DK175975B1 - NANBV diagnostics and vaccines - Google Patents

Description

DK 175975 B1 NANBV diagnostics and vaccines

Technical Field 5 The present invention relates to materials and methodologies for controlling the spread of non-A, non-B hepatitis virus (NANBV) infection. More specifically, it relates to diagnostic DNA fragments, diagnostic proteins, diagnostic antibodies and protective antibodies against an etiologic agent for NANB hepatitis, i.e. hepatitis C virus.

10

The prior art features of the application

Barr el al. (1986), Biotechniques 4: 428.

Botstein (1979), Gene 8:17.

Brinton, M.A. (1986) in THE VIRUSES: THE TOGAVIRIDAE AND FLAVIVIRIDAE (Series eds. Fraenkel-Conrat and Wagner, vol. Eds.

Schlesinger and Schlesinger, Plenum Press), pages 327-374.

Broach (1981) in: Molecular Biology of the Yeast Saccharomyces, Volume 1, page 445, Cold Spring Harbor Press.

Broach et al. (1983), Meth. Enz. 101: 307th

Chang et al. (1977), Nature 198: 1056.

Chirgwin et al. (1979), Biochemistry 18: 5294.

Chomczynski and Sacchi (1987), Analytical Biochemistry 162: 156.

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Clewell (1972), J. Bacteriol. 110: 667th

Cohen (1972), Proc. Natl. Acad. Sci. USA 69: 2110.

Cousens et al. (1987), Genes 61: 265.

De Boer et al. (1983), Proc. Natl. Acad. Sci. USA 292: 128.

Dreesman et al. (1985), J. Infect. Disease 151: 761.

30 Feinstone, S.M. and Hoofnagle, J.H. (1984), New Engl. J. Med. 311: 185th

Fields & Knipe (1986), FUNDAMENTAL VIROLOGY (Raven Press, N.Y.).

2 DK 175975 B1

Fiers et al. (1978), Nature 273: 113.

Gerety, R.J. et al., in VIRAL HEPATITIS AND LIVER DISEASE (Vyas, B.N.,

Tuesday, J.L., and Hoofnagle, J.H., eds,

Grune and Stratton, Inc., (1984) pages 23-47.

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10 Hammerling etal. (1981), MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS.

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15 Holland etal. (1978). Biochemistry 17: 4900.

The Netherlands (1981), J. Biol. Chem. 256: 1385.

Houghton et al. (1981), Nucleic Acids Res. 9: 247

Hunyh, T.V. et al. (1985) in DNA CLONING TECHNIQUES; A PRACTICAL

APPROACH (D. Glover, Ed., IRL Press, Oxford, U.K.) pages 49-78.

Immune. Rev. (1982) 62: 185.

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Kennet et al. (1980) MONOCLONAL ANTIBODIES.

Laemmli (1970), Nature 227, 680.

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25 Maniatis.T., Et al. (1982) MOLECULAR CLONING; A LABORATORY MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).

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20 Roehrig (1986) in THE VIRUSES: THE TOGAVIRIDAE AND FLAVIVIRIDAE (Series eds. Fraenkel-Conrat and Wagner, vol. Eds. Schlesinger and Schlesinger, Plenum Press)

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5 Tsu and Herzenberg (1980), in SELECTED METHODS IN CELLULAR IMMUNOLOGY (W.H. Freeman and Co.) pages 373-391.

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Patent Specifications: US Patent No. 4,341,761 US 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. Pat. 4,491,632 US Patent No. 4,493,890 to DK 175975 B1

The prior art

Non-A, non-B hepatitis (NANBH) is a transmissible disease or disease family believed to be virus-induced and distinguishable from 5 other types of virus-related liver diseases, including the known hepatitis virus-caused diseases, i.e. hepatitis A virus (HAV), hepatitis B virus (HBV), and delta hepatitis virus (HDV), as well as hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV).

NANBH was first identified in individuals who had received transfusion.

10 Human transmission to chimpanzees and serial transmission in chimpanzees provided evidence that NANBH is due to a transmitted infection agent (s). However, the transmissible agent responsible for NANBH is still unidentified and the number of agents causing the disease is unknown.

15

Epidemiological evidence suggests that there may be three types of NANBH: the epidemic type transmitted through water, the blood or cannula type and the occasional (community acquired) type. However, the number of agents that may be causing NANBH is unknown.

20

Clinical diagnosis and identification of NANBH is primarily achieved by the exclusion of other viral markers. Among the methods used to detect putative NANBV antigens and antibodies are agar diffusion, contraimmunoelectrophoresis, immunofluorescence microscopy, immune electron microscopy, radioimmunoassay and enzyme-linked immunosorbent assay.

However, none of these assays have been found to be sufficiently sensitive, specific and reproducible for use as a diagnostic test for NANBH. .

30 There has so far been no clarity or agreement as to the identity or specificity of the antigen / antibody systems associated with 'NANBH agents. This is due, at least in part, to prior or simultaneous infection with HBV and NANBV in individuals, as well as the known complexity of the soluble and particulate antigens associated with HBV, as well as the integration of HBV DNA into the liver cell genome. In addition, there is a possibility that NANBH is due to more than one 'infection agent' as well as the possibility that NANBH has been misdiagnosed. It is further unclear what the serological analyzes show in the serum of patients with NANBH. It has been claimed that the agar gel diffusion and counterimmunoelectrophoresis assays detect autoimmune reactions or non-specific protein interactions that may occasionally occur between serum samples and do not represent specific NANBV antigen antibody reactions. Immunofluorescence- and enzyme-linked immunosorbent and radioimmunoassays appear to detect low levels of a rheumatoid factor-like substance frequently present in serum in patients with NANBH as well as in patients with other liver and non-liver diseases. Some of the detected reactivity may represent antibody to host-determined cytoplasmic antigens.

There are a number of NANBV candidates. See, e.g. the statements by Prince 20 (1983), Feinstone and Hoofnagle (1984) and Overby (1985, 1986, 1987) and the article by Iwarson (1987). But there is no evidence that any of these candidates represent the etiological agent of NANBH.

There is a significant need for sensitive, specific methods to screen and identify carriers of NANBV and NANBV-contaminated blood or blood products. Posttransfusion hepatitis (PTH) occurs in ca. 10% of patients who received transfusion and NANBH account for up to 90% of these cases. The main problem with this disease is the frequent development of chronic liver damage (25 - 55%).

30 7 DK 175975 B1

Patient care, as well as prevention of transmission of NANBH with blood and blood products or by close personal contact, requires reliable diagnostic and prognostic tools for the detection of nucleic acids, antigens and antibodies associated with NANBV. In addition, there is also a need for effective vaccines and immunotherapeutic therapeutics to prevent and / or treat the disease.

Description of the Invention The invention generally relates to the use of a newly discovered etiologic agent for NANBH, hepatitis C virus (HCV). In particular, the invention provides a family of cDNA replicas of portions of the HCV genome encoding an HCV antigen determinant containing a continuous sequence of at least 8 amino acids that can be used to detect antibodies to HCV in vitro.

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Furthermore, the invention relates to a fusion protein comprising the above polypeptide (s), wherein the continuous sequence is fused into a non-HCV amino acid sequence selected from the group consisting of super oxide dismutase sequence, beta-galactosidase sequence, sequence which enables secretion, sequence enabling particle formation, hepatitis B surface precursor, and hepatitis B surface antigen sequence.

The invention also relates to an immunoassay kit comprising the above polypeptide.

25

The invention also relates to immunoassay for in vitro determination of antibodies which comprise the following steps: (a) providing a polypeptide comprising an antigenic determinant capable of binding to anti-HCV antibodies, the antigenic determinant comprising an amino acid sequence encoded in an of Figures 47, 14, 26, 8, or in the Iambda-gt11 cDNA library deposited in the ATCC under accession number 40394, and comprises at least 8 amino acids, (b) incubating an in vitro biological sample with the polypeptide under conditions allowing forming an antibody-antigen complex, and (c) determining whether an antibody-antigen complex containing the polypeptide is formed.

Furthermore, the invention relates to an antibody composition which is an anti-HCV antibody composition comprising antibodies which bind to an HCV antigenic determinant in any of the above polypeptides, and which antibodies are (a) a pure product of polyclonal antibodies, (b) a product of monoclonal antibodies.

According to the invention, there is also provided a composition wherein the anti-HCV antibodies are bound to a solid substrate, as well as immunoassay kits comprising one of the above-mentioned polypeptides in a suitable container.

The invention also relates to immunoassay demonstrating the presence of HCV antigen in a sample which comprises: (a) providing an anti-HCV antibody composition mentioned above, (b) incubating a biological sample with the anti-HCV antibody composition under conditions such as enabling the formation of an antibody-antigen complex, and (c) determining whether the antibody-antigen complex comprising the anti-HCV antibody is formed.

Finally, the invention relates to substantially isolated polypeptide for use in preparing an anti-HCV antibody composition comprising HCV antigenic determinant and containing a continuous sequence of at least 8 amino acids encoded as in the sequence shown in Figures 47, 14, 26 or 32.

Studies of the nature of the HCV genome using probes derived from the HCV cDNA as well as sequence information contained in the HCV cDNA suggest that HCV is a flavivirus or a flavi-like virus.

Portions of the HCV-derived cDNA sequences are well suited as probes to diagnose the presence of viruses in samples and to isolate naturally occurring variants of the virus. These cDNAs also make polypeptide sequences of HCV antigens encoded in the HCV * genome (HCV genomes) available and enable polypeptides to be prepared as standards or reagents in diagnostic studies and / or as vaccine components. Both polyclonal and monoclonal antibodies directed against HCV epitopes contained in these polypeptide sequences are also well suited for diagnostic studies, as therapeutics, for screening antivirals, and for isolating the NANBV agent from which these cDNAs are derived. By using probes derived from these cDNAs, it is further possible to isolate and sequence other parts of the HCV genome, thus producing additional probes and polypeptides suitable for diagnosis and / or treatment, both prophylactically and therapeutically, by NANBH.

in

Brief Description of the Drawings 25 i

Figure 1 shows the double-stranded nucleotide sequence of the HCV cDNA insert ion in clone 5-1-1 and the putative amino acid sequence of the polypeptide encoded therein.

Figure 2 shows the homologies between the overlapping HCV cDNA sequences in clones 5-1-1.81, 1-2 and 91.

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Figure 3 shows an HCV cDNA composite sequence derived from overlapping clones 81, 1-2 and 91 and the amino acid sequence encoded therein.

Figure 4 shows the double-stranded nucleotide sequence of the HCV cDNA insert ion of clone 81 and the putative amino acid sequence of the polypeptide encoded therein.

Figure 5 shows the HCV cDNA sequence in clone 36, the segment that overlaps with the NANBV cDNA in clone 81 and the polypeptide sequence encoded in clone 36.

Figure 6 shows the total ORF or HCV cDNAs of clones 36 and 81 and the polypeptide encoded therein.

Figure 7 shows the HCV cDNA sequence of clone 32, the segment overlapping clone 81 and the polypeptide encoded therein.

Figure 8 shows the HCV cDNA sequence of clone 35, the segment overlapping clone 36 and the polypeptide encoded therein.

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Figure 9 shows the total ORF of HCV cDNAs in clones 35, 36, 81 and 32 and the polypeptide encoded therein.

Figure 10 shows clone 37b * s HCV cDNA sequence, the segment overlapping clone 35 and the polypeptide encoded therein.

Figure 11 shows the HCV cDNA sequence of clone 33, the segment that overlaps with clone 32 and the polypeptide encoded therein.

Figure 12 shows the HCV cDNA sequence of clone 40b, the segment overlapping clone 37b and the polypeptide encoded therein.

L _______-— --——-----! ---- 11 DK 175975 B1

Figure 13 shows the HCV cDNA sequence of clone 25c, the segment overlapping clone 33b and the polypeptide encoded therein.

Figure 14 shows the nucleotide sequence and the polypeptide encoded therein in ORF, which extends throughout the HCV cDNAs of clones 40b, 37b, 35, 36, 81,32, 33b and 25c.

Figure 15 shows the HCV cDNA sequence in clone 33c, the segment that overlaps with clones 40b and 33c and the amino acids encoded therein.

Figure 16 shows the HCV cDNA sequence in clone 8h, the segment overlapping clone 33c and the amino acids encoded therein.

Figure 17 shows the HCV cDNA sequence in clone 7e, the segment overlapping clone 8h and the amino acids encoded therein.

Figure 18 shows the HCV cDNA sequence in clone 14c, the segment overlapping clone 25c and the amino acids encoded therein.

20

Figure 19 shows the HCV cDNA sequence of clone 8f, the segment overlapping clone 14c and the amino acids encoded therein.

Figure 20 shows the HCV cDNA sequence in clone 33f, the segment that overlaps with clone 8f and the amino acids encoded therein.

Figure 21 shows the HCV cDNA sequence in clone 33g, the segment overlapping clone 33f and the amino acids encoded therein.

Figure 22 shows the HCV cDNA sequence in clone 7f, the segment that overlaps the sequence in clone 7e and the amino acids encoded therein.

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Figure 23 shows the HCV cDNA sequence in clone 11b, the segment overlapping the sequence in clone 7f and the amino acids encoded therein.

Figure 24 shows the HCV cDNA sequence in clone 14i, the segment that overlaps the sequence in clone 11b and the amino acids encoded therein.

Λ

Figure 25 shows the HCV cDNA sequence in clone 39c, the segment overlapping the sequence in clone 33g and the amino acids encoded therein.

10

Figure 26 shows a composite HCV cDNA sequence derived from the sequenced cDNAs of clones 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f and 33g, the amino acid sequence of the polypeptide encoded in the extended ORF of the derived sequence is also shown.

15

Figure 27 shows the sequence of the HCV cDNA in clone 12f, the segment overlapping clone 14i and the amino acids encoded therein.

Figure 28 shows the sequence of the HCV cDNA in clone 35f, the segment that overlaps with clone 39c and the amino acids encoded therein.

Figure 29 shows the HCV cDNA sequence in clone 19g, the segment overlapping clone 35f and the amino acids encoded therein.

Figure 30 shows the clone 26g sequence, the segment overlapping clone 19g and the amino acids encoded therein.

Figure 31 shows the clone 15e sequence, the segment overlapping clone 26g and the amino acids encoded therein.

30 DK 175975 B1 i 13

Figure 32 shows the sequence of a composite cDNA derived by sequencing the clones 12f-15e in the 5 'to 3' direction, also shown in the continuous ORF encoded amino acids.

Figure 33 shows a photograph of Western blots of a fusion protein, SOD-NANBso-r, with chimpanzee serum from chimpanzees infected with BB-NANB, HAV and HBV.

Figure 34 shows a photograph of Westem blots of a fusion protein, 10 SOD-NANB5-1-1 · with serum from humans infected with NANBV, HAV, HBV and serum from control subjects.

Figure 35 is a map showing the significant features of the pAB24 vector.

Figure 36 shows the putative carboxyl terminal amino acid sequence of the fusion polypeptide C100-3 and the nucleic acid sequence encoding it.

Figure 37A is a photograph of a coomassie blue colored polyacrylamide gel identifying C100-3 expressed in yeast.

20

Figure 37B shows a Western blot of C100-3 with serum from a NANBV-infected human.

Figure 38 shows an autoradiography of a Northern blot of RNA isolated from the liver of a BB-NANBV-infected chimpanzee probed with clone 81 BB-NANBV cDNA.

Figure 39 shows an autoradiography of NANBV nucleic acid treated with RNase A or DNase I and probed with clone 81 BB-NANBV cDNA.

30 1 14 DK 175975 B1

Figure 40 shows an autoradiography of nucleic acid extracted from NANBV particles captured from infected plasma with anti-NANBs.vi and probed with 32P-labeled NANBV cDNA from clone 81.

Figure 41 shows autoradiographs of filters containing isolated NANBV nucleic acids probed with 32P-labeled plus and minus strand DNA probes derived from NANBV cDNA in clone 81.

Figure 42 shows the homologies between a polypeptide encoded in HCV cDNA and a Dengue flavivirus NS protein.

Figure 43 shows a histogram of the HCV infection distribution in stochastic samples as determined by an ELISA screening.

Figure 44 shows a histogram of the HCV infection distribution in stochastic samples using two configurations of immunoglobulin enzyme conjugate in an ELISA assay.

Figure 45 shows the sequences of a primer mix derived from a conserved 20 sequence in NS1 from flaviviruses.

Figure 46 shows the HCV cDNA sequence in clone k9-1, the segment overlapping the cDNA of Figure 26 and the amino acids encoded therein.

Figure 47 shows the sequence of a composite cDNA that was derived by sequencing clones k9-1-15e in the 5 'to 3' direction; the amino acids encoded in the continuous ORF are also shown.

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Embodiments of the Invention I. Definitions 5 The term "hepatitis C virus" has been reserved by scientists in the art for a novel etiologic agent for NANBH. Accordingly, "hepatitis C virus" (HCV) as used herein refers to an agent causing NANBH previously referred to as NANBV and / or BB-NANBV. The terms HCV, NANBV and BB-NANBV are used with the same meaning 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 can be used herein with the same meaning.

The term "HCV", as used herein, denotes a virus species causing NANBH and attenuated strains or defective interfering particles derived therefrom. As shown below, the HCV genome is constituted by RNA. It is known that RNA-containing viruses have relatively high spontaneous mutation rates, i.e. allegedly in the range of 10'3 -10-4 pr. nucleotide (Fields & Knipe 20 (1986)). Therefore, there are a plurality of strains within the HCV species described below. The compositions and methods described herein enable the propagation, identification, detection and isolation of the various related strains. In addition, they also enable the preparation of diagnostic and vaccines for the various strains and are useful in screening procedures for antiviral agents for pharmacological use, inhibiting HCV replication.

The information provided herein, although derived from one HCV line, hereinafter referred to as CDC / HCV1, is sufficient to enable a virus taxonomist to identify other strains that fall within the species. As described herein, it has been discovered that HCV is a flavivirus or flavi-like virus. The morphology and composition of flavivirus particles is known and discussed in Brinton (1986). The flaviviruses generally contain, for morphology, a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of approx. 40 - 50 nm. Their cores are approx. 25-30 5 nm in diameter. Along the outer surface of the virion sheath are projections that are approx. 5-10 nm long with terminal buds that are approx. 2 nm in diameter.

HCV encodes an epitope that is immunologically identifiable with an epitope in the HCV genome, from which the cDNAs described herein are derived, the epitope 10 being preferably encoded in the cDNA described herein. The epitope is unique to HCV compared to other known flaviviruses. The unique nature of the epitope can be determined by its immunological reactivity with HCV and lack of immunologic reactivity with other flaviviruses. Methods for determining immunological reactivity are known in the art, e.g. radioimmunoassay, ELISA assay, hemaggiutination, and several examples of suitable assay techniques are provided herein.

In addition to the above, the following parameters are applicable, either alone or in combination, for the identification of an HCV strain. Since HCV lines 20 are evolutionarily related, it is expected that the general homology of the genomes at the nucleotide level will be approx. 40% or more, preferably approx.

60% or more and even more preferably approx. 80% or more, and in addition there will be corresponding consecutive sequences of at least approx. 13 nucleotides. The correspondence between the putative HCV strain genome sequence and the CDC / CH1 HCV cDNA sequence can be determined by techniques known in the art. For example, it can be determined by a direct comparison of the sequence information of the putative HCV polynucleotide and the HCV cDNA sequence (s) described herein.

For example, they can also be determined by polynucleotide hybridization under 30 conditions which form stable duplexes between homologous regions (e.g., those which could be used prior to Si digestion), followed by digestion with single stranded specific nuclease ( nucleases) and followed by sizing of the digested fragments.

Because of the evolutionary kinship of the HCV lines, the putative HCV-5 lines are identifiable by their polypeptide level homology. Generally, HCV lines are more than approx. 40% homologous, preferably more than ca. 60% homologous and even more preferably more than approx. 80% homologous at the polypeptide level. The techniques for determining amino acid sequence homology are known in the art. For example, the amino acid sequence can be directly determined and compared with the sequences provided herein. For example, the nucleotide sequence of the putative HCV genome material can also be determined (usually via a cDNA intermediate), the amino acid sequence encoded therein can be determined and the corresponding regions compared.

15

As used herein, a polynucleotide "derived from" refers to a given sequence, e.g. The HCV cDNA, in particular, those exemplified in FIGS. 1 - 32, or from an HCV genome, to a polynucleotide sequence comprised of a sequence of approximately at least ca. 6 nucleotides, and preferably is at least ca. And preferably more than at least about 20 nucleotides. 10-12 nucleotides and even more preferably are at least 15-20 corresponding nucleotides, i.e. are homologous to or complementary to a region of the indicated nucleotide sequence. The sequence of the region from which the polynucleotide is derived is preferably homologous to or complementary to a sequence unique to.

25 for an HCV genome. Whether or not a sequence is unique to the HCV genome can be determined by techniques known to those skilled in the art. For example, the sequence can be compared to sequences in databases, e.g.

Genebank, to determine if it is present in the uninfected host or other organisms. The sequence can also be compared with the known sequences of the other 30 viral agents, including those known to induce hepatitis, e.g. HAV, HBV, and HDV, and with other members of flavi- i in 18 DK 175975 B1 viridae. The correspondence or non-correspondence of the derived sequence with other sequences can also be determined by hybridization under appropriate stringency conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art and are discussed below. See also e.g. Maniatis et al. (1982). In addition, failure of duplex polynucleotides formed by hybridization can be determined by known techniques, e.g. comprises digestion with a nuclease, such as S1, which specifically digests single-stranded regions into duplex polynucleotides.

Areas from which typical DNA sequences can be "derived" include, but are not limited to, e.g. areas encoding specific epitopes, as well as non-transcribed and / or untranslated regions.

The derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but can be produced in any way, including e.g. chemical synthesis or DNA replication or reverse transcription or transcription, based on that of the base sequences in the region (s) from which the polynucleotide is derived, provided information.

In addition, combinations of regions corresponding to regions of the specified sequence may be modified to conform to a intended use in ways known in the art.

Similarly, a polypeptide or amino acid sequence "derived" refers to a given nucleic acid sequence, e.g. 1 to 32, or from an HCV genome, to a polypeptide having an amino acid sequence identical to the amino acid sequence 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 still more preferably at least 11-15 amino acids or which are immunologically identifiable with a polypeptide encoded in the sequence.

19 DK 175975 B1

A recombinant or derived polypeptide is not necessarily translated from a specified nucleic acid sequence, e.g. the sequences in Figures 1-26 or from an HCV genome, it can be produced in any way, including e.g. chemical synthesis or expression of a recombinant expression system or isolation from mutated HCV.

The term "recombinant polynucleotide" as used herein refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin, 10 by virtue of its origin or manipulation: (1) not associated with all or part of the polyriucleotide with which it is linked in nature or in the form of a library, and / or (2) is bound to a polynucleotide other than that to which it is bound 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 modified, e.g. by methylation and / or by "capping", and unmodified forms of the polynucleotide.

As used herein, the term "HCV containing a sequence corresponding to a cDNA" means that the HCV contains a polynucleotide sequence which is homologous to or complementary to a sequence in the specified DNA, the degree of homology or complementarity to The cDNA will be approx. 50% or more, preferably at least approx. 70% and even more preferably at least approx. 90%. The corresponding sequences will be at least approx. 70 nucleotides, preferably at least ca. 80 nucleotides and even more, preferably at least approx. 90 nucleotides long. The correspondence between the HCV sequence and the cDNA can be determined by techniques known in the art, including e.g. a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single-stranded nucleases followed by sizing of the digested fragments.

5

The term "purified viral polynucleotide" refers to an HCV genome or fragment thereof which is substantially free, i.e. contains less than approx.

50%, preferably less than about 50%. 70% and even more preferably less than approx. 90% polypeptides to which the viral polynucleotide is naturally associated. IN

Techniques for purifying virus polynucleotides from virus particles are known in the art and include, e.g. disruption of the particle with a chaotropic agent and separation of the polynucleotide (polynucleotides) and polypeptides by ion exchange chromatography, affinity chromatography, and density sedimentation.

15

The term "purified virus polypeptides" refers to an HCV polypeptide or fragment thereof which is substantially free, 50%, preferably less than about 50%. 70% and even more preferably less than approx. 90% of cell components to which the viral polypeptide is naturally linked. Techniques for purifying virus polypeptides are known in the art and examples thereof are discussed below.

"Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures" and other such designations for microorganisms or higher eukaryotic cell lines grown as single cell units refer to cells which can be used or have been used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell that has been transfected. It will be understood that the progeny of a single parent cell are not necessarily completely identical in morphology 30 or in genomic or total DNA complement to the original parent cell due to random or intentional mutation. Offspring of the parent cell having sufficient similarity to parent cells to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the offspring contemplated by this term and covered by the above designation. .

5

A "replicon" is any genetic element, e.g. a plasmid, one chromosome, a virus that behaves as an autonomous entity for polynucleotide replication within a cell, i.e. is capable of replication under its own control.

10

A "vector" is a replicon in which another polynucleotide segment is attached, so that replication and / or expression of the attached segment is performed.

"Control sequence" refers to polynucleotide sequences necessary to perform the expression of coding sequences to which they are ligated. The nature of such control sequences varies with the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such 20 control sequences generally include promoters, terminators and, in some cases, enhancers. The term "control sequences" should at least include all constituents whose presence is required for expression, and may also include additional constituents whose presence is advantageous, e.g. leader sequences.

"Functionally bonded" refers to an assembly in which the components so described are in a relationship which allows them to function in the intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequences.

22 DK 175975 B1

An "open reading frame" (ORF) is a region of a polynucleotide sequence encoding a polypeptide; this region may represent part of a coding sequence or the total coding sequence.

5

A "coding sequence" is a polynucleotide sequence that is transcribed into mRNA and / or translated into polypeptide when placed under the control of appropriate regulatory sequences. The boundary regions of the coding sequence are determined by a translation start codon at the S 'terminal and a translation stop codon at the 3' terminal. A coding sequence may include, but is not limited to, mRNA, cDNA and recombinant polynucleotide sequences.

"Immunologically identifiable with / as" refers to the presence of an epitope 15 (epitopes) and polypeptide (polypeptides) which are also present in and unique to the stated polypeptide (polypeptides), usually HCV proteins. Immunological identity can be determined by antibody binding and / or competitive binding; these techniques are known to those of ordinary skill in the art and are also illustrated below. The unique character of an epitope can also be determined by computer searches in known databases, e.g. Genebank, following the polynucleotide sequences encoding the epitope, and by amino acid sequence comparisons with other known proteins.

As used herein, "epitope" refers to an antigenic determinant of a polypeptide; an epitope may comprise three amino acids in a spacious conformation unique to the epitope, which usually consists of at least 5 such amino acids and more usually of at least 8-10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include e.g. X-ray crystallography, and 30 two-dimensional nuclear magnetic resonance.

23 DK 175975 B1

A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained in the polypeptide. Immunological reactivity can be determined by antibody binding, more specifically by antibody binding kinetics and / or by competitive binding 5 under The use of a known polypeptide (polypeptides) containing an epitope against which the antibody is directed which competitively binds The techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

As used herein, the term "immunogenic polypeptide containing an HCV epitope" includes naturally occurring HCV polypeptides or fragments thereof, as well as polypeptides prepared by other means, e.g. by chemical synthesis or by expression of the polypeptide in a recombinant organism.

15

The term "polypeptide" refers to a molecular chain of amino acids and does not refer to ten! a specific length of the product, thus peptides, oligopeptides and proteins are included in the definition of the polypeptide. This term also does not refer to post-expression modifications of the polypeptide, e.g. glycosylations, acetylations, phosphorylations and the like.

"Transformation" as used herein refers to the insertion of an exogenous polynucleotide in a host cell, regardless of the method of insertion used, e.g. direct uptake, transduction or f-bacterial junction. The exogenous polynucleotide can be maintained as a non-integral vector, e.g. a plasmid, or alternatively may be integrated into the host genome.

"Treatment" as used herein refers to prophylaxis and / or therapy.

24 DK 175975 B1

An "individual" as used herein refers to vertebrates, especially members of the mammalian species, and includes, but is not limited to, domestic animals, sport animals, primates and humans.

As used herein, the "plus strand" of a nucleic acid contains the sequence encoding the polypeptide. The "minus string" contains a sequence that is complementary to the "plus string".

As used herein, "a positive stranded genome" is a virus, one in which the genome, whether RNA or DNA, is single-stranded and encodes a viral polypeptide (polypeptides). Examples of positively closed RNA viruses include Togaviridae, Coronaviridae, Retroviridae, Picornaviridae and Caliciviridae. Flaviviridiae previously classified as Togaviradae are also included. See Fields & Knipe (1986).

15

As used herein, "antibody-containing body component" refers to a component of an individual's body that is a source of the antibodies of interest. Antibody-containing body constituents are known in the art and include, but are not limited to, e.g. plasma, serum, spinal fluid, lymph fluid, outer parts of the respiratory tract, intestinal and urogenital tracts, tears, saliva, milk, white blood cells and myeloma.

As used herein, "purified HCV" refers to a preparation of HCV that has been isolated from the cellular components with which the virus is normally associated, and from other types of viruses that may be present in the infected tissue. The techniques for isolating viruses are known to those skilled in the art and include, e.g. centrifugation and affinity chromatography; a method of preparing purified HCV is described below.

