified regions;
- an HCV subtype 2d genomic sequence, more particularly the coding region of HCV
subtype 2d;
- an HCV type 4 genomic sequence, more particularly the coding region, more particularly the coding region of subtypes 4a, 4e, 4f, 4g, 4h, 4i, and 4j, - an HCV type 5 genomic sequence, more particularly the coding region of HCV
type 5, more particularly the regions encoding Core, El, E2, NS3, and NS4 with said nucleotide numbering being with respect to the numbering of HCV
nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV (type 1, type 2, and type 3) polynucleic acid sequences in the above-indicated regions, or the complement thereof.
It is to be noted that the nucleotide difference in the polynucleic acids of the invention may involve or not an amino acid difference in the corresponding amino acid sequences coded by said polynucleic acids.
According to a preferred embodiment, the present invention relates to a composition comprising or containi.ng at least one polynucleic acid encoding an HCV
polyprotein, with said polynucleic acid containing at least 5, preferably at least 8 nucleotides corresponding to at least part of an HCV nucleotide sequence encoding an HCV polyprotein, and with said HCV polyprotein containing in its sequence at least one of the following amino acid residues:
L7, Q43, M44, S60, R67, Q70, T71, A79, A87, N106, K115, A127, A190, S130, V134, 0142, I144, E152, A157, V158, P165, S177 or Y177, I178, V180 or E180 or F182, R184, 1186, H187, T189, A190, S191 or G191, Q192 or L192 or 1192 or V192 or E192, N193 or H193 or P193, W194 or Y194, H195, A197 or 1197 or V197 or T197, V202, I203 or L203, Q208, A210, V212, F214, T216, R217 or D217 or E217 or V217, H218 or N218, H219 or SllBSTITUTE SHEET (RULE 26) V219 or L219, L227 or I227, M231 or E231 or Q231, T232 or D232 or A232 or K232, Q235 or I235, A237 or T237, I242, I246, S247, S248, V249, S250 or Y250, I251 or V251 or M251 or F251, D252, 7254 or V254, L255 or V255, E256 or A256, M258 or F258 or V258, A260 or Q260 or S260, A261, T264 or Y264, M265, I266 or A266, A267, G268 or =
T268, F271 or M271 or V271, I277, M280 or H280, I284 or A284 or L84, V274, V291, N292 or S292, R293 or I293 or Y293, Q294 or R294, L297 or I297 or Q297, A299 or K299 or Q299, N303 or T303, T308 or L308, T310 or F310 or A310 or D310 or V310, L313, G317 or Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, 1403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, S532, V534, F536, F537, M539, I546, C1282, A1283, H1310, V1312, Q1321, P1368, V1372, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, D1556, N1567, M1572, Q1579, L1581, S1583, F1585, V1595, E1606 or T1606, M1611, V1612 or L1612, P1630, C1636, P1651, T1656 or 11656, L1663, V1667, V1677, A1681, H1685, E1687, G1689, V1695, A1700, Q1704, Y1705, A1713, A1714 or S1714, M1718, D1719, A1721 or T1721, R1722, A1723 or V1723, H1726 or G1726, E1730, V1732, F1735, 11736, S1737, R1738, T1739, G1740, Q1741, K1742, Q1743, A1744, T1745, L1746, E1747 or K1747, I1749, A1750, T1751 or A1751, V1753, N1755, K1756, A1757, P1758, A1759, H1762, T1763, Y1764, P2645, A2647, K2650, K2653 or L2653, S2664, N2673, F2680, K2681, L2686, H2692, Q2695 or L2695 or I2695, V2712, F2715, V2719 or Q2719, T2722, T2724, S2725, R2726, G2729, Y2735, H2739, 12748, G2746 or I2746, 12748, P2752 or K2752, P2754 or T2754, T2757 or P2757, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990.
Each of the above-mentioned residues can be found in any of Figures 2, 5, 7, 11 or 12 showing the new amino acid sequences of the present invention aligned with known sequences of other types or subtypes of HCV for the Core, El, E2, NS3, NS4, and NS5 regions.
More particularly, a polynucleic acid contained in the composition according to the present invention contains at least 5, preferably 8, or more contiguous nucleotides corresponding to a sequence of contiguous nucleotides selected from at least one of HCV
sequences encoding the following new HCV amino acid sequences:
- new sequences spanning amino acid positions 1 to 319 of the Core/El region of HCV
subtype 2d, type 3 (more particularly new sequences for subtypes 3a and 3c), new type 4 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 subtypes (more particularly new sequences for subtypes 4a, 4e, 4f, 4g, 4h, 4i and 4j) and type 5a, as shown in Figure 5;
- new sequences spanning amino acid positions 328 to 546 of the E 1/E2 region of HCV
. subtype 5a as shown in Figure 12;
- new sequences spanning amino acid positions 1556 to 1764 of the NS3/NS4 region of HCV type 3 (more particularly for new subtypes 3a sequences), and subtype 5a, as shown in Figure 7 or 11;
- new sequences spanning amino acid positions 2645 to 2757 of the NS5B region of HCV
subtype 2d, type 3 (more particularly for new subtypes 3a and 3c), new type 4 subtypes (more particularly subtypes 4a, 4e, 4f, 4g, 4h, 4i and 4j) and subtype 5a, as shown in Figure 2, Using the LiPA system mentioned above, Brazilian blood donors with high titer type 3 hepatitis C virus, Gabonese patients with high-titer type 4 hepatitis C virus, and a Belgian patient with high-titer HCV type 5 infection were selected. Nucleotide sequences in the core, El, NS5 and NS4 regions which have not yet been reported before, were analyzed in the frame of the invention. Coding sequences (with the exception of the core region) of any type 4 isolate are reported for the first time in the present invention. The NS5b region was also analyzed for the new type 3 isolates. After having determined the NS5b sequences, comparison with the Ta and Tb subtypes described by Mori et al. (1992) was possible, and the type 3 sequences could be identified as type 3a genotypes. The new type 4 isolates segregated into 10 subtypes, based on homologies obtained in the NS5 and El regions. New type 2 and 3 sequences could also be distinguished from previously described type 2 or 3 subtypes from sera collected in Belgium and the Netherlands.
The term "polynucleic acid" refers to a single stranded or double stranded nucleic acid sequence which may contain at least 5 contiguous nucleotides to the complete nucleotide sequence (f.i. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous nucleotides). A
polynucleic acid which is up till about 100 nucleotides in length is often also referred to as an oligonucleotide. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes. A polynucleic acid may also comprise a double stranded cDNA clone which can be used for cloning purposes, or for in vivo therapy, or prophylaxis.
The term "polynucleic acid composition" refers to any kind of composition comprising essentially said polynucleic acids. Said composition may be of a diagnostic or a therapeutic SUBSTITUTE SHEET (RULE 26) nature.
The expression "nucleotides corresponding to" refers to nucleotides which are homologous or complementary to an indicated nucleotide sequence or region within a specific HCV
sequence. =
The term "coding region" corresponds to the region of the HCV genome that encodes the HCV polyprotein. In fact, it comprises the complete genome with the exception of the 5' untranslated region and 3' untranslated region.
The term "HCV polyprotein" refers to the HCV polyprotein of the HCV-J isolate (Kato et al., 1990). The adenine residue at position 330 (Kato et al., 1990) is the first residue of the ATG codon that initiates the long HCV polyprotein of 3010 amino acids in HCV-J and other type lb isolates, and of 3011 amino acids in HCV-1 and other type la isolates, and of 3033 amino acids in type 2 isolates HC-J6 and HC-J8 (Okamoto et al., 1992).
This adenine is designated as position 1 at the nucleic acid level, and this methionine is designated as position 1 at the amino acid level, in the present invention. As type la isolates contain 1 extra amino acid in the NS5a region, coding sequences of type la and lb have identical numbering in the Core, El, NS3, and NS4 region, but will differ in the NS5b region as indicated in Table 1. Type 2 isolates have 4 extra amino acids in the E2 region, and 17 or 18 extra amino acids in the NS5 region compared to type 1 isolates, and will differ in numbering from type 1 isolates in the NS3/4 region and NS5b regions as indicated in Table 1.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT1EP94/01323 Region Positions Positions Positions Positions = described in described for described for described for the HCV-J HCV-1 HC-J6, HC-J8 present (Kato et aL, (Choo et al., (Okamoto et invention* 1990) 1991) al., 1992) Nucleotide NS5b 8023/8235 8352/8564 8026/8238 8433/8645 s 7932/8271 8261/8600 7935/8274 8342/8681 coding 330/9359 1/9033 342/9439 region of present invention Amino NS5b 2675/2745 2675/2745 2676/2746 2698/2768 Acids 2645/2757 2645/2757 2646/2758 2668/2780 Table 1: Comparison of the HCV nucleotide and amino acid numbering system used in the present invention (*) with the numbering used for other prototype isolates.
For example, 8352/8564 indicates the region designated by the numbering from nucleotide 8352 to nucleotide 8564 as described by Kato et al. (1990). Since the numbering system of the present invention starts at the polyprotein initiation site, the 329 nucleotides of the 5' untranslated region described by Kato et al.
(1990) have to be substracted, and the corresponding region is numbered from nucleotide 8023 ("8352-329") to 8235 ("8564-329").
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "HCV type" corresponds to a group of HCV isolates of which the complete genome shows more than 74% homology at the nucleic acid level, or of whicb the NS5 region between nucleotide positions 7932 and 8271 shows more than 74% homology at the nucleic acid level, or of which the complete HCV polyprotein shows more than 78%
homology at the =
amino acid level, or of which the NS5 region between amino acids at positions 2645 and 2757 shows more than 80% homology at the amino acid level, to polyproteins of the other isolates of the group, with said numbering beginning at the first ATG codon or first methionine of the long HCV polyprotein of the HCV-J isolate (Kato et al., 1990). Isolates belonging to different types of HCV exhibit homologies, over the complete genome, of less than 74% at the nucleic acid level and less than 78% at the amino acid level. Isolates belonging to the same type usually show homologies of about 92 to 95% at the nucleic acid level and 95 to 96% at the amino acid level when belonging to the same subtype, and those belonging to the same type but different subtypes preferably show homologies of about 79% at the nucleic acid level and 85-86% at the amino acid level.
More preferably the definition of HCV types is concluded from the classification of HCV
isolates according to their nucleotide distances calculated as detailed below:
(1) based on phylogenetic analysis of nucleic acid sequences in the NS5b region between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and 8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al., 1991), isolates belonging to the same HCV type show nucleotide distances of less than 0.34, usually less than 0.33, and more usually of less than 0.32, and isolates belonging to the same subtype show nucleotide distances of less than 0.135, usually of less than 0.13, and more usually of less than 0.125, and consequently isolates belonging to the same type but different subtypes show nucleotide distances ranging from 0.135 to 0.34, usually ranging from 0.1384 to 0.2477, and more usually ranging from 0.15 to 0.32, and isolates belonging to different HCV types show nucleotide distances greater than 0.34, usually greater that 0.35, and more usually of greater than 0.358, more usually ranging from 0.1384 to 0.2977.
(2) based on phylogenetic analysis of nucleic acid sequences in the core/El region between nucleotides 378 and 957, isolates belonging to the same HCV type show nucleotide distances of less than 0,38, usually of less than 0.37, and more usually of less than 0.364, and isolates belonging to the same subtype show nucleotide distances of less than 0.17, usually of less than 0.16, and more usually of less than 0.15, more usually less than 0.135, more usually less than 0.134, and consequently isolates belonging to the same type but different subtypes show SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 nucleotide distances ranging from 0.15 to 0.38, usually ranging from 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, more usually ranging from 0.133 to 0.379, =and isolates belonging to different HCV types show nucleotide distances greater than 0.34, 0.35, 0.36, usually more than 0.365, and more usually of greater than 0.37, (3) based on phylogenetic analysis of nucleic acid sequences in the NS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) or between nucleotides 4993 and 5621 (Kato et al., 1990) or between nucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging to the same HCV type show nucleotide distances of less than 0.35, usually of less than 0.34, and more usually of less than 0.33, and isolates belonging to the same subtype show nucleotide distances of less than 0.19, usually of less than 0.18, and more usually of less than 0.17, and consequently isolates belonging to the same type but different subtypes show nucleotide distances ranging from 0.17 to 0.35, usually ranging from 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, and isolates belonging to different HCV
types show nucleotide distances greater than 0.33, usually greater than 0.34, and more usually of greater than 0.35.
Table 2 : Molecular evolutionary distances Region Core/EI El NS5B NSSB
579 bp 384 bp 340 bp 222 bp Isolates 0.0017 - 0.1347 0.0026 - 0.2031 0.0003 - 0.1151 0.000 - 0.1323 (0.0750 0.0245) (0.0969 0.0289) (0.0637 0.0229) (0.0607 0.0205) Subtypes' 0.1330 - 0.3794 0.1645 - 0.4869 0.1384 - 0.2977 0.117 - 0.3538 (0.2786 0.0363) (0.3761 0.0433) (0.2219 0.0341) (0.2391 0.0399) Types' 0.3479 - 0.6306 0.4309 - 0.9561 0.3581 - 0.6670 0.3457 - 0.7471 (0.4703 0.0525) (0.6308 0.0928) (0.4994 0.0495) (0.5295 + 0.0627) ' Figures created by the PHYLIP program DNADIST are expressed as minimum to maximum (average standard deviation). Phylogenetic distances for isolates belonging to the same subtype ('isolates'), to different subtypes of the same type ('subtypes'), and to different types ('types') are given.
In a comparative phylogenetic analysis of available sequences, ranges of molecular evolutionary distances for different regions of the genome were calculated, based on 19,781 SUBSTITUTE SHEET (RULE 26) pairwise comparisons by means of the DNA DIST program of the phylogeny inference package PHYLIP version 3.5C (Felsenstein, 1993). The results are shown in Table 2 and indicate that although the majority of distances obtained in each region fit with classification of a certain isolate, only the ranges obtained in the 340bp NS5B-region are non-overlapping =
and therefor conclusive. However, as was performed in the present invention, it is preferable to obtain sequence information from at least 2 regions before final classification of a given isolate.
Designation of a number to the different types of HCV and HCV types nomenclature is based on chronological discovery of the different types. The numbering system used in the present invention might still fluctuate according to international conventions or guidelines. For example, "type 4" migbt be changed into "type 5" or "type 6".
The term "subtype" corresponds to a group of HCV isolates of which the complete polyprotein shows a homology of more than 90% both at the nucleic acid and amino acid levels, or of which the NS5 region between nucleotide positions 7932 and 8271 shows a homology of more than 90% at the nucleic acid level to the corresponding parts of the genomes of the other isolates of the same group, with said numbering beginning with the adenine residue of the initiation codon of the HCV polyprotein. Isolates belonging to the same type but different subtypes of HCV show homologies of more than 74 % at the nucleic acid level and of more than 78% at the amino acid level.
The term "BR36 subgroup" refers to a group of type 3a HCV isolates (BR36, BR33, BR34) that are 95 %, preferably 95.5 %, most preferably 96 % homologous to the sequences as represented in SEQ ID NO 1, 3, 5, 7, 9, 11 in the NS5b region from position 8023 to 8235.
It is to be understood that extremely variable regions like the El, E2 and NS4 regions will exhibit lower homologies than the average homology of the complete genome of the polyprotein.
Using these criteria, HCV isolates can be classified into at least 6 types.
Several subtypes can clearly be distinguished in types l, 2, 3 and 4 : la, ib, 2a, 2b, 2c, 2d, 3a, 3b, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i and 4j based on homologies of the 5' UR and coding regions including the part of NS5 between positions 7932 and 8271. An overview of most of the reported isolates and their proposed classification according to the typing system of the present invention as well as other proposed classifications is presented in Table 3.
SUBST{TUTE SHEET (RULE 26) T le3 HCV CLASSIFICATION
OKA- MORI NAKA CHA PROTOTYPE
la I I Pt GI HCV-l, HCV-H, HC-J1 lb II II KI GII HCV-J, HCV-BK, HCV-T, HC-JK1, HC-J4, HCV-CH.TNA
ic HC-G9 2a III III K2a GIII HC-J6 2b IV IV K2b GIII HC-J8 2c S83, ARG6, ARG8, 110, T983 2d NE92 3a V V K3 GIV E-bi, Ta, BR36, BR33, HD10, NZLI
3b VI K3 GIV HCV-TR, Th 3c BE98 4a Z4, GB809-4 4b - Zi 4c GB116, GB358, GB215, Z6, Z7 4d DK13 4e GB809-2, CAM600, CAM736 4f CAM622, CAM627 4g GB549 4h - GB438 4i CAR4/1205 4j CARl/501 4k EG29 5a GV SA3, SA4, SAl, SA7, SAli, BE95 6a HK I, HK2, HK3, HK4 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "complement" refers to a nucleotide sequence which is complementary to an indicated sequence and which is able to hybridize to the indicated sequences.
The composition of the invention can comprise many combinations. By way of example, the composition of the invention can comprise: - two (or more) nucleic acids from the same region or, - two nucleic acids (or more), respectively from different regions, for the same isolate or for different isolates, - or nucleic acids from the same regions and from at least two different regions (for the same isolate or for different isolates).
The present invention relates more particularly to a polynucleic acid composition as defined above, wherein said polynucleic acid corresponds to a nucleotide sequence selected from any of the following HCV type 3 genomic sequences:
- an HCV genomic sequence having a homology of at least 67%, preferably more than 69%, more preferably 71 %, even more preferably more than 73 %, or most preferably more than 76% to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD 10, BR36 or BR33 sequences) in the region spanning positions 417 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence having a homology of at least 65 %, preferably more than 67 %, preferably more than 69%, even preferably more than 70%, most preferably more than 74% to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD 10, BR36 or BR33 sequences) in the region spanning positions 574 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of at least 79%, more preferably at least 81 %, most preferably more than 83 % or more to any of the sequences as represented in SEQ ID NO 147 (representing positions 1 to 346 of the Core region of HVC type 3c, sequence BE98) in the region spanning positions 1 to 378 of the Core region as shown in Figure 3;
- an HCV genomic sequence of HVC type 3a having a homology of at least 74 %, more preferably at least 76 %, most preferably more than 78 % or more to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HDIO, BR36 or sequences) in the region spanning positions 417 to 957 in the Core/El region as shown in Figure 4;
- an HCV genomic sequence of HCV type 3a as having a homology of at least 74 %, SUBSTITUTE SHEET (RULE 26) preferably more than 76 %, most preferably 78 % or more to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD10, BR36 or BR33 sequences) in the region spanning positions 574 to 957 in the El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of more than 73.5 %, preferably more than 74 %, most preferably 75 % homology to the sequence as represented in SEQ
ID NO
29 (HCC153 sequence) in the region spanning positions 4664 to 4730 of the NS3 region as shown in figure 6;
- an HCV genomic sequence having a homology of more than 70%, preferably more than 72%, most preferably more than 74% homology to any of the sequences as represented in SEQ ID NO 29, 31, 33, 35, 37 or 39 (HCC153, HD10, BR36 sequences) in the region spanning positions 4892 to 5292 in the NS3/NS4 region as shown in Figure 6 or 10;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a as having a homology of more than 95%, preferably 95,5%, most preferably 96% homology to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in the region spanning positions 8023 to 8235 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a as having a homology of more than 96%, preferably 96.5 %, most preferably 97% homology to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in the region spanning positions 8023 to 8192 of the NS5B region as shown in Figure 1;
- an HCV genomic sequence of HCV type 3c being characterized as having a homology of more than 79 %, more preferably more than 81 %, and most preferably more than 83 % to the sequence as represented in SEQ ID NO 149 (BE98 sequence) in the region spanning positions 7932 to 8271 in the NS5B region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- an HCV genomic sequence being characterized as having a nucleotide distance of less than 0.44, preferably of less than 0.40, most preferably of less than 0.36 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 spanning positions 417 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence being characterized having a nucleotide distance of less than 0.53, preferably less than 0.49, most preferably of less than 0.45 to any of the sequences as represented in SEQ ID NO 19, 21, 23, 25 or 27 in the region spanning positions 574 =
to 957 of the E1 region as shown in Figure 4;
- an HCV genomic sequence characterized having a nucleotide distance of less than 0.15, preferably less than 0.13, and most preferably less than 0.11 to any of the sequences as represented in SEQ ID NO 147 in the region spanning positions 1 to 378 of the Core region as shown in Figure 3;
- an HCV genomic sequence of HVC type 3a being characterized as having a nucleotide distance of less than 0.3, preferably less than 0.26, most preferably of less than 0.22 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region spanning positions 417 to 957 in the Core/El region as shown in Figure 4;
- an HCV genomic sequence of HCV type 3a being characterized as having a nucleotide distance of less than 0.35, preferably less than 0.31, most preferably of less than 0.27 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region spanning positions 574 to 957 in the El region as shown in Figure 4;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a being characterized as having a nucleotide sequence of less than 0.0423, preferably less than 0.042, preferably less than 0.0362 to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 in the region spanning positions 8023 to 8235 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence of HCV type 3c being characterized as having a nucleotide distance of less than 0.255, preferably of less than 0.25, more preferably of less than 0.21, most preferably of less than 0.17 to the sequence as represented in SEQ ID NO
149 in the region spanning positions 7932 to 8271 in the NS5B region as shown in Figure 1.
In the present application, the El sequences encoding the antigenic ectodomain of the El protein, which does not overlap the carboxyterminal signal-anchor sequences of El disclosed by Cha et al. (1992; WO 92/19743), in addition to the NS4 epitope region, and a part of the NS5 region are disclosed for 4 different isolates: BR33, BR34, BR36, HCC153 and HD10, all belonging to type 3a (SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 or 39).
Also within the present invention are new subtype 3c sequences (SEQ ID NO 147, 149 of the isolate BE98 in the Core and NS5 regions (see Figures 3 and 1).
SUBSTITkJTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 Finally the present invention also, relates to a new subtype 3a sequence as represented in SEQ ID NO 217 (see Figure 1) Also included within the present invention are sequence variants of the polynucleic acids = as selected from any of the nucleotide sequences as given in any of the above mentioned SEQ
ID numbers, with said sequence variants containing either deletions and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 3 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type 3 as shown in Figure 1(NS5 region), Figure 3 (Core region), Figure 4 (Core/El region), Figure 6 and 10 (NS3/NS4 region).
According to another embodiment, the present invention relates to a polynucleic acid composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 5 genomic sequences:
- an HCV genomic sequence as having a homology of more than 85 %, preferably more than 86%, most preferably more than 87% homology to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 (PC sequences) or 151 (BE95 sequence) in the region spanning positions 1 to 573 of the Core region as shown in Figure 9 and 3;
- an HCV genomic sequence as having a homology of more than 61 %, preferably more than 63 %, more preferably more than 65 % homology, even more preferably more than 66%
homology and most preferably more than 67 % homology (f. i. 69 and 71 %) to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 (PC
sequences), 153 or 155 (BE95, BE100 sequences) in the region spanning positions 574 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 76.5 %, preferably of more than 77%, most preferably of more than 78% homology with any of the sequences as represented in SEQ ID NO 55, 57, 197 or 199 (PC sequences) in the region spanning positions 3856 to 4209 of the NS3 region as shown in Figure 6 or 10;
, - an HCV genomic sequence having a homology of more than 68 %, preferably of more than 70 %, most preferably of more than 72 % homology with the sequence as represented in SEQ ID NO 157 (BE95 sequence) in the region spanning positions 980 to 1179 of the E1/E2 region as shown in Figure 13;
- an HCV genomic sequence having a homology of more than 57 %, preferably more than S1lBST(Tt1TE SHEET (RULE 26) 59%, most preferably more than 61 % homology to any of the sequences as represented in SEQ ID NO 59 or 61 (PC sequences) in the region spanning positions 4936 to 5296 of the NS4 region as shown in Figure 6 or 10;
- an HCV genomic sequence as having a homology of more than 93 %, preferably more than =
93.5%, most preferably more than 94% homology to any of the sequences as represented in SEQ ID NO 159 or 161 (BE95 or BE96 sequences) in the region spanning positions 7932 to 8271 of the NS5B region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- a nucleotide distance of less than 0.53, preferably less than 0.51, more preferably less than 0.49 for the El region to the type 5 sequences depicted above;
- a nucleotide distance of less than 0.3, preferably less than 0.28, more preferably of less than 0.26 for the Core region to the type 5 sequences depicted above;
- a nucleotide distance of less than 0.072, preferably less than 0.071, more preferably less than 0.070 for the NS5B region to the type 5 sequences as depicted above.
Isolates with similar sequences in the 5'UR to a group of isolates including SA1, SA3, and SA7 described in the 5'UR by Bukh et al. (1992), have been reported and described in the 5'UR and NS5 region as group V by Cha et al. (1992; WO 92/19743). This group of isolates belongs to type 5a as described in the present invention (SEQ ID NO 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 151, 153, 155, 157, 159, 161, 197 and 199).
Also included within the present invention are sequence variants of the polynucleic acids as selected from any of the nucleotide sequences as given in any of the above given SEQ ID
numbers with said sequence variants containing either deletion and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between different genotypes of HCV) by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 5 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type as shown in Figure 3 (Core region), Figure 4(Core/E1 region), Figure 10 (NS3 /
region), Figure 14 (E1/E2 region).
SUBS"T{TUTE SHEET (RULE 26) Another group of isolates including BU74 and BU79 having similar sequences in the 5'UR
to isolates including Z6 and Z7 as described in the 5'UR by Bukh et al.
(1992), have been described in the 5'UR and classified as a new type 4 by the inventors of this application (Stuyver et al., 1993). Coding sequences, including core, El and NS5 sequences of several new Gabonese isolates belonging to this group, are disclosed in the present invention (SEQ
ID NO 106, 108, 110, 112, 114, 116, 118, 120 and 122).
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 4 genomic sequences:
- an HCV genomic sequence having a homology of more than 66%, preferably more than 68%, most preferably more than 70% homology in the El region spanning positions 574 to 957 to any of the sequences as represented in SEQ ID NO 118, 120 or 122 (GB358, GB549, GB809 sequences) as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 71 %, preferably more than 72 %, most preferably more than 74 % homology to any of the sequences as represented in SEQ ID NO 118, 120 or 122 (GB358, GB549, GB809 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 92 %, preferably more than 93%, most preferably more than 94% homology to any of the sequences as represented in SEQ ID NO 163 or 165 (GB809, CAM600 sequences) in the region spanning positions 1 to 378 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4c) having a homology of more than 85 %, preferably more than 86 %, more preferably more than 86.5 % homology, most preferably more than 87, more than 88 or more than 89% homology to any of the sequences as represented in SEQ ID NO 183, 185 or 187 (GB116, GB215, GB809 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4a) having a homology of more than 81 %, preferably more than 83 %, most preferably more than 85 % homology to the sequence as represented in SEQ ID NO 189 (GB908 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4e) having a homology of more than 85 %, preferably more than 87 %, most preferably more than 89 % homology to any of the sequences as represented in SEQ ID NO 167 or 169 (CAM600, GB908 sequences) in the region SUBSTITUTE SHEET (RULE 26) spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4f) having a homology of more than 79%, preferably more than 81 %, most preferably more than 83 % homology to any of the sequences as represented in SEQ ID NO 171 or 173 (CAMG22, CAMG27 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4g) having a homology of more than 84 %, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 175 (GB549 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4h) having a homology of more than 83 %, preferably more than 85 %, most preferably more than 87% homology to the sequence as represented in SEQ ID NO 177 (GB438 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
-' an HCV genomic sequence (subtype 4i) as having a homology of more than 76%, preferably more than 78%, most preferably more than 80% homology to the sequence as represented in SEQ ID NO 179 (CAR4/1205 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4j?) having a homology of more than 84 %, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 181 (CAR4/901 sequence) in the region spanning positions 379 to 957 of the El region as shown in figure 4;
- an HCV genomic sequence as having a homology of more than 73 %, preferably more than 75 %, most preferably more than 77 % homology to any of the sequences as represented in SEQ ID NO 106, 108, 110, 112, 114, or 116 (GB48, GB116, GB215, GB358, GB549, GB809 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4c) having a homology of more than 88%, preferably more than 89%, most preferably more than 90% homology to any of the sequences as represented in SEQ ID NO 106, 108, 110, or 112 (GB48, GB 116, GB215, GB358 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4e) having a homology of more than 88 %, preferably more than 89 %, most preferably more than 90 % homology to any of the sequences as SUBSTITUTE SHEET (RULE 26) represented in SEQ ID NO 116 or 201 (GB809 or CAM 600 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4f) having a homology of more than 87%, preferably = more than 89 %, most preferably more than 90 % homology to the sequence as represented in SEQ ID NO 203 (CAMG22 sequence) in the region spanning positions 7932 to of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4g) as having a homology of more than 85 %, preferably more than 87 %, most preferably more than 89 % homology to the sequence as represented in SEQ ID NO 114 (GB549 sequence) in the region spanning positions to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4h) as having a homology of more than 86%, preferably more than 87%, more preferably more than 88% homology, more preferably more than 89% homology to the sequence as represented in SEQ ID NO 207 (GB437 sequence) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4i) having a homology of more than 84%, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 209 (CAR4/1205 sequence) in the region spanning positions 7932 to of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4j) having a homology of more than 81 %, preferably more than 83 %, most preferably more than 85 % homology to the sequence as represented in SEQ ID NO 211 (CARI/501 sequence) in the region spanning positions 7932 to of the NS5 region as shown in figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of , less than 0.52, 0.50, 0.4880, 0.46, 0.44, 0.43 or most preferably less than 0.42 in the region spanning positions 574 to 957 to any of the sequences as represented in SEQ ID NO
118, 120 or 122 in the region spanning positions 1 to 957 of the Core/E1 region as shown in Figure 4;
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of SUBSTffEtTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 less than 0.39, 0.36 0.34 0.32 or most preferably less than 0.31 to any of the sequences as represented in SEQ ID NO 118, 120 or 122 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4c) being characterized as having a nucleotide distance of less than 0.27, 0.26, 0.24, 0.22, 0.20, 0.18, 0.17, 0.162, 0.16 or most preferably less than 0.15 to any of the sequences as represented in SEQ ID NO 183, 185 or 187 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4a) being characterized as having a nucleotide distance of less than 0.30, 0.28, 0.26, 0.24, 0.22, 0.21 or most preferably of less than 0.205 to the sequence as represented in SEQ ID NO 189 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4e) being characterized as having a nucleotide distance of less than 0.26, 0.25, 0.23, 0.21, 0.19, 0.17, 0.165, most preferably less than 0.16 to any of the sequences as represented in SEQ ID NO 167 or 169 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4f) being characterized as having a nucleotide distance of less than 0.26, 0.24, 0.22, 0.20, 0.18, 0.16, 0.15 or most preferably less than 0.14 to any of the sequences as represented in SEQ ID NO 171 or 173 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4g) being characterized as having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17 or most preferably of less than 0.16 to the sequence as represented in SEQ ID NO 175 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4h) being characterized as having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17 and most preferably of less than 0.16 to the sequence as represented in SEQ ID NO 177 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4i) being characterized as having a nucleotide distance of less than 0.27, 0.25, 0.23, 0.21 and preferably less than 0.16 to the sequence as represented in SEQ ID NO 179 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4j?) being characterized as having a nucleotide distance of less than 0.19, 0.18, 0.17, 0.165 and most preferably of less than 0.16 to the SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 sequence as represented in SEQ ID NO 181 in the region spanning positions 379 to 957 of the El region as shown in figure 4;
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of less than 0.35, 0.34, 0.32 and most preferably of less than 0.30 to any of the sequences as represented in SEQ ID NO 106, 108, 110, 112, 114, or 116 in the region spann.ing positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4c) being characterized as having a nucleotide distance of less than 0.18, 0.16, 0.14, 0.135, 0.13, 0.1275 or most preferably less than 0.125 to any of the sequences as represented in SEQ ID NO 106, 108, 110, or 112 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4e) being characterized as having a nucleotide distance of less than 0.15, 0.14, 0.135, 0.13 and most preferably of less than 0.125 to any of the sequences as represented in SEQ ID NO 116 or 201 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4f) being characterized as having a nucleotide distance of less than 0.15, 0.14, 0.135, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 203 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4g) being characterized as having a nucleotide distance of less than 0.17, 0.16, 0.15, 0.14, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 114 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4h) being characterized as having a nucleotide distance of less than 0.155, 0.15, 0.145, 0.14, 0.135, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 207 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4i) being characterized as having a nucleotide distance of less than 0.17, 0.16, 0.15, 0.14, 0.13 or most preferably of less than 0.125 to the sequence as represented in SEQ ID NO 209 in the region spanning positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4j) being characterized as having a nucleotide distance of less than 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13 and most preferably of less than 0.125 to the sequence as represented in SEQ ID NO 211 in the region spanning SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCTlEP94101323 positions 7932 to 8271 of the NS5 region as shown in figure 1.
Also included within the present invention are sequence variants of the polynucleic acids as selected from any of the nucleotide sequences as given in any of the above given SEQ ID
numbers with said sequence variants containing either deletion and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between different genotypes of HCV) by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 4 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type 4 as shown in Figure 3 (Core region), Figure 4 (Core/El region), Figure 10 (NS3 / NS4 region), Figure 14 (E1/E2 region).
The present invention also relates to a sequence as represented in SEQ ID NO
193 (GB724 sequence).
After aligning NS5 or El sequences of GB48, GB, 116, GB215, GB358, GB549 and GB809, these isolates clearly segregated into 3 subtypes within type 4: GB48, GB116, GB215 and GB358 belong to the sybtype designated 4c, GB549 to subtype 4g and GB809 to subtype 4e. In NS5, GB809 (subtype 4e) showed a higher nucleic acids homology to subtype 4c isolates (85.6 - 86. 8%) than to GB549 (subtype 4g, 79.7 %), while GB549 showed similar homologies to both other subtypes (78.8 to 80% to subtype 4c and 79.7% to subtype 4e). In El, subtype 4c showed equal nucleic acid homologies of 75.2% to subtypes 4g and 4e while 4g and 4e were 78.4 % homologous. At the amino acid level however, subtype 4e showed a normal homology to subtype 4c (80.2%), while subtype 4g was more homologous to 4c (83.3 %) and 4e (84.1 %).
