PT89041B - METHOD OF PREPARATION OF NANBV DIAGNOSTICS AND VACCINES - Google Patents

Abstract

Purified hepatitis C virus (HCV) and purified or recombinant HCV nucleic acids and polypeptides are claimed. Also claimed are recombinant HCV nucleic acid vectors and expression systems, cells transformed with them, anti-HCV antibodies, and HCV-infected cells grown in tissue culture.

Description

VIVIRIDAE (Seriss eds, Fraenkel-Conrat and Wagner, vol. Eds, Schlesinger and Schlesinger, Plenurn Press), pp. 327-374.

Broach (1981) in: Molecular Biology of the East 5accharomyees Vol 1, p, 445, Cold Spring Harbor Press,

Broach et ^ 1. (1983), Meth. Enz. 101: 307.

Chang et al, (1989), Nature 198-1056.

Chirgwin et al, (l979), Biochernistry 18; 5294.

Chomczynski and Sacchi (1987), Analytical Biochernistry 162: 156. Clewell et al. , (1969), Proc. Natl. Acad. Sci, U.S., 62; 1159 «Clewell (1972), J, Baoteriol. 110: 667.

Cohen (1972), Proc. Natl, Acad. Know. U.S., 69: 2110.

Cousens et al, (1987), gene 6l: -265 «

De Boer et al. (1983), Prop. Natl. Acad, Sei. U.S. 292: 128. Dreesman et al. (1985), J. Infect. Disease 151 - 76l.

Feinstone, S.M. and Hoofnagle, J.H. (1984), New Engl. J. Med,

331: 185.

Fields & Knipe (1986), FUNDAMENTAL VIROLOGY (Raven Press, N.Y.) Fiers et al. (1978), Nature 273: 113.

Gerety, R.J. et al., In VIRAL HEPATITIS AND LIVER DISEASE (Vyas, B.N., Dienstag, J.L., and Hoofnagle, J.H., eds. Grune and Stratton, Inc., 1984) pp. 23-47.

Goeddel et al, (ΐ9θθ), Nucleic Acids Res. 8: 4-057,

Graham and Van der Eb (1978), Virology 52 ² : 546.

Grunstein and Hogness (1975), Proc. Natl. Acad. I know, USA, A. 73? : 39ol.

Grych et al, (1985), Nature 316: 74,

Gubler and Hoffman (1983), Gene 2: 263.

Hammerling * et al. (1998), MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOWAS.

Hess et al. (1968), J. Adv. Enzyme Reg (7,: 149.

Hinnen et al, (ΐ97θ) »Proc. Natl. Acad, Sci., 75: 1929.

Hitzeman et al (1980), J. Biol, Chem, 255: 5073.

Holland et al. (1978), Biochemistry 17; 4900 ♦

Holland (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; PRACTI

CAL APPROACH (D. Glover, Ed., IRL Press, Oxford, United Kingdom) pgs. 49-78.

Immun, Rev, (1982) 62; 1885.

Iwarson (1987), British Medical J, 295: 946 ..

Kennett et al. (1980) MONOCLONAL ANTIBODIES,

Laemmli (1970), Nature 227, 680,

Lee et al, (1988), Science 239: 1288.

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

Mayer and Walker, eds, (198?), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London).

Maxam et al, (ΐ9θθ), Methods in Enzymology 65; 499 ·

MacNamara et al. (1984), Science 226: 1353.

Messing et al, (ΐ9θΐ), Nucleic Acids Res, 2. ^{ 3θ9 ·

Messing (1983), Methods in Enzymology 101; 20-37 »

METHODS IN ENZYMOLOGY (Academic Press),

Michelle et al., Int. Symposium on Virai Hepatitis,

Monath (1986) in THE VIRUSES: THE TOGAVIRADAE AND FLAVIVIRIDAE (Series eds, Praenkel-Conrat and Wagner, vol.eds, Schlesinger and Schlesinger, Plenum Press), pp. 375-440. Nagahuma et al. (1984), Anal. Biochem, 14; 74,

Neurath et al. (1984), Science 224; 392.

Nisonoff et al, (1998), Clin. Immunol. Immunopathol. 21: 397-406.

Overby, L.R. (1985), Curr. Hepatol. 5:49.

Overby, L.R. (1986), Curr, Hepatol. 6:65,

Overby, L.R. (1987), Curr, Hepatol, 7:35.

Peleg (1969), Nature 221: 193.

Pfefferkorn and Shapiro (1974), in COMPREHENSIVE VTROLOGY, Vol, (Fraenkel-Conrat & Wagner, eds ,, Plenum, N.Y.) p. 171

-23O.

Prince, A.M. (1983) »Annu. Rev. Microbiol, 37: 217.

Rice et al. (1986), in THE 'VIRUSES: THE TOGAVIRIDAE ANI) FLAVI

VIRIDAE (Series eds. Fraenkel-Conrat & Wagner, vol. Eds.

Schlesinger and Schlesinger, Plenum Press), pp. 279-328. Roehrig (1986) in THE VIRUSES; THE TOGAVIRIDAE AND FLAVIVIRIDAE (Series eds, Fraenkel-Conrat & Wagner, vol. Eds. Schle singer and Schlesinger, Plenum Press).

Sadler et al. (1980), Gene 8, 279.

Saiki et al. (1986), Nature 3241163,

Sanger et al, (1977), Proc, Natl. Acad. Scl, USA, 74: 5463. Schlesinger et al. (1986), J, Virol »60: 1153 ♦

Schreier, M., et al, (ΐ9θθ) HYBRIDOMA TECHNIQUES.

Scopes (1984), PROTEIN PURJFICATION, PRINCIPLES AND PRACTICE,

SECOND EDITION (Springer-Verlag, N, Y.).

Shimatake et al. (1998), Nature 292; 128.

Steimer et al. (1986), J. Virol. 58: 9.

Stollar (ΐ98θ), in THE TOGAVIRUSES (R.W.Schlinger, ed, Aca demic Press, N.Y.) pages, 584-622,

Taylor et al, (1976), Biochem, Biophys. Act 442: 324,

Towbin et al. (1979), Proc. Natl. Acad. Sei, USA, A, 76, 4350, Tsu and Herzenherg (1980), in SELECTED METHODS IN CELLULAR WIU

NOLOGY (W.H, Freeman and Co.) p. 373-391.

Vytdehaag et al, (1985), J. Immunol. 134: 1225,

Valenzuela, P., et al. (1982), Nature 298: 344.

Valenzuela, P. ,, et al. (1984), in HEPATITIS B (Millman, I ,, et al., Ed, Plenum Press) pages, 225-236,

Warner (1984), DNA 3.:401,

Wu and Grossman (1987), Methods in Enzymology Vol, 154, RECQMBI

NANT DNA, Part E.

Wu (1987), Methods in Enzymology vol. 155, RECOMBINANT DNA, part F,

Zoller (1982), Nucleic Acids Res, 10: 6487,

Cited Patents

Patent

Patent

Patent

Patent

Patent

Patent

Patent

US Patent No, North American No, North American No, North American No.

North American No.

North American No, North American No.

North American No.

4,341,761 4,399,121 4,427,783 4,444,887 4,466,917 4,472,5OO 4,491,632 4,493,890

Prior Art

Hepatitis Non-A and NSo-B (NANBH) is a disease or family

of communicable diseases believed to be induced by viruses, which are distinguishable from other forms of liver disease associated with viruses, including those caused by the known hepatitis viruses, that is, hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis delta virus (HDV), as well as cytomegalovirus-induced hepatitis (CMV) or Epstein-Barr virus (EBV), NANBH was first identified in inoculated individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provides evidence that NANBH is due to a transmissible infectious agent or agents. However, the responsible transmissible agent p <e t

la NANBH is not yet identified and the number of agents that cause the disease is unknown,

Epidemiological evidence suggests that there can be three types of NANBH: the epidemic type carried by water; the type associated with blood or needle; and the type (community acquired) that occurs sporadically. However, the number of agents that can cause NANBH is unknown.

The clinical diagnosis and identification of NANBH has been carried out mainly by excluding other viral markers. Among the methods used to detect supposed NANBH antigens and antibodies are agar-gel diffusion, counterimmunoelectrophoresis, immunofluorescence microscopy, immune electron microscopy, radioimmunity assay and enzyme-linked immunosorbent assay. However, none of these tests proved to be sufficiently sensitive, specific and

- 6 reproducible to be used as a diagnostic test for NANBH.

So far there has been no clarity or agreement regarding the identity or specificity of the antibody antigen systems associated with NANBH agents. This is due, at least in part, to the previous or co-infection of HBV with NANBV in individuals, and to the known complexity of the soluble, 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 may be caused by more than one infectious agent, as well as the possibility that NANBH was undiagnosed. Furthermore, it is not clear that serological tests detect in the serum of patients with NANBH. It has been admitted that agar-gel diffusion assays and electron counterimmunoassay detect aytoimmune responses or non-specific protein interactions that sometimes occur between so, ro specimens, and that they do not represent specific antigenic reactions. NANBH antibody. Immunofluorescence, enzyme-linked immunosorbent, and radioimmunity assays appear to detect low levels of a material similar to the rheumatoid factor. that it is often present in the serum of patients with NANBH, as well as in patients with liver and non-liver disease, part of the detected reactivity may represent an antibody to cytoplasmic antigens of a given host.

There are a number of NANBV candidates. See, for example, the magazines by Prince (1983), Feinstone and Hoof nagle (1984), and Overby (1985, 1986, 19θ7) θ the article by Iwar- 7 a son

(1987). However, there is no evidence that any of these candidates represents the etiological agent of NANBH,

The search for specific, sensitive methods for screening to identify NANBV vehicles and NANBV-contaminated blood or blood products is significant. Post-transfusion hepa titis (PTH) occurs in approximately 10% of inoculated patients, and NANBH is limited to 90% of these cases. The biggest problem in this disease is the frequent progression to chronic liver damage (25- $ 55),

Care for the patient as well as the prevention of transmission of NANBH by blood and blood products or by close personal contact requires safe diagnosis and prognostic tools to detect NANBV-related nucleic acids, antigens and antibodies. In addition, there is also a need effective vaccines and therapeutic immunotherapeutic agents for the prevention and / or treatment of the disease.

Disclosure of the Invention

The invention concerns the isolation and characterization of a recently discovered etiological agent of NANBH, hepatitis virus, C (HCV). More specifically, the invention provides a family of cDNA replicas of portions of the HCV genome. These cDNA replicates were isolated by a technique; that included a new phase of scresning of expression products from cDNA libraries created from a particulate agent in tissue infected with sera from patients with NANBH, to detect newly synthesized antigens derived from the genome of the viral agent so far not isolated and not characterized, and the chosen clones which produce products that react immunologically only as sera from infected individuals, compared to uninfected individuals.

Studies of the nature of the HCV genome, using probes derived from HCV cDNA, as well as sequence information contained within the HCV cDNA, are suggestive that HCV is a Flavivirus or a Flavi virus analog.

Portions of HCV-derived cDNA sequences are useful as probes for diagnosing the presence of viruses in samples, and for isolating naturally occurring virus variants. These cDNA’s also make sequences of

I HCV antigen polypeptide encoded within the genome (s) and allow the production of polypeptides that are useful as standards or reagents in diagnostic tests and / or as components of vaccines. Antibodies, both polyclonal and monoclonal, directed against HCV epitopes contained within these polypeptide sequences are also useful for diagnostic testing, as therapeutic agents, for screening of antiviral agents, and for the isolation of the NANBV agent from which these cDNAs are found. * s derive. In addition, using probes derived from these cDNA's it is possible to isolate and sequence other portions of the HCV genome, thus giving rise to additional probes and polypeptides that are useful in the diagnosis and / or treatment, both prophylactic and therapeutic, of NANBH, ·

Accordingly, in relation to polynucleotides ^

Some aspects of the invention are: a purified HCV polynucleotide; a recombinant HCV polynucleotide; a recombinant polynucleotide that comprises a sequence derived from the HCV genome or HCV cDNA; a recombinant polynucleotide encoding an HCV epitope; a recombinant vector containing any of the above recombinant polynucleotides, and a host cell transformed by any of these vectors.

Other aspects of the invention are: a recombinant expression system comprising an open reading structure (ORF) of DNA derived from an HCV genome or HCV cDNA, in which the ORF is operably linked to a t sequence

control compatible with a desired host, a cell transformed by the recombinant expression system, and a polypeptide produced by the transformed cell.

Still other aspects of the invention are: purified HCV, a polypeptide preparation from purified HCV; a purified HCV polypeptide; a purified polypeptide comprising an epitope that is immunologically identifiable with an epitope contained in HCV,

Aspects included in the invention are a recombinant HCV polypeptide; a recombinant polypeptide composed of a sequence derived from an HCV genome or HCV cDNA. _a recombinant polypeptide consisting of an HCV epitope; and a fusion polypeptide consisting of an HCV polypeptide.

Also included in the invention are a monoclonal antibody directed against an HCV epitope; and a purified preparation of polyclonal antibodies directed against an HCV epitope.

Another aspect of the invention is a particle that is immunogenic against HCV infection comprising a non-HCV polypeptide having an amino acid sequence capable of forming a particle when said sequence is produced in a eukaryotic host, and an HCV epitope,

Yet another aspect of the invention is a polynucleotide probe for HCV,

Aspects of the invention that belong to sets are those intended for: analysis samples for the presence of HCV-derived polynucleotides comprising a polynucleotide probe, containing an HCV nucleotide sequence of about 8 or more nucleotides, in a suitable container ; analysis samples for the presence of an HCV antigen comprising an antibody directed against the HCV antigen to be detected, in a suitable container; analysis samples for the presence of an antibody directed against an HCV antigen comprising a polypeptide containing an HCV epitope present on the HCV antigen, in a suitable container.

Other aspects of the invention are: a polypeptide comprising an HCV epitope attached to a solid substrate; and an antibody to an HCV epitope, attached to a soli substrate. of.

Still other aspects of the invention are: a method for producing a polypeptide containing an HCV epitope comprising incubation host cells transformed with an expression vector containing a sequence encoding a polypeptide containing an HCV epitope under conditions that allow expression of the said polypeptide; and a polypeptide containing an HCV epitope produced by this method.

The invention also includes a method for detecting HCV nucleic acids in a sample that includes reactive nucleic acids from the sample with a probe for an HCV polynucleotide, under conditions that allow the formation of a duplex polynucleotide between the probe and the HCV nucleic acid. , from the sample, and detecting a duplex polynucleotide that contains the probe.

Immunoassays are also included in the invention. These include an immunoassay to detect an HCV antigen which comprises incubating a sample suspected of containing an HCV antigen with a probe antibody directed against the HCV antigen to be detected, under conditions that allow formation of an antigen-antibody complex and detecting an antigen-antibody complex containing the probe antibody. An immunoassay to detect antibodies directed against an HCV antigen comprising incubating a sample suspected of containing anti-HCV antibodies with a probe polypeptide that contains an HCV epitope, under conditions that allow the formation of an antibody-antigen complex; and detecting the antibody-antigen complex that contains the probe antigen.

Also included in the invention are vaccines for treating HCV infection comprising an immunogenic peptide containing an HCV epitope, or an inactivated HCV preparation, or an attenuated HCV preparation.

Another aspect of the invention is a cell developed in tissue culture, infected with HCV,

Yet another aspect of the invention is a method for producing antibodies to HCV which comprises administering to an individual an isolated immunogenic polypeptide containing an HCV epitope, in an amount sufficient to produce an immune response.

Another aspect of the invention is a method for isolating cDNA derived from the genome of an unidentified infectious agent, comprising: (a) providing host cells transformed with expression vectors containing a cDNA library prepared from nucleic acids isolated from infected tissue with the agent _? and developing said host cells under conditions that allow expression of the polypeptide (s) encoded in the cDNA; (b) interacting the cDNA expression products with an antibody containing the body component of an individual infected with said infectious agent, under conditions that allow an immunoreaction, and detecting antibody-antigen complexes formed as a result of the interaction; (c) developing host cells that express polypeptides that form antibody-antigen complexes in step (b) under conditions that allow them to grow as individual clones and isolate said clones; (d) developing cells from clones of (c) under conditions that allow expression of encoded polypeptide (s) within the cDNA, and interacting with antibody expression products containing body components from different individuals the individual in stage (a) who are infected with the infectious agent and control subjects not infected with the agent, and detecting antibody-antigen complexes formed as a result of the interaction; (e) to develop host cells that express polypeptides that form antibody-antigen complexes with an antibody containing body components of infected individuals and individuals Suspected of being infected, and not with the said components of control individuals, under conditions that allow the their development as individual clones, and isolating said clones; and (f) isolating such cDNA from clones of the host cell of (e).

Brief Description of Drawings

Fig. 1 shows the HCV cDNA double fiber nucleotide sequence inserted in clone 5-1-1, and the putative amino acid sequence of the polypeptide encoded therein.

Fig. 2 shows the liomologies of the HCV cDNA sequences that overlap in clones 5-1-1, 81, 1-2, and 91.

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

Fig. 4 shows the duplex fiber nucleotide sequence of the HCV cDNA inserted in clone 81, and the pivotal sequence of the amino acid of the polypeptide encoded therein,

Fig. 5 shows the HCV cDNA sequence in clone 36, the segment that overlaps the NANBV cDNA of clone 8l, and the polypeptide sequence encoded within clone 36,

Fig. 6 shows the combined ORF of HCV cDNAs in clones 36 and 8l, and the polypeptide encoded in them,

Fig. 7 shows the HCV cDNA sequence in clone 32, the segment that overlaps clone 81, and the co polypeptide. in them.

Fig. 8 shows the HCV cDNA sequence in clone 35, the segment that overlaps with clone 36, and the codified polypeptide in the medians.

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

Fig. 10 shows the HCV cDNA sequence in clone 37b, the segment that overlaps clone 35, θ the polypeptide encoded in them,

Fig. 11 shows the HCV cDNA sequence in clone 33b, the segment that overlaps clone 32, and the polypeptide encoded therein,

Fig. 12 shows the HCV cDNA sequence in clone 40b, the segment that overlaps with clone 37b, and the polypeptide encoded therein,

Fig. 13 shows the HCV cDNA sequence in clone 25c, the segment that overlaps clone 33b, and the polypeptide encoded therein.

Fig. 14 shows the nucleotide sequence and the polypeptide encoded in the same ORF extending through HCV cDNAs in clones 40b, 37b, 35, 36, 8l, 32, 33b, and 25c.

Fig. 15 shows the HCV cDNA sequence in clone 33c, the segment that overlaps clones 40b and 33c, and amd. noacids encoded in them,

Fig. 16 shows the HCV cDNA sequence in clone 8h, the segment that overlaps clone 33c, and the amino acids encoded in them,

Fig. 17 shows the HCV cDNA sequence in the clone

7θ, the segment that overlaps clone 8h, and the amino acids encoded in them.

Fig. 18 shows the HCV cDNA sequence in clone 14c, the segment that overlaps clone 25c, and the amino acids encoded therein.

Fig. 19 shows the HCV cDNA sequence in clone 8f, the segment that overlaps clone 14c, and the amino acids encoded therein.

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

Fig. 21 shows the HCV cDNA sequence in clone 33g, the segment that overlaps clone 33f, and the amino acids encoded therein.

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

Fig. 23 shows the HCV cDNA sequence in clone 11b, the segment that overlaps the sequence in clone 7f, θ the amino acids encoded therein.

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

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

Figure 26 shows a composite HCV cDNA sequence derived from aligned cDNAs in clones 14i, 11b, 7f, 7®,

8h, 33c, 40b, 37b, 35, 36, 8l, 32, 33b, 25c, Luc, 8f, 33f, and is also shown 33gJ polipe ^the amino acid sequence encoded £ taken in the extended ORF in the derived sequence.

Fig. 27 shows the HCV cDNA sequence in clone 12f, the segment that overlaps clone 14i, and the amino acids. encoded in them, '

Fig. 28 shows the HCV cDNA sequence in heat. ne 35f, the segment that overlaps clone 39c, and the amino acids of those encoded therein.

Fig. 29 shows the «HCV cDNA equation in clg 19g, the segment that overlaps clone 35f, ® the amino acids. encoded in them. '

Fig. 30 shows the sequence of clone? 6g, the segment that overlaps clone 19g, © ^the amino acids encoded in them,

Fig. 31 shows the sequence of clone 15θ, the segment that overlaps clone 26g, and the amino acids oodifi. in them,

Figure 32 shows the sequence in a composite cDNA which was derived by aligning clones 12f to 15®, in the 5 'to 3''it also shows ^the amino acids of the ORF encodes continues,

Fig. 33 shows a photograph of Western blots of a fusion protein, SOD-NANB4 with chimpanzee serum, from chimpanzees infected with B6-NAN8, HAV and HBV.

ί - *

Fig. 34 shows a photograph of Oci spots. teeth of a fusion protein, SOD-NANB ^ _{1 lf} with serum from humans infected with NANBV, HAV, HBV, and from control humans.

