LV10306B - Nanbv diagnostics and vaccines - Google Patents

Abstract

Hepatitis C virus (HCV) new DNS sequence and poly-peptides are described. Application of these sequences and poly-peptides in immunological analyses, diagnostical probes, anti-HCV anti-agents production, PCR technology and recombinant DNS technology is described.

Description

LV10306

NANBV DIAGNOSTICS AND VACCINES

Technical Field

The invention relates to materiāls and methodologies for managing the spread of non-A, non-B hepatitis virus (NANBV) infection. More specifically, it relates to polynucleOtides derived from the genome of an etiologic aģent of NANBH, hepatitis C virus (HCV), to polypeptides encoded therein, and to antibodies directed to the polypeptides. These reaģents are useful as screening aģents for HCV and its infection, and as protective aģents against the disease.

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Cited Patents EPO Pub. No. 318,216 PCT Pub. No. WO 89/04669 u.s. Patent No.4,341,761 u.s. Patent No.4,399,121 u.s. Patent No.4,427,783 u.s. Patent No.4,444,887 u.s. Patent No.4,466,917 u.s. Patent No.4,472,500 u.s. Patent No.4,491,632 u.s. Patent No.4,493,890 5

Background Art

Non-A, Non-B hepatitis (NANBH) is a transmissible disease or family of diseases that are believed to be virai -induced, and that are distinguishable from other fonus of virai-associated liver diseases, including that caused by the known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (HBV), and delta hepatitis virus (HDV), as well as the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). NANBH was first identified in transfused individuāls. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that NANBH is due to a transmissible Infectious aģent or aģents.

Epidemiologic evidence is suggestive that there may be three types of NANBH: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of aģents which may be the causative of NANBH are unknovm.

Clinical diagnosis and identification of NANBH has been accomplished primarily by exclusion of other virai markers. Among the methods used to detect putative NANBV antigens and antibodies are agar-gel diffusion, counterimmunoelectrophoresis,immunofluorescence microscopy, immune electron microscopy, radioimmunoassay, and enzyme-linked immunosorbent assay. However, none of these assays has proved to be sufficiently sensitive, specific, and reproducible to be used as a diagnostic tēst for NANBH. 6 LV10306

Previously there was neither clarity nor agreement as to the identity or specificity of the antigen antibody systems associated with aģents of NANBH. This was due, at least in part, to the prior or co-infection of HBV with NANBV in Individuāls, and to the known complexity of the soluble and particulate antigens associated with HBV, as well as to the integration of HBV DNA into the genome of liver celis. In addition, there is the possibility that NANBH is caused by more than one infectious aģent, as well as the possibility that NANBH has been misdiagnosed. Moreover, it is unclear what the aerological assays detect in the serum of patients with NANBH. It has been postulated that the agar-gel diffusion and counterimmunoelectrophoresis assays detect autoimmune responses or nonspecific protein interactions that sometimes occur between serum specimens, and that they do not represent specific NANBV antigen-antibody reactions. The immunofluorescence, and enzyme-linked immtinosorbent, and radioimmunoassays appear to detect low Ievels of a rheumatoid-factor-like material that is frequently present in the serum of patients with NANBH as well as in patients with other hepatic and nonhepatic diseases. Some of the reactivity detected may represent antibody to hostdetermined cytoplasmic antigens.

There have been a number of candidate NANBV. See, for example the reviews by Prince (1983), Feinstone and Hoofnagle (1984), and Overby (1985, 1986, 1987) and the article by Iwarson (1987). However, there is no proof that any of these candidates represent the etiological aģent of NANBH.

The demand for sensitive, specific methods for screening and identifying carriers of NANBV and NANBV 7 contaminated blood or blood products is significant. Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfused patients, and NANBH accounts for up to 90% of these cases. The major problem in this disease is the £requent progression to chronic liver damage (25-55%) .

Patient care as well as the prevention of transmission of NANBH by blood and blood products or by close personai contact reguire reliable screening, diagnostic and prognostic tools to detect nucleic acids, antigens and antibodies related to NANBV. In addition, there is also a need for effective vaccines and immunotherapeutic therapeutic aģents for the prevention and/or treatment of the disease.

Applicant discovered a new virus, the Hepatitis C virus (HCV), which has proven to be the major etiologic aģent of blood-borne NANBH (BB-NANBH). Applicant*s initial work, including a partial genomic sequence of the prototype HCV isolate, CDC/HCV1 (also called HCV1) / is described in EPO Pub. No. 318,216 (published 31 May 1989) and PCT Pub. No. WO 89/04669 (published.1 June 1989). The disclosures of these patent applications, as well as any corresponding national patent applications, are incorporated herein by reference. These applications teach, inter alia, recombinant DNA methods of cloning and expressing HCV sequences, HCV polypeptides, HCV immunodiagnostic techniques, HCV probe diagnostic techniqu.es, anti-HCV antibodies, and methods of isolating new hCV sequences, including sequences of new HCV isolates. 8 LV 10306

Disclosure of the Invention

The present invention is based, in part, on new HCV sequences and polypeptides that are not disclosed In EPO Pub. No. 318,216, or in PCT Pub. No. WO 89/04669. Included within the invention is the application of these new seguences and polypeptides in, inter alia, immunodiagnostics, probe diagnostics, anti-HCV antibody production, PCR technology and recombinant DNA technology. Included within the invention, also, are new immunoassays based upon the immunogenicity of HCV polypeptides disclosed herein. The new subject matter claimed herein, while developed using technigues described in, for example, EPO Pub. No. 318,216, has a priority datē which antecedes that publication, or any counterpart thereof. Thus, the invention provides novel compositions and methods useful for screening samples for HCV antigens and antibodies, and useful for treatment of HCV infections.

Accordingly, one aspect of the invention Is a recombinant polynucleotide comprising a seguence derived from HCV CDNA, wherein the HCV CDNA is in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b, or clone pil4a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16jh, or wherein the HCV CDNA is of a seguence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17.

Another aspect of the invention is a purified polypeptide comprising an epitope encoded within HCV CDNA wherein the HCV CDNA is of a seguence Indicated by 9 nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17. ·

Yet another aspect of the invention is an immunogenic polypeptide produced by a celi transformed with a recombinant expression vector comprising an ORF of DNA derived from HCV CDNA, wherein the HCV CDNA is comprised of a sequence derived from the HCV CDNA sequence In clone CA279a, or clone CA74a, or clone 13if or clone CA290a, or clone 33C or clone 40b, or clone 33b, or clone 25c, or clone 14c, or clone Bf, or clone 33f, or clone 33g, or clone 39c, or clone 15e, and wherein the ORF is operably linked to a control sequence compatible with a desired host.

Another aspect of the invention is a peptide comprising an HCV epitope, wherein the peptide is of the formula AAx-AAy1 wherein x and y designate amino acid numbers shown in Fig. 17, and wherein the peptide is selected from the group consisting of AA1-AA25, AA1-AAS0, AA1-AA94, AA9-AA177, AA1-AA10, AA5-AA20, AA20-AA25, AA35-AA45, AA50- AA100, AA40-AA90, AA45-AA65, AA65-AA75, AA80-90, AA99- AA120, AA95-AA110, AA105-AA120, AA100-AA150, AA150-AA200, AA155-AA170, AA190-AA210, AA200-AA250, AA220-AA240,. AA245-AA265. AA250-AA300, AA290-AA330, AA290-305, AA30 0-AA3 50, AA310-AA330, AA350-AA400, AA380-AA395, AA405-AA495, AA400-AA450, AA405-AA415, AA415-AA425, AA425-AA435, AA437-AA582, AA450-AA500, AA440-AA460, AA460-AA470, AA475-AA495, AA500-AASSO, AAS11-AA690, AA515-AA550, AA550-AA600, AA550-AA625t AA575-AA605, 10 LV10306 AA5BS-AA600, AA600-AA650, AA600-AA625, AA635-AA665t AA650-AA700, AA645-AA680, AA700-AA750, AA700-AA725, AA700-AA750, AA7 2 5-AA7 75t AA770-AA790, AA750-AA800i AA800-AA815, AA825-AAB50, AA850-AA875, AA800-AA850, AA920-AA990, AA850-AA900Ī AA920-AA945, AA940-AA965, AA970-AA990, AA950-AA1000, AA1000-AA1060, AA1000-AA1025, AA1000-AA1050, AA1025-AA1040, AA1040-AA1055, AA1075-AA1175, AA1050-AA1200, AA1070-AA1100, AA1100-AA1130, AA1140-AA1165, AA1192-AA1457, AA1195-AA1250#1 AA1200-AA1225, AA1225-AA1250, AA1250-AA1300, AA1260-AA1310, AA1260-AA1280, AA1266-AA1428, AA1300-AA1350, AA1290-AĀ1310, AA1310-AA1340, AA1345AA1405, AA1345-AA1365, AA1350-AA1400, AA1365-

AA1380, AA1380-AA1405, AA1400-AA1450, AA1450-AA15OOF AA14 6 0-AA147 5, AA1475-AA1515, AA1475-AA1500, AA1500-AA1550, AA1500-AA1515, AA1515-AA15500 AA1550-AA1600, AA1545-AA1560, AA1569-AA1931, AA1570-AA1590, ΑΆ1595-ΆΑ1610, AA1590-AA1650, AA1610-AA1645, ΑΑ1650-ΑΆ1690, AA1685-AA1770, AA1689- AA1805, AA1690-AA1720, ΑΑ1694-ΑΆ1735, ΑΑ1720-ΆΆ1745, AA1745-AA1770, AA1750-AA1800, AA1775-AA1810, AA1795-AA1850, AA1850-AA1900, AAl900-ΆΆ1950, AA1900-AA1920, AA1916-AA2021, AA1920-AA1940, AA1949-AA2124, AA1950-AA2000, ΑΆ1950-ΑΑ1985, AA1980-AA2000, AA2000-AA2050, AA2005-AA2025, AA2020-AA2045, AA2045-AA2100, AA2045-AA2070, AA2054-AA2223, AA2070-AA2100, AA2100-AA2150, AA2150-AA2200, AA2200-AA2250, AA2200-AA2325, AA2250-AA2330, AA2255-AA2270, AA2265-AA2280, AA2280-AA2290, AA2287-AA2385, AA2300- AA2 350, AA2290-AA2310, AA2310-AA2330/ 11 AA2330-AA2350, AA2350-AA2400, AA2348-AA2464 AA2345-AA2415, AA2345-AA2375, AA2370-AA2410 AA2371-AA2502, AA2400-AA2450, AA2400-AA2425 AA2415-AA2450, AA2445-AA2500, AA2445-AA2475 AA2470-AA2490, AA2500-AA2550, AA2505-AA2540 AA2535-AA2560, AA2550-AA2600, AA2560-AA2580 AA2 600-AA2 650, AA2605-AA2620, AA2620-AA2650 AA2640-AA2660, AA2650-AA2 700t AA2655-AA2670 AA2670-AA2700, AA2700-AA2750, AA2740-AA2760 AA2 7 5 0-AA2 800, AA2755-AA2780, AA2780-AA2830, AA2785-AA2810, AA2796-AA2886 ΑΑ2810-ΑΑ2825, ΑΑ2800-ΑΑ2850, ΑΑ2850-ΑΑ29009,ΑΑ2850-ΑΑ2865, ΑΑ2885-ΑΑ2905, ΑΑ2900-ΑΑ2950, ΑΑ-2910-ΑΑ.2930, ΑΑ2925-ΑΑ2950, AA2945-end(C' termiņai).

Stili another aspect of the invention is a monoclonal antibody directed against an epitope encoded in HCV cDNA, wherein the HCV cDNA is of a sequence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or is the seguence present in clone 13i, or clone 26j, or clone 59a, or clone 84a. or clone CA156e, or clone 167b, or clone pil4a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16jh.

Yet another aspect of the invention is a preparation of purified polyclonal antibodies directed against a polypeptide comprised of an epitope encoded within HCV cDNA, wherein the HCV cDNA is of a sequence Indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or is the sequence present in in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b, or clone pil4a, or clone CA216a, 12 LV 10306 or clone CA290a, or clone ag30a, or clone 205a, or clone lgg, or clone 16jh.

Stili another aspect of the invention is a polynucleotide probe for HCV, wherein the probe is comprised of an HCV seguence derived from an HCV CDNA seguence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or from the complement of the HCV CDNA seguence.

Yet another aspect of the invention is a kit for analysing samples for the presence of polynucleotides from HCV comprising a polynucleotide probe containing a nucleotide sequence of about 8 or - more nucleotides, wherein the nucleotide seguence is derived from HCV CDNA which is of a seguence indicated by nucleotide numbers 319 to 1348 or 8659 to 8866 in Fig. 17, wherein the polynucleotide probe is in a suitable Container.

Another aspect of the Invention is a kit for analysing samples for the presence of an HCV antigen comprising an antibody which reacts immunologically with an HCV antigen, vrtierein the antigen contains an epitope encoded within HCV CDNA which is of a seguence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or wherein the HCV cDNA is in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b. or clone pil4a, or clone CA216a, or clone CA290a. or clone ag30a, or clone 205a, or clone 18g, or clone 16jh.

Yet another aspect of the invention is a kit for analysing samples for the presence of an HCV antibody comprising an antigenic polypeptide containing an HCV epitope encoded within HCV cDNA which is of a seguence indicated by nucleotide numbers -319 to 1348 or 8659 to 13 8866 in Fig. 17, or is in clone 131, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b. or clone pll4a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16jh.

Another aspect of the invention is a kit for analysing samples for the presence of an HCV antibody comprising an antigenic polypeptide expressed from HCV CDNA in clone CA279a, or clone CA74a, or clone 13i, or clone CA290a, or clone 33C or clone 40b, or clone 33b, or clone 25c, or clone 14c, or clone 8f, or clone 33f, or clone 33g, or clone 39c, or clone 15e, wherein the antigenic polypeptide is present in a suitable Container.

Stili another aspect of the invention is a method for detecting HCV nucleic acids In a sample comprising: (a) reacting nucleic acids of the sample with a polynucleotide probe for HCV, wherein the probe is comprised of an HCV sequence derived from an HCV cDNA seguence Is of a sequence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, and wherein the reacting is under conditions which allow the formation of a polynucleotide duplex between the probe and the HCV nucleic acid from the sample; and (b) detecting a polynucleotide duplex which contains the probe, formed in step (a).

Yet another aspect of the invention is an immunoassay for detecting an HCV antigen comprising: (a) incubating a sample suspected of containing an HCV antigen with an antibody directed against an HCV epitope encoded in HCV cDNA, wherein the HCV cDNA Is of a sequence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or is the 14 LV10306 sequence present in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b, or clone pil4a·, or clone CA216a, or clone CA290a, or clone ag30a. or clone 205a, or clone 18g, or clone 16jh, and wherein the incubating is under conditions which allow formation of an antigenantibody complex; and (b) detecting an antibody-antigen complex formed in step (a) which contains the antibody.

Stili another aspect of the invention is an lmmunoassay for detecting antibodies directed against an HCV antigen comprising: (a) incubating a sample suspected of containing anti-HCV antibodies with an antigen polypeptide containing an epitope encoded in HCV cDNA, wherein the HCV cDNA is of a secpience indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17. or is the seguence present in clone 13i, or clone 26 j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b, or clone pil4a, or clone CA216a. or clone CA290a, or clone ag30a, or clone 205a, or clone lgg, or clone 16 jh, and wherein the incubating is under conditions which allow formation of an antigenantibody complex; and detecting an antibody-antigen complex formed in step (a) which contains the antigen polypeptide.

Another aspect of the invention is a vaccine for treatment of HCV infection comprising an immunogenic polypeptide containing an HCV epitope encoded in HCV cDNA, vrtierein the HCV cDNA is of a sequence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17 or is the secpience present in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 167b, or clone pll4a, or clone CA216a, or clone CA290a, 15 or clone ag30a, or clone 205a, or clone 18g, or clone 16 jh, and wherein the immunogenic polypeptide Is present in a pharmacologically effective dose in a pharmaceutically acceptable excipient.

Yet another aspect of the Inventlon is a method for producing antibodies to HCV comprising administering to an individual an isolated immunogenic polyeptide containing an HCV epitope encoded in HCV CDNA, wherein the HCV CDNA is o£ a seguence indicated by nucleotide numbers -319 to 1348 or 8659 to 8866 in Fig. 17, or is of the seguence present in clone CA279a, or clone CA74a, or clone 13i, or clone CA290a, or clone 33C or clone 40b, or clone 33b, or clone 25c, or clone 14c, or clone 8f, or clone 33f, or clone 33g, or clone 39c, or clone 15e, and wherein the immunogenic polypeptide is present In a pharmacologically effective dose in a pharmaceutically acceptable excipient.

Stili another aspect of the Invention is an antisense polynucleotide derived from HCV CDNA, wherein the HCV CDNA is that shown in Fig. 17.

Yet another aspect of the invention is a method for preparing purified fusion polypeptide C100-3 comprising: (a) providing a crude celi lysate containing polypeptide CIO0-3, (b) treating the crude celi lysate with an amount of acetone which causes the polypeptide to precipitate, (c) isolating and solubilizing the precipitated material, (d) isolating the C100-3 polypeptide by anion exchange chromatography, and 16 LV10306 (e) further isolating the C100-3 polypeptide of step (d) by gel filtration.

Brief Description of the Drawings

Fig. 1 shows the seguence of the HCV cDNA in clone 12f, and the amino aclds encoded thereln.

Fig. 2 shows the HCV cDNA seguence in clone k91, and the amino acids-encoded therein.

Fig. 3 shows the sequence of clone 15e, and the amino acids encoded therein.