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 2d genomic sequences:
- an HCV genomic sequence as having a homology of more than 78 %, preferably more than 80%, most preferably more than 82% homology to the sequence as iepresented in SEQ
ID NO (NE92) 143 in the region spanning positions 379 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of more than 74 %, preferably more than 76%, most preferably more than 78% homology to the sequence as represented in SEQ
ID NO 143 (NE92) in the region spanning positions 574 to 957 as shown in Figure 4;
SUBSTTrUTE SHEET (RULE 26) - an HCV genomic sequence as having a homology of more than 87%, preferably more than 89 %, most preferably more than 91 % homology to the sequence as represented in SEQ
ID NO 145 (NE92) in the region spanning positions 7932 to 8271 of the NS5B
region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- a nucleotide distance of less than 0.32, preferably less than 0.31, more preferably less than 0.30 for the El region (574 to 957) to any of the above specified sequences;
- a nucleotide distance of less than 0.08, preferably less than 0.07, more preferably less than 0.06 for the Core region (1 to 378) to any of the above given sequences - a nucleotide distance of less than 0.15, preferantially less than 0.13, more preferentially less than 0.12 for the NS5B region to any of the above-specified sequences.
Polynucleic acid sequences according to the present invention which are homologous to the sequences as represented by a SEQ ID NO can be characterized and isolated according to any of the techniques known in the art, such as amplification by means of type or subtype specific primers, hybridization with type or subtype specific probes under more or less stringent conditions, serological screening methods (see examples 4 and 11) or via the LiPA typing system.
Polynucleic acid sequences of the genomes indicated above from regions not yet depicted in the present examples, figures and sequence listing can be obtained by any of the techniques known in the art, such as amplification techniques using suitable primers from the type or subtype specific sequences of the present invention.
The present invention relates also to a composition as defined above, wherein said polynucleic acid is liable to act as a primer for amplifying the nucleic acid of a certain isolate belonging to the genotype from which the primer is derived.
An example of a primer according to this embodiment of the invention is HCPr 152 as shown in table 7 (SEQ ID NO 79).
The term "primer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products.
SUBSTITEJTE SHEET (RULE 26) WO 94/25601 PCTlEP94/01323 Preferably the primer is about 5-50 nucleotides. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
The fact that amplification primers do not have to match exactly with corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR;
Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 1989;
Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of QB replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules using primer extension. During amplification, the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplification reaction is repeated between 20 and 80 times, advantageously between 30 and 50 times. -The present invention also relates to a composition as defined above, wherein said polynucleic acid is able to act as a hybridization probe for specific detection andlor classification into types of a nucleic acid containing said nucleotide sequence, with said oligonucleotide being possibly labelled or attached to a solid substrate.
The term "probe" refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence of the HCV
genotype(s) to be detected.
Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about to 25 nucleotides.
The term "solid support" can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead).
Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fiucation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/El'94/01323 genomes present in a sample. The term "universal HCV primer" refers to oligonucleotide sequences complementary to any of the conserved regions of the HCV genome.
The expression "appropriate" hybridization and washing conditions are to be understood as stringent and are generally known in the art (e.g. Maniatis et al., Molecular Cloning: A
Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982).
However, according to the hybridization solution (SSC, SSPE, etc.), these probes should be hybridized at their appropriate temperature in order to attain sufficient specificity.
The term "labelled" refers to the use of labelled nucleic acids. This may include the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art.
The process of the invention comprises the steps of contacting any of the probes as defined above, with one of the following elements:
- either a biological sample in which the nucleic acids are made available for hybridization, - or the purified nucleic acids contained in the biological sample - or a single copy derived from the purified nucleic acids, - or an amplified copy derived from the purified nucleic acids, with said elements or with said probes being attached to a solid substrate.
The expression "inferring the presence of one or more HCV genotypes present from the observed hybridization pattern" refers to the identification of the presence of HCV genomes in the sample by analyzing the pattern of binding of a panel of oligonucleotide probes. Single probes may provide useful information concerning the presence or absence of HCV genomes in a sample. On the other hand, the variation of the HCV genomes is dispersed in nature, so rarely is any one probe able to identify uniquely a specific HCV genome.
Rather, the identity of an HCV genotype may be inferred from the pattern of binding of a panel of oligonucleotide probes, which are specific for (different) segments of the different HCV
genomes. Depending on the choice of these oligonucleotide probes, each known HCV
genotype will correspond to a specific hybridization pattern upon use of a specific combination of probes. Each HCV genotype will also be able to be discriminated from any other HCV genotype amplified with the same primers depending on the choice of the oligonucleotide probes. Comparison of the generated pattern of positively hybridizing probes for a sample containing one or more unkown HCV sequences to a scheme of expected SUBSTITEJTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 hybridization patterns, allows one to clearly infer the HCV genotypes present in said sample.
The present invention thus relates to a method as defined above, wherein one or more hybridization probes are selected from any of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, = 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61, 106, 108, 110, 112, 114, 116, 118, 120, 122, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 198, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 222, 269 or sequence variants thereof, with said sequence variants containing deletions and/or insertions of one or more nucleotides, mainly at their extremities (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between genotypes) by others (including modified nucleotides or inosine), or with said variants consisting of the complement of any of the above-mentioned oligonucleotide probes, or with said variants consisting of ribonucleotides instead of deoxyribonucleotides, all provided that said variant probes can be caused to hybridize with the same specificity as the oligonucleotide probes from which they are derived.
In order to distinguish the amplified HCV genomes from each other, the target polynucleic acids are hybridized to a set of sequence-specific DNA probes targetting HCV
genotypic regions located in the HCV polynucleic acids.
Most of these probes target the most type-specific regions of HCV genotypes, but some can be caused to hybridize to more than one HCV genotype.
According to the hybridization solution (SSC, SSPE, etc.), these probes should be stringently hybridized at their appropriate temperature in order to attain sufficient specificity.
However, by slightly modifying the DNA probes, either by adding or deleting one or a few nucleotides at their extremities (either 3' or 5'), or substituting some non-essential nucleotides (i.e. nucleotides not essential to discriminate between types) by others (including modified nucleotides or inosine) these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e. the same temperature and the same hybridization solution). Also changing the amount (concentration) of probe used may be beneficial to obtain more specific hybridization results. It should be noted in this context, that probes of the same length, regardless of their GC content, will hybridize specifically at approximately the same temperature in TMACI solutions (Jacobs et al., 1988).
Suitable assay methods for purposes of the present invention to detect hybrids formed between the oligonucleotide probes and the nucleic acid sequences in a sample may comprise SUBSTITkJTE SHEET (RULE 26) }
WO 94/25601 = PCT/EP94/01323 any of the assay formats known in the art, such as the conventional dot-blot format, sandwich hybridization or reverse hybridization. For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being bound to a membrane, the membrane being incorporated with at least one labelled probe under suitable ' hybridization and wash conditions, and the presence of bound probe being monitored.
An alternative and preferred method is a "reverse" dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropriate stringent hybridization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.
According to an advantageous embodiment, the process of detecting one or more HCV
genotypes contained in a biological sample comprises the steps of contacting amplified HCV
nucleic acid copies derived from the biological sample, with oligonucleotide probes which have been immobilized as parallel lines on a solid support.
According to this advantageous method, the probes are immobilized in a Line Probe Assay (LiPA) format. This is a reverse hybridization format (Saiki et al., 1989) using membrane strips onto which several oligonucleotide probes (including negative or positive control oligonucleotides) can be conveniently applied as parallel lines.
The invention thus also relates to a solid support, preferably a membrane strip, carrying on its surface, one or more probes as defined above, coupled to the support in the form of parallel lines.
The LiPA is a very rapid and user-friendly hybridization test. Results can be read 4 h.
after the start of the amplification. After amplification during which usuaily a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybridization is carried out for about 1 to 1,5 h hybridized polynucleic acid is detected. From the hybridization pattern generated, the HCV type can be deduced either visually, but preferably using dedicated software. The LiPA format is completely compatible with commercially available scanning devices, thus =
rendering automatic interpretation of the results very reliable. All those advantages make the LiPA format liable for the use of HCV detection in a routine setting. The LiPA
format should be particularly advantageous for detecting the presence of different HCV
genotypes.
The present invention also relates to a method for detecting and identifying novel HCV
SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCTIEP94/01323 genotypes, different from the known HCV genomes, comprising the steps of:
- determining to which HCV genotype the nucleotides present in a biological sample belong, according to the process as defined above, in the case of observing a sample which does not generate a hybridization pattern compatible with those defined in Table 3, sequencing the portion of the HCV
genome sequence corresponding to the aberrantly hybridizing probe of the new HCV genotype to be determined.
The present invention also relates to the use of a composition as defined above, for detecting one or more genotypes of HCV present in a biological sample liable to contain them, comprising the steps of:
(i) possibly extracting sample nucleic acid, (ii) amplifying the nucleic acid with at least one of the primers as defined above, (iii) sequencing the amplified products (iv) inferring the HCV genotypes present from the determined sequences by comparison to all known HCV sequences.
The present invention also relates to a composition consisting of or comprising at least one peptide or polypeptide comprising a contiguous--sequence of at least 5 amino acids corresponding to a contiguous amino acid sequence encoded by at least one of the HCV
genomic sequences as defined above, having at least one amino acid differing from the corresponding region of known HCV (type 1 and/or type 2 and/or type 3) polyprotein sequences as shown in Table 3, or muteins thereof.
It is to be noted that, at the level of the amino acid sequence, an amino acid difference (with respect to known HCV amino acid sequences) is necessary, which means that the polypeptides of the invention correspond to polynucleic acids having a nucleotide difference (with known HCV polynucleic acid sequences) involving an amino acid difference.
The new amino acid sequences, as deduced from the disclosed nucleotide sequences (see SEQ ID NO 1 to 62 and 106 to 123 and 143 to 218, 223 and 270), show homologies of only 59.9 to 78 % with prototype sequences of type 1 and 2 for the NS4 region, and of only 53.9 to 68.8 % with prototype sequences of type 1 and 2 for the El region. As the NS4 region is known to contain several epitopes, for example characterized in patent application EP-A-0 489 968, and as the El protein is expected to be subject to immune attack as part of the viral envelope and expected to contain epitopes, the NS4 and El epitopes of the new type 3, 4 and isolates will consistently differ from the epitopes present in type I and 2 isolates. This is SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 examplified by the type-specificity of NS4 synthetic peptides as presented in example 4, and the type-specificity of recombinant E1 proteins in example 11.
After aligning the new subtype 2d, type 3, 4 and 5 (see SEQ ID NO 1 to 62 and 106 to 123 and 143 to 218, 223 and 270) amino acid sequences with the prototype sequences of type la, lb, 2a, and 2b, type- and subtype-specific variable regions can be delineated as presented in Figure 5 and 7.
As to the muteins derived from the polypeptides of the invention, Table 4 gives an overview of the amino acid substitutions which could be the basis of some of the muteins as defmed above.
The peptides according to the present invention contain preferably at least 5 contiguous HCV amino acids, preferably however at least 8 contiguous amino acids, at least 10 or at least 15 (for instance at least 9, 11, 12, 13, 14, 20 or 25 amino acids) of the new HCV
sequences of the invention.
SUBSTlTM SHEET (RULE 26) ------------------------------------------------------Amino acids Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R) Arg, His, Lys, Glu, Gln Leu (L) Leu; Ile, Met, Phe, Val, Tyr Pro (P) Pro, Ala, Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A) Ala, Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G) Gly, Ala, Thr, Pro, Ser Ile (1) Ile, Met, Leu, Phe, Val, Ile, Tyr Phe (F) Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, I1e, Val, Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln, Arg, Lys, Glu, Thr Gln (Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn (N) Asn, Asp, Ser, Gln Lys (K) Lys, Arg, Glu, Gln, His Asp (D) Asp, Asn, Glu, Gin Glu (E) Glu, Gin, Asp, Lys, Asn, His, Arg Met (M) Met, Iie, Leu, Phe, Val The polypeptides of the invention, and particularly the fragments, can be prepared by classical chemical synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled "Methode der organischen chemie"
(Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).
The polypeptides according to this invention can be prepared by means of recombinant DNA techniques as described by Maniatis et al., Molecular Cloning: A
Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982).
The present invention relates particularly to a polypeptide or peptide composition as defined above, wherein said contiguous sequence contains in its sequence at least one of the following amino acid residues:
St1BSTITM SHEET (RULE 26) L7, Q43, M44, S60, R67, Q70, T71, A79, A87, N106, K115, A127, A190, S130, V134, G142, I144, E152, A157, V158, P165, S177 or Y177, I178, V180 or E180 or F182, R184, I186, H187, T189, A190, S191 or G191, Q192 or L192 or I192 or V192 or E192, N193 or H193 or P193, W194 or Y194, H195, A197 or I197 or V197 or T197, V202, I203 or L203, Q208, A210, V212, F214, T216, R217 or D217 or E217 or V217, H218 or N218, H219 or V219 or L219, L227 or I227, M231 or E231 or Q231, 7232 or D232 or A232 or K232, Q235 or I235, A237 or T237, I242, 1246, S247, S248, V249, S250 or Y250, I251 or V251 or M251 or F251, D252, T254 or V254, L255 or V255, E256 or A256, M258 or F258 or V258, A260 or Q260 or S260, A261, 7264 or Y264, M265, 1266 or A266, A267, G268 or T268, F271 or M271 or V271, I277, M280 or H280, 1284 or A284 or L84, V274, V291, N292 or S292, R293 or I293 or Y293, Q294 or R294, L297 or I297 or Q297, A299 or K299 or Q299, N303 or T303, T308 or L308, T310 or F310 or A310 or D310 or V310, L313, G317 or Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, S532, V534, F536, F537, M539, I546, C1282, A1283, H1310, V1312, Q1321, P1368, V1372, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, D1556, N1567, M1572, Q1579, L1581, S1583, F1585, V1595, E1606 or T1606, M1611, V1612 or L1612, P1630, C1636, P1651, T1656 or I1656, L1663, V1667, V1677, A1681, H1685, E1687, G1689, V1695, A1700, Q1704, Y1705, A1713, A1714 or S1714, M1718, D1719, A1721 or T1721, R1722, A1723 or V1723, H1726 or G1726, E1730, V1732, F1735, I1736, S1737, R1738, T1739, G1740, Q1741, K1742, Q1743, A1744, T1745, L1746, E1747 or K1747, I1749, A1750, T1751 or A1751, V1753, N1755, K1756, A1757, P1758, A1759, H1762, T1763, Y1764, P2645, A2647, K2650, K2653 or L2653, S2664, N2673, F2680, K2681, L2686, H2692, Q2695 or L2695 or I2695, V2712, F2715, V2719 or Q2719, 72722, T2724, S2725, R2726, G2729, Y2735, H2739, I2748, G2746 or I2746, I2748, P2752 or K2752, P2754 or T2754, 72757 or P2757, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 as shown in Table 1(comparison with other isolates). See also the numbering in Figures 2, 5, 7, and 11 (alignment amino acid sequences).
Within the group of unique and new amino acid residues of the present invention, the following residues were found to be specific for the following types of HCV
according to the SUBST(Tk1TE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 HCV classification system used in the present invention:
- Q208, R217, E231, I235, I246, T264, 1266, A267, F271, K299, L2686, Q2719 which are specific for the HCV subtype 2d sequences of the present invention as shown in Fig. 5 and 2;
- Q43, S60, R67, F182, I186, H187, A190, S191, L192, W194, V202, L203, V219, Q231, D232, A237, T254, M280, Q299, T303, L308, and/or L313 which are specific for the Core/El region of HCV type 3 of the invention as shown in Fig.
5;
- D1556, Q1579, L1581, S1584, F1585, E1606, V1612, P1630, C1636, T1656, L1663, H1685, E1687, G1689, V1695, Y1705, A1713, A1714, A1721, V1723, H1726, R1738, Q1743, A1744, E1747,11749, A1751, A1759 and/or H1762 which are specific for the NS3/4 region of HCV type 3 sequences of the invention as shown in Fig. 7;
- K2665, D2666, R2670 which are specific for the NS5B region of HCV type 3 of the invention as shown in Fig. 2;
- L7, A79, A127, S130, E152, V158, S177 or Y177, V180 or E180, R184, T189, Q192 or E192 or I192, N193 or H193, 1197 or V197, 1203, A210, V212, E217, H218, H219, L227, A232, V249, 1251 or M251, D252, L255 or V255, E256, M258 or V258 or F258, A260 or Q260, M265, T268, V271, V274, M280, I284, N292 or S292, Q294, L297 or I297, T308, A310 or D310 or V310 or T310, and G317 which are specific for the core/El region of HCV type 4 sequences of the present invention as shown in Fig. 5;
- P2645, K2650, K2653, G2656, V2658, T2668, N2673 or N2673, K2681, H2686, D2691, L2692, Q2695 or L2695 or I2695, Y2704, V2712, F2715, V2719, I2722, S2725, G2729, Y2735, G2746 or I2746, P2752 or K2752, Q2753, P2754 or T2754, T2757 or P2757 which are specific for the NS5B region of the HCV type 4 sequences of the present invention as shown in Fig. 2;
- M44, Q70, A87, N106, K115, V137, G142, P165,1178, F251, A299, N303, Q317 which are specific for the Core/El region of the HCV type 4 sequences of the present invention as shown in Fig. 5;
- L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F102, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S532, V534, F537, M539, 1546 which are specific for SUBSTITUTE SHEET (RULE 26) the E1/E2 region of the HCV type 5 sequences of the present invention as shown in Fig. 12;
- C1282, A1283, V1312, Q1321, P1368, V1372, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, M1572, V1595, T1606, M1611, L1612, I1656, V1667, A1681, A1700, A1713, S1714, M1718, D1719, T1721, R1722, A1723, G1726, F1735, I1736, S1737, T1739, G1740, K1742, T1745, L1746, K1747, A1750, V1753, N1755, A1757, D1758, T1763, and Y1764 which are specific for the NS3/NS4 region of HCV
type 5 sequences of the invention as shown in Fig. 7;
- A2647, L2653, S2674, F2680, T2724, R2726, Y2730, H2739 which are specific for the NS5B region of the HCV type 5 sequences of the present invention as shown in Fig. 2;
- A256, P1631, V1677, Q1704, E1730, V1732, Q1741 and T1751 which are specific for the HCV type 3 and 5 sequences of the present invention as shown in Fig. 5 and 7;
- T71, A157, I227, T237, T240, Y250, V251, S260, M271, T2673, T2722, I2748 which are specific for the HCV type 3 and 4 sequences of the present invention as shown in Fig. 5 and 2, - V192, Y194, A197, P249, S250, R294 which are specific for the HCV type 4 and sequences of the present invention as shown in Fig. 5;
- 1293 which is specific for the HCV type 4 and subtype 2d sequence of the present invention as shown in Fig. 5;
- D217 and R294 which are specific for the HCV type 3, 4 and 5 sequences of the present invention as shown in Fig. 5;
- L192 which is specific for the HCV type 3 and subtype 2d sequences of the present invention as shown in Fig. 5;
- G191 and T197 which are specific for the HCV type 3, 4 and subtype 2d sequences of the present invention as shown in Fig. 5;
- K232 which is specific for the HCV subtype 2d en type 5 sequences of the present invention as shown in Fig. 5.
and with said notation being composed of a letter, unambiguously representing the amino acid by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 (see also Table 1 for comparison with other isolates), as well as Figure 2 (NS5 SUBSTITkITE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 region), Figure 5 (Core/El region), Figure 7 (NS3/NS4 region), Figure 12 (E1/E2 region).
Some of the above-mentioned amino acids may be contained in type or subtype specific epitopes.
For example M231 (detected in type 5) refers to a methionine at position 231.
A glutamine (Q) is present at the same position 231 in type 3 isolates, whereas this position is occupied by an arginine in type 1 isolates and by a lysine (K) or asparagine (N) in type 2 isolates (see Figure 5).
The peptide or polypeptide according to this embodiment of the invention may be possibly labelled, or attached to a solid substrate, or coupled to a carrier molecule such as biotin, or mixed with a proper adjuvant.
The variable region in the core protein (V-CORE in Fig. 5) has been shown to be useful for serotyping (Machida et at., I992). The sequence of the disclosed type 5 sequence in this region shows type-specific features. The peptide from amino acid 70 to 78 shows the following unique sequence for the sequences of the present inevntion (see figure 5):
QPTGRSWGQ (SEQ ID NO 93) RSEGRTSWAQ (SEQ ID NO 220) and RTEGRTSWAQ (SEQ ID NO 221) Another preferred V-Core spanning region is the peptide spanning positions 60 to 78 of subtype 3c with sequence:
SRRQPIPRARRTEGRSWAQ (SEQ ID NO 268) Five type-specific variable regions (Vi to V5) can be identified after aligning El amino acid sequences of the 4 genotypes, as shown in Figure 5.
Region V1 encompasses amino acids 192 to 203, this is the amino-terminal 10 amino acids of the El protein. The following unique sequences as shown in Fig. 5 can be deduced:
LEWRNTSGLYVL (SEQ ID NO 83) VNYRNASGIYHI (SEQ ID NO 126) QHYRNISGIYHV (SEQ ID NO 127) EHYRNASGIYHI (SEQ ID NO 128) IHYRNASGIYHI (SEQ ID NO 224) VPYRNASGIYHV (SEQ ID NO 84) VNYRNASGIYHI (SEQ ID NO 225) VNYRNASGVYHI (SEQ ID NO 226) VNYHNTSGIYHL (SEQ ID NO 227) SUBSTITUTE SHEET (RULE 26) QHYRNASGIYHV (SEQ ID NO 228) QHYRNVSGIYHV (SEQ ID NO 229) IHYRNASDGYYI (SEQ ID NO 230) LQVKNTSSSYMV (SEQ ID NO 231) Region V2 encompasses amino acids 213 to 223. The following unique sequences can be found in the V2 region as shown in Figure 5: VYEADDVILHT (SEQ ID NO 85) VYETEHHILHL (SEQ ID NO 129) VYEADHHIMHL (SEQ ID NO 130) VYETDHHILHL (SEQ ID NO 131) VYEADNLILHA (SEQ ID NO 86) VWQLRANLHV (SEQ ID NO 232) VYEADYHILHL (SEQ ID NO 233) VYETDNHILHL (SEQ ID NO 234) VYETENHILHL (SEQ ID NO 235) VFETVHHILHL (SEQ ID NO 236) VFETEHHILHL (SEQ ID NO 237) VFETDHHIMHL (SEQ ID NO 238) VYETENHILHL (SEQ ID NO 239) VYEADALILHA (SEQ ID NO 240) Region V3 encompasses the amino acids 230 to 242. The following unique V3 region sequences can be deduced from Figure 5:
VQDGNTSTCWTPV (SEQ ID NO 87) VQDGNTSACWTPV (SEQ ID NO 241) VRVGNQSRCWVAL (SEQ ID NO 132) VRTGNTSRCWVPL (SEQ ID NO 133) VRAGNVSRCWTPV (SEQ ID NO 134) EEKGNISRCWIPV (SEQ ID NO 242) VKTGNQSRCWVAL (SEQ ID NO 243) VRTGNQSRCWVAL (SEQ ID NO 244) VKTGNQSRCWIAL (SEQ ID NO 245) VKTGNVSRCWIPL (SEQ ID NO 247) VKTGNVSRCWISL (SEQ ID NO 248) SUBSTITUTE SHEET (RULE 26) VRKDNVSRCWVQI (SEQ ID NO 249) Region V4 encompasses the amino acids 248 to 257. The following unique V4 region sequences can be deduced from figure 5:
VRYVGATTAS (SEQ ID NO 89) APYIGAPLES (SEQ ID NO 135) APYVGAPLES (SEQ ID NO 136) AVSMDAPLES (SEQ ID NO 137) APSLGAVTAP (SEQ ID NO 90) APSFGAVTAP (SEQ ID NO 250) VSQPGALTKG (SEQ ID NO 251) VKYVGATTAS (SEQ ID NO 252) APYIGAPVES (SEQ ID NO 253) AQHLNAPLES (SEQ ID NO 254) SPYVGAPLEP (SEQ ID NO 255) SPYAGAPLEP (SEQ ID NO 256) APYLGAPLEP (SEQ ID NO 257) APYLGAPLES (SEQ ID NO 258) APYVGAPLES (SEQ ID NO 259) VPYLGAPLTS (SEQ ID NO 260) APHLRAPLSS (SEQ ID NO 261) APYLGAPLTS (SEQ ID NO 262) Region V5 encompasses the amino acids 294 to 303. The following unique V5 region peptides can be deduced from figure 5:
RPRRHQTVQT (SEQ ID NO 91) QPRRHWTTQD (SEQ ID NO 138) RPRRHWTTQD (SEQ ID NO 139) RPRQHATVQN (SEQ ID NO 92) RPRQHATVQD (SEQ ID NO 263) SPQHHKFVQD (SEQ ID NO 264) RPRRLWTTQE (SEQ ID NO 265) PPRIHETTQD (SEQ ID NO 266) The variable region in the E2 region (HVR-2) of type 5a as shown in Figure 12 spanning amino acid positions 471 to 484 is also a preferred peptide according to the present invention SUBSCITUTE SHEET (RULE 26) with the following sequence:
TISYANGSGPSDDK (SEQ ID NO 267) The above given list of peptides are particularly suitable for vaccine and diagnostic development.
Also comprised in the present invention is any synthetic peptide or polypeptide containing at least 5 contiguous amino acids derived from the above-defined peptides in their peptidic chain.
According to a specific embodiment, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV
amino acid type 3 sequences:
- a sequence having a homology of more than 72 %, preferably more than 74 %, more preferably more than 77 % and most preferably more than 80 or 84 % homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the region spanning positions 140 to 319 in the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 70%, preferably more than 72%, more preferably more than 75 % homology, most preferably more than 81 % homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the El region spanning positions 192 to 319 as shown in Figure 5;
- a sequence having a homology of more than 86%, preferably more than 88%, and most preferably more than 90% homology to the amino acid sequences as represented in SEQ
ID NO 148 (type 3c); BE98 in the region spanning positions 1 to 110 in the Core region as shown in Figure 5;
- a sequence having a homology of more than 76%, preferably more than 78%, most preferably more than 80% to any of the amino acid sequences as represented in SEQ ID
NO 30, 32, 34, 36, 38 or 40 (HCC153, HD 10, BR36 sequences) in the region spanning positions 1646 to 1764 in the NS3/NS4 region as shown in Figure 7 and 11;
- a sequence having a homology of more than 81 %, preferably more than 83 %, and most preferably more than 86% homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the region spanning positions 140 to 319 in the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 81.5 %, preferably more than 83 %, and most SIfBST{TUTE SHEET (RULE 26) preferably more than 86% homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD 10, BR36, BR33 sequences) in the El region spanning positions 192 to 319 as shown in Figure 5;
- a sequence having a homology of more than 86%, preferably more than 88%, most preferably more than 90% to the amino acid sequence as represented in SEQ ID
NO 150;
(type 3c BE98) in the region spanning positions 2645 to 2757 in the NS5B
region as shown in Figure 2.
According to yet another embodiment, the present invention relates to a composition as defmed above, wherein said contiguous sequence is selected from any of the following HCV
amino acid type 4 sequences:
- a sequence having a homology of more than 80%, preferably more than 82%, most preferably more than 84 % homology to any of the amino acid sequences as represented in SEQ ID NO 118, 120, and 122 (GB358, GB549, GB809 sequences) in the region spanning positions 127 to 319 of the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 73%, preferably more than 75%, most preferably more than 78 % homology in the El region spanning positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 118, 120, and 122 (GB358, GB549, GB809 sequences) in the region spanning positions 140 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 85 %, preferably more than 86%, most preferably more than 87 % homology to any of the amino acid sequences as represented in SEQ ID NO
118, 120 or 122 (GB358, GB549, GB809 sequences) in the region spanning positions 192 to 319 of El as shown in Figure 5;
- a sequence showing more than 73 %, preferably more than 74 %, most preferably more than 75 % homology to any of the amino acid sequences as represented in SEQ ID NO
106, 108, 110, 112, 114 or 116 (GB48, GB116, GB215, GB358, GB549, GB809 sequences) in the region spanning positions 2645 to 2757 of the NS5B region as shown in Figure 2;
- a sequence having any of the sequences as represented in SEQ ID NO 164 or 166 (GB809 and CAM600 sequences) in the Core/El region as shown in Figure 5;
- a sequence having any of the sequences as represented in SEQ ID NO 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 or 190 (CAM600, GB809, CAMG22, CAMG27, GB549, GB438, CAR4/1205, CAR4/901, GB116, GB215, GB958, GB809-4 sequences) in the El region as shown in Figure 5;
SUBSTiTUTE SHEET (RULE 26) - a sequence having any of the sequences as represented in SEQ ID NO 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212 (GB358, GB724, BE100, PC, CAM600, CAMG22, etc.) in the NS5B region.
The above-mentioned type 4 peptides polypeptides comprise at least an amino acid sequence selected from any HCV type 4 polyprotein with the exception of core sequence as disclosed by Simmonds et al. (1993, EG-29, see Figure 5).
According to yet another aspect, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV amino acid type 5 sequences:
- a sequence having more than 93 %, preferably more than 94 %, most preferably more than 95 % homology in the region spanning Core positions 1 to 191 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC
sequences) and SEQ ID NO 152 (BE95) as shown in Figure 5;
- a sequence having more than 73 %, preferably more than 74 %, most preferably more than 76% homology in the region spanning El positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC sequences) as shown in Figure 5 - a sequence having a more than 78%, preferably more than 80%, most preferably more than 83 % homology to any of the amino acid sequences as represented in SEQ ID
NO 42, 44, 46, 48, 50, 52, 54, 154, 156 (BE95, BE100) (PC sequences) in the region spanning positions 1 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 90%, preferably more than 91 %, most preferably more than 92 % homology to any of the amino acid sequences represented in SEQ ID NO 56 to 58 (PC sequences) in the region spanning positions 1286 to 1403 of the NS3 region as shown in Figure 7 or 11;
- a sequence having more than 66 %, more particularly 68 %, most particularly 70 % or more homology to any of the amino acid sequences as represented in SEQ ID NO 60 or 62 (PC
sequences) in the region spanning positions 1646 to 1764 of the NS3/4 region as shown in Figure 7 or 11.
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV amino acid type 2d sequences:
- a sequence having more than 83 %, preferably more than 85 %, most preferably more than SUBSTIT(JTE SHEET (RULE 26) 87 % homology to the amino acid sequence as represented in SEQ ID NO 144 (NE92) in the region spanning positions 1 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 79%, preferably more than 81 %, most preferably more than . 84% homology in the region spanning E1 positions 192 to 319 to the amino acid sequence as represented in SEQ ID NO 144 (NE92) as shown in Figure 12;
- a sequence having more than 95 %, more particularly 96 %, most particularly 97 % or more homology to the amino acid sequence as represented in SEQ ID NO 146 (NE92) in the region spanning positions 2645 to 2757 of the NS5B region as shown in Figure 2.
The present invention also relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral promoter sequence followed by the nucleotide sequences as defined above, with said recombinant vector allowing the expression of any one of the HCV
type 2 and/or HCV type 3 and/or type 4 and/or type 5 derived polypeptides as defined above in a prokaryotic, or eukaryotic host or in living mammals when injected as naked DNA, and more particularly a recombinant vector allowing the expression of any of the following HCV
type 2d, type 3, type 4 or type 5 polypeptides spanning the following amino acid positions:
- a polypeptide starting at position 1 and ending at any position in the region between positions 70 and 326, more particularly a polypeptide spanning positions 1 to 70, 1 to 85, positions 1 to 120, positions 1 to 150, positions 1 to 191, positions 1 to 200, for expression of the Core protein, and a polypeptide spanning positions 1 to 263, positions 1 to 326, for expression of the Core and El protein;
- a polypeptide starting at any position in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, for expression of El, or forms that have the putative membrane anchor deleted (positions 264 to 293 plus or minus 8 amino acids);
- a polypeptide starting at any position in the region between positions 1556 and 1688, and ending at any position in the region between positions 1739 and 1764, for expression of the NS4 regions, more particularly a polypeptide starting at position 1658 and ending at position 1711 for expression of the NS4a antigen, and more particularly, a polypeptide starting at position 1712 and ending between positions 1743 and 1972, for example 1712-1743, 1712-1764, 1712-1782, 1712-1972, 1712 to 1782 and 1902 to 1972 for expression of the NS4b protein or parts thereof.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "vector" may comprise a plasmid, a cosmid, a phage, or a virus.
In order to carry out the expression of the polypeptides of the invention in bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out:
- transformation of an appropriate cellular host with a recombinant vector, in which a nucleotide sequence coding for one of the polypeptides of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence. In the case of an eukaryotic host any artificial signal sequence or pre/pro sequence might be provided, or the natural HCV signal sequence might be employed, e.g. for expression of El the signal sequence starting between amino acid positions 117 and 170 and ending at amino acid position 191 can be used, for expression of NS4, the signal sequence starting between amino acid positions 1646 and 1659 can be used, - culture of said transformed cellular host-under conditions enabling the expression of said insert.
The present invention also relates to a composition as defined above, wherein said polypeptide is a recombinant polypeptide expressed by means of an expression vector as defined above.
The present invention also relates to a composition as defined above, for use in a method for immunizing a mammal, preferably humans, against HCV comprising administring a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response, more particularly a vaccine composition including HCV type 3 polypeptides derived from the Core, El or the NS4 region and/or HCV
type 4 and/or HCV type 5 polypeptides and/or HCV type 2d polypeptides.