Fig. 35 is a map showing the significant characteristics of the pAB24 vector,

Fig. 36 shows the putative amino acid sequence of the carboxy-terminus of the C100-3 fusion polypeptide, and the nucleotide sequence encoding it.

Fig. 37 θ is a photograph of a polyacrylamide gel stained with coomas'sie blue that identifies C100-3 expressed in yeast.

Fig. 37 shows a Western spot of C100-3 with serum from a human infected with NANBV.

Fig. 38 shows an autoradiography of a Northern RNA spot isolated from the liver of an i.n chimpanzee infected with BB-NANBV, probed with BB-NANBV cDNA from clone 8l,

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

Fig. 40 shows an autoradiography of nucleic acids extracted from NANBV particles captured from plasma infected with anti-NANB ^ χ χ ' ^and probed with NANBV cDNA 32 labeled p, from clone 81.

Fig. 4l shows radiographs of filters containing isolated NANBV nucleic acids, probed _with s

DNA probes with more and less classified fibers

3P 'P derived from NANBV cDNA in clone 8l,

Fig. 42 shows the homologies between a polypeptide encoded in HCV cDNA and an NS protein from flavi. Dengue virus,

Fig. 43 shows a histogram of the distribution of HCV infection in random samples, as determined by an ELISA screening,

Fig. 44 shows a · histogram of the distribution of HCV infection in random samples using two conjugated immunoglobulin-enzyme configurations in the ELISA assay,

Fig. 45 shows the sequences in a primary mixture, derived from a conserved sequence in NS1 of flavivirus.

Modes for Carrying Out the Invention

I, Definitions

The expression hepatitis C virus was reserved by professionals in this field for an etiological agent hitherto unknown in NANBH. Accordingly, as used here, hepatitis C virus (HCV) refers to a people that causes NANBH, which was previously referred to as NANBV and / or BB-NANBV, The terms HCV, NANBV, and BB-NANBV they are used interchangeably here. As an extension of this terminology, the disease caused by HCV, previously called NANB hepatitis (NANBH), is called hepatitis C. The terms NANBH and hepatitis C can be used interchangeably here.

HCV, as used herein, denotes a viral species that causes NANBH, and attenuated strains or defective interference particles derived from them. As shown below, the HCV genome is made up of RNA. It is known that RNA containing viruses has speeds relatively him. spontaneous mutations, that is, presumably in the order of 10 to 10 per nucleotide (Fields & Knipe (1986)). Therefore, there are multiple strains within the HCV species described below. The compositions and methods described herein, allow for the propagation, identification, detection, and isolation of the various related strains. In addition, they also allow the preparation of diagnoses and vaccines for the various strains, and are useful in screening processes, for anti-viral agents for pharmacological use, as they inhibit HCV replication.

The information provided here, although derived from that of an HCV strain, hereinafter referred to as CDC / / HCV1, is sufficient to allow a taxonomy to identify other strains that are comprised within the species. As described herein, it was found that HCV is a Flavi virus or a similar flavi virus. Flavivirus particle morphology and composition are known, and are discussed in Brinton (1986). Generally, in terms of morphology, Flaviviruses contain a central nucleoside surrounded by a lipid bilayer. The virions are spherical and have a diameter of about 40-50 nm. Their nuclei are about 25-30 nm in diameter. Along the outer surface of the virion shell are projections that are about 5-10 nm in co-length, with rounded end protrusions about 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; preferably the epitope is encoded in a cDNA described herein, the epitope is unique to HCV when compared to the other known Flaviviruses,

The uniqueness of the epitope can be determined by its immunological reactivity with HCV and lack of immunological reactivity.

I deal with other species of Flavivirus. Methods for determining immune reactivity are known in the art, for example, i

by radioimmunoassay, by Elisa assay, by hemagglutination, and several examples of techniques suitable for assays are provided herein.

In addition to what happens, the following parameters are applicable, either alone or in combination in the identification of a strain as HCV. Since HCV strains are progressively related, tc>

Such genome at the nucleotide level is about 40% or more, preferably about 60% or more, and even more preferably about 80% or more; and in addition, that there are corresponding contiguous sequences of at least about 1'3 nucleotides. The correspondence between the putative HCV genomic sequence and the CDC / CH1 HCV cDNA sequence can be determined by techniques known in the art, For example, they can be determined by a direct comparison of the polynucleotide sequence information from the putative HCV, and the HCV cDNA sequence (s) described herein. For example, too, they can be determined by hybridizing the polynucleotides under conditions that form stable duplexes between homologous regions (for example, those that could be used prior to S4 digestion), followed by digestion with specific nuclease (s) ( s) simple fiber, followed by the determination of the size of the macerated fragments.

Due to the evolutionary relationship of HCV strains, putative HCV strains are identifiable by their polypeptide level, Geralmerite, HCV strains are more than about 40% homologous, preferably more than about 60% homologous, and even more preferably more than about 80 $ ⁵ homologai the polypeptide level. Techniques for determining the homology of the amino acid sequence are known in the art. For example, the amino acid sequence can be determined directly and compared to the sequences provided herein. For example, too, the nucleotide sequence of the putative HCV genomic material can be terminated (usually via a cDNA intermediate); the amino acid sequence encoded therein can be determined, and the corresponding regions compared.

As used herein, a polynucleotide is derived from a designated sequence, for example HCV cDNA, particularly those exemplified in Pigs. 1-32, or from an HCV genome, refers to a polynucleotide sequence that is composed of a sequence of approximately at least about 6 nucleotides, preferably has at least about 8 nucleotides, is preferably at least least about 10-12 nucleotides, and even more preferably at least

about 15-20 corresponding nucleotides, i.e., homologous to / or complementary to, a region of the designated nucleotide sequence. Preferably, the sequence of the ^ region from which the polynucleotide is derived * is homologous to, or complementary to, a sequence that is unique to an HCV genome. It can be determined by techniques known to those of skill in the art whether a sequence is or not unique to the HCV genome. For example, the sequence can be compared with sequences in databases, for example Genebank, to determine whether it is present in the uninfected host or other organisms. The sequence can also be compared with the known sequences of other viral agents, including those that are known to induce hepatitis, for example, HAV, HBV, and HDV, and with other elements of FIVaviviridae. other sequences can also be determined by hybridization under appropriate stringent conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art, and are discussed below. See also, for example, Maniatis et.al, (1982). Furthermore, mismatches of duplex polynucleotides, formed by hybridization can be determined by known techniques, including, for example, digestion with a nuclease, such as Sl, which specifically macerates single fiber areas in polynucleoty. duplex. Regions from which typical DNA sequences can be derived include, but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and / or untranslated regions.

χΟ derived polynucleotide is not necessarily physically derived from the represented nucleotide sequence, but can be generated in any way, including, for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on the information provided by the base sequence in (s) ) region (s) from which the polynucleotide is derived. In addition, combinations of regions that correspond to those of the designated sequence may be hampered by forms known in the art compatible with the utility. intended use.

Similarly, a polypeptide sequence

or amino acid derived from a nucleic acid sequence. as designated, for example, the sequences in Figs. 1-32, or an HCV genome, refers to a polypeptide that has an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof the portion consists of at least. at 3-5 amino acids, and more preferably at least ten-10 amino acids, and even more preferably at least 11-15 amino acids, or that is immunologically identifiable with a polypeptide of the one encoded in the sequence.

A derived recombinant or polypeptide is not necessarily translated from a nucleic acid sequence designated, for example, the sequences in Figs, 1-32, or from an HCV genome; half is generated in any way, including, for example, chemical synthesis, or expression of a recombinant expression system, or isolation from

Mutated HCV.

The expression recombinant polynucleotide as

- 24 9

used here means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation! (l) it is not associated with all or a portion of the polynucleotide with which it is associated, in nature or in the form of a library; and / or (2) is linked to a polynucleotide other than that to which it is linked in nature.

Polynucleotide, as used herein, refers to a polymeric form of nucleotides of any length, whether ribunocleotides or deoxyribunocleotides. This term refers only to the primary structure of the molecule.

t

Thus, this term includes DNA with double and single fiber ^ as well as RNA with double and single fiber. It also includes forms of the polynucleotide modified, for example, by methylation and / or by encapsulation, and unmodified, of the polynucleotide.

As used herein, the term HCV containing a sequence that corresponds to a cDNA means that HCV contains a polynucleotide sequence that is homologous to, or) complementary to, a sequence in the designated DNA; the degree of homology or complementarity of the cDNA will be approximately 50% or more, it will preferably be at least about 70%, and even more preferably it will be at least about 90%. The corresponding sequences will be at least about 70 nucleotides, preferably at least about 80 nucleotides, and even more preferably at least about 90 nucleotides in length. The correspondence between the HCV sequence and the cDNA can be determined by techniques known in the art, including, for example, a direct comparison of the se25 material.

quenced with the described cDNAs, or hybridization and digestion with single fiber nucleases, followed by the determination of the size of the macerated fragments.

The expression purified viral polynucleotide refers to an HCV genome or fragment thereof that is essentially free, that is, containing less than about 50%, preferably less than about 70%, and even more preferably less than about 90% polypeptides with which the well. viral linucleotide is naturally associated. Techniques for purifying viral polynucleotides from viral particles are known in the art, and include for example, breaking the particles with a chaotropic agent, and separating the polynucleotide (s) and polypeptides by ion exchange chromatography, chromatography affinity, and sedimentation according to density.

The term purified viral polypeptide refers to an HCV polypeptide, or fragment thereof, which is essentially free, that is, it contains less than about 50%, preferably less than about 70%, and even more preferably less than about 90% of cellular components with which the viral polypeptide is naturally associated. Techniques for purifying viral polypeptides are known in the art, and examples of these techniques are discussed below,

Recombinant host cells, host cells, cells, cell lines, cell cultures and other similar expressions designating microorganisms or larger eukaryotic cell lines, cultured

as single-celled entities refer to cells that can be, or have been, used as recipients for recombinant vector or other DNA transfer, and include the progeny of the original cell that was transfected. It is understood that the progeny of a simple parent cell may not necessarily be completely identical in morphology or in the genomics or total DNA complement to the original parent, due to the accidental or deliberate mutation, The progeny of the parent cell that is sufficiently similar to that of the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, is included in the progeny intended by this definition, and is covered by the above expressions.

A replicon is any genetic element, for example a plasmid, a chromosome, a virus, which behaves as an autonomous polynucleotide replication unit within a cell; that is, capable of replication under its own control.

A vector is a replicon to which another polynucleotide segment is attached, in order to effect replication and / or expression of the linked segment.

Control sequence refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include a promoter, ribos27-binding site XI υ

evil, and finishers; in eukaryotes, generally, such control sequences include promoters, finalizers and, in some cases, intensifiers. The term control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose advantageous θ presence, for example, guide sequences.

Operatively linked refers to justja i

position in which the components thus described are in a relationship allowing them to function in their intended manner. A control sequence operably linked to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

An open reading frame (ORF) is a region of a polynucleotide sequence that encodes a polypeptide. This region can represent a portion of a coding sequence or a total coding sequence.

A coding sequence is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5'-terminus, and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA , and recombinant polynucleotide sequences.

J

Immunologically identifiable with / as it refers to the presence of epitope (s) and polypeptide (s) that are also present in and are unique to the polypeptide (s), usually called HCV protein, The identity immunological po. de aer determined by antibody binding and / or competition in binding; these techniques are known to those of ordinary skill in the art, and are also illustrated below. The singularity of an epitope can also be determined by computer searches of known databases, for example Genjs bank, for 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 can comprise 3 amino acids in a spatial conformation that is unique to the epitope; generally an epitope consists of at least 5 of such amino acids, and more usually, consists of at least 8-10 of such amino acids. Methods for determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance.

A polypeptide is immunologically reactive with an antibody when it binds to an antibody due to the antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity can be terminated by antibody binding, more particularly by the magnetic binding of the antibody binding, and / or by competition in binding using a known polypeptide or polypeptides as a competitor (s) against which or against the epitope. which the antibody is targeted. 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, for example, chemical synthesis, or expression of the polypeptide in a recombinant organism.

term polypeptide ”refers to a molecular chain of amino acids. and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to post-expressed modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like,

Transformation, as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for insertion, for example, direct survey, transduction, or f-union. The exogenous polynucleotide can be maintained as a vector not integrated, for example, a plasmid or, alternatively, can be integrated into a host genony.

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

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

As used herein, the fiber more than one nucleic acid contains the sequence encoding the polypeptide. Afibra less contains a sequence that is complementary to the sequence of the more fiber.

As used herein, a virus-positive genome with a virus is one in which the genome, whether RNA or DNA, is single-fiber and encodes a viral polypeptide or polypeptides. Examples of RNA-positive fibrous viruses include Togaviridae, Coronaviridae, Retroviridae, Picornaviridae and Caliciviridae, Also included are Flaviviridae, which were previously classified as Togaviridae. See Fields & Knipe (1986).

As used herein, antibody containing body component refers to an individual body component, which is a source of the antibodies of interest. Antibodies containing body components are known in the art, and include, but are not limited to, for example, plasma, serum, spinal fluid, lymphatic fluid, the outer sections of the respiratory, intestinal and genito-urinary tracts, tears, saliva, milk, white blood cells and tnielotnas,

As used herein, purified HCV refers to an HCV preparation that has been isolated from the cellular constituents with which the virus is normally associated, and from other virus species that may be present in the infected tissue. Techniques for isolating viruses are known to those of skill in the art, and include, for example, centrifugation and affinity chromatography; a method for preparing purified HCV is discussed below.

II, Description of the Invention

The practice of the present invention will use, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Such techniques are fully explained in the literature.

See for example, Maniatis, Fitsch & Sambrook, MOLECULAR CLO

NING; A LABORATORY MANUAL (1982); DNA CLONING, VOLUMES I AND II (D. N. Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESI3 (M.J. Gait ed, 1984) 5 NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S,

J. Higgins eds. 1984); TRANSCRIPTION AND TRANSLATION (B.D. Hames & S.J. Higgins eds. 1984); ANIMAL CELL CULTURE (R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (iRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (JH Miller and MP, Calos eds, 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds ,, respectively), Mayer and Walker , eds, (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London) Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, NY), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (DM

Weir and C, C, Blackwell eds. 1986),

All patents, patent applications, and publications mentioned herein, both before and after, are therefore hereby incorporated by reference.

The useful materials and processes 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 to genomic DNA in humans or chimpanzees, from uninfected individuals, since the nucleotides in 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 has no significant homology to sequences contained within the HBV genome.

The sequence of a family member, contained within clone 5-1-1, has a continuous open reading frame (ORF) that encodes a polypeptide of approximately 50 amino acids. Sera from HCV-infected humans contains antibodies that bind to this polypeptide, whereas sera from uninfected humans do not contain antibodies to this polypeptide. Finally, while sera from uninfected chimpanzees does not contain antibodies to this polypeptide, antibodies are induced in chimpanzees following acute NANBH infection.

In addition, antibodies to this polypeptide are not detected in chimpanzees and humans infected with HAV and HBV. By this criterion, the sequence is a cDNA for a viral sequence, i

where the virus causes or is associated with NANBH; this cDNA sequence is shown, in Fig. 1, as discussed below, the cDNA sequence in clone 5-1-1. differs from that of the other isolated cDNAs in that it contains 28 extra base pairs. A composite with other elements identified in the cDNA family, which were isolated using a synthetic sequence equivalent to a cDNA fragment in clone 5-1-1 as a probe, is shown in Fig. 3, A member of the cDNA family that was isolated using a synthetic sequence derived from the cDNA in clone 81 is shown in Fig. 5, θ θ composite of this sequence with that of clone 81 is shown in Fig. 6. Other members of the cDNA family, including those present in clones l4i, llb, 7f, 7e, 8H, 33c, 40b, 37b, 35, 36, 81, 32, 33b,

25c, 14c, 8f, 33f, 33g, θ 39c are described in Section IV.À. A composite of the cDNAs in these clones is described in Section IV.A.18, l

and shown in Fig. 26. The cDNA composite shows that it contains a continuous ORF, and thus encodes a polyprotein. This finding is consistent with the suggestion, discussed later, that HCV is a flavivirus or flavi virus analog.

The availability of this family of cDNAS shown in Figs. 1-32, inclusive ', allows the construction of DNA probes and polypeptides useful for diagnosing NANBH due to HCV infection, and in the separation of blood donors as well as donated blood and blood products for infection. For example,

I from the sequences it is possible to synthesize DNA oligomers fljB CBifCA - 8-10 nucleotides, or larger, which are useful as hybridization probes to detect the presence of the viral genome in, for example, sera from subjects suspected of harboring the virus, or for screening donated blood for the presence of the virus, The cDNA sequence family also allows the design and production of specific HCV polypeptides that are useful as diagnostic reagents for the presence of antibodies raised during NANBH, Antibodies to purified derived polypeptides of cDNAs can also be used to detect viral antigens in infected individuals and in the blood, knowledge of these cDNA sequences also allows the design and production of polypeptides that can be used as vaccines against HCV and also for the production of antibodies, which in turn can be used for protection against disease, and / or for therapy of individuals infected with HCV.

In addition, the cDNA sequence family also allows for the characterization of the HCV genome. Polynucleotide probes derived from these sequences can be used for screening ¹ cDNA library for additional overlapping of cDNA sequences, which, in turn, can be used to obtain more overlapping sequences. Unless the genome is segmented, and the segments have no common sequences, this technique can be used to gain the total genome sequence. However, if the genome is segmented, other segments of the genome can be obtained by repeating the lambda-gtll serological screening process used to isolate the cDNA clones described herein, or alternatively isolating the genome from purified HCV particles.

The cDNA sequence family and the _A polypeptides derived from these sequences, as well as the antibodies directed against these polypeptides are also useful in the isolation and identification of the BB-NANBV agent (s). For example, antibodies directed against HCV epitopes dotted on polypeptides derived from the cDNAs can be used in processes based on chromatographic affinity to isolate the virus. Anternatively, antibodies can be used to identify viral particles isolated by other techniques. The viral antigens and the genetic material within the isolated viral particles can then be further characterized.

The information obtained from subsequent sequencing of the HCV genome (s), as well as from subsequent characterization of HCV antigens and characterization of the genome, allows the design and synthesis of additional probes and polypeptides and antibodies that can be used for diagnosis, prevention, and therapy for NANBH induced by

HCV, and for screening infected blood and related blood products,

The availability of probes for HCV, including antigens and antibodies, and polynucleotides derived from ge-. noma from which the cDNA family is derived also allows the development of tissue culture systems that will be the main use in elucidating HCV biology. This, in turn, can lead to the development of new treatment regimes based on · In antiviral compounds

I which preferably inhibit the replication of, or infection by,

HCV, t

The method used to identify and isolate the etiologic agent for NANBH is new, and the same may be applicable to the identification and / or isolation of agents not characterized so far that contain a genera, and that are associated with a variety of diseases, including those induced by viruses, viroids, bacteria, fungi and parasites. In this method, a cDNA library was created from nucleic acids present in infected tissue from an infected individual.

The librarian was created in a vector that allowed the expression of polypeptides encoded in the cDNA, Clones of host cells containing the vector, which expresses an immunologically reactive fragment of a polypeptide of the etiologic agent, were selected by immunological screening of the expression products of the librarian with an antibody containing body component from another individual previously infected with the putative agent. The stages in the immunological screening technique include inter-cloning the expression products of the cDNA containing vectors with the antibody that contains the body component of a second infected individual and detecting the formation of antibody-antigen complexes between the ) expression product (s) and antibodies from the second infected individual. The isolated clones are then immunologically sieved by interaction of their expression products with the antibody containing body components of other individuals infected with the putative agent and with control subjects not infected with the putative agent, and detecting the formation of antigen-complexes. antibody with antibodies from infected individuals; the vectors containing. cDNAs encoding polypeptides that react immunologically with antibodies from infected individuals and individuals suspected of being infected with the agent, but not with control individuals, are isolated. The infected individuals used for the construction of the cDNA library, and for immunological oscraening ”do not need to be of the same species.

The cDNAs isolated as a result of this method, and their expression products, and antibodies directed against the expression products, are useful in characterizing and / or capturing the etiologic agent. As described in more detail below, this method has been used successfully to isolate a family of cDNAs derived from the HCV genome.

II, Α. Preparation of the cDNA Sequence

Serum collected from a chimpanzee with chronic HCV infection and containing a high virus titer, that is, at least 10 infectious doses of chimpanzee / ml (ICD / ml) was used to isolate viral particles; Nucleic acids isolated from these particles were used as a standard in the construction of a cDNA · library for the viral genome. The procedures for the isolation of putative HCV particles and for the construction of the lambda-gtll cDNA library are discussed in Section IV.A.l. Lambda-gt11 is a vector which has been developed specifically to express insidious cDNAs as beta-galactosidase fusion polypeptides and to screen large numbers of recombinant anti-phage. 'specific adsorbs raised against a defined antigen,

The lambda-gtll cDNA library generated from a cDNA crop containing approximately 200 base pair medium size cDNA was sieved to encode epitopes that can specifically bind with sera derived from patients who had previously experienced NANB hepatitis, Huynh, TV et al , (1985). Screening was performed at approximately 10 ^ fages, and 5 positive fages were identified purified, and then tested for specificity of binding to sera, from different humans and chimpanzees previously infected with the HCV agent. One of the fages, 5-1-1, bound 5 of the 8 human sera tested. This link appears to be selective for sera derived from patients with previous NANB hepatitis infections, since sera from 7 normal blood donors did not show such a link.