Fig. 4 shows the nucleotide sequence of HCV cDNA in clone 13i, the amino acids encoded therein, and the seguences which overlap with clone 12f.

Fig. 5 shows the nucleotide seguence of HCV cDNA in clone 2 6j, the amino acids encoded therein, and the sequences which overlap clone 13i.

Fig. 6 shows the nucleotide seguence of HCV cDNA In clone CA59a, the amino acids encoded therein, and the sequences which overlap with clones 26J and K9-1.

Fig. 7 shows the nucleotide sequence of HCV cDNA in clone CAS4a, the amino acids encoded therein, and the seguences which overlap with clone CA59a.

Fig. 8 shows the nucleotide seguence of HCV cDNA in clone CA156e, the amino acids encoded therein, and the seguences which overlap with CA84a.

Fig. 9 shows the nuclootide seguence of RCV cDNA in clone CA167b, the amino acids encoded therein, and the seguences which overlap CA156e.

Fig. 10 shows the nuclootide seguence of HCV cDNA In clone CA216a, the amino acids encoded therein, and the overlap with clone CA167b. 17

Fig. 11 shows the nuc lootide seguence of IICV cDNA In clone CA290ai the amino aclds encoded thereiny and the overlap with clone CA216a.

Fig. 12 shovm the nucleotide seguence of HCV cDNA in clone ag30a and the overlap with clone CA290m.

Fig. 13 shows the nuclootide seguence of RCV cDNA In clone CA205a, and the overlap with the HCV cDNA seguence In clone CA290a.

Fig. 14 shovm the nuclootide seguence of HCV

cDNA In clone I8g, and the overlap with the HCV cDNA seguence in clone ag30a.

Fig. 15 shows the nucleotide seguence of HCV cDNA In clone 16Jh, the amino acids encoded therein, and the overlap of nucleotides with the HCV cDNA seguence In clone 15o.

Fig. 16 shovm the ORP of HCV cDNA derived from clones,pil4at CA167b, CA156c, CAB4a# CAS9at X9-li 12ft I4it llbr 7£, 7e. Oh, 33c·# 40b# 37bf 3SF 36# 81# 32t 33bi 25c, 14c# Of, 33£, 33g, 39c, 35f, lggp 26gt and 15e. Fig. 17 shovm the sense strand of the compiled HCV cDNA seguence derived from the above-described clones and the compiled HCV cDNA seguence published In EPO Pub. No. 318.216. The clones from which the seguence was derive.d are bll4a, 18g, ag30a, CA205a, CA290a, CA216a, pil4a, CA167b, CAl56e, CA84a, CAS9a, K9-1 (also called k9- 1) / 26 j , 13i, 12f , 14i, 11b, 7f, 7e, 8h,, 33c, 40b, 37b, 35, 36, 81, 32, 33b. 25c, 14c. 8f, 33f, 39c, 35f, 19g, 26g, 15e, b5a, and 16jh. In the figurē the three horizontal dashes above the seguence Indicate the position of the putative Initiator nothionine codon; the two vertical daehee Indicate the first and last nucleotides of the published seguence. Also shovm in the 18 LV10306 figurē In the amino acid sequence of the putative polyprotein encoded In the RCV cDNA.

Fig. 18 is a diagram of the Immunological colony screening method used In antigenic mapping studies.

Fig. 19 shows the hydrophobicity profilēs of polyproteint encoded in HCV and In West Nile vīrus.

Fig. 20 In a tracing of the hydrophilicity/ hydrophobicity profilē and of the antigenic Index of the putative HCV polyprotein.

Fig. 21 shovm. the conserved co-linear peptides In HCV and Flaviviruses.

Modes forCarrying Out the Invention I. Definitions

The term "hepatitis C virus" has been reserved by workers In the field for an heretofore unknown otiologic aģent of NANBH. Accordingly, an used herein, '-•hepatitis C virus". (HCV) refers to an aģent causitive of NMBH, which was formerly referred to as NANBV and/or BBNANBV. The terms HCV, NANBV, and BB-NANBV are used Interchangeably herein. As an extension of this terminology, the disease caused by HCV, formerly called NANB -hepatitis (NANBH) , is called hepatitis C. The terms NANBH and hepatitis C may be used interchangeably herein. The term "HCV", as used herein, denotes a virai species of which pathogenic strains cause NANBH, and attenuated strains or defective Interfering pārticies derived therefrom. An shovm infra.g the HCV genome is comprised of RNA. It is known that RNA containing viruses have 19 relatively high rātes of spontaneous mutation, i.e., reportedly on the order of 10"3 to 10"4 per nucleotide (Fields & Knipe (1986)). Therefore, there are multiple strains, which may be virulent or avirulent, within the HCV species described infra. The compositions and methods described herein, enable the propagation, Identification, detection, and isolatlon of the various HCV strains or isolates. Moreover, the disclosure herein allows the preparation of diagnostics and vaccines for the various strains, as well as compositions and methods that have utility in screening procedures for anti-virai aģents for pharmacologic use, such as aģents that inhibit replication of HCV.

The Information provided herein, although derived from the prototype strain or isolate of HCV, hereinafter referred to as CDC/HCV1 (also called HCVl), is sufficient to allow a virai taxonomist to identify other strains which fall within the species. The Information provided herein allows the belief that HCV Is a Flavi-like virus. The morphology and composition of Flavivirus pārticies are known, and are discussed in Brinton (1986). Generally, with respect to morphology, Flaviviruses contain a Central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections that are about 5-10 nm long with termiņai knobs about 2 nm in diameter.

Different strains or isolates of HCV are expected to contain variations at the amino acid and nucleic acids compared with the prototype isolate, HCVl. Many Isolates are expected to show much (I.e. more than about 40%) 20 LV10306 homology in the total amino acid seguence compared with HCVl. However, it may also be found that other less homologous -HCV isolates. These would be defined as HCV strains according to various criteria such as an ORF of approximately 9,000 nucleotides to approximately 12,000 nucleotidest encoding a polyprotein similar in size to that of HCVl, an encoded polyprotein of similar hydrophobic and antigenic character to that of HCVl, and the presence of co-linear peptide sequences that are conserved with HCVl. In addition, the genome would be a positive-stranded RNA. HCV encodes at least one epitope which is immunologically identifiable with an epitope in the HCV genome from which the cDNAs described herein are derived; preferably the epitope is contained an amino acid sequence described herein. The epitope is unique to HCV when compared to other known Flaviviruses. The uniqueness of the epitope may be determined by its immunological reactivity with anti-HCV antibodies and lack of immunological reactivity with antibodies to other Flavivirus species. Methods for determining immunological reactivity are known in the art, for example, by radioimmunoassay, by Elisa assay, by hemagglutination, and several examples of suitable techniques for assays are provided herein.

In addition to the above, the following parameters of nucleic acid homology and amino acid homology are applicable, either alone or in combination, in identifying a strain or isolate as HCV. Since HCV strains and isolates are evolutionarily related, it is expected that the overall homology of the genomes at the nucleotide Ievel probably will be about 40% or greater. 21 probably about 60% or greater, and even more probably about 80% or greater; and in addition that there will be corresponding contiguous sequences of at least about 13 nucleotides. The correspondence between the putative HCV strain genomic seguence and the CDC/HCVl cDNA seguence can be determined by technigues known in the art. For example they can be determined by a direct comparison of the seguence Information of the polynucleotide from the putative HCV, and the HCV CDNA seguences described herein. For example, also, they can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous reģions (for example, those which would be used prior to Sx digestion), followed by digestion with single stranded specific nuclease(s), followed by size determination of the digested fragments.

Because of the evolutionary relationship of the strains or isolates of HCV, putative HCV strains or Isolates are identifiable by their homology at the polypeptide Ievel. Generally, HCV strains or isolates are expected to be more than about 40% homologous, probably more than about 70% homologous, and even more probably more than about 80% homologous, and some may even be more. than about 90% homologous at the polypeptide Ievel. The techniques for determining amino acid sequence homology are known in the art. For example, the amino acid seguence may be determined directly and compared to the seguences provided herein. Alternatively the nucleotide seguence of the genomic material of the putative HCV may be determined (usually via a cDNA intermediate), the amino acid seguence encoded therein 22 LV10306 can be determined, and the corresponding reģions compared.

As used herein, a polynucleotide "derived from" a designated sequence refers to a polynucleotide seguence which is comprised of a sequence of approximately at least about 6 nucleotides, preferably at least about 8

nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding to a region of the designated nucleotide seguence. "Corresponding" means homologous to or complementary to the designated sequence. Preferably, the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a seguence which is unique to an HCV

genome. Whether or not a sequence Is unique to the HCV genome can be determined by technigues known to those of skill in the art. For example, the seguence can be compared to seguences in databanks, e.g., Genebank, to determine whether it Is present in the uninfected host or other organisms. The seguence can also be compared to the known seguences of other virai aģents, including those which are known to inducē hepatitis, e.g., HAV, HBV, and HDV, and to other members of the Flaviviridae. The correspondence or noncorrespondence of the derived seguence to other seguences can also be determined by hybridization under the appropriate stringency conditions. Hybridization technigues for determining the complementarity of nucleic acid seguences are knovm in the art, and are discussed infra. See also, for example, Maniatis et al. (1982). In addition, mismatches of duplex polynucleotides formed by hybridization can be determined by knovm technigues, including for example, digestion 23 with a nuclease such as S1 that specifically digests single-stranded a-reas in duplex polynucleotides. Reģions from which 1:ypical DNA seguences may be "derived" include but are not limited to, for example, reģions encoding specific epitopes, as well as non-transcribed and/or non-translated reģions.

The derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but may be generated in any manner, including for example, Chemical synthesis or DNA replication or reverse transcription or transcription. In addition, combinations of reģions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.

Similarly, a polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the seguence. A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid seguence, for example, the HCV cDNA seguences described herein, or from an HCV genome; it may be generated in any manner, including for example, Chemical synthesis, or expression of a recombinant expression system, or isolation from mutated HCV. A recombinant or derived polypeptide may include one or more analogs of amino acids or unnatural amino acids in its seguence. Methods 24 LV10306 of inserting analogs of amino acids into a sequence are known in the art. It also may include one or more labels, which are known to those of skill in the art.

The term "recombinant polynucleotiden as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation which: (1) is not associated with ali or a portion of a polynucleotide with which it is associated iņ nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, as well as double- and single stranded RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution,of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with unchanged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metāls, radioactive metāls, boron, oxidative metāls, etc.), those containing alkylators, those with modified linkages (e.g., alpha 25 anomeric nucleic acids, etc.), as well as unmodified formš of the polynucleotide.

The term "purified virai polynucleotide" refers to an HCV genome or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more pre£erably less than about 90% of polypeptides with which the virai polynucleotide is naturally associated. Technicpies for purifying virai polynucleotides from virai pārticies are knovm in the art, and include for example, disruption of the particle with a chaotropic aģent, differential extraction and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density.

The term "purified virai polypeptide" refers to an HCV polypeptide or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more preferably less than about 90%, of cellular components with which the virai polypeptide is naturally associated. Techniques for purifying virai polypeptides are known in the art, and examples of these technigues are discussed infra. The term "purified virai polynucleotide" refers to an HCV genome or fragment thereof which is essentially free, i.e., contains less than about 20%, preferably less than about 50%, and even more preferably less than about 70% of polypeptides with which the virai polynucleotide is naturally associated. Techniques for purifying virai polynucleotides from virai pārticies are known in the art, and include for example, disruption of the particle with a chaotropic aģent, and separation of the polynucleotide (s) and polypeptides by ion-exchange 26 LV10306 chromatography, affinity chromatography, and sedimentation according to density. "Recombinant host celis", "host celis", "celis", "celi lines", "celi cultures", and other such terms denoting microorganisms or higher eukaryotic celi lines cultured as unicellular entities refer to celis which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original celi which has been transfected. It is understood that the progeny of a single parental celi may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, duo to natūrai, accidental, or deliberate mutation. A "replicon" is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc. that behaves as an autonomous unit of polynucleotide replication within a celi; i.e., capable of replication under its own control. A "vector", is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. "Control sequence" refers to polynucleotide seguences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally, such control seguences include promoters, terminators and, in some instances, enhancers. 27

The term "control seguences" is intended to include, at a minimum, ali components whose presence is necessary for expression,· and may also include additional components whose presence is advantageous, for example, leader seguences. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function In their intended manner. A control sēquence "Operably linked" to a coding sequēnce is ligated in such a way that expression of the coding sequence Is achieved under conditions compatible with the control sequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding seguence. A "coding sequence" is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control .of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'terminus. A coding sequence can include, but is not limited to mRNA, cDNA, and recombinant polynucleotide seguences. "Immunologically identifiable with/as" refers to the presence of epitope(s) and polypeptides(s) which are also present in the designated polypeptide(s), usually HCV proteīns. Immunological identity may be determined by antibody binding and/or competition in binding; these techniques are known to those of average skill in the art, and are also illustrated lnfra. 28 LV 10306

As used herein, "epitope" refers to an antigenic determinant of a polypeptide; an epitope could comprise 3 amino acids· in a spatial conformation which is unique to the epitope, generally an epitope consists of at least 5 such amino acids, and more usually, consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of amino acids are knovm in the art, and include, for example, x-ray crystallography and 2dimensional nuclear magnetic resonance. A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitors) a knovm polypeptide(s) containing an epitope against which the antibody is directed. The techniques for determining vrhether a polypeptide is immunologically reactive with an antibody are known in the art.

As used herein, the term "immunogenic polypeptidell is a polypeptide that elicits a cellular and/ or humoral response, vrhether alone or linked to a carrier in the presence or abšence of an adjuvant.

The term ,,polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteīns are included vrithin the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, 29 polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. "Transformation", as used herein, refers to the insertion of an erogenous polynucleotide into a host celi, irrespective of the method used for the insertion, v for example, direct uptake, transduction, f-mating or electroporation. The erogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome. "Treatment" as used herein refers to prophylaxis and/or therapy.

An "individual", as used herein, refers to vertebrates, particularly members of the mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humāns.

As used herein, the, "sense strand" of a nucleic acid contains the sequence that has sequence homology to that of MRNA. The "anti-sense strand", contains a seguence which is complementary to that of the "sense strand".

As used herein, a "positive stranded genome" of a virus is one in which the genome, whether RNA or DNA, is single-stranded and which encodes a virai polypeptide(s). Examples of positive stranded RNA viruses include Togaviridae, Coronaviridae, Retroviridae,

Picornaviridae, and Caliciviridae. Included also, are the Flaviviridae, which were formerly classified as

Togaviradae. See Fields & Knipe (1986). 30 LV10306

As used herein, ,,antibody-containing body component" refers to a component of an individuāls body which is a source of the antibodies of interest. Antibody containing body component s are known in the art, and include but are not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, white blood celis, and myelomas.

As used herein, "purified HCV" refers to a preparation of HCV which has been isolated from the cellular constituents with which the virus is normally associated, and from other types of viruses which may be present in the infected tissue. The technigues for Isolating viruses are knovm. to those of skill in the art, and include, for example, centrifugation and affinity chromatography; a method of preparing purified HCV is discussed infra.

The term ''HCV pārticies" as used herein include entire virion as well as pārticies which are intermediates in virion formation. HCV pārticies generally have one or more HCV proteīns associated with the HCV nucleic acid.

As used herein, the term "probe" refers to a polynucleotide which forms a hybrid structure with a sequence in a target region, due to complementarity of at least one seguence in the probe with a sequence in the target region. The probe, however, does not contain a sequence complementary to sequences used to prime the polymerase chain reaction.

As used herein, the term "target region" refers to a region of the nucleic acid which is to be amplified and/or detected. 31 which

As used herein, the term "virai RNA", includes HCV RNA, refers to RNA from the virai genome, fragments ‘thereof, transcripts thereof, and mutant seguences derived therefrom.

As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from an individual, Including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood celis, tumors, orgāns, and also samples of An vitro celi culture constituents (including but not limited to conditioned medium resulting from the growth of celis in celi culture medium, putatively virally Infected celis, recombinant celis, and celi components). II · Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such technigues are explained fully in the literature. See e.g,, Maniatis, Fitsch & Sambrook, MOLECULAR CLONING; A LABORATORĪ MANUAL (1982); DNA CLONING, VOLUMES I AND II (D.N Glover ed. 1985); OLIGONUCLEOTIDE SĪNTHESIS (M.J. Gait ed, 1984); NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. Higgins eds. 1984); TRANSCRIPTION AND TRANSLATION (B.D. Hames & S.J. Higgins. eds. 1984); ANIMAL CELL CULTURE (R.I. Freshney ed. 1986); IMMOBILIZED ČELLS AND ENZīMES (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); 32 LV10306 the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN ČELLS (J.H. Miller and M.P. Čalos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN.CELL AMD MOLECULAR BIOLOGY (Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, Ν.Υ.), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGYR VOLUMES I-IV (D.M. Weir and C. C. Blackwell eds 1986). Ali patents, patent applications, and publications mentioned herein, both supra and Infra, are hereby incorporated herein by reference.

The useful materiāls and processes of the present invention are made possible by the provision of a family of nucleotide sequences isolated from CDNA libraries which contain HCV cDNA sequences. These cDNA librar-ies were derived from nucleic acid sequences present in the plasma of an HCV-infected chimpanzee. The construction of one of these libraries, the "c" library (ATCC No. 40394), was reported in EPO Pub. No. 318.216. Several of the clones containing HCV cDNA reported herein were obtained from the "c" library. Although other clones reported herein were obtained from other HCV cDNA libraries, the presence of clones containing the sequences in the "c" library was confirmed. As discussed in EPO Pub. No. 318,216, the family of HCV cDNA seguences isolated from the "c" library are not of human or chimpanzee origin, and show no significant homology to sequences contained within the HBV genome.