The present invention also relates to an antibody raised upon immunization with a composition as defined above by means of a process as defined above, with said antibody being reactive with any of the polypeptides as defined above, and with said antibody being preferably a monoclonal antibody.
The monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from SUBSTITUTE SHEET (RULE 26) a mouse or rat, immunized against the HCV polypeptides according to the invention, or muteins thereof, or fragments thereof as defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.
The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA
technology, departing from parts of mouse andlor human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
Alternatively the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies. These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with type 3, type 4 or type 5 HCV, or vaccinated against HCV. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al. 1992).
The invention also relates to the use of the proteins of the invention, muteins thereof, or peptides derived therefrom for the selection of recombinant antibodies by the process of repertoire cloning (Persson et al., 1991).
Antibodies directed to peptides derived from a certaing genotype may be used either for the detection of such HCV genotypes, or as therapeutic agents.
The present invention also relates to the use of a composition as defined above for incorporation into an immunoassay for detecting HCV, present in biological sample liable to contain it, comprising at least the following steps:
(i) contacting the biological sample to be analyzed for the presence of HCV
antibodies with any of the compositions as defined above preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said polypeptide can be a biotinylated polypeptide which is covalently bound to a solid substrate by means of streptavidin or avidin complexes, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, which SUBSTITUTE SHEET (RULE 26) specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immunecomplexes visually or by means of densitometry and inferring the HCV serotype present from the observed hybridization pattern.
The present invention also relates to the use of a composition as defined above, for incorporation into a serotyping assay for detecting one or more serological types of HCV
present in a biological sample liable to contain it, more particularly for detecting El and NS4 antigens or antibodies of the different types to be detected combined in one assay format, comprising at least the following steps:
(i) contacting the biological sample to be analyzed for the presence of HCV
antibodies or antigens of one or more serological types, with at least one of the compositions as defined above, an immobilized form under appropriate conditions which allow the formation of an immunecomplex, (ii) removing unbound components, (iii) incubating the immunecomplexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immunecomplexes visually or by means of densitometry and inferring the presence of one or more HCV serological types present from the observed binding pattern.
The present invention also relates to the use of a composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above.
The present invention thus also relates to a kit for determining the presence of HCV
genotypes as defined above present in a biological sample liable to contain them, comprising:
- possibly at least one primer composition containing any primer selected from those defined above or any other HCV type 3 and/or HCV type 4, and/or HCV type 5, or universal HCV primers, SUBSTITETTE SHEET (RULE 26) - at least one probe composition as defined above, with said probes being preferentially immobilized on a solid substrate, and more preferentially on one and the same membrane strip, a buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the possibly amplified products to be carried out, means for detecting the hybrids resulting from the preceding hybriziation, - possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed hybridization pattern.
The genotype may also be detected by means of a type-specific antibody as defined above, which is linked to any polynucleotide sequence that can afterwards be amplified by PCR to detect the immune complex formed (Immuno-PCR, Sano et al., 1992);
The present invention also relates to a kit for determining the presence of HCV antibodies as defined above present in a biological sample liable to contain them, comprising:
- at least one polypeptide composition as defined above, preferentially in combination with other polypeptides or peptides from HCV type 1, HCV type 2 or other types of HCV, with said polypeptides being preferentially immobilized on a solid substrate, and more preferentially on one and the same membrane strip, - a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides and the antibodies against HCV present in the biological sample, means for detecting the immunecomplexes formed in the preceding binding reaction, - possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed binding pattern.
SUBSTITUTE SHEET (RULE 26) Figure Legends Figure 1 Alignment of consensus nucleotide sequences for each of the type 3a isolates BR34, BR36, and BR33, deduced from the clones with SEQ ID NO 1, 5, 9; type 4 isolates GB48, GB116, GB215, GB358, GB549, GB809, CAM600, CAMG22, GB438, CAR411205, CAR1/501 (SEQ ID
NO. 106, 108, 110, 112, 114, 116, 201, 203, 205, 207, 209 and 211); type 5a isolates BE95 and BE96 (SEQ ID NO 159 and 161) and type 2d isolate NE92 (SEQ
ID NO
145) from the region between nucleotides 7932 and 8271, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, HC-J8, TI and T9, and others as shown in Table 3.
Figure 2 Alignment of amino acids sequences deduced from the nucleic acid sequences as represented in Figure 1 from the subtype 3a clones BR34 (SEQ ID NO 2, 4), BR36 (SEQ ID
NO 6, 8) and BR33 (SEQ ID NO 10, 12), the subtype 3c clone BE98 (SEQ ID NO
150), and the type 4 clones GB48 (SEQ ID NO 107), GB1 16 (SEQ ID NO 109), GB215 (SEQ ID
NO
111), GB358 (SEQ ID NO 113), GB549 (SEQ ID NO 115) GB809 (SEQ ID NO 117);
CAM600, CAMG22, GB438, CAR4/1205, CAR1/501 (SEQ ID NO 202, 204, 206, 208, 210, 212); the type 5a clones BE95 and BE96 (SEQ ID NO 160 and 162); as well as the subtype 2d isolate NE92 (SEQ ID NO 146) from the region between amino acids 2645 to 2757 with known sequences from the corresponding region of isolates HCV-I, HCV-J, HC-J6, and HC-J8, T1 and T9, and other sequences as shown in Table 3.
Figure 3 Aligment of type 2d, 3c, 4 and 5a nucleotide sequences from isolates NE92, BE98, GB358, GB809, CAM600, GB724, BE95 (SEQ ID NO 143, 147, 191, 163, 165, 193 and 151) in the Core region between nucleotide positions 1 and 500, with known sequences from the corresponding region of type 1, type 2, type 3 and type 4 sequences.
Figure 4 Alignment of nucleotide sequences for the subtype 2d isolate NE92 (SEQ ID NO
143), the type 4 isolates GB358 (SEQ ID NO 118 and 187), GB549 (SEQ ID NO 120 and 175), and SUBSTiTUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 GB809-2 (SEQ ID NO 122 and 169), GB 809-4, BG116, GB215,- CAM600, CAMG22, CAMG27, GB438, CAR4/1205, CAR4/901(SEQ ID NO 189, 183, 185, 167, 171, 173, 177, 179, 181), sequences for each of the subtype 3a isolates HD10, BR36, and BR33, (SEQ ID
NO 13, 15, 17 (HD 10), 19, 21 (BR36) and 23, 25 or 27 (BR23) and the subtype 5a isolates BE95 and BE100 (SEQ ID NO 143 and 195) from the region between nucleotides 379 and 957, with known sequences from the corresponding region of type 1 and 2 and 3.
Figure 5 Alignment of amino acid sequences deduced from the new HCV nucleotide sequences of the Core/El region of isolates BR33, BR36, HD10, GB358, GB549, and GB809, PC
or BE95, CAM600, and GB724 (SEQ ID NO. 14, 20, 24, 119 or 192, 121, 123 or 164, 54 or 152, 166 and 194) from the region between positions 1 and 319, with known sequences from type la (HCV-1), type lb (HCV-J), type 2a (HC-JG), type 2b (HC-J8), NZL1, HCV-TR, positions 7-89 of type 3a (E-bl), and positions 8-88 of type 4a (EG-29). V-Core, variable region with type-specific features in the core protein, V1, variable region 1 of the El protein, V2, variable region 2 of the E1 protein, V3, variable region 3 of the El protein, V4, variable region 4 of the El protein, V5, variable region 5 of the El protein.
Fignre 6 Alignment of nucleotide sequences of isolates HCCL53, HD10 and BR36, deduced from clones with SEQ ID NO 29, 31, 33, 35, 37 and 39, from the NS314 region between nucleotides 4664 to 5292, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8, EB1, EB2, EB6 and EB7.
Fi re 7 Alignment of amino acid sequences deduced from the new HCV nucleotide sequences of the NS3/NS4 region of isolate BR36 (SEQ ID NO 36, 38 and 40) and BE95 (SEQ ID
NO
270). NS4-1, indicates the region that was synthesized as synthetic peptide I
of the NS4 region, NS4-5, indicates the region that was synthesized as synthetic peptide 5 of the NS4 region; NS4-7, indicates the region that was synthesized as synthetic peptide 7 of the NS4 region.
Fiugre8 SUBSTITUTE SHEET (RULE 26) Reactivity of the three LIPA-selected (Stuyver et al., 1993) type 3 sera on the Inno-LIA
HCV Ab II assay (Innogenetics) (left), and on the NS4-LIA test. For the NS4-LIA test, NS4-1, NS4-5, and NS4-7 peptides were synthesized based on the type 1 (HCV-1), type 2(HC-J6) and type 3 (BR36) prototype isolate sequences as shown in Table 4, and applied as parallel lines onto a membrane strip as indicated. 1, serum BR33, 2, serum HD10, 3, serum DKH.
Fiizure 9 Nucleotide sequences of Core/El clones obtained from the PCR fragments PC-2, PC-3, and PC-4, obtained from serum BE95 (PC-2-l (SEQ ID NO 41), PC-2-6 (SEQ ID NO
43), PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47), PC-3-4 (SEQ ID NO 49), and PC-3-(SEQ ID NO 51)) of subtype 5a isolate BE95.
A consensus sequence is shown for the Core and El region of isolate BE95, presented as PC C/E1 with SEQ ID NO 53. Y, C or T, R, A or G, S, C or G.
Figure 10 Alignment of nucleotide sequences of clones with SEQ ID NO 197 and 199 (PC
sequences, see also SEQ ID NO 55, 57, 59) and SEQ ID NO 35, 37 and 39 (BR36 sequences) from the NS3/4 region between nucleotides 3856 to 5292, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8.
Figure 11 Alignment of amino acid sequences of subtype 5a BE95 isolate PC clones with SEQ ID
NO 56 and 58, from the NS3/4 region between amino acids 1286 to 1764, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8.
Figure 12 Aligment of amino acid sequences of subtype 5a isolate BE95 (SEQ ID NO 158) in the El/E2 region spanning positions 328 to 546, with known sequnces from the corresponding region of isolates HCV-1, HCV-J, HC-J6, HC-J8, NZLI and HCV-TR (see Table 3).
Figure 13 Alignment of the nucleotide sequences of subtype 5a isolate BE95 (SEQ ID NO
157) in the El/E2 region with known HCV sequences as shown in Table 3.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 EXAMPLES
Example 1: The NS5b region of HCV type 3 Type 3 sera, selected by means of the INNO-LiPA HCV research kit (Stuyver et al., 1993) from a number of Brazilian blood donors, were positive in the HCV antibody ELISA
(Innotest HCV Ab II; Innogenetics) and/or in the INNO-LIA HCV Ab 11 confirmation test (Innogenetics). Only those sera that were positive after the first round of PCR reactions (Stuyver et al., 1993) were retained for further study.
Reverse transcription and nested PCR: RNA was extracted from 50 l serum and subjected to eDNA synthesis as described (Stuyver et al., 1993). This cDNA was used as template for PCR, for which the total volume was increased to 50 l containing 10 pmoles of each primer, 3 41 of lOx Pfu buffer 2 (Stratagene) and 2.5 U of Pfu DNA polymerase (Stratagene). The cDNA was amplified over 45 cycles consisting of 1 min 94 C, 1 min 50 C and 2 min 72 C.
The amplified products were separated by electrophoresis, isolated, cloned and sequenced as described (Stuyver et al., 1993).
Type 3a and 3b-specific primers in the NS5 region were selected from the published sequences (Mori et al., 1992) as follows:
for type 3a:
HCPr161(+): 5'-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3' (SEQ ID NO 63) and HCPr162(-): 5'-GGGCTGCTCTATCCTCATCGACGCCATC-3' (SEQ ID NO 64);
for type 3b:
HCPr163(+): 5'-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3' (SEQ ID O 65) and HCPr164(-): 5'-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3' (SEQ ID NO 66) Using the Line Probe Assay (LiPA) (Stuyver et al., 1993), seven high-titer type 3 sera were selected and subsequently analyzed with the primer sets HCPr161/162 for type 3a, and HCPr163/164 for type 3b. None of these sera was positive with the type 3b primers. NS5 PCR fragments obtained using the type 3a primers from serum BR36 (BR36-23), serum BR33 (BR33-2) and serum BR34 (BR34-4) were selected for cloning. The following sequences were obtained from the PCR fragments :
From fragment BR34-4:
BR34-4-20 (SEQ ID NO 1), BR34-4-19 (SEQ ID NO 3) From fragment BR36-23:
BR36-23-18 (SEQ ID NO 5), BR36-23-20 (SEQ ID NO 7) SUBSTITUTE SHEET (RULE 26) From fragment BR33-2:
BR33-2-17 (SEQ ID NO 9), BR33-2-21 (SEQ ID NO 11) An alignment of sequences with SEQ ID NO 1, 5 and 9 with known sequences is given in Figure 1. An alignment of the deduced amino acid sequences is shown in Figure 2. The 3 isolates are very closely related to eacb other (mutual homologies of about 95 %) and to the published sequences of type 3a (Mori et al., 1992), but are only distantly related to type 1 and type 2 sequences (Table 5). Therefore, it is clearly demonstrated that NS5 sequences from LiPA-selected type 3 sera are indeed derived from a type 3 genome.
Moreover, by analyzing the NS5 region of serum BR34, for which no 5'UR sequences were determined as described in Stuyver et al. (1993), the excellent correlation between typing by means of the LiPA and genotyping as deduced from nucleotide sequencing was further proven.
Example 2: The Core/El reEion of HCV type 3 After aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992), PCR primers were chosen in those regions of little sequence variation. Primers HCPr23(+): 5'-CTCATGGGGTACATTCCGCT-3' (SEQ ID NO 67) and HCPr54(-): 5'-TATTACCAGTTCATCATCATATCCCA-3' (SEQ ID NO 68), were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). This set of primers was selected to amplify the sequence from nucleotide 397 to 957 encoding amino acids 140 to 319 (Kato et al., 1990):
52 amino acids from the carboxyterminus of core and 128 amino acids of El (Kato et al., 1990). The amplification products BR36-9, BRR33-1, and HD10-2 were cloned as described (Stuyver et al., 1993). The following clones were obtained from the PCR
fragments:
From fragment HD10-2:
HD10-2-5 (SEQ ID NO 13), HD10-2-14 (SEQ ID NO 15), HD10-2-21 (SEQ ID NO 17) From fragment BR36-9:
BR36-9-13 (SEQ ID NO 19), BR36-9-20 (SEQ ID NO 21), From fragment BR33-1:
BR33-1-10 (SEQ ID NO 23), BR33-1-19 (SEQ ID NO 25), BR33-1-20 (SEQ ID NO 27), An alignment of the type 3 El nucleotide sequences (HD10, BR36, BR33) with SEQ
ID
NO 13, 19 and 23 with known El sequences is presented in Figure 4. Four variations were detected in the El clones from serum HD10 and BR36, while only 2 were found in BR33.
All are silent third letter variations, with the exception of mutations at position 40 (L to P) SUBSTiTk1TE SHEET (RULE 26) and 125 (M to I). The homologies of the type 3 El region (without core) with type I and 2 prototype sequences are depicted in Table 5.
In total, 8 clones covering the core/El region of 3 different isolates were sequenced and the El portion was compared with the known genotypes (Table 3) as shown in Figure 5.
After computer analysis of the deduced amino acid sequence, a signal-anchor sequence at the core carboxyterminus was detected which might, through analogy with type lb (Hijikata et al., 1991), promote cleavage before the LEWRN sequence (position 192, Fig. 5).
The L-to-P
mutation in one of the HD10-2 clones resides in this signal-anchor region and potentially impairs recognition by signal peptidase (computer prediction). Since no examples of such substitutions were found at this position in previously described sequences, this mutation might have resulted from reverse transcriptase or Pfu polymerase misincorporation. The 4 amino-terminal potential N-linked glycosylation sites, which are also present in HCV types la and 2, remain conserved in type 3. The N-glycosylation site in type lb (aa 250, Kato et al., 1990) remains a unique feature of this subtype. All El cysteines, and the putative transmembrane region (aa 264 to 293, computer prediction) containing the aspartic acid at position 279, are conserved in all three HCV types. The following hypervariable regions can be delineated: V 1 from aa 192 to 203 (numbering according to Kato et al., 1990), V2 (213-223), V3 (230-242), V4 (248-257), and V5 (294-303). Such hydrophilic regions are thought to be exposed to the host defense mechanisms. This variability might therefore have been induced by the host's immune response. Additional putative N-linked glycosylation sites in the V4 region in all type lb isolates known today and in the V5 region of HC-J8 (type 2b) possibly further contribute to modulation of the immune response. Therefore, analysis of this region, in the present invention, for type 3 and 4 sequences has been instrumental in the delineation of epitopes that reside in the V-regions of El, which will be critical for future vaccine and diagnostics development.
Example 3: The NS3/NS4 region of HCV Tyne 3 For the NS3/NS4 border region, the folllowing sets of primers were selected in the regions of little sequence variability after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) (smaller case lettering is used for nucleotides added for cloning purposes):
set A:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) SUBSTITLTTE SHEET (RULE 26) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) setB:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr 118(-): 5' -actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set C:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set D:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set E:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set F:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set G: -HCPr131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set H:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set I:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set J:
HCPr 131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) set K:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) SUBSTITUTE SHEET (RULE 26) set L:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set M:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr4(-): 5'-GACATGCATGTCATGATGTA-3 (SEQ ID NO 78) set N:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr11 8(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set 0:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTI'CCACAT-3' (SEQ ID NO 70) No PCR products could be obtained with the sets of primers A, B, C, D, E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained from type 3 sera. With the primer set 0, no fragment could be amplified from type 3 sera. However, a smear containing a few weakly stainable bands was obtained from serum BR36. After sequence analysis of several DNA fragments, purified and cloned from the area around 300 bp on the agarose gel, only one clone, HCC153 (SEQ ID NO 29), was shown to contain HCV information. This sequence was used to design primer HCPr152.
A new primer set P was subsequently tested on several sera.
set P:
HCPr152(+): 5'-TACGCCTCTTCTATATCGGTTGGGGCCTG-3' (SEQ ID NO 79) and HCPr66(-): 5'-CTATTATTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) The 464-bp HCPr152/66 fragment was obtained from serum BR36 (BR36-20) and serum HD10 (HD10-1). The following clones were obtained from these PCR products:
From fragment HD10-1:
HD10-1-25 (SEQ ID NO 31), HD10-1-3 (SEQ ID NO 33), From fragment BR36-20:
BR36-20-164 (SEQ ID NO 35), BR36-20-165 (SEQ ID NO 37), BR36-20-166 (SEQ ID
NO 39), The nucleotide sequences obtained from clones with SEQ ID NO 29, 31, 33, 35, 37 or 39 are shown aligned with-the sequences of prototype isolates of other types-of HCV in Figure 6. In addition to one silent 3rd letter variation, one 2nd letter mutation resulted in an SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 E to G substitution at position 175 of the deduced amino acid sequence of BR36 (Fig. 7).
Serum HD 10 clones were completely identical. The two type 3 isolates were -nearly 94%
homologous in this NS4 region. The homologies with other types are presented in Table 5.
Example 4: Analysis of the anti-NS4 response to tXpe-specific peptides As the NS4 sequence contains the information for an important epitope cluster, and since antibodies towards this region seem to exhibit little cross-reactivity (Chan et al., 1991), it was worthwhile to investigate the type-specific antibody response to this region.
For each of the 3 genotypes, HCV-1 (Choo et al., 1991), HC-J6 (Okamoto et al., 1991) and BR36 (present invention), three 20-mer peptides were synthesized covering the epitope region between amino acids 1688 and 1743 (as depicted in table 6). The synthetic peptides were applied as parallel lines onto membrane strips. Detection of anti-NS4 antibodies and color development was performed according to the procedure described for the INNO-LIA HCV Ab Ii kit (Innogenetics, Antwerp). Peptide synthesis was carried out on a 9050 PepSynthesizer (Millipore). After incubation with 15 LiPA-selected type 3 sera, 9 samples showed reactivity towards NS4 peptides of at least 2 different types, but a clearly positive reaction was observed for 3 sera (serum BR33, HD30 and DKH) on the type 3 peptides, while negative (serum BR33 and HD30) or indeterminate (serum DKH) on the type 1 and type 2 peptides; 3 sera tested negative for anti-NS4 antibodies (Figure 8). Using the same membrane strips coated with the 9 peptides as indicated above and as shown in Figure 8, 38 type 1 sera (10 type la and 28 type 1b), 11 type 2 sera (10 type 2a and 1 type 2b), 12 type 3a sera and 2 type 4 sera (as determined by the LiPA procedure) were also tested. As shown in Table 8, the sera reacted in a genotype-specific manner with the NS4 epitopes. These results demonstrate that type-specific anti-NS4 antibodies can be detected in the sera of some patients. Such genotype-specific synthetic peptides might be employed to develop serotyping assays, for example a mixture of the nine peptides as indicated above, or combined with the NS4 peptides from the HCV type 4 or 6 genotype or from new genotypes corresponding to the region between amino acids 1688 and 1743, or synthetic peptides of the NS4 region between amino acids 1688 and 1743 of at least one of the 6 genotypes, combined with the El protein or deletion mutants thereof, or synthetic El peptides of at least one of the genotypes.
Such compositions could be further extended with type-specific peptides or proteins, including for example the region between amino acids 68 and 91 of the core protein, or more preferably the region between amino acids 68 and 78. Furthermore, such type-specific SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 antigens may be advantageously used to improve current diagnostic screening and confirmation assays and/or HCV vaccines.
Example 5 The Core and El regions of HCV type 5 Sample BE95 was selected from a group of sera that reacted positive in a prototype Line Probe Assay as described earlier (Stuyver et al., 1993), because a high-titer of HCV RNA
could be detected, enabling cloning of fragments by a single round of PCR. As no sequences from any coding region of type 5 has been disclosed yet, synthetic oligonucleotides for PCR
amplification were chosen in the regions of little sequence variation after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), HC-J8 (Okamoto et al., 1992), and the new type 3 sequences of the present invention HD10, BR33, and BR36 (see Figure 5, Example 2). The following sets of primers were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems):
Set 1:
HCPr52(+): 5'-atgTTGGGTAAGGTCATCGATACCCT-3' (SEQ ID NO 80) and HCPr54(-): 5'-ctattaCCAG77CATCATCATATCCCA-3' (SEQ ID NO 78) Set 2:
HCPr4i(+): 5'-CCCGGGAGGTCTCGTAGACCGTGCA-3' (SEQ ID NO 81) and HCPr40(-): 5'-ctattaAAGATAGAGAAAGAGCAACCGGG-3'(SEQ ID NO 82) Set 3:
HCPr41(+): 5'-CCCGGGAGGTCTCGTAGACCGTGCA-3' (SEQ ID NO 81) and HCPr54(-): 5'-ccattaCCAGTTCATCATCATATCCCA-3' (SEQ ID NO 78) The three sets of primers were employed to amplify the regions of the type 5 isolate PC
as described (Stuyver et al., 1993). Set I was used to amplify the El region and yielded fragment PC-4, set 2 was designed to yield the Core region and yielded fragment PC-2. Set 3 was used to amplify the Core and El region and yielded fragment PC-3. These fragments were cloned as described (Stuyver et al., 1993). The following clones were obtained from the PCR fragments:
From fragment PC-2:
PC-2-1 (SEQ ID NO 41), PC-2-6 (SEQ ID NO 43), - From fragment PC-4:
PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47), SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94101323 From fragment PC-3:
PC-3-4 (SEQ ID NO 49), PC-3-8 (SEQ ID NO 51) An alignment of sequences with SEQ ID NO 41, 43, 45, 47, 49 and 51, is given in Figure 9. A consensus amino acid sequence (PC C/El; SEQ ID NO 54) can be deduced from each of the 2 clones cloned from each of the three PCR fragments as depicted in Figure 5, which overlaps the region between nucleotides 1 and 957 (Kato et al., 1990). The 6 clones are very closely related to each other (mutual homologies of about 99.7%).
An alignment of nucleotide sequence with SEQ ID NO 53 or 151 (PC C/El from isolate BE95) with known nucleotide sequences from the Core/El region is given in Figure 3. The clone is only distantly related to type 1, type 2, type 3 and type 4 sequences (Table 5).
Example 6: NS3/NS4 region of HCV type 5 Attempts were undertaken to clone the NS3/NS4 region of the isolate BE95, described in example 5. The folllowing sets of primers were selected in the regions of little sequence variability after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J
(Kato et al., 1991), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) and of the sequences obtained from type 3 sera of the present invention (SEQ ID NO 31, 33, 35, 37 and 39); smaller case lettering is used for nucleotides added for cloning purposes:
set A:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 66) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set B:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set C:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set D:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr 118 (-): 5' -actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (S EQ ID NO 71) set E:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) SUBSTITUTE SHEET (RULE 26) set F:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set G:
HCPr131(+): 5'-ggaattcta.gaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set H:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set I:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set J:
HCPr131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID 74) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set K:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGGIACIGTITGYGC-3' (SEQ ID NO 75) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set L:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set M:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr4(-): 5'-GACATGCATGTCATGATGTA-3' (SEQ ID NO 78) set N:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) set 0:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) No PCR products could be obtained with the sets of primers A, B, C, D, E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained from type 3 sera.
However, SUBSTITk1TE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 set 0 yielded what appeared to be a PCR artifact fragment estimated about 1450 base pairs, instead of the expected 628 base pairs. Although it is not expected that PCR artifact fragments contain information of the gene or genome that was targetted in the experiment, efforts were put in cloning of this artifact fragment, which was designated fragment PC-1. =
The following clones, were obtained from fragment PC-i:
PC-1-37 (SEQ ID NO 59 and SEQ ID NO 55), PC-1-48 (SEQ ID NO 61 and SEQ ID NO
57) The sequences obtained from the 5' and 3' ends of the clones are given in SEQ
ID NOS
55, 57, 59, and 61, and the complete sequences with SEQ ID NO 197 and 199 are shown aligned with the sequences of prototype isolates of other types of HCV in Figure 10 and the alignment of the deduced amino acid sequences is shown in Figure 11 and 7.
Surprisingly, the PCR artifact clone contained HCV information. The positions of the sequences within the HCV genome are compatible with a contiguous HCV sequence of 1437 nucleotides, which was the estimated size of the cloned PCR artifact fragment. Primer HCPr66 primed correctly at the expected position in the HCV genome. Therefore, primer HCPr3 must have incidentally misprimed at a position 809 nucleotides upstream of its legitimate position in the HCV genome. This could not be expected since no sequence information was available from a coding region of type S.
Example 7: The E2 region of HCV type 5 Serum BE95 was chosen for experiments aimed at amplifying a part of the E2 region of HCV
type 5.
After aligning the sequences of HCV-1 (2), HCV-J(1), HC-J6 (3), and HC-J8 (4), PCR
primers were chosen in those regions of little sequence variation.
Primers HCPr109(+): 5'-TGGGATATGATGATGAACTGGTC-3' (SEQ ID NO 141) and HCPr14(-): 5'-CCAGGTACAACCGAACCAATTGCC-3' (SEQ ID NO 142) were combined to amplify the aminoterminal region of the E2/NS 1 region, and were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). With primers HCPr109 and HCPrl4, a PCR
fragment of 661 bp was generated, containing 169 nucleodtides corresponding to the El carboxyterminus and 492 bases from the region encoding the E2 aminoterminus.
An alignment of the type 5 E1/E2 sequences with seq ID NO. 158 with known sequences is presented in Figure 10. The deduced protein sequence was compared with the different SUBSTITUTE SHEET (RULE 26) genotypes (Fig. 12, amino acids 328-546). In the El region, there were no extra structural important motifs found. The aminoterminal part of E2 was hypervariable when compared with the other genotypes. All 6 N-glycosylation sites and all 7 cysteine residue's were conserved in this E2 region. To preserve alignment, it was necessary to introduce a gap between aa 474 and 475 as for type 3a, but not between aa 480 and 481, as for type 2.
Example 8 : The NS5b region of HCV type 4 Type 4 sera GB48, GB116, GB215, and GB358, selected by means of the line probe assay (LiPA, Stuyver et al., 1993), as well as sera GB549 and GB809 that could not be typed by means of this LiPA (only hybridization was observed with the universal probes), were selected from Gabonese patients. All these sera were positive after the first round of PCR
reactions for the 5' untranslated region (Stuyver et al., 1993) and were retained for further study.
RNA was isolated from the sera and cDNA synthesized as described in example 1.
Universal primers in the NS5 region were selected after alignment of the published sequences as follows:
HCPr206(+) : 5' -TGGGGATCCCGTATGATACCCGCTGCTTTGA-3' (SEQ ID NO. 124) and HCPr207(-): 5'-GGCGGAATTCCTGGTCATAGCCTCCGTGAA-3' (SEQ ID NO. 125);
and were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). Using the Line Probe Assay (LiPA), four high-titer type 4 sera and 2 sera that could not be classified were selected and subsequently analyzed with the primer set HCPr206/207. NS5 PCR
fragments obtained using these primers from serum GB48 (GB48-3), serum GB 116 (GB 116-3), serum GB215 (GB215-3), serum GB358 (GB358-3), serum GB549 (GB549-3), and serum GB809 (GB809-3), were selected for cloning. The following sequences were obtained from the PCR
fragments:
From fragment GB48-3 : GB48-3-10 (SEQ ID NO. 106) From fragment GB116-3: GB116-3-5 (SEQ ID NO. 108) From fragment GB215-3: GB215-3-8 (SEQ ID NO. 110) From fragment GB358-3: GB358-3-3 (SEQ ID NO. 112) From fragment GB549-3: GB549-3-6 (SEQ ID NO. 114) SUBSTITUTE SHEET (RULE 26) From fragment GB809-3: GB809-3-1 (SEQ ID NO. 116) An alignment of nucleotide sequences with SEQ ID NO. 106, 108, 110, 112, 114, and 116 with known sequences is given in Figure 1. An alignment of deduced amino acid sequences with SEQ ID NO. 107, 109, 111, 113, 115, and 117 with known sequences is given in Figure =
2. The 4 isolates that had been typed as type 4 by means of LiPA are very closely related to each other (mutual homologies of about 95 %), but are only distantly related to type 1, type 2, and type 3 sequences (e.g. GB358 shows homologies of 65.6 to 67.7% with other genotypes, Table 4). The sequence obtained from sera GB549 and GB809 also show similar homologies with genotypes 1, 2, and 3 (65.9 to 68.8% for GB549 and 65.0 to 68.5% for GB809, Table 4), but an intermediate homology of 79.7 to 86. 8%(often observed between subtypes of the same type) exists between GB549 or GB809 with the group of isolates consisting of GB48, GB116, GB215, and GB358, or between GB549 and GB809. These data indicate the discovery of 3 new subtypes within the HCV genotype 4: in the present invention, these 3 subtypes are designated subtype 4c, represented by isolates GB48, GB 116, GB215, and GB358, subtype 4g, represented by isolate GB549, and subtype 4e, represented by isolate GB809. Although the homologies observed between subtypes in the NS5 region seem to indicate a closer relationship between subtypes 4c and 4e, the homologies observed in the El region indicate that subtypes 4g and 4e show the closest relation (see example 8).
Example 9: The Core/E1 region of HCV type 4 From each of the 3 new type 4 subtypes, one representative serum was selected for cloning experiments in the Core/El region. GB549 (subtype 4g) and GB809 (subtype 4e) were analyzed together with isolate GB358 that was chosen from the subtype 4c group.
Synthetic oligonucleotides:
After aligning the sequences of HCV-1 (2), HCV-J(1), HC-J6 (3), and HC-J8 (4), PCR
primers were chosen in those regions of little sequence variation.
Primers HCPr52(+): 5'-atgTTGGGTAAGGTCATCGATACCCT-3', HCPr23(+): 5'-CTCATGGGGTACATTCCGCT-3', and HCPr54(-); 5'-CTATTACCAGTTCATCATCATATCCCA-3', were synthesized on a 392 DNA/RNA
synthesizer (Applied Biosystems). The sets of primers HCPr23/54 and HCPr52/54 were used, but only with the primer set HCPr52/54, PCR fragments could be obtained. This set of primers amplified the sequence from nucleotide 379 to 957 encoding amino acids 127 to 319:
amino acids from the carboxyterminus of core and 128 amino acids of E1. The SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 amplification products GB358-4, GB549-4, and GB809-4 were cloned as described in example 1. The following clones were obtained from the PCR fragments:
From fragment GB358-4: GB358-4-1 (SEQ ID NO 118) . From fragment GB549-4: GB549-4-3 (SEQ ID NO 120) From fragment GB809-4: GB809-4-3 (SEQ ID NO 122) ' An alignment of the type 4 Core/E1 nucleotide sequences with seq ID NO. 118, 120, and 122 with known sequences is presented in Figure 4. The homologies of the type 4 El region (without core) with type 1, type 2, type 3, and type 5 prototype sequences are depicted in Table 4. Homologies of 53 to 66% are observed with representative isolates of non-type 4 genotypes. Observed homologies in the El region within type 4, between the different subtypes, ranges from 75.2 to 78.4%. The recently disclosed sequences of the core region of Egyptian type 4 isolates (for example EG-29 in Figure 3) described by Simmonds et al.
(1993) do not allow alignment with the Gabonese sequences (as described in the present invention) in the NSB region and may belong to different type 4 subtypes(s) as can be deduced from the core sequences. The deduced amino acid sequences with SEQ ID
NO 119, 121, and 123 are aligned with other prototype sequences in Figure 5. Again, type-specific variation mainly resides in the variable V regions, designated in the present invention, and therefore, type-4-specific amino acids or V regions will be instrumental in diagnosis and therapeutics for HCV type 4.