The sequence of recombinant phage 5-1-1 cDNA has been determined, and is represented in Fig. 1. The polypeptide encoded by this cloned cDNA, which is in the same translational structure as the N-terminal beta-Galactosidase half of the polypeptide of fusion is represented above the nucleotide sequence. This translational ORF, therefore, encodes an epitope (s) specifically recognized by sera from patients with NANB hepatitis infections.

The availability of the cDNA in the recombinant phage 5-1-1 allowed isolation of other clones containing additional segments and / or alternative segments of cDNA up to the viral genome. The lambda-gt11 cDNA library above was sieved using synthetic polynucleotide derivative from the cloned 5-1-1 cDNA sequence. This screening provided three other clones, which were identified as 81, 1-2 and 91; the cDNAs contained within these clones were sequenced. See Sections IV.A.3 and IV, A.4, The homologies between the four independent clones are shown in Fig. 2, where the homologies are indicated by the vertical lines. Nucleotide sequences present only in clones 5-1-1, 8l, and 91 are indicated by lowercase letters.

The cloned cDNAs present in recombinant enifages in clones 5-1-1, 8l, 1-2, and 91 are highly homologous and differ only in two regions. First, the nude

Cleftide number 67 in clone 1-2 is a thymidine, while the other three clones contain a cytidine residue at this position. This substitution, however, does not alter the nature of the encoded amino acid.

The second difference between clones is that clone 5-1-1 contains 28 base pairs in its 5'-terminus that are not present in the other clones. The extra sequence p £ being a cloning artifact ^first 5 'terminal;5'-terminal clonan artifacts are commonly seen in all cDNA products.

Synthetic sequences derived from the 5 'and 3' region of the HCV rio clone 8l cDNA were used to sieve and isolate cDNAs from the lambda-gtll library NANBV cDNA, which overlaps clone 8l cDNA (Section IV.A.5, ). The sequences of the resulting cDNAs, which are in clone 36 and clone 32, respectively, are represented in Fig. 5 ® in Fig, 7 ·

Similarly, a syntactic polynucleotide, based on the 5 'region of clone 36, was used for screening and to isolate lambda gt-11 NANBV cDNA cDNAs that overlap with 36 cDNA clone (Section IV.A.8J. One purified cDNA clone containing recombinant phage which, hybridized to the synthetic polynucleotide probe, was designated clone 35, and the NANBV cDNA sequence contained in this clone, is shown in Figure 8.

Using the technique of isolating the overlapping cDNA sequences, clones containing HCV cDNA sequences upstream and downstream were obtained. The isolation of these clones is described later in Section IV.A

Analysis of the nucleotide sequences of the HCV cDNAs encoded within the isolated clones shows that composite cDNA contains a long continuous ORF. Fig. 26 shows the sequence of the composite cDNA from these clones, together with the putative HCV polypeptide encoded in the system.

The description of the method for restoring cDNA sequences is, in most cases, of historical interest. The resulting sequences (and their complements) are provided herein, and the sequences, or any portion thereof, can be prepared using synthetic methods, or by a combination of synthetic methods with restoration of partial sequences using methods similar to those described herein.

Lambda-gtll strains replicated from the HCV cDNA library and from clones 5-1-1, 81,

1-2 and 91 were deposited under the terms of the Treaty of Budapejste at the American Type Culture Collection (àTCC), 12301 Park lawn Dr., Rockville, Marilândia 20852, and received the following Accession Numbers.

Lambda-gtll ATCC No. Paw of Deposit

Library HCV cDNA 40394 Dec 1 1987 clone 81 40388 Nov 17, 1987 clone 91 40389 Nov 17, 1987 clone 1-2 '40390 Nov 17, 1987 clone 5-1-1 40391 Nov. 18, 1987

After granting and publishing this application as a United States Patent, all restrictions on the availability of these deposits will be irrevocably removed, and access to designated deposits will be available pending the aforementioned application, by someone determined by the Commissioner, to authorized under the code 37 CFR l.l4 and 35 USC 1.22. In addition, designated deposits will be held for a period of thirty (30) years from the date of deposit, or for five (5) years after the last filing petition; or during the mandatory life of the US patent, whatever its duration. These deposits and other materials deposited herein are intended for convenience only, and are not required for the practice of the present invention in view of the present description. HCV cDNA sequences in all deposited materials are hereby incorporated by reference.

The above description of the walking of the genome, isolating overlapping cDNA sequences from the HCV lambda gt-11 library provides a method by which cDNAs corresponding to the total HCV genome can be isolated. However, given the information provided here, other methods for isolating these cDNAs are obvious to anyone skilled in the art. Some of these methods are described in Section IV.A, below.

II.B. Preparation of Polypeptides and Viral Fragments

The availability of cDNA sequences, both those isolated using cDNA sequences in

Figs. 1-32, as discussed later, as well as the cDNA sequences in these figures, allows the construction of expression vectors antigenically encoding active regions of the polypeptide encoded in its fiber. · These antigenically active regions can be derived from coat or envelope antigens or core antigens, including, for example, polynucleotide binding proteins, polynucleotide polymerase (s), and other viral proteins required for virus particle replication and / or assembly. Fragments encoding the desired polypeptides are derived from cDNA clones using conventional restriction maceration or by synthetic methods, and are linked in vector that can, for example, contain portions of fusion sequences such as beta-Galactosidase or superoxide dismutase (SOD ), preferably SOD, Methods and vectors that are useful for the production of polypeptides that contain SOD fusion sequences are described in Publi. European Patent Application number 0196056, published on October 1, 1986. Vectors encoding fu polypeptides are SOD and HCV polypeptides, i.e. NANE ^^, ΝΑΝΒ _θ1 , and C100-3, which are encoded in a composite of HCV cDNAs , are described in Sections IV, Bl, IV, B, 2, and IV.B, 4, respectively. Any desired portion of HCV cDNA containing an open reading frame, in either fiber direction, can be obtained as a recombinant polypeptide, such as a mature or fusion protein; alternatively, a polypeptide encoded in the cDNA can be provided by chemical synthesis.

DNA encoding the desired polypeptide, either in the fused or matured form, and whether or not it contains one.

signal sequence to allow secretion, can be linked in expression vectors to any suitable host. Both eukaryotic and prokaryotic host systems are presently used in the formation of recombinant polypeptides, and a summary of some of the most common control systems and host cell lines is given in Section III, A., below, the polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent necessary for its intended use. Purification can be carried out by techniques known in the art, for example, salt fractionation, chromatography on heat exchange resins. ions, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology, for a variety of methods for protein purification. Such polypeptides can be used as dialysis, or those that give rise to neutralization of antibodies can be formulated in vaccines. Antibodies raised against these polypeptides can also be used as diagnostics, or for passive immunotherapy. In addition, as discussed in Section II.J. further, antibodies to these polypeptides are useful for isolating and identifying HCV particles.

HCV antigens can also be isolated from HCV virions. Virions can be germinated in HCV infected cells in tissue culture, or in an infected host.

II. C. Preparation of Antigenic Polypeptides and Conjugation with a Vehicle

An antigenic region of a polypeptide is generally relatively small - typically 8 to 10 amino acids, or less, in length. Fragments as small as 5 amino acids can characterize an antigenic region. These segments can correspond to regions of the HCV antigen. Accordingly, using HCV cDNAs as a base,

DNAs encoding short segments of HCV polypeptides can be expressed recombinantly, either as fusion proteins or as isolated polypeptides. In addition, short amino acid sequences can be conveniently obtained by chemical synthesis. In cases where the synthesized polypeptide is correctly configured to provide the correct epitope, but is too 'small for its immunogenicity, the polypeptide can be linked to a suitable vehicle.

A number of techniques for obtaining such a bond are known in the art, including the formation of disulfide bonds using N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC) obtained from Pierce Company, Rockford, Illinois, (if the peptide does not have a sulfhydryl group, this can be provided by the addition of a cysteine residue). These reagents create a disulfide bond between themselves, and peptide cysteine residues on one protein, and an amide bond via epsilon-amino on one lysine, or other free amino group on the other. A variety of such disulfide / amide forming agents are known, see, for example, Immun. Rev, (1982) 02.:185. Other bifunctional linkers form a thioether instead of a disulfide bond. Many of these thioether forming agents are commercially available and include reactive esters of 6-ma.leimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, and analogues. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The above list is not intended to be exhaustive, and modifications of the compounds mentioned can clearly be used.

Any vehicle can be used that does not itself induce the production of antibodies harmful to the host. Suitable vehicles are typically large macromolecules, slowly metabolized, such as proteins; polysaccharides, such as latex functionalized sepharose, agarose, cellulose, cellulose grains and the like; polymeric amino acids, such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and injectable virus particles, see, for example, section II.D, Especially useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art.

II.D, Preparation of Particulate Hybrid Immunogens

Containing HCV Epitopes. The immunogenicity of HCV epitopes can also be enhanced by preparing them in mammals or yeast systems fused or assembled with particle-forming proteins, such as, for example, those associated with

with hepatitis B surface antigen. Constructions in which the NANBV epitope is directly linked to the particle-forming protein, encoding sequences, produce hybrids that are immunogenic to the HCV epitope. In addition, all prepared vectors include specific epitopes for HCV, having varying degrees of immunogenicity, such as, for example, the pre-S peptide. Thus, particles constructed from the particle-forming protein that include HCV sequences are immunogenic in relation to HCV and HBV.

hepatitis surface antigen (HBSAg) has been shown to be formed and assembled into particles in S, cerevisiae (Valenzuela et al, (1982)), as well as, for example, mammalian cells (Valenzuela, P., et al (1984)), the formation of such particles has been shown to enhance the immunogenicity of the monomer subunit. The constructs may also include the HBSAg immuno-dominant epitope, comprising the 55 amino acids of the pre-surface region (pre -S), Neurath et al. (1984). Constructions of the yeast-expressable pre-S-HBSAg particle are disclosed in EPO No. 174,444, published on March 19, 1986; hybrids including heterologous viral sequences for yeast expression are disclosed in EPO n? 175 * 261, published on March 26, 1966. Both requests are transmitted to the assignee of the present, and are incorporated here for 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)).

In addition, the portions of the sequence encoding a. - 47 particle-forming protein can be replaced by codons that encode an HCV epitope. In this substitution, regions that are not required to mediate the aggregation of the units to form immunogenic particles in yeast or mammals can be deleted, thus eliminating additional HBU antigenic sites from competition with the HCV epitope,

II. E. Vaccine Preparation

Vaccines can be prepared from one or more immunogenic polypeptides, derived from HCV cDNA as well as from the cDNA sequences in Figs. 1-32, or from the HCV genome, to which they correspond, The observed homology between HCV and Flavivirus provides information regarding the polypeptides that are likely to be most effective, such 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 genomic RNA flavivirus is believed to be the only species of specific mRNA virus, and it is translated into three viral structural proteins, namely C. M, and E, as well as two large proteins non-structural, NV4 and NV5, and a complex set of smaller non-structural proteins. It is known that the largest neutralization epitopes for Flavivirus reside in protein E (envelope) (Roehrig (1986)). The corresponding HCV E gene, and the region encoding the polypeptide, can be diagnosed, based on the homology of the Flavivirus. Thus, vaccines can consist of recombinant polypeptides containing HCV E epitopes. These polypeptides can be β

Expressed in bacteria, yeast, or mammalian cells, or alternatively, they can be isolated from viral preparations. It is also anticipated that other structural proteins may also contain epitopes that give rise to protective anti-HCV antibodies. Thus, polypeptides containing the epitopes of E, C, and M can be used, either alone or in combination, in HCV vaccines,

In addition to what has been described above, immunization with NSl (non-structural protein 1) has been shown to result in protection against yellow fever (Schlesinger et al. (1986). This is true although immunization does not neutralizing antibodies, particularly because this protein appears to be highly conserved among flaviviruses, HCV NS1 is also likely to be protective against HCV infection, and even shows that non-structural proteins can provide protection against viral pathogenicity, even if they do not produce neutralizing antibodies.

In view of the above, multivalent HCV vaccines may consist of one or more structural proteins, and / or one or more proteins not

Structural I. These vaccinans can consist of, for example, recombinant HCV polypeptides and / or polypeptides isolated from virions. In addition, it may be possible to use inactivated HCV in vaccines; inactivation can be carried out by preparing viral lysates, or by

-49 _

other means known in the art to cause flavivirus inactivation, for example 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.

Some of the proteins in Flaviviruses are known to contain highly conserved regions; thus, some immunological cross-reactivity is expected between HCV and other Flaviviruses,, It is possible that epitopes shared between Flaviviruses and HCV will give rise to protective antibodies against one or more of the disorders caused by these pathogens. Thus, it may be possible to design multipurpose vaccines based on this knowledge.

The preparation of vaccines containing immunogenic polypeptide (s) as active ingredients is known to those of skill in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid before injection can also be prepared. The preparation can also be emulsified, or the protein encased in liposomes. The immunogenic active ingredients are sometimes mixed with excipients which are pharmaceutical. acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, brine, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, agents pH buffer, and / or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants that can be effective include, but are not limited to: aluminum hydroxide, N-acetyl-mura mil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- alanyl-D-isoglutamine (CGP 11Ó37, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy ) -ethyl mine (CGP 19835-A, referred to as MTP-PE), and R1BI, which contains three components extracted from ^ bacteria, monophosphoryl lipid A, trehalose dimicolate and cell wall skeleton (MPL + TDM + CWS · ) in a 2% emulsion of squalene / / Tween 80. The effectiveness of an adjuvant can be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an HCV antigenic sequence that results from the administration of this polypeptide in vaccines that are also constituted by various adjuvants.

Vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include supositories and, in some cases, oral formulations. For top sites, traditional binders and vehicles can include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of from 0.5% to 10%, preferably 1% to -2%. Oral formulations include such standard excipients

Most commonly employed, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, formulations or delayed-release feet and contain 10% -95% active ingredient, preferably 25% -7%.

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

11. F, Dosage and Vaccine Administration

Vaccines are administered in a manner compatible with the dosage formulation, and in an amount that will be prophylactic and / or therapeutically effective,

The amount to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject being treated, the capacity of the immune system

I read the subject to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each subject.

The vaccine can be given on a single dose schedule, or preferably on a multiple dose schedule.

A multiple dose schedule is one in which a primary course of vaccination can be with i-10 separate doses, followed by other doses given at subsequent intervals necessary to maintain and / or reinforce the immune response, for example, 1- 4 months for a second dose and, if necessary, a subsequent dose (s) after several months. The dosage regimen will also be, at least in part, determined by the individual's need and will be dependent on the judgment of the physician.

In addition, the vaccine containing the immunogenic HCV antigen (s) can be administered in conjunction with other immunoregulatory agents, for example, immune globulins.

’Preparation of HCV Epitope Antibodies

The immunogenic polypeptides prepared as described above are used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (eg, rat, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide that contains an HCV epitope (s), the serum pro- 53!

from the immunized animal is collected and treated according to known procedures. If the serum containing poly antibodies to an HCV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for the production and processing of polyclonal antisera are known in the art, see for example Mayer and Walker (1987),

Alternatively, polyclonal antibodies can be isolated from a mammal that has been previously infected with HCV. An example of a method for purifying antibodies to HCV epitopes, from serum from an infected individual, based on afi chromatography. nity and using a SOD fusion polypeptide and a polypeptide encoded within the 5-1-1 cDNA clone, is shown in Section V.E.

Monoclonal antibodies directed against HCV epitopes can also be easily produced by whom, anyone skilled in the art, The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by fusing cells, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, for example, M. Schreier et al. (1981); Hammerling et al. (1981); Kennett et al. (1980); See also, . US patents 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.

Monoclonal antibody panels produced against epitopes

- 54 HCV can be sieved for various properties, that is, for isotype, epitope affinity, etc.

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

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

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

11. Η, Probes and Diagnostic Oligonucleotide Sets

Using the revealed portions of HCV cDNAs as a base, including those represented in Eigs. 1-32, oligetera of approximately 8 nucleotides or more can be prepared, either by excision or synthetically, which hybridize with the HCV genome and are useful in the identification of the viral agent (s), later characterization of the (s) viral genome (s), as well as in the detection of the virus (s) in sick individuals. The probes for IICV polynucleotides (natural or derived) are of a length that allows the detection of the unique viral sequences by hybridization. Although 6-8 nucleotides may be a usable length, sequences of 10-12 nucleotides are preferred, and about 20 nucleotides appear to be optimal. Preferably, these sequences will be derived from regions that have no heterogeneity. These probes can be prepared using routine methods, including synthetic self-ligated oligonucleotide methods. Among the useful probes, for example, are clone 5-1-1 and the additional clones disclosed here, as well as the various oligomers useful for probing cDNA librarians, listed below. A complement to any single portion of the HCV genome will be satisfactory. For use as probes, complete complementarity · is desirable, although this may be unnecessary as the fragment is enlarged.

For the use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, is treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample can be stained with dots without dimension separation. The probes are then classified. Suitable classifications, and methods for classifying probes are known in the art, and include, for example, radioactive classifications incorporated by incision or kinasing translation, biotin, fluorescent probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the classified probe under suitable stringent hybridization conditions.

The probes can be completely complementary to the HCV genome. Therefore, high stringency conditions are usually desired in order to avoid false positives. However, high stringency conditions should be used only if the probes are complementary to regions of the viral genome that are not heterogeneous. Hybridization stringency is determined by a number of factors during hybridization and during the washing process, including temperature, ionic resistance, time span, and formamide concentration. These factors are summarized in, for example, Maniatis, T, (1982),

Generally, HCV genome sequences are expected to be present in the serum of infected individuals at relatively low levels, that is, at approximately

3 λ r

-IO ⁵ sequences per ml. This level may require amplification techniques to be used in hybridization assays. Such techniques are known in the art. For example, the Bio-Bridge system from Enzo Biochemical Corpòration uses terminal deoxynucleotide transferase to add unmodified 3'-poH -dT tails to a DNA probe. The poly dT tail probe is hybridized to the target nucleotide sequence, and then to the modified biotin poly-A. PCT order 84/03520 and

ΕΡΑ124-221 describe a DNA hybridization assay in which: (l) analyte is quenched to a single fiber DNA probe that is complementary to a classified enzyme oligonucleotide; (2) the resulting tailed duplex is hybridized to a classified enzyme cleetide oligonu. EPA 204-510 describes a DNA hybridization test in which the DNA analyte is contacted with a probe that has a tail, such as a poly-dT tail, an amplifying fiber that has a sequence that hybridizes to the probe tail, such as a poly-A sequence, and which is capable of binding a plurality of classified fibers. A té <? particularly desirable technique may first involve amplifying the target HCV sequences in sera approximately 10,000 times, i.e., up to approximately 10 sequences / ml. This can be accomplished, for example, by the technique of Saiki et al. (1986). The amplified sequence (s) can then be detected using a hybridization assay that is described in the co-pending U.S. patent application, Attorney Docket No. 2300-0171, which was filed on October 15, 2010. 1987, θ transmitted to the assignee of the present, and is hereby incorporated by reference. This hybridization assay, which should detect sequences at the level of 10 µg / ml, uses nucleic acid multimers that bind to a single fiber nucleic acid analyte, and which also bind to a fine, single-fiber classified oligonucleotide. A suitable solution phase sandwich assay that can be used with classified polynucleotide probes, and the methods for preparing probes are described in EPO 225,807, published on June 16, 1987, which was transmitted to the assignee. , and which is incorporated here as a reference.

The probes can be packaged in diagnostic kits. Diagnostic kits include the DNA probe that can be classified; alternatively, the DNA son may be unclassified and the ingredients for classification may be included in the kit, the kit may also contain other packaged reagents and materials required for the particular hybridization protocol, for example, rules, as well as instructions for conducting the test, ί

II.I. Immunoassay and Diagnostic Sets

Both polypeptides that react immunologically with serum containing HCV antibodies, for example, those derived from, or encoded within, the clones described in Section

I

IV.A., and composites thereof (see section IV.A.) and antibodies raised against HCV-specific epitopes in these polypeptides, see for example Section IV.E, are useful in immunoassays to detect the presence of antibodies to HCV, or the presence of viruses and / or viral antigens, in biological samples, including, for example, blood samples or serum · θ immunoassay design is subject to a large number of variations, and a variety of these are known in art. For example, the immunoassay can use a viral antigen, for example, a polypeptide derived from either heat. nes containing the HCV cDNA described in Section IV.A., or the composite cDNAs derived from the cDNAs in these clones, or from the HCV genome from which the cDNA 'in these clones is derived; al

In fact, the itnunoassay can use a combination of viral antigens derived from these sources. The same can use, for example, a monoclonal antibody directed to a viral epitope (s), a combination of monoclonal antibodies directed to a viral antigen, monoclonal antibodies directed to different viral antigens, polyclonal antibodies directed to the same viral antigen, or polyclonal antibodies directed to different viral antigens. Protocols can be based, for example, on competition, or direct reaction, or sandwich tests.