25 DK 175975 B1 II. Description of the Invention

The practice of the present invention will, unless otherwise stated, include conventional molecular biological, microbiological, recombinant DNA 5 and immunological techniques which are within the skill of the art. Such techniques are extensively explained in the literature. See, e.g. Maniatis, Fitsch & Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL (1982); DNA CLONING, BIND I AND II (D.N. Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed. 1984); NUCLEIC ACID HYBRIDIZATION (B.D.

10 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 CELLS AND ENZYMES (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the METHODS IN ENZYMOLOGY series (Academic Press, Inc.); GENE 15 TRANSFER VECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P.

Calos eds. 1987, Cold Spring Harbor Laboratory); Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, respectively, Wu, eds.); Mayer and Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London); Scopes, (1987), 20 PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, BIND I - IV (D.M. Weir and C.C. Blackwell eds. 1986).

All patents, patent applications and publications cited above, both above 25 and below, are hereby incorporated herein by reference.

The suitable materials of the present invention are made possible by the provision of a family of closely homologous nucleotide sequences isolated from a cDNA library derived from nucleic acid sequences present in the plasma of an HCV-infected chimpanzee. This family of nucleotide sequences is not of human or chimpanzee origin, as it does not hybridize with human or chimpanzee genomic DNA from uninfected individuals, as nucleotides of this sequence family are present only in the liver and plasma of chimpanzees with HCV. infection, and the sequence is not present in Genebank. In addition, the sequence family shows no significant homology to sequences contained in the HBV genome. The sequence of a family member contained in clone 5-1-1 has a continuous open reading frame (ORF) encoding a polypeptide of approx. 50 amino acids. HCV-infected human sera contain antibodies that bind to this polypeptide, whereas non-infected human sera do not contain antibodies to this polypeptide. Finally, the antibodies in chimpanzees are subsequently induced by acute NANBH infection, whereas sera from uninfected chimpanzees do not contain antibodies to this polypeptide. In addition, antibodies to this polypeptide have not been detected in chimpanzees and humans infected with HAV and HBV. From these criteria, the sequence is a cDNA of a virus sequence whereby the virus causes or is associated with NANBH; this cDNA sequence is shown in Figure 1. As discussed below, the cDNA sequence in clone 5-1-1 differs from the sequences of the other isolated cDNAs by containing 28 additional base pairs.

A composite of other identified members of the cDNA family isolated using a probe as synthetic equivalent sequence for cDNA fragment in clone 5-1-1 is shown in Figure 3. A member of the cDNA family that was isolated using a synthetic sequence derived from the cDNA of clone 81 is shown in Figure 5 and the composite of this sequence with the sequence in clone 81 is shown in Figure 6. Other members of the cDNA family include those members, present in clones 12f, 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f and 33g, 39c, 35f, 19g, 26g and 15e are described in section IV.A. A composite of the cDNAs in these clones is described in Section IV.A. 19 and shown in Figure 32. The composite cDNA 30 shows that it contains a continuous ORF and thus encodes a polyprotein. This information is consistent with the suggestions below that HCV is a flavivirus or flavi-like virus.

The availability of this family of cDNAs, shown in Figures 1-32 5 inclusive, allows DNA probes and polypeptides to be engineered to diagnose NANBH due to HCV infection and to screen for infection. blood donors as well as donated blood and blood products. For example, from the sequences it is possible to synthesize DNA oligomers of ca. 8-10 nucleotides or greater suitable as 10 hybridization probes for detecting the presence of the viral genome in e.g. sera from patients suspected of carrying the virus or screening donated blood for the presence of the virus. Families of cDNA sequences also enable the construction and preparation of HCV-specific polypeptides suitable as diagnostic reagents for the presence of antibodies produced under NANBH. Antibodies to purified polypeptides derived from the cDNAs can also be used to detect viral antigens in infected individuals and in blood.

Knowledge of these cDNA sequences also allows for the construction and preparation of polypeptides that can be used as vaccines against HCV and also for the production of antibodies which in turn can be used to protect against the disease and / or for the therapy of HCV-infected individuals.

The family of cDNA sequences allows further characterization of the HCV-25 genome.

in

Polynucleotide probes derived from these sequences can be used to screen cDNA libraries for additional overlapping cDNA sequences, which in turn can be used to obtain multiple overlapping sequences. Unless the genome is sequenced and the segments lack common sequences, this technique can be used to obtain the entire genome sequence. However, if the genome 28 is segmented, other genome segments can be obtained by repeating the Iambda-gt11 serological screening procedure used to isolate cDNA clones described herein, or alternatively by isolating the genome from purified HCV particles .

5

The family of cDNA sequences and polypeptides derived from these sequences, as well as antibodies directed against these polypeptides, are also well suited for isolating and identifying the BB-NANBV agent (s).

in

For example, antibodies directed against HCV epitopes contained in polypeptides derived from the cDNAs can be used in processes based on affinity chromatography to isolate the virus. Alternatively, the antibodies may be used to identify viral particles isolated by other techniques. The viral antigens and the genomic material in the isolated viral particles can then be further characterized.

15

The information obtained by further sequencing of the HCV genome (s), as well as by further characterization of the HCV antigens and characterization of the genome, allows the construction and synthesis of additional probes and polypeptides and antibodies useful for diagnosis. prevention and for therapy of HCV-induced NANBH and for screening for infected blood and blood-related products.

The availability of HCV, including antigens and antibodies, and polynucleotides derived from the genome from which the family of cDNAs are derived, also enables the development of tissue culture systems which will be of greater importance in elucidating HCV biology. This in turn may lead to the development of new treatment regimens based on antiviral compounds that preferentially inhibit the replication of or infection with HCV.

The method used to identify and isolate the etiologic agent of NANBH is novel and can be used for the identification and / or isolation of novel uncharacterized agents containing a genome and associated with a variety of diseases, including the diseases induced by viruses, viroids, bacteria, fungi and parasites.

According to this method, a cDNA library was generated from the 5 nucleic acids present in infected tissue from an infected individual. The library was generated in a vector that allowed expression of polypeptides encoded in the cDNA. Host cell clones containing the vector expressing an immunologically reactive fragment of a etiologic agent polypeptide were selected by immunologically screening the library's 10 expression products with an antibody-containing body component from another individual previously infected with the putative agent. The steps of the immunological screening technique included interaction of the expression products of the cDNA-containing vectors with the antibody-containing body component of another infected individual, as well as detection of the formation of antibody-antigen complexes between the expression product (s) and antibodies of the other infected individual. The isolated clones are further immunologically screened by interaction of their expression products with the antibody-containing body component of other individuals infected with the putative agent and with 20 control individuals not infected with the putative agent, and by detection of the formation of antigen-antibody complexes. with antibodies from the infected individuals; the cDNA-containing vectors encoding polypeptides that react immunologically with antibodies from infected individuals and individuals believed to be infected with the agent but not with 25 control individuals are isolated. The infected individuals used for the construction of the cDNA library and for the immunological screening need not be of the same kind.

The cDNAs isolated as a result of this method and their expression products and antibodies directed against the expression products are well suited for characterizing and / or capturing the etiologic agent.

30 DK 175975 B1

As described in more detail below, this method has been successfully used to isolate a family of cDNAs derived from the HCV genome.

II.A. Preparation of the cDNA sequence

Combined serum from a chimpanzee with chronic HCV infection and containing a high titer of the virus, i.e. at least 106 chimpanzee infectious doses / ml (CID / ml) were used to isolate viral particles; nucleic acids isolated from the particles were used as a template in the construction of a cDNA library for the viral genome. The methods for isolating putative HCV particles and for constructing the cDNA library of Iambda-gt11 are discussed in Section IV.A.1. Lambda-gt11 is a vector that has been specifically developed to express inserted cDNAs as beta-galactosidase fusion polypeptides and to screen large numbers of recombinant phages with specific antisera produced against a defined antigen. The Lambda-gt11 cDNA library generated from a cDNA pool containing approximately 200 base pairs of cDNA was screened for encoded epitopes that could be specifically linked to sera derived from 20 patients previously had NANB hepatitis. Huynh, T.V. et al (1985). Ca.

106 phages were screened and 5 positive phages were identified, purified and then tested for binding specificity to sera from various humans and chimpanzees previously infected with the HCV agent. One of these subjects, 5-1 -1, bound 5 of the 8 human sera tested. This binding 25 appeared selective with respect to sera derived from patients with previous NANB hepatitis infections, with 7 normal donor sera showing no such binding.

cDNA The sequence of recombinant phage 5-1-1 was determined and is shown in Figure 1.

The polypeptide encoded by this cloned cDNA, which is in the same translation frame as the N-terminal beta-galactosidase portion of the fusion polypeptide, is shown over the nucleotide sequence. This transitional ORF therefore encodes an epitope (epitopes) specifically recognized by sera from patients with NANB hepatitis infections.

5 The availability of cDNA in recombinant phage 5-1-1 has enabled the isolation of other clones containing additional segments and / or alternative segments of the viral genome cDNA. The Lambda-gt11 cDNA library described above was screened using a synthetic polynucleotide derived from the sequence of the cloned 5-1-1 cDNA. This screening yielded 3 other clones identified as 81, 1-2 and 91; the cDNAs contained in these clones were sequenced. See sections VI.A.3. and IV.A.4. The homologies between the 4 independent clones are shown in Figure 2, where the homologies are indicated by the vertical lines. Nucleotide sequences present exclusively in clones 5-1-1, 81 and 91 are indicated by lowercase letters.

The cloned cDNAs present in recombinant phages in clones 5-1-1, 81, 1-2 and 91 are highly homologous and differ in only two regions. First, nucleotide # 67 in clone is 1-2 thymidine, whereas the other three clones 20 contain a cytidine residue at this position. However, this substitution does not change the nature of the encoded amino acid.

The other difference between the clones is that clone 5-1-1 contains 28 base pairs at the 5 'terminal that are not present in the other clones. The additional sequence 25 may be a 5 'terminal cloning artifact; 5 'terminal cloning artifacts are commonly observed in the products of cDNA methods.

Synthetic sequences derived from the 5 region and 3 region of HCV cDNA in clone 81 were used to screen and isolate the cDNAs from the lambda-30 gt11 NANBV cDNA library that overlapped clone 81 cDNA (section 32 DK 175975 B1 IV.A.5.). The sequences of the resulting cDNAs, which are in clone 36 and clone 32, respectively, are shown in Figure 5 and Figure 7.

Similarly, a synthetic polynucleotide based on the 5'-5 region of clone 36 was used to screen and isolate the cDNAs from the lambda gt-11 NANBV cDNA library that overlapped clone 36 cDNA (Section IV.A 0.8.).

A purified clone of recombinant phage-containing cDNA that hybridizes with the synthetic polynucleotide probe is referred to as clone 35, and the NANBV cDNA sequences contained in this clone are shown in Figure 8.

10

Using the isolation technique for overlapping cDNA sequences, clones have been obtained that contain additional upstream and downstream HCV cDNA sequences. The isolation of these clones is described below in Section IV.A.

Analysis of the HCV cDNA nucleotide sequences encoded in the isolated clones shows that the composite cDNA contains a long continuous ORF.

Figure 26 shows the sequence of the composite DNA from these clones together with the putative HCV polypeptide encoded therein.

20 The description of the method for retrieving the cDNA sequences is most of historical interest. The resulting sequences (and their complements) are provided therein, and the sequences or any part thereof could be prepared using synthetic methods or by a combination of synthetic methods with the recovery of partial sequences using methods similar to the described herein.

Lambda-gt11 strains replicated from the HCV cDNA library and from clones 5-1-11.81, 1-2 and 91 have been deposited under the terms of the Budapest Treaty at American Type Culture Collection (ATCC), 12301 Parklawn, 30 Dr . Rockville, Maryland 20852 and has been assigned the following landfill numbers.

33 DK 175975 B1

Iambda-at11_ATCC No._Depository Date 5 HCV cDNA Library 40: 394 December 1, 1987 Clone 81 40388 November 17, 1987 Clone 91 40: 389 November 17, 1987 Clone 1-240: 390 November 17, 1987 Clone 5-1 -1 40: 391 November 18, 1987 10

Upon granting and issuing this application as a U.S. patent, all restrictions on the availability of these deposits will be irrevocably removed; and access to the designated deposits will be possible while the above application is pending for a person who has been authorized to do so under 15: 37 CFR 1.14 and: 35 USC 1.22 as appropriate. In addition, the stated deposits will be maintained over a 30 year period from the date of filing or 5 years after the last filing request or in the lifetime of US patents, whichever is longer. These deposits and other materials deposited herein are for convenience only and are not required to carry out the present invention in view of the present disclosure. The HCV cDNA sequences in all the deposited materials are included herein.

The above description of "genome walking" by isolating overlapping 25 cDNA sequences from the HCV lambda gt11 library provides a method by which cDNAs corresponding to the entire HCV genome can be isolated.

However, with the information provided herein, other methods of isolating these cDNAs will be apparent to those skilled in the art. Some of these methods are described in Section IV A. below.

30! i i j

- I

34 DK 175975 B1 II.B. Preparation of Viral Polypeptides and Fragments 5 The availability of cDNA sequences, either those isolated using the cDNA sequences of Figures 1 - 32, as discussed below, as well as the cDNA sequences of these figures, enable the construction of expression vectors which encodes antigenically active regions of the polypeptide encoded in both strands. These antigenically active regions may be derived from envelope antigens or from nuclear antigens, including e.g. polynucleotide-binding proteins, polynucleotide polymerase (polymerases), and other viral proteins required for replication and / or assembly of the virus particle. The fragments encoding the desired polypeptides are derived from the cDNA clones using usual restriction digestion or by synthetic methods and ligated into vectors such as e.g. may contain portions of fusion sequences such as beta-galactosidase or superoxide dismutase (SOD), preferably (SOD). Methods and vectors suitable for the preparation of polypeptides containing fusion sequences of SOD are described in EPO publication no. 0196056, published October 1, 1986. Vectors encoding 20 fusion polypeptides of SOD and HCV polypeptides, i.e. NANB1, NANBei, and C100-3 encoded in a composite of HCV cDNAs are described in sections IV.B.1, IV.B.2 and IV.B.4, respectively. Any desired portion of HCV cDNAs containing an open reading frame in any of the strands can be obtained as a recombinant polypeptide, such as a mature or a fusion protein; alternatively, by chemical synthesis, a polypeptide encoded in the cDNA may be provided.

The DNA encoding the desired polypeptide, whether in fused or mature form, and whether or not it contains a signal sequence that allows secretion, can be ligated into expression vectors suitable for any appropriate host. Both eukaryotic and prokaryotic host systems are used for the generation of recombinant polypeptides for the time being and are given in section III.A. Below is a summary of some of the more common management systems and host cell lines. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent necessary for its intended use. The purification may be by known techniques, e.g. salt fractionation, ion exchange resin chromatography, affinity chromatography, centrifugation and the like. See, e.g. "Methods in Enzymology" concerning a variety of methods for purifying proteins. Such polypeptides can be used as diagnostics or those which produce neutralizing antibody production can be formulated for vaccines. Antibodies produced against these polypeptides can also be used as diagnostics or for passive immunotherapy. In addition, antibodies against these polypeptides are suitable for isolating and identifying HCV particles, as described in the following section M.J.

The HCV antigens can also be isolated from HCV virions. The virions can be cultured in HCV-infected cells in tissue culture or in an infected host.

II.C. Preparation of Antigenic Polypeptides and Conjugation with Carrier 20

An antigenic region of a polypeptide is generally relatively small - typically 8-10 amino acids or less in length. Fragments of as few as 5 amino acids may characterize an antigenic region. These segments may correspond to regions of HCV antigen. DNAs encoding short HCV-25 polypeptide segments can therefore, using HCV cDNAs as a basis, be recombinantly expressed either as fusion proteins or as isolated polypeptides. In addition, short amino acid sequences can conveniently be obtained by chemical synthesis. In cases where the synthesized polypeptide is properly configured to provide the correct epitope but is too small to be immunogenic, the polypeptide can be bound to an appropriate carrier.

36 DK 175975 B1

A number of techniques are known in the art for obtaining such bonding, including forming disulfide bonds using N-succinimidyl 3- (2-pyridylthio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC) available from Pierce 5 Company, Rockford, Illinois (if the peptide lacks a sulfhydryl group, this can be provided by the addition of a cysteine residue). These reagents produce a disulfide bond between themselves and peptide cysteine residues on one protein and an amide bond through the epsilonamino group on one lysine or other free amino groups on the other. A number of such disulfide / amide forming agents are known. See, e.g. Immum. Rev. (1982) 62: 185th Other double-functional coupling agents form a thioether rather than a disulfide bond. Many of these thioether forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, the sodium salt. The foregoing list is not intended to be exhaustive and, of course, modifications may be made to the said compounds.

Any carrier which does not in itself induce production of antibodies harmful to the host may be used. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, 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 particles, see e.g. Section II.D. Particularly suitable protein substrates are serum albumin, hemocyanin from the keyhole limpet (a species of elbow peel), immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid and other proteins well known in the art.

30 37 DK 175975 B1 ILD. Preparation of Hybrid Particle Immunogens Containing HCV Epitopes The immunogenicity of HCV epitopes may also be enhanced by preparing them in mammalian or yeast systems fused with or combined with particle-forming proteins, such as e.g. the protein associated with hepatitis B surface antigen. Constructs in which the NANBV epitope is linked directly to the particle-forming protein coding sequences produce hybrids that are immunogenic with respect to the HCV epitope. In addition, all of the prepared vectors include epitopes that are specific to HBV and have varying degrees of immunogenicity, such as e.g. the pre-S peptide. Thus, particles constructed from particle-forming protein comprising HCV sequences are immunogenic with respect to HCV and HBV.

15

It has been found that hepatitis surface antigen (HBSAg) is formed and assembled into particles in S. cerevisiae. (Valenzuela et al. (1982)), as well as in e.g. mammalian cells (Valenzuela, P., et al. (1984)). It has been found that the formation of such particles enhances the immunogenicity of the monomer subunit.

The constructs may also comprise the immunodominant epitope of HBSAg, including the 55 amino acids in the pre-S (pre-S) region. Neurath et al. (1984). Constructs of the pre-S-HBSAg particle which can be expressed in yeast are described in EPO No. 174,444, issued March 19, 1986; hybrids comprising heterologous viral sequences for yeast expression are disclosed in EPO 175,261, filed March 26, 1966. Both applications are assigned to the present applicants and are incorporated herein by reference. These constructs can also be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells using an SV40 dihydrofolate reductase vector (Michelle et al. (1984)).

30 38 DK 175975 B1

In addition, parts of the sequence encoding the particle-forming protein can be replaced by codons encoding an HCV epitope. In this exchange, areas that are not required to mediate the aggregation of the units to form immunogenic particles in yeast or mammals may be omitted, thereby excluding additional HBV antigenic sites from competition with the HCV epitope.

II.E. Preparation of Vaccines 10 Vaccines may be prepared from one or more immunogenic polypeptides derived from the HCV cDNA as well as from the cDNA sequences in Figures 1-32 or from the corresponding HCV genome. The observed homology between HCV and flaviviruses provides information regarding the polypeptides likely to be most effective as vaccines, as well as the regions of the genome in which they are encoded. The general structure of the flavivirus genome is discussed in Rice et al. (1986). The flavivirus RNA genome is thought to be the only virus-specific mRNA species and it translates into three viral structural proteins, i.e. C, M and E as well as two large non-structural proteins, NV4 and NV5, as well as a complex of smaller non-structural proteins. It is known that greater 20 neutralizing epitopes for flaviviruses are in the E (mantle) protein ((Roéhrig 1986)). The corresponding HCV E gene and polypeptide coding region can be predicted based on the homology of the flaviviruses. Vaccines may thus comprise recombinant polypeptides containing epitopes of HCV E. These polypeptides may be expressed in bacteria, yeast or mammalian cells or they may alternatively be isolated from viral preparations. In addition, it is expected that the other structural proteins may also contain epitopes that cause the production of protective anti-HCV antibodies. Thus, polypeptides containing the epitopes of E, C and M can also be used in HCV vaccines, either alone or in combination.

30 39 DK 175975 B1

In addition to the above, immunization with NS1 (non-structural protein 1) has been shown to lead to protection against yellow fever (Schlesinger et al.

(1986)). This is true even if the immunization does not cause the production of the neutralizing antibodies. In particular, because this protein appears to be highly conserved among flaviviruses, it is likely that HCV NS1 will also protect against HCV infection. It also shows that non-structural proteins can provide protection against viral pathogenicity even if they do not cause neutralizing antibody production.

In view of the above, multivalent HCV vaccines may comprise one or more structural proteins and / or several non-structural proteins. The vaccines may comprise e.g. recombinant HCV polypeptides and / or polypeptides isolated from the virions. In addition, it is possible to use inactivated HCV in vaccines; inactivation may occur by the preparation of viral lysates or by other known methods which cause inactivation of flaviviruses, e.g. treatment with organic solvents or detergents or treatment with formalin.

In addition, vaccines can also be prepared from attenuated HCV strains. The preparation of attenuated HCV strains is described below.

It is known that some of the proteins in flaviviruses contain highly conserved regions and thus some immunological cross-reactivity between HCV and other flaviviruses is expected. It is possible that common epitopes between the flaviviruses and HCV will give rise to the production of protective antibodies against one or more of the diseases caused by these pathogenic agents. Thus, it is possible to construct vaccines for multiple purposes on the basis of this knowledge.

The preparation of vaccines containing an immunogenic polypeptide (polypeptides) as active ingredients is known to those skilled in the art. Such vaccines are typically prepared in injectable form, either in the form of liquid solutions or as suspensions; they may also be prepared in solid form suitable for solution or suspension in liquid prior to injection. The preparation can also be emulsified or the protein can be encapsulated in liposomes. The active immunogenic components are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Own excipients include e.g. water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of adjuvants such as wetting or emulsifying agents, pH buffering agents and / or adjuvants which enhance the efficacy of the vaccine. Examples of adjuvants that may be effective include, but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl -D-isoglutamine (CGP 11637, designated nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) - ethylamine (CGP 19835A, designated MTP-PE), and RIBI containing three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene / Tween 80 emulsion. The effectiveness of an adjuvant can be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an HCV antigen sequence resulting from administration of the polypeptide in vaccines comprising the various adjuvants.

The vaccines are usually administered parenterally by injection, e.g. either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include suppositories and in some cases oral formulations. For suppositories, traditional binders and carriers may include e.g. polyalkylene glycols or triglycerides, such suppositories may be formed from mixtures containing the active ingredient in an amount of from 0.5% to 10%, preferably 1% to 2%. Oral formulations include the normally used excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. The compositions take the form of delays, suspensions, tablets, pills, capsules, delayed release or powder formulations and contain 10% - 95% active ingredient, preferably 25% - 70%.

The proteins can be formulated in the vaccine as neutral or in salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and formed with inorganic acids such as e.g. hydrochloric or phosphoric or organic acids such as acetic, oxalic, tartaric, maleic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxides and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

II.F. Dosage and administration of vaccines

Vaccines are administered in a manner compatible with the dosage formulation and in an amount that is prophylactic and / or therapeutically effective. The amount to be administered, which usually ranges from 5 pm to 250 pm 20 antigen per day. the dose depends on the patient to be treated, the patient's immune system capacity to synthesize antibodies, and the desired degree of protection. The exact amount of active ingredient to be administered may depend on the practitioner's assessment and may be individual to patient.

25

The vaccine may be given as a single dose or preferably as multiple doses. A multidose schedule is characterized by a primary course of vaccination which may consist of 1 to 10 separate doses followed by other doses given at subsequent intervals that may be required for maintenance or enhancement of the immune response, e.g. after an interval of 1 - 4 months for the second dose and, if necessary, a subsequent dose (doses) after several months. The dosage pattern will also, at least in part, be determined by the individual's needs and depend on the practitioner's assessment.

In addition, the vaccine containing the immunogenic HCV antigen (antigens) can be administered with other immunoregulatory agents, e.g. immunoglobulins.

II.G. Preparation of antibodies against HCV epitopes 10

The immunogenic polypeptides prepared as described above are used to produce antibodies that can be both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide carrying an HCV epitope (epitopes). Serum from the immunized animal is collected and treated according to known methods. If serum containing polyclonal antibodies to an HCV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for the preparation and further treatment of 20 polyclonal antisera are known in the art, see e.g. Mayer and Walker (1987).

Alternatively, polyclonal antibodies can be isolated from a mammal that has previously been infected with HCV. An example of a method for purifying 25 antibodies against HCV epitopes from serum of an infected individual based on affinity chromatography using a fusion polypeptide of SOD and a polypeptide encoded in cDNA clone 5-1-1 is given in section VE

Monoclonal antibodies directed against HCV epitopes may also be readily prepared by one of ordinary skill in the art. The general methodology for producing monoclonal antibodies in hybridomas is well known. Sustainable B1 antibody producing cell lines can be produced by cell fusion and also by other techniques 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 5, U.S. Patent Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,887, 4,466,917, 4,472,500, 4,491,632, and 4,493,890. Reference groups of monoclonal antibodies produced against HCV epitopes can be screened for various properties, e.g. isotype, epitope affinity, etc.

10 Both monoclonal and polyclonal antibodies directed against HCV epitopes are particularly well suited for diagnosis and those that neutralize are suitable for passive immunotherapy. In particular, monoclonal antibodies can be used to produce anti-idiotype antibodies.

Anti-idiotype antibodies are immunoglobulins that carry an "inner image" of the antigen of the infectious agent, whereas protection is desired. See, e.g.

Nisonoff, A. et al (1981) and Dreesman et al. (1985).

Techniques for producing anti-idiotype 20 antibodies are known in the art. See, e.g. Grzych (1985), MacNamara et al. (1984) and Uytdehaag et al. (1985). These anti-idiotype antibodies may also be effective in the treatment of NANBH, as well as in elucidating the immunogenic regions of the HCV antigens.

II.H. Diagnostic oil nucleotide probes and assay kits i

Using the described portions of the isolated HCV cDNAs as the basis, including those referred to in Figures 1 - 32, oligomers of ca. Eight nucleotides or more are produced either by cutting or synthetic, which 30 oligomers hybridize to the HCV genome and are suitable for identification: of the viral agent (s), further characterization of the viral genome 44, and for the detection of the virus (viruses) in sick individuals. The probes for HCV polynucleotides (natural or derived) have a length that allows detection of unique viral sequences by hybridization. Although 6-8 nucleotides may be a useful length, the sequences of 5-10-12 nucleotides and ca. 20 nucleotides appear optimal. These sequences will preferably be derived from regions which lack heterogeneity. The probes can be prepared routinely by methods involving automated oligonucleotide synthesis methods. Examples of suitable probes are the clone 5-1-1 and the further clones described herein are the various 10 oligomers suitable for probing cDNA libraries, as discussed below. A complement to any unique part of the HCV genome will be satisfactory. Complete complementarity is required for use as probes, although it may be unnecessary as the length of the fragment is increased.

15

For use of the probes as diagnostics, the biological sample to be assayed, such as blood or serum, is processed, 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 can be dot exposed without size separation.

The probes are then labeled. Suitable labels and methods for labeling probes are known in the art and include e.g. radioactive labels incorporated by "nick translation" or treatment with kinase, biotin, fluorescent probes and chemiluminescent probes. The nucleic acids extracted from sample 25 are then treated with the labeled probe under hybridization conditions of appropriate stringency.

The probes can be made completely complementary to the HCV genome. Therefore, high stringency conditions are usually desired to prevent false results. However, high stringency conditions should only be used if the probes are complementary to those regions of the viral genome that lack heterogeneity.

The hybridization stringency is determined by a number of factors during the hybridization and during the washing procedure, including temperature, ionic strength, duration and formamide concentration. These factors are outlined in e.g. Maniatis, T. (1982).

It is generally expected that the HCV genome sequences will occur at 10 relatively low levels in the serum of infected individuals, i.e. in quantities of approx. 102-103 sequences per ml. This level may require the use of amplification techniques in the hybridization assays. Such techniques are known in the art. For example, in the Enzo Biochemical Corporation "Bio-Bridge" system, terminal deoxynucleotide transferase is used to add unmodified, 3'-poly-dT tails to a DNA probe. The poly-dT tail probe is hybridized to the target nucleotide sequence and then to a biotin-modified poly-A. PCT application no. 84/03520 and EP A 124221 a DNA hybridization assay wherein: (1) "analyte" is cured on a single stranded DNA probe complementary to an enzyme labeled oligonucleotide and (2) the resulting duplex with tail is hybridized with an enzyme tag oligonucleotide. EPA No. 204,510 discloses a DNA hybridization assay in which "analyte" DNA is contacted with a probe having a tail, such as a poly-dT tail, an amplifier strand having a sequence that hybridizes to the tail of the probe. , such as a poly-A sequence, and 25 capable of binding a plurality of labeled strands. A particularly desired technique may first comprise reinforcement of approx. 10,000 fold of the target HCV sequences in sera, e.g. to approx. 106 sequences / ml. This can be eg obtained by the technique of Saiki et al. (1986). The amplified sequence (s) can then be detected using a hybridization assay described in pending U.S. patent application (Prosecutor's Ref. No. 2300-0171) filed October 15, 1987, assigned to 46 DK 175975 B1. the present applicant, and is incorporated herein by reference.