Examnle 10 = The Core/El and NS5b regions of new HCV tvne 2 3 and 4 subtypes Samples NE92 (subtype 2d), BE98 (subtype 3c), CAM600 and GB809 (subtype 4e), CAMG22 and CAMG27 (subtype 4f), GB438 (subtype 4h), CAR4/1205 subtype (4i), CAR1/501 (subtype 4j), CAR1/901 (subtype 4?), and GB724 (subtype 4?) were selected from a group of sera that reacted positive but aberrantly in a prototype Line Probe Assay as described earlier (Stuyver et al., 1993). Another type 5a isolate BE100 was also analyzed in the C/El region, and yet another type 5a isolate BE96 in the NS5b region. A
high-titer of HCV RNA could be detected, enabling cloning of fragments by a single round of PCR. As no sequences from any coding region of these subtypes had been disclosed yet, synthetic oligonucleotides for PCR amplification were chosen in the regions of little sequence variation after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J(Kato et al., 1990), HC-J6 (Okamoto et al., 1991), HC-J8 (Okamoto et al., 1992), and the other new sequences of the present invention.
SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCTIEP94/01323 The above mentioned sets 1, 2 and 3 (see example 5) of primers were used, but only with set 1, PCR fragments could be obtained from all isolates (except for BE98, GB724, and CAR1/501). This set of primers amplified the sequence from nucleotide 379 to 957 encoding amino acids 127 to 319: 65 amino acids from the carboxyterminus of core and 128 amino acids of E1. With set 3, the core/E1 region from isolate NE92 and BE98 could be amplified, and with set 2, the core region of GB358, GB724, GB809, and CAM600 could be amplified. }
The amplification products were cloned as described in example 1. The following clones were obtained from the PCR fragments:
From isolate GB724, the clone with SEQ ID NO 193 from the core region.
From isolate NE92, the clone with SEQ ID NO 143 From isolate BE98, the clone from the core/El region of which part of the sequence has been analyzed and is given in SEQ ID NO 147, From isolate CAM600, the clone with SEQ ID NO 167 from the El region, or SEQ
ID NO
165 from the Core/El region as shown in Figure 3, From isolate CAMG22, the clone with SEQ ID NO 171 from the El region as shown in Figure 4, from isolate GB358, the clone with SEQ ID NO 191 in the core region,.
from isolate CAMG27, the clone with SEQ ID NO 173 from the core/El region, from isolate GB438, the clone with SEQ ID NO 177 from the core/ El region, from isolate CAR4/1205, the clone with SEQ ID NO 179 from the core/El region, from isolate CAR1/901, the clone with SEQ ID NO 181 from the core/ El region, from isolate GB809, the clone GB809-4 with SEQ ID NO 189 from the core/El region, clone GB809-2 with SEQ ID NO 169 from the core/El region and the clone with SEQ ID
NO 163 from the core region, and from isolate BE100, the clone with SEQ ID NO 155 from the Core/El region as shown in Figure 4.
An alignment of these Core/El sequences with known Core/El sequences is presented in Figure 4. The deduced amino acid sequences with SEQ ID NO 144, 148, 164, 168, 170, 172, 174, 178, 180, 182, 190, 192, 194, 156, 166 are aligned with other prototype sequences in Figure 5. Again, type-specific variation mainly resides in the variable V
regions, designated in the present invention, and therefore, type 2d, 3c and type 4-specific amino acids or V
regions will be instrumental in diagnosis and therapeutics for HCV type (subtype) 2d, 3c or the different type 4 subtypes.
SUBSTITUTE SHEET (RULE 26) WO 94125601 PCTlEP94/01323 The NS5b region of isolates NE92, BE98, CAM600, CAMG22, GB438, CAR4/1205, CAR1/501, and BE96 was amplified with primers HCPr206 and HCPr207 (Table 7).
The corresponding clones were cloned and sequenced as in example 1 and the corresponding . sequences (of which BE98 was partly sequenced) received the following identification numbers:
' NE92: SEQ ID NO 145 BE98: SEQ ID NO 149 CAM600: SEQ ID NO 201 CAMG22: SEQ ID NO 203 GB438: SEQ ID NO 207 CAR4/1205: SEQ ID NO 209 CAR1/501: SEQ ID NO 211 BE95: SEQ ID NO 159 BE96: SEQ ID NO 161 An alignment of these NS5b sequences with known NS5b sequences is presented in Figure 1. The deduced amino acid sequences with SEQ ID NO 146, 150, 202, 204, 206, 208, 210, 212, 160, 162 are aligned with other prototype sequences in Figure 2. Again, subtype-specific variations can be observed, and therefore, type 2d, 3c and type 4-specific amino acids or V
regions will be instrumental in diagnosis and therapeutics for HCV type (subtype) 2d, 3c or the different type 4 subtypes.
Example 11 : Genotype-secafic reactivity of anti-El antibodies (SerotvninQ) El proteins were expressed from vaccinia virus constructs containing a core/El region extending from nucleotide positions 355 to 978 (Core/El clones described in previous examples including the primers HCPr52 and HCPr54), and expressed proteins from (after the initiator methionine) to W326 of the HCV polyprotein. The expressed protein was modified upon expression in the appropriate host cells (e.g. HeLa, RK13, HuTK-, HepG2) by cleavage between amino acids 191 and 192 of the HCV polyprotein and by the addition of high-mannose type carbohydrate motifs. Therefore, a 30 to 32 kDa glycoprotein could be observed on western blot by means of detection with serum from patients with hepatitis C.
As a reference, a genotype lb clone obtained form the isolate HCV-B was also expressed in an identical way as described above, and was expressed from recombinant vaccinia virus vvHCV-11A.
SUBSTITUTE SHEET (RULE 26) A panel of 104 genotyped sera was first tested for reactivity with a cell lysate containing type lb protein expressed from the recombinant vaccinia virus vvHCV-11A, and compared with cell lysate of RK13 cells infected with a wild type vaccinia virus ('E1/WT'). The lysates were coated as a 1/20 dilution on a normal ELISA microtiter plate (Nunc maxisorb) and left to react with a 1/20 diluation of the respective sera. The panel consisted of 14 type la, 38 type ib, 21 type 2, 21 type 3a, and 9 type 4 sera. Human antibodies were subsequently detected by a goat anti-human IgG conjugated with peroxidase and the enzyme activity was detected. The optical density values of the El and wild type lysates were divided and a factor 2 was taken as the cut-off. The results are given in the table A. Eleven out of 14 type la sera (79 %), 25 out of 38 type lb sera (66 %), 6 out of 21 (29 %), 5 out of 21 (24 %), and none of the 9 type 4 or the type 5 serum reacted (0 %). These experiments clearly show the high prevalence of anti-El antibodies reactive with the type 1 El protein in patients infected with type 1 (36/52 (69%)) (either type la or type ib), but the low prevalence or absence in non-type 1 sera (11/52 (21 %)).
TABLE A
serum E1/WT
type la 3748 3.15 3807 3.51 5282 1.99 9321 3.12 9324 2.76 9325 6.12 9326 10.56 9356 1.79 9388 3.5 8366 10.72 8380 2.27 10925 4.02 10936 5.04 10938 1.36 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . pCT/EP94/01323 type lb 5205 2.25 5222 1.33 5246 1.24 5250 13.58 5493 0.87 5573 1.75 8243 1.77 8244 2.05 8316 1.21 8358 5.04 9337 14.47 9413 5.51 10905 1.26 10919 5.00 10928 8.72 .10929 8.26 10931 2.3 10932 4.41 44 2.37 45 3.14 46 4.37 47 5.68 48 2.97 49 1.18 50 9.85 51 4.51 52 1.11 53 5.20 54 0.98 55 1.48 56 1.06 57 3.85 58 7.6 59 3.28 60 3.23 61 7.82 62 1.92 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 type 2 23 0.91 24 1.16 25 2.51 26 0.96 27 1.20 =
28 0.96 29 2.58 30 8.05 31 0.92 32 0.82 33 5.75 34 0.79 35 0.86 36 0.85 37 0.76 38 0.92 - 39 1.08 40 2.33 41 2.83 42 1.21 43 0.91 type 3 1 6.88 2 1.47 3 3.06 4 6.52 10.24 6 2.72 7 1.11 8 1.54 9 1.60 1.21 11 1.07 12 1.00 13 0.85 14 0.96 =
0.51 16 1.00 17 1.09 18 0.99 19 1.04 1.04 21 0.96 SUBSTITUTE SHEET (RULE 26) type 4 22 0.87 GB48 0.49 - GB113 0.68 GB116 0.73 GB215 0.52 GB358 0.56 GB359 0.71 GB438 1.08 GB516 1.04 type 5 BE95 0.86 Core/El clones of isolates BR36 (type 3a) and BE95 (type 5a) were subsequently recombined into the viruses wHCV-62 and vvHCV-63, respectively. A genotyped panel of sera was subsequently tested onto cell lysates obtained from RK13 cells infected with the recombinant viruses wHCV-62 and wHCV-63. Tests were carried out as described above and the results are given in the table given below (TABLE B). From these results, it can clearly be seen that, although some cross-reactivity occurs (especially befween type I and 3), the obtained values of a given serum are usually higher on its homologous El protein than on an El protein of another genotype. For type 5 sera, none of the 5 sera were reactive on type 1 or 3 El proteins, while 3 out of 5 were shown to contain anti-El antibodies when tested on their homologous type 5 protein. Therefore, in this simple test system, a considerable number of sera can already be serotyped. Combined with the reactivity to type-specific NS4 epitopes or epitopes derived from other type-specific parts of the HCV polyprotein, a serotyping assay may be developed for discriminating the major types of HCV. To overcome the problem of cross-reactivity, the position of cross-reactive epitopes may be determined by someone skilled in the art (e.g. by means of competition of the reactivity with synthetic peptides), and the epitopes evoking cross-reactivity may be left out of the composition to be included in the serotyping assay or may be included in sample diluent to outcompete cross-reactive antibodies.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94101323 TABLE B
serum E11b/WT E13a/WT E158/WT
type lb 8316 0.89 0.59 0.80 ~
8358 2.22 2.65 1.96 9337 1.59 0.96 0.93 9410 16.32 9.60 3.62 9413 9.89 2.91 2.85 10905 1.04 0.96 1.05 10919 3.17 2.56 2.96 10928 4.39 2.28 2.07 10929 2.95 2.07 2.08 10931 3.11 1.49 2.11 0.86 0.86 0.96 6 3.48 1.32 1.32 7 6.76 4.00 3.77 8 10.88 3.44 4.04 9 1.76 1.88 1.58 9.88 7.48 7.20 11 8.48 8.99 8.45 12 0.76 0.72 0.76 13 5.04 5.67 5.37 14 10.48 10.54 11.22 5.18 1.62 1.65 type 3 8332 3.39 4.22 0.66 10907 3.24 4.39 0.96 10908 0.99 0.94 0.98 10934 0.86 0.90 0.90 10927 2.58 2.71 2.44 8210 0.82 0.80 0.86 8344 1.09 6.66 1.17 8351 1.21 1.29 1.22 30 0.85 4.11 0.98 32 0.85 2.16 1.04 type 5 0.78 0.95 1.54 BE110 0.79 1.01 4.95 BE95 0.47 0.52 0.65 BE111 0.71 0.75 8.33 BE112 1.01 1.27 2.37 BEII3 1.11 1.35 1.60 SIjBSTITEJfE SHEET (RULE 26) Table 5. Homologies of new HCV sequences with other known HCV types Region Isolate la lb 2a 2b 3a 3b (nucleotides) (type) HCV-l HCV-J HC-J6 HC-J8 T1 T7 T9 Ti0 Core (1-573) PC (5) 83.8 (91.6) 84.8 (92.1) 82.6 (90.1) 82.4 (89.0) t1 (574-957) I-ID10 (3) 61.5 (68,0) 64.6 (68.8) 57.8 (55.5) 56.3 (59.4) BR36 (3) 62.0 (66.4) 62.5 (67.2) 56.5 (53.9) 55.2 (58.6) BR33 (3) 60.7 (67.2) 63.3 (68.0) 56.5 (54.7) 56.0 (58.6) PC (5) 61.4 (64,0) 62.4 (64.8) 54.1 (49.6) 53.3 (47.2) GB358 (4a) 62.5 (69.1) 62.8 (65.9) 59.4 (54.0) 54.4 (54.0) GB549 (4b) 66.0 (72.2) 62,8 (69.8) 59.1 (56,4) 56.5 (54.0) GB809 (4c) 63.3 (69.1) 60.7 (64.3) 56.7 (53.2) 53.0 (51.6) NS3 PC (5) 74.7 (89) 76.1 (86.4) 76.1 (89.8) 78.0 (89.0) (3856-4209) NS4 BR36 (3) 67.8 (78.5) 69.8 (75.1) 62.0 (67.5) 61.7 (66.0) (4892-5292) HD 10 (3) 69.8 (74.6) 66.6 (69.7) 57.8 (59.9) 59.1 (59.9) NS4 PC (5) 61.3 (62.2) 63.0 (65.5) 52.9 (46.2) 54.3 (43.7) (4936-5292) NS5b BR34 (3) 65.7 66.7 63.9 64.3 94.8 93.9 75.6 77.0 (8023-8235) BR36 (3) 64.3 67.6 64.8 66.7 94.8 93.4 75.1 76.5 BR33 (3) 65.7 67.1 64.3 64.8 94.8 93.9 76.0 77.5 GB358 (4a) 67.7 (76.1) 65.6 (77.0) 66.5 (70.8) 65.6 (71,7) GB549 (4b) 68.8 (76.1) 67.1 (77.0) 65.9 (71.7) 65.9 (74.4) GB809 (4c) 68.5 (73.5) 65.0 (73.5) 67.7 (69.9) 67.7 (73.5) Shown are the nucleotide homologies (the amino-acid homology is given between brackets) for the region indicated in the left column.
SUBSTITUTE SHEET (RULE 26) Table 6. NS4 sequences of the different Lyenotypes prototype TYPE SYNTHETiC PEPTIDE NS4-1 SYNTHEIIC PEPTIDE NS4-5 SYNTHETlC
(NS4a) (NS4b) (NS4b) position-> 9 0 2 3 3 4 , = 4 = -4 t~ 4 = = i M = = -t HCV-1 la LSG KPAIIPDREV LYREFDE SQHLPYIEQ GMGLAEQFKQ K LAEQPKQ KALGLLQTAS RQA
HCV-7 lb LSG RPAVIPDREV LYQEFDE ASHLPYIEQ GMQ_LAEQFKg K LAEQFKQ KALGLLQTAT KQA
HC-76 2a VNQ RAWAPDKEV LYEAFDE ASRaAT.iF-? GQRIABMLKS K TAEMLBS KIQGLLQQAS KQA
HC J8 2b LND RWVAPDR'EI LY_'AFDE ASRAALIEE GQRMAIIvILi{S K MAp.hAxc K1QGLyQQAT
RQA
I BR36 3a LGG KPAIVPDKEV LYQQ YDE SQAAPYIEQ AQ_VIAHQFKE K IALIQFKE KV LGLLQRAT
QQQ
V
residues conserved in every genotype. Underlined amino acids are type-specific, amino acids in italics are unique to type 3 and 5 sequences.
SUBSTITUTE SHEET (RULE 26) Table 7 SEQ ID Primer NO Sequence from 5' to 3' NO (polarity) 63 HCPr161(+) 5'-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3' 64 HCPr162(-) 5'-GGGCTGCTCTATCCTCATCGACGCCATC-3' 65 HCPr163(+) 5'-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3' 66 HCPr164(-) 5'-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3' 67 HCPr23(+) 5'-CTCATGGGGTACATTCCGCT-3' 68 HCPr54(-) 5'-CTATTACCAGTTCATCATCATATCCCA-3' 69 HCPr116(+) 5'-ttttAAATACATCATGRCITGYATG-3' 70 HCPr66(-) 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' 71 HCPr 118(-) 5' actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' 72 HCPr 117(+) 5' -ttttAAATACATCGCIRCITGCATGCA-3' 73 HCPr119(-) 5'-actagtcgactaRTTIGCIATIAGCCKRTTCATCCAYTG-3' 74 HCPr131(+) 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' 75 HCPrl 30(+) 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' 76 HCPr 134(+) 5' -CATATAGATGCCCACTTCCTATC-3' 77 HCPr3(+) 5'-GTGTGCCAGGACCATC-3' 78 HCPr4(-) 5'-GACATGCATGTCATGATGTA-3' 79 HCPr 152(+) 5' -TACGCCTCTTCTATATCGGTTGGGGCCTG-3' 80 HCPr52(+) 5'-atgTTGGGTAAGGTCATCGATACCCT-3' 81 HCPr41(+) 5' -CCCGGGAGGTCTCGTAGACCGTGCA-3' 82 HCPr40(-) 5'-ctattaAAGATAGAGAAAGAGCAACCGGG-3' 124 HCPR206 5'-tggggatcccgtaxgatacccgctgctttga-3' 125 HCPR207 5'-ggcggaattcctggtcatagcctccgtgaa-3' 141 HCPR 109 5' -tgggatatgatgatgaactggtc-3' 142 HCPR14 5' -ccaggtacaaccgaaccaattgcc-3' SUBSTI7UTE SHEET (RULE 26) t~ M'-+MNN+ ~NMM
4t ~
.-i + ~ i M + + N + ~
l~ M N M M N N N N M
`e!
kn --~ i+ N N -+ N+ N+
N
C3.
K1 N M M M'-~ M M M M
Z 1/') M,~ M M M~-+ M M M M
Qy ~ F~ H M~~ M M M M M M M
~
~
z ~
pp ~ ~ p ~ N M ~Y v1 ~O [~ oo !T O
~ O O O O O O O O O~
r. .-. .-. .-~ .~ .-. .-F
SUBSTI7UTE SHEET (RULE 26) [~ , f+1 M M M~" , t''1 N ! f~l ~~ N
tt ~
z .
FD-.
N N
N~-+ N m N N N N N N
Z ~!1 + +++-~I-`I+N.--.-.~
l~ , M M M M M , M N M M~ N M
CA
Z
i/) + N c~ M M c~ , N N M M M c~ M
N N M M M~ + , M M K1 M
---Fi i. ~
[Y W) `O I'- oo Cn O --~ N tn ~t ~, - --+r+ NNNN N
SUBSTITUTE SHEET (RULE 26) 1~ M+- ++
~
`~' V~ =--, =-= + + +
~ N ~ + N N + + ~--~ ~-- M N N
t- M *~ ~-+ -- rM , , + cM , N N + M + , ~ N N
'ef z 1n + + + .-. , rl ~--~ =--~ , , , + ~ + , , ~ 'I' ~" 'I' M M N , =-~ =--= M + M +
C- c'*1 N + K1 M ~ '-' ~ K1 N M M M
Z f*1 N N M C''f N~""
rl -G.1 N
y N
~' v~ ~D l~ 00 CT O~ N M ~t v> ~D t~ 00 põ rn C:) ~-, N MV- v-) ~O C- o0 N N N N N M M M M M M M M M ~ M Ci' d' :r 114, 'f qT 'cl- V- Q-r. .~. r+ r-. ..r .~r .-~ ~-r .... .r ~ .-. ~r ~ .-. .~ ~ ~ ..-, r-. .-. .-=+
.-, .-~
SUBST(TUTE SHEET (RULE 26) t~ + M N f+1 M r N N cn M N
z + M r i~~^~ , N N M N M , r N ~ N M r N~= r+ M r r+ r+
N N , i N+ ,~-+ r+
z tfJ + N + +
r.~t M + r r r r r r+ r r r , , i r r CA
Z , + ~ , i =- N
.-y ~
t~. N M
p, a, Q~--~ N M d' xn "D l- 00 ON 0~ N M
~ ~ D, ~l Vl V~ ~/'1 ~/1 ~1 r/1 V1 N v1 \D ~ ~, ~O ~D
SUBSTITLlTE SHEET (RULE 26) REFERENCES
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SUBSf ITM SHEET (RULE 26)
THERAPEUTIC AND DIAGNOSTIC AGENTS
---------------------The invention relates to new sequences of hepatitis C virus (HCV) genotypes and their use as therapeutic and diagnostic agents.
' The present invention relates to new nucleotide and amino acid sequences corresponding to the coding region of a new type 2 subtype 2d, type-specific sequences corresponding to HCV type 3a, to new sequences corresponding to the coding region of a new subtype 3c, and to new sequences corresponding to the coding region of HCV type 4 and type 5 subtype 5a;
a process for preparing them, and their use for diagnosis, prophylaxis and therapy.
The technical problem underlying the present invention is to provide new type-specific sequences of the Core, the El, the E2, the NS3, the NS4 and the NS5 regions of HCV type 4. and type 5, as well as of new variants of HCV types 2 and 3. These new HCV
sequences are useful to diagnose the presence of type 2 and/or type 3 and/or type 4 and/or type 5 HCV
genotypes in a biological sample. Moreover, the availability of these new type-specific sequences can increase the overall sensitivity of HCV detection and should also prove to be useful for therapeutic purposes. -Hepatitis C viruses (HCV) have been found to be the major cause of non-A, non-B
hepatitis. The sequences of cDNA clones covering the complete genome of several prototype isolates have been determined (Kato et al., 1990; Choo et al., 1991; Okamoto et al., 1991;
Okamoto et al., 1992). Comparison of these isolates shows that the variability in nucleotide sequences can be used to distinguish at least 2 different genotypes, type 1(HCV-1 and HCV-J) and type 2(HC-J6 and HC-J8), with an average homology of about 68%. Within each type, at least two subtypes exist (e.g. represented by HCV-1 and HCV-J), having an average homology of about 79%. HCV genomes belonging to the same subtype show average homologies of more than 90% (Okamoto et al., 1992). However, the partial nucleotide sequence of the NS5 region of the HCV-T isolates showed at most 67 % homology with the ~= previously published sequences, indicating the existence of a yet another HCV type (Mori et al., 1992). Parts of the 5' untranslated region (UR), core, NS3, and NS5 regions of this type 3 have been published, further establishing the similar evolutionary distances between the 3 major genotypes and their subtypes (Chan et al., 1992).
The identification of type 3 genotypes in clinical samples can be achieved by means of PCR with type-specific primers for the NS5 region. However, the degree to which this will SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCT/EP941t1323 be successful is largely dependent on sequence variability and on the virus titer present in the serum. Therefore, routine PCR in the open reading frame, especially for type 3'and the new type 4 and 5 described in the present invention and/or group V (Cha et al., 1992) genotypes can be predicted to be unsuccessful. A new typing system (LiPA), based on variation in the highly conserved 5' UR, proved to be more useful because the 5 major HCV
genotypes and their subtypes can be determined (Stuyver et al., 1993). The selection of high-titer isolates enables to obtain PCR fragments for cloning with only 2 primers, while nested PCR requires that 4 primers match the unknown sequences of the new type 3, 4 and 5 genotypes.
New sequences of the 5' untranslated region (5'UR) have been listed by Bukh et al.
(1992). For some of these, the El region has recently been described (Bukh et al., 1993).
Isolates with similar sequences in the 5'UR to a group of isolates including DK12and HK10 described by Bukh et al. (1992) and E-b 1 to E-b8 described and classified as type 3 by Chan et al. (1991), have been reported and described in the 5'UR, the carboxyterminal part of El, and in the NS5 region as group IV by Cha et al. (1992; WO 92/19743), and have also been described in the 5'UR for isolate BR56 and classified as type 3 by the inventors of this application (Stuyver et al., 1993).
The aim of the present invention is to provide new HCV nucleotide and amino acid sequences enabling the detection of HCV infection.
Another aim of the present infection is to provide new nucleotide and amino acid HCV
sequences enabling the classification of infected biological fluids into different serological groups unambiguously linked to types and subtypes at the genome level.
Another aim of the present invention is to provide new nucleotide and amino acid HCV
sequences ameliorating the overall HCV detection rate.
Another aim of the present invention is to provide new HCV sequences, useful for the design of HCV vaccine compositions.
Another aim of the present invention is to provide a pharmaceutical composition consisting of antibodies raised against the polypeptides encoded by these new HCV
sequences, for therapy or diagnosis.
The present invention relates more particularly to a composition comprising or consisting of at least one polynucleic acid containing at least 5, and preferably 8 or more contiguous nucleotides selected from at least one of the following HCV sequences:
- an HCV type 3 genomic sequence, more particularly in any of the following regions:
SUBSTITUTE SHEET (RULE 26) - the region spanning positions 417 to 957 of the Core/El region of HCV
subtype 3a, - the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3, - the region spanning positions 4892 to 5292 of the NS3/4 region of HCV
type 3, - the region spanning positions 8023 to 8235 of the NS5 region of the BR36 subgroup of HCV subtype 3a, - an HCV subtype 3c genomic sequence, more particularly the coding regions of the above-specified regions;
- an HCV subtype 2d genomic sequence, more particularly the coding region of HCV
subtype 2d;
- an HCV type 4 genomic sequence, more particularly the coding region, more particularly the coding region of subtypes 4a, 4e, 4f, 4g, 4h, 4i, and 4j, - an HCV type 5 genomic sequence, more particularly the coding region of HCV
type 5, more particularly the regions encoding Core, El, E2, NS3, and NS4 with said nucleotide numbering being with respect to the numbering of HCV
nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV (type 1, type 2, and type 3) polynucleic acid sequences in the above-indicated regions, or the complement thereof.
It is to be noted that the nucleotide difference in the polynucleic acids of the invention may involve or not an amino acid difference in the corresponding amino acid sequences coded by said polynucleic acids.
According to a preferred embodiment, the present invention relates to a composition comprising or containi.ng at least one polynucleic acid encoding an HCV
polyprotein, with said polynucleic acid containing at least 5, preferably at least 8 nucleotides corresponding to at least part of an HCV nucleotide sequence encoding an HCV polyprotein, and with said HCV polyprotein containing in its sequence at least one of the following amino acid residues:
L7, Q43, M44, S60, R67, Q70, T71, A79, A87, N106, K115, A127, A190, S130, V134, 0142, I144, E152, A157, V158, P165, S177 or Y177, I178, V180 or E180 or F182, R184, 1186, H187, T189, A190, S191 or G191, Q192 or L192 or 1192 or V192 or E192, N193 or H193 or P193, W194 or Y194, H195, A197 or 1197 or V197 or T197, V202, I203 or L203, Q208, A210, V212, F214, T216, R217 or D217 or E217 or V217, H218 or N218, H219 or SllBSTITUTE SHEET (RULE 26) V219 or L219, L227 or I227, M231 or E231 or Q231, T232 or D232 or A232 or K232, Q235 or I235, A237 or T237, I242, I246, S247, S248, V249, S250 or Y250, I251 or V251 or M251 or F251, D252, 7254 or V254, L255 or V255, E256 or A256, M258 or F258 or V258, A260 or Q260 or S260, A261, T264 or Y264, M265, I266 or A266, A267, G268 or =
T268, F271 or M271 or V271, I277, M280 or H280, I284 or A284 or L84, V274, V291, N292 or S292, R293 or I293 or Y293, Q294 or R294, L297 or I297 or Q297, A299 or K299 or Q299, N303 or T303, T308 or L308, T310 or F310 or A310 or D310 or V310, L313, G317 or Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, 1403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, S532, V534, F536, F537, M539, I546, C1282, A1283, H1310, V1312, Q1321, P1368, V1372, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, D1556, N1567, M1572, Q1579, L1581, S1583, F1585, V1595, E1606 or T1606, M1611, V1612 or L1612, P1630, C1636, P1651, T1656 or 11656, L1663, V1667, V1677, A1681, H1685, E1687, G1689, V1695, A1700, Q1704, Y1705, A1713, A1714 or S1714, M1718, D1719, A1721 or T1721, R1722, A1723 or V1723, H1726 or G1726, E1730, V1732, F1735, 11736, S1737, R1738, T1739, G1740, Q1741, K1742, Q1743, A1744, T1745, L1746, E1747 or K1747, I1749, A1750, T1751 or A1751, V1753, N1755, K1756, A1757, P1758, A1759, H1762, T1763, Y1764, P2645, A2647, K2650, K2653 or L2653, S2664, N2673, F2680, K2681, L2686, H2692, Q2695 or L2695 or I2695, V2712, F2715, V2719 or Q2719, T2722, T2724, S2725, R2726, G2729, Y2735, H2739, 12748, G2746 or I2746, 12748, P2752 or K2752, P2754 or T2754, T2757 or P2757, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990.
Each of the above-mentioned residues can be found in any of Figures 2, 5, 7, 11 or 12 showing the new amino acid sequences of the present invention aligned with known sequences of other types or subtypes of HCV for the Core, El, E2, NS3, NS4, and NS5 regions.
More particularly, a polynucleic acid contained in the composition according to the present invention contains at least 5, preferably 8, or more contiguous nucleotides corresponding to a sequence of contiguous nucleotides selected from at least one of HCV
sequences encoding the following new HCV amino acid sequences:
- new sequences spanning amino acid positions 1 to 319 of the Core/El region of HCV
subtype 2d, type 3 (more particularly new sequences for subtypes 3a and 3c), new type 4 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 subtypes (more particularly new sequences for subtypes 4a, 4e, 4f, 4g, 4h, 4i and 4j) and type 5a, as shown in Figure 5;
- new sequences spanning amino acid positions 328 to 546 of the E 1/E2 region of HCV
. subtype 5a as shown in Figure 12;
- new sequences spanning amino acid positions 1556 to 1764 of the NS3/NS4 region of HCV type 3 (more particularly for new subtypes 3a sequences), and subtype 5a, as shown in Figure 7 or 11;
- new sequences spanning amino acid positions 2645 to 2757 of the NS5B region of HCV
subtype 2d, type 3 (more particularly for new subtypes 3a and 3c), new type 4 subtypes (more particularly subtypes 4a, 4e, 4f, 4g, 4h, 4i and 4j) and subtype 5a, as shown in Figure 2, Using the LiPA system mentioned above, Brazilian blood donors with high titer type 3 hepatitis C virus, Gabonese patients with high-titer type 4 hepatitis C virus, and a Belgian patient with high-titer HCV type 5 infection were selected. Nucleotide sequences in the core, El, NS5 and NS4 regions which have not yet been reported before, were analyzed in the frame of the invention. Coding sequences (with the exception of the core region) of any type 4 isolate are reported for the first time in the present invention. The NS5b region was also analyzed for the new type 3 isolates. After having determined the NS5b sequences, comparison with the Ta and Tb subtypes described by Mori et al. (1992) was possible, and the type 3 sequences could be identified as type 3a genotypes. The new type 4 isolates segregated into 10 subtypes, based on homologies obtained in the NS5 and El regions. New type 2 and 3 sequences could also be distinguished from previously described type 2 or 3 subtypes from sera collected in Belgium and the Netherlands.
The term "polynucleic acid" refers to a single stranded or double stranded nucleic acid sequence which may contain at least 5 contiguous nucleotides to the complete nucleotide sequence (f.i. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous nucleotides). A
polynucleic acid which is up till about 100 nucleotides in length is often also referred to as an oligonucleotide. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes. A polynucleic acid may also comprise a double stranded cDNA clone which can be used for cloning purposes, or for in vivo therapy, or prophylaxis.
The term "polynucleic acid composition" refers to any kind of composition comprising essentially said polynucleic acids. Said composition may be of a diagnostic or a therapeutic SUBSTITUTE SHEET (RULE 26) nature.
The expression "nucleotides corresponding to" refers to nucleotides which are homologous or complementary to an indicated nucleotide sequence or region within a specific HCV
sequence. =
The term "coding region" corresponds to the region of the HCV genome that encodes the HCV polyprotein. In fact, it comprises the complete genome with the exception of the 5' untranslated region and 3' untranslated region.
The term "HCV polyprotein" refers to the HCV polyprotein of the HCV-J isolate (Kato et al., 1990). The adenine residue at position 330 (Kato et al., 1990) is the first residue of the ATG codon that initiates the long HCV polyprotein of 3010 amino acids in HCV-J and other type lb isolates, and of 3011 amino acids in HCV-1 and other type la isolates, and of 3033 amino acids in type 2 isolates HC-J6 and HC-J8 (Okamoto et al., 1992).
This adenine is designated as position 1 at the nucleic acid level, and this methionine is designated as position 1 at the amino acid level, in the present invention. As type la isolates contain 1 extra amino acid in the NS5a region, coding sequences of type la and lb have identical numbering in the Core, El, NS3, and NS4 region, but will differ in the NS5b region as indicated in Table 1. Type 2 isolates have 4 extra amino acids in the E2 region, and 17 or 18 extra amino acids in the NS5 region compared to type 1 isolates, and will differ in numbering from type 1 isolates in the NS3/4 region and NS5b regions as indicated in Table 1.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT1EP94/01323 Region Positions Positions Positions Positions = described in described for described for described for the HCV-J HCV-1 HC-J6, HC-J8 present (Kato et aL, (Choo et al., (Okamoto et invention* 1990) 1991) al., 1992) Nucleotide NS5b 8023/8235 8352/8564 8026/8238 8433/8645 s 7932/8271 8261/8600 7935/8274 8342/8681 coding 330/9359 1/9033 342/9439 region of present invention Amino NS5b 2675/2745 2675/2745 2676/2746 2698/2768 Acids 2645/2757 2645/2757 2646/2758 2668/2780 Table 1: Comparison of the HCV nucleotide and amino acid numbering system used in the present invention (*) with the numbering used for other prototype isolates.
For example, 8352/8564 indicates the region designated by the numbering from nucleotide 8352 to nucleotide 8564 as described by Kato et al. (1990). Since the numbering system of the present invention starts at the polyprotein initiation site, the 329 nucleotides of the 5' untranslated region described by Kato et al.
(1990) have to be substracted, and the corresponding region is numbered from nucleotide 8023 ("8352-329") to 8235 ("8564-329").