Protocols can also, for example, use supports, solid, or can be performed by immunoprecipitation. Most assays involve the use of classified antibody or polypeptide; classifications can be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify signals from the probe are also known; examples of which are assays using biotin and avidin, and classified enzyme and mediated immunoassays, such as ELISA assays, Flavivirus model for HCV allows diagnoses in relation to the probable location of the diagnostic epitopes for the virion structural proteins, The C domains, pre -M, Μ, and E are all likely to contain epitopes of significant potential to detect viral antigens, and particularly for diagnosis. Similarly, the non-structural protein domains are supposed to contain important diagnostic epitopes (for example, NS5 encoding a putative polymerase; and NS1 encoding

- 60 a putative complement-binding antigen). Recombinant polypeptides, or viral polypeptides, that include epitopes from these specific domains can be useful for the detection of viral antibodies in blood donors with infections and infected patients.

In addition, antibodies directed against E and / or M proteins can be used in immunoassays for the detection of viral antigens in patients with HCV NANBH, and in infectious blood donors. In addition, these antibodies will be extremely useful for detecting data, patients and patients in the acute phase.

Kits suitable for immunodiagnosis and containing the appropriate classified reagents are constructed by packaging the appropriate materials, including the polypeptides of the invention containing HCV epitopes or antibodies directed against HCV epitopes in suitable containers, together with the remaining reagents and materials necessary for driving of the test, as well as an appropriate set of test instructions.

II, J, Later Characterization of the HCV Genome, Virions, and Viral Antigen Using cDNA-derived Probes for the Viral Genome

The HCV cDNA sequence information in the clones described in Section IV, A., As depicted in Figs, 1-32 inclusive, can be used to acquire additional information about the HCV genome sequence, and for identification and isolation of the HCV agent, and thus will assist in your ca61

characterization including the nature of the genome, the structure of the viral particle, and the nature of the antigens of which it is composed. This information, in turn, can lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and. antibodies directed against HCV epitopes that would be useful for the diagnosis and / or treatment of NAN.BM caused by MCU,

The cDNA sequence information in the aforementioned clones is useful for the design of probes for the isolation of additional cDNA sequences that are derived from regions not yet identified, the HCV genome (s) from which the cDNAs in clones described in Section IV.A, are derived. For example, classified trams containing a se. approximately 8 or more nucleotides, and preferably 20 or more nucleotides, which are derived from regions close to the 5 '”terminus or 3 ¹ -termini of the HCV cDNA sequence family shown in Figs. 1, 3, 6, 9, 14 and 32 po. can be used to isolate overlapping cDNA sequences from HCV cDNA libraries. These sequences that overlap the cDNAs in the aforementioned clones, but which also contain sequences derived from regions of the genome from which the cDNA in the aforementioned clones are not derived, can then be used to synthesize probes to identify other fragments that are overlap which do not necessarily overlap cDNAs in the clones described in Section IV.A. Unless the HCV genome is sequenced and the segments lack common sequences, it is possible to sequence the total gene (s) using the technique of isolating overlapping cDNAs derived from the ma gene (s) viral (s). Although it is unlikely, if the genome is a segmented genome that lacks common sequences, the sequence of the genome can be determined by serologically sieved lambda-gtll HCV cDNA libraries, as used to isolate the .5-1-1 clone, sequencing isolated cDNAs, and using isolated cDNAs to isolate overlapping fragments, using the technique described for the isolation and sequencing of the clones described in Section IV.A, Alternatively, the characterization of the genomic segments can be done from the viral genome (s) i, sole (s) from purified HCV particles. Methods for purifying HCV particles and for detecting them during the purification process are described hereinafter. Processes for isolating polynucleotide genomes from viral particles are known in the art, and a process that can be used is shown in Example IV, A.1. The isolated genomic segments can then be cleaved and sequenced. Then, with the information provided here, it is possible to clone and sequence the HCV genome (s) regardless of its nature.

Methods for building cDNA libraries are known in the art, and are discussed above and below; a method for constructing HCV cDNA libraries in lambda-gtll is discussed later in Section IV.A. However, cDNA libraries that are useful for screening with nucleic acid probes can also be constructed in other vectors known in the art, for example, lambda-gtO (Huynh et al. (1985).). The HCV derivative of cDNA detected by the probes is derived

- 63 of those of the cDNAs in Figs. 1-32, and from probes synthesized from polynucleotides derived from these cDNAs, can be isolated from the clone by digestion of the polynucleotide isolated with the appropriate restriction enzyme (s), and sequenced. See, for example, Section IV.A.3 · © IV.A.4 for techniques used for the isolation and sequencing of HCV cDNA that overlaps HCV cDNA in clone 5-1-1, Sections IV.A. 5-IV, A, 7 for the isolation and sequencing of HCV cDNA that overlaps with that in clone 8l, and Section IV.A.8 and IV.A.9 for the isolation and sequencing of a clone that overlaps another clone ( clone 36), which overlaps clone 8l, i

The sequence information derived from these overlapping HCV cDNAs is useful for determining areas of homology and heterogeneity within the viral genome (s), which may indicate the presence of strains other than the genome, and / or defective particle populations. It is also useful for the design of hybridization probes to detect HCV or HCV antigens or HCV nucleic acids in biological samples ^

I, and during HCV isolation (discussed below), using the techniques described in Section II.G. In addition, overlapping cDNAs can be used to create expression vectors for polypeptides derived from the genome (s) ) of HCV that also encode the polypeptides encoded in clones 5-1-1, 36, 81, 91, θ 1-2, and in the other clones described in Section IV.A, The techniques for creating these polypeptides, which contain epitopes HCV, and for antibodies directed against HCV epitopes contained within them, as well as their uses, are analogous to those described for polypeptides.

derivatives of NANBV cDNA sequences contained within clones 5-1-1, 32, 35, 36, 1-2, 8l, and 91, and discussed above and below.

Encoded within the family of cDNA sequences contained within clones 5-1-1 »32, 35» 36, 8l,

91, 1-2, and other clones described in Section IV.A. are antigen (s) containing epitopes that appear to be unique to HCV; is. that is, antibodies directed against these antigens are absent in individuals infected with HAV or HBV, and in individuals not infected with HCV (see the serological data in Section IV.B,). The sequence of these cDNAs with the sequences of HAV, HBV, HDV, and with the genomic sequences in Genebank indicates that there is a homoloid. minimal gap between these cDNAs and the polynucleotide sequences from those sources. Thus, antibodies directed against antigens encoded within the cDNAs of these clones can be used to identify BB-NANBV particles isolated from infected individuals. In addition, they are also useful for the isolation of NANBH agent (s),

HCV particles can be isolated from sera from individuals infected with BB-NANBV or from cell cultures by any of the methods all known in the art, including for example, techniques based on size discrimination, such as sedimentation methods or exclusion, or density-based techniques such as ultra-centrifugation on density gradients, or precipitation with agents such as polyethylene glycol, or chromatography on a variety of materials such as anionic or cationic exchange materials, and bonding materials due to hydrophobicity, as well as affinity columns. During the isolation process, the presence of HCV can be detected by hybridization analysis of the extracted genome, using probes derived from the HCV cDNAs described above, or by immunoassay (see Section II.i) using antibodies directed against antigens from HCV encoded within the family of cDNA sequences in Figs. 1-32, and also directed against HCV antigens encoded within the overlapping HCV cDNA sequences discussed above.

i

The antibodies can be monoclonal, or polyclonal, and it may be desirable to purify the antibodies before use, in the immunoassay. A purification process for poly antibodies. lyclonal directed against antigen (s) encoded with clone 5-l? -l is described in Section IV.E; analogous purification processes can be used for antibodies directed against other HCV antigens,

Antibodies directed against HCV antigens encoded within the family of cDNAs represented in Figs, 1-32, as well as those encoded within the overlapping HCV cDNAs, which are attached to solid supports, are useful for the isolation of HCV by immunoaffinity chromatography . Techniques for immunoaffinity chromatography are known in the art, including techniques for affixing antibodies to solid supports so that they retain their immunoselective activity; the techniques may be those in which the antibodies are adsorbed to the support (see, for example, Kurstak in ΕΝΖΥΜΕ IMMUNODIAGNOS1S, pages 31-37), as well as those in which the antibodies are covalently bound to the support. Generally, the techniques are similar to those used for covalently attaching antigens to a solid support, which are generally described in Section II, C}, however, spacer groups can be included in the bifunctional binding agents so that the antigen that binds the local to the antibody remains accessible,

During the purification process the pre. HCV sensitivity can be detected, and / or verified by nucleic acid hybridization, using as polynucleotides probes derived from the family of HCV cDNA sequences represented in Figs, 1-3 ² , as well as from overlapping HCV cDNA sequences, described earlier. In this case, the fractions are treated under conditions that may cause the breakdown of the viral particles, for example, with detergents in the presence of chelification agents, and in the presence of

Viral nucleic acid I determined by hybridization techniques described in 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, following the determination of whether the NANBH symptoms result from infection.

Viral particles from purified preparations can then be further characterized. The genomic nucleic acid was purified. Based on its sensitivity to RNase, not DNase I, it appears that the virus'

- 67 composed of an RNA genome. See Example IV.C, 2., Below, The ability to form fibers and the circularity or non-circularity can be determined by techniques known in the art, including, for example, their visualization by electron microscope, their migration in gradients density, and its sedimentation characteristics. Based on the hybridization of the HCV genome captured to HCV negative fibers from the HCV cDNAs, it appears that HCV may consist of a positive RNA genome with fibers (see Section IV.Hl), Techniques such as these are described in, for example, METHODS IN ΕΝΖΥΜΟ LOGY. In addition, the purified nucleic acid can be cleaved and sequenced by known techniques, including reverse transcription since _; p genomic material and RNA. See, for example, Maniatis (1982), and Glover (1985). Using the nucleic acid derived from the viral particles, it is possible to sequence the entire genome, whether or not it is segmented.

examination of the homology of the polypeptide encoded within the continuous ORF of combined clones 14 to 39c (See Fig. 26), shows that the HCV polypeptide contains regions of honoraria with the corresponding proteins in conserved flavivirus regions. An example of this is described in Section IV.H.3. This discovery has many important ramifications. First of all, this evidence, together with the results showing that HCV contains a genome with positive fibers, the size of which is approximately 10,000 nucleotides, is consistent with the suggestion that HCV is a flavivirus, or an analogous virus . Generally, flavivirus virions and their genomes have a relatively consistent structure and organization, which are known. See Rice et al, (1986), and Brinton, MA (1988). Thus, the structural genes encoding the C, pre-M / M, and E polypeptides may be located at the 5'-terminus of the genorn upstream of clone l4i . In addition, using the comparison with other flaviviruses, prognoses can be made as to the precise location of the sequences that encode these proteins, isolation of the sequences upstream of those in clone 14i can be performed in several ways that, given the information here, will be obvious to anyone in the art. For example, the walking technique of the genorna, can be used to isolate other sequences that are 5 'for those in clone 14i, but that overlap that clone; this in turn leads to the isolation of additional sequences. This technique is extensively demonstrated later in Section IV.A .. For example, too, flaviviruses are known to have conserved epitopes and regions of conserved nucleic acid sequences. Polynucleotides

I containing the conserved sequences can be used as probes that bind the HCV genorn, thus allowing its isolation. In addition, these conserved sequences, together with those derived from HCV cDNAs represented in Eig. 22, can be used to design primers for use in systems that amplify sequences of genera upstream from those in clone 14i, using polymerase chain reaction technology. An example of this is described later.

The structure of HCV can also be determined and its components isolated, The morphology and size can be determined by, for example, electron microscopy, The identification and location of specific viral polypeptide antigens such as coating or envelope antigens, or internal antigens, such as nucleic acid binding proteins, nucleic antigens, and polynucleotide polymerase (s) can also be determined by, for example, determining whether the antigens are present as major or minor viral components, as well as by the use of antibodies directed against specific antigens encoded within isolated cDNAs as probes. This information is useful when designing vaccines; for example, it may be preferable to include an external antigen in a vaccine preparation. Multivalent vaccines can be used, for example, for a polypeptide derived from the genome encoding a structural protein, for example, E, so

I as a polypeptide from another portion of the genome, for example, a non-structural or structural polypeptide.

II, K, Cell Culture Systems and Model Systems

Animal for HCV Replication

The suggestion that HCV is a flavivirus ·. or flavi analogue virus also provides information on methods for the germination of HCV. The term flavi analogue means that the virus has a significant amount of homology to the known conserved regions of flavivirus and that most of the genome is a single ORF.

Methods for growing flaviviruses are known to those of skill in the art (See, for example, the magazines by Brinton (1986) and Stollar, V, (1980)). In general, cells or cell lines suitable for cultivating HCV can include those that are known to support replication of Fia vivirus, for example, the following: monkey kidney cell lines (for example MKg, VERO); porcine kidney cell lines (for example PS); baby hamster kidney cell lines (e.g. BHK); murine macrofage cell lines (for example, P388D1, Mkl, Mml); human macrofage cell lines (for example, U-937); peripheral blood leukocytes from humans; adherent monocytes of humans; hepatocytes or hepatocyte cell lines (for example, HUH7, HEPG2); embryo or embryonic cells (for example, chick embryo fibroblasts); or cell lines derived from invertebrates, preferably from insects (for example drosophyll cell lines), or more preferably from arthropods, for example mosquito cell lines (for example, A. Albopictus, Aedes aegypti, Cutex tritaeniorhynchus) or mite cell lines (for example RML-14 Dermacentor pa rumapertus),

It is possible that primary hepatocytes can be cultured, and then infected with HCV5 or alter natively, hepatocyte cultures can be derived from livers of infected individuals (eg humans or chimpanzees), the latter case being an example of a cell that is infected in vivo being passed in vitro, furthermore, go71

Several methods of immortalization can be used to obtain cell lines derived from cultures of hepatocytes. For example, primary liver cultures (before and after the hepatocyte population enrichment) can be fused to a variety of cells to maintain stability. For example, too, cultures can be infected with transformation viruses, or transfected with transformation genes in order to create permanent> or semi-permanent cell lines. In addition, for example, cells in liver cultures can be fused to establish cell lines (eg, HepG-2). Methods for cell fusion are known in the art, and include, for example, the use of fusion agents such as pplethylene glycol, Sendai virus, and Epstein-Barr virus.

As was discussed earlier, HCV is a Flavivirus or Flavi virus analog. Therefore, it is likely that HCV infection of cell lines can be performed by techniques known in the art to infect cells with Flavivirus. These include, for example, incubating cells with viral preparations under conditions that allow viruses to enter the cell. In addition, it may be possible to obtain viral production by transfecting cells with isolated polynucleotides, viral toxins. Togavirus and Flavivirus RNAs are known to be infectious in a variety of vertebrate cell lines (Pfefferkorn and Shapiro (197 ^)), θ in a mosquito cell line (peleg (1969)) · Methods for transfection. Tissue culture cells with RNA duplexes, RNAs with positive fibers, and DNAs (including cDNAs) are known in the art, and include, for example, techniques that use electroporation, and precipitation with DEAE-Dextran or calcium phosphate. An abundant 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 spread of the virus.

In addition for cultured cells, animal model systems can be used for viral replication; animal systems in which flaviviruses are present are known to those of skill in the art (see, for example, the magazine by Monath (1986)). Thus, HCV replication can occur not only in chimpanzees, but also, for example, marmosets and suckling rats. '

11. L. Screening for HCV Anti-Viral Agents

The viability of cell culture and animal model systems for HCV also makes screening for anti-viral agents that inhibit HCV replication possible, and particularly for those agents that preferably allow cell development and multiplication while inhibiting viral replication. These screening methods are known to those of skill in the art. Generally, anti-viral agents are tested at a variety of concentrations, for their effect on preventive viral replication in cell culture systems that support viral replication, and then for an inhibition of infection.

viral activity or pathogenicity (and a low level of toxicity) in an animal model system.

The methods and compositions provided here for detecting HCV antigens and HCV polynucleotides are useful for the screening of anti-viral agents in which they provide an alternative, and perhaps more sensitive means, to detect the effect of the agent on viral replication, that the test of

of the HCV-polynucleotide described herein can be used to quantify the amount of viral nucleic acid produced in the cell culture. This can be accomplished, for example, by hybridization or competition hybridization of nucleic acids from cells infected with a classified HCV polynucleotide probe. For example, too, anti-HCV antibodies can be used to identify and quantify HCV antigen (s) in cell culture using the immunoassays described herein. In addition, since it may be desirable to quantify HCV antigens in the infected cell culture by a competition assay, the polypeptides encoded within the HCV cDNAs described herein are useful in these competition assays. Generally, a recombinant HCV polypeptide derived from HCV cDNA should be classified, and the inhibition of binding of this classified polypeptide to an HCV polypeptide due to the antigen produced in the cell culture system must be controlled. Furthermore, these techniques are particularly useful in cases where HCV may be able to replicate in a cell line without causing cell death.

- 74 II.Μ. Preparation of Attenuated HCV Strains

In addition to the above, using tissue culture systems and / or animal model systems, it may be possible to isolate attenuated strains of HCV. These strains will be suitable for vaccines, or for the isolation of viral antigens. The attenuated strains are isolable after multiple passages in cell culture and / or animal model. The detection of an attenuated strain in an infected cell or individual is achievable by techniques known in the art, and may include, for example, the use of antibodies to one or more epitopes encoded in HCV as a probe or the use of a polynucleotide containing an HCV sequence of at least about 8 nucleotides' as a probe. Alternatively, or in addition. Tion, an attenuated strain can be constructed using the HCV genomic information provided herein, and using recombinant techniques. Generally, one can attempt to remove the region from the genome by encoding, for example, a polypeptide related to pathogenicity, but which allows for viral replication. In addition, the genome construction will allow the expression of an epitope that gives rise to neutralization of antibodies to HCV. The altered genome can then be used to transform cells that allow HCV to replicate, and the cells develop under conditions to allow viral replication. Attenuated HCV strains are useful not only for vaccine purposes, but also as sources for commercial production of viral antigens, as processing these viruses would require less stringent protective measures for employees involved in viral production,

III. General Methods

The general techniques used in extracting the genome from a virus, preparing and probing a cDNA library, sequencing clones, building expression vectors, transforming cells, performing immunological assays such as radioimmunoassays and ELISA assays, for developing cells in culture , and analogues are known in the art and laboratory manuals are available, which describe these techniques. However, as a general guide, the following shows some sources currently available for such procedures, and for materials useful for carrying out them.

III, A, Host and Expression Control Sequences

Host cells both prokaryotic and eukaryotic can be used for expression of the desired coding sequences when appropriate control sequences are used that are compatible with the designated host. 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 commonly derived from, for example, pBR322, a plasmid containing operons that confer resistance - 76 associates ampicillin β tetracycline, and the various pUC vectors, which also contain sequences that confer antibiotic resistance markers. These markers can be used to obtain successful transformants by selection. If commonly used prokaryotic control sequences include Beta-lactamase (penicillinase) and lactose-promoting systems (Chang et al, (1977)) »the tryptophan (trp) promoter system (Goeddel et al.) (1980)) and the promoter lambda-derivative and ribosome binding site N gene (Shimatake et al. (ΐ9θΐ)) and the hybrid tac promoter (De Doer et al. (1983)) derived from the trp and lac UV5 promoter sequences. The previous systems are particularly compatible with E, coli; if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas can be used, with corresponding control sequences.

Eukaryotic hosts include yeast and mammalian cells in culture systems. Saccharomyces cervisiae and Saccharomyces carlsbergensis are the most commonly used yeast hosts, and are hosts. convenient fungi. Compatible yeast vectors support markers that allow successful selection of transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metals over wild-type strains. Compatible yeast vectors can employ the 2-micron origin of replication (Broach et al. (1983)), the combination of CEN3 and ARSI or other means to ensure replication, such as the sequences that will result in the incorporation of a fragment into the genome of the host cell. The control strings

- 77 '"Μ le for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al. (1968)} Holland et al, (ΐ97θ))» including the promoter for 3 phosphoglycerate kinase (Hitzeman (1980)). Finalizers can also be included, such as those derived from the enolase gene (Holland (1998)). Particularly useful control systems are those that comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or the regulable alcohol dehydrogenase (ADH) promoter, finalizers also derived from GAPDH, and if secretion is desired, the guide sequence from the alpha factor of yeast. In addition, the transcriptional regulatory region and the transcriptional initiation region that are operatively linked may be such that they are not associated in the wild type organism. These systems are described in detail in EPO 120.551 'published on October 3, 1984; EPO 116,201, published August 22, 1984; and EPO 164,556, published on December 18, 1985 »which were all transmitted to the present assignee, and are incorporated herein by reference.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including HeLa cells, Chinese hamster ovary (CHO) cells, kidney cells baby hamster (ΒΗΚ) and a number of other cell lines. Promoters suitable for mammalian cells are also known in the art and include viral promoters such as those obtained from Siroian Virus 4θ (SV4O) (Fiers (1978)), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma (BPV). Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences that increase expression may also be included, and sequences that cause amplification of the gene may also be desirable. These sequences are known in the art. Vectors suitable for replication in mammalian cells can include viral replicons, or sequences that ensure integration of the appropriate sequences encoding NANBV epitopes in the host genome.