This hybridization assay intended to detect sequences at the level of 106 / ml uses nucleic acid multimers which bind to single-stranded analyte-nucleic acid and which also bind to a plurality of labeled single-stranded 5 oligonucleotides. A suitable solution phase sandwich assay that can be used with labeled polynucleotide probes and the methods of producing the probes is disclosed in EPO No. 225.807, published June 16, 1987, assigned to the present applicant, and is hereby incorporated herein by reference.

10

The samples can be packaged in diagnostic assay kits. Diagnostic assay kits include the DNA probe which may be labeled; alternatively, the DNA probe may be unlabeled and the components of the label may be present in the assay kit. The assay kit may also contain other suitable packaged reagents and materials needed for the particular hybridization protocol, e.g. standards and instructions for performing the test.

II.I Immunoassay and Diagnostic Assay Kits 20 Both the polypeptides that react immunologically with serum containing HCV antibodies, e.g. those derived from or encoded in the clones described in Section IV.A and their composites (see Section IV.A), and the antibodies produced from the HCV-specific eptitopes of these polypeptides, see e.g. section IV.E, are well suited in immunoassays to detect the presence of HCV antibodies or the presence of the virus and / or viral antigens in biological samples, including e.g. blood or serum samples. The design of the immunoassays undergoes a number of variations, and a number of these are known in the art. For example, in the immunoassay a viral antigen may be used, e.g. a polypeptide derived from any of the clones containing HCV cDNA described in section IV.A or from the composite cDNAs derived from cDNA ' eme in the clones, or from the HCV genome from which the cDNA of these clones is derived; alternatively, in the immunoassay, a combination of viral antigens derived from these sources may be used. For example, a monoclonal antibody directed against a viral epitope (epitopes), a combination of monoclonal antibodies directed against a viral antigen, monoclonal antibodies directed against various viral antigens, polyclonal antibodies directed against it, the same viral antigen or polyclonal antibodies directed against different viral antigens. For example, protocols may be based on competition or direct response 10 or sandwich type analyzes. For the protocols, e.g. solid substrates are also used or they can be by immunoprecipitation. Most assays include the use of labeled antibody or polypeptide; the labels can e.g. be fluorescent, chemiluminescent, radioactive or color molecules. Also known are assays which amplify the signals from the probe; Examples include assays using biotin and avidin and enzyme labeled and mediated immunoassays such as ELISA assays.

The HCV flavivirus model makes it possible to predict the likely location of diagnostic epitopes for the virion structural proteins. The C, pre-M, M and E domains probably contain all epitopes with significant potential for detection of viral antigens and especially for diagnosis. Similarly, the non-structure proteins domains are expected to contain important diagnostic epitopes (e.g., NS5 encoding a putative polymerase and NS1 encoding an putative complement-binding antigen). Recombinant polypeptides or viral polypeptides containing epitopes from these specific domains may be suitable for the detection of viral antibodies in infectious blood donors and infected patients.

30 48 DK 175975 B1

In addition, antibodies directed against the E and / or M proteins can be used in immunoassays to detect viral antigens in patients with HCV-induced NANBH and in infectious blood donors. Certain antibodies will further be extremely useful for the detection of acute phase donors and 5 patients.

Assay kits suitable for immunoassay containing suitably labeled reagents are constructed by packing the appropriate materials, including the polypeptides of the invention containing HCV epitopes 10 or antibodies directed against HCV epitopes, into suitable containers with the other reagents and materials , which should be used for performing the analysis, as well as an appropriate set of analysis instructions.

IIJ. Further characterization of the HCV oenome. viruses and viral antigens using probes derived from cDNA of the viral aenome HCV cDNA Sequence Information in the sections of Section IV.A. Clones described, as shown in Figures 1-32 inclusive, can be used to obtain additional information on the HCV genome sequence and to identify and isolate the HCV agent, thus contributing to its characterization, including the nature of the genome, the structure of the viral particles. and the nature of the antigens from which it is composed. This information in turn may lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and antibodies directed against HCV epitopes that may be suitable for diagnosis and / or treatment of HCV-induced NANBH.

The cDNA sequence information in the above clones is suitable for constructing probes to isolate additional cDNA sequences derived from hitherto undefined regions of the HCV genome (s), from which the 30 cDNAs in the clones described in Section IV .A. is derived. For example, labeled probes containing a sequence of ca. 8 or more nucleotides and preferably 20 or more nucleotides derived from regions close to 5 'termini or 3' termini of the family of HCV cDNA sequences shown in Figures 1, 3, 6, 9, 14 and 32 are used to isolate overlapping cDNA sequences from HCV cDNA libraries. These sequences, which overlap the cDNAs of the above clones, but which also contain sequences derived from regions of the genome from which the cDNA of the above clones are not derived, can then be used to synthesize probes for identification of other overlapping fragments that do not necessarily overlap the cDNAs of those of Section IV.A. described clones. With the 10 smaller HCV genome segmented and the segments lacking common sequences, it is possible to sequence the entire viral genome (s) using the isolation technique for overlapping cDNAs derived from the viral genome (genomes). Although it is unlikely if the genome is a segmented genome lacking common sequences, the sequence of the genome can be determined by screening serological Iambda-gt11 HCV cDNA libraries, as used to isolate clone 5-1-1, by sequencing cDNA isolates, and using the isolated cDNAs to isolate overlapping fragments using the technique described for isolating and sequencing the clones described in Section 20 IV.A. Alternatively, the characterization of the genomic segments may be based on the viral genome (genomes) isolated from purified HCV particles. Methods for purifying HCV particles and for detecting them during the purification procedure are described below. Methods for isolating polynucleotide genomes from viral particles are known in the art and a useful method is shown in Example IV.A.1. The isolated genomic segments can then be cloned and sequenced. Thus, with the information provided herein, it is possible to clone and sequence the HCV genome (s) regardless of their nature.

Methods for constructing cDNA libraries are known in the art as discussed above and below; a method for constructing 50 HC 175975 B1 HCV cDNA libraries in Iambda-gt11 is discussed below in section IV.A. However, cDNA libraries suitable for screening with nucleic acid probes may also be constructed in other vectors known in the art, e.g. Iambda-gt10 (Huynh et al. (1985)). The HCV-derived cDNA detected by the probes derived from the cDNAs of Figs. 1 - 32 and from the probes synthesized from polynucleotides derived from the cDNAs can be isolated from the clone by digestion. of the isolated polynucleotide with the appropriate restriction enzyme (restriction enzymes) and sequenced. See, e.g. Sections IV.A.3 and IV.A.4. regarding the techniques used for isolating and sequencing HCV cDNA overlapping HCV cDNA in clone 5-1-1, section IV.A.5. and IV.A.7 for isolating and sequencing HCV cDNA overlapping HCV cDNA in clone 81 and section IV.A.8. and IV.A.9 for isolating and sequencing a clone overlapping another clone (clone 36) overlapping clone 81.

15

The sequence information derived from these overlapping HCV cDNAs is well suited for determining regions of homology and heterogeneity within the viral genome (genomes) that could indicate the presence of different strains of the genome and / or populations of defective particles.

The sequence information is also suitable for constructing hydration probes for detection of HCV or HCV antigens or HCV nucleic acid in biological samples, and during isolation of HCV (discussed below) using the techniques described in Section 11. G. The overlapping cDNAs can further be used to create expression vectors for polypeptides derived from the HCV genome (s), which also encode those in clones 5-1-1, 36, 81, 91 and 1- 2 encoded polypeptides and in the other clones described in section IV.A. The techniques for producing the polypeptides containing HCV epitopes and antibodies directed against HCV epitopes contained herein, as well as uses thereof, are analogous to the techniques described for polypeptides derived from 51 N 175B75 B1 from NANBV cDNA sequences contained in clones 5-1-1, 32, 35, 36, 1-2, 81 and 91 discussed above and below.

There are in the family of cDNA sequences contained in clones 5-1-1, 32, 5 35, 36, 81, 91, 1-2 and the others in Section IV.A. described clones encoded antigen (antigens) containing epitopes which appear to be unique to HCV; i.e. antibodies directed against these antigens are lacking in individuals infected with HAV or HBV and in individuals not infected with HCV (see the serological data presented in section IV.B.). A comparison of the sequence information of these cDNAs with the sequences of HAV, HBV, HDV and with the Genebank genomic sequences further shows that minimal homology exists between these cDNAs and the source polynucleotide sequences. Thus, antibodies directed against the antigens encoded in the cDNAs of these clones can be used to identify BB-NANBV particles isolated from infected individuals. In addition, they are also suitable for isolation of the NANBH agent (s).

The HCV particles can be isolated from sera from BB-NANBV-infected individuals or from cell cultures by any of the methods known in the art, including e.g. techniques based on size discrimination such as sedimentation or exclusion methods or techniques based on density such as ultracentrifugation, identity gradients or precipitation with agents such as polyethylene glycol, or chromatography on a variety of materials such as anion or cation exchange materials and materials which binds due to hydrophobicity, as well as affinity columns. During the isolation procedure, the presence of HCV can be detected by hydration analysis of the extracted genome using probes derived from the HCV cDNAs described above or by immunoassay (see 30 section II.I) using antibodies directed as probes. against HCV antigens encoded in the family of cDNA sequences shown in Figures 1 - 32 and 52 DK 175975 B1 also directed to HCV antigens encoded in the overlapping HCV cDNA sequences discussed above . The antibodies may be monoclonal or polyclonal and it may be desirable to purify the antibodies prior to use in the immunoassay. A purification procedure for polyclonal antibodies directed against antigen (antigens) encoded in clone 5-1-1 is described in Section IV.E .; analogous purification procedures can be used for antibodies directed against other HCV antigens.

Antibodies directed against HCV antigens encoded in the family of cDNAs, 10 shown in Figures 1 - 32, as well as those encoded in the overlapping HCV cDNAs attached to solid substrates, are suitable for isolating HCV by immunoaffinity chromatography. There are known in the art techniques for performing immunoaffinity chromatography, including techniques for attaching antibodies to solid substrates so as to retain their immunoselective activity; these may be the techniques in which the antibodies are adsorbed on the substrate (see, for example, Ceiling Racks in ENZYME IMMUNO-DIAGNOSIS, pages 31 - 37) as well as those in which the antibodies are covalently bound to the substrate. The techniques are generally similar to those used for covalently binding antigens to a solid support, as generally described in 20 Section II.C .; but spacer groups may be included in the dual-functional coupling agents so that the antigen binding site of the antibody remains accessible.

During the purification procedure, the presence of HCV can be detected and / or verified by nucleic acid hybridization using polynucleotides as probes, which polynucleotides are derived from the family of HCV cDNA sequences shown in Figures 1-32, as well as from overlapping HCV cDNA sequences described above. In this case, the fractions are treated under conditions that can cause the bursting of viral particles, e.g. with detergents in the presence of gelling agents and viral nucleic acid determined by the hybridization techniques described in 53 II 175975 B1 Section II.H. Further confirmation that the isolated particles are the agents that induce HCV can be obtained by infecting chimpanzees with the isolated virus particles followed by determining whether the NANBH symptoms originate from the infection.

5

Virus particles from the purified preparations can then be further characterized. The genomic nucleic acid has been purified. Based on its sensitivity to RNase, and not DNase I, the virus appears to be composed of an RNA genome. See Example IV.C.2. below. The severity and the circularity or non-circularity can be determined by techniques known in the art, including e.g. visualization of the genome by electron microscopy, its migration in density gradients, and its sedimentation characteristics. Based on the hybridization of the captured HCV genome to the negative of the HCV cDNAs, it appears that HCV may consist of a positive 15-stranded RNA genome (see section IV.H.1). Techniques such as these are described in e.g. METHODS OF ENZYMOLOGY. The purifying nucleic acid can be further cloned and sequenced by known techniques, including reverse transcription, the genomic material being RNA. See, e.g. Maniatis (1982) and Glover (1985). Using the nucleic acid derived from the viral 20 particles, it is possible to sequence the entire genome, whether segmented or not.

Homology examination of the polypeptide encoded in the continuous ORF of the combined clones 14i-39c (see Figure 26) shows that the HCV-25 polypeptide contains homology regions with the corresponding proteins in conserved regions of flaviviruses. An example of these is described in Section IV.H.3. This finding has many important culprits. First is this testimony, together with the results showing that HCV contains a positive stranded genome, the size of which is approx. 10,000 nucleotides, consistent with the assumption that HCV is a flavivirus or flavi-like virus. In general, flavivirus viruses and their genomes have a relatively consistent structure and organization that is known. See Rice et al.

(1986) and Brinton, M.A. (1988). The structural genes encoding the polypeptides C, pre-M / M and E may be located in the 5 'terminus of the genome upstream of clone 14i. When compared to other flaviviruses 5, the precise localization of the sequences encoding these proteins can be further predicted.

Isolation of the sequences upstream of the sequences of clone 14i can be accomplished in a variety of ways that are readily apparent to one skilled in the art from the information provided herein. For example, the "genome walking" technique can be used to isolate other sequences that are 5 'to the sequences in clone 14i but overlap with this clone; this in turn can lead to isolation of additional sequences. This technique is fully demonstrated below in Section IV.A. It is e.g. also known that the flaviviruses have conserved epitopes and regions of 15 conserved nucleic acid sequences. Polynucleotides containing the conserved sequences can be used as probes that bind the HCV genome, thus enabling isolation thereof. These conserved sequences can further be used in conjunction with the sequences derived from the HCV cDNAs shown in Figure 22 to construct primers for use in systems that amplify the genome sequences upstream of the sequences in clone 14i using polymerase chain reaction technology. An example of this is described below.

The HCV structure can also be determined and its components isolated.

The morphology and size may e.g. determined by electron microscopy. Identification and localization of specific viral polypeptide antigens such as envelope antigens or internal antigens such as nucleic acid binding proteins, chemistry antigens and polynucleotide polymerase (polymerases) may also be determined, e.g. by determining whether the antigens are present as 30 major or minor viral components, and by using antibodies directed against the specific antigens encoded in isolated cDNAs as probes. This information is useful for the construction of vaccines; it can for example. is preferred to include an outer antigen in a vaccine preparation. Multivalent vaccines can e.g. consist of a polypeptide derived from the genome encoding a structural protein, e.g. E as well as a polypeptide 5 from another part of the genome, e.g. a non-structural or structural polypeptide.

II.K. Cell culture systems and animal model systems for HCV replication

The assumption that HCV is a flavivirus or flavi-like virus also provides information on methods of cultivating HCV. The term "flavi-like" means that the virus shows a significant amount of homology to the known conserved regions of flaviviruses and that the majority of the genome is a single ORF. Methods for cultivating flaviruses are known to those skilled in the art. (See, for example, the statements by Brinton (1986) and Stollar, V. (1980)). In general, suitable cells or cell lines for culturing HCV may include those known to support flavivirus replication, e.g. the following: monkey kidney cell lines (eg MK2, VERO); pig kidney cell lines (e.g., PS); baby hamster ant cell lines (e.g. BHK); mouse macrophage cell lines (e.g., P388D1, MK1, Mm1); human macrophage cell lines (e.g., U-937); human peripheral blood leukocytes; 20 human adherent immunocytes; hepatocytes or hepatocyte cell lines (e.g.

HUH7, HEPG2); embryos or embryo cells (e.g., chicken embryo fibroblasts); or cell lines derived from invertebrates, preferably from insects (e.g., drosophila cell lines) or, more preferably from arthropods, e.g. mosquito cell lines (e.g., A. albopictus, Aedes aegypti, Culex 25 tritaeniorhynchus) or mid-cell lines (e.g., RML-14 Dermacentor parumapertus).

It is possible that primary hepatocytes may be cultured and then infected with HCV; or alternatively, the hepatocyte cultures may be derived from the liver from 30 infected individuals (e.g., humans or chimpanzees). The latter case is an example of a cell infected in vivo transferred in vitro.

j DK 175975 B1 56

In addition, various methods can be used to obtain sustained cell lines derived from hepatocyte cultures. Eg. For example, primary liver cultures (before and after enrichment of the hepatocyte population) can be fused with a variety of cells to maintain stability. Cultures can, for example, also 5 are infected with transforming viruses or transfected with transforming genes to create permanent or semi-permanent cell lines. In addition, e.g. cells in liver cultures are fused to established cell lines (e.g.

HepG2). Methods of cell fusion are known in the art, including e.g. use of fusion agents such as polyethylene glycol, Sendai Virus and Epstein-Barr virus.

As mentioned above, HCV is a flavivirus or flavi-like virus. Therefore, it is likely that HCV infection of cell lines can be obtained by techniques known in the art for infecting cells with flaviviruses. These 15 include e.g. incubating the cells with viral preparations under conditions that allow virus entry into the cell. In addition, it may be possible to achieve virus production by transfecting the cells with isolated viral polynucleotides. Togavirus and flavivirus RNAs are known to be infectious in a variety of vertebrate cell lines (Pfefferkom and Shapiro 20 (1974)) and in a mosquito cell line (Peleg (1969)).

Methods for transfecting tissue culture cells with RNA duplexes, positively stranded RNAs and DNAs (including cDNAs) are known in the art and include, for example, techniques using electroporation and precipitation with DEAE-Dextran or calcium phosphate.

A rich source of HCV RNA can be obtained by performing in vitro transcription of an HCV cDNA corresponding to the complete genome. Transfection with this material or with cloned HCV cDNA should result in viral replication and in vitro propagation of the virus.

30 57 DK 175975 B1

In addition to cultured cells, animal model systems can be used for virus replication; animal systems in which flaviviruses are known to those skilled in the art (see, for example, Monath (1986)). Thus HCV Replication can occur not only in chimpanzees, but also e.g. in squirrels and teasing mice.

5

II.L. Screening for HCV anti-viral agents

The availability of cell culture and animal model systems for HCV also allows screening for antiviral agents that inhibit HCV replication, and in particular for those agents that preferentially allow cell growth and proliferation while inhibiting viral replication. These screening methods are known to those skilled in the art. The antivirals are generally tested at a range of concentrations for their efficacy in preventing viral replication in cell culture systems that support viral replication, and then for an inhibition of infectivity or viral pathogenicity (and a low level of toxicity) in an animal model system.

The methods and compositions described herein for detecting HCV antigens and HCV polynucleotides are well suited for screening antiviral agents in that they provide an alternative, and perhaps more sensitive, means for detecting the effect of the agent on virus replication than the cell plaque assay or analysis. Eg. For example, the HCV polynucleotide probes described herein can be used to quantify the amount of viral nucleic acid produced in a cell culture. This can be eg is obtained by hybridization or competitive hybridization of the infected cell nucleic acids with a labeled HCV polynucleotide probe. Eg. For example, anti-HCV antibodies can also be used to identify and quantify HCV antigen (antigens) in the cell culture using the immunoassays described herein. Furthermore, since it is desirable to quantify HCV antigens in the infected cell culture by a competitive assay, the polypeptides encoded in the HCV cDNAs described herein are well suited for these competitive assays. In general, a recombinant HCV polypeptide derived from the HCV cDNA will be labeled and the inhibition of binding of this labeled polypeptide to an HCV polypeptide due to the antigens produced in the cell culture system will be monitored. In addition, these techniques are particularly suitable in cases where the HCV may be able to replicate in a cell line without causing cell death.

II.M. Preparation of attenuated HCV strains In addition to the above, it may be possible to isolate attenuated HCV strains using the tissue culture systems and / or animal model systems. These strains will be suitable for vaccines or for isolation of viral antigens. It is possible to isolate attenuated strains following a plurality of cell culture transfers and / or an animal model. Detection of an attenuated strain in an infected cell or individual can be accomplished by techniques known in the art, and may e.g. include the use of antibodies against one or more epitopes encoded in HCV as a probe, or the use of a polynucleotide containing an HCV sequence of at least ca. 8 nucleotides as a probe. An attenuated strain can be constructed as an alternative or an addition using the HCV genomic information provided herein and using recombinant techniques. Generally, an attempt will be made to delete an area of the genome, e.g. encodes a polypeptide that relates to pathogenicity but which permits viral replication. In addition, the genome construction will allow expression of an epitope that induces production of neutralizing antibodies to HCV. The altered genome can then be used to transform cells that allow HCV replication and the cells are grown under conditions that allow virus replication. Attenuated HCV strains are not only suitable for vaccine purposes, but also as sources of commercial viral antigen production, the processing of these viruses would require less stringent protective precautions for the employees involved in virus production and / or the production of viral products.

ML_General Procedures in .5

The common techniques used to extract the genome from a virus, manufacture and probe a cDNA library, sequencing clones, constructing expression vectors, transforming cells, performing immunological assays such as radioimmunoassays and ELISA assays for culturing cells in culture and the like are known in the art and laboratory manuals describing these techniques are available. However, in the following, as a general guide, some sources currently available for such procedures, as well as materials suitable for carrying them out, are provided.

15 'III.A. Hosts and expression control sequences

Both prokaryotic and eukaryotic host cells can be used to express desired coding sequences when appropriate control sequences compatible with the indicated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters optionally containing operator moieties and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are generally derived from e.g. pBR322, a plasmid containing operons that transmit ampicillin and tetracycline resistance, as well as the various pUC vectors that also contain sequences that transmit antibiotic resistance markers. The markers can be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include 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. - 60 DK 175975 B1 promoter and the N-gene ribosome binding site (Shimatake et al. (1981)) and the hybrid tac promoter (De Boer et al. (1983)) derived from sequences of the tg} and lac UV5 promoters. The previous systems are particularly compatible with É. coli; if desired, other prokaryotic hosts, such as 5 strains of Bacillus or Pseudomonas, may be used with similar control sequences.

Eukaryotic hosts include yeast and mammalian cells in culture systems. Saccharomyces cerevisiae and Saccharomyces carlsberaensis are the most commonly used yeast hosts and are suitable fungal hosts. Yeast-compatible vectors carry markers that enable selection of successful transformants by transfer of prototrophy to auxotrophic mutants or resistance to heavy metals in wild-type strains. Yeast compatible vectors may use the 2 micron replication origin (Broach et al. (1983)), the combination of CEN3 and ARS1, or other means to ensure replication, such as sequences that will lead to the incorporation of a suitable fragment into the host cell genome. Control sequences 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 of 3-phosphoglycerate kinase (Hitzeman (1980)). Terminators such as those derived from the enolase gene (Holland (1981)) may also be included. Particularly suitable control systems are those comprising the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) -regulable promoter, terminators are also derived from GAPDH and if secretion is desired, the yeast alpha factor leader sequence. The transcriptionally regulating region and the transcriptionally initiating region that are operably linked may be such that they are not naturally associated in the wild-type organism. These systems are described in detail in EPO No. 120,551, filed October 3, 1984; EPO No. 116.201, presented 22.

30 August 1984 and EPO No. 164,556, submitted December 18, 1985, all of which are assigned to the present applicant and are incorporated herein by reference.

Known in the art are mammalian cell lines available as expression hosts, and they include many sustained cell lines available from The American Type Culture Collection (ATCC), including HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster ants (BHK). ) cells and a variety of other cell lines. Suitable mammalian cell promoters are also known in the art and the 10 include viral promoters such as the promoter of Simian Virus 40 (SV40) (Fiers (1978)), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papillomavirus (BPV). Mammalian cells may also require terminator sequences and poly-A addition sequences; amplifier sequences that increase expression may also be included and sequences causing amplification of the gene may also be desirable. These sequences are known in the art. Vectors suitable for mammalian cell replication may include viral replicas or sequences ensuring the integration of the appropriate sequences encoding NANBV epitopes into the host genome.

III.B, Transformations

The transformation can take place by any known method of introducing polynucleotides into a host cell, including e.g. packing the polynucleotide into a virus and transduction of a host cell with the virus, as well as by direct uptake of the polynucleotide. The transformation procedure used depends on the host to be transformed. Eg. In the example below, transformation of the E. coli host cells with Iambda-gt11 containing BB-NANBV sequences is mentioned. Transformation of bacteria by direct uptake generally involves treatment with 30 calcium or rubidium chloride (Cohen (1972); Maniatis (1982)). The direct yeast transformation can be performed using 62 DK 175975 B1

Hinnen et al. (1978) method. Mammalian transformation by direct uptake can be performed using the calcium phosphate precipitation method of Graham and Van der Eb (1978), or the various known modifications thereof.

III.C. vector Construction

Techniques known in the art are used to construct vectors. Site-specific DNA cleavage is performed by treatment with suitable restriction enzymes under conditions generally specified by the manufacturer of these commercially available enzymes. In general, about 1 µg of plasmid or DNA sequence of 1 enzyme unit for approx. 20 µl buffer solution by incubation for 1-2 hours at 37 ° C. After incubation with the restriction enzyme, the protein is removed by phenol / chloroform extraction and the DNA is recovered by ethanol precipitation. The cleaved fragments can be separated using polyacrylamide or agarose gel electrophoresis techniques according to the general procedures found in "Methods in Enzymology" (1980) 65: 499-560.

Cleavage fragments with adhesive ends can be blunt ended using E. coli DNA polymerase I (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease may also be used, leading to hydrolysis of any single stranded DNA moiety.

25

Ligations are performed with standard buffer and under temperature conditions using T4 DNA ligase and ATP; ligations of adhesive ends require less ATP and less ligase than ligations of blunt ends.

When the vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal phosphatase to remove the 5 'phosphate and thus prevent the religation of the vector; alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation.

Ligation mixtures are transformed into suitable cionic hosts such as E 5 coli and successful transformants are selected, e.g. antibiotic resistance and they are screened for proper construction.

III.D. Construction of Desired DNA Sequences Synthetic oligonucleotides can be prepared using an automatic oligonucleotide synthesizer as described by Warner (1984). If desired, the synthetic strands can be labeled with 32P by treatment with polynucleotide kinase in the presence of 32P-ATP using standard conditions of the reaction.

DNA sequences, including those isolated from cDNA libraries, can be modified by known techniques, including e.g. site-directed mutagenesis as described by Zoller (1982). Briefly, the DNA to be modified is packed into a phage as a single-stranded sequence and converted to a 20-stranded DNA with DNA polymerase, using a synthetic oligonucleotide as a primer, which oligonucleotide is complementary to the DNA portion. which must be modified and have the desired modification included in its own sequence. The resulting double-stranded DNA is transformed into a phage supporting host bacterium. Cultures of the 25 transformed bacteria containing replications of each strand of the phage are plated in agar to obtain plaque. 50% of the new plaques contain theoretical subjects with the mutated sequence and the remaining 50% have the original sequence. Replicates of these plaques are hybridized with a labeled synthetic probe at temperatures and conditions that allow for hybridization with the correct strand but not with the unmodified 64 Se 175975 B1 sequence. The sequences identified by hybridization are recovered and cloned.

III.E. Hybridization with Probe 5 DNA Libraries can be probed using the Grunstein and Hogness (1975) procedure. 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. / volume) of each of bovine serum albumin, polyvinylpyrrolidone and

Ficoll, 50 mM Na phosphate (pH 6.5), 0.1% SDS and 100 µg / 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, depend on the required stringency. Oligomeric probes requiring lower stringency conditions are generally used with low percentages of the formamide, lower temperatures and longer hybridization times. Probes containing more than 30 or 40 nucleotides, such as those derived from cDNA or genomic sequences, generally use higher temperatures, e.g. ca. 40-42 ° C and a high percentage, e.g. 50%, formamide.

After prehybridization, 5'-32P-labeled oligonucleotide probe is added to the buffer and the filters are incubated in this mixture under hybridization conditions. After 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.

III.F. Verification of construction and sequencing

For routine vector constructs, ligation mixtures are transformed into E. coli strain HB101 or other suitable host and successful transformants 30 are selected for antibiotic resistance or other markers. Plasmids from the transformants are then prepared according to the method of Clewell et al. (1969), usually after chloramphenicol enhancement (Clewell (1972)). The DNA is usually isolated and analyzed by restriction enzyme analysis and / or sequencing. Sequencing can be done by Sanger et al. (1977) method of videoxy, further described by Messing et al. (1981) or at 5 Maxam et al. (1980) method. The band compression problems often observed in GC-rich areas were overcome by T-deazoguanosine according to Barr et al. (1986).

III.G. Enzyme-linked immunosorbent assay 10

The enzyme-linked immunosorbent assay (ELISA) can be used to measure either antigen or antibody concentrations. This method depends on conjugation of an enzyme to either antigen or antibody and the bound enzyme activity is used as a quantitative label. For measurement of antibody 15, the known antigen is fixed to a solid phase (e.g., a microplate or plastic cup), incubated with test serum dilutions, washed, incubated with anti-immunoglobulin labeled with an enzyme, and washed again. Suitable enzymes for labeling are known in the art and include e.g. horseradish peroxidase. Enzyme activity bound to the solid phase is measured by adding the specific substrate and determining product formation and substrate utilization colorimetrically. The bound enzyme activity is a direct function of the amount of bound antibody.

For the measurement of antigen, a known specific antibody is fixed to the solid phase, which is added to the test material containing antigen, after incubation, the solid phase is washed and a secondary enzyme labeled antibody is added. After washing, the substrate is added and the enzyme activity is evaluated colorimetrically and related to antigen concentration.

30 66 DK 175975 B1 IV. examples

Examples of the present invention are provided below which are provided for illustrative purposes only and not to limit the scope of the present invention. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those skilled in the art. The procedures described, e.g. in Section IV.A. may, if desired, be repeated, but need not be, as techniques are available for constructing the desired nucleotide * 10 sequences based on the information provided by the invention. Expression is exemplified in E. coli; but other systems are available as more fully set out in Section III.A. Additional epitopes derived from the genome structure can also be produced and these can be used to generate antibodies as described below.