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "HCV type" corresponds to a group of HCV isolates of which the complete genome shows more than 74% homology at the nucleic acid level, or of whicb the NS5 region between nucleotide positions 7932 and 8271 shows more than 74% homology at the nucleic acid level, or of which the complete HCV polyprotein shows more than 78%
homology at the =
amino acid level, or of which the NS5 region between amino acids at positions 2645 and 2757 shows more than 80% homology at the amino acid level, to polyproteins of the other isolates of the group, with said numbering beginning at the first ATG codon or first methionine of the long HCV polyprotein of the HCV-J isolate (Kato et al., 1990). Isolates belonging to different types of HCV exhibit homologies, over the complete genome, of less than 74% at the nucleic acid level and less than 78% at the amino acid level. Isolates belonging to the same type usually show homologies of about 92 to 95% at the nucleic acid level and 95 to 96% at the amino acid level when belonging to the same subtype, and those belonging to the same type but different subtypes preferably show homologies of about 79% at the nucleic acid level and 85-86% at the amino acid level.
More preferably the definition of HCV types is concluded from the classification of HCV
isolates according to their nucleotide distances calculated as detailed below:
(1) based on phylogenetic analysis of nucleic acid sequences in the NS5b region between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and 8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al., 1991), isolates belonging to the same HCV type show nucleotide distances of less than 0.34, usually less than 0.33, and more usually of less than 0.32, and isolates belonging to the same subtype show nucleotide distances of less than 0.135, usually of less than 0.13, and more usually of less than 0.125, and consequently isolates belonging to the same type but different subtypes show nucleotide distances ranging from 0.135 to 0.34, usually ranging from 0.1384 to 0.2477, and more usually ranging from 0.15 to 0.32, and isolates belonging to different HCV types show nucleotide distances greater than 0.34, usually greater that 0.35, and more usually of greater than 0.358, more usually ranging from 0.1384 to 0.2977.
(2) based on phylogenetic analysis of nucleic acid sequences in the core/El region between nucleotides 378 and 957, isolates belonging to the same HCV type show nucleotide distances of less than 0,38, usually of less than 0.37, and more usually of less than 0.364, and isolates belonging to the same subtype show nucleotide distances of less than 0.17, usually of less than 0.16, and more usually of less than 0.15, more usually less than 0.135, more usually less than 0.134, and consequently isolates belonging to the same type but different subtypes show SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 nucleotide distances ranging from 0.15 to 0.38, usually ranging from 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, more usually ranging from 0.133 to 0.379, =and isolates belonging to different HCV types show nucleotide distances greater than 0.34, 0.35, 0.36, usually more than 0.365, and more usually of greater than 0.37, (3) based on phylogenetic analysis of nucleic acid sequences in the NS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) or between nucleotides 4993 and 5621 (Kato et al., 1990) or between nucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging to the same HCV type show nucleotide distances of less than 0.35, usually of less than 0.34, and more usually of less than 0.33, and isolates belonging to the same subtype show nucleotide distances of less than 0.19, usually of less than 0.18, and more usually of less than 0.17, and consequently isolates belonging to the same type but different subtypes show nucleotide distances ranging from 0.17 to 0.35, usually ranging from 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, and isolates belonging to different HCV
types show nucleotide distances greater than 0.33, usually greater than 0.34, and more usually of greater than 0.35.
Table 2 : Molecular evolutionary distances Region Core/EI El NS5B NSSB
579 bp 384 bp 340 bp 222 bp Isolates 0.0017 - 0.1347 0.0026 - 0.2031 0.0003 - 0.1151 0.000 - 0.1323 (0.0750 0.0245) (0.0969 0.0289) (0.0637 0.0229) (0.0607 0.0205) Subtypes' 0.1330 - 0.3794 0.1645 - 0.4869 0.1384 - 0.2977 0.117 - 0.3538 (0.2786 0.0363) (0.3761 0.0433) (0.2219 0.0341) (0.2391 0.0399) Types' 0.3479 - 0.6306 0.4309 - 0.9561 0.3581 - 0.6670 0.3457 - 0.7471 (0.4703 0.0525) (0.6308 0.0928) (0.4994 0.0495) (0.5295 + 0.0627) ' Figures created by the PHYLIP program DNADIST are expressed as minimum to maximum (average standard deviation). Phylogenetic distances for isolates belonging to the same subtype ('isolates'), to different subtypes of the same type ('subtypes'), and to different types ('types') are given.
In a comparative phylogenetic analysis of available sequences, ranges of molecular evolutionary distances for different regions of the genome were calculated, based on 19,781 SUBSTITUTE SHEET (RULE 26) pairwise comparisons by means of the DNA DIST program of the phylogeny inference package PHYLIP version 3.5C (Felsenstein, 1993). The results are shown in Table 2 and indicate that although the majority of distances obtained in each region fit with classification of a certain isolate, only the ranges obtained in the 340bp NS5B-region are non-overlapping =
and therefor conclusive. However, as was performed in the present invention, it is preferable to obtain sequence information from at least 2 regions before final classification of a given isolate.
Designation of a number to the different types of HCV and HCV types nomenclature is based on chronological discovery of the different types. The numbering system used in the present invention might still fluctuate according to international conventions or guidelines. For example, "type 4" migbt be changed into "type 5" or "type 6".
The term "subtype" corresponds to a group of HCV isolates of which the complete polyprotein shows a homology of more than 90% both at the nucleic acid and amino acid levels, or of which the NS5 region between nucleotide positions 7932 and 8271 shows a homology of more than 90% at the nucleic acid level to the corresponding parts of the genomes of the other isolates of the same group, with said numbering beginning with the adenine residue of the initiation codon of the HCV polyprotein. Isolates belonging to the same type but different subtypes of HCV show homologies of more than 74 % at the nucleic acid level and of more than 78% at the amino acid level.
The term "BR36 subgroup" refers to a group of type 3a HCV isolates (BR36, BR33, BR34) that are 95 %, preferably 95.5 %, most preferably 96 % homologous to the sequences as represented in SEQ ID NO 1, 3, 5, 7, 9, 11 in the NS5b region from position 8023 to 8235.
It is to be understood that extremely variable regions like the El, E2 and NS4 regions will exhibit lower homologies than the average homology of the complete genome of the polyprotein.
Using these criteria, HCV isolates can be classified into at least 6 types.
Several subtypes can clearly be distinguished in types l, 2, 3 and 4 : la, ib, 2a, 2b, 2c, 2d, 3a, 3b, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i and 4j based on homologies of the 5' UR and coding regions including the part of NS5 between positions 7932 and 8271. An overview of most of the reported isolates and their proposed classification according to the typing system of the present invention as well as other proposed classifications is presented in Table 3.
SUBST{TUTE SHEET (RULE 26) T le3 HCV CLASSIFICATION
OKA- MORI NAKA CHA PROTOTYPE
la I I Pt GI HCV-l, HCV-H, HC-J1 lb II II KI GII HCV-J, HCV-BK, HCV-T, HC-JK1, HC-J4, HCV-CH.TNA
ic HC-G9 2a III III K2a GIII HC-J6 2b IV IV K2b GIII HC-J8 2c S83, ARG6, ARG8, 110, T983 2d NE92 3a V V K3 GIV E-bi, Ta, BR36, BR33, HD10, NZLI
3b VI K3 GIV HCV-TR, Th 3c BE98 4a Z4, GB809-4 4b - Zi 4c GB116, GB358, GB215, Z6, Z7 4d DK13 4e GB809-2, CAM600, CAM736 4f CAM622, CAM627 4g GB549 4h - GB438 4i CAR4/1205 4j CARl/501 4k EG29 5a GV SA3, SA4, SAl, SA7, SAli, BE95 6a HK I, HK2, HK3, HK4 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "complement" refers to a nucleotide sequence which is complementary to an indicated sequence and which is able to hybridize to the indicated sequences.
The composition of the invention can comprise many combinations. By way of example, the composition of the invention can comprise: - two (or more) nucleic acids from the same region or, - two nucleic acids (or more), respectively from different regions, for the same isolate or for different isolates, - or nucleic acids from the same regions and from at least two different regions (for the same isolate or for different isolates).
The present invention relates more particularly to a polynucleic acid composition as defined above, wherein said polynucleic acid corresponds to a nucleotide sequence selected from any of the following HCV type 3 genomic sequences:
- an HCV genomic sequence having a homology of at least 67%, preferably more than 69%, more preferably 71 %, even more preferably more than 73 %, or most preferably more than 76% to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD 10, BR36 or BR33 sequences) in the region spanning positions 417 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence having a homology of at least 65 %, preferably more than 67 %, preferably more than 69%, even preferably more than 70%, most preferably more than 74% to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD 10, BR36 or BR33 sequences) in the region spanning positions 574 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of at least 79%, more preferably at least 81 %, most preferably more than 83 % or more to any of the sequences as represented in SEQ ID NO 147 (representing positions 1 to 346 of the Core region of HVC type 3c, sequence BE98) in the region spanning positions 1 to 378 of the Core region as shown in Figure 3;
- an HCV genomic sequence of HVC type 3a having a homology of at least 74 %, more preferably at least 76 %, most preferably more than 78 % or more to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HDIO, BR36 or sequences) in the region spanning positions 417 to 957 in the Core/El region as shown in Figure 4;
- an HCV genomic sequence of HCV type 3a as having a homology of at least 74 %, SUBSTITUTE SHEET (RULE 26) preferably more than 76 %, most preferably 78 % or more to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD10, BR36 or BR33 sequences) in the region spanning positions 574 to 957 in the El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of more than 73.5 %, preferably more than 74 %, most preferably 75 % homology to the sequence as represented in SEQ
ID NO
29 (HCC153 sequence) in the region spanning positions 4664 to 4730 of the NS3 region as shown in figure 6;
- an HCV genomic sequence having a homology of more than 70%, preferably more than 72%, most preferably more than 74% homology to any of the sequences as represented in SEQ ID NO 29, 31, 33, 35, 37 or 39 (HCC153, HD10, BR36 sequences) in the region spanning positions 4892 to 5292 in the NS3/NS4 region as shown in Figure 6 or 10;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a as having a homology of more than 95%, preferably 95,5%, most preferably 96% homology to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in the region spanning positions 8023 to 8235 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a as having a homology of more than 96%, preferably 96.5 %, most preferably 97% homology to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in the region spanning positions 8023 to 8192 of the NS5B region as shown in Figure 1;
- an HCV genomic sequence of HCV type 3c being characterized as having a homology of more than 79 %, more preferably more than 81 %, and most preferably more than 83 % to the sequence as represented in SEQ ID NO 149 (BE98 sequence) in the region spanning positions 7932 to 8271 in the NS5B region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- an HCV genomic sequence being characterized as having a nucleotide distance of less than 0.44, preferably of less than 0.40, most preferably of less than 0.36 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 spanning positions 417 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence being characterized having a nucleotide distance of less than 0.53, preferably less than 0.49, most preferably of less than 0.45 to any of the sequences as represented in SEQ ID NO 19, 21, 23, 25 or 27 in the region spanning positions 574 =
to 957 of the E1 region as shown in Figure 4;
- an HCV genomic sequence characterized having a nucleotide distance of less than 0.15, preferably less than 0.13, and most preferably less than 0.11 to any of the sequences as represented in SEQ ID NO 147 in the region spanning positions 1 to 378 of the Core region as shown in Figure 3;
- an HCV genomic sequence of HVC type 3a being characterized as having a nucleotide distance of less than 0.3, preferably less than 0.26, most preferably of less than 0.22 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region spanning positions 417 to 957 in the Core/El region as shown in Figure 4;
- an HCV genomic sequence of HCV type 3a being characterized as having a nucleotide distance of less than 0.35, preferably less than 0.31, most preferably of less than 0.27 to any of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in the region spanning positions 574 to 957 in the El region as shown in Figure 4;
- an HCV genomic sequence of the BR36 subgroup of HCV type 3a being characterized as having a nucleotide sequence of less than 0.0423, preferably less than 0.042, preferably less than 0.0362 to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3, 9 or 11 in the region spanning positions 8023 to 8235 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence of HCV type 3c being characterized as having a nucleotide distance of less than 0.255, preferably of less than 0.25, more preferably of less than 0.21, most preferably of less than 0.17 to the sequence as represented in SEQ ID NO
149 in the region spanning positions 7932 to 8271 in the NS5B region as shown in Figure 1.
In the present application, the El sequences encoding the antigenic ectodomain of the El protein, which does not overlap the carboxyterminal signal-anchor sequences of El disclosed by Cha et al. (1992; WO 92/19743), in addition to the NS4 epitope region, and a part of the NS5 region are disclosed for 4 different isolates: BR33, BR34, BR36, HCC153 and HD10, all belonging to type 3a (SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37 or 39).
Also within the present invention are new subtype 3c sequences (SEQ ID NO 147, 149 of the isolate BE98 in the Core and NS5 regions (see Figures 3 and 1).
SUBSTITkJTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 Finally the present invention also, relates to a new subtype 3a sequence as represented in SEQ ID NO 217 (see Figure 1) Also included within the present invention are sequence variants of the polynucleic acids = as selected from any of the nucleotide sequences as given in any of the above mentioned SEQ
ID numbers, with said sequence variants containing either deletions and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 3 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type 3 as shown in Figure 1(NS5 region), Figure 3 (Core region), Figure 4 (Core/El region), Figure 6 and 10 (NS3/NS4 region).
According to another embodiment, the present invention relates to a polynucleic acid composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 5 genomic sequences:
- an HCV genomic sequence as having a homology of more than 85 %, preferably more than 86%, most preferably more than 87% homology to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 (PC sequences) or 151 (BE95 sequence) in the region spanning positions 1 to 573 of the Core region as shown in Figure 9 and 3;
- an HCV genomic sequence as having a homology of more than 61 %, preferably more than 63 %, more preferably more than 65 % homology, even more preferably more than 66%
homology and most preferably more than 67 % homology (f. i. 69 and 71 %) to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 (PC
sequences), 153 or 155 (BE95, BE100 sequences) in the region spanning positions 574 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 76.5 %, preferably of more than 77%, most preferably of more than 78% homology with any of the sequences as represented in SEQ ID NO 55, 57, 197 or 199 (PC sequences) in the region spanning positions 3856 to 4209 of the NS3 region as shown in Figure 6 or 10;
, - an HCV genomic sequence having a homology of more than 68 %, preferably of more than 70 %, most preferably of more than 72 % homology with the sequence as represented in SEQ ID NO 157 (BE95 sequence) in the region spanning positions 980 to 1179 of the E1/E2 region as shown in Figure 13;
- an HCV genomic sequence having a homology of more than 57 %, preferably more than S1lBST(Tt1TE SHEET (RULE 26) 59%, most preferably more than 61 % homology to any of the sequences as represented in SEQ ID NO 59 or 61 (PC sequences) in the region spanning positions 4936 to 5296 of the NS4 region as shown in Figure 6 or 10;
- an HCV genomic sequence as having a homology of more than 93 %, preferably more than =
93.5%, most preferably more than 94% homology to any of the sequences as represented in SEQ ID NO 159 or 161 (BE95 or BE96 sequences) in the region spanning positions 7932 to 8271 of the NS5B region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- a nucleotide distance of less than 0.53, preferably less than 0.51, more preferably less than 0.49 for the El region to the type 5 sequences depicted above;
- a nucleotide distance of less than 0.3, preferably less than 0.28, more preferably of less than 0.26 for the Core region to the type 5 sequences depicted above;
- a nucleotide distance of less than 0.072, preferably less than 0.071, more preferably less than 0.070 for the NS5B region to the type 5 sequences as depicted above.
Isolates with similar sequences in the 5'UR to a group of isolates including SA1, SA3, and SA7 described in the 5'UR by Bukh et al. (1992), have been reported and described in the 5'UR and NS5 region as group V by Cha et al. (1992; WO 92/19743). This group of isolates belongs to type 5a as described in the present invention (SEQ ID NO 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 151, 153, 155, 157, 159, 161, 197 and 199).
Also included within the present invention are sequence variants of the polynucleic acids as selected from any of the nucleotide sequences as given in any of the above given SEQ ID
numbers with said sequence variants containing either deletion and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between different genotypes of HCV) by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 5 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type as shown in Figure 3 (Core region), Figure 4(Core/E1 region), Figure 10 (NS3 /
region), Figure 14 (E1/E2 region).
SUBS"T{TUTE SHEET (RULE 26) Another group of isolates including BU74 and BU79 having similar sequences in the 5'UR
to isolates including Z6 and Z7 as described in the 5'UR by Bukh et al.
(1992), have been described in the 5'UR and classified as a new type 4 by the inventors of this application (Stuyver et al., 1993). Coding sequences, including core, El and NS5 sequences of several new Gabonese isolates belonging to this group, are disclosed in the present invention (SEQ
ID NO 106, 108, 110, 112, 114, 116, 118, 120 and 122).
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 4 genomic sequences:
- an HCV genomic sequence having a homology of more than 66%, preferably more than 68%, most preferably more than 70% homology in the El region spanning positions 574 to 957 to any of the sequences as represented in SEQ ID NO 118, 120 or 122 (GB358, GB549, GB809 sequences) as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 71 %, preferably more than 72 %, most preferably more than 74 % homology to any of the sequences as represented in SEQ ID NO 118, 120 or 122 (GB358, GB549, GB809 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence having a homology of more than 92 %, preferably more than 93%, most preferably more than 94% homology to any of the sequences as represented in SEQ ID NO 163 or 165 (GB809, CAM600 sequences) in the region spanning positions 1 to 378 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4c) having a homology of more than 85 %, preferably more than 86 %, more preferably more than 86.5 % homology, most preferably more than 87, more than 88 or more than 89% homology to any of the sequences as represented in SEQ ID NO 183, 185 or 187 (GB116, GB215, GB809 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4a) having a homology of more than 81 %, preferably more than 83 %, most preferably more than 85 % homology to the sequence as represented in SEQ ID NO 189 (GB908 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4e) having a homology of more than 85 %, preferably more than 87 %, most preferably more than 89 % homology to any of the sequences as represented in SEQ ID NO 167 or 169 (CAM600, GB908 sequences) in the region SUBSTITUTE SHEET (RULE 26) spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4f) having a homology of more than 79%, preferably more than 81 %, most preferably more than 83 % homology to any of the sequences as represented in SEQ ID NO 171 or 173 (CAMG22, CAMG27 sequences) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4g) having a homology of more than 84 %, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 175 (GB549 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4h) having a homology of more than 83 %, preferably more than 85 %, most preferably more than 87% homology to the sequence as represented in SEQ ID NO 177 (GB438 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
-' an HCV genomic sequence (subtype 4i) as having a homology of more than 76%, preferably more than 78%, most preferably more than 80% homology to the sequence as represented in SEQ ID NO 179 (CAR4/1205 sequence) in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4j?) having a homology of more than 84 %, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 181 (CAR4/901 sequence) in the region spanning positions 379 to 957 of the El region as shown in figure 4;
- an HCV genomic sequence as having a homology of more than 73 %, preferably more than 75 %, most preferably more than 77 % homology to any of the sequences as represented in SEQ ID NO 106, 108, 110, 112, 114, or 116 (GB48, GB116, GB215, GB358, GB549, GB809 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4c) having a homology of more than 88%, preferably more than 89%, most preferably more than 90% homology to any of the sequences as represented in SEQ ID NO 106, 108, 110, or 112 (GB48, GB 116, GB215, GB358 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4e) having a homology of more than 88 %, preferably more than 89 %, most preferably more than 90 % homology to any of the sequences as SUBSTITUTE SHEET (RULE 26) represented in SEQ ID NO 116 or 201 (GB809 or CAM 600 sequences) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4f) having a homology of more than 87%, preferably = more than 89 %, most preferably more than 90 % homology to the sequence as represented in SEQ ID NO 203 (CAMG22 sequence) in the region spanning positions 7932 to of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4g) as having a homology of more than 85 %, preferably more than 87 %, most preferably more than 89 % homology to the sequence as represented in SEQ ID NO 114 (GB549 sequence) in the region spanning positions to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4h) as having a homology of more than 86%, preferably more than 87%, more preferably more than 88% homology, more preferably more than 89% homology to the sequence as represented in SEQ ID NO 207 (GB437 sequence) in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4i) having a homology of more than 84%, preferably more than 86 %, most preferably more than 88 % homology to the sequence as represented in SEQ ID NO 209 (CAR4/1205 sequence) in the region spanning positions 7932 to of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4j) having a homology of more than 81 %, preferably more than 83 %, most preferably more than 85 % homology to the sequence as represented in SEQ ID NO 211 (CARI/501 sequence) in the region spanning positions 7932 to of the NS5 region as shown in figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of , less than 0.52, 0.50, 0.4880, 0.46, 0.44, 0.43 or most preferably less than 0.42 in the region spanning positions 574 to 957 to any of the sequences as represented in SEQ ID NO
118, 120 or 122 in the region spanning positions 1 to 957 of the Core/E1 region as shown in Figure 4;
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of SUBSTffEtTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 less than 0.39, 0.36 0.34 0.32 or most preferably less than 0.31 to any of the sequences as represented in SEQ ID NO 118, 120 or 122 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4c) being characterized as having a nucleotide distance of less than 0.27, 0.26, 0.24, 0.22, 0.20, 0.18, 0.17, 0.162, 0.16 or most preferably less than 0.15 to any of the sequences as represented in SEQ ID NO 183, 185 or 187 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4a) being characterized as having a nucleotide distance of less than 0.30, 0.28, 0.26, 0.24, 0.22, 0.21 or most preferably of less than 0.205 to the sequence as represented in SEQ ID NO 189 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4e) being characterized as having a nucleotide distance of less than 0.26, 0.25, 0.23, 0.21, 0.19, 0.17, 0.165, most preferably less than 0.16 to any of the sequences as represented in SEQ ID NO 167 or 169 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4f) being characterized as having a nucleotide distance of less than 0.26, 0.24, 0.22, 0.20, 0.18, 0.16, 0.15 or most preferably less than 0.14 to any of the sequences as represented in SEQ ID NO 171 or 173 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4g) being characterized as having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17 or most preferably of less than 0.16 to the sequence as represented in SEQ ID NO 175 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4h) being characterized as having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17 and most preferably of less than 0.16 to the sequence as represented in SEQ ID NO 177 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4i) being characterized as having a nucleotide distance of less than 0.27, 0.25, 0.23, 0.21 and preferably less than 0.16 to the sequence as represented in SEQ ID NO 179 in the region spanning positions 379 to 957 of the El region as shown in Figure 4;
- an HCV genomic sequence (subtype 4j?) being characterized as having a nucleotide distance of less than 0.19, 0.18, 0.17, 0.165 and most preferably of less than 0.16 to the SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 sequence as represented in SEQ ID NO 181 in the region spanning positions 379 to 957 of the El region as shown in figure 4;
- an HCV genomic sequence (type 4) being characterized as having a nucleotide distance of less than 0.35, 0.34, 0.32 and most preferably of less than 0.30 to any of the sequences as represented in SEQ ID NO 106, 108, 110, 112, 114, or 116 in the region spann.ing positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4c) being characterized as having a nucleotide distance of less than 0.18, 0.16, 0.14, 0.135, 0.13, 0.1275 or most preferably less than 0.125 to any of the sequences as represented in SEQ ID NO 106, 108, 110, or 112 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4e) being characterized as having a nucleotide distance of less than 0.15, 0.14, 0.135, 0.13 and most preferably of less than 0.125 to any of the sequences as represented in SEQ ID NO 116 or 201 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4f) being characterized as having a nucleotide distance of less than 0.15, 0.14, 0.135, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 203 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4g) being characterized as having a nucleotide distance of less than 0.17, 0.16, 0.15, 0.14, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 114 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4h) being characterized as having a nucleotide distance of less than 0.155, 0.15, 0.145, 0.14, 0.135, 0.13 or most preferably less than 0.125 to the sequence as represented in SEQ ID NO 207 in the region spanning positions 7932 to 8271 of the NS5 region as shown in Figure 1;
- an HCV genomic sequence (subtype 4i) being characterized as having a nucleotide distance of less than 0.17, 0.16, 0.15, 0.14, 0.13 or most preferably of less than 0.125 to the sequence as represented in SEQ ID NO 209 in the region spanning positions 7932 to 8271 of the NS5 region as shown in figure 1;
- an HCV genomic sequence (subtype 4j) being characterized as having a nucleotide distance of less than 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13 and most preferably of less than 0.125 to the sequence as represented in SEQ ID NO 211 in the region spanning SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCTlEP94101323 positions 7932 to 8271 of the NS5 region as shown in figure 1.
Also included within the present invention are sequence variants of the polynucleic acids as selected from any of the nucleotide sequences as given in any of the above given SEQ ID
numbers with said sequence variants containing either deletion and/or insertions of one or more nucleotides, mainly at the extremities of oligonucleotides (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between different genotypes of HCV) by others (including modified nucleotides an/or inosine), for example, a type 1 or 2 sequence might be modified into a type 4 sequence by replacing some nucleotides of the type 1 or 2 sequence with type-specific nucleotides of type 4 as shown in Figure 3 (Core region), Figure 4 (Core/El region), Figure 10 (NS3 / NS4 region), Figure 14 (E1/E2 region).
The present invention also relates to a sequence as represented in SEQ ID NO
193 (GB724 sequence).
After aligning NS5 or El sequences of GB48, GB, 116, GB215, GB358, GB549 and GB809, these isolates clearly segregated into 3 subtypes within type 4: GB48, GB116, GB215 and GB358 belong to the sybtype designated 4c, GB549 to subtype 4g and GB809 to subtype 4e. In NS5, GB809 (subtype 4e) showed a higher nucleic acids homology to subtype 4c isolates (85.6 - 86. 8%) than to GB549 (subtype 4g, 79.7 %), while GB549 showed similar homologies to both other subtypes (78.8 to 80% to subtype 4c and 79.7% to subtype 4e). In El, subtype 4c showed equal nucleic acid homologies of 75.2% to subtypes 4g and 4e while 4g and 4e were 78.4 % homologous. At the amino acid level however, subtype 4e showed a normal homology to subtype 4c (80.2%), while subtype 4g was more homologous to 4c (83.3 %) and 4e (84.1 %).
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said polynucleic acids correspond to a nucleotide sequence selected from any of the following HCV type 2d genomic sequences:
- an HCV genomic sequence as having a homology of more than 78 %, preferably more than 80%, most preferably more than 82% homology to the sequence as iepresented in SEQ
ID NO (NE92) 143 in the region spanning positions 379 to 957 of the Core/El region as shown in Figure 4;
- an HCV genomic sequence as having a homology of more than 74 %, preferably more than 76%, most preferably more than 78% homology to the sequence as represented in SEQ
ID NO 143 (NE92) in the region spanning positions 574 to 957 as shown in Figure 4;
SUBSTTrUTE SHEET (RULE 26) - an HCV genomic sequence as having a homology of more than 87%, preferably more than 89 %, most preferably more than 91 % homology to the sequence as represented in SEQ
ID NO 145 (NE92) in the region spanning positions 7932 to 8271 of the NS5B
region as shown in Figure 1.
Preferentially the above-mentioned genomic HCV sequences depict sequences from the coding regions of all the above-mentioned sequences.
According to the nucleotide distance classification system (with said nucleotide distances being calculated as explained above), said sequences of said composition are selected from:
- a nucleotide distance of less than 0.32, preferably less than 0.31, more preferably less than 0.30 for the El region (574 to 957) to any of the above specified sequences;
- a nucleotide distance of less than 0.08, preferably less than 0.07, more preferably less than 0.06 for the Core region (1 to 378) to any of the above given sequences - a nucleotide distance of less than 0.15, preferantially less than 0.13, more preferentially less than 0.12 for the NS5B region to any of the above-specified sequences.
Polynucleic acid sequences according to the present invention which are homologous to the sequences as represented by a SEQ ID NO can be characterized and isolated according to any of the techniques known in the art, such as amplification by means of type or subtype specific primers, hybridization with type or subtype specific probes under more or less stringent conditions, serological screening methods (see examples 4 and 11) or via the LiPA typing system.
Polynucleic acid sequences of the genomes indicated above from regions not yet depicted in the present examples, figures and sequence listing can be obtained by any of the techniques known in the art, such as amplification techniques using suitable primers from the type or subtype specific sequences of the present invention.
The present invention relates also to a composition as defined above, wherein said polynucleic acid is liable to act as a primer for amplifying the nucleic acid of a certain isolate belonging to the genotype from which the primer is derived.
An example of a primer according to this embodiment of the invention is HCPr 152 as shown in table 7 (SEQ ID NO 79).
The term "primer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products.
SUBSTITEJTE SHEET (RULE 26) WO 94/25601 PCTlEP94/01323 Preferably the primer is about 5-50 nucleotides. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
The fact that amplification primers do not have to match exactly with corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR;
Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 1989;
Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of QB replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules using primer extension. During amplification, the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplification reaction is repeated between 20 and 80 times, advantageously between 30 and 50 times. -The present invention also relates to a composition as defined above, wherein said polynucleic acid is able to act as a hybridization probe for specific detection andlor classification into types of a nucleic acid containing said nucleotide sequence, with said oligonucleotide being possibly labelled or attached to a solid substrate.
The term "probe" refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence of the HCV
genotype(s) to be detected.
Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about to 25 nucleotides.
The term "solid support" can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead).
Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fiucation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/El'94/01323 genomes present in a sample. The term "universal HCV primer" refers to oligonucleotide sequences complementary to any of the conserved regions of the HCV genome.
The expression "appropriate" hybridization and washing conditions are to be understood as stringent and are generally known in the art (e.g. Maniatis et al., Molecular Cloning: A
Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982).
However, according to the hybridization solution (SSC, SSPE, etc.), these probes should be hybridized at their appropriate temperature in order to attain sufficient specificity.
The term "labelled" refers to the use of labelled nucleic acids. This may include the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art.
The process of the invention comprises the steps of contacting any of the probes as defined above, with one of the following elements:
- either a biological sample in which the nucleic acids are made available for hybridization, - or the purified nucleic acids contained in the biological sample - or a single copy derived from the purified nucleic acids, - or an amplified copy derived from the purified nucleic acids, with said elements or with said probes being attached to a solid substrate.
The expression "inferring the presence of one or more HCV genotypes present from the observed hybridization pattern" refers to the identification of the presence of HCV genomes in the sample by analyzing the pattern of binding of a panel of oligonucleotide probes. Single probes may provide useful information concerning the presence or absence of HCV genomes in a sample. On the other hand, the variation of the HCV genomes is dispersed in nature, so rarely is any one probe able to identify uniquely a specific HCV genome.
Rather, the identity of an HCV genotype may be inferred from the pattern of binding of a panel of oligonucleotide probes, which are specific for (different) segments of the different HCV
genomes. Depending on the choice of these oligonucleotide probes, each known HCV
genotype will correspond to a specific hybridization pattern upon use of a specific combination of probes. Each HCV genotype will also be able to be discriminated from any other HCV genotype amplified with the same primers depending on the choice of the oligonucleotide probes. Comparison of the generated pattern of positively hybridizing probes for a sample containing one or more unkown HCV sequences to a scheme of expected SUBSTITEJTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 hybridization patterns, allows one to clearly infer the HCV genotypes present in said sample.
The present invention thus relates to a method as defined above, wherein one or more hybridization probes are selected from any of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, = 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61, 106, 108, 110, 112, 114, 116, 118, 120, 122, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 198, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 222, 269 or sequence variants thereof, with said sequence variants containing deletions and/or insertions of one or more nucleotides, mainly at their extremities (either 3' or 5'), or substitutions of some non-essential nucleotides (i.e. nucleotides not essential to discriminate between genotypes) by others (including modified nucleotides or inosine), or with said variants consisting of the complement of any of the above-mentioned oligonucleotide probes, or with said variants consisting of ribonucleotides instead of deoxyribonucleotides, all provided that said variant probes can be caused to hybridize with the same specificity as the oligonucleotide probes from which they are derived.
In order to distinguish the amplified HCV genomes from each other, the target polynucleic acids are hybridized to a set of sequence-specific DNA probes targetting HCV
genotypic regions located in the HCV polynucleic acids.
Most of these probes target the most type-specific regions of HCV genotypes, but some can be caused to hybridize to more than one HCV genotype.
According to the hybridization solution (SSC, SSPE, etc.), these probes should be stringently hybridized at their appropriate temperature in order to attain sufficient specificity.
However, by slightly modifying the DNA probes, either by adding or deleting one or a few nucleotides at their extremities (either 3' or 5'), or substituting some non-essential nucleotides (i.e. nucleotides not essential to discriminate between types) by others (including modified nucleotides or inosine) these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e. the same temperature and the same hybridization solution). Also changing the amount (concentration) of probe used may be beneficial to obtain more specific hybridization results. It should be noted in this context, that probes of the same length, regardless of their GC content, will hybridize specifically at approximately the same temperature in TMACI solutions (Jacobs et al., 1988).
Suitable assay methods for purposes of the present invention to detect hybrids formed between the oligonucleotide probes and the nucleic acid sequences in a sample may comprise SUBSTITkJTE SHEET (RULE 26) }
WO 94/25601 = PCT/EP94/01323 any of the assay formats known in the art, such as the conventional dot-blot format, sandwich hybridization or reverse hybridization. For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being bound to a membrane, the membrane being incorporated with at least one labelled probe under suitable ' hybridization and wash conditions, and the presence of bound probe being monitored.
An alternative and preferred method is a "reverse" dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropriate stringent hybridization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.
According to an advantageous embodiment, the process of detecting one or more HCV
genotypes contained in a biological sample comprises the steps of contacting amplified HCV
nucleic acid copies derived from the biological sample, with oligonucleotide probes which have been immobilized as parallel lines on a solid support.
According to this advantageous method, the probes are immobilized in a Line Probe Assay (LiPA) format. This is a reverse hybridization format (Saiki et al., 1989) using membrane strips onto which several oligonucleotide probes (including negative or positive control oligonucleotides) can be conveniently applied as parallel lines.
The invention thus also relates to a solid support, preferably a membrane strip, carrying on its surface, one or more probes as defined above, coupled to the support in the form of parallel lines.