III.B. Transformations

Transformation can be carried out by any method of introducing polynucleotides into a hoe cell, including, for example, packaging the polynucleotide into a virus and transducting the host cell with the virus, and by directly raising the polynucleotide. The transformation procedure used depends on the host to be transformed. For example, the transformation of E. coli host cells with lambda-gt11 containing BB-NANBV sequences is discussed in the Example section, below. Bacterial transformation by direct survey generally employs treatment with calcium or rubidium chloride (Cohen (197 ² )} Maniatis (1982)). The transformation of yeast by direct survey can be performed using the method of Hinnen et al. (1978). Transformations in mammals by direct survey can be conducted using the phosphate precipitation method

Graham's and Van der Eb's ο (ΐ97θ) »the various known modifications of the same.

III.C, Vector Construction

Vector construction employs techniques that are known in the art. DNA cleavage at the specific site is performed by treating with suitable restriction enzymes under conditions that are generally specified by the manufacturer of these commercially available enzymes. In general, about 1 microgram of plasmid or DNA sequence is cleaved by an enzyme unit in about 20 microliters of buffer solution by incubation for 1-2 hours at 37 ° C. After incubation with the restriction enzyme, the protein is removed by extraction with phenol / chloroform and DNA recovered by ethanol precipitation. The cleaved fragments can be separated using polyacrylamide or agar gel electrophoresis techniques, according to the general procedures found in Methods in Enzymology (1980) 65: 499-560.

The viscous terminated cleavage fragments can be terminated without cutting using E. coli DNA polymerase I (Klenow) in the presence of the appropriate deoxy-nuoleotide triphosphates (dNTPs) present in the mixture. The SI nuclease treatment can also be used, resulting in the hydrolysis of any single fiber DNA portions.

Ligations are performed using standard buffers and temperature conditions using T4 DNA and ATP ligase; viscous terminal connections require less ATP and less li. gas that blunt terminal connections. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove 5'-phosphate and thus prevent rewiring of the vector; alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent binding.

The ligation mixtures are transformed into suitable cloning hosts, such as E. coli. and successful transformants chosen for, for example, antibiotic resistance, and sieved for correct construction.

III.D. Construction of Desired DNA Sequences

Synthetic oligonucleotides can be prepared using an automated oligonucleotide synthesizer as described by Warner (1984). If desired, 32 synthetic fibers can be classified with P, by treatment with 32 polynucleotide kinase in the presence of P-ATP, using standard conditions for the reaction.

DNA sequences, including those isolated from cDNA library, can be modified by known techniques, including, for example, site-directed mutagenesis, as described by Zoller (1982). Briefly, the DNA to be modified is packaged in the phage as a single fiber sequence, and converted to a double-fiber DNA with DNA polymerase using, as a primer, a synthetic synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence

cia. The resulting double-fiber DNA is transformed into a phage

I supporting host bacteria. Cultures of the transformed bacteria, which contain replications of each fiber of the phage, are laminated on agar to obtain plaques. Theoretically, 50% of the new plates contain phage having the sequence mutated, and the remaining 50% have the original sequence. The replicates of the piacas are hybridized to a synthetic probe classified as having characteristics and conditions that allow hybridization with the correct fiber, but not with the unmodified sequence. Sequences that have been identified by hybridization are recovered and cloned.

III.E. Hybridization with Probe

DNA libraria can be probed using the procedure of Grunste.in and Hogness (l975) · In short, in this procedure, the DNA to be probed is immobilized on nitrocellulose filters, denatured, and pre-hybridized with a buffer containing 0-50% of formamide, (^ 75 M NaCl, 75 mM Na citrate, 0.02% (w / v) each of bovine serum albumin, polyvinyl pyrrolidone, and Picoll, 50 mM Na phosphate (pH 6.5 ), 0.1% SDS, and 100 micrograms / ml of denatured DNA vehicle.

The percentage of formamide in the buffer, as well as the time and temperature conditions of the pre-hybridization and subsequent hybridization steps depends on the required stringency. Oligomeric probes that require low stringency conditions are generally used with low percentages of formamide, lower temperatures, and long periods of hybridization. Probes containing more than 3θ or Uo nucleotides such as those derived from cDNA b

or genomic sequences generally employ high temperatures, for example about 40-42 ° C, and a high percentage, for example 5θ%, of formamide. Following prehybridization, the 5 * - P-classified 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; the DNA in corresponding locations on the original agar plates is used as the source of the desired DNA

III. F. Construction Check and Sequencing

For routine vector constructs, ligation mixtures are transformed into strain HB101 of E, coli or another suitable host, and successful transformants chosen for antibiotic resistance or other markers. Plasmids from the transformants are then prepared according to method of Clewell et al, (1969), usually following the amplification of chloramphenicol (Clewell (1972)), DNA is isolated and analyzed, usually by restriction enzyme analysis and / or sequencing. Sequencing can be carried out by the dideoxy method of Sanger et al. (1977) as further described by Messing et al, (1981), or by the method of Maxam et al. (1980). The problems with tape compression, which are sometimes seen in rich GC regions, have been overcome by the use of T-deazoguanosine according to Barr et al, (1986).

III.G, Bnzima-Bound Immunosorbent Assay enzyme-bound immunosorbent assay

83 (ELISA) can be used to measure and concentrations of either antigen or antibody. This method depends on the conjugation of an enzyme with either antigen or an antibody, and uses the activity of the bound enzyme as a quantitative classification. To measure the antibody, the known antigen is fixed to a solid phase (for example, a microplate or plastic bowl) incubated with dilutions of test serum, washed, incubated with anti-immunoglobulin classified with an enzyme, and washed again . Enzymes suitable for classification are known in the art, and include, for example, horseradish peroxidase. The activity of the enzyme bound to the solid phase is measured by adding the specific substrate, and product formation or use of substrate is determined colorimetrically. The binding of enzyme activity is a direct function of the amount of antibody binding.

To measure the antigen, a specific known antibody is attached to the solid phase, test material containing antigen is added, then an incubation of the solid and washed phase, and a second sified enzyme-class antibody is added. After washing, substrate is added, and activation. enzyme is estimated colorimetrically, and referred to the antigen concentration.

IV. Examples

Described below are examples of the present invention that are provided for illustrative purposes only, and not to limit the scope of the present invention. In the light of the present description, numerous embodiments den84

within the scope of the claims will become apparent to those normally understood in the art. The procedures indicated, for example, in Sections IV.A. they can, if desired, be repeated but need not be, as techniques are available for constructing the desired nucleotide sequences based on the information provided by the invention. The expression is exemplified in E. coli} however, other systems are available, as indicated more fully in Section III.A. Additional epitopes derived from the geometrical structure can also be produced, and used to generate antibodies as indicated below.

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

IV.A.1. Preparation of HCV cDNA

The source of the NANB agent was a plasma reservoir derived from a chimpanzee with chronic NANBH, the chimpanzee had been experimentally infected with blood from another chimpanzee with chronic NANBH resulting from infection with

I HCV in a contaminated factor 8 concentrate bath derived from harvested human sera. The chimpanzee plasma reservoir was made by combining many individual plasma samples containing high levels of alanine aminotransferase activity; this activity results from liver injury due to infection with HCV, Since 1 ml of a 10 ~ ^ dilution of this collected serum gave IV NANBH provoked in another chimpanzee, its CID was at least 10 ^ / ml, that is, the he even had a high degree of infectious virus.

A cDNA library from the high titer plasma pool was generated as follows. First, the viral particles were isolated from the plasma; a 90 ml aliquot was diluted with 310 ml of a solution containing 50 mM Tris-HCl, pH 8.0, ImM EDTA, 100 mM NaCl. Fragments were removed by centrifugation for 20 minutes at 15,000 xg at 20 ° C. The viral particles in the resulting supernatant were then transformed into microspheres by centrifugation in a Beckman SW28 rotor at 28,000 rpm for 5 hours at 20 ° C. To release the genome the particles were broken by suspending the microspheres in 15 ml of solution containing 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, also containing 2 mg / ml of proteinase k, followed by incubation at 45 ° C for 90 minutes. Nucleic acids were isolated by adding 0.8 micrograms of bacterial MS2 RNA as a carrier, and extracting the mixture four times with a 1: 1 mixture of phenol: chloroform (phenol saturated with 0.5 ^M Tris-HCl), pH 7.5, 0.1 $ (v / v) d® beta-mercaptoethanol, 0.1 $ (w / v) hydroxyquinolone, followed by extraction twice with chloroform. The aqueous phase was concentrated with 1 -butanol before precipitation with 2.5 volumes of absolute ethanol overnight at -20 ° C. The nucleic acid was recovered by centrifugation in a Beckman SW4l rotor at 40,000 rpm for 90 minutes at 4 ° C, and dissolved in water that had been treated with 0.05% (v / v) of diethylpyrocarbonate and placed in an autoclave.

nucleic acid obtained by the above procedure (<2 micrograms) was denatured with 17.5 mM CH3 HgOH; the cDNA was synthesized using this denatured nucleic acid as a standard, and was cloned into the EcoR1 site of the lainbda86 '3 fage

-gtll using methods described by Huynh (1985), except that the random primers replaced oligo (dT) 12-18 during the synthesis of the first cDNA fiber by reverse transcriptase (Taylor et al. (1976)). The resulting double fiber cDNAs were fractionated according to size on a column of

I

Sepharose CL-4B} The eluted material of approximate size 400, 300, 200, and 100 base pairs were collected in cDNA reservoirs 1, 2, 3 and 4, respectively. The lambda-gtll cDNA was generated from the cDNA in reservoir 3.

The lambda-gtll cDNA generated from reservoir 3 was sieved for epitopes that can specifically bind with serum derived from a patient who had previously tried NANBH. About 10 ^ pages were sieved with patient sera using the methods of Huynh et al. (1985), except that human antibody binding was detected with sheep anti-human Xg antisera that 12 Ç had been radio-classified with I. Five positive fages were identified and purified. The five positive phage were then tested for specificity of binding to sera from 8 different humans previously infected with the NANBH agent, using the same method. Four of the fages encoded a polypeptide that immunologically queried with only one human serum, that is, the only one that was used for primary screening of the fage book, the fifth (5-1 — 1) encoded a polypeptide that reacted immunologically with 5 of the 8 of the tested sera. Furthermore, this polypeptide did not react immunologically with sera from 7 donors

----- - normal. Therefore, it appears that clone 5-1-1 encodes a polypeptide that is specifically recognized immunologically by sera from NANB patients,

IV, A, 2, HCV cDNA Sequences in Recombinant Phage 5-1-1 'and Encoded Polypeptide Within the Sequence, cDNA in recombinant phage 5-1-1 was sequenced by the method of Sanger et al. (1977). Essentially, the cDNA was excised with EcoRI, isolated by size fractionation using gel electrophoresis. The EcoRI restriction fragments were subcloned into vectors M13, mpl8 and mpl9 (Messing (1983)) and sequenced using the dideoxy chain termination method of Sanger et al, (1977)). The obtained sequence is shown in Fig. 1.

Pig-encoded polypeptide. 1 that is encoded in the HCV cDNA is in the same translational structure as the N-terminal beta-galactosidase half into which it is fused. As shown in Section IV.A., the 5—1—1 translational open reading frame (ORF) encodes epitope (s) specifically recognized by sera from patients and chimpanzees with NANBH infections.

IV, A, 3. Isolation of HCV cDNA that overlaps cDNA in Clone 5-1-1

The HCV cDNA overlapping the cDNA in the clone was obtained by screening the same lambda-gtll library, created as described in Section XV, A. 1., with a synthetic polynucleotide derived from the HCV cDNA sequence in clones 5-1-1, as shown in Fig. 1. The sequence of the polynu—

ο cleotide used for screening was:

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-gtll library was sieved with this probe, using the method described in Huynh (1985). Approximately 1 in 50,000 clones hybridized with the probe. Three clones containing cDNAs that hybridize to the synthetic probe were numbered 81, 1-2 and 91.

IV.A.4. Nucleotide sequences of HCV cDNAs that overlap the 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 in relation to the HCV cDNA sequence in phage 5-1-1 are shown in Fig. 2, which shows the fiber encoding the detected HCV epitope, and where the homologies in the nucleotide sequences are indicated by the vertical lines between the sequences.

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

The second difference in that 5-1-1 con clone has 28 base pairs that are not present in the other three clones. These base pairs occur at the beginning of the 5-1-1 cDNA sequence ^and are indicated by lowercase letters. Based on the radioimmunoassay data, which will be discussed later in Section IV.D., it is possible that an HCV epitope could be encoded in this 28 bp region,

The absence of the 28 base pairs 5-1-1 in clones 81, 1-2 and 91 may mean that the cDNA in these clones was derived from the defective HCV genomesj alternatively, the 28 bp region could be 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 were not found in other cDNAs because the cDNAs that overlap these regions have not yet been iso.

sides.

A composite HCV cDNA sequence derived from the overlapping cDNAs in clones 5-1-1, 8l ,. 1-2 and 91 is, shown in Fig. 3. However, in this figure the only 28 base pairs of clone 5-1-1 have been omitted. The figure also shows the sequence of the.polypeptide encoded within the HCF of the HCV cDNA composite,

IV.A.5. Isolation of HCV cDNAs that overlap cDNA

Clone 81.

isolation of HCV cDNA sequences upstream of, and overlapping those in, clone 81 cDNA was performed as follows. The lambda-gtll library cDNA pre-prepared as described in Section IV.A.l. was sieved by hybridization with a synthetic polynucleotide probe that was homologous to a 5 'terminal sequence of clone 81. The sequence of clone 81 is shown in Figure 4. The sequence of the synthetic polynucleotide 9'0 used for' 'screening was :

5 'CTG TCA GGT ATG ATT GCC GGC TTC CCG GAC 3'.

The methods were essentially as described in Huynh (1985) »except that the library filters were given two washes under strict conditions, that is, the washes in 5 x SSC, 0.1% SDS at 55 ° C for 30 minutes each. Approximately 1 in 50,000 clones hybridized with the probe. A positive recombinant phage containing cDNA that hybridized to the sequence was isolated and purified. This fage was numbered as clone 36.

The downstream cDNA sequences, which overlap with the carboxyl terminal sequences in clone cDNA 81, were isolated using a procedure similar to that used for isolating the upstream cDNA sequences, except that a synthetic oligonucleotide probe was prepared, which is homologation of a 3 'terminal sequence of clone 81. The sequence of the synthetic polynucleotide used for screening was:

5 'TTT GGC TAG TGG TTÁ GTG GGC TGG TGA CAG 3'

A recombinant positive fage, which contained cDNA that hybridized to the latter sequence, was isolated and purified, and was numbered clone 3 ² »

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.

The double-fibered cDNA sequence, its region of overlap with the HCV cDNA in clone 81, and the polypeptide encoded by _ 91_

ORF are shown in Fig. 5.

The ORF in clone 36 is in the same translational structure as the HCV antigen encoded in clone 8l. Thus, in combination, the ORFs in clones 36 and 81 encode a polypeptide that represents part of a large HCV antigen. The sequence of this putative HCV polypeptide and the double-stranded DNA sequence encoding the same, which is derived from the combined ORFs of the HCV cDNAs of clones 36 and 81, as shown in Fig. 6.

IV, A.7 HCV cDNA nucleotide sequences in Clone 32

The nucleotide sequence of the cDNA in clone 32 was determined essentially as described in Section IV.A.2 for the sequence of clone 5-1-1. The sequence data indicated that the cDNA in recombinant phage clone 32 was derived from two different sources. A cDNA fragment was composed of 418 nucleotides derived from the HCV genome; the other fraction consisted of 172 nucleotides derived from the bacteriophage genome MS2, which had been used as a vehicle during the preparation of the lambda-gtll plasma cDNA library.

The sequence of the cDNA in clone 3 ² corresponding to the HCV genome is shown in Figure 7. The region of the sequences which overlap with clone 81, and the polypeptide encoded by the ORF are also indicated in the figure. This sequence contains a continuous ORF that is in the same translational structure as the HCV antigen encoded by clone 81,

IV, A, 8 Isolation of HCV cDNA that Overlaps cDNA in Clone

2nd isolation of HCV cDNA sequences upstream

- 92 te de, and overlapping those of clone 36 cDNA was performed as described in Section IV, A.5, for those overlapping clone 81 cDNA, except that the synthetic polynucleotide was based on the 5 'region of the clone 36 · The sequence of the synthetic polynucleotide used for screening was:

5 'AAG CCA CCG TGT GCG CTA GGG CTC AAGCCC 3 ’

Approximately 1 in 50,000 clones hybridized with the probe. The purified clone isolated from the recombinant phage containing cDNA that hybridized to this sequence was designated clone 35

IV.A.9 HCV cDNA Nucleotide Sequence in Clone 35

The nucleotide sequence of the cDNA in clone 35 was determined essentially as described in Section IV.A.2.

The sequence, its region of overlap with that of the cDNA in clone 36, and the putative polypeptide encoded therein, are shown in Fig. 8.

clone 35 apparently contains a single, continuous ORF encoding a polypeptide in the same translational structure as that encoded by clone 36, clone 8l, and clone 32. Fig. 9 shows the sequence of the ORF continuous length that extends through clones 35 »36, 81 and 32, together with the putative HCV polypeptide encoded in them, this combined sequence was confirmed using other independent cDNA clones derived from the same lambda-gtll cDNA library,

IV.A, 10. Isolation of HCV cDNA that overlaps cDNA in Clo0 Isolation of HCV cDNA sequences upstream, and overlapping those of clone 35 cDNA was performed with £

- 93 m 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 sequence of the synthetic polynucleotide used for screening was:

5 'CAG GAT GCT GTC TCC CGC ACT CAA CGT 3'

Approximately 1 in 50,000 clones hybridized with the probe. The isolated, purified clone of the recombinant phage containing cDNA that hybridized to this sequence was designated clone 37h.

IV, A.11. HCV Nucleotide Sequence in Clone 37b

The nucleotide sequence of the cDNA in clone 37b was determined essentially as described in Section IV.A.2. The sequence, its region of overlap with that of the cDNA in clone 35, and the putative polypeptide encoded therein, are represented in Fig. 10.

5'-terminal nucleotide of clone 35 θ a T, whereas the corresponding nucleotide in clone 37b is an A. The cDNAs from three other independent clones that were isolated during the procedure in which clone 37b was isolated, described in Section IV.A.10, were also sequenced. The cDNAs from these clones also contain an A in this position. Thus, the 5'-T-terminus in clone 35 may be an artifact of the cloning procedure. It is known that artifacts often rise at the 5'-termini of cDNA molecules.

clone 37b apparently contains a continuous ORF encoding a polypeptide that is a continuation of the lip94 peptide encoded in the ORF that extends through overlapping clones 35, 36, 81 and 32.

IV.A.12 Isolation of the HCV cDNA that overlaps the cDNA in Clone 22 isolation of the HCV cDNA sequences downstream of clone 32 was performed as follows. First, clone cia was isolated using a synthetic hybridization probe that was 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 there

5 · AGT GCA GTG GAT GAA CCG GCT GAT AGC CTT 3 '.

Using the nucleotide sequence from the clone, another synthetic nucleotide was synthesized, which had the following sequence:

5 'TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3'.

screening of the lambda-gtll library using the sequence derived from the clone cia as a probe produced approximately 1 in 50 · 00θ positive colonies. One isolated, purified clone that hybridized to this probe was designated clone 33b.

IV.A.13 HCV cDNA Nucleotide Sequence in Clone 33b

The nucleotide sequence of the cDNA in clone 33b was determined essentially as described in Section XV.A.2. The sequence, its region of overlap with that of the cDNA in clone 32, and the putative polypeptide encoded in it, are shown in Fig. 11, clone 33b apparently contains a continuous ORF that is an extension of the ORFs in the clones 37b, 35, 3 ^, 81 and 32 that overlap, the polypeptide encoded in clone 33b is in the same translational structure as that encoded in the extended ORF of these overlapping clones,

IV. A. 14 Isolation of HCV cDNAs that Overlap Clone cDNA 37b and cDNA in Clone 33b

In order to isolate the HCV cDNAs that overlap the cDNAs in clone 37b and in clone 33b, the following synthetic oligonucleotide probes, which were derived from the cDNAs in those clones, were used to sieve the lambda-gtll library, using essentially the method described in Section IV, A, 3. The probes used were;

5 'CAG GAT GCT GTC TCC CGC ACT CAA CGT C 3' e

5 'TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3' to detect colonies containing HCV cDNA sequences that overlap with clones 37b and 33t> »respectively. Approximately 1 in 50,000 colonies were detected on each probe. A clone that contained cDNA that was upstream of, and that clasped the cDNA in clone 37b, was designated clone 40b. A clone that contained cDNA that was downstream of, and that overlapped, the cDNA in clone 33b was designated clone 25c.