15

IV.A. Preparation, Isolation and Sequencing of HC cDNA

IV.A.1._ Preparation of HCV cDNA

The source of the NANB agent was a plasma pool derived from a chronic NANBH chimpanzee. The chimpanzee had been experimentally infected with blood from another chronic NANBH chimpanzee, which was derived from HCV infection in a contaminated batch of factor 8 concentrate derived from unified human sera. The chimpanzee pool was prepared by combining many individual plasma samples containing high levels of alanine aminotransferase activity; this activity results from liver damage due to the HCV infection. As 1 ml of a 10-6 dilution of this unified serum given i.v. caused NANBH in another chimpanzee, its CID was at least 106 / ml, ie. it had a high infectious virus titer.

30 67 DK 175975 B1

A cDNA library from the high-titer plasma pool was generated as follows. First, viral particles were isolated from the plasma; a 90 ml sample was diluted with 310 ml of solution containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl. Residues were removed by centrifugation for 20 min at 5,000 x g at 20 ° C. Viral particles in the resulting supernatant were then pelleted by centrifugation in a Beckman SW28 rotor at 28,000 rpm for 5 hours at 20 ° C. To release the viral genome, the particles were disrupted by suspending pellets in 15 ml of solution containing 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, also 10 containing 2 mg / ml proteinase k, followed by incubation at 45 ° C for 90 min. Nucleic acid was isolated by adding 0.8 µg of MS2 bacteriophage RNA as a carrier and extracting the mixture 4x with a 1: 1 mixture of pheno-chloroform (phenol saturated with 0.5 M Tris-HCl, pH 7.5, 1% (v / v) beta-mercaptoethanol, 0.1% (w / v) hydroxyquinolone followed by extraction twice with chloroform). The aqueous phase was concentrated with 1-butanol prior to precipitation with 2.5 volumes of absolute ethanol overnight at -20 ° C. Nucleic acid was removed by centrifugation in a Beckman SW41 rotor at 40,000 rpm for 90 min at 4 ° C and dissolved in water that had been treated with 0.05% (v / v) diethyl pyrocarbonate and autoclaved. -

Nucleic acid obtained by the above procedure (<2 µg) was denatured with 17.5 mM CH 2 H 2 OH; cDNA was synthesized using the denatured nucleic acid as template and was cloned into the EcoRI site of 25 phage Iambda-gt11 using methods described by Huynh (1985) except that stochastic primers replaced oligo (dT ) 12-18 during the synthesis of the first cDNA strand by reverse transcriptase (Taylor et al. (1976)). The resulting double-stranded cDNA has been fractionated by size of a Sepharose CL-4B column; eluted material of 30 approx. Mean size 400, 300, 200 and 100 base pairs were pooled into cDNA pools 68. The Lambda-gt11 cDNA library was generated from cDNA in pool 3.

The Lambda-gt11 cDNA library generated from pool 3 was screened 5 for epitopes that can be specifically bound to serum derived from a patient previously exposed to NANBH. Ca. 106 phages were screened with patient sera by the methods of Huynh et al. (1985), except that bound human antibody was detected with sheep anti-human Ig antisera radiolabelled with 125 I. Five positive phages were identified and purified.

10 The five positive phages were then tested for binding specificity to sera from 8 different people previously infected with the NANBH agent using the same method. The four offenders encoded a polypeptide that reacted immunologically with only a human serum, ie. that used for primary screening of the subject library. The fifth phage (5-1-1) coded 15 for a polypeptide that reacted immunologically with 5 of 8 of the sera tested. Furthermore, this polypeptide did not react immunologically with sera from 7 normal blood donors. Therefore, clone 5-1-1 appears to encode a polypeptide specifically recognized immunologically by sera from NANB patients.

IV.A.2. HCV cDNA sequences in recombinant phage 5-1-1 and polypeptide sequences encoded in the sequence cDNA In recombinant phage 5-1-1 were sequenced by Sanger et al. (1977) -25 method. Essentially, the cDNA was cut with Eco RI isolated by size fractionation using gel electrophoresis. The Eco RI restriction fragments were subcloned into the M13 vectors, mp18 and mp19 (Messing (1983)), and sequenced using the dideoxy chain termination method of Sanger et al. (1977). The sequence obtained is shown in Figure 1.

I ------- DK 175975 B1 69

The polypeptide encoded in Figure 1 encoded in the HCV cDNA is in the same translation frame as the N-terminal beta-galactosidase moiety to which it is fused. As shown in Section IV.A. encodes the open translation reading frame (ORF) of 5-1-1 for epitopes specifically recognized by sera from 5 patients and chimpanzees with NANBH infections.

IV.A.3._Isolation of HCV cDNA overlapping with cDNA in clone 5-1-1

Overlapping HCV cDNA that overlaps with the cDNA in clone 5-1-1 was obtained by screening the same Iambda-gt11 library, generated as described in Section IV.A.1. with a synthetic polynucleotide derived from the HCV cDNA sequence of clone 5-1-1 as shown in Figure 1. The polynucleotide sequence used for screening was:

15 5'-TCC CTT GCT CGA TGT ACG GTA AGT GCT GAG AGC

ACT CTT CCA TCT CAT CGA ACT CTC GGT AGA GGA CTT CCC TGT CAG GT-3 *.

The Lambda-gt11 library was screened with this probe using the method described in Huynh (1985). Ca. 1 in 50,000 clones hybridized with the probe. Three clones containing cDNAs hybridizing to the synthetic probe have been numbered 81, 1-2 and 91.

IV.A.4. Nucleotide sequences of HCV cDNAs overlapping with cDNA in clone 5-1-1

The nucleotide sequences of the three cDNAs in clones 81, 1-2, and 91 were determined essentially as in Section IV.A.2. The sequences of these clones relative to the HCV cDNA sequence in phage 5-1-1 are shown in Figure 2, showing the strand encoding the detected HCV epitope and where the homologies in the nucleotide sequences are indicated by vertical lines between the sequences.

c 70 DK 175975 B1

The sequences of the cloned HCV cDNAs are highly homologous in the overlapping regions (see Figure 2). However, there are differences in two areas. Nucleotide 67 in clones 1-2 is thymidine, whereas the other three clones contain 5 a cytidine residue at this position. However, it should be noted that the same amino acid is encoded when either C or T occupies this position.

The other difference is that clone 5-1-1 contains 28 base pairs not found in the other three clones. These basepacs occur at the onset of cDNA sequence 10 in 5-1-1 and are indicated by lowercase letters. Based on radioimmunoassay data discussed below in Section IV.D., it is possible that an HCV epitope may be encoded in this 28 bp range.

Absence of the 28 base pairs in 5-1-1 from clones 81, 1-2, and 91 may mean that the 15 cDNAs in these clones were derived from defective HCV genomes; alternatively, the 28 bp region could be a terminal artifact in clone 5-1-1.

The lower case sequences in the nucleotide sequence of clones 81 and 91 simply indicate that these sequences have not been found in other cDNAs because so far 20 cDNAs overlapping these regions have not been isolated.

A composite HCV cDNA sequence derived from overlapping cDNAs in clones 5-1-1, 81 and 1-2 and 91 is shown in Figure 3.1. This figure is the unique 28 base pairs from clone 5-1-1 however, omitted. The figure also shows the sequence of the polypeptide encoded in the ORF of the composite HCV cDNA.

IV.A.5._Isolation of HCV cDNAs. that overlap with cDNA in clone 81

The isolation of HCV cDNA sequences upstream of and overlapping with those of clone 81 cDNA was obtained as follows. The Lambda-gt11 cDNA library, prepared as described in Section IV.A.1., Was screened by hybridization with a synthetic polynucleotide probe homologous to a 5 'terminal sequence in clone 81. The sequence in clone 81 is shown in Figure 4 The synthetic polynucleotide sequence used for screening was: 71 CT 175975 B1 5 5 'CTG TCA GGT ATG ATT GCC GGC TTC CCG GAC 3'.

The methods were essentially as described by Huynh (1985), with the exception that the library filters were washed twice under stringent conditions, i.e. they were washed in 5 x SSC, 01,% SDS at 55 ° C for 30 min each. Ca.

10 l of 50,000 clones hybridized with the probe. A positive recombinant phage containing cDNA hybridizing to the sequence was isolated and purified. This subject has been numbered clone 36.

Downstream cDNA sequences overlapping with the carboxylene sequences of clone 81 cDNA were isolated using a procedure similar to the isolation of upstream cDNA sequences except that a synthetic oligonucleotide probe homologous to a 3 'terminal sequence in clone 81. The synthetic polynucleotide sequence used for screening was: 20 5' TTT GGC TAG TGG TTA GTG GGC TGG TGA CAG 3 '.

A positive recombinant phage containing cDNA hybridizing to the latter sequence was isolated and purified and has been numbered clone 25 32.

IV.A.6. HCV cDNA nucleotide sequence in clone 36 The cDNA nucleotide sequence in clone 36 was determined essentially as described in Section IV.A.2. This double stranded sequence of this cDNA, its region overlapping with the HCV cDNA in clone 81, and the ORF encoded polypeptide is shown in Figure 5.

The ORF of clone 36 is in the same translation frame as the HCV antigen encoded in clone 81. Thus, the ORFs of clones 36 and 81 encode in combination for a polypeptide representing a portion of a larger HCV antigen. The sequence of this putative HCV polypeptide and its double stranded DNA sequence derived from the combined ORFs of the HCV cDNAs in clones 36 and 81 are shown in Figure 6.

IV.A.7 HCV cDNA Nucleotide Sequence in Clone 32 The cDNA nucleotide sequence in clone 32 was determined essentially as described in Section IV.A.2. for the sequence of clone 5-1-1. Sequence data showed that the 15 cDNA in clone 32 recombinant phage was derived from two different sources. A fragment of the cDNA was 418 nucleotides derived from the HCV genome; the second fragment was comprised of 172 nucleotides derived from the bacteriophage MS2 genome that had been used as a carrier during the preparation of the Iambda-gt11 plasmid cDNA library.

The cDNA sequence in clone 32 corresponding to the sequence of the HCV genome is shown in Figure 7. The region of the sequences overlapping the region of clone 81 and the polypeptide encoded by the ORF is also shown in the figure. This sequence contains a continuous ORF that is in the same translation frame as the HCV antigen encoded by clone 81.

IV.A.8._Isolation of HCV cDNA. that overlap with cDNA in clone 36

Isolation of HCV cDNA sequences upstream of and overlapping with those of clone 36 cDNA was performed as described in Section IV.A.5., Of those overlapping clone 81 cDNA, except that the synthetic polynucleotide was based on the 5 'region of clone 36. The synthetic polynucleotide sequence used for screening was: 73' AAG CCA CCG TGT GCG CTA GGG CTC AAG CCC 3 '.

5

Ca. 1 in 50,000 clones hybridized with the probe. The isolated purified recombinant phage clone containing cDNA hybridizing to this sequence was named clone 35.

IV.A.9._HCV cDNA Nucleotide Sequence in Clone 35 The cDNA nucleotide sequence in clone 35 was determined essentially as described in Section IV.A.2. The sequence, its overlap region with the cDNA of clone 36, and the putative polypeptide encoded therein are shown in Figs.

Clone 35 apparently contains a single, continuous ORF encoding a polypeptide in the same translation frame as the polypeptide encoded by clone 36, clone 81 and clone 32. Figure 9 shows the sequence of the long 20 continuous ORF extending over clones 35, 36, 81 and 32 together with the putative HCV polypeptide encoded therein. This combined sequence has been confirmed using other independent cDNA clones derived from the same Iambda-gt11 cDNA library.

25, IV.A.10. Isolation of HCV cDNA. that overlaps with cDNA in clone 35!

The isolation of HCV cDNA sequences upstream of and overlapping with the sequences of clone 35 cDNA was performed as described in Section IV.A.8., For those overlapping clone 36 cDNA, except that the synthetic polynucleotide was based on the 5 'region of clone 35. The synthetic polynucleotide sequence used for screening was.

74 DK 175975 B1 5 'CAG GAT GCT GTC TCC CGC ACT C AA CGT 3'.

5

Ca. 1 in 50,000 clones hybridized with the probe. The isolated purified recombinant phage clone containing cDNA hybridizing to this sequence was named clone 37b.

10 IV.A.11. HCV Nucleotide Sequence in Clone 37b The cDNA nucleotide sequence in clone 37b was determined essentially as described in Section IV.A.2. The sequence, its overlap region with the cDNA of clone 35 and the putative polypeptide encoded therein are shown in Figure 10.

The 5'-terminal nucleotide in clone 35 is T, while the corresponding nucleotide in clone 37b is A. The cDNAs From 3 other independent clones isolated during the procedure in which clone 37b was isolated, described in Section IV.A.10 ., has also been sequenced. The cDNAs From these clones, 20 also contains A in this position. Thus, the 5'-terrninal T in clone 35 may be an artifact of the cloning procedure. It is known that artifacts often occur at 5 'termini in cDNA molecules.

Clone 37b apparently contains a continuous ORF encoding a polypeptide which is an extension of the polypeptide encoded in the ORF that extends through the overlapping clones 35, 36, 81 and 32.

IV.A.12. Isolation of HCV cDNA overlapping with cDNA in clone 32 Isolation of HCV cDNA sequences downstream of clone 32 was performed as follows. First, clone c1a was isolated using a synthetic 75 'hybridization probe based on the nucleotide sequence of the HCV cDNA sequence in clone 32. The method was essentially that described in section IV.A.5. except that the sequence of the synthetic probe was: DK 175975 B1 5 5 'AGT GCA GTG GAT G AA CCG GCT GAT AGC CTT 3'.

Using the nucleotide sequence from clone c1a, another synthetic nucleotide was synthesized which had the sequence: 10 5'TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3 '.

Screening of the Iambda-gt11 library using the clone-derived sequence as a probe yielded ca. 1 in 50,000 positive colonies. An isolated purified clone that hybridized with this probe was called clone 33b.

15 IV.A.13. HCV cDNA nucleotide sequence in clone 33b The cDNA nucleotide sequence in clone 33b was determined essentially as described in Section IV.A.2. The sequence, its overlap region with the cDNA of 20 clone 32 and the putative polypeptide encoded therein are shown in Figure 11.

Clone 33b apparently contains a continuous ORF that is an extension of the ORFs in overlapping clones 37b, 35, 36, 81 and 32. The polypeptide encoded in clone 33b is in the same translation frame as the polypeptide encoded in the extended ORF of these overlapping clones.

IV.A. 14. Isolation of HCV cDNAs overlapping with cDNA in clone 37b and with cDNA in clone 33b 30 To isolate HCV cDNAs overlapping with cDNAs in clone 37b and in clone 33b, the following synthetic oligonucleotide probes were derived from the cDNA of these clones used to screen the Iambda-gt11 library essentially using the method described in Section IV.A.3. The probes used were: 5 5 'CAG GAT GCT GTC TCC CGC ACT CAA CGT C 3 * ° 9 5' TCC TG A GGC GAC TGC ACC AGT GGA TAA GCT 3 * 10 for detection of colonies containing HCV cDNA sequences , which overlaps with the sequences of clones 37b and 33b. Ca. 1 in 50,000 colonies was detected with each probe. A clone containing upstream cDNA that overlapped with the cDNA of clone 37b was referred to as clone 40b. A clone containing downstream cDNA that overlapped with the cDNA of clone 33b was referred to as clone 25c.

IV.A.15. HCV cDNA Nucleotide Sequences in Clone 40b and Clone 25c 20 The nucleotide sequences of the cDNAs of clone 40b and clone 25c were determined essentially as described in Section IV.A.2. The sequences of 40b and 25c, their overlap regions with the cDNA of clones 37b and 33b, and the putative polypeptides encoded therein are shown in Figure 12 (Clone 40b) and Figure 13 (Clone 25c).

However, the 5 'terminal nucleotide in clone 40b is the G. cDNAs From 5 other independent clones isolated during the procedure in which clone 40b was isolated, described in Section IV.A.14., Have also been sequenced. The cDNAs for these clones also contain T in this position. Thus, the GET may represent a cloning artifact (see the discussion in Section IV.A.11.).

77 DK 175975 B1

The 5 'terminus of clone 25c is ACT, but this region's sequence is in clone cla (sequence not shown) and in clone 33b is TCA. This difference may also represent a cloning artifact just like the extra 28: 5 'terminal nucleotides in clone 5-1-1.

5

Clones 40b and 25c apparently contain an ORF which is an extension of the continuous ORF of the previously sequenced clones. The nucleotide sequence of the ORF extending through clones 40b, 37b, 35, 36, 81, 32, 33b and 25c, and the amino acid sequence of the putative polypeptide encoded therein are shown in Figure 14. In the figure the potential artifacts are omitted from the sequence and the corresponding sequences in non-5 'terminal regions of multiple overlapping clones are shown instead.

IV.A.16. Preparation of a composite HCV cDNA from the cDNAs of the clones 15 36. 81 and 32

The composite HCV cDNA, C100, was constructed as follows. First, the cDNAs from clones 36, 81 and 32 were cut with EcoRI. The EcoRI fragment of cDNA from each clone. were individually cloned into the EcoRI site by the Vector 20 pGEM3-blue (Promega Biotec). The resulting recombinant vectors containing the cDNAs from clones 36, 81 and 32 were named pGEM3-blue / 36, pGEM3-blue / 81 and pGEM3-blue / 32. The properly oriented pGEM3-blue / 81 recombinant was digested with Nael and Narl, and the larger (-2,850 bp) fragment was purified and ligated with the smaller (-570 bp) 25 Nael / Narl purified restriction fragment from pGEM3-blue / 36. This composite of the cDNAs from clones 36 and 81 was used to generate a pGEM3 blue vector containing the continuous HCV ORF contained in the overlapping cDNA of these clones. The new plasmid was then digested with Pvull / EcoRI to release a fragment of ca. 680 bp, 30 which was then ligated to the smaller (580 bp) Pvull / EcoRI fragment isolated from the appropriately oriented pGEM3 blue / 32 plasmid and the composite cDNA of clones 36, 81 and 32 was ligated into the EcoRI linearized vector pSODcfl described in Section IV.B.1. and used to express clone 5-1-1 in bacteria. Recombinants containing approx. The 1,270 bp EcoRI fragment of composite HCV cDNA (C100) was selected, and the cDNA from the plasmids was cut with EcoRI and purified.

IV.A.17.solarization and nucleotide sequences of HCV cDNAs in clones 14i. 11b.

7f. 7th, 8h. 33c, 14c. 8f. 33f. 33q and 39c The HCV cDNAs of clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were isolated by the technique of isolating overlapping cDNA fragments from the Iambda gt11 library of HCV cDNAs, described in Section IV.A.1. The technique used was substantially consistent with that of Section IV.A.3. described except that the probes used were constructed from the nucleotide sequence of the last isolated clones from the 5 and 3 'ends of the combined HCV sequences. The frequency of clones that hybridized with the clones described below was approx. 1 in 50,000 in each case.

The HCV cDNA nucleotide sequences of clones 14i, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were determined essentially as described in Section IV.A.2., Except that it cut off cDNA from these phages replaced the cDNA isolated from clone 5-1 -1.

Clone 33c was isolated using a hybridization probe based on the nucleotide sequence of clone 40b. The nucleotide sequence of clone 40b is shown in Figure 12. The nucleotide sequence of the probe used to isolate 33c was: 30 'ATC AGG ACC GGG GTG AGA ACA ATT ACC ACT 3'.

79 DK 175975 B1

The sequence of HCV cDNA in clone 33c and overlapped with that of clone 40b is shown in Figure 15, which also shows the amino acids encoded therein.

Clone 8h was isolated using a probe based on the 5 nucleotide sequence of clone 33c. The nucleotide sequence of the probe was: 5 'AGA GAC AAC CAT GAG GTC CCC GGT GTT C 3'.

The HCV cDNA sequence in clone 8h and overlapped with that in clone 33c and the amino acids encoded therein are shown in Figure 16.

Clone 7e was isolated using a probe based on the nucleotide sequence of clone 8h. The nucleotide sequence of the probe was: 15 5 'TCG GAC CTT TAC CTG GTC ACG AGG CAC 3'.

HCV cDNA In clone 7e, overlapped with clone 8h and the amino acids encoded therein, is shown in Figure 17.

Clone 14c was isolated with a probe based on the nucleotide sequence I clone 35c. The sequence in clone 35c is shown in Figure 13. The probe for isolating clone 14c had the sequence: 5 'ACC TTC CCC ATT AAT GCC TAC ACC ACG GGC 3'.

The HCV cDNA sequence in clone 14c, its overlap with clone 25c, and the amino acids encoded therein are shown in Figure 18.

Clone 8f was isolated using a probe based on the nucleotide sequence of clone 14c. The nucleotide sequence of the probe was: in one 80 'TCC ATC TCT C AA GGC AAC TTG CAC CGC TAA 3'.

The HCV cDNA sequence in clone 8f, its overlap with clone 14c and the amino acids encoded therein are shown in Figure 19.

5

Clone 33f was isolated using a probe based on the nucleotide sequence present in clone 8f. The nucleotide sequence of the probes was: 5 'TCC ATG GCT GTC CGC TTC CAC CTC C AA AGT 3 *.

The HCV cDNA sequence in clone 33f, its overlap with clone 8f and the amino acids encoded therein are shown in Figure 20.

Clone 33g was isolated using a probe based on the nucleotide sequence of clone 33f. The nucleotide sequence of the probe was 5 'GCG ACA ATA CGA C AA CAT CCT CTG AGC CCG 3 \ HCV cDNA Sequence in clone 33g, its overlap with clone 33f and the 20 encoded amino acids are shown in Figure 21.

Clone 7f was isolated using a probe based on the nucleotide sequence of clone 7e. The nucleotide sequence of the probes was: 5 'AGC AGA C AA GGG GCC TCC TAG GGT GCA TAA T 3'.

The HCV cDNA sequence in clone 7f, its overlap with clone 7e and the amino acids encoded therein are shown in Figure 22.

Clone 11b was isolated using a probe based on the sequence of clone 7f. The nucleotide sequence of the probe was: 81 '175 CAC CTA TGT TTA TAA CCA TCT CAC TCC TCT 3'.

The HCV cDNA sequence in clone 11b, its overlap with clone 7f and the amino acids encoded therein are shown in Figure 23.

Clone 14i was isolated using a probe based on the nucleotide sequence of clone 11b. The nucleotide sequence of the probe was: 5'CTC TGT CAC CAT ATT ACA AGC GCT ATA TCA 3 '.

The HCV cDNA sequence in clone 14i, its overlap with 11b, and the amino acids encoded therein are shown in Figure 24.

Clone 39c was isolated using a probe based on the nucleotide sequence of clone 33g. The nucleotide sequence of the probe was: 5'CTC GTT GCT ACG TCA CCA CAA TTT GGT GTA 3 '.

The 20 HCV cDNA sequence in clone 39c, its overlap with clone 33g, and the amino acids encoded therein are shown in Figure 25.

IV.A. 18. The composite HCV cDNA sequence derived from isolated clones containing the HCV cDNA 25 The HCV cDNA sequences of the clones described above have been sequenced to create a composite HCV cDNA sequence ... isolated clones arranged in the 5 'to 3' direction are: 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81.32, 33b, 25c, 14c, 8f, 33f, 33g and 39c.

30 82 DK 175975 B1

A composite HCV cDNA sequence derived from the isolated clones and the amino acids encoded therein is shown in FIG. 26th

In generating the composite sequence, heterogeneities in the following sequence 5 are taken into account. Clone 33c contains an 800 base pair HCV cDNA that overlaps the cDNAs of clones 40b and 37c. However, in clone 33c as well as in five other overlapping clones, nucleotide is 789 G. In clone 37b (see Section IV.A.11), the corresponding nucleotide is A. This sequence difference creates an apparent heterogeneity in the 10 amino acids encoded therein. can be either CYS or TYR, for G or A, respectively.

This heterogeneity may have important culprits in terms of protein folding.

Nucleotide residue # 2 in clone 8h HCV cDNA is T. But as shown below, the corresponding residue in clone 7e is A; moreover, A has also been found in this position in three other isolated, overlapped clones. Thus, the T residue in clone 8h may represent a cloning artifact. Therefore, in FIG. 26 the remainder in this position A.

The 3'-terminal nucleotide in clone 8f HCV cDNA is G. But the corresponding residue in clone 33f and in two other overlapping clones is T. Therefore, the residue in this position is designated T in Figure 26.

The 3'-terminal sequence in clone 33f HCV cDNA is TTGC. But the 25 corresponding sequence in clone 33g and in two other overlapping clones is ATTC. Therefore, the corresponding region of FIG. 26 designated ATTC.

Nucleotide residue # 4 in clone 33g HCV cDNA is T. But in clone 33f and in two other overlapping clones, the corresponding residue is A. Therefore, the corresponding residue in FIG. 26 designated A.

The B1 3'-terminal in clone 14i is AA, while the corresponding dinucleotide in clone 11b and in three other clones is TA. Therefore, the TA residue is depicted in FIG. 26th

The solution of other sequence heterogeneities is discussed above.

5

A study of the composite HCV cDNA shows that it contains one major ORF. This suggests that the viral genome is translated into a larger polypeptide that is processed simultaneously with or subsequent translation.

IV.A. 19. Isolation and nucleotide sequences of HCV cDNAs in clones 12f, 35f. 1 9q. The 26q and 15e HCV cDNAs of clones 12f, 35f, 19g, 26g and 15e were essentially isolated by the techniques described in Section IV.A.17, except that the 15 groups were as shown below. The frequency of clones that hybridized with the probes was approx. 1 in 50,000 in each case. The HCV cDNA nucleotide sequences of these clones were determined essentially as described in Section IV.A.2, with the exception that the cDNA from the clones shown replaced the cDNA isolated from clone 5-1-1.

20

The isolation of clone 12f containing cDNA upstream of HCV cDNA in ftg.

26, was performed using a hybridization probe based on the nucleotide sequence of clone 14i. The nucleotide sequence of the probe was 5 'TGC TTG TGG ATG ATG CTA CTC ATA TCC C AA 3'.

The HCV cDNA sequence in clone 12f, its overlap with clone 14i and the amino acids encoded therein are shown in FIG. 27th

84 DK 175975 B1

Isolation of clone 35f containing cDNA downstream of the HCV cDNA of FIG. 26 was performed using a hybridization probe based on the nucleotide sequence of clone 39c. The nucleotide sequence of the probe was 5 'AGC AGC GGC GTC AAA AGT G AA GGC TAA CTT 3'.

The sequence of clone 35f, its overlap with the sequence of clone 39c and the amino acids encoded therein are shown in FIG. 28th

Isolation of clone 19g was performed using a hybridization probe based on the 3 sequence of clone 35f. The nucleotide sequence of the probe was: 5 'TTC TCG TAT GAT ACC CGC TGC TTT GAC TCC 3'.

The HCV cDNA sequence in clone 19g, its overlap with the sequence in clone 35f and the amino acids encoded therein are shown in FIG. 29th

Isolation of clone 26g was performed using a hybridization probe based on the 3 sequence of clone 19g. The nucleotide sequence of the probe was 20 5 'TGT GTG GCG ACG ACT TAG TCG TTA TCT GTG 3 \ HCV cDNA Sequence of clone 26g, its overlap with the sequence of clone 19g and the amino acids encoded therein are shown in FIG. 30th

25

Clone 15e was isolated using a hybridization probe based on the 3 sequence of clone 26g. The nucleotide sequence of the probe was 5 'CAC ACT CCA GTC AAT TCC TGG CTA GGC AAC 3'.

The HCV cDNA sequence of clone 15e, its overlap with the sequence of clone 26g and the amino acids encoded therein are shown in FIG. 31st

The clones described in this section are deposited with ATCC under the clauses of section 5 II.A. conditions described and have been assigned the following deposit numbers:

Lambda-qt11_ATCC No., Date of Deposit

Clone 12f 40514 10 Nov. 1988 10 Clone 35f 40511 10 Nov. 1988

Clone 15th 40513 10 Nov. 1988

Clone K9-1 40512 10 Nov. The 1988 HCV cDNA sequences in the clones described above have been placed in 15 rows to produce a composite HCV cDNA sequence. The isolated clones arranged in the 5 'to 3' direction are: 12f, 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f , 33g, 39c, 35f, 19g, 26g and 15e.

A composite HCV cDNA sequence derived from the isolated clones and the amino acids encoded therein is shown in FIG. 32nd

IV.A.20. Alternative method for isolating cDNA sequences upstream of the HCV cDNA sequence in clone 12f

Based mostly on the 5 'HCV sequence of FIG. 32, derived from HCV cDNA in clone 12f, less synthetic reverse oligonucleotide primers are synthesized and used to bind to the corresponding sequence in genomic HVC RNA to prime reverse transcription of the 30 upstream sequences. The primer sequences are proximal to the known 5-terminal sequence in clone 12f, but sufficiently downstream to allow in the construction of probe sequences upstream of the primer sequences. Known standard methods for priming and cloning are used. The resulting cDNA libraries are screened with sequences upstream of the priming sites (as deduced from the illustrated sequence in clone 12f). Genomic HCV RNA is obtained from either plasma or liver samples from chimpanzees with NANBH, or from analogous samples from humans with NANBH.

IV.A.21. Alternative method using tail attachment to isolate sequences from the 5 'terminal region of the HCV genome.