The LiPA is a very rapid and user-friendly hybridization test. Results can be read 4 h.
after the start of the amplification. After amplification during which usuaily a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybridization is carried out for about 1 to 1,5 h hybridized polynucleic acid is detected. From the hybridization pattern generated, the HCV type can be deduced either visually, but preferably using dedicated software. The LiPA format is completely compatible with commercially available scanning devices, thus =
rendering automatic interpretation of the results very reliable. All those advantages make the LiPA format liable for the use of HCV detection in a routine setting. The LiPA
format should be particularly advantageous for detecting the presence of different HCV
genotypes.
The present invention also relates to a method for detecting and identifying novel HCV
SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCTIEP94/01323 genotypes, different from the known HCV genomes, comprising the steps of:
- determining to which HCV genotype the nucleotides present in a biological sample belong, according to the process as defined above, in the case of observing a sample which does not generate a hybridization pattern compatible with those defined in Table 3, sequencing the portion of the HCV
genome sequence corresponding to the aberrantly hybridizing probe of the new HCV genotype to be determined.
The present invention also relates to the use of a composition as defined above, for detecting one or more genotypes of HCV present in a biological sample liable to contain them, comprising the steps of:
(i) possibly extracting sample nucleic acid, (ii) amplifying the nucleic acid with at least one of the primers as defined above, (iii) sequencing the amplified products (iv) inferring the HCV genotypes present from the determined sequences by comparison to all known HCV sequences.
The present invention also relates to a composition consisting of or comprising at least one peptide or polypeptide comprising a contiguous--sequence of at least 5 amino acids corresponding to a contiguous amino acid sequence encoded by at least one of the HCV
genomic sequences as defined above, having at least one amino acid differing from the corresponding region of known HCV (type 1 and/or type 2 and/or type 3) polyprotein sequences as shown in Table 3, or muteins thereof.
It is to be noted that, at the level of the amino acid sequence, an amino acid difference (with respect to known HCV amino acid sequences) is necessary, which means that the polypeptides of the invention correspond to polynucleic acids having a nucleotide difference (with known HCV polynucleic acid sequences) involving an amino acid difference.
The new amino acid sequences, as deduced from the disclosed nucleotide sequences (see SEQ ID NO 1 to 62 and 106 to 123 and 143 to 218, 223 and 270), show homologies of only 59.9 to 78 % with prototype sequences of type 1 and 2 for the NS4 region, and of only 53.9 to 68.8 % with prototype sequences of type 1 and 2 for the El region. As the NS4 region is known to contain several epitopes, for example characterized in patent application EP-A-0 489 968, and as the El protein is expected to be subject to immune attack as part of the viral envelope and expected to contain epitopes, the NS4 and El epitopes of the new type 3, 4 and isolates will consistently differ from the epitopes present in type I and 2 isolates. This is SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 examplified by the type-specificity of NS4 synthetic peptides as presented in example 4, and the type-specificity of recombinant E1 proteins in example 11.
After aligning the new subtype 2d, type 3, 4 and 5 (see SEQ ID NO 1 to 62 and 106 to 123 and 143 to 218, 223 and 270) amino acid sequences with the prototype sequences of type la, lb, 2a, and 2b, type- and subtype-specific variable regions can be delineated as presented in Figure 5 and 7.
As to the muteins derived from the polypeptides of the invention, Table 4 gives an overview of the amino acid substitutions which could be the basis of some of the muteins as defmed above.
The peptides according to the present invention contain preferably at least 5 contiguous HCV amino acids, preferably however at least 8 contiguous amino acids, at least 10 or at least 15 (for instance at least 9, 11, 12, 13, 14, 20 or 25 amino acids) of the new HCV
sequences of the invention.
SUBSTlTM SHEET (RULE 26) ------------------------------------------------------Amino acids Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R) Arg, His, Lys, Glu, Gln Leu (L) Leu; Ile, Met, Phe, Val, Tyr Pro (P) Pro, Ala, Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A) Ala, Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G) Gly, Ala, Thr, Pro, Ser Ile (1) Ile, Met, Leu, Phe, Val, Ile, Tyr Phe (F) Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, I1e, Val, Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln, Arg, Lys, Glu, Thr Gln (Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn (N) Asn, Asp, Ser, Gln Lys (K) Lys, Arg, Glu, Gln, His Asp (D) Asp, Asn, Glu, Gin Glu (E) Glu, Gin, Asp, Lys, Asn, His, Arg Met (M) Met, Iie, Leu, Phe, Val The polypeptides of the invention, and particularly the fragments, can be prepared by classical chemical synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled "Methode der organischen chemie"
(Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).
The polypeptides according to this invention can be prepared by means of recombinant DNA techniques as described by Maniatis et al., Molecular Cloning: A
Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982).
The present invention relates particularly to a polypeptide or peptide composition as defined above, wherein said contiguous sequence contains in its sequence at least one of the following amino acid residues:
St1BSTITM SHEET (RULE 26) L7, Q43, M44, S60, R67, Q70, T71, A79, A87, N106, K115, A127, A190, S130, V134, G142, I144, E152, A157, V158, P165, S177 or Y177, I178, V180 or E180 or F182, R184, I186, H187, T189, A190, S191 or G191, Q192 or L192 or I192 or V192 or E192, N193 or H193 or P193, W194 or Y194, H195, A197 or I197 or V197 or T197, V202, I203 or L203, Q208, A210, V212, F214, T216, R217 or D217 or E217 or V217, H218 or N218, H219 or V219 or L219, L227 or I227, M231 or E231 or Q231, 7232 or D232 or A232 or K232, Q235 or I235, A237 or T237, I242, 1246, S247, S248, V249, S250 or Y250, I251 or V251 or M251 or F251, D252, T254 or V254, L255 or V255, E256 or A256, M258 or F258 or V258, A260 or Q260 or S260, A261, 7264 or Y264, M265, 1266 or A266, A267, G268 or T268, F271 or M271 or V271, I277, M280 or H280, 1284 or A284 or L84, V274, V291, N292 or S292, R293 or I293 or Y293, Q294 or R294, L297 or I297 or Q297, A299 or K299 or Q299, N303 or T303, T308 or L308, T310 or F310 or A310 or D310 or V310, L313, G317 or Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, S532, V534, F536, F537, M539, I546, C1282, A1283, H1310, V1312, Q1321, P1368, V1372, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, D1556, N1567, M1572, Q1579, L1581, S1583, F1585, V1595, E1606 or T1606, M1611, V1612 or L1612, P1630, C1636, P1651, T1656 or I1656, L1663, V1667, V1677, A1681, H1685, E1687, G1689, V1695, A1700, Q1704, Y1705, A1713, A1714 or S1714, M1718, D1719, A1721 or T1721, R1722, A1723 or V1723, H1726 or G1726, E1730, V1732, F1735, I1736, S1737, R1738, T1739, G1740, Q1741, K1742, Q1743, A1744, T1745, L1746, E1747 or K1747, I1749, A1750, T1751 or A1751, V1753, N1755, K1756, A1757, P1758, A1759, H1762, T1763, Y1764, P2645, A2647, K2650, K2653 or L2653, S2664, N2673, F2680, K2681, L2686, H2692, Q2695 or L2695 or I2695, V2712, F2715, V2719 or Q2719, 72722, T2724, S2725, R2726, G2729, Y2735, H2739, I2748, G2746 or I2746, I2748, P2752 or K2752, P2754 or T2754, 72757 or P2757, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 as shown in Table 1(comparison with other isolates). See also the numbering in Figures 2, 5, 7, and 11 (alignment amino acid sequences).
Within the group of unique and new amino acid residues of the present invention, the following residues were found to be specific for the following types of HCV
according to the SUBST(Tk1TE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 HCV classification system used in the present invention:
- Q208, R217, E231, I235, I246, T264, 1266, A267, F271, K299, L2686, Q2719 which are specific for the HCV subtype 2d sequences of the present invention as shown in Fig. 5 and 2;
- Q43, S60, R67, F182, I186, H187, A190, S191, L192, W194, V202, L203, V219, Q231, D232, A237, T254, M280, Q299, T303, L308, and/or L313 which are specific for the Core/El region of HCV type 3 of the invention as shown in Fig.
5;
- D1556, Q1579, L1581, S1584, F1585, E1606, V1612, P1630, C1636, T1656, L1663, H1685, E1687, G1689, V1695, Y1705, A1713, A1714, A1721, V1723, H1726, R1738, Q1743, A1744, E1747,11749, A1751, A1759 and/or H1762 which are specific for the NS3/4 region of HCV type 3 sequences of the invention as shown in Fig. 7;
- K2665, D2666, R2670 which are specific for the NS5B region of HCV type 3 of the invention as shown in Fig. 2;
- L7, A79, A127, S130, E152, V158, S177 or Y177, V180 or E180, R184, T189, Q192 or E192 or I192, N193 or H193, 1197 or V197, 1203, A210, V212, E217, H218, H219, L227, A232, V249, 1251 or M251, D252, L255 or V255, E256, M258 or V258 or F258, A260 or Q260, M265, T268, V271, V274, M280, I284, N292 or S292, Q294, L297 or I297, T308, A310 or D310 or V310 or T310, and G317 which are specific for the core/El region of HCV type 4 sequences of the present invention as shown in Fig. 5;
- P2645, K2650, K2653, G2656, V2658, T2668, N2673 or N2673, K2681, H2686, D2691, L2692, Q2695 or L2695 or I2695, Y2704, V2712, F2715, V2719, I2722, S2725, G2729, Y2735, G2746 or I2746, P2752 or K2752, Q2753, P2754 or T2754, T2757 or P2757 which are specific for the NS5B region of the HCV type 4 sequences of the present invention as shown in Fig. 2;
- M44, Q70, A87, N106, K115, V137, G142, P165,1178, F251, A299, N303, Q317 which are specific for the Core/El region of the HCV type 4 sequences of the present invention as shown in Fig. 5;
- L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F102, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S532, V534, F537, M539, 1546 which are specific for SUBSTITUTE SHEET (RULE 26) the E1/E2 region of the HCV type 5 sequences of the present invention as shown in Fig. 12;
- C1282, A1283, V1312, Q1321, P1368, V1372, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, M1572, V1595, T1606, M1611, L1612, I1656, V1667, A1681, A1700, A1713, S1714, M1718, D1719, T1721, R1722, A1723, G1726, F1735, I1736, S1737, T1739, G1740, K1742, T1745, L1746, K1747, A1750, V1753, N1755, A1757, D1758, T1763, and Y1764 which are specific for the NS3/NS4 region of HCV
type 5 sequences of the invention as shown in Fig. 7;
- A2647, L2653, S2674, F2680, T2724, R2726, Y2730, H2739 which are specific for the NS5B region of the HCV type 5 sequences of the present invention as shown in Fig. 2;
- A256, P1631, V1677, Q1704, E1730, V1732, Q1741 and T1751 which are specific for the HCV type 3 and 5 sequences of the present invention as shown in Fig. 5 and 7;
- T71, A157, I227, T237, T240, Y250, V251, S260, M271, T2673, T2722, I2748 which are specific for the HCV type 3 and 4 sequences of the present invention as shown in Fig. 5 and 2, - V192, Y194, A197, P249, S250, R294 which are specific for the HCV type 4 and sequences of the present invention as shown in Fig. 5;
- 1293 which is specific for the HCV type 4 and subtype 2d sequence of the present invention as shown in Fig. 5;
- D217 and R294 which are specific for the HCV type 3, 4 and 5 sequences of the present invention as shown in Fig. 5;
- L192 which is specific for the HCV type 3 and subtype 2d sequences of the present invention as shown in Fig. 5;
- G191 and T197 which are specific for the HCV type 3, 4 and subtype 2d sequences of the present invention as shown in Fig. 5;
- K232 which is specific for the HCV subtype 2d en type 5 sequences of the present invention as shown in Fig. 5.
and with said notation being composed of a letter, unambiguously representing the amino acid by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 (see also Table 1 for comparison with other isolates), as well as Figure 2 (NS5 SUBSTITkITE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 region), Figure 5 (Core/El region), Figure 7 (NS3/NS4 region), Figure 12 (E1/E2 region).
Some of the above-mentioned amino acids may be contained in type or subtype specific epitopes.
For example M231 (detected in type 5) refers to a methionine at position 231.
A glutamine (Q) is present at the same position 231 in type 3 isolates, whereas this position is occupied by an arginine in type 1 isolates and by a lysine (K) or asparagine (N) in type 2 isolates (see Figure 5).
The peptide or polypeptide according to this embodiment of the invention may be possibly labelled, or attached to a solid substrate, or coupled to a carrier molecule such as biotin, or mixed with a proper adjuvant.
The variable region in the core protein (V-CORE in Fig. 5) has been shown to be useful for serotyping (Machida et at., I992). The sequence of the disclosed type 5 sequence in this region shows type-specific features. The peptide from amino acid 70 to 78 shows the following unique sequence for the sequences of the present inevntion (see figure 5):
QPTGRSWGQ (SEQ ID NO 93) RSEGRTSWAQ (SEQ ID NO 220) and RTEGRTSWAQ (SEQ ID NO 221) Another preferred V-Core spanning region is the peptide spanning positions 60 to 78 of subtype 3c with sequence:
SRRQPIPRARRTEGRSWAQ (SEQ ID NO 268) Five type-specific variable regions (Vi to V5) can be identified after aligning El amino acid sequences of the 4 genotypes, as shown in Figure 5.
Region V1 encompasses amino acids 192 to 203, this is the amino-terminal 10 amino acids of the El protein. The following unique sequences as shown in Fig. 5 can be deduced:
LEWRNTSGLYVL (SEQ ID NO 83) VNYRNASGIYHI (SEQ ID NO 126) QHYRNISGIYHV (SEQ ID NO 127) EHYRNASGIYHI (SEQ ID NO 128) IHYRNASGIYHI (SEQ ID NO 224) VPYRNASGIYHV (SEQ ID NO 84) VNYRNASGIYHI (SEQ ID NO 225) VNYRNASGVYHI (SEQ ID NO 226) VNYHNTSGIYHL (SEQ ID NO 227) SUBSTITUTE SHEET (RULE 26) QHYRNASGIYHV (SEQ ID NO 228) QHYRNVSGIYHV (SEQ ID NO 229) IHYRNASDGYYI (SEQ ID NO 230) LQVKNTSSSYMV (SEQ ID NO 231) Region V2 encompasses amino acids 213 to 223. The following unique sequences can be found in the V2 region as shown in Figure 5: VYEADDVILHT (SEQ ID NO 85) VYETEHHILHL (SEQ ID NO 129) VYEADHHIMHL (SEQ ID NO 130) VYETDHHILHL (SEQ ID NO 131) VYEADNLILHA (SEQ ID NO 86) VWQLRANLHV (SEQ ID NO 232) VYEADYHILHL (SEQ ID NO 233) VYETDNHILHL (SEQ ID NO 234) VYETENHILHL (SEQ ID NO 235) VFETVHHILHL (SEQ ID NO 236) VFETEHHILHL (SEQ ID NO 237) VFETDHHIMHL (SEQ ID NO 238) VYETENHILHL (SEQ ID NO 239) VYEADALILHA (SEQ ID NO 240) Region V3 encompasses the amino acids 230 to 242. The following unique V3 region sequences can be deduced from Figure 5:
VQDGNTSTCWTPV (SEQ ID NO 87) VQDGNTSACWTPV (SEQ ID NO 241) VRVGNQSRCWVAL (SEQ ID NO 132) VRTGNTSRCWVPL (SEQ ID NO 133) VRAGNVSRCWTPV (SEQ ID NO 134) EEKGNISRCWIPV (SEQ ID NO 242) VKTGNQSRCWVAL (SEQ ID NO 243) VRTGNQSRCWVAL (SEQ ID NO 244) VKTGNQSRCWIAL (SEQ ID NO 245) VKTGNVSRCWIPL (SEQ ID NO 247) VKTGNVSRCWISL (SEQ ID NO 248) SUBSTITUTE SHEET (RULE 26) VRKDNVSRCWVQI (SEQ ID NO 249) Region V4 encompasses the amino acids 248 to 257. The following unique V4 region sequences can be deduced from figure 5:
VRYVGATTAS (SEQ ID NO 89) APYIGAPLES (SEQ ID NO 135) APYVGAPLES (SEQ ID NO 136) AVSMDAPLES (SEQ ID NO 137) APSLGAVTAP (SEQ ID NO 90) APSFGAVTAP (SEQ ID NO 250) VSQPGALTKG (SEQ ID NO 251) VKYVGATTAS (SEQ ID NO 252) APYIGAPVES (SEQ ID NO 253) AQHLNAPLES (SEQ ID NO 254) SPYVGAPLEP (SEQ ID NO 255) SPYAGAPLEP (SEQ ID NO 256) APYLGAPLEP (SEQ ID NO 257) APYLGAPLES (SEQ ID NO 258) APYVGAPLES (SEQ ID NO 259) VPYLGAPLTS (SEQ ID NO 260) APHLRAPLSS (SEQ ID NO 261) APYLGAPLTS (SEQ ID NO 262) Region V5 encompasses the amino acids 294 to 303. The following unique V5 region peptides can be deduced from figure 5:
RPRRHQTVQT (SEQ ID NO 91) QPRRHWTTQD (SEQ ID NO 138) RPRRHWTTQD (SEQ ID NO 139) RPRQHATVQN (SEQ ID NO 92) RPRQHATVQD (SEQ ID NO 263) SPQHHKFVQD (SEQ ID NO 264) RPRRLWTTQE (SEQ ID NO 265) PPRIHETTQD (SEQ ID NO 266) The variable region in the E2 region (HVR-2) of type 5a as shown in Figure 12 spanning amino acid positions 471 to 484 is also a preferred peptide according to the present invention SUBSCITUTE SHEET (RULE 26) with the following sequence:
TISYANGSGPSDDK (SEQ ID NO 267) The above given list of peptides are particularly suitable for vaccine and diagnostic development.
Also comprised in the present invention is any synthetic peptide or polypeptide containing at least 5 contiguous amino acids derived from the above-defined peptides in their peptidic chain.
According to a specific embodiment, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV
amino acid type 3 sequences:
- a sequence having a homology of more than 72 %, preferably more than 74 %, more preferably more than 77 % and most preferably more than 80 or 84 % homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the region spanning positions 140 to 319 in the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 70%, preferably more than 72%, more preferably more than 75 % homology, most preferably more than 81 % homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the El region spanning positions 192 to 319 as shown in Figure 5;
- a sequence having a homology of more than 86%, preferably more than 88%, and most preferably more than 90% homology to the amino acid sequences as represented in SEQ
ID NO 148 (type 3c); BE98 in the region spanning positions 1 to 110 in the Core region as shown in Figure 5;
- a sequence having a homology of more than 76%, preferably more than 78%, most preferably more than 80% to any of the amino acid sequences as represented in SEQ ID
NO 30, 32, 34, 36, 38 or 40 (HCC153, HD 10, BR36 sequences) in the region spanning positions 1646 to 1764 in the NS3/NS4 region as shown in Figure 7 and 11;
- a sequence having a homology of more than 81 %, preferably more than 83 %, and most preferably more than 86% homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the region spanning positions 140 to 319 in the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 81.5 %, preferably more than 83 %, and most SIfBST{TUTE SHEET (RULE 26) preferably more than 86% homology to any of the amino acid sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD 10, BR36, BR33 sequences) in the El region spanning positions 192 to 319 as shown in Figure 5;
- a sequence having a homology of more than 86%, preferably more than 88%, most preferably more than 90% to the amino acid sequence as represented in SEQ ID
NO 150;
(type 3c BE98) in the region spanning positions 2645 to 2757 in the NS5B
region as shown in Figure 2.
According to yet another embodiment, the present invention relates to a composition as defmed above, wherein said contiguous sequence is selected from any of the following HCV
amino acid type 4 sequences:
- a sequence having a homology of more than 80%, preferably more than 82%, most preferably more than 84 % homology to any of the amino acid sequences as represented in SEQ ID NO 118, 120, and 122 (GB358, GB549, GB809 sequences) in the region spanning positions 127 to 319 of the Core/El region as shown in Figure 5;
- a sequence having a homology of more than 73%, preferably more than 75%, most preferably more than 78 % homology in the El region spanning positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 118, 120, and 122 (GB358, GB549, GB809 sequences) in the region spanning positions 140 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 85 %, preferably more than 86%, most preferably more than 87 % homology to any of the amino acid sequences as represented in SEQ ID NO
118, 120 or 122 (GB358, GB549, GB809 sequences) in the region spanning positions 192 to 319 of El as shown in Figure 5;
- a sequence showing more than 73 %, preferably more than 74 %, most preferably more than 75 % homology to any of the amino acid sequences as represented in SEQ ID NO
106, 108, 110, 112, 114 or 116 (GB48, GB116, GB215, GB358, GB549, GB809 sequences) in the region spanning positions 2645 to 2757 of the NS5B region as shown in Figure 2;
- a sequence having any of the sequences as represented in SEQ ID NO 164 or 166 (GB809 and CAM600 sequences) in the Core/El region as shown in Figure 5;
- a sequence having any of the sequences as represented in SEQ ID NO 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 or 190 (CAM600, GB809, CAMG22, CAMG27, GB549, GB438, CAR4/1205, CAR4/901, GB116, GB215, GB958, GB809-4 sequences) in the El region as shown in Figure 5;
SUBSTiTUTE SHEET (RULE 26) - a sequence having any of the sequences as represented in SEQ ID NO 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212 (GB358, GB724, BE100, PC, CAM600, CAMG22, etc.) in the NS5B region.
The above-mentioned type 4 peptides polypeptides comprise at least an amino acid sequence selected from any HCV type 4 polyprotein with the exception of core sequence as disclosed by Simmonds et al. (1993, EG-29, see Figure 5).
According to yet another aspect, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV amino acid type 5 sequences:
- a sequence having more than 93 %, preferably more than 94 %, most preferably more than 95 % homology in the region spanning Core positions 1 to 191 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC
sequences) and SEQ ID NO 152 (BE95) as shown in Figure 5;
- a sequence having more than 73 %, preferably more than 74 %, most preferably more than 76% homology in the region spanning El positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC sequences) as shown in Figure 5 - a sequence having a more than 78%, preferably more than 80%, most preferably more than 83 % homology to any of the amino acid sequences as represented in SEQ ID
NO 42, 44, 46, 48, 50, 52, 54, 154, 156 (BE95, BE100) (PC sequences) in the region spanning positions 1 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 90%, preferably more than 91 %, most preferably more than 92 % homology to any of the amino acid sequences represented in SEQ ID NO 56 to 58 (PC sequences) in the region spanning positions 1286 to 1403 of the NS3 region as shown in Figure 7 or 11;
- a sequence having more than 66 %, more particularly 68 %, most particularly 70 % or more homology to any of the amino acid sequences as represented in SEQ ID NO 60 or 62 (PC
sequences) in the region spanning positions 1646 to 1764 of the NS3/4 region as shown in Figure 7 or 11.
According to yet another embodiment, the present invention relates to a composition as defined above, wherein said contiguous sequence is selected from any of the following HCV amino acid type 2d sequences:
- a sequence having more than 83 %, preferably more than 85 %, most preferably more than SUBSTIT(JTE SHEET (RULE 26) 87 % homology to the amino acid sequence as represented in SEQ ID NO 144 (NE92) in the region spanning positions 1 to 319 of the Core/El region as shown in Figure 5;
- a sequence having more than 79%, preferably more than 81 %, most preferably more than . 84% homology in the region spanning E1 positions 192 to 319 to the amino acid sequence as represented in SEQ ID NO 144 (NE92) as shown in Figure 12;
- a sequence having more than 95 %, more particularly 96 %, most particularly 97 % or more homology to the amino acid sequence as represented in SEQ ID NO 146 (NE92) in the region spanning positions 2645 to 2757 of the NS5B region as shown in Figure 2.
The present invention also relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral promoter sequence followed by the nucleotide sequences as defined above, with said recombinant vector allowing the expression of any one of the HCV
type 2 and/or HCV type 3 and/or type 4 and/or type 5 derived polypeptides as defined above in a prokaryotic, or eukaryotic host or in living mammals when injected as naked DNA, and more particularly a recombinant vector allowing the expression of any of the following HCV
type 2d, type 3, type 4 or type 5 polypeptides spanning the following amino acid positions:
- a polypeptide starting at position 1 and ending at any position in the region between positions 70 and 326, more particularly a polypeptide spanning positions 1 to 70, 1 to 85, positions 1 to 120, positions 1 to 150, positions 1 to 191, positions 1 to 200, for expression of the Core protein, and a polypeptide spanning positions 1 to 263, positions 1 to 326, for expression of the Core and El protein;
- a polypeptide starting at any position in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, for expression of El, or forms that have the putative membrane anchor deleted (positions 264 to 293 plus or minus 8 amino acids);
- a polypeptide starting at any position in the region between positions 1556 and 1688, and ending at any position in the region between positions 1739 and 1764, for expression of the NS4 regions, more particularly a polypeptide starting at position 1658 and ending at position 1711 for expression of the NS4a antigen, and more particularly, a polypeptide starting at position 1712 and ending between positions 1743 and 1972, for example 1712-1743, 1712-1764, 1712-1782, 1712-1972, 1712 to 1782 and 1902 to 1972 for expression of the NS4b protein or parts thereof.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 The term "vector" may comprise a plasmid, a cosmid, a phage, or a virus.
In order to carry out the expression of the polypeptides of the invention in bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out:
- transformation of an appropriate cellular host with a recombinant vector, in which a nucleotide sequence coding for one of the polypeptides of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence. In the case of an eukaryotic host any artificial signal sequence or pre/pro sequence might be provided, or the natural HCV signal sequence might be employed, e.g. for expression of El the signal sequence starting between amino acid positions 117 and 170 and ending at amino acid position 191 can be used, for expression of NS4, the signal sequence starting between amino acid positions 1646 and 1659 can be used, - culture of said transformed cellular host-under conditions enabling the expression of said insert.
The present invention also relates to a composition as defined above, wherein said polypeptide is a recombinant polypeptide expressed by means of an expression vector as defined above.
The present invention also relates to a composition as defined above, for use in a method for immunizing a mammal, preferably humans, against HCV comprising administring a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response, more particularly a vaccine composition including HCV type 3 polypeptides derived from the Core, El or the NS4 region and/or HCV
type 4 and/or HCV type 5 polypeptides and/or HCV type 2d polypeptides.
The present invention also relates to an antibody raised upon immunization with a composition as defined above by means of a process as defined above, with said antibody being reactive with any of the polypeptides as defined above, and with said antibody being preferably a monoclonal antibody.
The monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from SUBSTITUTE SHEET (RULE 26) a mouse or rat, immunized against the HCV polypeptides according to the invention, or muteins thereof, or fragments thereof as defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.
The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA
technology, departing from parts of mouse andlor human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
Alternatively the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies. These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with type 3, type 4 or type 5 HCV, or vaccinated against HCV. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al. 1992).
The invention also relates to the use of the proteins of the invention, muteins thereof, or peptides derived therefrom for the selection of recombinant antibodies by the process of repertoire cloning (Persson et al., 1991).
Antibodies directed to peptides derived from a certaing genotype may be used either for the detection of such HCV genotypes, or as therapeutic agents.
The present invention also relates to the use of a composition as defined above for incorporation into an immunoassay for detecting HCV, present in biological sample liable to contain it, comprising at least the following steps:
(i) contacting the biological sample to be analyzed for the presence of HCV
antibodies with any of the compositions as defined above preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said polypeptide can be a biotinylated polypeptide which is covalently bound to a solid substrate by means of streptavidin or avidin complexes, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, which SUBSTITUTE SHEET (RULE 26) specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immunecomplexes visually or by means of densitometry and inferring the HCV serotype present from the observed hybridization pattern.
The present invention also relates to the use of a composition as defined above, for incorporation into a serotyping assay for detecting one or more serological types of HCV
present in a biological sample liable to contain it, more particularly for detecting El and NS4 antigens or antibodies of the different types to be detected combined in one assay format, comprising at least the following steps:
(i) contacting the biological sample to be analyzed for the presence of HCV
antibodies or antigens of one or more serological types, with at least one of the compositions as defined above, an immobilized form under appropriate conditions which allow the formation of an immunecomplex, (ii) removing unbound components, (iii) incubating the immunecomplexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immunecomplexes visually or by means of densitometry and inferring the presence of one or more HCV serological types present from the observed binding pattern.
The present invention also relates to the use of a composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above.
The present invention thus also relates to a kit for determining the presence of HCV
genotypes as defined above present in a biological sample liable to contain them, comprising:
- possibly at least one primer composition containing any primer selected from those defined above or any other HCV type 3 and/or HCV type 4, and/or HCV type 5, or universal HCV primers, SUBSTITETTE SHEET (RULE 26) - at least one probe composition as defined above, with said probes being preferentially immobilized on a solid substrate, and more preferentially on one and the same membrane strip, a buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the possibly amplified products to be carried out, means for detecting the hybrids resulting from the preceding hybriziation, - possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed hybridization pattern.
The genotype may also be detected by means of a type-specific antibody as defined above, which is linked to any polynucleotide sequence that can afterwards be amplified by PCR to detect the immune complex formed (Immuno-PCR, Sano et al., 1992);
The present invention also relates to a kit for determining the presence of HCV antibodies as defined above present in a biological sample liable to contain them, comprising:
- at least one polypeptide composition as defined above, preferentially in combination with other polypeptides or peptides from HCV type 1, HCV type 2 or other types of HCV, with said polypeptides being preferentially immobilized on a solid substrate, and more preferentially on one and the same membrane strip, - a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides and the antibodies against HCV present in the biological sample, means for detecting the immunecomplexes formed in the preceding binding reaction, - possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed binding pattern.
SUBSTITUTE SHEET (RULE 26) Figure Legends Figure 1 Alignment of consensus nucleotide sequences for each of the type 3a isolates BR34, BR36, and BR33, deduced from the clones with SEQ ID NO 1, 5, 9; type 4 isolates GB48, GB116, GB215, GB358, GB549, GB809, CAM600, CAMG22, GB438, CAR411205, CAR1/501 (SEQ ID
NO. 106, 108, 110, 112, 114, 116, 201, 203, 205, 207, 209 and 211); type 5a isolates BE95 and BE96 (SEQ ID NO 159 and 161) and type 2d isolate NE92 (SEQ
ID NO
145) from the region between nucleotides 7932 and 8271, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, HC-J8, TI and T9, and others as shown in Table 3.
Figure 2 Alignment of amino acids sequences deduced from the nucleic acid sequences as represented in Figure 1 from the subtype 3a clones BR34 (SEQ ID NO 2, 4), BR36 (SEQ ID
NO 6, 8) and BR33 (SEQ ID NO 10, 12), the subtype 3c clone BE98 (SEQ ID NO
150), and the type 4 clones GB48 (SEQ ID NO 107), GB1 16 (SEQ ID NO 109), GB215 (SEQ ID
NO
111), GB358 (SEQ ID NO 113), GB549 (SEQ ID NO 115) GB809 (SEQ ID NO 117);
CAM600, CAMG22, GB438, CAR4/1205, CAR1/501 (SEQ ID NO 202, 204, 206, 208, 210, 212); the type 5a clones BE95 and BE96 (SEQ ID NO 160 and 162); as well as the subtype 2d isolate NE92 (SEQ ID NO 146) from the region between amino acids 2645 to 2757 with known sequences from the corresponding region of isolates HCV-I, HCV-J, HC-J6, and HC-J8, T1 and T9, and other sequences as shown in Table 3.
Figure 3 Aligment of type 2d, 3c, 4 and 5a nucleotide sequences from isolates NE92, BE98, GB358, GB809, CAM600, GB724, BE95 (SEQ ID NO 143, 147, 191, 163, 165, 193 and 151) in the Core region between nucleotide positions 1 and 500, with known sequences from the corresponding region of type 1, type 2, type 3 and type 4 sequences.
Figure 4 Alignment of nucleotide sequences for the subtype 2d isolate NE92 (SEQ ID NO
143), the type 4 isolates GB358 (SEQ ID NO 118 and 187), GB549 (SEQ ID NO 120 and 175), and SUBSTiTUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 GB809-2 (SEQ ID NO 122 and 169), GB 809-4, BG116, GB215,- CAM600, CAMG22, CAMG27, GB438, CAR4/1205, CAR4/901(SEQ ID NO 189, 183, 185, 167, 171, 173, 177, 179, 181), sequences for each of the subtype 3a isolates HD10, BR36, and BR33, (SEQ ID
NO 13, 15, 17 (HD 10), 19, 21 (BR36) and 23, 25 or 27 (BR23) and the subtype 5a isolates BE95 and BE100 (SEQ ID NO 143 and 195) from the region between nucleotides 379 and 957, with known sequences from the corresponding region of type 1 and 2 and 3.
Figure 5 Alignment of amino acid sequences deduced from the new HCV nucleotide sequences of the Core/El region of isolates BR33, BR36, HD10, GB358, GB549, and GB809, PC
or BE95, CAM600, and GB724 (SEQ ID NO. 14, 20, 24, 119 or 192, 121, 123 or 164, 54 or 152, 166 and 194) from the region between positions 1 and 319, with known sequences from type la (HCV-1), type lb (HCV-J), type 2a (HC-JG), type 2b (HC-J8), NZL1, HCV-TR, positions 7-89 of type 3a (E-bl), and positions 8-88 of type 4a (EG-29). V-Core, variable region with type-specific features in the core protein, V1, variable region 1 of the El protein, V2, variable region 2 of the E1 protein, V3, variable region 3 of the El protein, V4, variable region 4 of the El protein, V5, variable region 5 of the El protein.
Fignre 6 Alignment of nucleotide sequences of isolates HCCL53, HD10 and BR36, deduced from clones with SEQ ID NO 29, 31, 33, 35, 37 and 39, from the NS314 region between nucleotides 4664 to 5292, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8, EB1, EB2, EB6 and EB7.