The availability of the HCV cDNAs described herein permits the construction of polynucleotide probes which 33 are reaģents useful for detecting virai polynucleotides in biological samples, including donated blood. For example, fr-om the seguences it is possible to synthesize DNA oligomers of about 8-10 nucleotides, or larger, which are useful as hybridization probes to detect the presence of HCV RNA in, for example, donated blood, sera of subjects suspected of harboring the virus, or celi culture systems in which the virus is replicating. In addition, the cDNA seguences also allow,the design and production of HCV specific polypeptides which are useful as diagnostic reaģents for the presence of antibodies raised during HCV infection. Antibodies to purified polypeptides derived from the cDNAs may also be used to detect virai antigens in biological samples, including, for example, donated blood samples, sera from patients with NANBH, and in tissue culture systems being used for HCV replication. Moreover, the immunogenic polypeptides disclosed herein, which are encoded in portions of the ORF of HCV cDNA shown in Fig. 17, are also useful for t,HCV screening, diagnosis, and treatment, and for raising antibodies which are also useful for these purposes. in addition, the novel cDNA seguences described herein enable further characterization of the HCV genome. Polynucleotide probes and primers derived from these seguences may be used to amplify seguences present in CDNA libraries, and/or to screen cDNA libraries for additional overlapping cDNA seguences, which, in turn, may be used to obtain more overlapping seguences. As indicated lnfra. and in EPO Pub. No. 318,216, the genome of HCV appears to be RNA comprised primarily of a large open reading frame (ORF) which encodes a large polyprotein. 34 LV10306

The HCV cDNA sequences provided hereiii/ the polypeptides derived from these sequences, and the immunogenic· polypeptides described herein, as well as antibodies directed against these polypeptides are also useful in the isolation and Identification of the blood-borne NABV (BB-NANBV) aģent(s). For example, antibodies directed against HCV epitopes contained in polypeptides derived from the cDNAs may be used in processes based upon affinity chromatography to isolate the virus. Alternatively, the antibodies may be used to identify virai pārticies isolated by other techniques. The virai antigens and the genomic material within the isolated virai pārticies may then be further characterized.

In addition to the above, the Information provided infra allows the identification of additional HCV strains or isolates. The isolation and characterization of the additional HCV strains or isolates may be accomplished by isolating the nucleic acids from body components which contain virai pārticies and/or virai RNA, creating cDNA libraries using polynucleotide probes based on the HCV cDNA probes described infra., screening the libraries for clones containing HCV cDNA seguences described infra., and comparing the HCV cDNAs from the new isolates with the cDNAs described infra. The polypeptides encoded therein, or in the virai genome, may be monitored for immunological cross-reactivity utilizing the polypeptides and antibodies described supra. Strains or isolates which fit within the parameters of HCV, as described In the Definitions section, supra, are readily Identifiable. Other methods for identifying HCV strains will be obvious to those of skill in the art, based upon the Information provided herein. 35

Isolation of the HCV CDNA Seguences

The novel HCV cDNA seguences described infra. extend the seguence of the cDNA to the HCV genome reported in EPO Pub. No. 318,216. The seguences which are present in clones bll4a, 189, ag30a, CA205a, CA290a, CA216a, pil4a, CA167b, CAl56e, CA84a, and CA59a lie upstream of the reported seguence, and when compiled, yield nucleotides nos. -319 to 1348 of the composite HCV cDNA seguence. (The negative number on a nucleotide indicates its distance upstream of the nucleotide which starts the putative initiator MET codon.) The seguences which are present in clones b5a and 16jh lie dovmstream of the reported seguence, and yield nucleotides nos. 8659 to 8866 of the composite seguence. The composite HCV cDNA seguence which includes the seguences in the aforementioned clones, is shown in Fig. 17.

The novel HCV cDNAs described herein were isolated from a number of HCV cDNA libraries, including the "c" library present in lambda gtll (ATCC No. 40394) . The HCV cDNA libraries were constructed using pooled serum from a chimpanzee with chronic HCV Infection and containing a high titer of the virus, I.e., at least 10 6 chimp infectious doses/ml (CID/ml) . The pooled serum was used to isolate virai pārticies; nucleic acids isolated from these pārticies was used as the template in the construction of cDNA libraries to the virai genome. The procedures for isolation of putative HCV pārticies and for constructing the "c" HCV cDNA library is described in EPO Pub. No. 318,216. other methods for constructing HCV cDNA libraries are knovm in the art, and some of these methods are described infra., in the Examples. Isolation 36 of the sequences was by screening the libraries using synthetic polynucleotide probes, the seguences of which were derived from the 5',-region and the 3'-region of the known HCV cDNA sequence. The description of the method to retrive the cDNa seguences is mostly of historical interest. The resultant sequences (and their complements) are provided herein, and the seguences, or any portion thereof, could be prepared using synthetic methods, or by a combination of synthetic methods with retrieval of partial seguences using methods similar to those described herein.

Preparation of Virai Polypeptides and Fragments

The availability of HCV cDNA seguences, or nucleotide seguences derived therefrom (including segments and modifications of the seguence), permits the construction of expression vectors encoding antigenically active reģions of the polypeptide encoded in either strand. These antigenicaļly active reģions may be derived from coat or envelope antigens pr frpm cpre antigens, pr from antigens which are non-structural including, for example, polynucleotide binding proteīns, polynucleotide polymerase(s), and other virai proteins reguired for the replication and/or assembly of the virus particle. Fragments encoding the desired polypeptides are derived from the cDNA clones using conventional restriction digestion or by synthetic methods, and are ligated into vectors which may, for example, contain portions of fusion seguences such as beta-galactosidase or superoxide dismutase (SOD), preferably SOD. Methods and vectors 37 which are useful for the production of polypeptides which contain fusion sequences of SOD are described in European Patent Office Publication number 0196056, published October 1, 1986. Vectors for the exprešsion of fusion polypeptides of SOD and HCV polypeptides encoded in a number of HCV clones are described infra., in the Examples. Any desired portion of the HCV CDNA containing an open reading frame, in either sense strand, can be obtained as a recombinant polypeptide, such as a mature or fusion protein; alternatively, a polypeptide encoded in the CDNA cah be provided by Chemical synthesis.

The DNA encoding the desired polypeptide, whether in fused or mature form, and whether or not containing a signal seguence to permit secretion, may be ligated into expression vectors suitable for any convenient host. Both eukaryotic and prokaryotic host systems are presently used in forming recombinant polypeptides, and a summary of some of the more common control systems and host celi lines is given infra. The polypeptide is then isolated from lysed celis or from the culture medium and purified to the extent needed for its intended use. Purification may be by techniques known in the art, for example, differential extraction, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variēty of methods for purifying proteins. Such polypeptides can be used as diagnostics, or those which give rise to neutralizing antibodies may be formulated into vaccines. Antibodies raised against these polypeptides can also be used as diagnostics, or for passive immunotherapy. In addition, as discussed infra., antibodies to these 38 LV 10306 polypeptides are useful for isolating and identifying HCV pārticies.

Preparation of Antigenic Polypeptides and Conjugation with Carrier

An antigenic region of a polypeptide is generally relatively small-typically 8 to 10 amino acids or less in length. Fragments of as few as 5 amino acids may characterize an antigenic region. These segments may correspond to reģions of HCV antigen. Accordingly, using the cDNAs of HCV as a basis, DNAs encoding short segments of HCV polypeptides can be expressed recombinantly either as fusion proteins, or as isolated polypeptides. In addition, short amino acid seguences can be conveniently obtained by Chemical synthesis. In instances wherein the synthesized polypeptide is correctly configured so as to provide the correct epitope, but is too small to be immunogenic, the polypeptide may be linked to a suitable carrier. A number of technigues for obtaining such linkage are known in the art, including the formation of disulfide linkages using N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) obtained from Pierce Company, Rockford, Illinois, (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue.) These reaģents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilonamino on a lysine, or other free amino group in the other. A variety of such disulf ide/amide-forming 39 aģents are knovm. See, for example, Immun. Rev. (1982) 62:185. Other bifunctional coupling aģents form a thioether rather than a disulfide linkage. Many of these thio-etherforming aģents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid, and the like. The carboxyl groups can be activated by combining them with succinimide or l-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Additional methods of coupling antigens employs the rotavirus/"binding peptide" system described in EPO Pub. No. 259,149, the disclosure of which is incorporated herein by reference. The foregoing list is not meant to be exhaustive, and modifications of the named compounds can clearly be used.

Any carrier may be used which does not itself inducē the production of antibodies harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteīns; polysaccharides, such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids, such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus pārticies. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteīns well known to those skilled in the art.

In addition to full-length virai proteīns, polypeptides comprising truncated HCV amino acid seguences encoding at least one virai epitope are useful immunological reaģents. For example, polypeptides comprising such truncated seguences can be used as 40 reaģents in an immunoassay. These polypeptides also are candidate subunit antigens in compositions for antiserum produetion or vaccines. While these truncated seguences can be produced by various known treatments of native virai protein, it is generally preferred to make synthetic or recombinant polypeptides comprising an HCV sequence. Polypeptides comprising these truncated HCV seguences can be made up entirely of HCV seguences (one or more epitopes, either contiguous or noncontiguous), or HCV seguences and heterologous seguences in a fusion protein.

Useful heterologous seguences include seguences that provide for secretion from a recombinant host, enhance the immunological reactivity of the HCV epitope(s), or facilitate the coupling of the polypeptide to an immunoassay support or a vaccine carrier. See, e.g., EPO Pub. No. 116,201; U.S. Pat. No. 4,722,840; EPO Pub. No. 259,149; U.S. Pat. No. 4,629,783, the disclosures of which are incorporated herein by reference.

The size of polypeptides comprising the truncated HCV seguences can vary widely, the minimum size being a seguence of sufficient size to provide an HCV epitope, while the maximum size is not critical. For convenience, the maximum size usually is not substantially greater than that reguired to provide the desired HCV epitopes and function(s) of the heterologous seguence, if any. Typically, the truncated HCV amino acid seguence will range from about 5 to about 100 amino acids in length. More typically, hovever, the HCV seguence will be a maximum of about 50 amino acids in length, preferably a maximum of about 30 amino acids. It is usually desirable to select HCV seguences of at least about 10, 12 or 15 41 amino acids, up to a maximum of about 20 or 25 amino acids.

Trunca-ted HCV amino acid seguences comprising epitopes can be identified in a number of ways. For example, the entire virai protein seguence can be screened by preparing a series of short peptides that together span the entire protein seguence. An example of antigenic screening of the reģions of the HCV polyprotein is shown infra. In addition, by starting with, for example# lOOmer polypeptides, it would be routine to tēst each polypeptide for the presence of epitope(s) showing a desired reactivity, and then testing progressively smaller and overlapping fragments from an identified lOOmer to map the epitope of interest. Screening such peptides in an immunoassay is within the skill of the art. It is also known to carry out a Computer ahalysis of a protein sequence to identify potential epitopes, and then prepare oligopeptides comprising the identified reģions for screening. Such a Computer analysis of the HCV amino acid sequence Is shown in Fig. 20, where the hydrophilic/hydrophobic character is displayed above the antigen index. The amino acids are numbered from the starting MET (position 1) as shovm In Fig. 17. It is appreciated by those of skill in the art that such Computer analysis of antigenicity does not always identify an epitope that actually exists, and can also incorrectly identify a region of the protein as containing an epitope.

Exaxnples of HCV amino acid seguences that may be useful, which are expressed from expression vectors comprised of clones 5-1-1, 81, CA74a, 35f, 279a, C36, C33b, CA290a, C8f, C12f, 14c, 15e, C25c, C33c, C33f, 33g, 42 LV 10306 C39c, C40b, CA167b are described infra. Other examples of HCV amino acid sequences that may be useful as described herein are -set forth below. It is to be understood that these peptides do not necessarily precisely map one epitope, and may also contain HCV seguence that is not iīnmunogenic. These non-immunogenic portions of the seguence can be defined as described above using conventional technigues and deleted from the described sequences. Further, additional truncated HCV amino acid seguences that comprise an epitope or are iīnmunogenic can be identified as described above. The £ollowing seguences are given by amino acid number (i.e., "AAn") where n is the amino acid number as shovm, in Fig. 17: AA1-AA25; AA1-AA50; AA1-AA84; AA9-AA177; AA1-AA10; AA5- AA20; AA20-AA25; AA35-AA45; AA50-AA100; AA40-AA90; AA45-AA65; AA65-AA75; AA80- 90; AA99-AA120; AA95-AA110; AA105-AA120; AA100-AA150; AA150-AA200; AA155-AA170; AA190-AA210; AA200-AA250; AA220-AA240; AA245-AA265; AA250-AA300; AA290-AA330; AA290-305; AA300-AA350; AA310-AA330; AA350-AA400; AA380-AA395; AA405-AA495; AA400-AA450; AA405-AA415? AA415-AA425; AA425-AA435; AA437-AA582; AA450-AASOO; AA440-AA460; AA460-AA470; AA475-AA495; AA500-AA550; AA511-AA690; AA515-AA550; AASSO-AA600; AA550-AA625; AA575-AA605; AA585-AA600; AA6 0 0-AA650; AA600-AA625; AA635-AA665; AA650-AA700; AA645-AA680; AA700-AA750; AA700-AA725; AA700-AA750; AA725-AA775; AA770-AA790; AA750-AA800; AASOO-AA815; AA825-AA850; AA850-AA875; AA800-AA850; AA920-AA990; AAB50-AA900; AA920-AA945; AA940-AA965; AA970-AA990; AA950-AA1000; AA1000-AA1060; .AA1000-AA1025; AA1000-AA1050 ; AA1025-AA1040 ; AA1040-AA1055; AA1075-AA1175 ; AA1050-AA1200 ; AA1070-AA1100; 43 ΑΑ1100-ΑΑ1130; AA1195-AA1250? ΑΑ1250-ΑΑ1300; ΑΑ1266-ΑΑ1428; ΑΑ1310-ΑΑ1340; ΑΑ1350-ΑΑ1400; ΑΑ1400-ΑΑ1450; ΑΑ1475-ΑΑ1515; ΑΑ1500-ΑΑ1515; ΑΑΙ545-ΑΑ1560; ΑΑ1595-ΑΑ1610; ΑΑ1650-ΑΑ1690; ΑΑ1690-ΑΑ1720; ΑΑ1745-ΑΑ1770; ΑΑ1795-ΑΑ1850; AA1900-AA1920? ΑΑ1949-ΑΑ2124; ΑΑ1980-ΑΑ2000; ΑΑ2020-ΑΑ2045; ΑΑ2054-ΑΑ2223; ΑΑ2150-ΑΑ2200; ΑΑ2250-ΑΑ2330; ΑΑ2280-ΑΑ2290; ΑΑ2290-ΑΑ2310; ΑΑ2350-ΑΑ2400; ΑΑ2345-ΑΑ2375; ΑΑ2400-ΑΑ2450; AA2445-AA2500? ΑΑ2500-ΑΑ2550; ΑΑ2550-ΑΆ2600; AA2605-AA2620? ΑΑ2650-ΑΑ2700; ΑΑ1140-ΑΑ1165; ΑΑ1200-ΑΑ1225; ΑΑ1260-ΑΑ1310; ΑΑ1300-ΑΑ1350; ΑΑ1345-ΑΑ1405; ΑΑ1365-ΑΑ1380; ΑΑ1450-ΑΑ1500; ΑΑ1475-ΑΑ1500; ΑΑ1515-ΑΑ1550; ΑΑ1569-ΑΑ1931; ΑΑ1590-AA1650; AA1685-AA1770; AA1694-AA1735; AA1750-AA1800; AA1850-AA19 00; AA1916-AA2021; AA1950-AA2000; AA2000-AA2050; AA2045-AA2100; AA2070-AA2100; AA2200-AA2250; AA2255-AA2270? AA2287-AA2385; AA2310-AA2330; AA2348-AA2464; AA2370-AA2410; AA2400-AA2425? AA2445-AA2475; AA2505-AA2540; AA2560-AA2580; AA2620-AA2650; AA2655-AA2670; AA1192-AA1457 AA1225-AA1250 AA1260-AAI280 AA1290-AA1310 AA1345-AA1365 AA1380-AA1405 AA14 6 0-AA147 5 AA1500-AA1550 AA1550-AA1600 AA1570-AA1590 AA1610-AA1645 AA1689-AA1805 AA1720-AA1745 AA1775-AA1810 AA1900-AA1950 AA1920-AA1940 AA1950-AA1985 AA2005-AA2025 AA2045-AA2070 AA2100-AA2150 AA2200-AA2325 AA2265-AA2280 AA2300-AA2350 AA2330-AA2350 AA2345-AA2415 AA2371-AA2502 AA2415-AA2450 AA2470-AA2490 AA2535-AA2560 AA2600-AA2650 AA2640-AA2660 AA2 670-AA2 700 LV 10306 AA2700-AA2750; AA2740-AA2760; AA2750-AA2800; AA2755-AA2780; AA2780-AA2830; AA2785-AA2810; AA2796-AA2986; AA2810-AA2825; AA2800-AA2850; AA2850-AA2900; AA2850-AA2865; AA2885-AA2905; AA2900-AA2950;AA2910-AA2930; AA2925-AA2950; AA2945-end(C' termiņai).

The above HCV amino acid seguences can be prepared as discrete peptides or incorporated into a larger polypeptide, and may find use as described herein. Additional polypeptides comprising truncated HCV sequences are described in the examples.