10

To isolate the extreme 5'-terminal sequences of the HCV RNA genome, the first round of reverse transcription, duplexed with the template RNA, attaches an oligo C tail to the cDNA product. This is done by incubating the product with terminal transferase in the presence of CTP. The second round 15 of cDNA synthesis that provides the complement to the first cDNA strand is performed using oligo G as a primer for the reverse transcriptase reaction. The sources of HCV genomic RNA are as described in Section IV.A.20. The methods for loading tail with terminal transferase and for the reverse transcriptase reactions are as described in Maniatis et al. (1982).

The cDNA products are then cloned, screened and sequenced.

IV.A.22. Alternative method using tail application to isolate frequencies from the 3 'terminal region of the HCV genome.

This method is based on previously used methods for cloning flavivirus RNA cNDAs. In this method, the RNA is subjected to denaturing conditions to remove secondary structures at the 3 'terminus and then applied to the tail with Poly A polymerase using rATP as a substrate. The reverse transcription of the RNA with attached poly A tail 30 is catalyzed by reverse transcriptase using oligo dT as a primer.

87 DK 175975 B1 cDNA's no. two strands are synthesized, the cDNA products are cloned, screened and sequenced.

IV.A.23. Preparation of Lambda-at11 HCV cDNA libraries. containing 5 major cDNA insertions.

The method used to prepare and screen the Lambda-gt11 libraries is essentially as described in Section IV.A.1. with the exception that the library is generated from a pool of larger size cDNAs eluted from the Sepharose CL-4B column.

IV.A.24. Preparation of HCV cDNA Libraries Using Synthetic Oleomers as Primers.

15 New HCV cDNA libraries have been prepared from the RNA derived from the infectious chimpanzee plasma pool described in Section IV.A.1. and from the poly A + fraction derived from the liver of this infected animal. The cDNA was constructed essentially as described by Gubier and Hofmann (1983) with the exception that primers for the first cDNA strand synthesis were two synthetic oligomers based on the HCV genome sequence described above. Primers based on the sequence in sections 11 b and 7e were respectively: 5 'CTG GCT TGA AGA ATC 3' and 25 5 'AGT TAG GCT GGT GAT TAT GC 3 *.

The resulting cDNAs were cloned into lambda bacteriophage vectors and screened with various other synthetic oligomers whose sequences were based on the HCV sequence of FIG. 32nd

30 88 DK 175975 B1 IV.B. Expression of polypeptides encoded in HCV cDNAs and identification of the expression products as HCV-induced antigens IV.B.1. Expression of the polypeptide encoded in clone 5-1-1 The HVC polypeptide encoded in clone 5-1-1 (see Section IV.A.2, supra) was Expressed as a Fusion Polypeptide with Super Oxide Dismutase (SOD). This was done by subcloning the clone 5-1-1 cDNA insert into the expression vector pSODcfl (Steimer et al. (1986)) as follows.

10 First, DNA isolated from pSODcfl was treated with BamH1 and EcoRI and the following linker was ligated into the linear DNA created by the restriction enzymes: 15 5 'GAT CCT GGA ATT CTG ATA A 3' 3 'GA CCT TAA GAC TAT TFT AA 5 '

After the cloning, the plasmid containing the insert was isolated.

The plasmid containing the insert was restriction treated with Eco RI. The HCV cDNA insert in clone 5-1-1 was excised with EcoRI and ligated into this EcoRI linearized plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al. (1980)). Recombinants with the 5-1-1 cDNA in the correct orientation for expression of the ORF shown in FIG. 1, were identified by restriction mapping and nucleotide sequencing.

Recombinant bacteria from a clone were induced to express SOD-NANB5-1-1 polypeptide by culturing the bacteria in the presence of IPTG.

30 89 DK 175975 B1 IV.B.1. Expression of the polypeptide encoded in clone 81 The HCV cDNA contained in clone 81 was expressed as a SOD-NANBei fusion polypeptide. The method of preparing the vector encoding this fusion polypeptide was analogous to the method used to generate the vector encoding SOD-NANB5-1.1 except that the source of HCV cDNA clone 81 isolated and described in Section IV.A.3., for which the cDNA sequence was determined as described in Section IV.A.4. The HCV cDNA nucleotide sequence of clone 81 and the putative amino acid sequence of the polypeptide encoded therein are shown in FIG. 4th

The HCV cDNA insert in clone 81 was cut with EcoRI and ligated into the pSODcfl containing the linker (see IV.B.1) and linearized by EcoRI treatment. The DNA mixture was used to transform EL 15 coli strain D1210. Recombinants with the clone 81 HCV cDNA in the correct orientation for expression of the one in FIG. 4 was identified by restriction mapping and nucleotide sequencing.

Recombinant bacteria from a clone were induced to express SOD-20 NANBei polypeptide by culturing the bacteria in the presence of IPTG.

IV.B.3. Identification of the polypeptide encoded in clone 5-1-1 as an HCV-00 NANBH-associated antigen.

The polypeptide encoded in the HCV cDNA of clone 5-1-1 was identified as a NANBH-associated antigen by demonstrating that chimpanzee sera and humans infected with NANBH reacted immunologically with the fusion polypeptide, SOD-NANB5. -M, constituted by superoxide dismutase at the N-terminus and the "in-frame 5-1-1 antigen" at the C-terminus. This was achieved by Westem blotting (Towbin et al. (1979)) as follows.

90 DK 175975 B1

A recombinant bacterial strain transformed with an expression vector encoding the SOD-NANB5.M polypeptide, as described in Section IV.B.I., was induced to express the fusion polypeptide by culture in the presence of IPTG. Total bacterial lysate was electrophoresed through 5 polyacrylamide gels in the presence of SDS according to Laemmli (1970). The separated polypeptides were transferred to nitrocellulose filters (Towbin et al. (1979)). The filters were then cut into thin strips and the strips were incubated individually with the different chimpanzee and human sera. Bound antibodies were detected by further incubation with 125l-labeled 10 sheep anti-human Ig as described in section IV.A.1.

The characterization of the chimpanzee sera used for Western blots and the results shown in the photograph of the autoradiographed strips are shown in FIG. 33. Nitrocellulose strips containing polypeptides were incubated 15 with sera derived from chimpanzees at various times during acute NANBH (Hutchinson strain) infections (pathways 1-16), hepatitis A infections (pathways 17-24 and 26-33) and hepatitis B infections (pathways 34-44). Routes 25 and 45 show positive controls in which the immunoblots were incubated with serum of the patient 20 to identify the recombinant clone 5-1-1 in the original screening of the Lambda-gt11 cDNA library (see section IV.A. 1).

The visible band in the control walking paths, 25 and 46 of FIG. 23, reflects the binding of antibodies to the NANBs-M portion of the SOD fusion polypeptide.

The antibodies do not exhibit binding to SOD alone, as this has also been included as a negative control in the samples and would have emerged as a band that migrated significantly faster than the SOD-NANB5 m fusion polypeptide.

30 The walking paths 1-16 in FIG. 33 shows the binding of antibodies in serum samples from four chimpanzees; sera were obtained immediately prior to infection with 91 NANBH and sequentially during acute infection. As seen from the figure, antibodies that reacted immunologically with the SOD-NANBs-m polypeptide were missing in serum samples obtained prior to infectious HCV inoculum administration and during the early acute infection phase, while all four animals eventually induced circulating antibodies against this polypeptide in the latter part of or subsequently the acute phase. Further observed bands on the immunoblots with chimpanzee numbers 3 and 4 were due to background binding to host bacterial proteins.

Contrary to the results obtained with chimpanzee sera infected with NANBH, no development of antibodies against the NANB5.i.i portion of the fusion polypeptide was observed in four HAV-infected chimpanzees or three HBV-infected chimpanzees. The only binding in these cases was background binding to host bacterial proteins that also occurred in the HCV-15 infected samples.

The characterization of the human sera used for Western blots and the results shown in the photograph of the autoradiographed strips are shown in FIG. 34. Nitrocellulose strips containing polypeptides were incubated with 20 sera derived from humans at various times during infection with NANBH (routes 1-21), HAV (routes 33-40) and HBV (routes 41-49). Routes 25 and 50 show positive controls in which the immunoblots were incubated with serum from a patient used in the original screening of the Iambda-gt11 library described above.

Routes 22-24 and 26-32 show "uninfected" controls in which sera originate from "normal" blood donors.

As seen in FIG. 34 contained sera from nine NANBH patients, including the serum used for the screening of the Iambda-gt11 library, antibodies to the 30 NANB5 portion of the fusion polypeptide. Sera from three patients with NANBH did not contain these antibodies. It is possible that the anti-NANBs-in-antibodies will develop later in these patients. It is also possible that this lack of response was due to the fact that it was another NANBV agent that caused the disease in the individuals from which the non-responding serum was taken.

5

FIG. 34 also show that sera from many HAV and HBV infected patients did not contain anti-NANB5.M antibodies, nor were these antibodies found in sera from "normal" controls. Although one HAV patient (route 36) appears to contain anti-NANBs β antibody, it is possible that this patient has previously been infected with HCV, the NANBH incidence being very high and often subclinical.

These serological studies show that the cDNA in clone 5-1-1 encodes epitopes that are specifically recognized by sera from BB-NANBV-infected patients and animals. In addition, the cDNA does not appear to be derived from the primate genome. A hybridization probe prepared from clone 5-1-1 or from clone 81 did not hybridize with "Southern" blots of human and chimpanzee genomic control DNA from uninfected individuals under conditions where unique, single copy genes can be detected. with 20 Southern blots of bovine genomic DNA.

IV.B.4. Expression of the polypeptide encoded in a comoosite of the HCV cDNAs in the clones 36. 81 oo 32.

The HCV polypeptide encoded in the ORF extending through clones 36, 81 and 32 was expressed as a fusion polypeptide with SOD.

This was done by inserting the composite cDNA, C100, into an expression cassette containing the human superoxide dismutase gene, inserting the expression cassette into a yeast expression vector, and expressing the polypeptide in yeast.

93 DK 175975 B1

An expression cassette containing the composite C100 cDNA derived from clones 36, 81 and 32 was constructed by inserting the -270pb EcoRI fragment into the EcroRI site of the vector pS3-56 (also called pS356) yielding the plasmid pS3 The construction of the C100 is described in section 5 IV.A.16., Above.

The vector pS3-56, which is a pBR322 derivative, contains an expression cassette constituted by the ADH2 / GAPDH hybrid yeast promoter upstream of the human superoxide dismutase gene and a downstream GAPDH 10 transcription terminator. A similar cassette containing these controls and the superoxide dismutase gene has been described in Cousens et al. (1987) and in pending EP Patent Application No. 196,056, filed October 1, 1986, jointly owned by the present applicant. However, the cassette in pS3-56 differs from the cassette in Cousens et al.

15 (1987) know that the heterologous proinsulation and immunoglobulin hinge are deleted and that the gln145 of the superoxide dismutase is followed by an adapter sequence containing an EcoRI site. The adapter sequence is: 5'-ATT TTG GGA ATT CCA TAA TGA G -3 '

20 AC CCT TAA GGT ATT ACT CAG CT

The EcroRI site allows insertion of hetorologic sequences which, when expressed from a vector containing the cassette, yield polypeptides fused with superoxide dismutase via an oligopeptide linker containing the amino acid sequence -asn-leu-gly-ile-arg-.

On April 29, 1988, a sample of pS356 was deposited under the terms of the Budapest Treaty with the Amerian Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Maryland 20853, and assigned 9462 175975 B1 to the deposit number 67683. Terms of accessibility and access to the landfill and for landfill maintenance are the same as those in Section I LA. specified for strains containing NANBV cDNAs. This deposit is for convenience only and 5 is not a prerequisite for carrying out the present invention, given the present disclosure. The deposited materials are incorporated herein by reference.

Following the isolation of recombinants containing the C100 cDNA insert in the correct orientation, the expression cassette containing the C100 cDNA was cut from pS3-56cioo with BamHI and a ~ 3400bp fragment containing the cassette was isolated and purified. This fragment was then inserted into the BamHI site of the yeast vector pAB24.

15 Plasmid pAB24, whose significant features are shown in FIG. 35, is a yeast shuttle vector containing the complete 2 micron sequence for replication [Broach (1981)] and pBR322 sequences. It also contains the yeast URA3 gene derived from plasmid YEp24 [Botstein et al (1979)] and yeast LEU2d, the gene derived from plasmid pC1 / 1 EPO publication number 116.201. Plasmid pAB24 was constructed by digesting YEp24 with EcoRI and religating the vector to remove the partial 2 micron sequences. The resulting plasmid, YEP24deltaRI, was linearized by digestion with Clal and ligated with the complete 2 micron plasmid that had been linearized with Clal. The resulting plasmid, pCBou, was then digested with XbaI and the 8605 bp vector fragment was gel isolated. The isolated XbaI fragment was ligated with a 4460pb XbaI fragment containing the LEU2d gene isolated from pCI / 1; the orientation of the LEU2d gene has the same direction as the URA3 gene. Insertion of the expression occurred in the unique BamHI site of the pBR322 sequence, thus interrupting the bacterial resistance gene against tetracycline.

30 95 DK 175975 B1

The recombinant plasmid containing the SOD-C10O expression cassette, pAB24C 100-3, was transformed into yeast strain JSC 308 as well as into other yeast strains. The cells were transformed as described by Hinnen et al.

(1978) and plated on ura-selective plates. Single colonies were inoculated on 5 leu-selective media and cultured for saturation. The culture was induced to express the SOD-C100 polypeptide (called C100-3) by growing in YEP containing 1% glucose.

Strain JSC 308 has the genotype MAT @, Ie2, ura3 (part), DM1510 (GAP / ADR1) integrated at the ADR1 locus. In JSC 308, overexpression of the positive activator gene product, ADR1, results in hyper-depression (relative to an ADR1 wild-type control) and significantly higher yields of expressed heterologous proteins when such proteins are synthesized via an ADH2 UAS regulatory system. The construction of the yeast strain JSC 308 is described in pending patent application US No. (Prosecutor's Ref. No. 2300-0229), filed at the same time and which is incorporated herein by reference. A sample of JSC 308 was deposited on May 5, 1988, with the ATCC under the terms of the Budapest Treaty and has been assigned Accession number 20879. The conditions for accessibility and access to the landfill and for landfill maintenance are the same as specified in Section II.A. for strains containing HCV cDNAs.

The complete C100-3 fusion polypeptide encoded in pAB24C100-3 should contain 154 amino acids of human SOD at the amino terminal, 5 25 amino acid residues derived from the synthetic adapter containing the EcoRI site, 363 amino acid residues derived from the C100 cDNA and 5 carboxy terminal amino acids derived from the MS2 nucleotide sequence which. adjacent to the HCV cDNA sequence in clone 32. (See Section IV.A.7.). The putative amino acid sequence of the carboxy terminal of this polypeptide beginning at the penultimate Ala residue of SOD is shown in FIG. 36; the nucleotide sequence encoding this portion of the polypeptide is also shown.

96 DK 175975 B1 IV.B.5. Identification of the polypeptide encoded in C100 as a NANBH-associated antigen C100-3 The fusion polypeptide expressed from plasmid pAB24C100-3 in the yeast strain JSC 308 was characterized in size and the polypeptide encoded in C100 was identified as a NANBH gene. associated antigen by its immunological reactivity with serum of a human with chronic NANBH.

C100-3 The polypeptide expressed as described in Section IV.B.4. became

analyzed as follows. JSC 308 Yeast cells were transformed with pAB24 or with pAB24C100-3 and induced to express the heterologous plasmid-encoded polypeptide. The induced yeast cells in 1 ml of culture (OD650 nm ~ 20) were pelleted by centrifugation at 10,000 rpm for one minute and lysed 15 by vigorously treating them (10 x 1 min) with two volumes of solution and one volume of glass beads (0, 2 millimicrons in diameter). The solution contained 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin. Insoluble material in the lysate comprising the C100-3 peptide was collected by centrifugation 20 (10,000 rpm for 5 min) and dissolved by boiling for 5 min. in Laemmli SDS

sample buffer. [See Laemmli (1970)]. A polypeptide amount equivalent to the amount in 0.3 ml of the induced yeast culture was electrophoresed through 10% polyacrylamide gels in the presence of SDS according to Laemmli (1979). Protein standards were co-electrophoresed on the gels. Gels containing the expressed polypeptides were either stained with Coomassie brilliant blue or subjected to "Western" blot as described in Section IV.B.2. using serum from a patient with chronic NANBH to determine the immunological reactivity of the polypeptides expressed from pAB24 and pAB24C100-3.

30 _: _: ___ __ 97 DK 175975 B1

The results are shown in FIG. 37. In FIG. 37A, the polypeptides were stained with Coomassie brilliant blue. The insoluble polypeptide (polypeptides) of JSC 308 transformed with pAB24 and from two different colonies of JSC transformed with pAB24C100-3 are shown in pathway 1 (pAB24) and pathways 2 and 3, respectively. Pathways 2 and 3 with Pathway 1 show that the induced expression of a polypeptide corresponds to a molecular weight of -54,000 daltons from JSC 308 transformed with pAB24C100-3 not induced in JSC308 transformed with pAB24.

This polypeptide is shown by the arrow.

10

FIG. 37B shows the results of Western blots of the insoluble polypeptides expressed in JSC 308 transformed with pAB24 (pathway 1) or with pAB24C100-3 (pathway 2). The polypeptides expressed by pAB24 were not immunologically reactive with human serum of NANBH. However, as shown by the arrow, JSC 308 transformed with pAB324C100-3 expressed a polypeptide of -354,000 dalton molecular weight that reacted immunologically with the human NANBH serum. The other immunologically reactive polypeptides in pathway 2 may be degradation and / or aggregation products thereof. -354,000 dalton polypeptide.

IV.B.6. Purification of fusion polypeptide C100-3

The fusion peptide, C100-3, consisting of SOD at the N-terminus and "in-frame" C100 HCV polypeptide at the C-terminus was purified by differential extraction of the insoluble portion of the extracted host yeast cells in which the polypeptide was expressed.

The fusion polypeptide, C100-3, was expressed in yeast strain JSC 308 transformed with pAB24C100-3 as described in Section IV.B.4. The yeast cells were then lysed by homogenization, the insoluble material in the lysate was extracted at pH 12.0, and C100-3 in the remaining insoluble fraction was solubilized in buffer containing SDS.

The yeast lysate was prepared essentially according to Nagahuma et al. (1984).

A yeast cell suspension was made up consisting of 33% cells (v / v) suspended in a solution (buffer A) containing 20 mM Tris HCl, pH 8.0, 1 mM dithiotreitol and 1 mM phenylmethylsulfonyl fluoride (PMSF). A suspension sample (15 ml) was mixed with a corresponding volume of glass beads (0.45 - 0.50 mm / diameter) and the mixture 10 was vortexed at the highest speed of a supermixer (Lab Line Instruments, Inc.) for 8 min. The homogenate and glass beads were separated and the glass beads washed three times with the same volume of buffer A as the initially packed cells. After combining the washes and homogenate, the insoluble material in the lysate was obtained by centrifuging the homogenate at 7,000 x g for 15 min. at 4 ° C, resuspending pellets in Buffer A equal to 2 x the volume of initially packed cells, and repellating the material by centrifugation at 7,000 x g for 15 min. This washing procedure was repeated three times.

The insoluble material from the lysate was extracted at pH 12.0 as follows. The pellet was suspended in buffer containing 0.5 M NaCl, 1 mM EDTA, with the suspending volume equal to 1.8 times the volume of the initially packed cells. The pH of the suspension was adjusted by adding 0.2 volumes of 0.4 M Na phosphate buffer, pH 12.0. After mixture 25, the suspension was centrifuged at 7,000 x g for 15 min. at 4 ° C and the supernatant was removed. The extraction was repeated twice. The extracted pellets were washed by suspending them in 0.5 M NaCl, 1 mM EDTA using a suspension volume equal to two volumes of the initially packed cells, followed by centrifugation at 7,000 30 x g for 15 min. at 4 ° C.

The polypeptide in the extracted pellet was dissolved by treatment with SDS. Pellets were suspended in Buffer A corresponding to 0.9 volume of the initially packed cell volume and 0.1 volume of 2% SDS was added. After the suspension was mixed, it was centrifuged at 7,000 x g for 15 min. at 4 ° C. The resulting pellet was extracted an additional three times with SDS. The resulting supernatants containing C100-3 were combined.

This procedure purifies C100-3 more than 10 times from the insoluble fraction of the yeast homogenate, and the recovery of the polypeptide is greater than 10 50%.

The purified fusion polypeptide was analyzed by polyacrylamide gel electrophoresis according to Laemmli (1970). On the basis of this analysis, the polypeptide was more than 80% pure and had an apparent molecular weight of 15 ~ 54,000 daltons.

IV.C. Identification of RNA in infected individuals hybridizing with HCV cDNA

IV.C.1. Identification of RNA in the liver from a chimpanzee with NANBH. there

hybridizes with HCV cDNA

RNA From the liver of a chimpanzee that had NANBH was shown to contain a species of RNA that hybridized with the HCV cDNA contained in clone 81 by Northern blotting as follows.

RNA was isolated from a liver biopsy of the chimpanzee from which the high titer plasma was derived (section IV.A.1.), Using techniques described in Maniatis et al. (1982) for isolating total RNA from mammalian cells 30 and for their separation into poly A + and poly A 'fractions. The RNA fractions were electrophoresed on a formaldehyde / agarose gel (1% in 100 w / v) and transferred to nitrocellulose. [Maniatis et al. (1982)]. The nitrocellulose filters were hybridized with radiolabeled HCV cDNA from clone 81 (see Fig. 4 for the insertion nucleotide sequence). To prepare the radiolabeled probe, the HCV cDNA insert isolated from clone 81 was radiolabelled with 32p by nick translation using DNA polymerase I (Maniatis et al. (1982)). The hybridization took place over 18 hours at 42 ° C in a solution containing 10% (w / v) dextran sulfate, 50% (w / v) deionized formamide, 750 mM NaCl, 75 mM Na citrate, 20 mM Na 2 HPO 4, pH 6 , 5, 0.1% SDS, 0.02% (w / v) bovine serum albumin (BSA), 0.02% (w / v) ficoll-400, 0.02% (w / v) polyvinylpyrrolidone , 100 micrograms / ml salmon sperm DNA treated by sonication and denatured, and 106 CPM / ml nick-translated cDNA probe.

In FIG. 38 shows an autoradiography of the probed filter. Walkway 1 15 contains 32P-labeled restriction fragment markers. Hiking paths 2-4 j contain chimpanzee liver RNA as follows: Hiking pathway 2 contains 30 micrograms of total RNA, migration pathway 3 contains 30 micrograms of poly A 'RNA! and route 4 contains 20 micrograms of poly A * RNA. As shown in FIG. 38, the liver of the chimpanzee with NANBH contains a heterogeneous population of 20 related poly A + molecules that hybridize with the HCV cDNA probe and which appear to have a size of from ca. 5,000 nucleotides to approx.

11,000 nucleotides. This RNA hybridizing to HCV cDNA could represent viral genomes and / or specific transcripts of the viral genome.

25

The section IV.C.2 below. experiment described agrees with the assumption that HCV contains an RNA genome.

30 101 DK 175975 B1 IV.C.2. Identification of HCV-derived RNA in serum of infected individuals

Nucleic acid was extracted from particles isolated from high titer chimpanzee NANBH plasma as described in Section IV.A.1. Aliquots 5 (equivalent to 1 ml of original plasma) of the isolated nucleic acids were resuspended in 20 microliters of 50 mM Hepes, pH 7.5, 1 mM EDTA and 16 micrograms / ml soluble yeast RNA. The samples were denatured by boiling for 5 min. immediately followed by freezing and treated with RNase A (5 microliters containing 0.1 mg / ml RNase A in 25 mM EDTA, 40 mM Hepes, 10 pH 7.5) or with DNase I (5 microliters containing 1 unit of DNase li 10 mM MgCl, 25 mM Hepes, pH 7.5), control samples were incubated without enzyme.

After incubation, 230 microliters of ice cold 2XSSC containing 2 micrograms / milliliter of soluble yeast RNA was added and the samples were filtered on a nitrocellulose filter. The filters were hybridized with a cDNA probe from clone 81 which had been 32P labeled by nick translation. FIG. 39 shows an autoradiography of the • filter. Hybridization signals were detected in the DNase-treated samples and the control samples (migration paths 2 and 1, respectively), but were not detected in the RNase-treated sample (migration pathway 3). Thus, since RNase A treatment destroyed the nucleic acids isolated from the particles and DNase I treatment 20 had no effect, the material strongly suggests that the HCV genome consists of RNA.

IV.C.3. Detection of enhanced HCV nucleic acid sequences derived from HCV nucleic acid sequences in liver and plasma samples from chimpanzees with NANBH 25 HCV nucleic acid found in the liver and plasma of chimpanzees with NANBH and in control chimpanzees was substantially enhanced by using polymerase chain reaction , described by Saiki et al., (1986). The primer nucleotides were derived from the HCV cDNA sequence of clone 81 30 or clones 36 and 37. The amplified sequences were detected by gel electrophoresis and Southern blot using the appropriate 102 cDNA oligomer with a sequence from the region between , but not comprehensive, the two primers.

RNA samples containing HCV sequences to be examined by the amplification system were isolated from liver biopsies from three chimpanzees with NANBH and from two control chimpanzees. The RNA fraction was isolated by the guanidinium thiocyanate procedure of section IV.C.1.

RNA samples to be examined by the amplification system were also isolated from plasmas from two chimpanzees with NANBH and from a control chimpanzee, as well as from a plasma pool from control chimpanzees. One infected chimpanzee had a CID / ml equal to or greater than 106, and the other infected chimpanzee had a CID / ml equal to or greater than 105

The nucleic acids were extracted from the plasma as follows. Either 0.1 ml or 0.01 ml of plasma was diluted to a final volume of 1.0 ml with a TENB / proteinase K / SDS solution (0.05 M Tris-HCl, pH 8.0, 0.001 M EDTA, 1 M NaCl, 1 mg / ml proteinase K and 0.5% SDS) containing 10 20 micrograms / ml polyadenyl acid, and incubated at 37 ° C for 60 min. Following this proteinase K digestion, the resulting plasma fractions were deproteinized by extraction with TE (10.0 mM Tris-HCl, pH 8.0, 1 mM EDTA) -saturated phenol. The phenol phase was separated by centrifugation and re-extracted with TENB containing 0.1% SDS. The resulting 25 water phases from each extraction were combined and extracted twice with an equal volume of phenol / chloroform / isoamyl alcohol [(1: 1 (99: 2) 1) and then twice with an equal volume of a 99: 1 After phase separation by centrifugation, the aqueous phase was brought to a final concentration of 0.2 M Na-acetate and the nucleic acids 30 precipitated by the addition of two volumes of ethanol The precipitated nucleic acids were removed by an ultracentrifugation. 41 rotor at 38 K, for 60 minutes at 4 ° C.

In addition to the above, the high titer chimpanzee and the unified control plasma were alternatively extracted with 50 micrograms of poly A carrier by the procedure of Chomcyzski and Sacchi (1987). In this procedure, an acid guanidinium thiocyanate extraction is used. RNA was recovered by centrifugation at 10,000 RPM for 10 min. at 4 ° C in an Eppendorf microcentrifuge.

10

Prior to cDNA synthesis in the PCR reaction, the nucleic acids extracted from the plasma by the proteinase K / SDS / phenol method were further purified by binding to and elution from S and S Elutip-R columns. The process was carried out according to the manufacturer's instructions.

15

The template cDNA used for the PCR reaction was derived from the nucleic acids prepared according to the above (either total nucleic acid or RNA).

After ethanol precipitation, the precipitated nucleic acids were dried and resuspended in DEPC-treated distilled water. Secondary structures in the 20 nucleic acids were broken by heating to 65 ° C for 10 min. and the samples were immediately cooled on ice. cDNA was Synthesized using 1 to 3 micrograms total chimpanzee RNA from liver or from nucleic acid (or RNA) extracted from 10 to 100 ml of plasma. Reverse transcriptase was used in the synthesis and the synthesis was carried out in a 25 microlitre reaction using the protocol specified by the manufacturer, BRL. The primers for cDNA synthesis were those also used in the PCR reaction as described below. All cDNA synthesis reaction mixtures contained 23 units of RNase inhibitor, RNASIN® (Fisher / Promega). After the c-DNA synthesis, the reaction mixtures were diluted with water, boiled for 10 min. and quickly cooled 30 on ice.

The PCR reactions were performed essentially according to the manufacturer's instructions (Cetus-Perkin-Elmer) with the exception of the addition of 1 microgram RNase A. The reactions were carried out in a final volume of 100 ml. PCR was performed for 35 cycles using a regime of 37 ° C, 72 ° C and 94 ° C.

5

The primers for cDNA synthesis and for the PCR reactions were derived from the HCV cDNA sequences in either clone 81, clone 86 or clone 37b. (The HCV cDNA sequences in clones 81, 36 and 37b are shown in Figures 3, 5 and 10, respectively). The two 16-mer primers sequences, derived from clone 81, were: 10 5 'C AA TCA TAC CTG ACA G 3' and 5 'GAT ACC CTC TGC CTG A3'

The primer sequence from clone 36 was: 5 'GCA TGT CAT GAT GTA T 3'.