Fi re 7 Alignment of amino acid sequences deduced from the new HCV nucleotide sequences of the NS3/NS4 region of isolate BR36 (SEQ ID NO 36, 38 and 40) and BE95 (SEQ ID
NO
270). NS4-1, indicates the region that was synthesized as synthetic peptide I
of the NS4 region, NS4-5, indicates the region that was synthesized as synthetic peptide 5 of the NS4 region; NS4-7, indicates the region that was synthesized as synthetic peptide 7 of the NS4 region.
Fiugre8 SUBSTITUTE SHEET (RULE 26) Reactivity of the three LIPA-selected (Stuyver et al., 1993) type 3 sera on the Inno-LIA
HCV Ab II assay (Innogenetics) (left), and on the NS4-LIA test. For the NS4-LIA test, NS4-1, NS4-5, and NS4-7 peptides were synthesized based on the type 1 (HCV-1), type 2(HC-J6) and type 3 (BR36) prototype isolate sequences as shown in Table 4, and applied as parallel lines onto a membrane strip as indicated. 1, serum BR33, 2, serum HD10, 3, serum DKH.
Fiizure 9 Nucleotide sequences of Core/El clones obtained from the PCR fragments PC-2, PC-3, and PC-4, obtained from serum BE95 (PC-2-l (SEQ ID NO 41), PC-2-6 (SEQ ID NO
43), PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47), PC-3-4 (SEQ ID NO 49), and PC-3-(SEQ ID NO 51)) of subtype 5a isolate BE95.
A consensus sequence is shown for the Core and El region of isolate BE95, presented as PC C/E1 with SEQ ID NO 53. Y, C or T, R, A or G, S, C or G.
Figure 10 Alignment of nucleotide sequences of clones with SEQ ID NO 197 and 199 (PC
sequences, see also SEQ ID NO 55, 57, 59) and SEQ ID NO 35, 37 and 39 (BR36 sequences) from the NS3/4 region between nucleotides 3856 to 5292, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8.
Figure 11 Alignment of amino acid sequences of subtype 5a BE95 isolate PC clones with SEQ ID
NO 56 and 58, from the NS3/4 region between amino acids 1286 to 1764, with known sequences from the corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8.
Figure 12 Aligment of amino acid sequences of subtype 5a isolate BE95 (SEQ ID NO 158) in the El/E2 region spanning positions 328 to 546, with known sequnces from the corresponding region of isolates HCV-1, HCV-J, HC-J6, HC-J8, NZLI and HCV-TR (see Table 3).
Figure 13 Alignment of the nucleotide sequences of subtype 5a isolate BE95 (SEQ ID NO
157) in the El/E2 region with known HCV sequences as shown in Table 3.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 EXAMPLES
Example 1: The NS5b region of HCV type 3 Type 3 sera, selected by means of the INNO-LiPA HCV research kit (Stuyver et al., 1993) from a number of Brazilian blood donors, were positive in the HCV antibody ELISA
(Innotest HCV Ab II; Innogenetics) and/or in the INNO-LIA HCV Ab 11 confirmation test (Innogenetics). Only those sera that were positive after the first round of PCR reactions (Stuyver et al., 1993) were retained for further study.
Reverse transcription and nested PCR: RNA was extracted from 50 l serum and subjected to eDNA synthesis as described (Stuyver et al., 1993). This cDNA was used as template for PCR, for which the total volume was increased to 50 l containing 10 pmoles of each primer, 3 41 of lOx Pfu buffer 2 (Stratagene) and 2.5 U of Pfu DNA polymerase (Stratagene). The cDNA was amplified over 45 cycles consisting of 1 min 94 C, 1 min 50 C and 2 min 72 C.
The amplified products were separated by electrophoresis, isolated, cloned and sequenced as described (Stuyver et al., 1993).
Type 3a and 3b-specific primers in the NS5 region were selected from the published sequences (Mori et al., 1992) as follows:
for type 3a:
HCPr161(+): 5'-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3' (SEQ ID NO 63) and HCPr162(-): 5'-GGGCTGCTCTATCCTCATCGACGCCATC-3' (SEQ ID NO 64);
for type 3b:
HCPr163(+): 5'-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3' (SEQ ID O 65) and HCPr164(-): 5'-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3' (SEQ ID NO 66) Using the Line Probe Assay (LiPA) (Stuyver et al., 1993), seven high-titer type 3 sera were selected and subsequently analyzed with the primer sets HCPr161/162 for type 3a, and HCPr163/164 for type 3b. None of these sera was positive with the type 3b primers. NS5 PCR fragments obtained using the type 3a primers from serum BR36 (BR36-23), serum BR33 (BR33-2) and serum BR34 (BR34-4) were selected for cloning. The following sequences were obtained from the PCR fragments :
From fragment BR34-4:
BR34-4-20 (SEQ ID NO 1), BR34-4-19 (SEQ ID NO 3) From fragment BR36-23:
BR36-23-18 (SEQ ID NO 5), BR36-23-20 (SEQ ID NO 7) SUBSTITUTE SHEET (RULE 26) From fragment BR33-2:
BR33-2-17 (SEQ ID NO 9), BR33-2-21 (SEQ ID NO 11) An alignment of sequences with SEQ ID NO 1, 5 and 9 with known sequences is given in Figure 1. An alignment of the deduced amino acid sequences is shown in Figure 2. The 3 isolates are very closely related to eacb other (mutual homologies of about 95 %) and to the published sequences of type 3a (Mori et al., 1992), but are only distantly related to type 1 and type 2 sequences (Table 5). Therefore, it is clearly demonstrated that NS5 sequences from LiPA-selected type 3 sera are indeed derived from a type 3 genome.
Moreover, by analyzing the NS5 region of serum BR34, for which no 5'UR sequences were determined as described in Stuyver et al. (1993), the excellent correlation between typing by means of the LiPA and genotyping as deduced from nucleotide sequencing was further proven.
Example 2: The Core/El reEion of HCV type 3 After aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992), PCR primers were chosen in those regions of little sequence variation. Primers HCPr23(+): 5'-CTCATGGGGTACATTCCGCT-3' (SEQ ID NO 67) and HCPr54(-): 5'-TATTACCAGTTCATCATCATATCCCA-3' (SEQ ID NO 68), were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). This set of primers was selected to amplify the sequence from nucleotide 397 to 957 encoding amino acids 140 to 319 (Kato et al., 1990):
52 amino acids from the carboxyterminus of core and 128 amino acids of El (Kato et al., 1990). The amplification products BR36-9, BRR33-1, and HD10-2 were cloned as described (Stuyver et al., 1993). The following clones were obtained from the PCR
fragments:
From fragment HD10-2:
HD10-2-5 (SEQ ID NO 13), HD10-2-14 (SEQ ID NO 15), HD10-2-21 (SEQ ID NO 17) From fragment BR36-9:
BR36-9-13 (SEQ ID NO 19), BR36-9-20 (SEQ ID NO 21), From fragment BR33-1:
BR33-1-10 (SEQ ID NO 23), BR33-1-19 (SEQ ID NO 25), BR33-1-20 (SEQ ID NO 27), An alignment of the type 3 El nucleotide sequences (HD10, BR36, BR33) with SEQ
ID
NO 13, 19 and 23 with known El sequences is presented in Figure 4. Four variations were detected in the El clones from serum HD10 and BR36, while only 2 were found in BR33.
All are silent third letter variations, with the exception of mutations at position 40 (L to P) SUBSTiTk1TE SHEET (RULE 26) and 125 (M to I). The homologies of the type 3 El region (without core) with type I and 2 prototype sequences are depicted in Table 5.
In total, 8 clones covering the core/El region of 3 different isolates were sequenced and the El portion was compared with the known genotypes (Table 3) as shown in Figure 5.
After computer analysis of the deduced amino acid sequence, a signal-anchor sequence at the core carboxyterminus was detected which might, through analogy with type lb (Hijikata et al., 1991), promote cleavage before the LEWRN sequence (position 192, Fig. 5).
The L-to-P
mutation in one of the HD10-2 clones resides in this signal-anchor region and potentially impairs recognition by signal peptidase (computer prediction). Since no examples of such substitutions were found at this position in previously described sequences, this mutation might have resulted from reverse transcriptase or Pfu polymerase misincorporation. The 4 amino-terminal potential N-linked glycosylation sites, which are also present in HCV types la and 2, remain conserved in type 3. The N-glycosylation site in type lb (aa 250, Kato et al., 1990) remains a unique feature of this subtype. All El cysteines, and the putative transmembrane region (aa 264 to 293, computer prediction) containing the aspartic acid at position 279, are conserved in all three HCV types. The following hypervariable regions can be delineated: V 1 from aa 192 to 203 (numbering according to Kato et al., 1990), V2 (213-223), V3 (230-242), V4 (248-257), and V5 (294-303). Such hydrophilic regions are thought to be exposed to the host defense mechanisms. This variability might therefore have been induced by the host's immune response. Additional putative N-linked glycosylation sites in the V4 region in all type lb isolates known today and in the V5 region of HC-J8 (type 2b) possibly further contribute to modulation of the immune response. Therefore, analysis of this region, in the present invention, for type 3 and 4 sequences has been instrumental in the delineation of epitopes that reside in the V-regions of El, which will be critical for future vaccine and diagnostics development.
Example 3: The NS3/NS4 region of HCV Tyne 3 For the NS3/NS4 border region, the folllowing sets of primers were selected in the regions of little sequence variability after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) (smaller case lettering is used for nucleotides added for cloning purposes):
set A:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) SUBSTITLTTE SHEET (RULE 26) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) setB:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr 118(-): 5' -actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set C:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set D:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set E:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set F:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set G: -HCPr131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set H:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set I:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set J:
HCPr 131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) set K:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) SUBSTITUTE SHEET (RULE 26) set L:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set M:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr4(-): 5'-GACATGCATGTCATGATGTA-3 (SEQ ID NO 78) set N:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr11 8(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set 0:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTI'CCACAT-3' (SEQ ID NO 70) No PCR products could be obtained with the sets of primers A, B, C, D, E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained from type 3 sera. With the primer set 0, no fragment could be amplified from type 3 sera. However, a smear containing a few weakly stainable bands was obtained from serum BR36. After sequence analysis of several DNA fragments, purified and cloned from the area around 300 bp on the agarose gel, only one clone, HCC153 (SEQ ID NO 29), was shown to contain HCV information. This sequence was used to design primer HCPr152.
A new primer set P was subsequently tested on several sera.
set P:
HCPr152(+): 5'-TACGCCTCTTCTATATCGGTTGGGGCCTG-3' (SEQ ID NO 79) and HCPr66(-): 5'-CTATTATTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) The 464-bp HCPr152/66 fragment was obtained from serum BR36 (BR36-20) and serum HD10 (HD10-1). The following clones were obtained from these PCR products:
From fragment HD10-1:
HD10-1-25 (SEQ ID NO 31), HD10-1-3 (SEQ ID NO 33), From fragment BR36-20:
BR36-20-164 (SEQ ID NO 35), BR36-20-165 (SEQ ID NO 37), BR36-20-166 (SEQ ID
NO 39), The nucleotide sequences obtained from clones with SEQ ID NO 29, 31, 33, 35, 37 or 39 are shown aligned with-the sequences of prototype isolates of other types-of HCV in Figure 6. In addition to one silent 3rd letter variation, one 2nd letter mutation resulted in an SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 E to G substitution at position 175 of the deduced amino acid sequence of BR36 (Fig. 7).
Serum HD 10 clones were completely identical. The two type 3 isolates were -nearly 94%
homologous in this NS4 region. The homologies with other types are presented in Table 5.
Example 4: Analysis of the anti-NS4 response to tXpe-specific peptides As the NS4 sequence contains the information for an important epitope cluster, and since antibodies towards this region seem to exhibit little cross-reactivity (Chan et al., 1991), it was worthwhile to investigate the type-specific antibody response to this region.
For each of the 3 genotypes, HCV-1 (Choo et al., 1991), HC-J6 (Okamoto et al., 1991) and BR36 (present invention), three 20-mer peptides were synthesized covering the epitope region between amino acids 1688 and 1743 (as depicted in table 6). The synthetic peptides were applied as parallel lines onto membrane strips. Detection of anti-NS4 antibodies and color development was performed according to the procedure described for the INNO-LIA HCV Ab Ii kit (Innogenetics, Antwerp). Peptide synthesis was carried out on a 9050 PepSynthesizer (Millipore). After incubation with 15 LiPA-selected type 3 sera, 9 samples showed reactivity towards NS4 peptides of at least 2 different types, but a clearly positive reaction was observed for 3 sera (serum BR33, HD30 and DKH) on the type 3 peptides, while negative (serum BR33 and HD30) or indeterminate (serum DKH) on the type 1 and type 2 peptides; 3 sera tested negative for anti-NS4 antibodies (Figure 8). Using the same membrane strips coated with the 9 peptides as indicated above and as shown in Figure 8, 38 type 1 sera (10 type la and 28 type 1b), 11 type 2 sera (10 type 2a and 1 type 2b), 12 type 3a sera and 2 type 4 sera (as determined by the LiPA procedure) were also tested. As shown in Table 8, the sera reacted in a genotype-specific manner with the NS4 epitopes. These results demonstrate that type-specific anti-NS4 antibodies can be detected in the sera of some patients. Such genotype-specific synthetic peptides might be employed to develop serotyping assays, for example a mixture of the nine peptides as indicated above, or combined with the NS4 peptides from the HCV type 4 or 6 genotype or from new genotypes corresponding to the region between amino acids 1688 and 1743, or synthetic peptides of the NS4 region between amino acids 1688 and 1743 of at least one of the 6 genotypes, combined with the El protein or deletion mutants thereof, or synthetic El peptides of at least one of the genotypes.
Such compositions could be further extended with type-specific peptides or proteins, including for example the region between amino acids 68 and 91 of the core protein, or more preferably the region between amino acids 68 and 78. Furthermore, such type-specific SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 antigens may be advantageously used to improve current diagnostic screening and confirmation assays and/or HCV vaccines.
Example 5 The Core and El regions of HCV type 5 Sample BE95 was selected from a group of sera that reacted positive in a prototype Line Probe Assay as described earlier (Stuyver et al., 1993), because a high-titer of HCV RNA
could be detected, enabling cloning of fragments by a single round of PCR. As no sequences from any coding region of type 5 has been disclosed yet, synthetic oligonucleotides for PCR
amplification were chosen in the regions of little sequence variation after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), HC-J8 (Okamoto et al., 1992), and the new type 3 sequences of the present invention HD10, BR33, and BR36 (see Figure 5, Example 2). The following sets of primers were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems):
Set 1:
HCPr52(+): 5'-atgTTGGGTAAGGTCATCGATACCCT-3' (SEQ ID NO 80) and HCPr54(-): 5'-ctattaCCAG77CATCATCATATCCCA-3' (SEQ ID NO 78) Set 2:
HCPr4i(+): 5'-CCCGGGAGGTCTCGTAGACCGTGCA-3' (SEQ ID NO 81) and HCPr40(-): 5'-ctattaAAGATAGAGAAAGAGCAACCGGG-3'(SEQ ID NO 82) Set 3:
HCPr41(+): 5'-CCCGGGAGGTCTCGTAGACCGTGCA-3' (SEQ ID NO 81) and HCPr54(-): 5'-ccattaCCAGTTCATCATCATATCCCA-3' (SEQ ID NO 78) The three sets of primers were employed to amplify the regions of the type 5 isolate PC
as described (Stuyver et al., 1993). Set I was used to amplify the El region and yielded fragment PC-4, set 2 was designed to yield the Core region and yielded fragment PC-2. Set 3 was used to amplify the Core and El region and yielded fragment PC-3. These fragments were cloned as described (Stuyver et al., 1993). The following clones were obtained from the PCR fragments:
From fragment PC-2:
PC-2-1 (SEQ ID NO 41), PC-2-6 (SEQ ID NO 43), - From fragment PC-4:
PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47), SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94101323 From fragment PC-3:
PC-3-4 (SEQ ID NO 49), PC-3-8 (SEQ ID NO 51) An alignment of sequences with SEQ ID NO 41, 43, 45, 47, 49 and 51, is given in Figure 9. A consensus amino acid sequence (PC C/El; SEQ ID NO 54) can be deduced from each of the 2 clones cloned from each of the three PCR fragments as depicted in Figure 5, which overlaps the region between nucleotides 1 and 957 (Kato et al., 1990). The 6 clones are very closely related to each other (mutual homologies of about 99.7%).
An alignment of nucleotide sequence with SEQ ID NO 53 or 151 (PC C/El from isolate BE95) with known nucleotide sequences from the Core/El region is given in Figure 3. The clone is only distantly related to type 1, type 2, type 3 and type 4 sequences (Table 5).
Example 6: NS3/NS4 region of HCV type 5 Attempts were undertaken to clone the NS3/NS4 region of the isolate BE95, described in example 5. The folllowing sets of primers were selected in the regions of little sequence variability after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J
(Kato et al., 1991), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) and of the sequences obtained from type 3 sera of the present invention (SEQ ID NO 31, 33, 35, 37 and 39); smaller case lettering is used for nucleotides added for cloning purposes:
set A:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 66) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set B:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set C:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set D:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr 118 (-): 5' -actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (S EQ ID NO 71) set E:
HCPr116(+): 5'-ttttAAATACATCATGRCITGYATG-3' (SEQ ID NO 69) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) SUBSTITUTE SHEET (RULE 26) set F:
HCPr117(+): 5'-ttttAAATACATCGCIRCITGCATGCA-3' (SEQ ID NO 72) HCPr119(-): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3' (SEQ ID NO 73) set G:
HCPr131(+): 5'-ggaattcta.gaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID NO 74) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set H:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' (SEQ ID NO 75) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set I:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) set J:
HCPr131(+): 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' (SEQ ID 74) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set K:
HCPr130(+): 5'-ggaattctagACIGCITAYCARGGIACIGTITGYGC-3' (SEQ ID NO 75) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO 71) set L:
HCPr134(+): 5'-CATATAGATGCCCACTTCCTATC-3' (SEQ ID NO 76) HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO71) set M:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr4(-): 5'-GACATGCATGTCATGATGTA-3' (SEQ ID NO 78) set N:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr118(-): 5'-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' (SEQ ID NO
71) set 0:
HCPr3(+): 5'-GTGTGCCAGGACCATC-3' (SEQ ID NO 77) and HCPr66 (-): 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' (SEQ ID NO 70) No PCR products could be obtained with the sets of primers A, B, C, D, E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained from type 3 sera.
However, SUBSTITk1TE SHEET (RULE 26) WO 94125601 . PCT/EP94/01323 set 0 yielded what appeared to be a PCR artifact fragment estimated about 1450 base pairs, instead of the expected 628 base pairs. Although it is not expected that PCR artifact fragments contain information of the gene or genome that was targetted in the experiment, efforts were put in cloning of this artifact fragment, which was designated fragment PC-1. =
The following clones, were obtained from fragment PC-i:
PC-1-37 (SEQ ID NO 59 and SEQ ID NO 55), PC-1-48 (SEQ ID NO 61 and SEQ ID NO
57) The sequences obtained from the 5' and 3' ends of the clones are given in SEQ
ID NOS
55, 57, 59, and 61, and the complete sequences with SEQ ID NO 197 and 199 are shown aligned with the sequences of prototype isolates of other types of HCV in Figure 10 and the alignment of the deduced amino acid sequences is shown in Figure 11 and 7.
Surprisingly, the PCR artifact clone contained HCV information. The positions of the sequences within the HCV genome are compatible with a contiguous HCV sequence of 1437 nucleotides, which was the estimated size of the cloned PCR artifact fragment. Primer HCPr66 primed correctly at the expected position in the HCV genome. Therefore, primer HCPr3 must have incidentally misprimed at a position 809 nucleotides upstream of its legitimate position in the HCV genome. This could not be expected since no sequence information was available from a coding region of type S.
Example 7: The E2 region of HCV type 5 Serum BE95 was chosen for experiments aimed at amplifying a part of the E2 region of HCV
type 5.
After aligning the sequences of HCV-1 (2), HCV-J(1), HC-J6 (3), and HC-J8 (4), PCR
primers were chosen in those regions of little sequence variation.
Primers HCPr109(+): 5'-TGGGATATGATGATGAACTGGTC-3' (SEQ ID NO 141) and HCPr14(-): 5'-CCAGGTACAACCGAACCAATTGCC-3' (SEQ ID NO 142) were combined to amplify the aminoterminal region of the E2/NS 1 region, and were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). With primers HCPr109 and HCPrl4, a PCR
fragment of 661 bp was generated, containing 169 nucleodtides corresponding to the El carboxyterminus and 492 bases from the region encoding the E2 aminoterminus.
An alignment of the type 5 E1/E2 sequences with seq ID NO. 158 with known sequences is presented in Figure 10. The deduced protein sequence was compared with the different SUBSTITUTE SHEET (RULE 26) genotypes (Fig. 12, amino acids 328-546). In the El region, there were no extra structural important motifs found. The aminoterminal part of E2 was hypervariable when compared with the other genotypes. All 6 N-glycosylation sites and all 7 cysteine residue's were conserved in this E2 region. To preserve alignment, it was necessary to introduce a gap between aa 474 and 475 as for type 3a, but not between aa 480 and 481, as for type 2.
Example 8 : The NS5b region of HCV type 4 Type 4 sera GB48, GB116, GB215, and GB358, selected by means of the line probe assay (LiPA, Stuyver et al., 1993), as well as sera GB549 and GB809 that could not be typed by means of this LiPA (only hybridization was observed with the universal probes), were selected from Gabonese patients. All these sera were positive after the first round of PCR
reactions for the 5' untranslated region (Stuyver et al., 1993) and were retained for further study.
RNA was isolated from the sera and cDNA synthesized as described in example 1.
Universal primers in the NS5 region were selected after alignment of the published sequences as follows:
HCPr206(+) : 5' -TGGGGATCCCGTATGATACCCGCTGCTTTGA-3' (SEQ ID NO. 124) and HCPr207(-): 5'-GGCGGAATTCCTGGTCATAGCCTCCGTGAA-3' (SEQ ID NO. 125);
and were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). Using the Line Probe Assay (LiPA), four high-titer type 4 sera and 2 sera that could not be classified were selected and subsequently analyzed with the primer set HCPr206/207. NS5 PCR
fragments obtained using these primers from serum GB48 (GB48-3), serum GB 116 (GB 116-3), serum GB215 (GB215-3), serum GB358 (GB358-3), serum GB549 (GB549-3), and serum GB809 (GB809-3), were selected for cloning. The following sequences were obtained from the PCR
fragments:
From fragment GB48-3 : GB48-3-10 (SEQ ID NO. 106) From fragment GB116-3: GB116-3-5 (SEQ ID NO. 108) From fragment GB215-3: GB215-3-8 (SEQ ID NO. 110) From fragment GB358-3: GB358-3-3 (SEQ ID NO. 112) From fragment GB549-3: GB549-3-6 (SEQ ID NO. 114) SUBSTITUTE SHEET (RULE 26) From fragment GB809-3: GB809-3-1 (SEQ ID NO. 116) An alignment of nucleotide sequences with SEQ ID NO. 106, 108, 110, 112, 114, and 116 with known sequences is given in Figure 1. An alignment of deduced amino acid sequences with SEQ ID NO. 107, 109, 111, 113, 115, and 117 with known sequences is given in Figure =
2. The 4 isolates that had been typed as type 4 by means of LiPA are very closely related to each other (mutual homologies of about 95 %), but are only distantly related to type 1, type 2, and type 3 sequences (e.g. GB358 shows homologies of 65.6 to 67.7% with other genotypes, Table 4). The sequence obtained from sera GB549 and GB809 also show similar homologies with genotypes 1, 2, and 3 (65.9 to 68.8% for GB549 and 65.0 to 68.5% for GB809, Table 4), but an intermediate homology of 79.7 to 86. 8%(often observed between subtypes of the same type) exists between GB549 or GB809 with the group of isolates consisting of GB48, GB116, GB215, and GB358, or between GB549 and GB809. These data indicate the discovery of 3 new subtypes within the HCV genotype 4: in the present invention, these 3 subtypes are designated subtype 4c, represented by isolates GB48, GB 116, GB215, and GB358, subtype 4g, represented by isolate GB549, and subtype 4e, represented by isolate GB809. Although the homologies observed between subtypes in the NS5 region seem to indicate a closer relationship between subtypes 4c and 4e, the homologies observed in the El region indicate that subtypes 4g and 4e show the closest relation (see example 8).
Example 9: The Core/E1 region of HCV type 4 From each of the 3 new type 4 subtypes, one representative serum was selected for cloning experiments in the Core/El region. GB549 (subtype 4g) and GB809 (subtype 4e) were analyzed together with isolate GB358 that was chosen from the subtype 4c group.
Synthetic oligonucleotides:
After aligning the sequences of HCV-1 (2), HCV-J(1), HC-J6 (3), and HC-J8 (4), PCR
primers were chosen in those regions of little sequence variation.
Primers HCPr52(+): 5'-atgTTGGGTAAGGTCATCGATACCCT-3', HCPr23(+): 5'-CTCATGGGGTACATTCCGCT-3', and HCPr54(-); 5'-CTATTACCAGTTCATCATCATATCCCA-3', were synthesized on a 392 DNA/RNA
synthesizer (Applied Biosystems). The sets of primers HCPr23/54 and HCPr52/54 were used, but only with the primer set HCPr52/54, PCR fragments could be obtained. This set of primers amplified the sequence from nucleotide 379 to 957 encoding amino acids 127 to 319:
amino acids from the carboxyterminus of core and 128 amino acids of E1. The SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 amplification products GB358-4, GB549-4, and GB809-4 were cloned as described in example 1. The following clones were obtained from the PCR fragments:
From fragment GB358-4: GB358-4-1 (SEQ ID NO 118) . From fragment GB549-4: GB549-4-3 (SEQ ID NO 120) From fragment GB809-4: GB809-4-3 (SEQ ID NO 122) ' An alignment of the type 4 Core/E1 nucleotide sequences with seq ID NO. 118, 120, and 122 with known sequences is presented in Figure 4. The homologies of the type 4 El region (without core) with type 1, type 2, type 3, and type 5 prototype sequences are depicted in Table 4. Homologies of 53 to 66% are observed with representative isolates of non-type 4 genotypes. Observed homologies in the El region within type 4, between the different subtypes, ranges from 75.2 to 78.4%. The recently disclosed sequences of the core region of Egyptian type 4 isolates (for example EG-29 in Figure 3) described by Simmonds et al.
(1993) do not allow alignment with the Gabonese sequences (as described in the present invention) in the NSB region and may belong to different type 4 subtypes(s) as can be deduced from the core sequences. The deduced amino acid sequences with SEQ ID
NO 119, 121, and 123 are aligned with other prototype sequences in Figure 5. Again, type-specific variation mainly resides in the variable V regions, designated in the present invention, and therefore, type-4-specific amino acids or V regions will be instrumental in diagnosis and therapeutics for HCV type 4.
Examnle 10 = The Core/El and NS5b regions of new HCV tvne 2 3 and 4 subtypes Samples NE92 (subtype 2d), BE98 (subtype 3c), CAM600 and GB809 (subtype 4e), CAMG22 and CAMG27 (subtype 4f), GB438 (subtype 4h), CAR4/1205 subtype (4i), CAR1/501 (subtype 4j), CAR1/901 (subtype 4?), and GB724 (subtype 4?) were selected from a group of sera that reacted positive but aberrantly in a prototype Line Probe Assay as described earlier (Stuyver et al., 1993). Another type 5a isolate BE100 was also analyzed in the C/El region, and yet another type 5a isolate BE96 in the NS5b region. A
high-titer of HCV RNA could be detected, enabling cloning of fragments by a single round of PCR. As no sequences from any coding region of these subtypes had been disclosed yet, synthetic oligonucleotides for PCR amplification were chosen in the regions of little sequence variation after aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J(Kato et al., 1990), HC-J6 (Okamoto et al., 1991), HC-J8 (Okamoto et al., 1992), and the other new sequences of the present invention.
SUBSTITUTE SHEET (RULE 26) WO 94125601 . PCTIEP94/01323 The above mentioned sets 1, 2 and 3 (see example 5) of primers were used, but only with set 1, PCR fragments could be obtained from all isolates (except for BE98, GB724, and CAR1/501). This set of primers amplified the sequence from nucleotide 379 to 957 encoding amino acids 127 to 319: 65 amino acids from the carboxyterminus of core and 128 amino acids of E1. With set 3, the core/E1 region from isolate NE92 and BE98 could be amplified, and with set 2, the core region of GB358, GB724, GB809, and CAM600 could be amplified. }
The amplification products were cloned as described in example 1. The following clones were obtained from the PCR fragments:
From isolate GB724, the clone with SEQ ID NO 193 from the core region.
From isolate NE92, the clone with SEQ ID NO 143 From isolate BE98, the clone from the core/El region of which part of the sequence has been analyzed and is given in SEQ ID NO 147, From isolate CAM600, the clone with SEQ ID NO 167 from the El region, or SEQ
ID NO
165 from the Core/El region as shown in Figure 3, From isolate CAMG22, the clone with SEQ ID NO 171 from the El region as shown in Figure 4, from isolate GB358, the clone with SEQ ID NO 191 in the core region,.
from isolate CAMG27, the clone with SEQ ID NO 173 from the core/El region, from isolate GB438, the clone with SEQ ID NO 177 from the core/ El region, from isolate CAR4/1205, the clone with SEQ ID NO 179 from the core/El region, from isolate CAR1/901, the clone with SEQ ID NO 181 from the core/ El region, from isolate GB809, the clone GB809-4 with SEQ ID NO 189 from the core/El region, clone GB809-2 with SEQ ID NO 169 from the core/El region and the clone with SEQ ID
NO 163 from the core region, and from isolate BE100, the clone with SEQ ID NO 155 from the Core/El region as shown in Figure 4.
An alignment of these Core/El sequences with known Core/El sequences is presented in Figure 4. The deduced amino acid sequences with SEQ ID NO 144, 148, 164, 168, 170, 172, 174, 178, 180, 182, 190, 192, 194, 156, 166 are aligned with other prototype sequences in Figure 5. Again, type-specific variation mainly resides in the variable V
regions, designated in the present invention, and therefore, type 2d, 3c and type 4-specific amino acids or V
regions will be instrumental in diagnosis and therapeutics for HCV type (subtype) 2d, 3c or the different type 4 subtypes.
SUBSTITUTE SHEET (RULE 26) WO 94125601 PCTlEP94/01323 The NS5b region of isolates NE92, BE98, CAM600, CAMG22, GB438, CAR4/1205, CAR1/501, and BE96 was amplified with primers HCPr206 and HCPr207 (Table 7).
The corresponding clones were cloned and sequenced as in example 1 and the corresponding . sequences (of which BE98 was partly sequenced) received the following identification numbers:
' NE92: SEQ ID NO 145 BE98: SEQ ID NO 149 CAM600: SEQ ID NO 201 CAMG22: SEQ ID NO 203 GB438: SEQ ID NO 207 CAR4/1205: SEQ ID NO 209 CAR1/501: SEQ ID NO 211 BE95: SEQ ID NO 159 BE96: SEQ ID NO 161 An alignment of these NS5b sequences with known NS5b sequences is presented in Figure 1. The deduced amino acid sequences with SEQ ID NO 146, 150, 202, 204, 206, 208, 210, 212, 160, 162 are aligned with other prototype sequences in Figure 2. Again, subtype-specific variations can be observed, and therefore, type 2d, 3c and type 4-specific amino acids or V
regions will be instrumental in diagnosis and therapeutics for HCV type (subtype) 2d, 3c or the different type 4 subtypes.
Example 11 : Genotype-secafic reactivity of anti-El antibodies (SerotvninQ) El proteins were expressed from vaccinia virus constructs containing a core/El region extending from nucleotide positions 355 to 978 (Core/El clones described in previous examples including the primers HCPr52 and HCPr54), and expressed proteins from (after the initiator methionine) to W326 of the HCV polyprotein. The expressed protein was modified upon expression in the appropriate host cells (e.g. HeLa, RK13, HuTK-, HepG2) by cleavage between amino acids 191 and 192 of the HCV polyprotein and by the addition of high-mannose type carbohydrate motifs. Therefore, a 30 to 32 kDa glycoprotein could be observed on western blot by means of detection with serum from patients with hepatitis C.
As a reference, a genotype lb clone obtained form the isolate HCV-B was also expressed in an identical way as described above, and was expressed from recombinant vaccinia virus vvHCV-11A.
SUBSTITUTE SHEET (RULE 26) A panel of 104 genotyped sera was first tested for reactivity with a cell lysate containing type lb protein expressed from the recombinant vaccinia virus vvHCV-11A, and compared with cell lysate of RK13 cells infected with a wild type vaccinia virus ('E1/WT'). The lysates were coated as a 1/20 dilution on a normal ELISA microtiter plate (Nunc maxisorb) and left to react with a 1/20 diluation of the respective sera. The panel consisted of 14 type la, 38 type ib, 21 type 2, 21 type 3a, and 9 type 4 sera. Human antibodies were subsequently detected by a goat anti-human IgG conjugated with peroxidase and the enzyme activity was detected. The optical density values of the El and wild type lysates were divided and a factor 2 was taken as the cut-off. The results are given in the table A. Eleven out of 14 type la sera (79 %), 25 out of 38 type lb sera (66 %), 6 out of 21 (29 %), 5 out of 21 (24 %), and none of the 9 type 4 or the type 5 serum reacted (0 %). These experiments clearly show the high prevalence of anti-El antibodies reactive with the type 1 El protein in patients infected with type 1 (36/52 (69%)) (either type la or type ib), but the low prevalence or absence in non-type 1 sera (11/52 (21 %)).