The observed relationship of the putative polyproteins of HCV and the Flaviviruses allows some prediction of the putative domains of the HCV "nonstructural" (NS) proteīns. The locations of the individual NS proteīns in the putative Flavivirus precursor polyprotein are fairly well-known. Moreover, these also coincide with observed gross fluctuatioņs in the hydrophobicity profilē of the polyprotein. It is established that NS5 of Flaviviruses encodes the virion polymerase, and that NS1 corresponds with a complement fixation antigen which has been shown to be an effective vaccine in animals. Recently, it has been shown that a flaviviral protease function resides in NS3. Due to the observed similarities betwen HCV and the Flaviviruses, described infra., deductions concerning the approximate locations of the corresponding protein domains and functions in the HCV polyprotein are possible. The expression of polypeptides containing these domains in a variety of recombinant host celis, including, for example, bacteria, yeast, insect, and vertebrate celis, 45 should give rise to important immunological reaģents which can be used for diagnosis, detection, and vaccines.

Although the non-structural protein reģions of the putative polyproteins of the HCV isolate described herein and of Flaviviruses appear to have some similarity, there is less similarity between the putative structural reģions reģions which are towards the N-terminus. In this region, there is a greater divergence in sequence, and in addition, the hydrophobic profilē of the two reģions show less similarity. This "divergence" begins in the N-terminal region of the putative NS1 domain in HCV, and extends to the presumed N-terminus. Nevertheless, it may stili be possible to predict the approximate locations of the putative nucleocapsid (N-termiņai basie domain) and E (generally hydrophobic) domains within the HCV polyprotein. In the Examples the predietions are based on the changes observed in the hydrophobic profilē of the HCV polyprotein, and on a knowledge of the location and character of the flavivirai proteīns. From these predietions it may be possible to identify approximate reģions of the HCV polyprotein that could correspond with useful immunological reaģents. For example, the E and NS1 proteīns of Flaviviruses are known to have efficacy as protective vaccines. These reģions, as well as some which are shown to be antigenic in the HCV isolate described herein, for example those within putative NS3, C, and NS5, etc., should also provide diagnostic reaģents. Moreover, the location and expression of viral-encoded enzymes may also allow the evaluation of anti-virai enzyme inhibitors, i.e., for example, inhibitors which prevent enzyme activity by virtue of an interaction with the enzyme itself, or substances which may prevent 46 LV 10306

expression of the enzyme, (for example, anti-sense RNA# or other drugs which interfere with expression). Preparation of Hybrid Particle Iīnmunogens Containincr HCV

Epi-topgs

The immunogenicity of the epitopes of HCV may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteīns such as, for example, that associated with hepatitis B surface antigen. Constructs wherein the NANBV epitope is linked directly to the particle-forming protein coding seguences producē hybrids which are immunogenic with respect to the HCV epitope. In addition, ali of the vectors prepared include epitopes specific to HBV, having various degrees of immunogenicity/ such as, for example, the pre-S peptide. Thus, pārticies constructed from particle forming protein which include HCV seguences are immunogenic with respect to HCV and HBV.

Hepatitis surface antigen (HBSAg) has been shown to be formed and assembled into pārticies in S. cerevisiae (Valenzuela et al. (1982)), as well as in, for example, mammalian celis (Valenzuela, P., et al. (1984)). The formation of such pārticies has been shovm to enhance the immunogenicity of the monomer subunit. The constructs may also include the. immunodominant epitope of HBSAg, comprising the 55 amino acids of the presurface (pre-S) region. Neurath et al. (1984) . Constructs of the pre-S-HBSAg particle expressible in yeast are disclosed in EPO 174,444, published March 19, 1986; hybrids including heterologous virai seguences for yeast expression are disclosed in EPO 175,261, published March 26, 1966. These 47 constructs may also be expressed in mammalian celis such as Chinese hamster ovary (CHO) celis using an SV40-dihydrofola.te reductase vector (Michelle et al. (1984)).

In addition, portions of the particle-forming protein coding seguence may be replaced with codons encoding an HCV epitope. In this replacement, reģions which are not required to mediate the aggregation of the units to form immunogenic pārticies in yeast or mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the HCV epitope.

Preparation of Vaccines

Vaccines may be prepared from one or more immunogenic polypeptides derived from HCV cDNA, including the cDNA seguences described in the Examples. The observed homology between HCV and Flaviviruses provides Information concerning the polypeptides which may be most effective as vaccines, as well as the reģions of the genome in which they are encoded. The general structure of the Flavivirus genome is discussed in Rice et al (1986) . The flavivirus genomic RNA is believed to be the only virus-specific mRNA species, and it is translated into the three virai structural proteīns, i.e., C, M, and E, as well as two large nonstructural proteīns, NS4 and NS5, and a complex set of smaller nonstructural proteīns. It is known that major neutralizing epitopes for Flaviviruses reside in the E (envelope) protein (Roehrig (1986)). Thus, vaccines may be comprised of recombinant poly peptides containing epitopes of HCV E. These polypeptides may be expressed in bacteria, yeast, or mammalian celis, or alternatively may be isolated from 48 LV10306 virai preparations. It is also anticipated that the other strudtural proteīns may also contain epitopes which give rise to · protective anti-HCV antibodies. Thus, polypeptides containing the epitopes o£ E, C, and M may also be used, whether singly or in combination, in HCV vaccines.

In addition to the above, it has been shown that immunization with NS1 (nonstructural protein 1), results in protection against yellow fever (Schlesinger et al (1986)). This is true even though the Immunization does not give rise to neutralizing antibodies. Thus, particularly since this protein appears to be highly conserved among Flaviviruses, it is likely that HCV NS1 will also be protective against HCV infection. Moreover, it also shows that nonstructural proteīns may provide protection against virai pathogenicity, even if they do not cause the production of neutralizing antibodies.

The Information provided in the Examples concerning the immunogenicity of the polypeptides expressed from cloned HCV cDNAs which span the various reģions of the HCV ORF also allows predictions concerning their use in vaccines.

In view of the above, multivalent vaccines against HCV may be comprised of one or more epitopes from one or more structural proteīns, and/or one or more epitopes from one or more nonstructural proteīns. These vaccines may be comprised of, for example, recombinant HC polypeptides and/or polypeptides isolated from the virions. In particular, vaccines are contemplated comprising one or more of the following HCV proteīns, or subunit antigens derived therefrom: E, NS1, C, NS2, NS3, 49 NS4 and NS5. Particularly preferred are vaccines compris ing E and/or NS1, or subunits thereof.

The preparation of vaccines which contain an immunogenic polypeptide(s) as active ingredients, is know to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liguid Solutions or suspensions; solid forma suitable for solution in, or suspension in, liguid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogeni ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the activ ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying aģents, pH buffering aģents, and/or adjuvants vrtiich enhance the effectiveness of the vaccine. Examples of adjuvants which may be effec tive include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),

Nacetyl-nor-muram.yl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1*-21-dipalmitoyl-sn-glycero-3hydroxyhosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RĪBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and celi wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The ef fectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an līnmunogenic polypeptide containing an HCV antigenic seguence resulting from 50 LV10306 ādministration of this polypeptide in vaccines which are also comprised of the various adjuvants.

The vaccines are conventionally ādministered parenterally, by injection, for example, either subcutaneously or intramuscularly· Additional formulations which are suitable for other modes of ādministration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, .polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of Solutions, suspensions, tablete, pilis, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

The proteīns may be formulated into the,vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. 51

Dosaae and Ādministration o£ Vaccines

The vaccines are ādmini stered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be ādmini stered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject,s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be ādminis tered may depend on the judgment of the practitioner and may be peculiar to each subject.

The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervāls reguired to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subseguent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner. in addition, the vaccine containing the immunogenic HCV antigen(s) may be administered in conjunction with other immunoregulatory aģents, for example, immune globulins. 52 LV10306

Preparation of Antibodies Against HCV Epitopes

The immunogenic polypeptides prepared as described above are used to producē antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammai (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing an HCV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an HCV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by lmmunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987).

Alternatively, polyclonal antibodies may be isolated from a mammai which has been previously infected with HCV. An example of a method for purifying antibodies to HCV epitopes from serum from an infected individual, based upon affinity chromatography and utilizing a fusion polypeptide of SOD and a polypeptide encoded within CDNA clone 5-1-1, is presented in EPO Pub. No. 318,216.

Monoclonal antibodies directed against HCV epitopes can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well knovm. Immortal antibody-producing celi lines can be created by celi fusion, and also by other technigues such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. (1980); Hammerling et al. (1981); Kennett et al. (1980); see also. U.S. Patent Nos. 4,341,761; 53 4,399,121? 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against,HCV epitopes can be screened for various properties; i.e., for isotype, epitope affinity, etc.

Antibodies, both monoclonal and polyclonal, which are directed against HCV epitopes are particularly useful in diagnosis, and those which are neutralizing are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies.

Anti-idiotype antibodies are immunoglobulins which carry an "internai image" of the antigen of the infectious aģent against which protection is desired. See, for example, Nisonoff, A., et al. (1981) and Dreesman et al. (1985).

Techniques for raising anti-idiotype antibodies are knovrn. in the art. See, for example, Grzych (1985), MacNamara et al. (1984), and Uytdehaag et al. (1985). These anti-idiotype antibodies may also be useful .for treatment and/or diagnosis of NANBH, as well as for. an elucidation of the immunogenic reģions of HCV antigens.

It would also be recognized by one of ordinary skill in the art that a variety of types of antibodies directed against HCV epitopes may be produced. As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site. An "antibody combining site" or "binding domain" is formed from the folding of variable domains of an antibody molecules) to form threedimensional binding spaces with an internai surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction 54 LV10306 with the antigen. An antibody combining site may be formēd from a heavy and/or a light chain domain (VH and VL, respectively), which form hypervariable loops which contribute to antigen binding. The term ,,antibody,, includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalentantibodies, the Fab proteīns, and single domain antibodies. A "single domain antibody" (dAb) is an antibody which is comprised of an VH domain, which reacts immunologically with a designated antigen. A dAB does not contain a VL domain, but may contain other antigen binding domains known to exist in antibodies, for example, the kappa and lambda domains. Methods for preparing dABs are known in the art. See, for example, Ward et al. (1989).

Antibodies may also be comprised of VH and VL domains, as well as other known antigen binding domains. Examples of these types of antibodies and methods for their preparation are knovai in the art (see, e.g., U.S. Patent No. 4,816,467, which is Incorporated herein by reference), and include the following. For example, "vertebrate antibodies" refers to antibodies which are tetramers or aggregates thereof, comprising light aņd heavy chains which are usually aggregated in a "Y" configuration and which may or may not have covalent linkages between the chains. In vertebrate antibodies, the amino acid sequences of ali the chains of a particular antibody are homologous with the chains found in one antibody produced by the lymphocyte which producēs that antibody in situ, or in vitro (for example, .in hybridomas). Vertebrate antibodies typicallly include 55 native antibodies, for example, purified polyclonal antibodies and monoclonal antibodies. Exampl.es of the methods for the preparation of these antibodies are described infra. "Hybrid antibodies" are antibodies wherein one pair of heavy and light chains is homologous to those in a first antibodyf while the other pair of heavy and light chains is homologous to those in a different second antibody. Typically, each of these two pairs will bind different epitopes, particularly on different antigens. This results in the property of "divalencell, i.e., the ability to bind two antigens simultaneously. Such hybrids may a.Lso be formed using chimeric chains, as set forth below. "Chimeric antibodies", are antibodies in which the heāvy and/or light chains are fusion proteīns. Typically the constant domain of the chains is from one particular species and/or class, and the variable domains are from a different species and/or class. Also included is any antibody in which either or both of the heavy or light chains are composed of combinations of seguences mimicking the seguences in antibodies of different sources, whether these sources be differing classes, or different species of origin, ' and whether or not the fusion point is at the variable/constant boundary. Thus, It Is possible to producē antibodies in which neither the constant nor the variable region mimic known antibody seguences. It then becomes possible, for example, to construct antibodies whose variable region has a higher specific affinity for a particular antigen, or whose constant region can elicit enhanced complement fixation, 56 LV10306 or to make other improvements in properties possessed by a particular constant region.

Another example is "altered antibodies", which refers to antibodies in which the naturally occurring amino acid seguence in a vertebrate antibody has been varied. Utilizing recombinant DNA technigues, antibodies can be redesigned to obtain desired characteristics. The possible variations are many, and range from the changing of one or more amino aCids to the complete redesign of a region, for example, the constant region. Changes in the constant region, in general, to attain desired cellular process characteristics, e.g., changes in complement fixation, interaction with membranes, and other effector functions. Changes in the variable region may be made to alter antigen binding characeristics. The antibody may also be engineered to aid the specific delivery of a molecule or substance to a specific celi or tissue site. The desired alterations may be made by knovm techniques in molecular biology, e.g., recombinant technigues, site directed mutagenesis, etc.

Yet another example are "univalent antibodies", which are aggregates comprised of a heavy chainllight chain dimer bound to the Fc (i.e., constant) region of a second heavy chain. This type of antibody escapes antigenic moduļation. See, e.g., Glennie et al. (1982).

Included also within the definition of antibodies are "Fab" fragments of antibodies. The "Fab" region refers to those portions of the heavy and light chains which are roughly equivalent, or analogous, to the sequences which comprise the branch portion of the heavy and light chains, and which have been shown to exhibit immunological binding to a specified antigen, but which 57 lack the effector Fc portion . nFab" includes aggregates of bne heavy and one light Chain (commonly known as Fab') / as -well as tetramers containing the 2H and 2L chains (referred to as F(ab)2), which are capable of selectively reacting with a designated antigen or antigen family. "Fab11 antibodies may be divided into subsets analogous to those described above, i.e, 'vertebrate Fab", "hybrid Fab", "chimeric Fab", and "altered Fab.",. Methods of producing "Fab" fragments of antibodies are knovm within the art and include, for example, proteolysis, and synthesis by recombinant techniques. XI.H. Diagnostic Oligonucleotide Probes and Kits

Using the disclosed portions of the isolated HCV cDNAs as a basis, oligomers of approximately 8 nucleotides or more can be prepared, either by excision or synthetically, which hybridize with the HCV genome and are useful In Identification of the virai aģent(s), further characterization of the virai genome (s), as well asj:in detection of the virus(es) in diseased individuāls. The probes for HCV polynucleotides (natūrai or derived) are a length which allows the detection of unique virai seguences by hybridization. While 6-8 nucleotides may be a workable length, seguences of 10-12 nucleotides are preferred, and about 20 nucleotides appears optimal. Preferably, these seguences will derive from reģions which lack heterogeneity. These probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. Among useful probes, for example, are those derived from the newly isolated clones disclosed herein, as well as the various oligomers 58 LV10306 useful in probing CDNA libraries, set forth. below. A complement to any unique portion of the HCV genome will be satisfactory. For use as probes, complete c omplementarity is desirable, though it may be unnecessary as the length of the fragment is increased.

For use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively/ the nucleic acid sample may be dot blotted without ' size separation. The probes are then labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescerit probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies, and polynucleotide duplexes containing the probe are detected.

The probes can be made completely complementary to the HCV genome. Therefore, usually high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency should only be used if the probes are complementary to reģions of the virai genome which lack heterogeneity. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperatūre, ionic strength, length of time, and concentration of formamide. These factors are outlined in, for example, Maniatis, T. (1982). 59

Generally, it is expected that the HCV genome seguences will be present in senam of infected individuāls· at relatively low Ievels, i.e., at approximately 10 -10 chimp infectious doses (CID) per ml. This Ievel may reguire that amplification technigues be used in hybridization assays. Such technigues are known in the art. For example, the Ehzo Biochemical Corporation "BioBridge" system uses termiņai deoxynucleotide transferase to add unmodified 31-poly-dT-tails to a DNA probe. The poly dt-tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly-A. PCT application 84/03520 and EPA124221 describe a DNA hybridization assay in which: (1) analyte is annealed to a single-stranded DNA probe that is complementary to an enzyme-labeled oligonucleotide; and (2) the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide. EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dt tail, an amplifier strand that has a sequence that hybridizes to the tail of the probe, such as a poly-A seguence, and which is capable of binding a plurality of labeled strands. A particularly desirable technigue may first involve amplification of the target HCV sequences in sera approximately 10,000 fold, i.e., to approximately 10 6 sequences/ml. This may be accomplished, for example, by the polymerase chain reactions (PCR) technique described which is by Saiki et al. (1986), by Mullis, U.S. Patent No. 4,683,195, and by Mullis et al. U.S. Patent No. 4,683,202. The amplified seguences may then be detected using a hybridization assay which is described in EP 317,077, published May 24, 60 LV10306 1989. These hybridization assays, which should detect sequences at the Ievel of 10 6 /ml, utilizē nucleic acid multimers which bind to single-stranded analyte nucleic acid, and which also bind to a multiplicity of single-stranded labeled oligonucleotides. A suitable solution phase sandwich assay which may be used with labeled polynucleotide probes, and the methods for the preparation of probes is described in EPO 225,807, published June 16, 1987.

The probes can be packaged into diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit in separate containers. The kit may also contain other suitably packaged reaģents and materiāls needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the tēst.