IV, A, 15 HCV cDNA Nucleotide Sequences in clone 40b and clone 25c

The nucleotide sequences of the cDNAs in heat.

ne 40b and clone 25c were determined essentially as described in Section IV.A, 2. The 40b and 25c sequences, their regions of overlap with the cDNAs in clones 37b and 33b, and the putative polypeptides encoded therein, are represented in Fig. 12 (clone 40b) and in Fig. 13 (clone 25c).

5'-terminal nucleotide of clone 40b is a G. However, cDNAs from five other pendant indone clones that were isolated during the process in which clone 40b was isolated, described in Section IV.A.14, were also sequenced, The cDNAs from these clones also contain a T in their position. Thus, G can represent a cloning artifact (see the discussion in Section IV.A.11).

5'-terminus of clone 25c is ACT, but the sequence of this region in clone cia (sequence not shown) and in clone 33b is TCA. This difference can also represent a cloning artifact, as can the other 5'-terminal 28 nucleotides in clone 5 * 1-1.

Clones 40b and 25c each apparently contain an ORF which is an extension of the continuous ORF in the previously sequenced clones. The ORF nucleotide sequence that extends through clones 4-Ob, 37b, 35, 36, 8l, 32, 33b and 25c, and the amino acid sequence of the putative polypeptide codi. the same, are represented in Fig. 14. In the figure, the potential artifacts were omitted in the sequence, and instead of les, the corresponding sequences in the non-5'-terminal regions of multiple overlapping clones are represented.

- 97 IV.A.ló. Preparation of a HCV cDNA Composite from cDNAs in Clones 36, 81 and 32

HCV cDNA. composite, C100, was built as follows. First the cDNAs from clones 36, and 32 were excised with EcoRI. The EcoRI fragment of the cDNA from each clone was individually cloned into the EcoRI site of the pGEM3-blue vector (Promega Biotec). The resulting recombinant vectors containing the cDNAs from clones 36, 81 and 32 were designated pGEM3-blue / 33, pGEM3-blue / / 81 and pGEM3-blue / 32, respectively. The appropriately oriented recombinant of pGEM3-blue / 8l was macerated with Nael and NarI, and the large fragment (~ 2850 bp) was purified and ligated with the small restriction (~ 57O bp) restriction Nael / NarI purified from pGEM3 -blue / 36. This composite of the cDNAs from clones 36 and 81 was used to generate another pGEM3-blue vector containing the continuous HCV ORF contained within the overlapping cDNA within these clones. This new plasniid was then macerated with PvuII and EcoRI to release a fragment of approximately 680bp, which was then ligated with the small (580bp) fragment Pvuli / EcoRI isolated from the plasmid, appropriately oriented pGEM3-blue / 32, and the composite cDNA from the clones 36 * 81 and 32 were linked to the linearized pSODc 1 vector EcoRI, which is described in section IV.Bl, and which was used to express clone 5-1-1 in bacteria. The recombinants containing the ~ 127O bp EcoRI fragment of the composite HCV cDNA (ClOO) were selected and the cDNA from the plasmids was excised with EcoRI and purified.

- 98— cDNAs

IV.A.17. Isolation and Nucleotide Sequences from HCV in Clones 14i, 11b, 7f, 7®, 8h, 33c, 14c, 8f, 33f,

HCV cDNAs in clones l4i, 11b, 7f, 7®, 8b, 33c, 14c, 8f, 33f, 33g ® 39c were isolated by the technique of isolating overlapping cDNA fragments from the lambda-gtll library of the HCV cDNAs described in Section IV.A.1. The technique used was essentially as described in Section IV.A.3 ·, except that the probes used were designed from the nucleotide sequence of the last clones isolated from the 5 '® 3' end of the combined HCV sequences . The frequency of clones that hybridized with the probes described below was approximately 1 in 5θ · θθθ in each case.

The nucleotide sequences of HCV cDNAs in clones l4i, 8h, 33c, l4c, 8f, 33f, 33g β 39c were determined essentially as described in Section IV.A.2., Except that the excised cDNA from these fages was replaced by cDNA isolated from clone 5-1-1.

clone 33c was isolated using a hybridization probe based on the nucleotide sequence in clone 40b. The nucleotide sequence of clone 40b is shown in Fig. 12. The nucleotide sequence of the probe used to isolate 33c was

5 'ATC AGG ACC GGG GTG AGA ACA ATT ACC ACT 3'

The HCV cDNA sequence in clone 33c, and the overlap with that in clone 4-Ob, is shown in Fig. 15, which also shows the amino acids encoded therein.

clone 8h was isolated using a probe based on the nucleotide sequence in 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 the overlap with that in clone 33c, and the amino acids encoded therein are shown in Fig. 16, clone 7® was isolated using a probe based on the nucleotide sequence in clone 8h, A nucleotide sequence of the probe was

5 'TCG GAC CTT TAC CTG GTC ACG AGG CAC 3'.

The HCV cDNA sequence in clone 7®, the overlap with clone 8h, and the amino acids encoded therein, are shown in Fig. 17.

clone l4c was isolated with a probe based on the nucleotide sequence in clone 25®. The sequence of clone 25c

I is shown in Fig. 13. The probe in isolation of clone l4c 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 that of clone 25c, and the amino acids encoded therein are shown in Fig. 18.

clone 8f was isolated using a probe based on the nucleotide sequence in clone l4c. The probe's nucleotide sequence was

5 · TCC ATC TCT CAA GGC AAC TTG CAC CGC TAA 3 '.

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

clone 33f was isolated using a probe based on the nucleotide sequence present in clone 8f. The nucleotide sequence of the probe was

5 'TCC ATG GCT GTC CGC TTC CAC CTC CAA AGT 3'.

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

clone 33 g was isolated using a probe based on the nucleotide sequence in clone 33f. The probe's nucleotide sequence was

5 'GCG ACA ATA-CGA CAA CAT CCT CTG AGC CCG 3'.

The HCV cDNA sequence in clone 33g, its overlap with that of clone 33f, and the amino acids encoded therein are shown in Fig. 21, clone 7f was isolated using a probe based on the nucleotide sequence in clone 7® · A nucleotide sequence of the probe was

5 'AGC AGA CAA GGG GCC TCC TAG GGT GCA TAA T 3'.

The HCV cDNA sequence in clone 7f, its overlap with clone 7®, and the amino acids encoded therein are shown in Fig. 22, clone 11b was isolated using a probe based on the clone 7f sequence. probe nucleotide was

5 'CAC CTA TGT TTA TAA CCA TCT fíAG TCC TCT 3'.

The HCV cDNA sequence in clone 11b, its overlap with the

1Q1 clone 7f, and the amino acids encoded therein are represented in Fig. 23, clone 14i was isolated using a probe based on the nucleotide sequence in clone 11b. The probe's nucleotide sequence was

5 'CTC TGT CAC CAT ATT ACA AGC GCT ATA TCA 3'.

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

clone 39c was isolated using a probe based on the nucleotide sequence in clone 33g. The probe's nucleotide sequence was

5 'CTC GTT GCT ACG TCA CCA CAA TTT GGT GTA 3'.

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

IV, A, 18. The HCV cDNA sequence derived from clones

Isolates Containing HCV cDNA

The HCV cDNA sequences in the isolated clones described above were aligned to create a HCV cDNA sequence with postite. The isolated clones, aligned 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 .

A composite HCV cDNA sequence derived from the isolated clones, and the amino acids encoded therein, are shown in Fig. 26.

In creating the composite sequence, the following sequence heterogeneities were considered. Clone 33c con has an 800 base pair HCV cDNA, which overlaps the cDNAs in clones 40b and 37c. In clone 33c, as well as in 5 other overlapping clones, nucleotide ^ j = 7θ9 ® a G, However, in clone 37b (see Section IV.A.ii), the corresponding nucleotide is an A. This difference in The sequence creates an apparent heterogeneity in the amino acids encoded therein, which could be either CYS or TYR, for G or A, respectively. This heterogeneity can have important ramifications in terms of protein folding.

nucleotide residue 2 in clone 8h HCV cDNA is a T. However, as shown below, the corresponding residue in clone 7® ® an A; in addition, an A in this position is also found in 3 other isolated clones that overlap. Thus, residue T in clone 8h may represent a cloning artifact. Therefore, in Fig. 26, the residue in this position is designated as an A, 3'-terminal nucleotide in clone 8f HCV cDNA is a G, However, the corresponding residue in clone 33f, θ in two other overlapping clones is one T. Therefore, in Fig, 26, the residue in this position is designated as a T.

The 3'-terminal sequence in the 33f HCV cDNA clone is TTGC. However, the corresponding sequence in clone 33g and in 2 other overlapping clones is ATTC. Therefore, in Fig.26, the corresponding region is represented as ATTC.

nucleotide residue # 4 in clone 33g HCV cDNA is a T. However, in clone 33f and in 2 other clones that overlap the corresponding residue is an A. Therefore, in Fig.26, the corresponding residue is designated as an A.

3'-terminal of clone l4i is an AA, whereas

- X03 that the corresponding dinucleotide in clone 11b, and in three other clones, is TA. Therefore, in Fig. 26, the TA residue is drawn.

The resolution of other sequence heterogeneities is discussed above.

An examination of the composite HCV cDNA indicates that the HCV contains a large ORF. This suggests that the viral genorn is carried in a large polypeptide which is processed concurrently with, or subsequently to translation.

IV.A.19. Isolation and Nucleotide Sequences from HCV cDNAs in Clones 12f, 35f, 19ff, 26g and 15θ

HCV cDNAs in clones 12f, 35f, 19g, 26g and 15θ were isolated essentially by the technique described in Section IV.A.17, except that the probes were as indicated below. The frequency of clones that hybridized to the probes was approximately 1 in 50,000 in each case. The nucleotide sequences of the HCV cDNAs in these clones were determined essentially as described in Section IV, A.2., Except that the cDNA from the indicated clones was replaced by the cDNA isolated from clone 5-1-1.

Isolation of clone 12f, which contains cDNA upstream from the HCV cDNA in Fig. 26, was performed using a hybridization probe based on the nucleotide sequence in clone 14i. The probe's nucleotide sequence was

5 'TGC TTG TGG ATG ATG CTA CTC ATA TCC CAA 3'.

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

104 isolation of clone 35f, which contains cDNA downstream of the HCV cDNA in Fig. 26, was performed using a hybridization probe based on the nucleotide sequence in cl <3 n and 39c. The probe's nucleotide sequence was

5 'AGC AGC GGC GTC AAA AGT GAA GGC TAA CTT 3'.

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

isolation of clone 19g was performed using a hybridization probe based on the 3 'sequence of clone 35f. The probe's nucleotide sequence was

5 'TTC TCG TAT GAT ACC CGC TGC TTT GAC TCC 3'.

The HCV cDNA sequence of clone 19g, its overlap with the sequence in clone 35f, ® the amino acids encoded therein are shown in Fig. 29.

isolation of clone 26g was performed using a hybridization probe based on the 3 'sequence of clone 19g. The probe's nucleotide sequence was

5 'TGT GTG GCG ACG ACT TAG TCG TTA TCT GTG 3'.

The HCV cDNA sequence of clone 26g, its overlap with the sequence in clone 19g, θ the amino acids encoded therein are shown in Fig. 30.

clone 15θ was isolated using a hybridization probe based on the 3 * sequence of clone 26g. The probe's nucleotide sequence was

5 'CAC ACT CCA GTC AAT TCC TGG CTA GGC AAC 3'

The HCV cDNA sequence of clone 15θ, its overlap with

- 105 the sequence in clone 26g, and the amino acids encoded therein are shown in Fig. 31.

The clones described in this Section have been deposited with the ATCC under the terms and conditions described in Section II.A., and have been designated with the following Accession Numbers.

lambda-gtll ATCC No. Deposit Date clone 12f 40514 November 10, 1988 clone 35f 40515 tf clone 15e 40513 ff clone K9-1 40512 fl

The HCV cDNA sequences in the isolated clones described above, were aligned to create a composite HCV cDNA sequence. The isolated clones, aligned in the 5 'to 3' direction are: 12f, 14i,? F, 7θ, 8h, 33c, 40b, 37b, 35, 36, 8l, 32, 33b, 25c, l4c, 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, are shown in Fig. 32.

IV.A.20. Alternative Method of Isolating cDNA Sequences Upstream of HCV cDNA Sequence in Clone 12f

Based on most of the 5 'HCV sequence in Fig. 32, which is derived from the HCV cDNA in clone 12f, small reverse transcriptase synthetic oligonucleotide primers are synthesized and used to bind to the HCV RNA genomic color sequence, for primary reverse transcription of the upstream sequences. The primary sequences are close to the known 5'-terminal sequence of clone 12f, but sufficiently downstream to allow the design of the “106 υ sequences

ο of the probe upstream of the primary sequences. Standard loading and cloning methods are used. The resulting cDNA librarians are sieved with sequences upstream of the loading sites (as deduced from the sequence elucidated in clone 12f). The HCV genomic RNA is obtained from either plasma or from a chimpanzee liver sample with NANBH, or from analogous human samples with NANBH.

IV.A.21 Alternative Method Using Tail Trainer to Isolate Sequences from the 5'-Terminal Region of the

HCV Genome

In order to isolate the extreme 5'-terminal sequences of the HCV RNA genome, the cDNA product of the first reverse transcriptional ron, which is duplexed with the standard RNA, is tailed with the oligo C. This is done by incubating the product with terminal transferase in the presence of CTP. The second round of cDNA synthesis, which provides the complement of the first cDNA fiber, is carried out 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. Methods for tailing with terminal transferase, and for reverse transcriptase reactions are as indicated in Maniatis et al. (1982). The cDNA products are then spun, sieved, and sequenced,

IV.A.22, Alternative Method Using Tail Formation to Isolate Sequences from the 3'-Terminal Region of the HCV Genome

This method is based on the methods previously used to clone Flavivirus RNA cDNAs.

107 ~

All in all, the RNA is subjected to denaturation conditions to move secondary structures in the 3'-terminus, and is then endowed with Poly A polymerase using rATP as a substrate. Reverse transcription of RNA equipped with a P1 li A tail is catalyzed by reverse transcriptase using oli. go dT as a primer. The second cDNA fibers are synthesized, the cDNA products are cleaved, sieved, and sequenced.

, IV, A.23 Creation of Librarias Lambda-gtll HCV cDNA Containing

Largest cDNA Supplements>

The method for creating and sieving Lambda-gtll libraries is essentially as described in Section IV.A.1., except that the library is generated from a larger size cDNA collection eluted from the Sepharose CL-4B column.

IV, A.24 Creation of HCV cDNA Librarians Using Synthetic Oligomers as Primers

I New HCV libraries with cDNA Using oligomers were prepared from RNA derived from the collection of sinks; of an infectious chimpanzee described in Section IV, A, 1., and from the poly A ⁺ RNA fraction derived from the liver of this infected animal. The cDNA was constructed essentially as described by Gubler and Hoffman (1983), except that the primers for the first cDNA fiber synthesis were two synthetic oligomers. based on the HCV genome sequence described above. The primers based on the clone 11b and 7e sequence were, respectively,

108 5 'CTG GCT TGA AGA ATC 3' e

5 'AGT TAG GCT GGT GAT TAT GC 3 ’

The resulting cDNAs were cleaved into lambda, 'phage vectors' and sieved with various other syntactic oligomers, whose sequences were based on the HCV sequence in Fig. 32.

IV. B, Expression of Polypeptides Encoded Within HCV cDNAs and Identification of Products Expressed as

HCV-induced antigens.

IV.B.1. Expression of the Polypeptide Encoded in Clohe 5-1-1.

HCV polypeptide encoded within clone 5-1-1 (see Section IV, A.2 ', above) was expressed as a superoxide dismutase (SOD) fusion polypeptide. This was accomplished by subcloning the 5-1-1 cDNA clone supplement in the pSODcfl expression vector (Steimer et al. (1986)) as follows.

First, the DNA isolated from pSODcfl was treated with BamHI and EcoRI, and the following ligands were ligated into the linear DNA created by the restriction enzymes:

5 ’GAT CCT GGA ATT CTG ATA A 3’

3 'GA CCT TAA GAC TAT TTT AA 5'

After cloning, the plasmid containing the supplement was isolated.

plasmid containing the supplement was restricted with EcoRI. The HCV cDNA supplement in clone 5-1-1 was extracted with EcoRI, and ligated into this linearized plasmid EcoRI DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al. (1980)). Combinants with 5-1-1 cDNA in the correct orientation for ORF expression shown in Fig. 1 were identified by restriction mapping and nucleotide sequencing.

Recombinant bacteria from the cl <> ne were induced to express the SOD-NANB ^^ polypeptide! by germination of bacteria in the presence of IPTG,

IV.B.2. Expression of the Encoded Polypeptide in Clone 8l

HCV cDNA contained within clone 81 was expressed as a SOD-ΝΑΝΒθ ^ fusion polypeptide. The method for preparing the vector encoding this fusion polypeptide was analogous to that used for the creation of the vector encoding SOD-NANB ^ except that the source of the HCV cDNA was clone 81, which was isolated as described in Section IV .A.3, and for which the cDNA sequence was determined as described ^! in Section IV.A, 4. The nucleotide sequence of the HCV cDNA in clone 81, and the putative amino acid sequence of the polypeptide encoded therein are shown in Fig. 4.

HCV cDNA supplement in clone 81 was excised with EcoRI, and ligated into the pSODcfl which contained the linker (see IV.B.l.) and which was linearized by treatment with EcoRI.

The DNA mixture was used to transform E12 coli strain D1210. Recombinants as clone 8l HCV cDNA in the orien

110

correct expression for ORF expression represented in Fig. 4 were identified by restriction design and nucleotide sequencing.

Recombinant bacteria from a clone were induced to express the SOD-NANBg ^ polypeptide by germinating the bacteria in the presence of IPTG.

IV, B.3. Identification of the Encoded Polypeptide Within the

Clone 5-1-1 as an HCV and Antigene NANBH Associated.

polypeptide encoded within the HCV cDNA of clone 5-1-1 was identified as an associated NANBH antigen demonstrating that the sera of chimpanzees and humans infected with NANBH reacted immunologically with the fusion polypeptide, SOD-NANB ^ · ₁ which is constituted by superoxide dismutase in its N-terminus and antigen 5-1-1 in structure in its C-terminus. This was accomplished by the formation of Western blotches (Towbin et al. (1979)) as follows.

A recombinant strain of bacteria transformed with an expression vector encoding the SOD-NANB4 polypeptide described in Section IV.B.I., was induced to express the germinating fusion polypeptide in the presence of IPTG. The total bacterial lysate was electrophoresed through polyacrylamide gels in the presence of SDS according to Laemmli (1997) · The separated polypeptides were transferred on nitrocellulose filters (Towbin et al. (1979)). The filters were then cut into thin strips, and the strips were individually incubated with different sera from chimpanzees and humans. Limit antibodies fo.

111 à

5 were detected by further incubation with classified sheep anti-human I-Ig, as described in Section IV, A, 1.

The characterization of the chimpanzee sera used for Western stains and the results, shown in the photograph of the autoradiographed strips, are present in Fig. 33. The nitrocellulose strips containing polypeptides were incubated with chimpanzee-derived sera at different times during acute infections of NANBH (Hutchinson strain) (passages l-ló), hepatitis A infections (passages 17-24 and 26-33) θ hepatitis B infections (passages

Passages 25 and 45 show positive controls in which the immunoblots were incubated with serum from the patient used to identify the recombinant clone

5-1-1 in the original screening of the lambda-gt11 library cDNA (see Section IV.A.l.).

The band visible at the control passages, 25 and 45 'in Fig, 23 reflects the binding of the antibodies to the NANBH ^ 1 1 d ° SOD fusion polypeptide. These antibodies do not exhibit binding to isolated SOD, as this was also included as a negative control in these samples, and would have appeared as a band migrating significantly faster than the SOD-NANB fusion polypeptide ^

Passages 1-6 of Fig, 33 show the binding of antibodies in serum samples from 4 chimpanzees; the Sroses were obtained just before infection with NANBH, and sequentially during acute infection. As seen in the figure, considering that the antibodies that reacted immunologically

112

with the SOD-NANB _ _{Ί polypeptide} . were absent in the 5 - .1 -1 serum samples obtained prior to the administration of infectious HCV inoculum and during the previous acute phase of infection, all 4 animals eventually induced circulation antibodies to this polypeptide during the last part of, or thereafter to the acute phase. Additional strips observed in the immunostains in cases <4th chimpanzees numbers 3 and 4 were due to the experiment binding to proteins of host bacteria.