The primer sequence from clone 37b was 20 5 'ACA ATA CGT GTG TCA C 3'.

In the PCR reactions, the primer pairs consisted of either the two 16-mer derived from clone 81 or the 16-mer from clone 36 and the 16-mer from clone 37b.

The PCR reaction products were analyzed by separating the products by alkaline gel electrophoresis followed by Southern blot and detected by the amplified HCV cDNA sequences with a 32P-labeled internal oligonucleotide probe derived from a region of the HCV cDNA that does not overlap the primers.

The 30 PCR reaction mixtures were extracted with phenol / chloroform and the nucleic acids precipitated from the aqueous phase with salt and ethanol. The precipitated nucleic acids were collected by centrifugation and dissolved in distilled water. Portions of the samples were subjected to electrophoresis on 1.8% alkaline agarose gels. Single stranded DNAs of 60, 108 and 161 nucleotide lengths were co-electrophoresed on the gels as molecular weight markers. Following electrophoresis, the DNAs in the gel were transferred to Biorad Zeta Probe® paper. Prehybridization and hybridization and washing conditions were as specified by the manufacturer (Biorad).

The probes used for the hybridization detection of amplified HCV cDNA sequences 10 were as follows. When a PCR primer pair was derived from clone 81, the probe was a 108-mer having a sequence corresponding to that located in the region between the two primers' sequences. When the PCR primer pair was derived from clones 36 and 37b, the probe was the nick-translated HCV cDNA insert derived from clone 35. The primers are derived from nucleotides 155-170 of the clone 37b insert and 206-268 of clone 36 insert. The 3-end of the HCV cDNA insert in clone 35 overlaps the nucleotides 1-186 of the insert in clone 36; and the 5 'end of the clone 35 insert overlaps the nucleotides 207-269 of the insert in clone 37b. (Compare Figures 5, 8 and 10). Thus, the cDNA insert in clone 35 spans a portion of the region between the sequences of the primers derived from clones 36 and 37b and is well suited as a probe for the amplified sequences comprising these primers.

The RNA analysis from the liver samples was performed according to the above procedure 25 using both sets of primers and probes. The RNA From the liver of the three chimpanzees with NANBH yielded positive hybridization results for amplification sequences of the expected size (161 and 586 nucleotides for 81 and 36 and 37b, respectively), while the control chimpanzees gave negative hybridization results. The same results were obtained when the experiment was repeated three times.

106 DK 175975 B1

Analysis of the nucleic acids and RNA from plasma was also performed according to the above procedure using the primers and probe from clone 81. The plasmas derived from two chimpanzees with NANBH, from a control chimpanzee and from united plasma from control chimpanzees. Both NANBH-5 plasmas contained nucleic acid / RNA which gave positive results in the PCR enhanced analysis, while both control piases yielded negative results. These results have repeatedly been achieved several times.

IV.D. Radioimmunoassay to detect serum HCV antibodies from 10 infected individuals

Solid-phase radioimmunoassays to detect antibodies to HCV antigens were developed based on Tsu and Herzenberg (1980). Microtiter plates (Immulon 2, Removawell strips) are coated with purified polypeptides containing 15 HCV epitopes. The coated plates are incubated with either human serum samples suspected of containing antibodies to the HCV epitopes, or with appropriate controls. During incubation, any antibody present immunologically binds to the solid phase antigen. After removal of the unbound material and washing of the microtiter plates, 20 complexes of human antibody NANBV antigen are detected by incubation with 125 I-labeled sheep anti-human immunoglobulin. Unbound, labeled antibody is removed by aspiration and the plates washed. The radioactivity in the individual wells is determined; the amount of bound human anti-HCV antibody is proportional to the radioactivity in the well.

IV.D.1. Purification of fusion powder peptide SOD-NANB * .i.i

The fusion polypeptide SOD-NANB5-1-1, expressed in recombinant bacteria as described in Section IV.B.1., Was purified from the recombinant E. coli by differential extraction of the urea cell extracts followed by chromatography on anion and cation exchange columns as follows.

107 DK 175975 B1}

Thawed cells from 1 liter of culture were resuspended in 10 ml of 20% (w / v) sucrose containing 0.01 M Tris HCl, pH 8.0 and 0.4 ml of 0.5 M ETDA, pH 8.0 added. After 5 minutes at 0 ° C, the mixture was centrifuged at 4,000 x g for 10 minutes. The resulting pellet was suspended in 10 ml of 25% (w / v) sucrose containing 0.05 M Tris HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin A, followed by the addition of 0.5 ml of lysozyme (10 mg / ml) and incubation at 0 ° C for 10 min. After adding 10 ml of 1% (v / v) Triton X-100 in 0.05 M Tris HCl, pH 8.0, 1 mM EDTA, the mixture was incubated for 10 min. at 0 ° C 10 with occasional shaking. The resulting viscous solution was homogenized by passage 6 times through a sterile 20 needle cannula and centrifuged at 13,000 x g for 25 min. The pelleted material was suspended in 5 ml of 0.01 M Tris HCl, pH 8.0 and the suspension was centrifuged at 4,000 x g for 10 min. The pellet containing SOD-NANB5-1-1-15 fusion protein was dissolved in 5 ml of 6 M urea in 0.02 M Tris HCl, pH 8.0, 1 mM dithiotreitol (buffer A) and loaded onto a Q-Sepharose Fast Flow column equilibrated with buffer A. The polypeptides were eluted with a linear gradient of 0.0 to 0.3 M NaCl in buffer A. After elution, the fractions were analyzed by polyacrylamide gel electrophoresis in the presence of SDS to determine their content of SOD-NANB5.1. -1. Fractions containing this polypeptide were pooled and dialyzed against 6 M urea in 0.02 M sodium phosphate buffer, pH 6.0, 1 mM dithiotreitol (buffer B). The dialyzed sample was loaded onto an S-Sepharose Fast Flow column equilibrated with buffer B and the polypeptides eluted with a linear gradient of 0.0 to 0.3 M NaCl in buffer B. The fraction was analyzed by polyacrylamide gel electrophoresis in the presence of SOD-NANB5. .-m, and the proper fractions united.

The final preparation of SOD-NANB5.M polypeptide was investigated by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 80% pure.

108 DK 175975 B1 IV.D.2. Purification of fusion polypeptide SOD-NANBsi

The fusion polypeptide SOD-NANB8i expressed in recombinant bacteria as described in Section IV.B.2 was purified from recombinant E. coli by differential extraction of the cell extracts with urea followed by chromatography on anion and cation exchange columns using that for isolation of fusion polypeptide SOD .M procedure used (see section IV.D.1.).

The final preparation of SOD-NANB50-1 polypeptide was investigated by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 50% pure.

IV.D.3. Detection of antibodies against HCV epitopes by solid-phase radioimmunoassay

Serum samples from 32 patients diagnosed with NANBH were analyzed by radioimmunoassay (RIA) to determine whether antibodies to HCV epitopes present in fusion polypeptides SOD-NANB5-1.1 and SOD-20 NANB8i were detectable.

Microtiter plates were coated with SOD-NANB5.M or SOD-NANB8i partially purified according to Section IV.D.1. and IV.D.2. The analyzes were performed as follows.

25

One hundred microliters of portions containing 0.1 to 1.5 micrograms of SOD-NANB5-M or SOD-NANB8i in 0.125 M Na borate buffer, pH 8.3, 0.075 NaCl (BBS) was added to each well of a microtiter plate (Dynatech Immulon 2 Removawell strips). The plate was incubated at 4 ° C overnight in a humid 30 chamber, after which the protein solution was removed and the wells were washed three times with BBS containing 0.02% Triton X-100 (BBST). To prevent non-specific binding, the wells were coated with bovine serum albumin (BSA) by addition of 100 microliters of a 5 mg / ml solution of BSA in BBS followed by incubation at room temperature for one hour; after this incubation, the BSA solution was ironed. The polypeptides in the coated wells were reacted with 5 serum by adding 100 microliters of serum samples diluted 1: 100 in 0.01 M Na-phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) containing 10 mg / ml BSA and by incubation in serum-containing wells for 1 hour at 37 ° C. After incubation, the serum samples were removed by aspiration and the wells were washed 5 times with BBST. Anti-NANB 1 M and anti-NANB $ in bound to the fusion polypeptides 10 were determined by the binding of 125 I-labeled F (ab) 2 sheep anti-human IgG to the coated wells. 100 microliters aliquots of the labeled probe (specific activity 5-20 microcurie / micrograms) were added to each well and the plates were incubated at 37 ° C for one hour followed by removal of excess probe by aspirating and washing 5 times with BBST. The bound amount of activity in each 15 well was determined by counting in a counter that detects gamma radiation.

The results of the detection of anti-NANBs.!. and anti-NANB8i in individuals with NANBH are listed in Table 1.

Table 1 110 DK 175975 B1 Detection of anti-5-1-1 and anti-81 in sera from NANB, HAV and HBV hepatitis patients 5

Patient S / N

reference number. Diagnosis Anti-5-1-1 Anti-81 1,281 Chronic NANB, IVD2 0.77 4.20 10 Chronic NANB, IVD 1.14 5.14

Chronic NANB, IVD 2.11 4.05 2.291 AVH3, NANB, sporadic 1.09 1.05

Chronic, NANB 33.89 11.39 Chronic, NANB 36.22 13.67 3. 301 AVH, NANB, IVD 1.90 1.54

Chronic NANB, IVD 34.17 30.28

Chronic NANB, IVD 32.45 30.84 20 4.31 Chronic NANB, PT4 16.09 8.05 5.321 Late AVH NANB, IVD 0.69 0.94

Late AVH NANB, IVD 0.73 0.68 6.331 AVH, NANB, IVD 1.66 1.96 AVH, NANB, IVD 1.53 0.56

Table 1 (continued) 111 DK 175975 B1 Detection of anti-5-1-1 and anti-81 in sera from NANB, HAV and HBV hepatitis patients 5

Patient S / N

reference r. Diagnosis Anti-5-1-1 Anti-81 7,341 Chronic NANB, PT 34.40 7.55 10 Chronic NANB, PT 45.55 13.11 • Chronic NANB, PT 41.58 13.45

Chronic NANB, PT 44.20 15.48 8. 351 AVHNANB, IVD 31.92 31.95 15 Recently "cured" NANB, 6.87 4.45

AVH

9.36 Late AVH NANB PT 11.84 5.79 20 10.37 AVHNANB, IVD 6.52 1.33 11.38 Late AVH NANB, PT 39.44 39.18 12.39 Chronic NANB, PT 42.22 37.54 25 13.40 AVH, NANB , PT 1.35 1.17 14.41 Chronic NANB? PT 0.35 0.28 30 15.42 AVH, NANB, IVD 6.25 2.34

Table 1 (continued) 112 DK 175975 B1 Detection of anti-5-1-1 and anti-81 in sera from NANB, HAV and HBV hepatitis patients 5

Patient S / N

reference number. Diagnosis Anti-5-1-1 Anti-81 16.43 Chronic NANB, PT 0.74 0.61 10 17.44 AVH, NANB, PT 5.40 1.83 18.45 Chronic, NANB, PT 0.52 0.32 15 19.46 AVH , NANB 23.35 4.45 20.47 AVH, type A 1.60 1.35 21.48 AVH, type A 1.30 0.66 20 22.49 AVH, type A 1.44 0.74 23.50 Recently Swallowed AVH, 0.48 0.56

type A

24.51 AVH, type A 0.68 0.64

Swallowed AVH, type A 0.80 0.65 25.52 Recently swollen AVH, type A 1.38 1.04

Recently flown AVH, type A 0.80 0.65 30

Table 1 (continued) 113 DK 175975 B1 Detection of anti-5-1-1 and anti-81 in sera from NANB, HAV and HBV hepatitis patients 5

Patient S / N

reference number. Diagnosis Anti-5-1-1 Anti-81 26.53 AVH, type A 1.85 1.16 10 Recently swallowed AVH, type A 1.02 0.88 27. 54 AVH, type A 1.35 0.74 28. 55 Late AVH, HBV 0.58 0.55 15 29.56 Chronic HBV 0.84 1.06 30.57 Late AVH, HBV 3.20 1.60 20 31.58 Chronic HBV 0.47 0.46 32.591 AVH, HBV 0.73 0 , 60

Healed AVH, HBV 0.43 0.44 33.601 AVH, HBV 1.06 0.92

Healed AVH, HBV 0.75 0.68 34. 611 AVH, HBV 1.66 0.61

Healed AVH, HBV 0.63 0.36 30

Table 1 (continued) 114 DK 175975 B1 Detection of anti-5-1-1 and anti-81 in sera from NANB-HAV and HBV hepatitis patients 5

Patient S / N

reference number. Diagnosis Anti-5-1-1 Anti-81 35,621. AVH, HBV 1.02 0.73 Healed AVH, HBV 0.41 0.42 36.631 AVH, HBV 1.24 1.21

Healed AVH, HBV 1.55 0.45 37.641 AVH, HBV 0.82 0.79

Healed AVH, HBV 0.53 0.37 38.651 AVH, HBV 0.95 0.92

Healed AVH, HBV 0.70 0.50 39.661 AVH, HBV 1.03 0.68

Cured AVH, HBV 1.71 1.39 1 Sequential serum samples available from patients.

25 2 IVD = Intravenous drug intake 3 AVH = Acute viral hepatitis 30 A PT = Post-transfusion 115 DK 175975 B1

As seen in Table 1, 19 of 32 sera from patients diagnosed with NANBH were positive for antibodies directed against HCV epitopes present in SOD-NANB5.-m and SOD-NANBei.

However, the positive serum samples were not equally immunologically reactive with SOD-NANB5-1.1 and SOD-NANBei. Serum samples from patient # 1 were positive for SOD-NANBei, but not with SOD-NANB5.1.1 · Serum samples from patients # 10, 15 and 17 were positive for SOD-NANB5.10 but not with SOD-NANB8i . Serum samples from patients Nos. 3, 8, 11 and 12 10 reacted similarly with both fusion polypeptides, while serum samples from patients Nos. 2, 4, 7 and 9 had 2-3 times greater response to SOD-NANB5-1-1 than to SOD. -NANB8i. These results suggest that NANB ^.! and NANBei may contain at least three different epitopes; that is, it is possible that each polypeptide contains at least one unique epitope and that the two polypeptides share at least one epitope.

IV.D.4. Solid-phase RIA specificity for NANBH

The solid phase RIA specificity against NANBH was tested by applying the assay to serum from HAV or HBV infected patients and to sera from control subjects. Partially Purified SOD-NANB5.1.1 and SOD-NANBei were used in the analyzes, which were performed essentially as described in Section IV.D.3. with the exception that sera originate from patients who had previously been diagnosed with HAV or HBV, or from individuals who were 25 blood donors. The results for sera from HAV and HBV infected patients are presented in Table 1. RIA was tested using 11 serum samples from HAV infected patients and 20 serum samples from HBV infected patients. As shown in Table 1, none of these sera conferred any positive immunological response to fusion polypeptides containing BB-NANBV-30 epitopes.

116 DK 175975 B1

The RIA in which the NANBs-1 antigen is used is used to determine immunological serum reactivity of control subjects. Out of 230 serum samples obtained from the normal blood donor population, only 2 showed positive reactions in RIA (results not shown). It is possible that the two blood donors from which these 5 serum samples originated had previously been exposed to HCV.

IV.D.5. NANBs-in-i reactivity during NANBH infection

Prevalence of anti-NANB5.M antibodies during a NANBH infection in two 10 patients and four chimpanzees was followed using RIA as described in Section IV.D.3. RIA was also used to determine the presence or absence of anti-N AN B5.1.1 antibodies during a HAV and HBV infection in infected chimpanzees.

The results reported in Table 2 show that in chimpanzees and humans, anti-NANB5.i. Antibodies were detected after the onset of the acute phase of NANBH infection. No anti-NANBs-in-i antibodies were detected in chimpanzee serum samples infected with either HAV or HBV. Thus, anti-NANB5.i-in antibodies serve as a marker for a 20 subject exposure to HCV.

Table 2 117 DK 175975 B1

Seroconversion in sequential serum samples from hepatitis patients and chimpanzees using 5-1-1 antigen 5

Patient / Trial date (days) Hepatitis- Anti-5-1-1 ALT

chimpanzees (0 = inoculation daq1) viruses (S / N) (mu / mO

Patient 29 T * NANB 1.09 1180 10 T + 180 33.89 425 T + 208 36.22

Patient 30 T NANB 1.90 1830 T + 307 34.17 290 15 T + 799 32.45 276

Chimpanzee 1 0 NANB 0.87 9 76 0.93 71 118 23.67 19 20 154 32.41

Chimpanzee 2 0 NANB 1.00 5 21 1.08 52 73 4.64 13 25 138 25.01

Chimpanzees 3 0 NANB 1.08 8 1 43 1.44 205 53 1.82. 14 30 159 11.87 6 j j i! i i i

Table 2 (continued) 118 DK 175975 B1

Seroconversion in sequential serum samples from hepatitis patients and chimpanzees using 5-1-1 antigen 5

Patient / Trial date (days) Hepatitis- Anti-5-1-1 ALT

chimpanzees (Q = inoculation dacO viruses (S / ΝΠ (mu / ml))

Chimpanzees 4 -3 NANB 1.12 11 10 55 1.25 132 83 6.60 140 17.51

Chimpanzee 5 0 HAV 1.50 4 15 25 2.39 147 40 1.92 18 268 1.53 5

Chimpanzee 6 -8 HAV 0.85 20 15 - 106 41 0.81 10 129 1.33

Chimpanzee 7 0 HAV 1.17 7 25 22 1.60 83 115 1.55 5 139 1.60

Table 2 (continued) 119 DK 175975 B1

Seroconversion in sequential serum samples from hepatitis patients and chimpanzees using 5-1-1 antigen 5

Patient / Trial date (days) Hepatitis- Anti-5-1-1 ALT

chimpanzees (0 = inoculation daQ) viruses (S / N) (mu / ml)

Chimpanzee 8 0 HAV 0.77 15 10 26 1.98 130 74 1.77 8 205 1.27 5

Chimpanzee 9 -290 HBV 1.74 15 379 3.29 9 435 2.77 6

Chimpanzee 10 0 HBV 2.35 8 111-118 2.74 96-156 20 (pool) (pool) 205 2.05 9 240 1.78 13

Chimpanzee 0 HBV 1.82 11 25 28-56 1.26 8-100 (pool) (pool)! 169 9 223 0.52 10 30 * T = First trial day in 120 DK 175975 B1 IV.E. Purification of Polyclonal Serum Antibodies to NANBs-vi Based on the specific immunological reactivity of the SOD-NANe ^ -M polypeptide with the antibodies in serum samples from patients with NANBH, a method was developed to purify serum antibodies that react immunologically with the epitope (epitopes) in NANB5.M. In this method, affinity chromatography is used. Purified SOD-NANB5.M polypeptide (see Section IV.D.1.) Was attached to an insoluble substrate; the fortress is such that the immobilized polypeptide retains its affinity for antibody to NANB5.1-1. Antibody in serum samples is absorbed into the matrix-bound polypeptide.

After washing to remove non-specific bound materials and unbound materials, the bound antibody is released from the bound SOD-HCV polypeptide by pH change and / or by chaotropic agents, e.g. urea.

15

Nitrocellulose membranes containing bound SOD-NANB ^ .i were prepared as follows. A nitrocellulose membrane, 2.1 cm Sartorius of 0.2 µm (micron) pore size, was washed for three minutes three times with BBS. SOD-NANB5.V1 Was bound to the membrane by incubating the purified preparation in BBS at room temperature for two hours; alternatively, it was incubated at 4 ° C overnight. The solution containing unbound antigen was removed and the filter was washed three times with BBS for three minutes per minute. wash. The remaining active sites on the membrane were blocked with BSA by incubation with a 5 mg / ml BSA solution for 30 min. Excess BSA was removed by washing the membrane five times with BBS and three times with distilled water. The membrane containing the viral antigen and BSA was then treated with 0.5 M glycine hydrochloride, pH 2.5, 0.10 M NaCl (GlyHCl) for 15 minutes, followed by washing three times every three minutes with PBS.

Thirty polyclonal anti-NANBs-vi antibodies were isolated by incubating the membranes containing the fusion polypeptide with serum from a subject for two hours. After the incubation, the filter was washed 5 times with BBS and twice with distilled water. Bound antibodies were then eluted from each filter with 5 GlyHCI elutions at three minutes per minute. elution.

The pH of the eluates was adjusted to pH 8.0 by collecting each eluate in a test tube containing 2.0 M Tris HCl, pH 8.0. The recovery of the anti-NANBβ1 antibody after affinity chromatography is approx. 50%.

The nitrocellulose membranes containing the bound viral antigen can be used several times without any significant decrease in binding capacity. For reuse of the membranes after the antibodies have been eluted, the membranes are washed with BBS three times for three minutes. They are then stored in BBS at 4 ° C.

IV.F. Capture of HCV particles from infected plasma using 15 purified. human, polyclonal anti-HCV antibodies; hybridization of

the nucleic acid in the captured particles with HCV cDNA

IV.F.1. Capture of HCV particles from infected plasma using polyclonal human anti-HCV antibodies 20

Protein-nucleic acid complexes present in infectious plasma from a chimpanzee with NANBH were isolated using polyclonal human anti-HCV antibodies bound to polystyrene beads.

25 Polyclonal anti-NANB5_1.1 antibodies were purified from human serum by NANBH using the SOD-HCV polypeptide encoded in clone 5-1-1. The purification method used in section IV.E. described.

The purified anti-NANBs antibodies were bonded to polystyrene beads (6.1 in 30 mm diameter, mirror coating, Precision Plastic Ball Co.,

Chicago, Illinois) by incubating each at room temperature overnight with B1 ml of antibodies (1 microgram / ml in borate buffer saline, pH 8.5). After overnight incubation, the beads were washed once with TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% (v / v) Tween 20) and then with phosphate buffer saline (PBS) containing 10 mg / ml. ml of BSA.

5

Control beads were prepared in the same way except that the purified anti-NANB 50 antibodies were replaced with total human immunoglobulin.

Capture of HCV from NANBH-infected chimpanzee plasma using anti-NANBs-M antibodies bound to beads was obtained as follows. The plasma used from a chimpanzee with NANBH is described in section IV.A.1.

One portion (1 ml) of the NANBV infected chimpanzee plasma was incubated for 3 hours at 37 ° C with each of 5 beads coated with either 15 anti-NANBs-i-i antibodies or with control immunoglobulins. The beads were washed 3 times with TBST.

IV.F.2. Hybridization of the nucleic acid in the captured particles with NANBV cDNA

20

The nucleic acid component released from the particles captured with anti-NANB1 antibodies was analyzed for hybridization with HCV cDNA derived from clone 81.

25 HCV Particles were captured from NANBH-infected chimpanzee plasma as described in IV.F.1. To release the nucleic acids from the particles, the washed beads were incubated for 60 min. at 37 ° C with 0.2 ml per ml. bead of a solution containing proteinase k (1 mg / ml), 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.25% (w / v) SDS, 10 micrograms / ml soluble yeast RNA, and the supernatant solution was removed. The supernatant was extracted with phenol and chloroform and the nucleic acids precipitated with ethanol overnight at -20 ° C. The nucleic acid precipitate was collected by centrifugation, dried and dissolved in 50 mM Hepes, pH 7.5. Duplicate portions of the soluble nucleic acids from the samples obtained from the anti-NANBs.M antibody coated beads and control beads containing total human immunoglobulin were filtered on nitrocellulose filters. The filters were hybridized with a 32 P-labeled nick-translated probe prepared from the purified HCV cDNA fragment in clone 81. The methods for producing the probe and for hybridization are described in section IV.C.1.

10 Autoradiographs of a probe filter containing the nucleic acids from particles captured with beads containing anti-NANBs-i-i antibodies are shown in FIG. 40. The extract (Ai, A2) obtained using the anti-NANBs-i.i antibody gave clear hybridization signals relative to the control antibody extract (A3, A4) and to control yeast RNA (Bi, B2). Standards consisting of 1pg, 15pg and 10pg of the purified clone 81 cDNA fragment are shown in C1 to 3, respectively.

The results demonstrate that the particles captured from NANBH plasma by anti-NANB1 antibodies contain nucleic acid that hybridizes with HCV cDNA 20 in clone 81, thus providing further evidence that the cDNAs in these clones are derived from the etiologic agent of NANBH.

IV.G. Immunological reactivity of Cl00-3 with purified anti-NANBs.i.i antibodies 25

The immunological reactivity of C100-3 fusion polypeptide with anti-NANBs. µ antibodies were determined by radioimmunoassay in which the antigens bound to a solid phase were challenged with purified anti-NANBs-i-i antibodies and the antigen-antibody complex was detected with 125l-labeled sheep in 30 anti-human antibodies. The immunological reactivity of C100-3 polypeptide was compared to the reactivity of SOD-NANB5-1-1 antigen.

124 DK 175975 B1

The fusion polypeptide C100-3 was synthesized and purified as described in Section IV.B.5, respectively. and in Section IV.B.6 .. The fusion polypeptide SOD-NANBs-m was synthesized and purified as described in Section IV.B.1. respectively, Section IV.D.1. Purified anti-NANBs-i-i antibodies were obtained as described in section IV.E.

One hundred microliters of portions containing varying amounts of purified C100-3 antigen in 0.125M Na-borate buffer, pH 8.3, 0.075M NaCl (BBS) were added to each well of a microtiter plate (Dynatec Immulon 20 Removawell Strips). The plate was incubated at 4 ° C overnight in a humid chamber, then the protein solution was removed and the wells washed 3 times with BBS containing 0.02% Triton X-100 (BBST). To prevent non-specific binding, the wells were coated with BSA by the addition of 100 microliters of a 5 mg / ml solution of BSA in BBS followed by incubation at 15 room temperature for one hour, then excess BBS solution was removed. The polypeptides in the coated wells were reacted with purified anti-NANBs.i.i antibodies by addition of 1 microgram antibody / well and incubation of the samples for one hour at 37 ° C. After incubation, excess solution was removed by aspiration and the wells were washed 5 times with BBST. Anti-20 ΝΑΝΒδ-Ί-ι bound to the fusion polypeptides was determined by the binding of 125 I-labeled F '(ab) 2 sheep anti-human IgG to the coated wells. Aliquots of 100 microliters of the labeled probe (specific activity 5-20 microcuries / microgram) were added to each well and the plates were incubated at 37 ° C for one hour followed by removal of excess probe by aspiration and 5 times wash with BBST. The bound amount of radioactivity in each well was determined by counting in a counter detecting gamma radiation.

The results of the immunological reactivity of C100 with purified anti-NANB5-1-1 compared to reactivity of NANB5-1-1 with the purified 30 antibodies are shown in Table 3.

125 DK 175975 B1

Table 3

Immunological reactivity of C100-3 compared to NANB5.M by radioimmunoassay 5 RIA (cpm / analysis) AGfnol 400 320 240 160_60_0 NANB5-V1 7332 6732 4954 4050 3051 57 10 C100-3 7450 6985 5920 5593 4096 67

The results in Table 3 show that anti-NANBs-i-i recognizes an epitope (epitopes) in the C100 portion of the C100-3 polypeptide. NANB5.1-1 and C100 therefore share a common 15 epitope (epitopes). The results suggest that the cDNA sequence encoding this NANBV epitope (epitopes) is present in both clone 5-1-1 and in clone 81.

IV.H. Characterization of HCV

IV.H.1. Characterization of the Severity of the HCV Genome The HCV genome was characterized in terms of its severity by isolating the nucleic acid fraction from trapped particles on anti-NANBs-II antibody-coated polystyrene beads and determining whether the isolated 25 nucleic acid hybridized with plus and / or minus strands of HC .

Particles were captured from HCV infected chimpanzee plasma using polystyrene beads coated with immunopurified anti-NANBs-i-i antibody as described in section IV.F.1. The nucleic acid component 30 of the particles was released using the method described in section IV.F.2. Portions of the isolated genomic nucleic acid equivalent to 3 ml of high titer plasma were exposed to nitrocellulose filters. As controls, portions of denatured HCV cDNA from clone 81 (2 picograms) were also blotted onto the same filters. The filters were probed with 32P-labeled mixture of plus or minus strands of single-stranded DNA cloned from HCV-5 cDNAs; The cDNAs were cut from clones 40b, 81 and 25c.

The single-stranded probes were obtained by cutting off the HCV cDNAs from clones 81, 40b and 25c with EcoRI, and cloning the cDNA fragments into M13 vectors, MP18 and MP19 [Messing (1983)]. The M13 clones were sequenced to determine whether they contained the plus or minus strands of DNA derived from the HCV cDNAs. Sequencing was by the dideoxy chain termination method of Sanger et al. (1977).

Each set of duplicate filters containing portions of the HCV genome isolated from the captured particles was hybridized with either plus or minus strand probes defatted from the HCV cDNAs. FIG. 41 shows the autoradiographs obtained from probing the NANBV genome with the mixture of probes derived from clones 81, 40b and 25c. This mixture was used to increase the sensitivity of the hybridization assay. The samples in panel I 20 were hybridized with the plus strand probe mixture. The samples in Panel II were probed by hybridization with the minus strand probe mixture. The composition of the samples in the immunoblot panels is given in Table 4.

127 DK 175975 B1

Table 4

A B

Migration pathway 5 1 HCV genome * 2 10 3 * cDNA 81 4 - cDNA 81 * is an undescribed sample.