Immunoassay and Diagnostic Kits

Both the polypeptides which react immunologically with serum containing HCV antibodies, for example, those detected by the antigenic screening method described infra* in the Examples, as well those derived from or encoded within the isolated clones described in the Examples, and composites thereof, and the antibodies raised against the HCV specific epitopes in these polypeptides, are useful in immunoassays to detect presence of HCV antibodies, or the presence of the vīrus and/ or virai antigens, in biological samples. Design of the immunoassays is subject to a great deal of variation, 61 and a variety of these are known in the art. Por example, the ' imm.unoassay may utilizē one virai epitope; alternative-ly, the inununoassay may use a combination of virai epitopes derived from these sources; these epitopes may be derived from the same or from different virai polypeptides, and may be in separate recombinant or natūrai polypeptides, or together in the same recombinant polypeptides. It may use, for example, a monoclonal antibody directed towards a virai epitope(s), a combination of monoclonal antibodies directed towards epitopes of one virai antigen, monoclonal antibodies directed towards epitopes of different virai antigens, polyclonal antibodies directed towards the same virai antigen, or polyclonal antibodies directed towards different virai antigens. Protocole may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signāls from the probe are also known; examples of which are assays which utilizē biotin and avidin, and enzymelabeled and mediated immunoassays, such as ELISA assays.

Some of the antigenic reģions of the putative polyprotein have been mapped and identified by screening the antigenicitiy of bacterial expression products of HCV cDNAs which encode portions of the polyprotein. See the Examples. Other antigenic reģions of HCV may be detected by expressing the portions of the HCV cDNAs in other expression systems, including yeast systems and cellular 62 LV10306 systems derived from insects and vertebrates. In addition, studies giving rise to an antigenicity index and hydrophobicity/hydrophilicity profilē give rise to Information concerning the probability of a region‘s antigenicity.

The studies on antigenic mapping by expression of HCV cDNAs showed that a number of clones containing these cDNAs expressed polypeptides vrtvich were ixnmunologically reactive with serum from individuāls with NANBH. No single polypeptide was immunologically reactiv with ali sera. Pive of these polypeptides were very ļmmunogenic in that antibodies to the HCV epitopes in these polypeptides were detected in many different patient sera, although the overlap In detection was not complete. Thus, the results on the immunogenicity of the polypeptides encoded in the various clones suggest that effecient detection systems may include the use of panels of epitopes. The epitopes in the panei may be constructed Into one or multiple polypeptides... Kits suitable for immunodiagnosis and containing the appropriate labeled reaģents are constructed by packaging the appropriate materiāls, including the polypeptides of the invention containing HCV epitopes or antibodies directed against HCV epitopes in suitable containers, along with the remaining reaģents and materiāls required for the conduct of the assay, as well as a suitable set of assay instructions. 63

Further Characterization of the HCV Genome,

Virions . and y-irĀl_Antiggns Using Proves Perived. Prom cDNA-to the Virai Genome

The HCV cDNA sequence Information in the newly isolated clones described in the Examp3.es may be used to gain further Information on the sequence of the HCV genome, and for Identification and isolation of the HCV aģent, and thus will aid in its characterization including the nature of the genome, the structure of the virai particle, and the nature of the antigens of which it is composed. This information, in turn, can lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and antibodies directed against HCV epitopeo which would be useful for the diagnosis and/or treatment of HCV caused NANBH.

The cDNA sequence information in the abovementioned clones is useful for the design of probes for the isolation Of additional cDNA seguences which are derived from as yet undefined reģions of the HCV genome(s) from which the cDNAs in clones described herein and in EP 0,316,218 are derived. For example, labeled probes containing a seguence of approximately 8 or more nucleotides, and preferably 20 or more nucleotides, v/hich are derived from reģions close to the 5'-termini or 3'-termini of the composite HCV cDNA seguence shown In Fig. 17 may be used to isolate overlapping cDNA sequences from HCV CDNA libraries. Alternatively, characterization of the genomic segments could be from the virai genome (s) Isolated from purified HCV pārticies. Methods for purifying HCV pārticies and for detecting them during the purification procedure are described herein, infra. 64 LV10306

Procedures for Isolating polynucleotide genomes from virai pārticies are known in the art, and one procedure which may be used is that described in EP 0,218.316. The isolated genomic segments could then be cloned and seguenced. An example of this technigue, which utilizēs amplification of the seguences to be cloned, is provided infra., and yielded clone 16jh.

Methods for constructing cDNA libraries are known In the art, and are discussed supra and infra; a method for the construction of HCV CDNA libraries in lambda-gtll is discussed in EPO Pub. No. 318,216. However, cDNA libraries which are useful for screening with nucleic acid probes may also be constructed in other vectors known in the art, for example, lambda-gtlO (Huynh et al. (1985)).

Screening for Anti-Virai Aģents for HCV

The availability of celi culture and animal modei systems for HCV makes it possible to screen for anti-virai aģents which inhibit HCV replication, and particularly for those aģents which preferentially allow celi growth and multiplication while inhibiting virai replication. These screening methods are known by those of skill in the art. Generally, the anti-viral aģents are tested at a variety of concentrations, for their effect on preventing virai replication in celi culture systems which support virai replication, and then for an inhibition of infectivity or of virai pathogenicity (and a low Ievel of toxicity) in an animal modei system.

The methods and compositions provided herein for detecting HCV antigens and HCV polynucleotides are useful 65 for screening of anti-virai aģents in that they provide an alternative, and perhaps more sensitive means, for detecting the aģent's effect on virai replication than the celi plague assay or IDS0 assay. For example, the HCVpolynucleotide probes described herein may be used to quantitate the amount of virai nucleic acid produced in a celi culture. This could be accomplished, for example, by hybridization or competition hybridization of the infected celi nucleic acids with a labeled HCV-polynucleotide probe. For example, also, anti-HCV antibodies may be used to identify and quantitate HCV antigen(s) in the celi culture utilizing the immunoassays described herein. In addition, since it may be desirable to quantitate HCV antigens in the infected celi culture by a competition assay, the polypeptides encoded within the HCV cDNAs described herein are useful in these competition assays. Generally, a recombinant HCV polypeptide derived from the HCV cDNA would be labeled, and the Inhibition of binding of this labeled polypeptide to an HCV polypeptide due to the antigen produced in the celi culture system would be monitored. Moreover, these technigues are particularly useful in cases where the HCV may be able to replicate in a celi line without causing celi death. *

The anti-virai aģents which may be tested for efficacy by these methods are known in the art, and Include, for example, those which interact with virion components and/or cellular components which are necessary for the binding and/or replication of the vīrus. Typical anti-viral aģents may include, for example, inhibitors of virion polymerase and/or protease(s) necessary for cleavage of the precursor polypeptides. Other anti-viral 66 LV10306 aģents may include those which act with nucleic acids to prevent virai replication, for example, anti-sense polynucleotides, etc.

Antisense polynucleotides molecules are comprised of a complementary nucleotide seguence which allows them to hybridize speci£ically to designated reģions of genomes or RNAs. Antisense polynucleotides may include, for example, molecules that will block protein translation by binding to mRNA, or may be molecules which prevent replication of virai RNA by transcriptase. They may also include molecules which carry aģents (noncovalently attached or covalently bound) which cause the virai RNA to be inactive by causing, for example, scissions in the virai RNA. They may also bind to cellular polynucleotides which enhance and/or are required for virai infectivity, replicative ability, or chronicity. Antisense molecules which are to hybridize to HCV derived RNAs may be designed based upon the sequence Information of.the HCV cDNAs provided herein. The antiviral aģents based upon anti-sense polynucleotides for HCV may be designed to bind with high specificity, to be of increased solubility, to be stable, and to have low toxicity. Hence, they may be delivered in specialized systems, for example, liposomes, or by gene therapy. In addition, they may include analogs, attached proteīns, substituted or altered bonding between bases, etc.

Other types of drugs may be based upon polynucleotides which "mimic" important control reģions of the HCV genome, and which may be therapeutic due to their interactions with key components of the system responsible for virai infectivity or replication. 67

General Methods

The general techniques used in extracting the genome from a virus, preparing and probing a cDNA library, sequencing clones, constructing expression vectors/ transforming celis, performing immunological assays such as radioimmunoassays and ELISA assays, for growing celis in culture, and the like are known in the art and laboratory manuals are available describing these techniques. However, as a general guide, the following sets forth some sources currently available for such procedures, and for materiāls useful in carrying them out.

Both prokaryotic and eukaryotic host celis may be used for expression of desired coding seguences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control secļuences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sibēs. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the Beta-lactamase (penicillinase) and lactose promoter systems (Chang et al. (1977)), the tryptophan (trp) promoter system (Goeddel et al. (1980)) and the lambda-derived PL promoter and N gene ribosome binding site (Shimatake et al. 68 LV10306 (1981)) and the hybrid tac promoter (De Boer et al. (1983)) derived from sequences of the trp and lac UV5 promoters. · The foregoing systems are particularly compatible with JĒ^. coli; if desired, other prokaryotic hosts such as stralns of Bacillus or Pseudomonas may be used, with corresponding control seguences.

Eukaryotic hosts include yeast and mammalian celis in culture systems. Saccharomyces cerevisiae and Saccharc>myces carlsbergensis are the most commonly used yeast hosts, and are convenient fungal hosts. Yeast compatible vectors carry markers which permit selection of successful transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metāls on wildtype strains. Yeast compatible vectors may employ the 2 micron origin of replication (Broach et al. (1983)), the combination of CEN3 and ARSI or other means for assuring replication, such as seguences which will result in incorporation of an appropriate fragment into the host celi genome. Control seguences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al. (1968);,Holland et al. (1978)), including the promoter for 3 phosphoglycerate kinase (Hitzeman (1980)). Terminators may also be included, such as those derived from the enolase gene (Holland (1981)). Particularly useful control systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, leader seguence from yeast alpha factor. In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they 69 are not naturally associated in the wild-type orgānism. Thesē systems are described in detail in EPO 120,551, published October 3, 1984; EPO 116,201, published August 22, 1984; and EPO 164,556, published December 18, 1985, ali of which are assigned to the herein assignee, and are hereby incorporated herein by reference.

Mammalian celi lines available as hosts for expression are knovm in the art and include many immortalized celi lines available from the American Type Culture Collection (ATCC), including HeLa celis, Chinese hamster ovary (CHO) celis, baby hamster kidney (BHK) celis, and a number of other celi lines. Suitable promoters for mammalian celis are also knovm in the art and include virai promoters such as that from Simian Virus 40 (SV40) (Fiers (1978)), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian celis may also reguire terminator seguences and poly A addition sequences; enhancer sequences which increase expression may also be included, and seguenpes which cause amplif ication of the gene may alsoī^be desirable. These seguences are knovm. in the art. Vectors suitable for replication in mammalian celis may include virai replicons, or seguences which insure integration of the appropriate seguences encoding NANBV epitopes into the host genome.

Trans forma tion may be by any knovm method for introducing polynucleotides into a host celi, including, for example packaging the polynucleotide In a virus and transducing a host celi with the virus, and by direct uptake of the polynucleotide. The transformation procedure used depends upon the host to be transformed. For example, transformation of the E. coli host celis with lambda-gtll 70 LV10306 containing BB-NANBV sequences is discussed in the Example section, infra. Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride (Cohen (1972); Maniatis (1982)). Yeast transformation by direct uptake may be carried out using the method of Hinnen et al. (1978) . Mammalian transformations by direct uptake may be conducted using the calcium phosphate precipitation method of Graham and van der Eb (1978), or the various knovm. modifications thereof.

Vector construction employs techniques which are known in the art. Site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. In general, about 1 microgram of plasmid or DNA seguence is cleaved by 1 unit of enzyme in about 20 microliters buffer solution by incubation of 1-2 hr at 37°C. After incubation with the restriction enzyme, protein is removed by phenol/ chloroform extraction and the DNA recovered by precipitation with ethanol. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis technigues, according to the general procedures found in Methods in Enzymology (1980) £1:499-560.

Sticky ended cleavage fragments may be blunt ended using Ē. coli DNA polymerase I (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with Sl nuclease may also be used, resulting in the hydrolysis of any single stranded DNA portions. 71

Ligations are carried out using Standard buffer and temperatūre conditions using T4 DNA ligase and ATP; sticky end· ligations require less ATP and less ligase than blunt end ligations. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 51-phosphate and thus prevent religation of the vector; alternatively/ restriction enzyme digestion of unwanted fragments can be used to prevent ligation.

Ligation mixtures are transformed into suitable cloning hosts, such as E. coli. and successful transformants selected by, for examplef antibiotic resistance, and screened for the correct construction.

Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer as described by Warner (1984). If desired the synthetic strands may be labeled with 32P by treatment with polynucleotide kinase in the presence of 32P-ATP, using Standard conditions for the reaction. **;· DNA seguences, Including those isolated from CDNA libraries, may be modified by knovm techniques, including, for example site directed mutagenesis, as described by Zoller (1982). Briefly, the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its ovm, sequence. The resulting double stranded DNA is transformed into a phage supporting host bacterium. Cultures of the transformed bacteria, which 72 LV10306 contain replications of each strand of the phage, are platēd In agar to obtain plaques. Theoretically,,50% of the new plagues contain phage having the mutated seguence, and the remaining 50% have the original seguence. Replicates of the plagues are hybridized to labeled synthetic probe at temperatures and conditions which permit hybridization with the correct strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned. DNA libraries may be probed using the procedure of Grunstein and Hogness (1975) . Briefly, in this procedure, the DNA to be probed is immobilized on nitrocellulose filters, denatured, and prehybridized with a buffer containing 0-50% formamide, 0.75 M NaCl, 75 mM Na citrate, 0.02% (wt/v) each of bovine serum albumin, polyvinyl pyrollidone, and Ficoll, 50 mM Na Phosphate (pH 6.5), 0.1% SDS, and 100 micrograms/ml carrier denatured DNA. The percentage of formamide In the buffer, as well as the time and temperature conditions of the prehybridization and subsequent hybridization steps depends on the stringency required. Oligomeric probes which require lower stringency conditions are generally used with low percentages of formamide, lower temperatures, and longer hybridization times. Probes containing more than 3 0 or 40 nucleotides such as those derived from CDNA or genomic sequences generally employ higher temperatures, e.g., about 40-42°C. and a high percentage, e.g., 50%, formamide. Following prehybridization, 5'- 32P-labeled oligonucleotide probe is added to the buffer, and the filters are incubated in this mixture under hybridization conditions. After 73 washing, the treated filters are subjected to autoradiography to show the location of the hybridized probe; DNA· in corresponding locations on the original agar plates is used as the source of the desired DNA.

For routine vector constructions, ligation mixtures are transformed into E. coli strain HB101 or other suitable host, and successful transformants selected by antibiotic resistance or other markers. Plasmide from the transformants are then prepared according to the method of Clewell et al. (1969), usually following chloramphenicol amplification (Clewell (1972)). The DNA is isolated and analyzed, usually by restriction enzyme analysis and/or seguencing. Sequencing may be by the dideoxy method of Sanger et al. (1977) as further described by Messing et al. (1981), or by the method of Maxam et al. (1980) . Problems with band compression, which are sometimes observed in GC rich reģions, were overcome by use of T-deazoguanosine according to Barr et al. (1986).

The enzyme-linked immunosorbent assay (ELISA) can be used to measure either antigen or antibody concentrations. This method depends upon conjugation of an enzyme to either an antigen or an antibody, and uses the bound enzyme activity as a quantitative label. To measure antibody, the knovm antigen is fixed to a solid phase (e.g., a microplate or plastic cup), incubated with tēst serum dilutions, washed, incubated with antiimmunoglobulin labeled with an enzyme, and washed again. Enzymes suitable for labeling are knovm In the art, and include, for example, horseradish peroxidase. Enzyme activity bound to the solid phase is measured by adding the specific substrate, and determining product 74 LV 10306 fonnation or substrate utilization colorimetrically. The enzyme activity bound is a direct funetion of the amount of antibody· bound.

To measure antigen, a known specific antibody is fixed to the solid phase, the tēst material containing antigen is added, after an incubation the solid phase is washed, and a second enzyme-labeled antibody is added. After washing, substrate is added, and enzyme activity is estimated colorimetrically, and related to antigen concentration.

Examples

Described below are examples of the present invention which are provided only for illustrative purposes, and not to limit the scope of the present invention. In light of the present disclosure, numerous embodiments within the scope of the elaims will be apparent to those of ordinary skill in the art.

Isolation and Seguence of Overlapping HCV CDNA Clones 13i. 26j. CA59a. CA84a, CA156e and CA167b

The clones 13i, 26j, CA59a, CA84a, CA156e and CA167b were isolated from the lambda-gtll library which contains HCV cDNA (ATCC No. 40394). the preparation of which is described in EPO Pub. No. 318,216 (published 31 May 1989) , and W0 89/04669 (published 1 June 1989) . Screening of the library was with the probes described infra., using the method described in Huynh (1985). The 75 freguencies with which positive clones appeared with the respective probes was about 1 in 50,000.

The isolation of clone 13i was accomplished using a synthetic probe derived from the sequence of clone 12f. The seguence of the probe was: 5' GAA CGT TGC GAT CTG GAA GAC AGG GAC AGG 3 ' .

The isolation of clone 26 J was accomplished using a probe derived from the 51-region of clone K9-1. The seguence of the probe was: 5' TAT CAG TTA TGC CAA CGG AAG CGG CCC CGA 3'.

The isolation procedures for,clone 12f and for clone k9-l (also called K9-1) are described in EPO Pub. No. 318,216, and their seguences are shown in Figs. 1 and 2, respectively. The HCV CDNA seguences of clones 131 and 26j, are shown in Figs. 4 and 5, respectively. Also shown are the amino acids encoded therein, as well as the overlap of clone 13i with clone 12f, and the overlap of clone 26J with clone 13i. The seguences for these clones confirmed the seguence of clone K9-1. Clone K9-1 had been isolated from a different HCV CDNA library (See EP 0,218,316).