In contrast to the results obtained with sera from chimpanzees infected with NANBH, the development of antibodies to the NANB4 half of the fusion polypeptide was not observed in 4 chimpanzees infected with HAV or 3 chimpanzees infected with HBV. The only link in these cases was the experimental link to these host bacterial proteins, which also occurred in samples infected with HCV.

The characterization of the human sera used for Western stains, and the results, which are represented in the photograph of the autoradiographed strips, are present. shown in Fig. 3 ^. Nitrocellulose strips containing polypeptides were incubated with sera derived from humans at different times during infections with NANBH (passages 1-21) ,, HAV (passages 33- ^ 0) θ HBV (passages 41-49). Passages 25 and 5θ show positive controls in which the immunostains were incubated with serum from the patient used in the original screening of the lambda library113

-gtll, described above. Passages 22-24 and 26-32 show uninfected controls in which the sera came from normal blood donors.

As seen in Fig., The sera from nine patients with NANBH, including the serum used for screening lambda-gtll library, contained antibodies to the NANB4 half of the fusion polypeptide. Sera from three patients with NANBH do not contain these antibodies. It is possible that anti-NANB_ antibodies,

5-1-1 - will return at a future date in these patients. It is also possible that this lack of reaction was the result of a different NANBV agent that causes the disease in individuals from whom the unresponsive serum was collected.

Fig. 3 ^ shows that the sera from many patients infected with HAV and HBV do not contain anti-NANB ^ χ ^and H ^u antibodies ^and these antibodies were also not present in sera from normal controls Although a patient with HAV (passage 36 ) appears to contain anti-NANB ^ χ χ »® antibodies possible that this patient may have previously been infected with HCV, since the incidence of NANBH is very high and since it is often sub-clinical.

These serological studies indicate that the cDNA in clone 5-1-1 encodes epitopes that are specifically recognized by sera from patients and animals infected with BB-NANBV. Furthermore, the cDNA does not appear to be derived from that of the primary genome. A hybridization probe made from clone 5-1-1 or from clone 81 did not hybridize the South DNA genetically control patches of humans and chimpanzees from uninfected individuals under conditions where single, copy genes -Unique are detectable. These probes also do not hybridize the southern spots of the bovine, genomic control DNA.

IV, B, 4. Expression of the Polypeptide Encoded in a Composite of HCV cDNAs in Clones 36, 81 and 32 HCV polypeptide that is encoded in the ORE that extends through clones 36, 81 and 32 was expressed as a SOD fusion polypeptide. This was accomplished by inserting the cDNA composite, C 100, 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.

An expression cassette containing 0 C100 cDNA with powder derived from clones 36, 8l and 32, was constructed by inserting the ~ 1270bp EcoRI fragment into the EcoRI site of the pS3-5Ó vector (also called pS35ú), providing the plasmid pS3-5Úg ^ QQ. The construction of C100 is described in Section IV.A, 16, above.

The pS3-5U vector, which is a pBR322 derivative, contains an expression cassette that consists of the hybrid yeast promoter ADH2 / GAPDH upstream of the human superoxide dismutase gene, and a downstream GAPDH transcription terminator. A similar cassette, which contains these control elements and the superoxide dismutase gene, has been described in Cousens et al. (1987), and in EPO order 196,056

115 co-pending, published on October 1, 1986, which is commonly owned by the assignee of the present application for pa. try. The pS3-5ú cassette, however, differs from that of Cousens et al. (1987) for the fact that the heterologous proinsulin gene and the immunoglobulin joint are removed, and because the glomerulin. the superoxide dismutase is followed by an adapter sequence that contains an EcoRI site. The adapter string is

5’-AAT TTG GGA ATT CCA TAA TGA G -3 '

AC CCT TAA GGT ATT ACT CAG CT site EcoRI allows the insertion of heterorologous sequences that, when expressed from a vector containing the cassette, provides polypeptides that are fused with superoxide dismutase through an oligopeptide linker containing the amino acid sequence:

-asn-leu-gly-ile-arg-.

A sample of pS35ú was deposited on April 29, 1988 under the conditions of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Marilândia 20853, θ was designated Accession No. 67,683, The terms and conditions for availability and access to the deposit, and for maintaining the deposit are the same as those specified in Section II.A, for strains containing NANBV-cDNAs. This deposit is made for convenience only, and is not required for the practice of By virtue of the description given herein, the deposited material is therefore incorporated herein by reference.

- Il6 After the recombinants containing the C100 cDNA supplement in the correct orientation were isolated, the expression cassette containing the C100 cDNA was excised from Ρ ^ 5-56θ ^ θθ with BamHI, and a ~ 3 ^ OObp fragment containing the cassette was isolated and purified. This fragment was then inserted into the BamHI site of the yeast vector pAB2U.

plasmid pAB24, whose significant characteristics are shown in Fig. 35, ® a span vector. comes from yeast containing the complete 2 micron sequence for J_ Broach replication (l98lj_7 and pBR3 ²² · 0 sequences it also contains the lévedurà URA3 gene derived from plasmid YE _p 24 / jBotstein et al. (1979.27, and the yeast gene LEU ^ derived from plasmid pCl / l, EPO pub No. 116,201, plasmid pAB24 was constructed by macerating YEp2U with EcoRI and rewiring the vector to remove partial 2 micron sequences. The resulting plasmid, YEP2àdeltaRI, was linearized by digestion with Ciai and ligated with the complete 2 micron plasmid that had been linearized with ClaI, the resulting plasmid, pCBou, was then macerated with Xbal and the 8605 bp vector fragment was gel isolated This isolated Xbal fragment was ligated with a 4 ^ fragment 60 bp Xbal containing the 2d, LEU gene isolated from pCl / l; the orientation of the 2d 'gene

LEU is in the same direction as the URA 3 gene. The expression insertion was in the only BamHI site of the pBR322 sequence, thus interrupting the gene for bacterial resistance to tetra cyclin.

- 117 (,

'The recombinant plasmid that contained the SOD-C100 expression cassette, pAB24C100-3, was transformed into the yeast strain JSC 308, as well as other yeast strains.' The cells were transformed as described by Hinnen et'al. (ΐ97θ), θ coated on ura-selective plates. Simple colonies were inoculated in leu-selective media and germinated until saturation. The culture was induced to express the SOD-C100 polypeptide (called C100-3) by germination in YEP containing 1% glucose.

The JSC strain 308 is of the genotype MAT, leu2, ura3 (del) DM15 (GAP / ADR1) integrated in the ADR1 site. In JSC 3O8, overexpression of the positive activator gene product, ADR1, results in hyperdepression (relative to a wild-type control ADR1) and significantly higher yields of heterologous proteins expressed when such proteins are synthesized through a system ADH2 UAS regulator. The construction of the yeast strain JSC 308 is disclosed in the US co-pending application Serial No. (Attorney Docket No.23OO-O229) filed simultaneously with the present, which is hereby incorporated by reference. A sample of JSC 308 was deposited on 5 May 1988 at the ATCC under the terms of the Budapest Treaty, and was assigned to Acejs are No. 20879. The terms and conditions for availability and access to the deposit, and for deposit maintenance are the same as those specified in Section II.A, for strains containing HCV cDNAs.

complete C100-3 fusion polypeptide encoded in pAB24C100-3 must contain 154 human SOD amino acids in the

118

amino-terminal, 5 amino acid residues derived from the synthetic adapter containing the EcoRI site, 363 amino acid residues derived from C100 cDNA, and 5 carboxy-terminal amino acids derived from the MS2 nucleotide sequence by joining the HCV cDNA sequence in clone 32. ( See Section IV.A.7). The putative amino acid sequence of the carboxy-terminal of this polypeptide, starting at the penultimate residue Ala de SOD, is represented in Fig. 3ú; Also depicted is the nucleotide sequence encoding this portion of the polypeptide.

IV.B.5. Identification of the C100-Encoded Polypeptide as a NANBH-Associated Antigen C100-3 fusion polypeptide expressed from the plasmid pAB24C100-3 in the yeast strain JSC 308 was characterized in relation to size, and the C100-encoded polypeptide was identified as a NANBH-associated antigen for its immunological reactivity with serum from a human with chronic NANBH, C100-3 polypeptide, which was expressed as described in Section IV.B.4, was analyzed as follows. YSC 3θθ yeast cells were transformed with pAB24, or pAB2UciOO-3, and were induced to express the encoded heterologous polypeptide plasmid. The yeast cells introduced in 1 ml of culture (0D / „ _n ~ 20) were transformed into microspheres by centrifugation at 10,000 rpm for 1 minute, and were lysed by swirling them vigorously (10 x x 1 min.) With 2 volumes of solution and 1 volume of glass microspheres (0.2 millimicron in diameter). The solution contained 50

119 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 microgram / ml of pepstatin. The lysate insoluble material, which includes the C100-3 polypeptide, was pooled by centrifugation (10,000 rpm for 5 minutes) and was dissolved by boiling for 5 minutes in a Laemmli SDS buffer sample. / See Laemmli (197.0.17 · An equivalent amount of Kquela polypeptides in 0.3 ml of the culture of an induced seal was electrophoresed through 10% polyacrylamide gels in the presence of SDS according to Laemmli (ΐ97θ) Standards. of protein were subjected to co-electrophoresis on the gels.-Gels containing the expressed polypeptides were stained with bright blue Coomassie, either subjected to Western blots 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.

The results are shown in Fig. 37. In Fig. 37A the polypeptides were stained with brilliant blue Coomassie. The insoluble polypeptide (s) from 3SC 308 transformed with pAB24 and from two different JSC colonies transformed with pAB24C100-3 are represented in passage 1 (pAB24), and passages 2 and 3 'respectively. A comparison of passages 2 θ 3 with passage 1 shows the, induced expression of a polypeptide corresponding to a molecular weight of ~ 54,000 daltons from JSC 308 transformed with pAB24C100-3 »which is not induced in JSC 308 transformed with pAB24. This polypeptide is indicated by

120 arrow

Fig. 37B shows the results of the Western blot of the insoluble polypeptides expressed in JSC 308 transformed with pAB24 (passage 1), or with pAB24clOO-3 (passage 2). The polypeptides expressed from pAB24 were not immunologically reactive with serum from a human with NANBH. However, as indicated by the arrow, JSC 308 transformed with pAB24C100-3 expressed a ~ 54,000 dalton molecular weight polypeptide that reacted immunologically with human NANBH serum. The other immunologically reactive polypeptides in passage 2 can be products of degradation and / or aggregation of this polypeptide ~ 54,000 dalton.

IV.B, 6. Purification of the C100-3 Fusion Polypeptide fusion polypeptide, C100-3 »consisting of SOD at the N-terminus and in structure of the ClOO HCV polypeptide at the C-terminus was purified by differential extraction of the insoluble fraction of the host yeast cells extracted in that the polypeptide was expressed.

fusion polypeptide, C100-3, was expressed in yeast strain JSC 3θθ transformed with pAB24C100-3, as described in Section IV, B.4. The yeast cells were then lysed by homogenization, the lysate insoluble material was extracted at pH 12.0, and the C100-3 in the remaining insoluble fraction was solubilized in the buffer containing SDS, ¹ yeast lysate was prepared essential121 -

according to Nagahuma et al. (1984). A yeast cell suspension was prepared with 33% cells (v / / v) suspended in a solution (Buffer A) containing 20mM Tris HCl, pH 8.0, 1mM dithiothreitol, and 1mM phenylmethylsulfonyl fluoride (PMSF) . An aliquot part of the suspension (15 ml) was mixed with an equal volume of glass microspheres (0.45-0.50 mm in diameter), and the mixture was swirled at the top speed of a Super Mixer (Lab Line Instruments, Inc,) for 8 min, the homogenate and the glass microspheres were separated, and the glass microspheres were washed 3 times with the same volume of Buffer A as the original packed cells. After combining the washes and the homogenate, the lysate insoluble material was obtained by centrifugation of the homogenate at 7,000 xg for 15 minutes at 4 ° C, resuspending the microspheres in Buffer A equal to twice the volume of the original packed cells , and turning the material back into microspheres by centrifugation at 7,000 xg for 15 min. This washing process was repeated 3 times.

insoluble material from lysate was extracted at pH 12.0 as follows. The microspheres were suspended in a buffer containing 0.5 M NaCl, 1 mM EDTA, where the suspension volume was 1.8 times that of the original packaged cells. The pH of the suspension was adjusted by adding 0.2 volumes of 0.4 M Na phosphate buffer, pH 12.0. After mixing, the suspension was centrifuged at 7,000 x g for 15 min, at 4 ° C, and the supernatant removed. The extraction was repeated 2 times. The extracted microspheres were there ’

- 122 vows suspending them in 0.5 M NaCl, 1 mM EDTA, using a suspension volume equal to two volumes of the original packed cells, followed by centrifugation at 7,000 x g for 15 min, at 4 ° C.

C100-3 polypeptide in the extracted microspheres was solubilized by treatment with SDS. The microspheres were suspended in Buffer Λ equal to 0.9 volumes of the volume of original packed cells, and 0.1 volumes of 2% SDS were added. After the suspension was mixed, it was centrifuged at 7,000 x g for 15 min. at 4 ° C. The resulting microspheres were extracted 3 times more with SDS. The resulting supernatants, which contained C100-3, were placed in a reservoir.

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

The purified preparation of the fusion polypeptide was analyzed by polyacrylamide gel electrophoresis according to Laemmli (1970). Based on this analysis, the polypeptide was superior in purity to 80%, and had an apparent molecular weight of * ”54,000 daltons.

IV, C. Identification of RNA in infected individuals that Hi-. bridizes for HCV cDNA

IV.C.1. Identification of RNA in the Liver of a Chimpanzee With NANBH that hybridizes to HCV cDNA.

RNA from a chimpanzee's liver

- 123a χ 'ο

that had .NANBH was shown to contain a species of RNA that hybridizes to the HCV cDNA contained within clone 81 by creating spots in the North, as follows.

RNA was isolated from a chimpanzee liver biopsy from which the high titer plasma was derived (see Section IV, A.1.) Using the techniques described in Maniatis et al. (1982) for the isolation of total RNA from mammalian cells, and for their separation into poly A ⁺ and poly A fractions. These RNA fractions were electrophoresed on a formaldehyde / agarose gel (1% w / v), and transferred to nitrocellulose. (Maniatis et al. (1982)). The nitrocellulose filters were hybridized with HCV cDNA radio-classified from clone 81 (see Fig.

for the nucleotide sequence of the supplement). To prepare the radio-classified probe, the HCV cDNA supplement isolated from clone 81 was radio-classified with ^J p by incision translation using DNA Polymerase 'I (Maniatis et al. (1982)), Hybridization was carried out for 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 NagHPO ^, pH 6.5, 0.1% SDS, 0.02% (w / v) of bovine serum albumin (BSA), 0.02% (w / v) of Ficoll-400, 0.02% ( w / v) polyvinylpyrrolidone, 100 micrograms / ml of salmon sperm DNA that had been sheared by sonication and denatured, and 10 æ CPM / ml of the incision-translated cDNA probe.

An autoradiography of the polled filter is shown in Fig. 38. Passage 1 contains fragments markers

124 -

p-classified constraint. Passages 2-4 contain chimpanzee liver RNA as follows: passage 2 contains 30 micrograms of total RNA; passage 3 contains 30 micrograms of poly A RNA and conduit 4 contains 20 micrograms of poly A ⁺ RNA. As shown in Fig. 3θ, θ chimpanzee liver with NANBH contains a heterogeneous population of moz 4 ·. related cells that hybridize to the HCV cDNA probe, and which appear to be from about 5,000 nucleotides to about 11,000 nucleotides in size. This RNA that hybridizes to the HCV cDNA, could represent viral genomes and / or transcripts specific to the viral genome.

The experiment described in Section IV.C.2, below, is consistent with the suggestion that HCV contains an RNA genome,

IV.C.2. Identification of HCV-Derived RNA in Serum from Infected Individuals.

Nucleic acids were extracted from particles isolated from NANBH plasma from high-tier chimpanzees as described in Section IV.Al. Aliquot parts (equivalent to 1 ml of original plasma) of the isolated nucleic acids were resuspended in 20 microliters of 50 mM Ilepes, pH 7.5, I mm EDTA and 16 micrograms / ml of yeast soluble RNA. The samples were denatured by boiling for 5 minutes followed by immediate refrigeration, and were treated with RNase A (5 microliters containing 0.1 mg / ml RNase A in 25 mM EDTA, 40 mM Hepes, pH 7.5) or with DNase I (5 microliters containing 1 DNasel unit in 10 mM of

125

MgCl ₂ , 25 mM Hepes, pH 7.5); control samples were incubated without enzyme. Following incubation, 230 microliters of 2XSSC ice cream containing 2 micrograms / ml of soluble RNA yeast were added, and the samples were filtered _; on a nitrocellulose filter. The filters were hybridized with a cDNA probe from clone 8l, which was classified ^J p by incision-translation. Fig. 39 shows an autoradiography of the filter. The hybridization signals were detected in the treated DNase and in the control samples (passages 2 and 1, respectively), but were not detected in the sample treated with RNase (passage 3). Thus, since treatment with RNase A destroyed the nucleic acids isolated from particles, and treatment with DNase I had no effect, the evidence strongly suggests that the HCV genome is composed of RNA.

IV.C, 3 »Detection of Extended HCV Nucleic Acid Sequences derived from HCV Nucleic Acid Sequences in the Liver and Plasma Specimens from Chimpanzees with NANBH

HCV nucleic acids present in the liver and plasma of chimpanzees with NANBH, and in chimpanzees of control. le, were amplified using essentially the polymerase chain reaction (PCR) technique described by Saiki et al, (1986). The primary oligonucleotides · were derived from the HCV cDNA sequences in clone 81, or clones 36 and 37 · The extended sequences were detected by gel electrophoresis and spotting in the south, using as probes the appropriate cDNA oligemere with a sequence from region

126 -

between, but not including, the two primaries.

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. Isolation of the RNA fraction was carried out by the gua nidinium thiocyanate process described in Section TV.C.l.

RNA samples that were to be examined by the amplification system were also isolated from plasmas of two chimpanzees with NANBH, and from a control chimpanzee, as well as from a control chimpanzee plasma reservoir. An infected chimpanzee had an ICD / ml equal to or greater than 10%. and the other infected chimpanzee had an ICD / ml equal to or greater than 10.

Nucleic acids were extracted from plasma as follows. Both 0.1 ml and 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, 0.1 M NaCl, 1 mg / ml proteinase K, and 0.5% SDS) containing 10 micrograms / ml polyadenic acid, and incubated at 37 ° C for 60 minutes. After 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) of saturated phenol. The phenol phase was separated by centrifugation, and was extracted again with TENB containing 0.1% SDS. The aqueous phases resulting from each extraction were placed in a reservoir, and extracted twice with an equal volume of fe127 nol / chloroform / isoarnyl alcohol / I: 1 (99i2j_7 »θ then twice with an equal volume of a 99: 1 mixture of chlorine, form / isoarnyl alcohol Following the phase separation by centrifugation, the aqueous phase was brought to the final concentration of 0.2 M Na acetate, and the nucleic acids were precipitated by the addition of two volumes of ethanol. precipitated nucleicles were recovered by ultra-centrifugation in a SW 4l rotor at 38 K, for 60 minutes at 4 ° C.

In addition to the above, the high-tier chimpanzee plasma and the control plasma in a reservoir were alternatively extracted with 5θ micrograms of poly A vehicle by the process of Chomcyzski and Sacchi (1987). This process uses an extraction; of acid guanidinium thiocyanate, the RNA was recovered by centrifugation at 10,000 rpm for 10 minutes at 4 ° C in an Eppendorf microfuge,

On two occasions, prior to cDNA synthesis in the PCR reaction, nucleic acids extracted from plasma by the proteinase K / SDS / phenol method were further purified by binding to and eluting from Columns S and S Elutip-R. The process followed was in accordance with the manufacturer's instructions.

cDNA used as a standard for the PCR reaction was derived from nucleic acids (either total nucleic acids or RNA) prepared as described above, Se. Following ethanol precipitation, nucleic acids precipitate. were dried, and resuspended in distilled water

128 treated with DEPC. The secondary structures in the nucleic acids were disrupted by heating at 65 ^o C for 10 minutes, and the samples were immediately cooled on ice. The cDNA was synthesized using 1 to 3 micrograms of total chimpanzee RNA from the liver, or acid, from the nucleic acids (or RNA) extracted from 10 to 100 microliters of plasma. The synthesis used reverse transcriptase, and was performed in a 25 microliter reaction, using the protocol specified by the manufacturer, BRL. Primers for .cDNA synthesis were also used in the PCR reaction, described below. All reaction mixtures for cDNA synthesis contained 23 units of the RNAase inhibitor, RNASIN ~ (Fisher / Promega). Following cDNA synthesis, the reaction mixtures were diluted with water, boiled for 10 minutes, and quickly cooled on ice.