15

As can be seen from the results in FIG. 41, only the minus strand DNA probe hybridizes to the isolated HCV genome. This result, in combination with the result showing that the genome is sensitive to RNase and DNase (see section IV.C.2.), Suggests that the NANBV genome is positively stranded RNA.

20

These data and data from other laboratories regarding the physical / chemical properties of one or more putative NANBVs (NANBVs) are consistent with the possibility that HCV is a member of the flaviviridae. However, the possibility that HCV represents a new class of viral agents is not excluded.

IV.H.2. Detection of sequences in captured particles when amplified by PCR. hybridizes with HCV cDNA derived from clone 81 The RNA in trapped particles was obtained as described in Section IV.H.1. The analysis for sequences hybridizing with HCV cDNA derived from clone 81, 128 DK 175975 B1 was performed using the PCR amplification procedure as described in Section IV.C.3, except that the hybridization probe was a kinase-treated oligonucleotide derived from clone 81 cDNA sequence. The results showed that the amplified sequences hybridize with the HCV cDNA probe derived from 5 clone 81.

IV.H.3. Homology between the Dengue flavivirus non-structural protein (MNWWVD1) and the HCV polypeptides. encoded in the combined ORF of clones 14i to 39c 10

The combined HCV cDNAs in clones 14i to 39c contain continuous ORF as shown in FIG. 26. The polypeptide encoded therein was analyzed for sequence homology with the Dengue flavivirus (MNWVD1) non-structural polypeptide region (s). In the analysis, the Dayhoff protein database was used and it was performed on computer. The results are shown in FIG. 42, where the symbol {:) shows an exact homology and the symbol (.) Shows a conservative replacement in the sequence; the dashes show interposed stays in the sequence to obtain the largest homologies. As seen from the figure, there is significant homology between the sequence encoded in the HCV cDNA and the Dengue virus non-structure 20 polypeptide (s). In addition to that shown in FIG. 42, the analysis of the polypeptide segment encoded in the region toward the 3-end of the cDNA also contained sequences homologous to sequences in the Dengue polymerase. Importantly, the recognized Gly-Asp-Asp (GDD) sequence, which is thought to be essential for RNA-dependent RNA polymerases, was found to be contained within the HCV cDNA encoded polypeptide having a localization consistent with the localization of the Dengue 2 virus. (Results not shown).

30 129 DK 175975 B1 IV.H.4. HCV DNA cannot be detected in NANBH infected tissue In two types of studies, results provide evidence that HCV DNA cannot be detected in tissue from a subject with NANBH. These results 5 compared with those in IV.C and IV.H.1. and IV.H.2. disclosed provides evidence that HCV is not a DNA containing virus and that replication thereof does not involve cDNA.

IV H.4.a. Southem blot procedure 10

To determine if NANBH-infected chimpanzees contain detectable HCV DNA (or HCV cDNA), restriction enzyme DNA fragments isolated from this source were Southern blotted, and the blots were probed with 32 P-labeled HCV cDNA. The results showed that the labeled HCV cDNA did not hybridize with the exposed DNA of the infected chimpanzee liver. It also did not hybridize with the control-exposed DNA of normal chimpanzee liver.

In a positive control, on the other hand, a labeled probe of the beta-interferon gene hybridized strongly with Southern blots of restriction enzyme digested human placental DNA. These systems were designed to detect a single copy of the gene to be detected with the labeled probe.

DNAs were isolated from the liver from two chimpanzees with NANBH. Control DNAs were isolated from uninfected chimpanzees and from human placentas. The DNA extraction procedure was performed essentially according to 25 Maniatis et al. (1982), and the DNA samples were treated with RNAse during the isolation procedure.

Each DNA sample was treated with either EcoRI, Mbol or Hindi (12 micrograms) according to the manufacturer's instructions. The digested DNAs were electrophoresed on 1% neutral agarose gels, Southern blotted on nitrocellulose, and the blotted material was hybridized with the appropriate nickel-probe probe cDNA (3 x 106 cpm / ml hybridization mix). The DNA From infected chimpanzee liver and normal liver was hybridized with 32P-labeled HCV cDNA from the clones 36 plus 81; The human placenta DNA was hybridized with 32 P-labeled DNA from the beta interferon gene. After hybridization, the blots were washed under stringent conditions, i.e. with a solution containing 0.1 x SSC, 0.1% SDS at 65 ° C.

The beta interferon gene DNA was prepared as described by Houghton et al (1981).

IV.H.4.b. Amplification by PCR technique

To determine if HCV DNA can be detected in liver from chimpanzees with NANBH, DNA was isolated from the tissue and subjected to the PCR technique for detection of amplification using primers and probe polynucleotides derived from clone 81. HVC cDNA as negative controls DNA samples were isolated from uninfected HepG2 tissue culture cells and from putative uninfected human placenta. As positive controls, samples of the negative control DNAs were used to which a known relatively small amount (250 molecules) of the HCV cDNA insert from clone 81 was added.

In addition, to confirm that RNA fractions isolated from the same liver of chimpanzees with NANBH contained complementary sequences to the HCV cDNA probe, the PCR amplification detection system was also used on the isolated RNA samples.

In the studies, the DNAs were isolated by the procedure described in Section IV.H.4.a and RNAs were extracted essentially as described by Chirgwin et al. (1981).

B 131 PNA Samples were isolated from two infected chimpanzee pupils, from uninfected hepG2 cells and from human placenta. One microgram of each DNA was digested with HindIII according to the manufacturers instructions. The digested samples were subjected to PCR amplification and detection of amplified HCV cDNA substantially as described in Section IV.C.3. with the exception that the reverse transcriptase step was omitted. The PCR primers and probe came from HCV cDNA clone 81 and are described in Section IV.C.3 .. Prior to amplification, for positive control, one microgram sample of each DNA was "spiked" by adding 250 molecules of HCV cDNA insert, isolated from clone 81.

10

To determine whether HCV sequences were present in RNA isolated from the liver of chimpanzees with NANBH, samples containing 0.4 micrograms of total RNA were subjected to the amplification procedure essentially as described in Section IV.C.3. with the exception that the reverse transcriptase was omitted from some of the samples as negative control. The PCR primers and probe came from HCV cDNA clone 81 as described above.

The results showed that enhanced complementary sequences for the HCV cDNA probe could not be detected in the DNA of infected chimpanzee liver, nor could the 20 be detected in the negative controls. On the contrary, the clone 81 sequence was detected in all positive control samples when the samples, including the DNA of infected chimpanzee liver, were "spiked" with the HCV cDNA prior to amplification. In addition, in the RNA studies, enhanced HCV cDNA clone 81 sequences were only detected when reverse transcriptase was used, which strongly suggests that the results were not due to a DNA contamination.

The results show that chimpanzee hepatocytes with NANBH contain no or undetectable levels of HCV cDNA. Based on the "spiking" study, the possibly present HCV DNA is present at a level well below 0.06 copies per hepatocyte. In contrast, the HCV sequences in total RNA from the same liver samples were immediately detected with The PCR technique.

Iv.i. ELISA determination of HCV infection using HCV C100-5 3 as test antigen

All samples were analyzed using HCV C100-3 ELISA. In this analysis, the HCV C100-3 antigen (which was synthesized and purified as described in Section IV.8.5) and a horseradish peroxidase (HRP) -10 conjugate of mouse monoclonal anti-human IgG are used.

Plates coated with the HCV C100-3 antigen were prepared as follows. A solution containing coating buffer (50mM Na-borate, pH 9.0), 21ml plate, BSA (25 micrograms / ml), C100-3 (2.50 micrograms / ml) was prepared immediately prior to addition to Removawell Immulon I plates (Dynatech Corp.). After mixing for 5 minutes, 0.2 ml / well of the solution was added to the plates, covered and incubated for 2 hours at 37 ° C, after which the solution was removed by aspiration. The wells were washed once with 400 microliters of wash buffer (100 mM sodium phosphate, pH 7.4, 140 20 mM sodium chloride, 0.1% (W / V) casein, 1% (W / V) Triton x-100, 0.01 % (W / V) Thimerosal). After removal of the washing solution, 200 microliters / well of Postcoat solution (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 0.1% (w / v) casein and 2 mM phenylmethylsulfonyl fluoride (PMSF)) were added to cover the plates to prevent evaporation and they were allowed to stand at room temperature for 30 minutes, then the contents of the wells were aspirated to remove the solution and freeze-dried overnight without shelf heating.The prepared plates can be stored at 2-8 ° C in sealed aluminum bags.

To perform the ELISA assay, 20 ml of serum sample or control sample was added to a well containing 200 microliters of sample diluent (100 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 1 mM EDTA, 0.1% (W / V) casein, 0.015 (W / V) therosal, 1% (W / V) triton x-100, 100 micrograms / ml yeast extract). The plates were sealed and incubated at 37 ° C for 2 hours, after which the solution was removed by aspiration and the wells were washed with 400 microliters of wash buffer (phosphate buffer saline (PBS) containing 0.05% Tween 20). The washed wells were treated with 200 microliters of mouse anti-human IgG-HRP conjugate contained in a solution of Ortho conjugate diluent (10mM sodium phosphate, pH 7.2, 150mM sodium chloride, 50% (W / V) bovine fetal serum, 1% (W / V) heat-treated horse serum, 1 mM K3 Fe (CN) 6, 0.05% (W / V) Tween 20, 0.02% (W / V) thimerosal). The treatment lasted one hour at 37 ° C and the solution was removed by aspiration and the wells were washed with wash buffer which was also removed by aspiration. To determine the amount of bound enzyme conjugate, 200 microliters of substrate solution (10 mg of o-phenylenediamine dihydrochloride per 5 ml of developer solution) was added. Developer solution contains 50 mM sodium citrate adjusted to pH 5.1 with phosphoric acid and 0.6 microliters / ml of 30% H2O2. The plates containing the substrate solution were incubated in the dark for 30 minutes at room temperature, the reactions were stopped by the addition of 50 microliters / ml of 4N 20 sulfuric acid and the ODs determined.

The examples provided below show that microtiter plate ELISA screening using HCV C100-3 antigen has a high degree of specificity, as shown at an initial reactivity rate of approx. 1% with a repeat reaction rate of approx. 0.5% on random donors. In the analysis, it can detect an immunoreaction in both the post-acute infection phase and during the chronic disease phase. In addition, the analysis is able to detect some samples that have negative scoring in the surrogate tests for NANBH; The samples come from individuals with NANBH history or from donors implicated in 30 NANBH transfer.

In the examples described below, the following abbreviations are used: ALT Alanine aminotransferase

Anti-HBc Antibody against HBc 5 Anti-HBsAg Antibody against HBsAg HBc Hepatitis B Nuclear Antigen ABsAg Hepatitis B Surface Antigen

IgG Immunoglobulin G

IgM Immunoglobulin M

10 IU / L International units / liter NA Not available NT Not tested in N Sample size

Neg Negative 15 OD Optical Density Us Positive S / CO Signal / Limit (SD: cutoff) SD Standard Deviation .v Average or Mean 20 WNL Within Normal Limits IV.1.1. HCV infection in a population of random blood donors

A group of 1056 samples (fresh sera) from random blood donors were obtained from Irwin Memorial Blood Bank, San Franscisco, California. The test results obtained with these samples are summarized in a histogram showing the distribution of OD values (Fig. 43). As seen in FIG. 43, 4 samples show values> 3, 1 sample show values between 1 and 3, 5 samples show values between 0.4 and 1, and the remaining 1046 samples show values <0.4 and over 30 90% of these samples show values <0.1.

135 DK 175975 B1

The results of the reactive random samples are shown in Table 5. Using a cutoff value equal to the mean plus 5 standard deviations, 10 samples out of the 1056 (0.95%) were initially reactive. Of these, 5 samples (0.47%) were repetitively reactive when analyzed a second time using ELISA. Table 5 also shows the ALT and anti-HBc status of each of the repetitive reactive samples. In particular, the fact that all 5 repetitive reactive samples were negative in both surrogate tests for NANBH while reacting positively in HCV-ELISA is interesting.

136 DK 175975 B1

Table 5

Results of reactive random samples 5 N = 1051 λ = 0.049 * SD = ± 0.074

Limit value: x + 5SD = 0.419 (0.400 + negative control) 10

Initially Gentagent Reactive Reactive Anti!

Samples OD OD ALT ** HBc *** (IU / L) (OD)

15 4227 0.462 0.084 NA NA

6292 0.569 0.294 NA NA

6188 0.699 0.326 NA NA

6157 0.735 0.187 NA NA

6277 0.883 0.152 NA NA

20 6397 1,567 1,392 30,14 1,433 6019> 3,000> 3,000 46,48 1,057 6651> 3,000> 3,000 48,53 1,343 6669> 3,000> 3,000 60,53 1,165 4003> 3,000 3,000 WNL **** Negative 25 10/1056 = 0.95% 5/1056 = 0.47% * Samples showing values> 1.5 were not included in the mean calculation and SD ** ALT = 68 IU / L is above normal limits.

30 *** AnthHBc = 0.535 (competitive analysis) is considered positive.

**** WNL: Within normal limits.

137 DK 175975 B1 IV.1.2. Chimpanzee serum samples

Serum samples from 11 chimpanzees were tested with HCV C100-3 ELISA. Four of these five chimpanzees were infected with NANBH from a contaminated factor VIII batch (presumably Hutchinson strain) according to an established procedure in collaboration with Dr. Daniel Bradley of the Centers for Disease Control. Four other chimpanzees were infected as controls with HAV and three with HBV.

Serum samples were obtained at different times after infection.

10

The results summarized in Table 6 show documented antibody seroconversion in all chimpanzees infected with the Hutchinson strain of NANBH. After the acute stage of infection (as shown by the significant increase and subsequent return to normal of ALT-15 levels), antibodies to HCV C100-3 could be detected in the sera of the 4/4 NANBH infected chimpanzees. As discussed in Section IV.B.3. 3 samples have previously been shown to be positive by a Western analysis and an RIA. In contrast, none of the control chimpanzees that had been infected with HAV or HBV showed evidence of reactivity in ELISA.

20 138 DK 175975 B1

Table 6

Chimpanzee Serum Samples

Inocule- Date of ALT Trans-5 OD S / CQ Date of Blood Test (IU / L) Fusion

Negative control 0.001

Positive control 1,504 10 Limit value 0.401

Chimpanzee 1 -0.007 0.00 24/05/84 24/05/84 9 NANB

0.003 0.01 07/08/84 71> 3,000> 7.48 18/09/84 19 15> 3,000> 7.48 24/10/84

Chimpanzees 2 - - 07/06/84 --- NANB

-0.003 0.00 31/05/84 5 -0,005 0.00 28/06/84 52 20 0.945 2.36 20/08/84 13> 3,000> 7.48 24/10/84

Chimpanzee 3 0.005 0.01 14/03/85 14/03/85 8 NANB

0.017 0.04 26/04/85 205 25 0.006 0.01 06/05/85 14 1,010 2.52 20/08/85 6

Chimpanzee 4 -0.006 0.00 11/03/85 11/03/85 11 NANB

0.003 0.01 09/05/85 132 30 0.523 1.31 06/06/85 1,574 3.93 01/08/85 139 DK 175975 B1

Table 6

Chimpanzee serum samples (continued)

Inoculum- Date of ALT Transod OD S / CO Date of Blood Test (IU / U fusion

Chimpanzee 5 -0.006 0.00 21/11/80 21/11/80 4 HAV

0.001 0.00 16/12/80 147 0.003 0.01 30/12/80 18 0.006 0.01 29 / 07-21 / 08/81 5 10

Chimpanzee 6 ...... 25/05/82 - - HAV '-0,005 0.00 17/05/82 0.001 0.00 10/06/82 106 -0,004 0.00 ~ 06/07/82 10 15 0.290 0.72 01/10/8 -

Chimpanzee 7 -0.008 0.00 25/05/82 25/05/82 7 HAV

-0.004 0.00 17/16/82 83 -0.006 0.00 16/09/82 5 20 0.005 0.01 09/10/82 -

Chimpanzee 8 -0.007 0.00 21/11/80 21/11/80 15 HAV

0.000 0.00 16/12/80 130 0.004 0.01 03/02/81 8 25 0.000 0.00 03 / 06-10 / 06/81 4.5

Chimpanzee 9 - - 24/07/80 - - HBV

0.019 0.05 22 / 08-10 / 10/79 - 11/03/81 57 30 0.015 0.04 01 / 07-05 / 08/81 9 0.008 0.02 01/10/81 6 140 DK 175975 B1

Table 6 *

Chimpanzee serum samples (continued)

Inoculum - Date of ALT Trans-5 OD S / CO Date of Blood Test (IU / L) Fusion

Chimpanzee 10 - - 12/05/82 - --- HBV

0.011 0.03 21 / 04-12 / 05/82 9 0.015 0.04 01 / 09-08 / 09/82 126 10 0.008 0.02 02/12/82 9 0.010 0.02 06/01/83 13

Chimpanzees - - 12/05/82 - - HBV

0,000 0.00 06/01/12/05/82 11 15 - 23/06/82 100 -0,003 0.00 09/06 - 07/07/82 -0,003 0,00 28/10/82 9 -0,003 0 , 00 20/12/82 10 20 IV.I.3. Reference group 1: Detected infectious sera from chronic human NANBH carriers

A reference group consisted of 22 unique coded samples, each in duplicate, with a total of 44 samples. The samples were from detected infectious sera from chronic 25 NANBH carriers, infectious sera from implicated donors as well as infectious sera from patients with acute phase NANBH. In addition, the samples were derived from high pedigree negative controls and other disease controls. This reference group was provided by Dr. H. Alter from "Department of Health and Human Services", National Institutes of Health, Bethesda, Maryland.

The reference group was constructed by Dr. Alter several years ago and has been used by Dr. Alter as a qualifying reference group for putative NANBH analyzes.

The entire reference group was analyzed twice with the ELISA assay and the 5 results were sent to Dr. Alter to inventory. The results of the inventory are shown in Table 7. Although the table shows only the results of one set of duplicates, the same values were obtained for each of the duplicate samples.

As shown in Table 7, 6 sera detected infectious in a 10 chimpanzee model were strongly positive. The seventh infectious serum corresponded to a sample from an acute NANBH case and did not respond in this ELISA analysis. A sample from an implicated donor with both normal ALT levels and ambiguous results in the chimpanzee studies was non-reactive in the analysis. Three other serial samples from an individual with acute NANBH were also not reactive. All 15 samples coming from the high-pedigree negative controls obtained from donors with at least 10 blood donations without hepatitis implication were non-reactive in the ELISA assay. Finally, 4 of the samples tested had previously shown positive in putative NANBH analyzes developed by others, but these analyzes could not be confirmed. These 4 samples were found to be negative in the HCV ELISA assay.

142 DK 175975 B1

Table 7 H. Alters reference group 1:

Referencegruppe_1. result 2. result 5 1) Detected infectious by chimpanzee transfer A. Chronic NANB: Post-Tx JF + + EB + + 10 PG ++

B. Implicated donors with elevated ALL

BC ++ JJ ++ BB ++ 15 C. Acute NANB: Post-Tx

WH

2) Ambiguously infectious by chimpanzee transmission

A. Implicated donor with normal ALT CC

20 3) Acute NANB: Post-Tx JLuge 1 - - JL week 2 JL week 3 4) Disease controls 25 A. Primary biliary cirrhosis ΕΚ

B. Alcoholic hepatitis during cure HB

30 143 DK 175975 B1

Table 7 H. Alters reference group 1: (continued) 5 Reference group_1. result 2. result 5) Stud book negative controls DM - -

DC

10 LV - -

ML AH

6) Potential NANB "antigens" 15 JS-80-01T-0 (Ishida)

Asterix (Trepo)

Zurtz (Arnold)

Becassdine (Trepo)

IV.I.4. Reference Group 2: Donor / recipient NANBH

The reference group consisted of 10 coded unambiguous donor-recipient cases of the transfusion-associated NANBH with a total of 188 samples. Each case consisted of samples from some or all of the recipient's donors and from serial samples 25 (sampled 3, 6 and 12 months after transfusion) from the recipient. Also included was a blood sample taken from the recipient prior to transfusion. The coded reference group was provided by Dr. H. Alter from NIH and the results were sent to him for inventory.

The results summarized in Table 8 show that in the ELISA assay antibody seroconversion was detected in 9 out of 10 cases of transfusion-associated NANBH.

144 DK 175975 B1

Samples from case # 4 (where no seroconversion was detected) consistently responded poorly in the ELISA assay. Two of the 10 recipient samples were reactive 3 months after transfusion. Six months later, 8 recipient samples were reactive and after 12 months, with the exception of Case # 4, all 5 samples were reactive. In addition, at least one antibody positive donor was found in 7 of the 10 cases, with case # 10 having 2 positive donors. In case # 10, the recipient's blood drain was also positive for HCV antibodies. The blood draw after one month from this recipient decreased to the limit level of response, while elevated to positive at the 4 and 10 month 10 blood draws. An S / CO of 0.4 is generally considered positive.

Thus, this case may represent a prior infection of the individual with HCV.

The ALT and HBc status of all the reactive, viz. positive, samples are summarized 15 in Table 9. As can be seen in the table, 1/8 donor samples were negative with respect to the surrogate markers and reactive in the HCV antibody ELISA assay. On the other hand, the recipient samples (followed up until 12 months after transfusion) had either elevated ALT, positive anti-HBc or both and.

145 DK 175975 B1

Table 8

Donor / recipient NANB reference group 5 H. Alter donor / recipient NANB reference group

Pre-bottling of

Blood from recipient Post-Tx trap Donor 3 months 6 months 12mdr.

no.

OD S / CO OD S / CO OD S / CO OD S / CO OD S / CO

1 - -, 032 0.07, 112 0.26> 3,000> 6.96> 3,000> 6.96 2 - -. , 059 0.14, 050 0.12 1,681 3.90> 3,000> 6.96 3, 403 0.94, 049 0.11, 057 0.13> 3,000> 6.96> 3,000> 6.96 4 -, 065 0.15, 073 0.17, 067 0.16, 217 0.50 5> 3,000> 6.96. , 0.34 0.08, 096 0.22> 3,000> 6.96> 3,000> 6.96 6> 3,000> 6.96, 056 0.13 1,475 3.44> 3,000> 6.96> 3,000> 6.96 7> 3,000> 6.96, 034 0.08, 056 0.13> 3,000> 6.96> 3,000> 6.96 8> 3,000> 6.96, 061 0.14, 078 0.18 2,262 5.28 > 3,000> 6.97 9> 3,000> 6.96, 080 0.19, 127 0.30, 055 0.13> 3,000> 6.96 10> 3,000> 6.96> 3,000> 6.96, 3171 0 , 74> 3,000 **> 6.96> 3,000 ***> 6.96> 3,000> 6.96 1 month, ** 4 months, *** 10 months 146 DK 175975 B1

Table 9

Alt and HBc status of reactive samples in H. Alter reference group 1 5 Samples Anti-Alt * HBc **

donors

Case # 3 Normal Negative 10 Case # 5 Elevated Positive

Case # 6 Elevated Positive

Case # 7 Not Available Negative

Case # 8 Normal Positive

Case # 9 Elevated Not Available 15 Case # 10 Normal Positive

Case # 11 Normal Positive

Recipients 20 Case # 1 6 months Increased Positive 12 months Elevated Not analyzed

Case # 2 6 months Increased Negative 12 months Increased Not analyzed 25

Case # 3 6 months Normal Not analyzed *** 12 months Increased Not analyzed ***

Case # 5 6 months Increased Not analyzed 30 12 months Increased Not analyzed 147 DK 175975 B1

Continued, from Table 9

Case # 6 3 months Increased Negative 6 months Increased Negative 12 months Increased Not analyzed 5

Case # 7 6 months Increased Negative 12 months Increased Negative

Case # 8 6 months Normal Positive 10 12 months Increased Not analyzed

Case No. 9 12 months Increased Not analyzed

Case # 10 4 months Increased Not analyzed 15 10 months Increased Not analyzed * ALT = 45 IU / L is above normal limits.

** Anti-HBc = 50% (competitive analysis) is considered positive.

*** Blood pre-dosing and 3-month samples were negative for 20 HBc.

IV.I.5. Detection of HCV infection in samples from high-risk groups

Samples from high-risk groups were monitored using the ELISA-25 assay to detect reactivity with HCV-C100-3 antigen. The samples were obtained from dr. Gary Tegtmeier, Community Blood Bank, Kansas City. The results are summarized in Table 10.

As indicated in the table, the highest reactivity was obtained in samples from softer 30 (76%). In addition, 51% reactive samples were found from individuals with elevated ALT and who were anti-HBc positive, a value consistent with the expected value based on clinical data and NANBH prevalence in this group. The incidence of HCV antibody was also greater in blood donors with elevated ALT alone, blood donors that are positive only for antibodies to hepatitis B chemistry, and in blood donors rejected for 5 reasons other than high ALT or anti-nuclear antibody levels. , when compared to random voluntary donors.

149 DK 175975 B1

Table 10 NANBH samples from high-risk rape Distribution 5 Group_N N_OD_% Reactive

Elevated ALT 35 3> 3000 11.4% 1 0.728 10 Anti-HBc 24 5> 3000 20.8%

Elevated ALL, anti- 33 12> 3000 51.5% HBc 1 2768 1 2324 15 1 0.939 1 0.951 1 0.906

Rejected donors 25 5> 3000 20.0% 20

Donors with previously diagnosed hepatitis 150 19> 3000 14.7% 1 0.837 25 1 0.714 1 0.469

Softer 50 31> 3000 76.0% 1 2568 30 1 2483 1 2000 1 · 1979 1 1495 1 1209 i 35 1 0.819 IV.1.6. Comparative studies using anti-IgG or anti-IgM monoclonal or polyclonal antibodies, as other antibody in the HCV-c100-3 ELISA assay 40 The sensitivity of the ELISA assay using anti-IgG monoclonal conjugate was compared to the sensitivity obtained obtained using either an anti-IgM monoclonal conjugate or by replacing both with a polyclonal antiserum which was to be both heavy and light chain specific. The following studies were performed.

IV.I.6.a. Serial samples from individuals with seroconversion

Serial samples from three NANB seroconversion cases were examined in the HCV-C100-3 ELISA assay using the enzyme conjugate of either the monoclonal anti-IgG alone or in combination with a monoclonal anti-IgM 10 or using a polyclonal antiserum. The tests were provided by dr. Cladd Stevens, N.V. Blood Center, N.Y.C., N.Y. The sample history is listed in Table 11.

The results obtained using an anti-IgG monoclonal antibody-enzyme conjugate 15 are listed in Table 12. The results show that initially strong reactivity was detected in samples 1-4, 2-8 and 3-5 of cases # 1, 2 and 3 respectively.

The results obtained using a combination of an anti-IgG monoclonal conjugate and an anti-IgM conjugate are listed in Table 13. Three different ratios of anti-IgG to anti-IgM were analyzed; The 1: 10,000 dilution of anti-IgG was constant all the time. The dilutions analyzed for monoclonal anti-IgM conjugate were 1: 30000, 1: 60000 and 1: 120000. The results show that, in accordance with the studies with anti-IgG alone, the initial strong reactivity is detected in samples 1-4, 2-8 and 3-5.

The results obtained with the ELISA assay using anti-IgG monoclonal conjugate (1: 10000 dilution) or Tago polyclonal conjugate (1: 80000 dilution) or Jackson polyclonal conjugate (1: 80000 dilution) are reported in Table 14. Results show that initial strong reactivity was detected in samples 1-4, 2-8 and 3-5 using all three configurations; Tago polyclonal antibodies gave the weakest signals.

The results presented above show that in all three configurations 5, reactive samples are detected at the same time after the acute phase of the disease (as demonstrated by the ALT elevation). The results further show that the sensitivity of the HCV-C100-3 ELISA assay using anti-IgG monoclonal enzyme conjugate is equal to or better than the results obtained using the other tested enzyme conjugate configurations.