Clone CA59a was isolated utilizing a probe based upon the seguence of the 51-region of clone 26j. The seguence of this probe was: 5' CTG GTT AGC AGG GCT TTT CTA TCA CCA CAA 3'. 76 LV10306 A probe derived from the sequen.ce of clone CA59ā was used to isolate clone CA84a. The sequence of the probe used for this isolation was: 5 AAG GTC CTG GTA GTG CTG CTG CTA TTT GCC 31.

Clone CAl56e was isolated uslng a probe derived from the sequence of clone CA84a. The sequence of the probe was: 5' ACT GGA CGA CGC AAG GTT GCA ATT GCT CTA 3'.

Clone CAl67b was isolated using a probe derived from the seguence of clone CA 156e. The sequence of the probe was: 5' TTC GAC GTC ACA TCG ATC TGC TTG TCG GGA 3 *.

The nucleotide sequences of the HCV cDNAs in clones CA59a, CA84a, CA156e, and CA167b, are sho\m Figs. 6r 7, 8, and 9, respectively. The amino acids encoded therein, as well as the overlap with the sequences of relevant clones, are also shown in the Figs.

Creation of "pi" HCV CDNA Library A library of HCV cDNA, the "pi" library, was constructed from the same batch of infectious chimpanzee plasma used to construct the lambda-gtll HCV cDNA library (ATCC No. 40394) described in EPO Pub. No. 318,216, and utilizing essentially the same technigues. However, construction of the pi library utilized a primer-extension method, in 77 which the primer for reverse transcriptase was based on the šequence of clone CAS9A. The seguence of the primer was: 5' GGT GAC GTG GGT TTC 3'.

Isolation and Seguence of Clone pi!4a

Screening of the "pi" HCV cDNA library described supra., with the probe used to isolate clone CA167b (See supra.) yielded clone pil4a. The clone contains about 800 base pairs of cDNA which overlaps clones CA167b, CA156e, CA84a and CA59a, which were isolated from the lambda gt-11 HCV cDNA library (ATCC No. 40394). In addition, pil4a also contains about 250 base pairs of DNA which are upstream of the HCV cDNA in clone CA167b.

Jgjplation and Seguence of Clones CA216a. CA290a flnd.jg3.0a

Based on the seguence of clone CA167b a synthetic probe was made having the following sequence: 5' GGC TTT ACC ACG TCA CCA ATG ATT GCC CTA 3'

The above probe was used to screen the L* vrtiich yielded clone CA216a, whose HCV seguences are shown in Fig. 10.

Another probe was made based on the seguence of clone CA216a having the following sequence: * Lambda-gtll library (ATCC #40394) 78 LV10306 5' TTT GGG TAA GGT CAT CGA TAC CCT TAC GTG 3'

Screening the lambda-gtll library (ATCC No. 40394) with this probe yielded clone CA290a, the HCV sequences therein being shown in Fig. 11.

In a parailei approach, a primer-extension cDNA library was made using nucleic acid extracted from the same infectious plasma used in the original lambda-gtll CDNA library described above. The primer used was based on the sequence of clones CA216a and CA290a: 5' GAA GCC GCA CGT AAG 3'

The cDNA library was made using methods similar to those described previously for libraries used in the Isolation of clones pil4a and k9-l. The probe used to screen this library was based on the seguence of clone CA290a: 51 CCG GCG TAG GTC GCG CAA TTT GGG TAA 3'

Clone ag30a was isolated from the new library with the above probe, and contained about 670 basepairs of HCV seguence. See Fig. 12. Part of this seguence overlaps the HCV seguence of clones CA216a and CA290a. About 300 basepairs of the ag30a seguence, however, is upstream of the seguence from clone CA290a. The non-overlapping seguence shows a start codon (*) and stop codons that may indicate the start of the HCV,0RF. AlsO indicated in Fig. 12 are putative small encoded peptides.,(#) which may play a role in regulating translation, as well as the putative first amino acid of the putative polypeptide (/), and dovmstream amino acids encoded therein. 79

Isolation and Seguence of Clone CA205a

Clone CA205a was isolated from the original lambda gt-11 library (ATCC No. 40394), using a synthetic probe derived from the HCV seguence in clone CA290a (Fig. 11) . The sequence of the probe was: 5' TCA GAT CGT TGG TGG AGT TTA CTT GTT GCC 3' .

The seguence of the HCV cDNA in CA205a, shown in Fig. 13, overlaps with the cDNA seguences in both clones ag30a and CA290a. T he overlap of the sequence with that of CA290a is shown by the dotted line above the seguence (the figurē also shows the putative amino acids encoded in this fragment).

As observed from the HCV cDNA seguences In clones CA205a and ag30a, the putative HCV polyprotein appears to begin at the ATG start codon; the HCV seguences in both clones contain an In-frame, contiguous double stop codon (TGATAG) forty two nucleotides upstream from this ATG. The HCV ORF appears to begin after these stop codons, and to extend for at least 8907 nucleotides (See the composite HCV CDNA sho\m in Fig. 17).

Isolation and Seguence of Clone 18g

Based on the seguence of clone ag30a (See Fig. 12) and of an overlapping clone from the original lambda gt-11 library (ATCC No. 40394), CA230a, a synthetic probe was made having the following seguence: 5' CCA TAG TGG TCT GCG GAA CCG GTG AGT ACA 3* 80 LV 10306

Screening of the original lambda-gtll HCV cDNA library with 'the probe yielded clone 18g, the HCV cDNA sequen.ce of which is shown in Fig. 14. Also shown in the figurē are the overlap with clone ag30a, and putative polypeptides encoded within the HCV cDNA.

The cDNA in clone lgg (C18g or 18g) overlaps that in clones ag30a and CA205a, described supra. The sequence of Clgg also contains the double stop codon region observed in clone ag30a. The polynucleotide region upstream of these stop codons presumably represents part of the S,-region of the HCV genome, which may contain short ORFS, and which can be confirmed by direct sequencing of the purified HCV genome. These putative small encoded peptides may play a regulatory role in translation. The region of the HCV genome upstream of that represented by C18g can be isolated for sequence analysis using essentially the technigue described in EPO Pub. No. 318,216 for isolating cDNA sequences upstream of the HCV cDNA seguence in clone 12f. Essentially, small synthetic olig onucleotide primers of reverse transcriptase, which are based upon the sequence of C18g. are synthesized and used to bind to the corresponding sequence in HCV genomic RNA. The primer sequences are proximal to the knovm 51terminal of C18g, but sufficiently dovmstream to allow the design of probe seguences upstream of the primer sequences. Known Standard methods of priming and cloning ar eused. The resulting cDNA libraries are screened with sequences upstream of the priming sites (as deduced from the elucidated sequence of Clgg) . The HCV genomic RNA is obtained from either plasma or liver samples from individuāls with NANBH. Since HCV appears to be a Flavilike virus, the 5'-terminus of the genome may be 81 modified with a "cap" structure. It is known that Flavivirus genomes contain 5'-termiņai "cap" structures. (Yellow Fever virus, Rice et al. (1988); Dengue virus, Hahn et al (1988); Japanese Encephalitis Virus (1987)).

Isolation and Seguence of Clones from the beta-HCV CDNA library

Clones containing cDNA representative of the 3' -termiņai region of the HCV genome were isolated from a cDNA library constructed from the original infectious chimpanzee plasma pool which was used for the creation of the HCV cDNA lambda-gtll library (ATCC No. 4.0394), described in EPO Pub. No. 318,216. In order to create the DNA library, RNA ešctracted from the plasma was "tailed" with poly rA using poly (rA) polymerase, and cDNA was synthesized using oligo (dt) 12_18 as a primer for reverse transcriptase. The resulting RNA:cDNA hybrid was digested with RNAase H, and converted to double stranded HCV cDNA. • ’ VI':·

The resulting HCV cDNA was cloned into lambda-gtlO, using essentially the technique described in Huynh (1985), yielding the beta (or b) HCV cDNA library. The procedures used were as follows.

An aliguot- (12ml) of the plasma was treated with proteinase K, and extracted with an egual volume of phenol saturated with 0.05M Tris-Cl, pH 7.5, 0.05% (v/v) betamercaptoethanol, 0.1% (w/v) hydroxyquinolone, 1 mM EDTA. The resulting aqueous phase was re-extracted with the phenol mixture, followed by 3 extractions with a 1:1 mixture containing phenol and chloroform:isoamyl alcohol (24:1), followed by 2 extractions with a mixture of chloroform and isoamyl alcohol (1:1). Subseguent to 82 LV 10306 adjustment of the aqueous phase to 200 mM with respect to NaCl, nucleic acids in the aqueous phase were precipitated overnight at -20°C, with 2.5 volumes of cold absolute ethanol. The precipitates were collected by'centrifugation at 10,000 RPM for 40 min., washed with 70% ethanol containing 20 mM NaCl, and with 100% cold ethanol, dried for 5 min. in a dessicator, and dissolved in water.

The isolated nucleic acids from the infectious chimpanzee plasma pool were tailed with poly rA utilizing poly-A polymerase in the presence of human placenta ribonuclease inhibitor (HPRI) (purchased from Amersham Corp.), utilizing MS2 RNA as carrier. Isolated nucleic acids equivalent to that in 2 ml of plasma were incubated in a solution containing TMN (50 mM Tris HC1, pH 7.9, 10 mM MgCl2 / 250 mM NaCl, 2.5 mM MnC12 , 2 mM dithiothreltol (DTT)) , 40 micromolar alpha-[ 32P] ATP, 20 units HPRI (Amersham Corp.), and about 9 to 10 units of RNase free poly-A polymerase (BRL) . Incubation was for 10 min. at 37°C, and the reactions were stopped with EDTA (final concentration about 250 mM) . The solution was extracted with an equal volume of phenol-chloroform, and with an egual volume of chloroform, and nucleic acids were precipitated overnight at -20°C with 2.5 volumes of ethanol in the presence of 200 mM NaCl.

Isolation of Clone b5a

The beta HCV cDNA library was screened by hybridization using a synthetic probe, which had a seguence based upon the HCV cDNA seguence in clone 15e. The isolation of clone 15e is described in EPO Pub. No. 83 318,216, and Its seguence Is shown in Fig. 3. The sequēnce of the synthetic probe was: 5' ATT GCG AGA TCT ACG GGG CCT GCT ACT CCA 3'.

Screening of the library yielded clone beta-5a (b5a), which contains an HCV CDNA region of approximately 1000 base pairs. The 5' -region of this cDNA overlaps clones 35f, 19g, 26g, and 15e (these clones are described supra). The region between the 3'-termiņai poly-A sequence and the 3'-sequence which overlaps clone 15e, contains approximately 200 base pairs. This clone allows the identif ication of a region of the 31-te.rminal seguence the HCV genome.

The sequence of b5a is contained within the sequence of the HCV cDNA in clone 16jh (described infra). Moreover, the sequence is also present in CC34a. isolated from the original lambda-gtll library (ATCC No. 40394). (The original lambda-gtll library is referred to herein as the "C" library) . 5?

Isolation and Seguence of Clones Generated by PCR Amplification of the 31-Region of the HCV

Genome

Multiple cDNA clones have been generated which contain nucleotide seguences derived from the 3'-region of the HCV genome. This was accomplished by amplifying a targeted region of the genome by a polymerase chain reaction technigue described In Saiki et al. (1986). and in Saiki et al. (1988), which was modified as described below. The HCV RNA which was amplified was obtained from the original infectious chimpanzee plasma pool which was 84 LV10306 used for the creation of the HCV cDNA lambda-gtll library (ATCC No. 40394) described In EPO Pub. No. 318,216. Isolation of the HCV RNA was as described supra. The isolated RNA was tailed at the 3' -end with ATP by E. coli poly-A polymerase as described in Sippel (1973). except that the nucleic acids isolated from chimp serum were substituted for the nucleic acid substrate. The tailed RNA was then reverse transcribed into cDNA by reverse transcriptase, using an oligo dt-primer adapter, essentially as described by Han (1987), except that the components and secpience of the primer-adapter weres

Stuffer Noti SP6 Promoter Primer AATTC GCGGCCGC CATACGATTTAGGTGACACTATAGAA T is The resultant cDNA was subjected to amplification by PCR using two primers:

Seguence ATAGCGGCCGCCCTCGATTGCGAGATCTAC AATTCGGGCGGCCGCCATACGA

Primer JH32 (30mer) JH11 (20mer)

The JH32 primer contained 20, nucleotide sequences hybridizable to the 5' -end of the target region in the cDNA, with an estimated Tm, of 66°C. The JH11 was derived from a portion of the oligo dt-primer adapter; thus, it is specific to the 3' -end of the cDNA with a Tm of 64°C. Both primers were designed to have a recognition site for the restriction enzyme, Noti, at the 5' -end, for use in subsecpient cloning of the amplified HCV cDNA.

The PCR reaction was carried out by suspending the cDNA and the primers in 100 microliters of reaction mixture containing the four deoxynucleoside 85 triphosphates, buffer salts and mētai ions, and a thermostable DNA polymerase isolated from Thermus aguaticus (Taq polymerase), which are in a Perkin Elmer Cetus PCR kit (N801-0043 or N801-0055) . The PCR reaction was performed for 35 cycles in a Perkin Elmer Cetus DNA thermal cycler. Each cycle consisted of a 1.5 min denaturation step at 94°C, an annealing step at 60°C for 2 min, and a primer extension step at 72°C for 3 min. The PCR products were subjected to Southern blot analysis using a 30 nucleotide probe, JH34, the sequence of which was based upon that of the 3·-termiņai region of clone 15e. The sequence of JH34 is: 5' CTT GAT CTA CCT CCA ATC ATT CAA AGA CTC 3' .

The PCR products detected by the HCV cDNA probe ranged in size from about 50 to about 400 base pairs.

In order to clone the amplified HCV cDNA, the PCR products were cleaved with Noti and size selected- by polyacrylamide gel electrophoresio. DNA larger than 300 base pairs was cloned into the Noti site of pUC18S The vector pUC18S is constructed by Including a Noti polylinker cloned between the EcoRI and Sali sites of pUC18. The clones wore screened for HCV cDNA using the JH34 probe. A number of positive clones were obtained and seguenced. The nucleotide seguence of the HCV cDNA insert in one of these clones, 16jh, and the amino acids encoded therein, are shown in Fig. 15. A nucleotide heterogeneity, detected in the sequence of the HCV cDNA in clone 16jh as compared to another clone of this region, is indicated in the figurē. 86 LV10306

Compiled HCV cDNA Secmenogg.

Ah HCV cDNA sequence has been compiled from a series of overlapping clones derived from the various HCV cDNA libraries described supra.. In this sequence, the compiled HCV cDNA sequence obtained from clones bll4a, 18g, ag30a, CA205a, CA290a, CA216a, pll4a. CAl67b, CA156e, CA84a, and CA59a is upstream of the compiled HCV CDNA segnence published in EPO Pub. No. 318,216, which is shown in Fig. 16. The compiled HCV cDNA sequence obtained from clones b5a and 16jh downstream of the compiled HCV cDNA sequence published in EPO Pub. No. 318,216.

Fig. 17 shows the compiled HCV cDNA sequence derived from the above-described clones and the compiled HCV cDNA seguence published in EPO Pub. No. 318,216. The clones from which the sequence was derived are bll4a, 18g, ag30a, CA205a, CA290a, CA216a, pil4a, CAl67b, CA156e, CA84a, CAS9a, K9-1 (also called k9-l),26j , 131, 12f, 141, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c, 35f, 19g, 26g, 15e, b5a, and 16jh. In the figurē the three dashes above t he sequence indicate the position of the putative initiator methionine codon.

Clone bll4a was obtained using the cloning procedure described for clone b5a, supra., except that the probe was the synthetic probe used to detect clone lgg, supra. Clone bll4a overlaps with clones 18g, ag30a, and CA205a, except that clone bll4a contains an extra two nucleotides upstream of the sequence in clone 18g (I.e., 5'-CA). These extra two nucleotides have been included in the HCV genomic seguence shown In Fig. 17. 87

It should be noted that although several of the clones described supra. have been obtained from libraries other than· the original HCV cDNA lambda-gtll C library (ATCC No. 40394), these clones contain HCV cDNA sequences which overlap HCV cDNA sequences in the original librrary . Thus, essentially ali of the HCV sequence is derivable from the original lambda-gtll C library (ATCC No. 40394) which was used to isolate the first HCV cDNA clone (5-1-1). The isolation of clone 5-1-1 Is described In EPO Pub. No. 318,216.

Purification of Fusion Polypeptide C100-3 ... (Alternate method)

The fusion polypeptide, CIO0-3 (also called HCV clOO-3 and alternatively, cl00-3), Is comprised of superoxide dismutase (SOD) at the N-terminus an in-frame C100 HCV polypeptide at the C-terminus. A method for preparing the polypeptide by expression in yeast, * and differential extraction of the Insoluble fraction of”' the extracted host yeast celis, Is described In EPO Pub. No. 318,216. An alternative method for the preparation of this fusion polypeptide is described below. In this method the antigen is precipitated from the crude celi lysate with acetone; the acetone precipitated antigen is then subjected to ion-exchange chromatography, and further purified by gel filtration.

The fusion polypeptide, C100-3 (HCV clOO-3), is expressed in yeast strain JSC 308 (ATCC No. 20879) transformed with pAB24C100-3 (ATCC No. 67976); the transformed yeast are grown under conditions which allow expression (i.e., by growth in ΥΕΡ containing 1% glucose) . (See EPO Pub. No. 88 LV 10306 318,216). A celi lysate is prepared by suspending the celis in Buffer A (20 mM Tris HC1, pH 8.0, 1 mM EDTA, 1 mM PMSF. The celis are brofcen by grinding with glass beads in a Dynomill type homogenizer or its equivalent. The extent of celi breakage is monitored by counting celis under a microscope with phase optics. Broken celis appear dark, while viable celis are lightcolored. The percentage of broken celis is determined.