TABLE A
serum E1/WT
type la 3748 3.15 3807 3.51 5282 1.99 9321 3.12 9324 2.76 9325 6.12 9326 10.56 9356 1.79 9388 3.5 8366 10.72 8380 2.27 10925 4.02 10936 5.04 10938 1.36 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . pCT/EP94/01323 type lb 5205 2.25 5222 1.33 5246 1.24 5250 13.58 5493 0.87 5573 1.75 8243 1.77 8244 2.05 8316 1.21 8358 5.04 9337 14.47 9413 5.51 10905 1.26 10919 5.00 10928 8.72 .10929 8.26 10931 2.3 10932 4.41 44 2.37 45 3.14 46 4.37 47 5.68 48 2.97 49 1.18 50 9.85 51 4.51 52 1.11 53 5.20 54 0.98 55 1.48 56 1.06 57 3.85 58 7.6 59 3.28 60 3.23 61 7.82 62 1.92 SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94/01323 type 2 23 0.91 24 1.16 25 2.51 26 0.96 27 1.20 =
28 0.96 29 2.58 30 8.05 31 0.92 32 0.82 33 5.75 34 0.79 35 0.86 36 0.85 37 0.76 38 0.92 - 39 1.08 40 2.33 41 2.83 42 1.21 43 0.91 type 3 1 6.88 2 1.47 3 3.06 4 6.52 10.24 6 2.72 7 1.11 8 1.54 9 1.60 1.21 11 1.07 12 1.00 13 0.85 14 0.96 =
0.51 16 1.00 17 1.09 18 0.99 19 1.04 1.04 21 0.96 SUBSTITUTE SHEET (RULE 26) type 4 22 0.87 GB48 0.49 - GB113 0.68 GB116 0.73 GB215 0.52 GB358 0.56 GB359 0.71 GB438 1.08 GB516 1.04 type 5 BE95 0.86 Core/El clones of isolates BR36 (type 3a) and BE95 (type 5a) were subsequently recombined into the viruses wHCV-62 and vvHCV-63, respectively. A genotyped panel of sera was subsequently tested onto cell lysates obtained from RK13 cells infected with the recombinant viruses wHCV-62 and wHCV-63. Tests were carried out as described above and the results are given in the table given below (TABLE B). From these results, it can clearly be seen that, although some cross-reactivity occurs (especially befween type I and 3), the obtained values of a given serum are usually higher on its homologous El protein than on an El protein of another genotype. For type 5 sera, none of the 5 sera were reactive on type 1 or 3 El proteins, while 3 out of 5 were shown to contain anti-El antibodies when tested on their homologous type 5 protein. Therefore, in this simple test system, a considerable number of sera can already be serotyped. Combined with the reactivity to type-specific NS4 epitopes or epitopes derived from other type-specific parts of the HCV polyprotein, a serotyping assay may be developed for discriminating the major types of HCV. To overcome the problem of cross-reactivity, the position of cross-reactive epitopes may be determined by someone skilled in the art (e.g. by means of competition of the reactivity with synthetic peptides), and the epitopes evoking cross-reactivity may be left out of the composition to be included in the serotyping assay or may be included in sample diluent to outcompete cross-reactive antibodies.
SUBSTITUTE SHEET (RULE 26) WO 94/25601 . PCT/EP94101323 TABLE B
serum E11b/WT E13a/WT E158/WT
type lb 8316 0.89 0.59 0.80 ~
8358 2.22 2.65 1.96 9337 1.59 0.96 0.93 9410 16.32 9.60 3.62 9413 9.89 2.91 2.85 10905 1.04 0.96 1.05 10919 3.17 2.56 2.96 10928 4.39 2.28 2.07 10929 2.95 2.07 2.08 10931 3.11 1.49 2.11 0.86 0.86 0.96 6 3.48 1.32 1.32 7 6.76 4.00 3.77 8 10.88 3.44 4.04 9 1.76 1.88 1.58 9.88 7.48 7.20 11 8.48 8.99 8.45 12 0.76 0.72 0.76 13 5.04 5.67 5.37 14 10.48 10.54 11.22 5.18 1.62 1.65 type 3 8332 3.39 4.22 0.66 10907 3.24 4.39 0.96 10908 0.99 0.94 0.98 10934 0.86 0.90 0.90 10927 2.58 2.71 2.44 8210 0.82 0.80 0.86 8344 1.09 6.66 1.17 8351 1.21 1.29 1.22 30 0.85 4.11 0.98 32 0.85 2.16 1.04 type 5 0.78 0.95 1.54 BE110 0.79 1.01 4.95 BE95 0.47 0.52 0.65 BE111 0.71 0.75 8.33 BE112 1.01 1.27 2.37 BEII3 1.11 1.35 1.60 SIjBSTITEJfE SHEET (RULE 26) Table 5. Homologies of new HCV sequences with other known HCV types Region Isolate la lb 2a 2b 3a 3b (nucleotides) (type) HCV-l HCV-J HC-J6 HC-J8 T1 T7 T9 Ti0 Core (1-573) PC (5) 83.8 (91.6) 84.8 (92.1) 82.6 (90.1) 82.4 (89.0) t1 (574-957) I-ID10 (3) 61.5 (68,0) 64.6 (68.8) 57.8 (55.5) 56.3 (59.4) BR36 (3) 62.0 (66.4) 62.5 (67.2) 56.5 (53.9) 55.2 (58.6) BR33 (3) 60.7 (67.2) 63.3 (68.0) 56.5 (54.7) 56.0 (58.6) PC (5) 61.4 (64,0) 62.4 (64.8) 54.1 (49.6) 53.3 (47.2) GB358 (4a) 62.5 (69.1) 62.8 (65.9) 59.4 (54.0) 54.4 (54.0) GB549 (4b) 66.0 (72.2) 62,8 (69.8) 59.1 (56,4) 56.5 (54.0) GB809 (4c) 63.3 (69.1) 60.7 (64.3) 56.7 (53.2) 53.0 (51.6) NS3 PC (5) 74.7 (89) 76.1 (86.4) 76.1 (89.8) 78.0 (89.0) (3856-4209) NS4 BR36 (3) 67.8 (78.5) 69.8 (75.1) 62.0 (67.5) 61.7 (66.0) (4892-5292) HD 10 (3) 69.8 (74.6) 66.6 (69.7) 57.8 (59.9) 59.1 (59.9) NS4 PC (5) 61.3 (62.2) 63.0 (65.5) 52.9 (46.2) 54.3 (43.7) (4936-5292) NS5b BR34 (3) 65.7 66.7 63.9 64.3 94.8 93.9 75.6 77.0 (8023-8235) BR36 (3) 64.3 67.6 64.8 66.7 94.8 93.4 75.1 76.5 BR33 (3) 65.7 67.1 64.3 64.8 94.8 93.9 76.0 77.5 GB358 (4a) 67.7 (76.1) 65.6 (77.0) 66.5 (70.8) 65.6 (71,7) GB549 (4b) 68.8 (76.1) 67.1 (77.0) 65.9 (71.7) 65.9 (74.4) GB809 (4c) 68.5 (73.5) 65.0 (73.5) 67.7 (69.9) 67.7 (73.5) Shown are the nucleotide homologies (the amino-acid homology is given between brackets) for the region indicated in the left column.
SUBSTITUTE SHEET (RULE 26) Table 6. NS4 sequences of the different Lyenotypes prototype TYPE SYNTHETiC PEPTIDE NS4-1 SYNTHEIIC PEPTIDE NS4-5 SYNTHETlC
(NS4a) (NS4b) (NS4b) position-> 9 0 2 3 3 4 , = 4 = -4 t~ 4 = = i M = = -t HCV-1 la LSG KPAIIPDREV LYREFDE SQHLPYIEQ GMGLAEQFKQ K LAEQPKQ KALGLLQTAS RQA
HCV-7 lb LSG RPAVIPDREV LYQEFDE ASHLPYIEQ GMQ_LAEQFKg K LAEQFKQ KALGLLQTAT KQA
HC-76 2a VNQ RAWAPDKEV LYEAFDE ASRaAT.iF-? GQRIABMLKS K TAEMLBS KIQGLLQQAS KQA
HC J8 2b LND RWVAPDR'EI LY_'AFDE ASRAALIEE GQRMAIIvILi{S K MAp.hAxc K1QGLyQQAT
RQA
I BR36 3a LGG KPAIVPDKEV LYQQ YDE SQAAPYIEQ AQ_VIAHQFKE K IALIQFKE KV LGLLQRAT
QQQ
V
residues conserved in every genotype. Underlined amino acids are type-specific, amino acids in italics are unique to type 3 and 5 sequences.
SUBSTITUTE SHEET (RULE 26) Table 7 SEQ ID Primer NO Sequence from 5' to 3' NO (polarity) 63 HCPr161(+) 5'-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3' 64 HCPr162(-) 5'-GGGCTGCTCTATCCTCATCGACGCCATC-3' 65 HCPr163(+) 5'-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3' 66 HCPr164(-) 5'-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3' 67 HCPr23(+) 5'-CTCATGGGGTACATTCCGCT-3' 68 HCPr54(-) 5'-CTATTACCAGTTCATCATCATATCCCA-3' 69 HCPr116(+) 5'-ttttAAATACATCATGRCITGYATG-3' 70 HCPr66(-) 5'-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3' 71 HCPr 118(-) 5' actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3' 72 HCPr 117(+) 5' -ttttAAATACATCGCIRCITGCATGCA-3' 73 HCPr119(-) 5'-actagtcgactaRTTIGCIATIAGCCKRTTCATCCAYTG-3' 74 HCPr131(+) 5'-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3' 75 HCPrl 30(+) 5'-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3' 76 HCPr 134(+) 5' -CATATAGATGCCCACTTCCTATC-3' 77 HCPr3(+) 5'-GTGTGCCAGGACCATC-3' 78 HCPr4(-) 5'-GACATGCATGTCATGATGTA-3' 79 HCPr 152(+) 5' -TACGCCTCTTCTATATCGGTTGGGGCCTG-3' 80 HCPr52(+) 5'-atgTTGGGTAAGGTCATCGATACCCT-3' 81 HCPr41(+) 5' -CCCGGGAGGTCTCGTAGACCGTGCA-3' 82 HCPr40(-) 5'-ctattaAAGATAGAGAAAGAGCAACCGGG-3' 124 HCPR206 5'-tggggatcccgtaxgatacccgctgctttga-3' 125 HCPR207 5'-ggcggaattcctggtcatagcctccgtgaa-3' 141 HCPR 109 5' -tgggatatgatgatgaactggtc-3' 142 HCPR14 5' -ccaggtacaaccgaaccaattgcc-3' SUBSTI7UTE SHEET (RULE 26) t~ M'-+MNN+ ~NMM
4t ~
.-i + ~ i M + + N + ~
l~ M N M M N N N N M
`e!
kn --~ i+ N N -+ N+ N+
N
C3.
K1 N M M M'-~ M M M M
Z 1/') M,~ M M M~-+ M M M M
Qy ~ F~ H M~~ M M M M M M M
~
~
z ~
pp ~ ~ p ~ N M ~Y v1 ~O [~ oo !T O
~ O O O O O O O O O~
r. .-. .-. .-~ .~ .-. .-F
SUBSTI7UTE SHEET (RULE 26) [~ , f+1 M M M~" , t''1 N ! f~l ~~ N
tt ~
z .
FD-.
N N
N~-+ N m N N N N N N
Z ~!1 + +++-~I-`I+N.--.-.~
l~ , M M M M M , M N M M~ N M
CA
Z
i/) + N c~ M M c~ , N N M M M c~ M
N N M M M~ + , M M K1 M
---Fi i. ~
[Y W) `O I'- oo Cn O --~ N tn ~t ~, - --+r+ NNNN N
SUBSTITUTE SHEET (RULE 26) 1~ M+- ++
~
`~' V~ =--, =-= + + +
~ N ~ + N N + + ~--~ ~-- M N N
t- M *~ ~-+ -- rM , , + cM , N N + M + , ~ N N
'ef z 1n + + + .-. , rl ~--~ =--~ , , , + ~ + , , ~ 'I' ~" 'I' M M N , =-~ =--= M + M +
C- c'*1 N + K1 M ~ '-' ~ K1 N M M M
Z f*1 N N M C''f N~""
rl -G.1 N
y N
~' v~ ~D l~ 00 CT O~ N M ~t v> ~D t~ 00 põ rn C:) ~-, N MV- v-) ~O C- o0 N N N N N M M M M M M M M M ~ M Ci' d' :r 114, 'f qT 'cl- V- Q-r. .~. r+ r-. ..r .~r .-~ ~-r .... .r ~ .-. ~r ~ .-. .~ ~ ~ ..-, r-. .-. .-=+
.-, .-~
SUBST(TUTE SHEET (RULE 26) t~ + M N f+1 M r N N cn M N
z + M r i~~^~ , N N M N M , r N ~ N M r N~= r+ M r r+ r+
N N , i N+ ,~-+ r+
z tfJ + N + +
r.~t M + r r r r r r+ r r r , , i r r CA
Z , + ~ , i =- N
.-y ~
t~. N M
p, a, Q~--~ N M d' xn "D l- 00 ON 0~ N M
~ ~ D, ~l Vl V~ ~/'1 ~/1 ~1 r/1 V1 N v1 \D ~ ~, ~O ~D
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SUBSf ITM SHEET (RULE 26)
Claims (42)
1. A polynucleic acid containing 8 or more contiguous nucleotides selected from any of the following HCV genomic sequences:
- an HCV genomic sequence having a homology of more than 85%, preferably of more than 86%, most preferably of more than 87% to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 or 151 in the region spanning positions 1 to 573 of the Core region;
- an HCV genomic sequence having a homology of more than 61%, preferably more than 63%, most preferably of more than 65% to any of the sequences as represented in SEQ ID NO 41, 43, 45, 57, 49, 51, 53, 153 or 155 in the region spanning positions 574 to 957 of the E1 region;
- an HCV genomic sequence having a homology of more than 76,5%, preferably of more than 77%, most preferably of more than 78% with any of the sequences as represented in SEQ ID NO 55, 57, 197 or 199 in the region spanning positions 3856 to 4209 of the NS3 region;
- an HCV genomic sequence having a homology of more than 68%, preferably of more than 70%, most preferably of more than 72% with the sequence as represented in SEQ ID NO 157 in the region spanning positions 980 to 1179 of the E1/E2 region;
- an HCV genomic sequence having a homology of more than 57%, preferably of more than 59%, most preferably of more than 61 % to any of the sequences as represented in SEQ ID NO 59 or 61 in the region spanning positions 4936 to 5296 of the NS4 region;
- an HCV genomic sequence having a homology of more than 93%, preferably of more than 93,5%, most preferably of more than 94% to any of the sequences as represented in SEQ ID NO 159 or 161 in the region spanning positions 7932 to in the NS5B region.
- an HCV genomic sequence having a homology of more than 85%, preferably of more than 86%, most preferably of more than 87% to any of the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49, 51, 53 or 151 in the region spanning positions 1 to 573 of the Core region;
- an HCV genomic sequence having a homology of more than 61%, preferably more than 63%, most preferably of more than 65% to any of the sequences as represented in SEQ ID NO 41, 43, 45, 57, 49, 51, 53, 153 or 155 in the region spanning positions 574 to 957 of the E1 region;
- an HCV genomic sequence having a homology of more than 76,5%, preferably of more than 77%, most preferably of more than 78% with any of the sequences as represented in SEQ ID NO 55, 57, 197 or 199 in the region spanning positions 3856 to 4209 of the NS3 region;
- an HCV genomic sequence having a homology of more than 68%, preferably of more than 70%, most preferably of more than 72% with the sequence as represented in SEQ ID NO 157 in the region spanning positions 980 to 1179 of the E1/E2 region;
- an HCV genomic sequence having a homology of more than 57%, preferably of more than 59%, most preferably of more than 61 % to any of the sequences as represented in SEQ ID NO 59 or 61 in the region spanning positions 4936 to 5296 of the NS4 region;
- an HCV genomic sequence having a homology of more than 93%, preferably of more than 93,5%, most preferably of more than 94% to any of the sequences as represented in SEQ ID NO 159 or 161 in the region spanning positions 7932 to in the NS5B region.
2. The polynucleic acid according to claim 1, wherein said polynucleic acid is able to act as a primer for amplifying the nucleic acid of a certain isolate belonging to the genotype from which the primer is derived.
3. The polynucleic acid according to claim 1, wherein said polynucleic acid is able to act as a hybridization probe for specific detection and/or classification into types of a nucleic acid containing said nucleotide sequence, with said polynucleic acid being possibly labelled or attached to a solid substrate.
4. The use of a polynucleic acid according to any one of claims 1 to 3 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, comprising at least the following steps:
- hybridizing the nucleic acids of the biological sample with one or more probes according to claim 3, - washing, - detecting the hybrids formed, - inferring the presence of one or more HCV genotypes present from the observed hybridization pattern.
- hybridizing the nucleic acids of the biological sample with one or more probes according to claim 3, - washing, - detecting the hybrids formed, - inferring the presence of one or more HCV genotypes present from the observed hybridization pattern.
5. The use of a polynucleic acid according to claim 4 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, further comprising the step of extracting sample nucleic acid.
6. The use of a polynucleic acid according to claim 4 or 5 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, further comprising the step of amplifying the nucleic acid with at least one of the primers according to claim 2 or any other HCV type 5 or universal primer.
7. The use of a polynucleic acid according to any one of claims 4 to 6 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, wherein said hybridizing is under denaturing conditions.
8. The use of a polynucleic acid according to any one of claims 4 to 7 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, wherein said nucleic acids are labelled during or after amplification.
9. The use of a polynucleic acid according to any one of claims 4 to 8 for in vitro detecting the presence of one or more HCV genotypes present in a biological sample liable to contain them, wherein said probes are attached to a solid substrate.
10. A peptide or polypeptide containing in its sequence a contiguous sequence of at least amino acids of an HCV polyprotein encoded by any one of the polynucleic acids according to claim 1.
11. The peptide or polypeptide according to claim 10, wherein said peptide or polypeptide contains in its sequence at least one amino acid which is unique for HCV type 5.
12. The peptide or polypeptide according to claim 10 or 11, wherein said contiguous sequence contains in its sequence at least one of the following amino acid residues:
M44, Q70, A87, N106, K115, V134, G142, P165, 1178, V192, P193, Y194, A197, D217, N218, L219, M231, T232, K232, 1242, S247, S250, F251, V254, A256, A261, Y264, A266, G268, L284, Y293, R294, Q297, A299, N303, Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, F536, M539, I546, V1312, Q1321, P1368, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, M1572, V1595, T1606, M1611, L1612, P1630, I1656, V1667, V1677, A1681, A1700, Q1704, A1713, S1714, M1718, D1719, T1721, R1722, A1723, G1726, E1730, V1732, F1735, I1736, S1737, T1739, G1740, Q1741, K1742, T1745, L1746, K1747, A1750, T1751, V1753, N1755, A1757, T1763, Y1764, A2647, L2653, S2664, F2680, T2724, R2726, Y2730, H2739 or K2752, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 as shown in Table 1.
M44, Q70, A87, N106, K115, V134, G142, P165, 1178, V192, P193, Y194, A197, D217, N218, L219, M231, T232, K232, 1242, S247, S250, F251, V254, A256, A261, Y264, A266, G268, L284, Y293, R294, Q297, A299, N303, Q317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386, I387, S392, I399, F402, I403, R405, D454, A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, F536, M539, I546, V1312, Q1321, P1368, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529, I1543, N1567, M1572, V1595, T1606, M1611, L1612, P1630, I1656, V1667, V1677, A1681, A1700, Q1704, A1713, S1714, M1718, D1719, T1721, R1722, A1723, G1726, E1730, V1732, F1735, I1736, S1737, T1739, G1740, Q1741, K1742, T1745, L1746, K1747, A1750, T1751, V1753, N1755, A1757, T1763, Y1764, A2647, L2653, S2664, F2680, T2724, R2726, Y2730, H2739 or K2752, with said notation being composed of a letter representing the amino acid residue by its one-letter code, and a number representing the amino acid numbering according to Kato et al., 1990 as shown in Table 1.
13 The peptide or polypeptide according to any one of claims 10 to 12, wherein said contiguous sequence is selected from any one of the following HCV amino acid sequences:
- a sequence having more than 93%, preferably more than 94%, most preferably more than 95% homology in the region spanning Core positions 1 to 191 to any of the amino acid sequences in SEQ ID NO 42, 44, 46, 48, 50, 52, 54, or 152;
- a sequence having more than 73%, preferably more than 74%, most preferably more than 76% homology in the region spanning E1 positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52, 54, 154 or 156;
- a sequence spanning positions 1286 to 1403 of the NS3 region, with said sequence being characterized as having more than 90%, preferably more than 91%, most preferably more than 92% homology to any of the amino acid sequences represented in SEQ ID NO 56 to 58;
- a sequence spanning positions 1646 to 1764 of the NS3/4 region, with said sequence being characterized as having more than 66%, more particularly 68%, most particularly 70% or more homology to any of the amino acid sequences as represented in SEQ ID NO 60 or 62.
- a sequence having more than 93%, preferably more than 94%, most preferably more than 95% homology in the region spanning Core positions 1 to 191 to any of the amino acid sequences in SEQ ID NO 42, 44, 46, 48, 50, 52, 54, or 152;
- a sequence having more than 73%, preferably more than 74%, most preferably more than 76% homology in the region spanning E1 positions 192 to 319 to any of the amino acid sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52, 54, 154 or 156;
- a sequence spanning positions 1286 to 1403 of the NS3 region, with said sequence being characterized as having more than 90%, preferably more than 91%, most preferably more than 92% homology to any of the amino acid sequences represented in SEQ ID NO 56 to 58;
- a sequence spanning positions 1646 to 1764 of the NS3/4 region, with said sequence being characterized as having more than 66%, more particularly 68%, most particularly 70% or more homology to any of the amino acid sequences as represented in SEQ ID NO 60 or 62.
14. The peptide or polypeptide according to any one of claims 10 to 13, wherein said contiguous sequence is selected from the following peptides:
QPTGRSWGQ (SEQ ID NO 93) VPYRNASGIYHV (SEQ ID NO 84) VYEADNLILHA (SEQ ID NO 86) VRKDNVSRCWVQI (SEQ ID NO 249) APSLGAVTAP (SEQ ID NO 90) APSFGAVTAP (SEQ ID NO 250) RPRQHATVQN (SEQ ID NO 92) RPRQHATVQD (SEQ ID NO 263) TISYANGSGPSDDK (SEQ ID NO 267)
QPTGRSWGQ (SEQ ID NO 93) VPYRNASGIYHV (SEQ ID NO 84) VYEADNLILHA (SEQ ID NO 86) VRKDNVSRCWVQI (SEQ ID NO 249) APSLGAVTAP (SEQ ID NO 90) APSFGAVTAP (SEQ ID NO 250) RPRQHATVQN (SEQ ID NO 92) RPRQHATVQD (SEQ ID NO 263) TISYANGSGPSDDK (SEQ ID NO 267)
15. A recombinant vector, particularly for cloning orexpression, or both, with said recombinant vector comprising a vector sequence, an [appropriate] prokaryotic, eukaryotic or viral promoter sequence followed by the polynucleic acid sequences as defined in claim 1, with said recombinant vector allowing the expression of an HCV type 5 derived polypeptide according to any of one claims 10 to 14 in a prokaryotic or eukaryotic host, or in living mammals when injected as naked DNA, and more particularly a recombinant vector allowing the expression of an HCV type 5 polypeptide spanning the following amino acid positions :
- a polypeptide starting at position 1 and ending at any position in the region between positions 70 and 326, more particularly a polypeptide spanning positions 1 to 70, 1 to 85, positions 1 to 120, positions 1 to 150, positions 1 to 191, positions 1 to 200, for expression of the Core protein, and positions 1 to 263, positions 1 to 326, for expression of the Core and E1 protein;
- a polypeptide starting at any position in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, more particularly from positions 119 to 326, for expression of E1, or forms that have the putative membrane anchor deleted;
- a polypeptide starting at any position in the region between positions 1556 to 1688, and ending at any position between positions 1739 and 1764, for expression of the NS4 regions, more particularly a polypeptide starting at position 1658 and ending at position 1711 for expression of the NS4a antigen, and more particularly, a polypeptide starting at position 1712 and ending between positions 1743 and 1972, for example 1712-1743, 1712-1764, 1712-1782, 1712-1972, 1712 to 1782 and 1902 to 1972 for expression of the NS4b protein or parts thereof.
- a polypeptide starting at position 1 and ending at any position in the region between positions 70 and 326, more particularly a polypeptide spanning positions 1 to 70, 1 to 85, positions 1 to 120, positions 1 to 150, positions 1 to 191, positions 1 to 200, for expression of the Core protein, and positions 1 to 263, positions 1 to 326, for expression of the Core and E1 protein;
- a polypeptide starting at any position in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, more particularly from positions 119 to 326, for expression of E1, or forms that have the putative membrane anchor deleted;
- a polypeptide starting at any position in the region between positions 1556 to 1688, and ending at any position between positions 1739 and 1764, for expression of the NS4 regions, more particularly a polypeptide starting at position 1658 and ending at position 1711 for expression of the NS4a antigen, and more particularly, a polypeptide starting at position 1712 and ending between positions 1743 and 1972, for example 1712-1743, 1712-1764, 1712-1782, 1712-1972, 1712 to 1782 and 1902 to 1972 for expression of the NS4b protein or parts thereof.
16. The recombinant vector allowing the expression of an HCV type 5 polypeptide according to claim 15, wherein said HCV type 5 polypeptide starting at any position in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, more particularly from positions 119 to 326, for expression of E1, wherein the putative anchor is deleted at positions 264 to 293 plus or minus 8 amino acids.
17. The peptide or polypeptide according to any one of claims 10 to 14, wherein said polypeptide is a recombinant polypeptide expressed by means of an expression vector as defined in claim 15 or 16.
18. A composition consisting of or comprising at least one peptide or polypeptide according to or as defined in any of claims 10 to 14 and 17.
19. The peptide or polypeptide according to any one of claims 10 to 14 and 17, or a composition according to claim 18, for use in a method for immunizing a mammal against HCV comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response, more particularly a vaccine composition including HCV type 5 polypeptides.
20. The peptide or polypeptide according to any one of claims 10 to 14 and 17, or a composition according to claim 18, for use in a method for immunizing humans, against HCV comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response, more particularly a vaccine composition including HCV type 5 polypeptides.
21. An antibody raised upon immunization with a peptide or polypeptide according to any one of claims 10 to 14 and 17 by means of a process according to claim 19, with said antibody being specifically reactive with any of the polypeptides as defined in any one of claims 10 to 14 and 17.
22. Process for detecting in vitro HCV present in a biological sample liable to contain it, the process comprising at least the following steps:
- contacting the biological sample to be analyzed for the presence of HCV
antibodies with any one of the peptides or polypeptides according to any one of claims 10 to 14 and 17 under conditions which allow the formation of an immune complex, - removing unbound components, - incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies being conjugated to a detectable label, - detecting the presence of said immune complexes visually or by means of densitometry and inferring the presence of HCV.
- contacting the biological sample to be analyzed for the presence of HCV
antibodies with any one of the peptides or polypeptides according to any one of claims 10 to 14 and 17 under conditions which allow the formation of an immune complex, - removing unbound components, - incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies being conjugated to a detectable label, - detecting the presence of said immune complexes visually or by means of densitometry and inferring the presence of HCV.
23. The process according to claim 22, wherein said peptides or polypeptides are in an immobilized form.
24. The process according to claim 23, wherein said polypeptide is in the form of a biotinylated polypeptide and is covalently bound to a solid substrate by means of streptavidin or avidin complexes.
25. The use of a peptide or polypeptide according to any one of claims 10 to 14 and 17, for incorporation into a serotyping assay for detecting one or more serological types of HCV
present in a biological sample liable to contain it, more particularly for detecting NS4 antigens or antibodies of the different types to be detected combined in one assay format, comprising at least the following steps:
- contacting the biological sample to be analyzed for the presence of HCV
antibodies or antigens of one or more serological type, with at least one of the peptides or polypeptides according to any one of claims 10 to 14 and 17, in an immobilized form under conditions which allow the formation of an immune complex, - removing unbound components, - incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies being conjugated to a detectable label, - detecting the presence of said immune complexes visually or by means of densitometry and inferring the HCV serological types present from the observed binding pattern.
present in a biological sample liable to contain it, more particularly for detecting NS4 antigens or antibodies of the different types to be detected combined in one assay format, comprising at least the following steps:
- contacting the biological sample to be analyzed for the presence of HCV
antibodies or antigens of one or more serological type, with at least one of the peptides or polypeptides according to any one of claims 10 to 14 and 17, in an immobilized form under conditions which allow the formation of an immune complex, - removing unbound components, - incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies being conjugated to a detectable label, - detecting the presence of said immune complexes visually or by means of densitometry and inferring the HCV serological types present from the observed binding pattern.
26. The use according to claim 25, wherein said polypeptide is in the form of a biotinylated polypeptide and is covalently bound to a solid substrate by means of streptavidin or avidin complexes.
27. A kit for determining the presence of HCV genotypes present in a biological sample liable to contain them, comprising:
- at least one probe according to claim 3, - a buffer or components necessary for producing the buffer enabling a hybridization reaction between these probes and the possibly amplified products to be carried out, - a means for detecting the hybrids resulting from the preceding hybridization,
- at least one probe according to claim 3, - a buffer or components necessary for producing the buffer enabling a hybridization reaction between these probes and the possibly amplified products to be carried out, - a means for detecting the hybrids resulting from the preceding hybridization,
28. The kit according to claim 27, further comprising at least one primer composition containing any primer selected from those defined in claim 2 or any other HCV
type 5 or universal HCV primer,
type 5 or universal HCV primer,
29. The kit according to claim 27 or 28, wherein said at least one probe according to claim 3 is combined with other probes from HCV type 1, type 2 or other types of HCV.
30. The kit according to any one of claims 27 to 29, wherein said probes are immobilized on a solid substrate.
31. The kit according to claim 30, wherein said probes are immobilized on one and the same membrane strip.
32. The kit according to any one of claims 27 to 31, further comprising an automated scanning and interpretation device for inferring the HCV genotype(s) present in the sample from the observed hybridization pattern.
33. A kit for determining the presence of HCV antibodies present in a biological sample liable to contain them, comprising:
- at least one peptide or polypeptide according to any one of claims 10 to 14 and 17, - a buffer or components necessary for producing the buffer enabling binding reaction between these peptides or polypeptides and the antibodies against HCV present in the biological sample, - a means for detecting the immune complexes formed in the preceding binding reaction.
- at least one peptide or polypeptide according to any one of claims 10 to 14 and 17, - a buffer or components necessary for producing the buffer enabling binding reaction between these peptides or polypeptides and the antibodies against HCV present in the biological sample, - a means for detecting the immune complexes formed in the preceding binding reaction.
34. The kit according to claim 33, wherein said polypeptides are immobilized on a solid substrate.
35. The kit according to claim 34, wherein said polypeptides are immobilized on one and the same membrane strip.
36. The kit according to any one of claims 33 to 35, further comprising an automated scanning and interpretation device for inferring the HCV genotype(s) present in the sample from the observed pattern.
37. A method for in vitro detecting or screening for HCV genotypes present in a biological sample, comprising the following steps:
(i) providing a sample nucleic acid (ii) determining the presence of a polynucleic acid according to claim 1.
(i) providing a sample nucleic acid (ii) determining the presence of a polynucleic acid according to claim 1.
38. The method according to claim 37, further comprising the step of inferring from the presence of one or more of these HCV polynucleic acids of step (ii) the HCV
genotype(s) present in said sample.
genotype(s) present in said sample.
39. A method for detecting or screening for HCV genotypes present in a biological sample, comprising the following steps:
(i) providing a sample nucleic acid (ii) specifically amplifying a polynucleic acid according to claim 1.
(i) providing a sample nucleic acid (ii) specifically amplifying a polynucleic acid according to claim 1.
40. The method according to claim 39, further comprising the step of inferring from the presence of one or more of these HCV polynucleic acids of step (ii) the HCV
genotype(s) present in said sample.
genotype(s) present in said sample.
41. A kit for determining the presence of HCV genotypes comprising a polynucleic acid according to any of one claims 1 to 3.
42. A composition comprising a polynucleic acid according to any one of claims 1 to 3.
Applications Claiming Priority (5)
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| EP93402019 | 1993-08-05 | ||
| EP93401099.2 | 1993-08-05 | ||
| EP93402019.9 | 1993-08-05 | ||
| PCT/EP1994/001323 WO1994025601A2 (en) | 1993-04-27 | 1994-04-27 | New sequences of hepatitis c virus genotypes and their use as therapeutic and diagnostic agents |
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| CA002139100A Expired - Lifetime CA2139100C (en) | 1993-04-27 | 1994-04-27 | New sequences of hepatitis c virus genotypes and their use as therapeutic and diagnostic agents |
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- 1994-04-27 US US08/362,455 patent/US7157226B1/en not_active Expired - Fee Related
- 1994-04-27 AT AT94915550T patent/ATE373093T1/en active
- 1994-04-27 JP JP6523877A patent/JPH07508423A/en active Pending
- 1994-12-21 NO NO944967A patent/NO944967D0/en not_active Application Discontinuation
- 1994-12-23 FI FI946066A patent/FI946066A0/en unknown
-
2001
- 2001-07-06 US US09/899,046 patent/US7195765B2/en not_active Expired - Fee Related
- 2001-11-21 JP JP2001356707A patent/JP2002233389A/en active Pending
- 2001-11-21 JP JP2001355654A patent/JP2002233388A/en active Pending
-
2003
- 2003-07-08 JP JP2003272022A patent/JP2004041206A/en not_active Withdrawn
- 2003-07-09 JP JP2003272151A patent/JP2004041207A/en not_active Withdrawn
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