152 DK 175975 B1

Table 11

Description of samples from Cladd Steven's reference group _Date_HBsAq Anti-HBs Anti-HBc ALT Bilirubin 5

Case No. 1 1-1 5/8/81 1.0 91.7 12.9 40.0 -1.0 1-2 2/9/81 1.0 121.0 15.1 274.0 1, 4 10 1-3 17/10/81 1.0 64.0 23.8 261.0 0.9 1-4 19/11/81 1.0 67.3 33.8 75.0 0.9 1- 5 15/12/81 1.0 50.5 27.6 71.0 1.0

Case # 2 15 2- 1 19/10/81 1.0 1.0 116.2 17.0 -1.0 2-2 17/11/81 1.0 0.8 89.5 46.0 1 , 1 2-3 02/12/81 1.0 1.2 78.3 63.0 1.4 2-4 14/12/81 1.0 0.9 90.6 152.0 1.4 20 2 -5 23/12/81 1.0 0.8 93.6 624.0 1.7 2-6 20/1/82 1.0 0.8 92.9 66.0 1.5 2-7 15 / 2/82 1.0 0.8 86.7 70.0 1.3 2-8 17/3/82 1.0 0.9 69.8 24.0 -1.0 2-9 21/4/82 1.0 0.9 67.1 53.0 1.5 25 2-10 19/5/82 1.0 0.5 74.8 95.0 1.6 2- 11 14/6/82 1.0 0, 8 82.9 37.0 -1.0

Case # 3 30 3-1 7/4/81 1.0 1.2 88.4 13.0 -1.0 3- 2 12/5/81 1.0 1.1 126.2 236.0 0 , 3 3-3 1/5/81 1.0 0.7 99.9 471.0 0.2 3-4 9/6/81 1.0 1.2 110.8 315.0 0.4 3- 5 6/7/81 1.0 1.1 89.9 273, 0.4 35 3-6 10/8/81 1.0 1.0 118.2 158.0 0.4 3-7 8/9 / 81 1.0 1.0 112.3 84.0 0.3 3-8 14/10/81 1.0 0.9 102.5 180.0 0.5 3-9 11.11.81 1.0 1 , 0 84.6 154.0 0.3 153 DK 175975 B1

Table 12 ELISA Assay Results Obtained Using a Monoclonal Anti-IgG Conjugate Sample No. Dato_ALT_OD_S / CO_

Negative control 0.076 limit value PC 0.476 (1: 128) 1.390 10

Case # 1 1-1 05/08/91 40.0 0.178 0.37 1-2 02/09/81 274.0 0.154 0.32 15 1-3 07/10/81 261.0 0.129 0.27 1-4 19/11/81 75.0 0.937 1.97 1- 5 15/12/81 71.0> 3,000> 6.30

Case # 2 20 2- 1 19/10/81 17.0 0.058 0.12 2-2 17/11/81 46.0 0.050 0.11 2-3 02/12/81 63.0 0.047 0.10 2-4 14/12/81 152,0 0,059 0,12 25 2-5 23/12/81 624,0 0,070 0,15 2-6 20/01/82. 66.0 0.051 0.11 2-7 15/02/82 70.0 0.139 0.29 2-8 17/03/82 24.0 1.867 3.92 2-9 21/04/82 53.0> 3,000 > 6.30 30 2-10 19/05/82 95.0> 3,000> 6.30 2- 11 14/06/82 37.0> 3,000> 6.30

Case # 3 35 3-1 07/04/81 13.0 0.090 0.19 3- 2 12/05/81 236.0 0.064 0.13 3-3 30/05/81 471.0 0.079 0.17 3-4 09/06/81 315.0 0.211 0.44 3-5 06/07/81 273.0 1,707 3.59 40 3-6 10/08/81 158.0> 3,000> 6.30 3- 7 08/09/81 84,0> 3,000> 6.30 3-8 14/10/81 180,0> 3,000> 6.30 3-9 11.11.81 154.0> 3,000> 6.30 154 DK 175975 B1

Table 13

ELISA assay results obtained using monoclonal anti-log

and anti-LaM Conjugate 5 NANB ELISA Assays

Monoclonal Monoclonal Monoclonal IgG 1: 1 OK IgG 1: 1 OK IgG 1: 1 OK

IgM 1: 30K IgM 1: 60K IgM 1: 120K

10

Sample No. Date ALT OD C / SO OD S / CO OD S / CO

Negative 0.100 '0.08 0.079

control limit value PC

(1: 128) 1,083 1,328 1,197

Case # 1 20 1-1 05/08/81 40 0.173 0.162 0.70 1-2 02/09/81 274 0.194 0.141 0.079 1-3 07/10/81 261 0.162 0.129 0.063 1-4 19/11/ 81 75 0.812 0.85 0.709 1-5 15/12/81 71> 3.00> 3.00> 3.00 25

Case # 2 1- 2 19/10/81 17 0.442 0.045 0.085 2- 2 17/11/81 46 0.102 0.029 0.030 30 2-3 02/12/81 63 0.059 0.036 0.027 2-4 14/12/81 152 0.065 0.041 0.025 2-5 23/12/81 624 0.082 0.033 0.032 2-6 20/01/82 66 0.102 0.042 0.027 2-7 15/02/82 70 0.188 0.068 0.096 35 2-8 17/03/82 24 1,728 1,668 1,541 2-9 21/04/82 53> 3.00 2.443> 3.00 2-10 19/05/82 95> 3.00> 3.00> 3.00 2- 11 14/06/82 37 > 3.00> 3.00> 3.00 40 Case No. 3 3- 1 07/04/81 13 0.193 0.076 0.049 3-2 12/05/81 236 0.201 0.051 0.038 155 DK 175975 B1 3-3 30 / 05/81 471 0.132 0.067 0.052 3-4 09/06/81 315 0.175 0.155 0.140 3-5 06/07/81 273 1,335 1,238 1,260 3-6 10/08/81 158> 3.00> 3.00> 3 , 00 5 3-7 08/09/81 84> 3.00> 3.00> 3.00 3-8 14/10/81 180> 3.00> 3.00> 3.00 3-9 11 / 11/81 154> 3.00> 3.00> 3.00 in 156 DK 175975 B1

Table 14 ELISA Assay Results Obtained Using Polyclonal Conjugates _ NANB ELISA Assays_ 5 Monoclonal Tago Jackson

1: 1 OK 1: 80K 1: 80K

Sample no. Date ALT OD S / CO OD S / CO OD S / CO

10 Negative Control 0.076 0.045 0.154 Limit PC 0.476 0.545 0.654 (1: 128) (Eng. 1,390 0.727 2,154 cutoff) 15 Case No. 1 1-1 05/08/81 40 0.178 0.37 0.067 0.12 0.153 0, 23 1-2 02/09/81 274 0.154 0.32 0.097 0.18 0.225 0.34 1-3 07/10/81 261 0.129 0.27 0.026 0.05 0.167 0.26 20 1-4 19/11 / 81 75 0.937 1.97 0.324 0.60 0.793 1.21 1- 5 12/15/81 71> 3.00> 6.30 1.778 3.27> 3.00> 4.59

Case no, 2 25 2-1 19/10/81 17 0.058 0.12 0.023 0.04 0.052 0.08 2- 2 17/11/81 46 0.050 0.11 0.018 0.03 0.058 0.09 2-3 02/12/81 63 0,047 0,10 0,020 0,04 0,0 0,0 0,0 2-4 14/12/81 152 0,059 0,12 0,025 0,05 0,054 0,08 2-5 23/12/81 624 0,070 0 , 0.026 0.05 0.074 0.11 30 2-6 20/01/82 66 0.051 0.11 0.018 0.03 0.058 0.09 2-7 15/02/82 70 0.139 0.29 0.037 0.07 0.146 0.22 2-8 17/03/82 24 1.867 3.92 0.355 0.65 1.429 2.19 2-9 21/04/82 53> 3.00> 6.30 0.748 1.37> 3.00> 4.59 2- 10 19/05/82 95> 3.00> 6.30 1.025 -1.88> 3.00> 4.59 35 2-11 14/06/82 37> 3.00> 6, 0.917 1.68> 3.00> 4.59

Case No. 3 3- 1 04/04/81 13 0.090 0.19 0.049 0.09 0.138 0.21 40 3-2 12/05/81 236 0.064 0.13 0.040 0.07 0.094 0.14 3-3 30/05/81 471 '0.079 0.17 0.045 0.08 0.144 0.22 3-4 09/06/81 315 0.211 0.44 0.085 0.16 0.275 0.42 3-5 06/07/81 273 1,707 3.59 0.272 0.50 1.773 2.71 3-6 10/08/81 158> 3.00> 6.30 1.347 2.47> 3.00> 4.59 45 3-7 08/09/81 84 > 3.00> 6.30 2.294 4.21> 3.00> 4.59 3-8 14/10/81 180> 3.00> 6.30> 3.00> 5.50> 3.00> 4.59 3-9 11/11/81 154> 3.00> 6.30> 3.00> 5.50> 3.00> 4.59 157 DK 175975 B1 IV.I.6.b._Samples from random blood donors

Samples from random blood donors (see section IV.1.1.) Were screened for HCV infection using the HCV-C100-3 ELISA assay, in which the antibody-enzyme conjugate was either a monoclonal anti-IgG conjugate or a polyclonal conjugate. The total number of screened samples was 1077 and 1056 for the polyclonal conjugate and the monoclonal conjugate, respectively. A summary of the screening results is given in Table 15 and the sample distributions are shown in the histogram of Figure 44.

10

Mean and standard deviation calculations were performed excluding samples yielding a signal greater than 1.5, i.e., 1073 OD values were used for calculations using the polyclonal conjugate and 1051 for the monoclonal anti-IgG conjugate. As seen in Table 15, the mean value ranged from 0.0493 to 0.0931 when the polyclonal conjugate was used and the standard deviation increased from 0.074 to 0.0933. Moreover ! the results also show that when the x + 5SD criterion is used to define the assay limit value, the polyclonal enzyme conjugate configuration in the ELISA assay assumes a higher limit value. This suggests a reduced assay specificity compared to the monoclonal system.

Furthermore, as depicted in the histogram of Figure 44, a greater separation of results between negative and positive distributions occurs when random blood donors are screened in an ELISA assay using it; monoclonal anti-IgG conjugate compared to the assay using a commercial polyclonal label.

. i t i 158 DK 175975 B1

Table 15

Comparison of two ELISA analogue configurations by analysis of random blood donor samples

5 Conjugate_Polvclonal_Monoclonal anti-IgG

(Jackson)

Sample number 1073 1051

Mean (.Y) 0.0931 0.04926 10 Standard Deviation 0.0933 0.07427 5 SD 0.4666 0.3714

Threshold (5 SD + x) 0.5596 0.4206 15 IV.J. Detection of HCV seroconversion in NANBH patients from a variety of geographical locations

Sera from patients suspected of having NANBH, based on elevated ALT levels and responding negatively in HAV and HBV assays, were screened using RIA essentially as described in Section IV.D. with the exception that the HCV-C100-3 antigen was used as the screening antigen in the microtiter plates. As can be seen from the results shown in Table 16, positive samples were detected with RIA in a large percentage of cases.

25

Table 16

Seroconversion rates for anti-C100-3 among NANBH patients in different countries 30 Land_Holland_Italien_Japan__

Number surveyed 5 36 26

Number of positive 3 29 19% positive 60 80 73 35 159 DK 175975 B1

IV. K. Detection of HCV Seroconversion in Patients with Co-Acquired NAN Bra

Sera obtained from 100 patients with NANBH for whom no apparent pathway of transmission was found (eg, no transfusions, intravenous drug use, promiscuity, etc. as risk factors) were provided by Dr. H. Alter from "Center for Disease Control" and dr. J. Dienstag from Harvard University. The samples were screened using RIA substantially as described in Section IV.D except that the HCV-C100-3 antigen was used as the screening antigen attached to the microtiter plates. The results showed that of the 100 serum samples, they contained 55 antibodies that reacted immunologically with the HCV-C100-3 antigen.

15 The results described above suggest that the "community business" NANBH is also caused by HCV. Furthermore, since it is shown that HCV is related to the flaviviruses, most of which are transmitted by arthropods, it is further suggested that HCV transmission in the "community acquired" cases also originates from arthropod transmission.

20 IV.L, Comparison of HCV antibody incidence and surrogate markers in donors implicated in the NANBH transmission

A future study was performed to determine if 25 seroconversion to positive anti-HCV antibody occurred in recipients of blood from putative NANBH positive donors developing NANBH. The blood donors were analyzed for the surrogate marker abnormalities currently used as markers for NANBH infection, i.e. elevated ALT levels and presence of antigenic antibody. The donors were additionally analyzed for 30 occurrence of anti-HCV antibodies. The determination of the presence of anti-HCV antibodies was determined using a radioimmunoassay as described in section IV.K. The results of the study are presented in Table 17, which shows: the patient number (column 1); presence of anti-HCV antibodies in patient serum (column 2); the number of blood donations received by the patient, each donation being from a different donor (column 3); presence of anti-HCV antibodies 5 in donor serum (column 4) and donor surrogate abnormality (column 5), (NT or - means "not assayed"), (ALT denotes elevated transaminase, and anti-HBc denotes antibody antibody) -

The results in Table 17 show that the HCV antibody assay is more accurate at 10 detection of infected blood donors than the surrogate marker assays. 9 Out of 10 patients who developed NANBH symptoms showed positive assays regarding anti-HCV antibody seroconversion. Of the 11 suspected donors, (patient # 6 received blood donations from 2 different individuals suspected of being NANBH carriers), 9 were positive for anti-15 HCV antibodies and 1 was borderline positive, and therefore ambiguous ( donor for patient # 1). In contrast, 6 out of 10 donors were negative in the elevated ALT assay and in the antibody antibody assay 5 out of 10 donors were found to be negative. However, it is of greater importance that the ALT assay and anti-HBC assay in three cases (donors to patient nos. 8, 9 and 20 10) yielded inconsistent results.

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* Q ^^ 3 2 • «-CMcotj-tncD scoa ° Jf5 162 DK 175975 B1 IV.M. Amplification of Cloning of HCV cDNA Sequences Using PCR and Primers Derived from Conserved Areas of Flavivirus Genome Sequences 5 The results presented above suggesting that HCV is a flavivirus or flavi-like virus allows a cloning strategy for characterized HCV cDNA sequences using the PCR technique and primers derived from the regions encoding conserved amino acid sequences in flaviviruses. Generally, one of the primers is derived from a defined HCV genome sequence and the other primer flanking a region of non-sequenced HCV polynucleotide is derived from a conserved region of the flavivirus genome. The flavirus genomes are known to contain conserved sequences within the NS1 and E polypeptides encoded in the 5 region of the flavivirus genome. Corresponding sequences encoding these regions lie 15 upstream of the HCV cDNA sequence shown in Figure 26. Therefore, to isolate cDNA sequences derived from this region of the HCV genome, upstream primers are constructed which are derived from the conserved sequences within these flavirus polypeptides. The downstream primers are derived from an upstream end of the known portion of the HCV cDNA.

Because of the codon of the code, it is likely that mismatches between the flavivus probes and the corresponding HCV genome sequence will occur. Therefore, a strategy similar to the strategy described by Lee (1988) is used. In the Lee procedure, the mixed oligonucleotide primers are used which are complementary to the reverse translation products of an amino acid sequence; the sequences of the mixed primers take into account any codon degeneration of the conserved amino acid sequence.

Three sets of primer mixtures are generated based on the amino acid rehomologies found in several flaviviruses, including Dengue-2.4 (D-2.4), Japanese Encephalitis Virus (JEV), yellow fever (YF) and West Nile virus 163 DK 175975 B1 (WN). The primer mixture derived from the most upstream conserved sequence (5-1) is based on the amino acid sequence gly-trp-gly, which is part of the conserved sequence asp-arg-gly-trp-gly-aspN found in E. protein of D-2, JEV, YF and WN. The next primer mixture (5-2) 5 is based on a downstream conserved sequence in E protein, phe-asp-gly-asp-ser-tyr-ileu-phe-gly-asp-ser-tyr-ileu, and is derived from phe-gly-asp; the conserved sequence is found in D-2, JEV, YF and WN. The third primer mixture (5'-3) is based on the amino acid sequence arg-ser-cys, which is part of the conserved sequence cys-cys-arg-ser-cys in the NS1 protein of D-2, 10 D-4, JEV , YF and WN. The individual primers constituting the mixture in 5'-3 are shown in Figure 45. In addition to the varied sequences derived from conserved region, each primer in each mixture also contains a constant region at the 5 'end, which region contains a sequence encoding the sites of the restriction enzymes, HindIII, Mbol and EcoRI.

15

The downstream primer, ssc5h20A, is derived from a clone 5h nucleotide sequence containing HCV cDNA with sequences overlapping those of clones 14i and 11b. The sequence of ssc5h20A is: 20 5'GTA ATA TGG TGA CAG AGT CA 3 '.

An alternative primer, ssc5h34A, may also be used. This primer is derived from a sequence in clone 5h and further contains nucleotides at the 5 'end, which create a restriction enzyme site and thereby facilitate cloning. The sequence of ssc5h34A is: 5 'GAT CTC TAG AG A AAT C AA TAT G GT GAC AGA GTC A 3'.

The PCR reaction originally described by Saiki et al. (1986), is performed essentially as described in Lee et al. (1988) with the exception that in the template for cDNA is RNA isolated from HCV-infected chimpanzee liver, as described in section IV.C.2., Or from viral particles isolated from HCV virus. infected chimpanzee serum, as described in section IV.A.1. In addition, the curing conditions are less stringent in the first amplification round (0.6M NaCl and 25 ° C), with the portion of the primer which will cure to the HCV sequence is only at 9 nucleotides and mismatching may occur. In addition, if ssc5h34A is used, the additional sequences that are not derived from the HCV genome will tend to destabilize the "primer template" hybrid. After the first amplification round, the curing conditions may be more stringent (0.066M NaCl and 32 ° C - 37 ° C), the amplified sequences now containing regions complementary to or duplexing the primers. In addition, the first ten amplification cycles are performed with Klenowenzyme I under appropriate PCR conditions for this enzyme. After completion of these cycles, the samples are extracted and performed with Taq polymerase, according to the assay instructions provided by Cetus / Perkin-Elmer.

After amplification, the amplified HCV cDNA sequences are detected by hydration using a probe derived from clone 5h. This probe is derived from sequences upstream of the sequences used for primer derivation and does not overlap with the sequences in the clone 5h derived primers. The sequence of the probe is: 5 'CCC AGC GGC GTA CGC GCT GGA CAC GGA GGT GGC CGC GTC GTG TGG CGG TGT TGT TCT CGT CGG GTT GAT GGC GC 3 *.

25

IV.N.1. Creation of HCV cDNA library from liver from a chimpanzee with infectious NANBH

An HCV cDNA library was generated from the liver of the chimpanzee from which the HCV cDNA library in section IV.A.1. was produced. The technique of creating the library is similar to that of Section IV.A.24. described with the exception of this second source of the RNA, and that a primer based on the HCV cDNA sequence in clone 11b was used. The primer sequence was: 5 'CTG GCT TG A AGA ATC 3' 5 IV.N.2. Isolation of nucleotide sequence of overlapping HCV cDNA in clone k9-1 with cDNA in clone 11b

Clone k9-1 was isolated from the HCV cDNA library generated from liver 10 of a NANBH-infected chimpanzee, as described in section IV.A.25. The library was screened for clones overlapping the sequence in clone 11b, using a clone overlapping clone 11b at the 5 'terminal, clone 11e.

The clone 11b sequence is shown in Figure 23. Positive clones were isolated at a frequency of one in 500,000. An isolated clone, k9-1, was subjected to 15 further studies. That the HCV cDNA in clone k9-1 was naturally overlapping with the 5 'end of the HCV cDNA sequence in Figure 26 was confirmed by probing the clone with clone Alex46; the latter clone contains an HCV cDNA sequence of 30 base pairs corresponding to the base pairs at the 5 'terminus of the HCV cDNA in clone 14i described above.

20

The nucleotide sequence of the HCV cDNA isolated from clone k9-1 was determined using the techniques described above. The sequence of the HCV cDNA in clone k9-1, the overlap with the HCV cDNA of Figure 26 and the amino acids encoded therein are shown in Figure 46.

The HCV cDNA sequence in clone k9-1 has been aligned with the sequences of those in Section IV.A.19. clones described to produce a composite HCV cDNA sequence, wherein the k9-1 sequence is located upstream of the sequence shown in Figure 32. The composite HCV cDNA comprising the k9-1 sequence 30 and the amino acids encoded therein is shown in Figure 47.

166 DK 175975 B1

The sequence of the amino acids encoded in the 5 'region of the HCV cDNA shown in Figure 47 has been compared to the corresponding region of one of the Dengue virus strands described above with respect to the profile of hydrophobic and hydrophilic regions. This comparison showed that the 5 HCV and Dengue polypeptides encoded in this region that correspond to the region encoding NS1 (or part thereof) have a similar hydrophobic / hydrophilic profile.

The information provided below makes it possible to identify HCV-10 strains. The isolation and characterization of other HCV strains can be performed by isolating the nucleic acids from body constituents containing viral particles, generating cDNA libraries using polynucleotide probes based on the HCV cDNA tubes described below, screening the libraries for clones containing 15 HCV cDNA sequences described below as well as comparing the HCV cDNAs from the novel isolates with the cDNAs described below. The polypeptides encoded therein, or in the viral genome, can be monitored for immunological cross-reactivity using the polypeptides and antibodies described above. Strains that match the HCV parameters, as described in the definition section above, can be immediately identified. Other methods for identifying HCV strains will be apparent to those skilled in the art based on the information provided herein.

Industrial Applicability 25

The invention has in the various embodiments described herein many industrial applications, some of which are described below. The HCV cDNAs Can be used for the construction of probes for detection of HCV nucleic acid in samples. The probes derived from the cDNAs can be used to detect HCV nucleic acid in e.g. chemical synthesis reactions. They can also be used in antiviral screening programs to determine the effect of agents on inhibiting viral replication in cell culture systems and animal model systems. The HCV polynucleotide probes are also well-suited for detecting viral nucleic acids in humans and thus can serve as a basis for diagnosing HCV infections in humans.

5

The cDNAs disclosed herein provide, in addition to the above information, a means for synthesizing polypeptides containing epitopes of HCV. The polypeptides provided by the present invention are well suited for the detection of antibodies to HCV antigens. There are described a number of 10 immunoassays for HCV infection based on recombinant polypeptides containing HCV epitopes, which will find commercial use in the diagnosis of HCV-induced NANBH, for screening blood donors for HCV-caused infectious hepatitis, and also for detection of contaminated blood from infectious blood donors. The viral antigens will also be useful for monitoring the efficacy of antiviral agents in animal model systems. In addition, the polypeptides described herein derived from the HCV cDNAs will be useful as vaccines for the treatment of HCV infections.

The polypeptides derived from the HCV cDNAs are, in addition to the applications mentioned above, suitable for the production of anti-HCV antibodies. Thus, they can be used in anti-HCV vaccines. However, the antibodies produced as a result of immunization with the HCV polypeptides are also suitable for detecting the presence of viral antigens in samples. Thus, they can be used for analysis for production of HCV polypeptides in chemical systems. The anti-HCV antibodies can also be used to monitor the efficacy of antiviral agents in screening programs where these agents are tested in tissue culture systems. They can also be used for passive immunotherapy and for the diagnosis of HCV-induced NANBH by enabling the detection of viral antigens (antigens) in both blood donors and recipients. Another important use for anti-HCV antibodies is for affinity chromatography to purify viruses and viral polypeptides. The purified virus and viral polypeptide preparations can be used in vaccines.

However, the purified virus can also be used to develop cell culture systems in which HCV is replicated.

5

Cell culture systems containing HCV-infected cells can have many purposes. They can be used for relatively large scale production of HCV. which is usually a low-titer virus. The systems may also be useful for elucidating the molecular biology of the virus and lead to the development of antiviral agents.

The cell culture systems will also be suitable for screening for the efficacy of antiviral agents. In addition, HCV permissive cell culture systems are well suited for the production of attenuated strains of HCV.

The anti-HCV antibodies and HCV polypeptides, whether natural or recombinant, can be suitably packaged in assay kits.

The method used to isolate HCV cDNA, which consists of producing a cDNA library derived from an individual's infected tissue, into an expression vector and selecting clones producing the expression products that react immunologically with antibodies in antibody-containing body constituents from other infected individuals and not from uninfected individuals may also be useful for isolating cDNAs derived from other hitherto uncharacterized disease-associated agents constituting a genomic component. This in turn could lead to isolation and characterization of these agents and to diagnostic reagents and vaccines against these other disease-associated agents.

Claims (22)

169 DK 175975 B1 Essentially isolated polypeptide, characterized in that it comprises an HCV antigenic determinant and contains a continuous sequence of at least 5 amino acids encoded as in the sequence of Figure 47 and can be used to detect antibodies to HCV (anti-HCV antibodies) in vitro. . 2. Essentially isolated polypeptide, characterized in that it comprises an HCV antigenic determinant and contains a continuous sequence of at least 10 amino acids encoded as in the sequence of Figure 14 and can be used to detect antibodies to HCV (anti-HCV antibodies). vitro. Essentially isolated polypeptide, characterized in that it comprises an HCV antigenic determinant and contains a continuous sequence of at least 15 amino acids encoded as in the sequence of Figure 26 and can be used to detect antibodies to HCV (anti-HCV antibodies). vitro. Essentially isolated polypeptide, characterized in that it comprises an HCV antigenic determinant and contains a continuous sequence of at least 20 amino acids encoded as in the sequence of Figure 32 and can be used to detect antibodies to HCV (anti-HCV antibodies) in vitro. Essentially isolated polypeptide, characterized in that it comprises an HCV antigenic determinant and contains a continuous sequence of at least 25 amino acids encoded in the Iambda-gt11 library deposited in the American Type Culture Colection (ATCC) under accession number 40394. A polypeptide according to any one of claims 1-5, characterized in that the continuous sequence comprises at least 10 amino acids. 30 170 DK 175975 B1 A polypeptide according to any one of claims 1-5, characterized in that the continuous sequence comprises at least 15 amino acids. A fusion protein characterized in that it comprises a polypeptide according to any one of claims 1-7, wherein the continuous sequence is fused into a non-HCV amino acid sequence selected from the group consisting of super-oxide dismutase sequence, beta-galactosidase sequence, sequence allowing secretion, sequence enabling particle formation, hepatitis B surface precursor, and hepatitis B surface antigen sequence. 10 An immunoassay kit characterized in that it comprises a polypeptide according to any one of claims 1-8 in a suitable container. Immunoassay for in vitro determination of antibodies, characterized in that it comprises the following steps: (a) providing a polypeptide comprising an antigenic determinant capable of binding to anti-HCV antibodies, the antigenic determinant comprising an amino acid sequence encoded by the sequence in Figure 47 and comprising at least 8 amino acids, (b) incubating an in vitro biological sample with the polypeptide under conditions allowing formation of an antibody-antigen complex, and (c) determining whether an antibody-antigen complex containing the polypeptide, is formed. Immunoassay for in vitro determination of antibodies, characterized in that it comprises the following steps: (a) providing a polypeptide comprising an antigenic determinant capable of binding to anti-HCV antibodies, wherein the antigenic determinant comprises a continuous sequence of at least 8 amino acids encoded in (B) incubating an in vitro biological sample with the polypeptide under conditions allowing formation of an antibody-antigen complex, and (c) the Iambda-gt11 cDNA library deposited in the American Type Culture Collection (ATCC) under accession number 40394, 171; ) determining whether an antibody-antigen complex containing the polypeptide is formed. 5 Immunoassay for in vitro determination of antibodies, characterized in that it comprises the following steps: (a) providing a polypeptide comprising an antigenic determinant capable of binding to anti-HCV antibodies, wherein said antigenic determinant 10 comprises an amino acid sequence encoded by the sequence of FIG. 14 and comprising at least 8 amino acids, (b) incubating an in vitro biological sample with the polypeptide under conditions allowing formation of an antibody-antigen complex, and (c) determining whether an antibody-antigen complex containing the polypeptide is formed. Immunoassay for in vitro determination of antibodies, characterized in that it comprises the following steps: (a) providing a polypeptide comprising an antigenic determinant 20 capable of binding to anti-HCV antibodies, the antigenic determinant comprising an amino acid sequence encoded by the sequence of FIG. 26 and comprising at least 8 amino acids, (b) incubating an in vitro biological sample with the polypeptide under conditions allowing formation of an antibody-antigen complex, and (c) determining whether an antibody-antigen complex containing the polypeptide is formed. Immunoassay for in vitro determination of antibodies, characterized in that it comprises the following steps: (a) providing a polypeptide comprising an antigenic determinant capable of binding to anti-HCV antibodies, wherein said antigenic determinant comprises an amino acid sequence encoded of the sequence of Figure 32 and comprising at least 8 amino acids, (b) incubating an in vitro biological sample with the polypeptide under conditions allowing formation of an antibody-antigen complex, and (c) determining whether an antibody-antigen complex containing the polypeptide is formed. An antibody composition characterized in that it is an anti-HCV antibody composition comprising antibodies which bind to an HCV antigen determinant in a polypeptide according to any one of claims 1-8, and which antibodies are (a) a pure product of polyclonal antibodies. , (b) a product of monoclonal antibodies. Composition according to claim 15, characterized in that the anti-HCV 15 antibodies are bound to a solid substrate. Immunoassay kit, characterized in that it comprises a polypeptide according to claim 15 or 16 in a suitable container. An immunoassay characterized in that it detects the presence of HCV antigen in a sample, comprising: (a) providing an anti-HCV antibody composition according to claim 15 or 16, (b) incubating a biological sample with anti-HCV the antibody composition under conditions which allow formation of an antibody-antigen complex, and (c) determining whether the antibody-antigen complex comprising the anti-HCV antibody is formed. 30 A substantially isolated polypeptide for use in preparing an anti-HCV antibody composition according to claim 15, characterized in that it comprises HCV antigenic determinant and contains a continuous sequence of at least 8 amino acids encoded as in the sequence of Figure 47. . A substantially isolated polypeptide for use in preparing an anti-5 HCV antibody composition according to claim 15, characterized in that it comprises HCV antigen determinant and contains a continuous sequence of at least 8 amino acids encoded as in the sequence of Figure 14. A substantially isolated polypeptide for use in preparing an anti-10 HCV antibody composition according to claim 15, characterized in that it comprises HCV antigen determinant and contains a continuous sequence of at least 8 amino acids encoded as in the sequence of Figure 26. A substantially isolated polypeptide for use in preparing an anti-15 HVC antibody composition according to claim 15, characterized in that it comprises HCV antigen determinant and contains a continuous sequence of at least 8 amino acids encoded as in the sequence of Figure 32.