When the percentage of broken celis is approximately 90% or greater, the broken celi debris Is separated from the glass beads by centrifugation, and the glass beads are washed with Buffer A. After combining the washes and homogenate, the insoluble material in the lysate is obtained by centrifugation. The material In the peliet is washed to remove soluble proteīns by suspension in Buffer B (50 mM glycine, pH 12.0, 1 mM DTT, 500 mM NaCl), followed by Buffer C (50 mM glycine, pH 10.Ot 1 mM DTT) . The insoluble material is recovered by centrifugation, and solubilized by suspension in Buffer C containing SDS. The extract solution may be heated in the presence of beta-mercaptoethanol and concentrated by

ultrafiltration. The HCV cl00-3 in the extract is precipitated with cold acetone. If desired, the precipitate may be stored at temperaturēs at,about or below -15°C. Prior to ion exchange chromatography, the acetone precipitated material is recovered by centrifugation, and may be dried under nitrogen. ' The precipitate is suspended in Buffer D (50 mM glycine, pH 10.0, 1 mM DTT, 7 M urea), and centrifuged to peliet insoluble material. The supernatant material is applied to an anion exchange column previously eguilibrated with 89

Buffer D. Fractions are collected and analyzed by ultraviolet absorbance or gel electrophoresis on SDS polyacrylamide gels. Those fractions containing the HCV clOO-3 polypeptide are pooled.

In order to purify the HCV clOO-3 polypeptide by gel filtration, the pooled fractions from the ion-exchange column are heated in the presence of beta-mercaptoethanol and SDS, and the eluate is concentrated by ultrafiltration. The concentrate Is applied to a gel filtration column previously equilibrated with Buffer E (20 mM Tris HC1, pH 7.0, 1 mM DTT, 0.1% SDS). The presence of HCV clOO-3 in the eluted fractions, as well as the presence of impurities, are determined by gel electrophoresis on polyacrylamide gels in the presence of SDS and visualization of the polypeptides. Those fractions containing purified HCV clOO-3 are pooled. Fractions high in HCV clOO-3 may be further purified by repeating the gel filtration process. If the removal of particulate material is desired, the HCV clOO-3 containing material may be filtered through a 0.22 micron filter.

Expression and Antigenicity of Polypeptides Encoded in HCV cDNA

Polypeptides Expressed in E. coli

The polypeptides encoded in a number of HCV cDNAs which span the HCV genomic ORF were expressed in Ē. coli. and tested for their antigenicity using serum obtained from a variety of individuāls with NANBH. The expression vectors containing the cloned HCV cDNAs were 90 LV10306 constructed from pSODcfl (Steimer. et al. (1986). In order to be certain that a correct reading frame would be achieved, -three separate expression vectors, pcflAB, pcflCD, and pcflEF were created by ligating either of three Tinkers, AB, CD, and EF to a BamHI-EcoRI fragment derived by dlgesting to completion the vector pSODcfl with EcoRI and BamHI, followed by treatment with alkaline phosphatase. The Tinkers were created from six oligomers, A, Bt Cf Df E, and F. Each oligomer was phosphorylated by treatment with kinase In the presence of ATP prior to annealing to its complementary oligomer. The seguences of the synthetic Tinkers were the following.

Name DNA Seguence (5* to 31)

A GATC CTG AAT TCC TGA TAA

B GAC TTA AGG ACT ATT TTA A

C GATC CGA ATT CTG TGA TAA

D GCT TAA GAC ACT ATT TTA A

E GATC CTG GAA TTC TGA TAA F GAC CTT AAG ACT ATT TTA A

Each of the three Tinkers destroys the original EcoRI site# and creates a new EcoRI site vithin the linker, but within a different reading frame. Hence, the HCV CDNA EcoRI fragments isolated from the clones when inserted into the expression vector/ were in three different reading frames. 91

The HCV cDNA fragments in the designated lambdagtll clones were excised by digestion with EcoRI; each fragment was inserted into pcflAB, pcflCD, and pcflEF. These expression constructs were then transformed into D1210 E. coli celis, the transformants vere cloned, and recombinant bacteria from each clone were induced to express the fusion polypeptides by growing the bacteria in the presence of IPTG.

Expression products of the indicated HCV cDNAs were tested for antigenicity by direct immunological screening of the colonies, using a modification of the method described in Helfman et al. (1983). Briefly, as shown in Fig. 18, the bacteria were plated onto nitrocellulose filters overlaid on ampicillin plates to give approximately 1,000 colonies per filter. Colonies were replica plated onto nitrocellulose filters, and the replicas were regrown overnight in the presence of. 2 mM IPTG and ampicillin. The bacterial colonies were lysed by suspending the nitrocellulose filters for about 15 to 20 min in an atmosphere saturated with CHC13 vapor. Each filter then was placed in an individual 100 mm Petri dish containing 10 ml of 50 mM Tris HC1, pH 7.5, 150 mM NaCl, 5 mM MgCl2 , 3% (w/v) BSA, 40 micrograms/ml lysozyme, and 0.1 microgram/ml DNase. The plates were agitated gently for at least 8 hours at room temperature. The filters were rinsed in TBST (50 mM Tris HC1, pH8.0, 150 mM NaCl, 0.005% Tween 20). After incubation, the celi residues were rinsed and incubated in TBS (TBST without Tween) containing 10% sheep serum; incubation was for 1 hour. The filters were then incubated with pretreated sera in TBS from individuāls with NANBH, which included: 3 chimpanzees; 8 patients with chronic NANBH whose sera 92 LV 10306 were positive with respect to antibodies to HCV C100-3 polypeptide (described in EPO Pub. No. 318,216, and supra.) (al-so called C100); 8 patients with chronic NANBH whose sera were negative for anti-CIOO antibodies; a convalescent patient whose serum was negative for antiCIOO antibodies; and 6 patients with community acquired NANBH, including one whose sera was strongly positive with respect to anti-CIOO antibodies, and one whose sera was marginally positive with respect to anti-CIOO antibodies. The sera, diluted in TBS, was pretrea ted by preabsorption with HSOD. Incubation of the filters with the sera was for at least two hours. After incubation, the filters were washed two times for 30 min with TBST. Labeling of expressed proteīns to which antibodies In the sera bound was accomplished by incubation for 2 hours with I-labeled sheep anti-human antibody. After washing, the filters were washed twice for 30 min with TBST, dried, and autoradiographed. A number of clones (see infra.) expressed polypeptides containing HCV epitopes which were immunologically reactive with serum from Individuāls with NANBH. Five of these polypeptides were very immunogenic in that antibodies to HCV epitopes in these polypeptides were detected in many different patient sera. The clones encoding these polypeptides, and the location of the polypeptide in the putative HCV polyprotein (wherein the amino acid numbers begin with the putative initiator codon) are the following: clone 5-1-1. amino acids 16941735; clone C100, amino acids 1569-1931; clone 33c, amino acids 1192-1457; clone CA279a, amino acids 1-84; and clone CA290a amino acids 9-177. The location of the 93 immunogenic polypeptides within the putative HCV polyprotein are shown immediately below.

Clones encoding polypeptides o£ proven _r.eac_tivity with sera from NANBH patients.

Clone Location within the HCV polyprotein (amino acid no. beginning with putative initiator methionine) CA279a 1-84 CA74a 437-582 13i 511-690 CA290a 9-177 33c 1192-1457 40b 1266-1429 5-1-1 1694-1735 81 1689-1805 33b 1916-2021 25c 1949-2124 14c 2054-2223 8f 2200-3325 33f 2287-2385 33g 2348-2464 39c 2371-2502 15e 2796-2896 clOO 1569-1931

The results on the immunogenicity o£ the polypeptides encoded in the various clones examined suggest efficient detection and immunization systems xnay include panels of HCV polypeptides/epitopes. 94 LV 10306

Expression of HCV Epitopes in Yeast

Three different yeast expression vectors which allow the insertion of HCV cDNA into three different reading frames are constructed. The construction of one of the vectors, PAB24C100-3 is described in EPO Pub. No. 318,216. In the studies below, the HCV cDNA from the clones listed in supra. in the antigenicity mapping study using the E. coli expressed products are substituted for the C100 HCV cDNA. The construction of the other vectors replaces the adaptor described in the above Ē. coli studies with one of the following adaptors:

Adaptor 1

ATT TTG AAT TCC TAA TGA G

AC TTA AGG ATT ACT CAG CT

Adaptor—2.

AAT TTG GAA TTC TAA TGA G AC CTT AAG ATT ACT CAG CT.

The inserted HCV cDNA is expressed in yeast transformed with the vectors, using the expression conditions described supra. for the expression of the fusion polypeptide, C100-3. The resulting polypeptides are screened using the sera from individuāls with NANBH, described supra. for the screening of immunogenic polypeptides encoded in HCV cDNAs expressed in JL*. coli. 95 -Comparis_on_o_f the Hydrophobic Profilēs o.£ HCV Polyproteins with West Nile Virus PolyT>ro£_ein and with Dengtie Virus NS1

The hydrophobicity profilē of an HCV polyprotein segment was compared with that of a typical Flavivirus, West Nile virus. The polypeptide sequence of the West Nile virus polyprotein was deduced from the known polynucleotide sequences encoding the non-structural proteins of that virus. The HCV polyprotein sequence was deduced from the sequence of overlapping cDNA clones. The profilēs were determined using an antigen program which uses a window of 7 amino acid width (the amino acid In guestion, and 3 residues on each side) to report the average hydrophobicity about a given amino acid residue. The parameters giving the reactive hydrophobicity for each amino acid residue are from Kyte and Doolittle (1982) . Fig. 19 shows the hydrophobic profilēs of the two polyproteins; the areas corresponding to the nonstructural proteins of West Nile virus, nsl through ns5, are indicated in the figurē. As seen in the figurē, there is a general similarity in the profilēs of the HCV polyprotein and the West Nile virus polyprotein.

The sequence of the amino acids encoded in the 5'-region of HCV cDNA shown in Fig. 16 has been compared with the corresponding region of one of the strains of Dengue virus, described supra., with respect to the profilē of reģions of hydrophobicity and hydrophilicity (data not shov/n) . This comparison indicated 1 that the polypeptides from HCV and Dengue encoded in this region, which corresponds to the region encoding NS1 (or a 96 LV10306 portion thereof), have a similar hydrophobic/hydrophilic profilē.

The similarity in hydrophobicity profilēs, in combination with the previously identified homologies in the amino acid seguences of HCV and Dengue Flavivirus in EP 0,218,316 suggests that HCV is related to these members of the Flavivirus family.

Characterization of the Putative Polypeptides Encoded Within the HCV ORF

The sequence of the HCV cDNA sense strand, shown in Fig. 17, was deduced from the overlapping HCV cDNAs in the various clones described In EPO Pub. No. 318,216 and those described supra. It may be deduced from theseguence that the HCV genome contains primāri ly one long continuous ORF, which encodes a polyprotein. In the seguence, nucleotide number 1 corresponds to the first nucleotide of the initiator MET codon; minus numbers indicate that the nucleotides are that distance away in the 5'-direction (upstream), while positive numbers indicate that the nucleotides are that distance away in the 31-direction (dovmstream). The composite sequence shows the "sense" strand of the HCV cDNA.

The amino acid seguence of the putative HCV polyprotein deduced from the HCV cDNA sense strand sequence is also shown in Fig. 17, where position 1 begins with the putative initiator methionine.

Possible protein domains of the encoded HCV polyprotein, as well as the approximate boundaries, are the following (the polypeptides identified within the 97 parentheses are those which are encoded in the Flavivirus domain):

Putative Domain Approxima-t_e_B.oundary (amino acid nos.) "C" (nucleocapsid protein) 1-120 "E" (Virion envelope protein(s) 120-400 and possibly matrix (M) proteins "NSl" (complement fixation 400-660 antigen?) ,,NS2H (unknovm function) 660-1050 nNS3" (protease?) 1050-1640 ,,NS4" (unknovm. function) 1640-2000 "NSS" (polymeraee) 2000-? end

It should be notedi however, that hydrophobicity profilēs (described lnfra), Indicate that HCV diverges from the Flavivirus modei, particularly with respect to the region upstream of NS52. Moreover, the boundaries indicated are not intended to show firm demarcations between the putative polypeptidta. 98 LV 10306

The Hydrophilic and Anticenic Profilē af the Polypeptide

Profilēs of the hydrophilicityl/hydrophobicity and the antigenic index of the putative poly .protein encoded In the HCV cDNA seguence shown in Fig. 16 were determined by Computer analysis. The program for hydrophilieityl/hydrophobicity was an described supra. The antigenic index results from a Computer program which relies on the following criteria 1) surface probability, 2) prediction of alpha-helicity by two different methods; 3) prediction of beta-sheet reģions by two different methods; 4) prediction of U-turns by two different methods; 5) hydrophilicityl/hydrophobicity; and flex-ibility.

The traces of the profilēs generated by the Computer analyses are shown In Fig. 20. In the hydrophilicity profilē, deflection above the abscissa indicates hydrophilicity, and below the abscissa Indicates hydrophobicity. The probability that a polypeptido region In antigenic Is usually considered to increase when there is a deflection upward from the abscissa in the hydrophilic and/or antigenic profilē. It should be noted, however, that these profilēs are not necessarily indicators of the strength of the immunogenicity of a polypeptide. 99

Identification of Co-linear Peptides in HCV and

Elaviviruses

The amino acid sequence of the putative polyprotein encoded in the HCV cDNA sense strand was compared with the known amino acid sequences of several members of Flaviviruses. The comparison shows that homology is slight, but due to the reģions in which it is found, it is probably significant. The conserved colinear reģions are shown In Fig. 21. The amino acid numbers listed below the sequences represent the number in the putative HCV polyprotein (See Fig. 17.)

The spacing of these conserved motifs is similar between the Flaviviruses and HCV, and implies that there is some similarity between HCV and these flaviviral aģents.

The following listed materiāls are on deposit under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr.,

Rockville, Maryland 20852, and have been assigned the following Accession Numbers. lambda-gtll ATCC No. DepositDate HCV cDNA library 40394 1 Dec. 1987 clone 81 40388 17 Nov. 1987 clone 91 40389 17 Nov. 1987 clone 1-2 40390 17 Nov.1987 clone 5-1-1 40391 18 Nov. 1987 clone 12f 40514 10 Nov. 1988 clone 35f 40511 10 Nov. 1988 clone 15e 40513 10 Nov. 1988 clone K9-1 40512 10 Nov. 1988 JSC 308 20879 5 May 1988 pS356 67683 29 April 1988 100 LV 10306

In addition, the following deposits vrere made on 11 May 1989.' gfccaln Linkers ATCC No,. D1210 (Cfl/5-1-1) EF 67967 D1210 (Cfl/81) EF 67968 D1210 (Cfl/CA74a) EF 67969 D1210 (Cfl/35f) AB 67970 D1210 (Cfl/279a) EF 67971 D1210 (Cfl/C36) CD 67972 D1210 (Cfl/13i) AB 67973 D1210 (Cf1/C33b) EF 67974 D1210 (Cfl/CA290a) AB 67975 HB101 (AB24/C100 #3R) 67976 The following derivatives of strain D1210 vrere deposited on 3 May 1989. ATCC No. 679.56 67952 67949 67954 67958 67953 67050 67951 67955 67957 67959

Strain Derivative pCFlCS/c8f pCFlAB/C12f pCFlEF/14c pCFlEF/15e pCFIAB/C25c pCFlEF/C33c pCFlEF/C33f pCFlCD/33g pCFlCD/C39c pCFlEF/C40b pCFIEF/CA167b

The following strains vrere deposited on May 12,1989. 101

Strain ATCC : Lambda gtll(C35) 40603 Lambda· gtlO (beta-5a) 40602 D1210 (C40b) 67980 D1210 (Ml6) 67981

The deposited materiāls mentioned herein are intended for convenience only, and are not required to practice the present invention in view of the descriptions herein, and in addition these materiāls are incorporated herein by reference.

Industrial Applicability

The invention, in the various manifestations disclosed herein, has many industrial uses, some of which are the £ollowing. The HCV cDNAs may be used for the design of probes for the detection of HCV nucleic acids in samples. The probes derived from the cDNAs may be used to detect HCV nucleic acids in, for example, Chemical synthetic reactions. They may also be used in screening programs for anti-viral aģents, to determine the effect of the aģents in inhibiting virai replication in celi culture systems, and animal modei systems. The HCV polynucleotide probes are also useful in detecting virai nucleic acids In humāns, and thus, may serve as a basis for diagnosis of HCV infections in humāns.

In addition to the above, the cDNAs provided herein provide Information and a means for synthesizing polypeptides containing epitopes of HCV. These polypeptides are useful in detecting antibodies to HCV antigens. A series of immunoassays for HCV infection, 102 LV10306 based on recombinant polypeptides containing HCV epitopes are described herein, and will find commercial use in diagnosing -HCV induced NANBH, in screening blood bank donors for HCV-caused infectious hepatitis, and also for detecting contaminated blood £rom infectious blood donors. The virai antigens will also have utility in monitoring the efficacy of anti-viral aģents In animal modei systems. In addition, the polypeptides derived from the HCV cDNAs disclosed herein will have utility as vaccines for treatment of HCV infections.

The polypeptides derived from the HCV cDNAs, besides the above stated uses, are also useful for raising anti-HCV antibodies. Thus, they may be used in anti-HCV vaccines. However, the antibodies produced as a result of immunization with the HCV polypeptides are also useful in detecting the presence of virai antigens in samples. Thus, they may be used to assay the production of HCV polypeptides in Chemical systems. The anti-HCV antibodies may also be used to monitor the ef£icacy of anti-viral aģents In screening programs where these aģents are tested in tissue culture systems. They may also be used for passive immuno the rapy, and to diagnose HCV caused NANBH by allowing the detection of virai antigen(s) in both blood donors and recipients. Another important use for anti-HCV antibodies is in affinity chromatography for the purification of virus and virai polypeptides. The purified virus and virai polypeptide preparations may be used in vaccines. However, the purified virus may also be useful for the development of celi culture systems in which HCV replicates.

Antisense polynucleotides may be used as inhibitors of virai replication. 103

For convenience, the anti-HCV antibodies and HCV polypeptides, whether natūrai or recombinant, may be packaged into kits. 104

Claims (34)

A recombinant polynucleotide comprising a sequence derived from a HCV cDNA located in clone 13i or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA216a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18gj or clone 16jh, or the HCV cDNA sequence corresponds to the nucleotide of FIG. 17 with numbers 319 to 1348 or 8659 to 8866. 2. A recombinant polynucleotide of claim 1 encoding an HCV epitope. A recombinant vector comprising a polynucleotide according to claim 1 or 2. 4. A host cell transformed with a vector according to item 3. 5. A recombinant expression system comprising the ORF structure of the correct reading sequence of DNA formed by recombinant polynucleotide according to claim 1 or 2, wherein the ORF is operably linked to a control sequence compatible with the desired host. 6. The cell. transformed by a recombinant expression system according to item 5. A polypeptide produced in a cell according to claim 6 A purified polypeptide comprising an HCV cDNA encoded epitope, wherein the HCV cDNA is a sequence corresponding to the nucleotide of FIG. 17 with numbers from -319 to 1358 or from 8659 to 8866. An immunogenic polypeptide produced in a cell transformed with a recombinant expression vector comprising a DNA ORF of HCV cDNA, wherein the HCV cDNA is a sequence derived from the HCV cDNA sequence in clone CA 279a or clone CA 74a or clone 13i. , or clone CA 290a, or clone 33c, or clone 40b, or clone 33b, or clone 25c, or clone 14c, or clone 8f, or clone 33f, or clone 33g, or clone 15e, and the ORF is operatively linked to the control sequence compatible with the desired host. A peptide comprising an HCV epitope, wherein the pept'id formula is AAx-AAy and X and Y denote amino acid sequence numbers as shown in FIG. 17, and the peptide 'taken from the group: AA1-AA25, AA1-AA50, AA-? -AA84, AAg-AA4, AA4-AA ^ g, AA5-AA2o »AA2o AA25, AA35-AA45, AA50-AA10o , AA4q "AAgg, AA45-AA65, AA65- aa75, AA80-AA90, AAg9-AA1O2» AA95-AA-AA0, AA105-AAi2o > AA10o " AAi50, AA150-AA2oo > AA155-AA17q, 90190-ΑΑ2ιοι AA2oo AA25o, AA22o AA24o, AA245-AA2651 AA250-AA300, AA29o-AA33o, Δ29o-ΑΑ305, AA300-AA350, AA31o-AA330, AA350-AA400, AA380-AA395, AA405-AA495, AA405-AA495, AA400-AA450, AA405.AA415, AA4-AA5, AA425, AA425, AA435, AA437, AA582, AA450-AA500, AA4O-AA40O, AA40O-AA47O, Δ475-Δ495, AA50q-AA550, Δ511-90690, AA515-AA55o, AA550-AA60o, AA55q "AA825, AA575-AA6051 AA585-AA6oo, AA600-AA650, AA600-AA625, AA635-AA665, AA750-AA750, AA70q-AA7251 AA725-AA775, ^ A770 " 790 »AA750-AA80o, ΑΑβοο'ΑΑβίδ, AA825-AA85o, AA850-AA875, 2 LV10306 ΑΑδΟΟ'ΑΑβδΟι AAg20, AA850-AAgoo, AAg20-AAg45, AA940-AA955, AAg70 " AAgg0 > AAggg-AAoOoOi AA " AA1060 > AA 000's "AA Q25» AA10oo "AA105o. AA1025-AA1040) AA104-AA1055, AAio75 " AA1175, AA1050-AA1200 »aa1070-aa1100 > AAnoo-AAl ^ AA, Allyl4o-AAlg5, AAl9292-AA457, 951195-ΑΑ125ο, AA12oo " AA1225 »AA 225- AA 250, AA250-AA430» AA126o AA1310, AA 260 ”AA 280» AA 266 " AAl 428, O 1n co < < 0 0 co < < AA2929 " AA13if AA1310-AA134o, AA345-AA405, AA 345-AA 365, AA1350 " AA1400 > AA 365-AAl 380, AA3838-AA1405, AA 4oo " AA 450, AA1450-AA-500, AA4eo-AAl475, AA4747-AA55, > > -P * > > cn 0 0 ΑΑ1500-ΑΑ155ο, AA5oo-AA15l5, AA1515-AA1550, AA155o-AAlgoo, AA 545-AA 1560, AA 569 ”A Al g31, AA57a-AAl590, AA595-AAigil, AA ^ go-Alesco, Algiers-AAlg45, AAlg50-AA1669, AA 685-AAl 670, AA-689 "AA8080 > AAlg9o-AA172o, AA 694-AA 735, AA 720-AA 745, AAi745-AA1770, AA7575-AA18oo, AA 775-AA 810, AA 795-AA 850, AA185o-AA1900, AA199_AAl95o, AA-900-AA 92o > AA169-AA2o2i. AA192o-AA1940, AA949-AA2124 > AAl95o-AA2ooo > AA 950-AA 985, A 98o ~ A Agooo > AA2000 "AA2050» The A2005-A A2025 > AA2020 ”AA2045 > AA2o45-AA21oo. AA2045 ”AA2070. AA2054-AA2223, AA2070 “AA2100 > AA2100 "AA2150, ΑΑ2150-2002200, AA2200" AA2250, AA22oo "AA2325, AA2250" AA233o, AA2255 "7227ο» ΑΑ2265-ΑΑ228ο, AA2280 " AA22901 AA2287 " The A2385, AA2300 “AA2350, A2222 " AA231 0 > AA2310 " AA2330. AA2330-AA2350, AA2350-AA2400, AA2348-AA2464, AA2345-AA24L5, AA2345-AA2375, AA2370-AA24L0, AA237l-AA2502, AA2400 'AA2450, AA24o0' A2424, AA245-AA2450, AA2445-AA2500, AA2445-AA2475, AA2470 -AA249o, AA25oo "AA2550, AA2505-AA2540, AA2535" AA255o, 5255ο-ΑΑ28οοι AA2560 "AA258o, AA2goo" AA265o, 602605 "ΑΑ2620. 3 ΑΑ2620 " ΑΑ2650 »ΑΑ2650-ΑΑ2700 · ΑΑ2655“ ΑΑ2670 > 402640 " ΑΑ2660 »ΑΑ2670 " ΑΑ2700 > ΑΑ27οο ~ ΑΑ2750. AA2740-AA27eo »AA2750 " AA28oo, ΑΑ278Ο " ΑΑ203Ο» ΑΑ2785 " ΑΑ2810 > 982798-882888, AA2gio-AA2825, ΑΑ20οο " ΑΑ285ο »85285ο-ΑΑ29οο. 85285ο-ΑΑ2885 > 882885 " ΑΑ29ο5 > 002900 " ΑΑ2950 > AA29i " ΑΑ2930 »ΑΑ2925-ΑΑ2950ι AA2945-GALS (C '-gals) A polypeptide comprising a peptide according to claim 10. An immunogenic polypeptide associated with a solid substrate, wherein the polypeptide is of one of claims 7-11, or comprises an HCV cDNA encoded epitope, wherein the HCV cDNA is a sequence corresponding to FIG. 17 with nucleotide numbers from -319 to 1348 or from 8659 to 8866. A monoclonal antibody to an epitope encoded by HCV cDNA, wherein the HCV cDNA sequence is a sequence corresponding to FIG. 17 or nucleotide numbers from -319 to 1348 or from 8659 to 8866, or the sequence in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16jhJ A polyclonal antibody purified preparation directed against a polypeptide comprising an HCV cDNA encoded epitope, wherein the HCV cDNA sequence is a sequence corresponding to FIG. 17 or nucleotide numbers from -319 to 1348 or from 8659 to 8866, or the sequence in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA216a, or clone 290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h. 15. A polynucleotide probe for HCV comprising an HCV sequence derived from a sequence of HCV cDNA corresponding to FIG. 17 to nucleotide 4 LV10306 numbers -319 to 1348 or 8659 to 8866 or a complementary sequence of HCV cDNA. A kit for sample analysis to determine the presence of HCV polynucleotides comprising a polynucleotide probe containing a nucleotide sequence of about 8 or more nucleotides taken from the HCV cDNA, wherein the HCV cDNA sequence is as shown in FIG. 17 in a suitable package as shown in nucleotide numbers -319 to 1348 or 8659 to 8866. A kit for sample analysis to determine the presence of an HCV antigen comprising an antibody immunologically acting on an HCV antigen, wherein the antigen contains an epitope encoded by HCV cDNA, wherein the HCV cDNA sequence is a sequence corresponding to FIG. 17 with nucleotide numbers from -319 to 1348 or from 8659 to 8866 or the sequence as it is in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone p14a, or clone CA216a or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h. A kit for sample analysis to determine the presence of HCV antibodies comprising an antigenic polypeptide comprising an HCV epitope encoded by HCV cDNA, wherein the HCV cDNA sequence is a sequence corresponding to FIG. 17 or nucleotide numbers from -319 to 1348 or from 8659 to 8866, or the sequence as in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone 4a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h. A kit for sample analysis to determine the presence of HCV antibodies comprising an antigenic polypeptide obtained by expression of HCV cDNA in clone 279a, or clone CA74a, or clone 13i, or clone CA290a, or clone 33C, or clone 40b, or clone pi33b, or clone 25c, or clone 14c, or clone 8f, or clone 33f, or clone 33g, or clone 39c, or clone 15e, and is contained in the appropriate packaging. 5 20. A method for detecting HCV nucleic acids in a sample, comprising: a) reacting a sample nucleic acid with a polynucleotide probe to HCV, wherein the probe comprises an HCV sequence derived from a sequence of HCV cDNA corresponding to FIG. 17 with nucleotide numbers from -319 to 1348 or from 8659 to 8866, and the reaction occurs under conditions allowing polynucleotide duplex formation between the probe and HCV nucleic acid in the sample; b) recording a duplex containing a probe-containing polynucleotide formed in step a). 21. An immunological assay for detecting HCV antigen, comprising: a) incubating a sample possibly containing HCV antigen with an anti-HCV epitope encoded by an HCV cDNA incubation, wherein the HCV cDNA sequence is a sequence; corresponding to fig. 17 depicted with nucleotide numbers from -319 to 1348 or from 8659 to 8866, or the sequence as in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16 [mu] g, under conditions allowing the formation of an antigen-antibody complex; " b) recording the antibody-containing antigen-antibody complex formed in step a). An immunological assay for detecting antibodies to HCV antigens, characterized in that: a) incubating the sample with a possible anti-HCV antibody content with an antigenic polypeptide containing an epitope encoded by HCV cDNA, the sequence of which corresponds to Fig. 17 depicted with nucleotide numbers -319 to 1348 or 8659 to 8866, or sequence 6 LV10306 as it is in clone 13i, or clone 26], or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone 4a, or clone CA216a, or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h, under conditions permitting the formation of an antigen-antiviral complex; b) recording the antigen-antibody of the complex comprising the antigenic polypeptide formed in step a). An immunological assay for the detection of antibodies to an HCV antigen, comprising: a) incubating the sample with a possible anti-HCV antibody content in a polypeptide according to claim 9 under conditions that ensure the formation of the antigen-antiviral complex; b) recording the antigen-antibody of the complex comprising the antigenic polypeptide formed in step a). A vaccine for the treatment of HCV infection comprising an immunogenic polypeptide comprising an HCV epitope encoded by an HCV cDNA having the sequence shown in FIG. 17 with nucleotide numbers from -319 to 1348 or from 8659 to 8866 or the sequence as it is in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone p14a, or clone CA216a or clone CA290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h, wherein the immunogenic polypeptide is taken at a pharmacologically effective dose and in a pharmaceutically acceptable carrier. A method for producing antibodies against HCV, characterized in that an immunogenic polypeptide comprising an HCV epitope encoded by an HCV cDNA having the sequence shown in FIG. 17 with nucleotide numbers -319 to 1348 or 8659 to 8866 or the sequence as it is in clone 279a, or clone CA74a, or clone 13i, or clone CA290a, or clone 33c, or clone 40b, or clone 33b, or clone 25c or clone 14c, or clone 8f, or clone 33f, or 7 clone 33g, or clone 39c, or clone 15e, wherein the immunogenic polypeptide is taken at a pharmacologically effective dose and in a pharmaceutically acceptable carrier. An anti-informative polynucleotide derived from HCV cDNA, wherein the HCV cDNA corresponds to FIG. 17 depicted. A process for preparing purified C100-3 purified protein, comprising: a) preparing a crude cell lysate containing the C100-3 polypeptide; b) treating the crude lysate with a quantity of acetone that precipitates the polypeptide; c) isolating and solubilizing the deposited material; d) isolating the C100-3 polypeptide by anion exchange chromatography; e) further isolating the polypeptide after d) by gel filtration. A method for producing an HCV polypeptide, characterized in that: a) preparing a host cell transformed with a recombinant expression vector containing a DHS ORF formed from a HCV cDNA, wherein the HCV cDNA sequence corresponds to the sequence in clone 13i or clone 26j; or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone 4a, or clone CA216a, or clone CA 290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h, or HCV cDNA The sequence corresponds to the nucleotides in FIG. 17 with numbers -319 to 1348 or 8659 to 8866, and the ORF is operatively associated with a control sequence compatible with the desired host; b) incubating host cells under conditions that allow expression of the NCV polypeptide. A method for producing an HCV polypeptide, characterized in that: a) preparing a host cell transformed with a recombinant expression vector containing a DHS ORF formed from HCV cDNA, wherein the HCV cDNA sequence corresponds to the sequence in clone CA 279a or clone CA, 74a or 8 LV10306 in clone 13i, or clone CA 290a, or clone 33C, or clone 40b, or clone 33b, or clone 25c, or clone 14c, or clone 8f, or clone 33f, or clone 33g, or clone 39c, or clone 15e, and the ORF operatively relates to a control sequence compatible with the desired host; b) incubating host cells under conditions that allow expression of the NCV polypeptide. A method for obtaining a host cell transformed with a recombinant polynucleotide comprising a sequence of HCV cDNA derived from the HCV cDNA sequence in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone. pi14a, or clone CA 216a, or clone CA 290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16ji, or the HCV cDNA sequence corresponds to nucleotide FIG. 17 with numbers -319 to 1348 or 8659 to 8866, characterized in that a) a) preparation of a transformer host cell is performed; b) preparing a recombinant polynucleotide; c) incubation of (a) and (b) under conditions that allow cell transformation with a polynucleotide. A method for obtaining a recombinant polynucleotide comprising a sequence of HCV cDNA, wherein the HCV cDNA sequence is derived from the sequence in clone 13i, or clone 26j, or clone 59a, or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA 216a, or clone CA 290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h, or the HCV cDNA sequence 'corresponds to nucleotide FIG. 17 having the numbers -319 to 1348 or 8659 to 8866, characterized in that; a) preparing a host cell transformed with recombinant polynucleotide; b) isolating the polynucleotide from said host cell. 32. A method of obtaining NCV free blood, characterized in that: a) prepare a blood sample with a possible NCV or NCV antibody content; b) preparing an immunogenic polypeptide according to claim 28 or 29; c) incubating the sample (a) with the immunogenic polypeptide (b) under conditions which allow the formation of HCV antibody-polypeptide complexes; d) identifying complexes formed in step (c); e) retaining blood samples that do not show complexes in step (d). A method of obtaining NCV free blood, characterized in that: a) preparing a nucleic acid from a blood sample with the possible presence of NCV polynucleotides; b) preparing a probe on HCV containing the sequence of HIV derived from the NCV cDNA determined by the nucleotide sequence from -319 to 1348 or from 8659 to 8866 by Fig. 17; c) contacting (a) with (b) under conditions permitting the formation of a polynucleotide duplex between the probe and the HCV nucleic acid from the sample: d) observing a probe-containing polynucleotide duplex formed in step (c); e) retaining blood samples that do not show complex formation in step (d). A method for producing hybridoma, which produces monoclonal antibodies to HCV, characterized in that: a) immunizes an individual with an immunogenic polypeptide comprising an epitope encoded by an HCV cDNA in a sequence such as clone 13i, or clone 26j, or clone 59a; or clone 84a, or clone CA156e, or clone 176b, or clone pi14a, or clone CA 216a, or clone CA 290a, or clone ag30a, or clone 205a, or clone 18g, or clone 16h, or the cDNA sequence of HCV 10 LV10306 for nucleotides fig. 17 with numbers -319 to 1348 or 8659 to 8866, or b) to immunize an individual with an immunogenic polypeptide according to item 29; c) providing anti-aging cells of the immunized individuals producing antibodies; d) selecting an aging cell that produces antibodies that react with an HCV epitope in an immunogenic polypeptide according to (a) or (b), and e) cultivate said non-aging cell. 11