The PCR reactions were carried out essentially according to the manufacturer's instructions (Cetus-Perkin-Elmer), except for the addition of 1 microgram of RNase A. The reactions were carried out in a final volume of 100 microliters. 0 PCR l

was performed for 35 cycles, using a regime of 37 ° C, 72 ° C, and 94 ° C.

The primers for cDNA synthesis and for PCR reactions were derived from HCV cDNA sequences in either clone 81, clone 36, or clone 37b. (The HCV cDNA sequences of clones 81, 36, and 37b are shown in Figs. 4, 5 θ 10, respectively). The sequences of the two primer primers derived from clone 81 were:

9. - = - 4 r

5 'CAA TCA TAC CTG ACA G 3 ’e

5 'GAT AAC CTC TGC CTG A 3' the primary sequence from clone 36 was:

5 ’GCA TGT CAT GAT. GTA T 3 ’

The primary sequence from clone 37b was:

5 'ACA ATA CGT GTG TCA C 3'.

In the PCR reactions, the primary pairs consisted of either two l6-mer derivatives of clone 81, or the l6-mer of clone 36 and the l6-mer of clone 37b.

The products of the PCR reaction were analyzed by separating the products by alkaline gel electrophoresis, followed by the creation of spots in the south, and detection of HCV-cDNA sequences with a cias32 internal oligonucleotide probe from a HCV cDNA region it does not overlap with the primaries. The 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 pooled by centrifugation, and dissolved in distilled water. Aliquots of the samples were electrophoresed on 1.8% alkaline agar gels. Single-fiber DNA of 6θ, 108, and long nucleotide lengths were electrophoresed together on the gels as molecular weight markers. After electrophoresis, the DNAs in the gel were transferred to Biorab Zeta Probe paper. Pre-hybridization and

hybridization, and washing conditions were those specified by the manufacturer (Biorad).

The probes used for the hybridization-detection of the applied HCV cDNA sequences were as follows. When the PCR primary pair was derived from clone 81, the probe was 108-mer with a sequence corresponding to that located in the region between the sequences of the two primaries. When the PCR primer pair was derived from clones 36 and 37b, the probe was the incision-translated HCV cDNA supplement derived from clone 35 »The primers are derived from nucleotides 155-170 of the clone 37b supplement, and the 206-268 supplement from clone 36. The 3'-end of the HCV cDNA supplement in clone 35 overlaps with nucleotides 1-186 of the supplement in clone 36; and the 5'-end of the supplement from clone 35 overlaps nucleotides 207-269 of the supplement in clone 37b. (Compare Figs, 5, 8 and Ιθ). Thus, the cDNA supplement in clone 35 spans part of the region between the primer sequences derived from clones 36 and 37b, and is useful as a probe for the extended sequences that include these primers.

The analysis of RNA from liver specimens was according to the above procedure using both sets of primers and probes. The RNA from the liver of the three chimpanzees with NANBH provided positive hybridization results for amplification sequences of the expected size (16 and 586 nucleotides for 81 and 36 and 37b,

I respectively), while control chimpanzees gave negative hybridization results. The same results were achieved when the experiment was repeated three times.

131 -

The analysis of nucleic acids and RNA from plasma was also performed according to the above procedure using the primers and probe from clone 81.

The plasmas came from two chimpanzees with NANBH, from a control chimpanzee, and plasmas in a reservoir from control chimpanzees. Both NANBH plasmas contained nucleic acids / RNA that tested positive in the amplified PCR assay, while both control plasmas tested negative. These results have been obtained repeatedly several times.

IV, D, Radioimmunoassay to Detect HCV Antibodies in Serum from Infected Individuals

Solid phase radioimmunoassays to detect antibodies to HCV antigens were developed based on Tsu and Herzenberg (1980). Microtiter plates (immulon 2, Removawell bands) are coated with purified polypeptides containing HCV epitopes. The coated plates were incubated with either human serum samples suspected of containing antibodies to HCV epitopes or for appropriate controls. During the incubation, the antibody, if present, is immunologically bound to the solid phase antigen. After removing the unbound material and washing the microtiter plates ”, human antibody-NANBV antigen complexes are detected by incubation with sheep anti-human immunoglobulin classifies

5, of I. The antibody classified as unbound and aspirated, and the plates are washed. Radioactivity in individual reservoirs is determined; the amount of human anti-HCV antibody bound and proportional to the radioactivity in the reservoir.

IV, D, 1. Purification of the SOD-NANB ^ Fusion Polypeptide. ₁ .

SOD-NANB ^ fusion polypeptide expressed in recombinant bacteria as described in Section IV.Bl ,, was purified from recombinant E, coli by differential extraction of urea cell extracts, followed by chromatography on anion and cation exchange columns. as follows.

Thawed cells, from 1 liter of culture, were resuspended in 10 ml of 20% (w / v) sucrose containing 0.01M Tris HCl, pH 8.0, and 0.4 ml 0.5M of EDTA, pH 8.0 were added. After 5 minutes at 0 ° C, the mixture was centrifuged at 4,000 x g for 10 minutes. The resulting microspheres were 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 llsozyme (10 mg / ml) and incubation at 0 ° C for 10 minutes. After adding 10 ml 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 with occasional stirring. The resulting viscous solution was homogenized by passing 6 times through a sterile 20 gauge hypodermic needle, and centrifuged at 13,000 x g for 25 minutes. The material in the form of microspheres was suspended in 5 ml of 0.01 M Tris HCl, pH 8.0, and the suspension centrifuged at 4,000 x g for 10 minutes. The microspheres, which contained the protein:

in the SOD-NANB fusion

p were dissolved in 5 ml of urea OM in 0.02 M of Tris HCl, pH 8.0, 1 mM of dithiothreitol (Size A), and were applied to a balanced Q-Sepharose column in Fast Flow ( Fast Flow) with Buffer A. The polypeptides were eluted with a linear gradient from 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 for determine its content of SOD-NANB ^ The fractions containing this polypeptide were placed in a reservoir, and dialysed against 6 M urea in 0.02 M sodium phosphate buffer, pH 6.0, 1 mM dithiothreitol (Buffer B) . The dialysed sample was applied to a fast flowing S-Sepharose column with Buffer B, and the polypeptide eluted with a linear gradient of 0.0 to 0.3 M NaCl in Buffer B. The fractions were analyzed by polyacrylamide gel electrophoresis for the presence of SOD-NANB4j and the appropriate fractions were placed in a reservoir.

The final preparation of the SOD-NANB4 polypeptide was examined by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 80% pure.

IV.D.2. Purification of the SOD-NANB ^ Fusion Polypeptide.

fusion polypeptide SOD-NANBg ^, expressed in recombinant bacteria as described in Section IV.B, 2, was purified from the recombinant E. coli by differential extraction of cell extracts with urea, followed by chromatography on anion exchange columns and cation use.

134 of the process described for the isolation of the fu polypeptide are SOD-NANB ₅ _ ₁ _ ₁ (See Section IV.Dl),

The final preparation of the SOD-NANBg ₁ polypeptide was examined 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 to HCV Epitopes by Solid Phase Radioimmunoassay

Serum samples from 32 patients who were diagnosed as having NANBH were analyzed by radioimmunoassay (RIA) to determine whether antibodies to the HCV epitopes present in the SOD-NANB fusion polypeptides.

5-1-1 and SOD-NANBqi were detected.

Microtiter plates were coated with SOD-NANB ,. ,,

5-1-1 or SOD-NANBg ^, which had been partially purified according to Sections IV.D.l. and 1V.D.2., respectively. The tests were conducted as follows.

One hundred parts. Microliter aliquots containing 0.1 to 0.5 micrograms of SOD-NANB ^ χ χ ^or SOD-ΝΑΝΒθ ^ in 0.125 M Na borate buffer, pH 8.3 »0.075 M NaCl ( BBS) was added to each reservoir of an ^M microtiter plate (Dynatech Immulon 2 Removawell Strips). The plate was incubated at 4 ° C overnight in a humid chamber, after which the protein solution was removed and the wells washed 3 times with BBS containing 0.02 # Triton X-100 (BBST). To avoid a non-specific connection, the reservoirs were covered with

with bovine serum albumin (BSA) by adding 100 ml of a 5 mg / ml solution of BSA in BBS followed by incubation at room temperature for 1 hour; after this incubation the BSA solution was removed. The polypeptides in the coated reservoirs were reacted with serum by adding 100 microliters of serum samples diluted 1: 100 in Ο, ΟΙΜ of Na phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) containing 10 mg / ml BSA, and incubating the serum-containing reservoirs for 1 hour at 37 ° C. After incubation, the serum samples were removed by aspiration, and the reservoirs were washed 5 times with BBST, The anti-NANB bond χ χ ^and Anti-NANBgp to the fusion polypeptides, was determined by the binding of sheep anti-human IgG F '(ab) 2 classified ^l2 ^ I to the coated reservoirs. 100 ml aliquots of the classified probe (specific activity 5-20 microcuries / microgram) were added to each well, and the plates were incubated at 37 ° C for 1 hour, followed by removal of the excess probe by aspiration, and 5 washes with BBST. The amount of radioactivity bound in each reservoir was determined by counting on a counter that detects gamma radiation.

ti-NANB _gl

1.

The results of the detection of anti-NANB ^ χ χ ^and S in individuals with NANBH are shown in Table

- 136 -

Table 1

Detection of Anti-5-1-1 ^and Anti-8l in Sera of Patients with Hepatitis NANB, HAV and HBV

Reference number Diagnosis s / n of patient Anti-5-1-1 Anti-8 1. 28 ¹ Chronic NANB, IVD ² 0.77 4.20 Chronic NANB, IVD 1.14 5.14 Chronic NANB, IVD 2.11 4.05 2. 29 ¹ AVH ^, NANB, Sporadic 1.09 1.05 NANB, Chronic 33.89 11.39 NANB, Chronic 36.22 13.67 3. 30 ¹ AVH, NANB, IVD 1.90 1.54 NANB, Chronic, IVD 34.17 30.28 NANB, Chronic, IVD 32.45 30.84 4. 31 NANB, Chronic, PT ^ 16.09 8.05 5. 32 ¹ AVH NANB, Late, IVD 0.69 0.94 AVH NANB, Late, IVD 0.73 0.68 6. 33 ¹ AVH, NANB, IVD 1.66 1.96 AVH, NANB, IVD 1.53 0.56 7. 34 ¹ NANB, Chronic, PT 34.40 7.55 NANB, Chronic, PT 45.55 13.11 NANB, Chronic, PT 41.58 13.45 NANB, Chronic, PT 44.20 15.48 8. 35 ¹ AVH NANB, IVD 31.92 31.95 Cured ”recent 6.87 4.45 NANB, AVH 9. 36 AVH NANB Late PT 11.84 5.79 10. 37 AVH NANB, IVD 6.52 1.33 11. 38 AVH NANB, late, PT 39.44 39.18 12. 39 Chronic NANB, PT 42.22 37.54 13. 40 AVH, NANB, PT 1.35 1.17

137

Table 1 (Cont.)

Number of

Reference Y / N

of patient Diagnosis Anti-5-1-1 Anti-8l 14. 41 NANB? Chronic PT 0.35 0.28 15. 42 AVH, NANB, IVD 6.25 2.34 16. 43 NANB, Chronic, PT 0.74 0.61 17. 44 AVH, NANB, PT 5.4 1.83 18. 45 NANB, Chronic, PT 0.52 0.32 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 22. 49 AVH, Type A 1.44 0.74 23. 50 AVH Recently determined, Type Á 0.48 0.56 24. 51 AVH, Type A 0.68 0.64 Determined AVH, Type A 0.8c 0.65 25. 52 AVH Recently Determined, Type A 1.38 1.04 AVH Recently Determined, Type A 0.80 0.65 26. 53 AVH, Type A 1.85 1.16 AVH Recently Determined, Type A 0.02 0.88 27. 54 AVH, Type A 1.35 0.74 28. 55 AVH, late HBV 0.58 0.55 29. 56 Chronic HBV 0.84 1.06 30. 57 AVH, late HBV 3.20 1.60 31. 58 Chronic HBV 0.47 0.46 32. 59 ¹ AVH, HBV 0.73 0.60 AVH, Cured HBV 0.43 0.44 33. 6th ¹ AVH, HBV ’1.06 0.92 AVH, Cured HBV 0.75 0.68

138

Table 1 (Cont.)

Number of

Reference Y / N

of patient Diagnosis Anti-5-1-1 Anti-8l 3 *. 61 ¹ AVH, HBV 1.66 o, 6i AVH, cured HBV 0.63 0.36 35. 62 ¹ AVH, HBV 1.02 0.73 AVII, cured HBV 0.4l 0.42 36. 63 ¹ AVH, HBV 1.24 1.31 AVH, cured HBV 1.55 0.45 37. 64 ¹ AVH, HBV 0.82 o, 79 AVH, cured HBV 0.53 0.37 38. 65 ¹ AVH, HBV 0.95 0.92 AVH, cured HBV 0.70 0.50 39. 66 ¹ AVH, HBV 1.03 0.68 AVH, cured HBV 1.71 1.39

¹ Sequential serum samples available from these patients

IVD = Intravenous Medication User 3

AVH »Acute viral hepatitis lt»

PT = Post-transfusion

139

As shown in Table 1, 19 of the 32 sera from patients diagnosed as having NANBH were positive for antibodies directed against HCV epitopes present in SOD-NANB ^ and SOD-ΝΑΝΒθ ^.

However, serum samples that were positive were not equally immunologically reactive with SOD-NANB ^ and SOD-NANBg ^ Serum samples from patient N ⁹ 1 were positive for SOD-NANBg ₁ but not for SOD-NANB ^ _{χ 4} , Serum samples from patients numbers 10, 15 and 17 were positive for SOD-NANB ^ but not for SOD-NANBg ^. Serum samples from patients numbers 3, 8, 11 and 12 reacted equally with both fusion polypeptides, whereas serum samples from patients numbers 2, 4, 7 θ 9 were 2-3 times higher in reaction to SOD-NANB ^ than to SOD-NANBg ^. These results suggest that NANB ^ jj θ NANBg ^ may contain at least 3 different epitopes, that is, it is possible that each con polypeptide has at least 1 single epitope, and that the two polypeptides with share at least 1 epitope,

IV.D.4, Specificity of the Solid Phase RIA for NANBH

The specificity of the solid phase RIAs for NANBH was tested using the assay on serum from patients infected with HAV or HBV and on sera from control subjects. The assays using partially purified SOD-NANB ^ _ ₁ _ ₁ and SOD-NANBg ₁ were conducted essentially as described in Section IV.D.3, except that the sera were from patients previously diagnosed as having HAV or HBV , or from individuals who were blood bank donors. The results for sera from patients infected with HAV and HBV are shown in Table 1. The RIA was tested using 11 specimens of serum from patients infected with HAV, and 20 specimens of serum from patients infected with HBV. As shown in Table 1, none of these compounds gave a positive immunological reaction with the fusion polypeptides containing BB-NANBV epitopes.

RIA using the NANB4 antigen was used to determine the immunological reactivity of serum from control subjects. Out of the 230 serum samples obtained from the normal blood donor population, only two gave positive reactions in the RIA (data not shown). It is possible that the two blood donors from which these serum samples originate previously been exposed to HCV.

IV.D.5. Reactivity of NANB ^,. During the Infection Course ................. 5 -i-ι —— tion NANBH.

The presence of anti-NANB antibodies during the course of NANBH infection of 2 patients and 4 chimpanzees was followed using RIA as described in Section IV.D.3. In addition, RIA was used to determine the presence or absence of anti-NANB4 antibodies during the course of HAV and HBV infection in infected chimpanzees.

The results, which are shown in Table 2, show that with chimpanzees and humans, the antibodies were detected following the beginning of the acute phase of NANBH infection. Anti-NANB antibodies.,,. were not

5- i — i detected in serum samples from chimpanzees infected with either HAV or HBV. Thus, anti d. -MANB ^ | | serve as a marker for an individual's exposure to HCV.

Table 2

Seroconversion in Sequential Serum Samples. from hepatitis in patients and chimpanzees using Antigene 5-1-1

Patient / 1 Sample Date (Days) Viruses of Anti-5-1-1 ALT (mu / ml) Chimp. 1 (0 = day inoculation) Hepatitis (y / n) Patient 29 T NANB 1.09 II80 T + 180 33.89 425 T + 208 36.22 —— Patient 30 T NANB 1.90 1830 T + 307 34.17 290 T + 799 32.45 276 Chimp. 1 0 NANB 0.87 9 76 0.93 71 118 23.67 19 154 32.41 - - Chimp. 2 0 NANB 1.00 5 21 1.08 52 73 4.64 13 138 25.01  ·· Chimp. 3 0 NANB 1.08 8 43 1.44 205 53 1.82 14 159 11.87 6 Chimp, 4 -3 NANB 1.12 11 55 1.25 132 83 6.60 - 140 17.51 Chimp. 5 0 25 HAV 1.50 2.39 4 147 40 1.92 18 268 1.53 5 Chimp. 6 -8 15 HAV 0.85 «W« * 106 41 0.81 10 129 1.33 W M,

142

Patient/ Chimp. Sample Date (Days) (0 = inoculation day) Viruses of Hepatitis Anti-5-1-1 (Y / N) ALT (mu / ml) Chimp. 7 0 HAV 1.17 7 22 1.60 83 115 1.55 5 139 1.60 - Chimp. 8 0 HAV 0.77 15 26 1.98 130 74 1.77 8 205 1.27 5 Chimp, 9 -290 HBV 1.7 * 1 - 379 3.29 9 435 2.77 6 Chimp. 10 0 HBV 2.35 8 111-118 (reservation- 2.74 96-156 thorium) (reservation thorium) 205 2.05 9 240 1.78 13 Chimp, 11 0 HBV 1.82 11 28-56 (reservation- thorium) 1.26 8-100 ( servant River) 169 - 9 223 0.52 10

* Τ = day of initial sampling

IV. Ε. Purification of Polyclonal Serum Antibodies for nanb _5- i_i

Based on the specific immunological reactivity of the SOD-NANB ^ polypeptide with the antibodies in serum samples from patients with NANBH, a method has been developed to purify serum antibodies that react immunologically with the epitope (s) in the NANB ^ This method uses affinity chromatography. The purified SOD-NANB4 polypeptide (see Section IV, D, 1) was attached to the insoluble support; the binding is such that the immobilized polypeptide retains its affinity for the antibody · the NANB ^ χ χ ’θ antibody in serum samples is absorbed up to the dye-binding polypeptide. After washing to remove non-specifically bound and unbound materials, the antibody is bound and released from the bound SOD-HCV polypeptide by change in pH, and / or chaotropic reagents, for example urea.

Nitrocellulose membranes containing SOD-NANB „ ₁ , bound were prepared as follows. A 2.1 cm nitrocellulose lose Sartorius membrane with a 0.2 micron pore size was washed for 3 minutes three times with BBS. The SOD-NANB ^ _ ₁ _ ₁ was attached to the membrane by incubating the preparation. purified on BBS at room temperature for 2 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 wash. The remaining active sites on the membrane were blocked with BSA by incubation with a 5 mg / ml solution of BSA for 30 minutes. , The excess BSA was removed by

144 washing the membrane 5 times with BBS and 3 times with distilled water. The membrane containing the viral antigen and the BSA was then treated with 0.05 M glycine hydrochloride, pH 2.5,

0.10 M NaCl (GlyHCl) for 15 minutes, followed by 3 washes of three minutes with PBS,

Polyclonal anti-NANB_ _Ί antibodies were _isolated

9— 1 - X ““ sides by incubating the membranes containing the fusion polypeptide with serum from an individual with NANBH for 2 hours. After incubation, the filters were washed 5 times with BBS, and twice with distilled water. Bound antibodies were then eluted from each filter with 5 GlyHCl elutions, 3 minutes by elution »The pH of the eluates was adjusted to pH 8.0 by pooling each eluate in a test tube containing 2.0 M Tris HCl , pH 8.0, Recovery of anti-NANB ^ _ ₁ antibody after affinity chromatography is approximately 5θ $ ·

The nitrocellulose membranes containing the bound viral antigen can be used several times without appreciable decrease in binding capacity. For the reuse of the membranes, after the antibodies have been eluted, the membranes are washed with BBS three times for 3 minutes. They are then stored in BBS at 4 ° C.

IV, F, A Capture of HCV Particles from Infected Plasma Using Purified Human Polyclonal Anti-HCV Antibodies; Hybridization of Nucleic Acid in Captured Particles for HCV cDNA

145 IV. F, 1. The capture of HCV Particles from infected Plasma Ih Using Polyclonal Anti-HCV Antibodies

Humans

Protein-nucleic acid complexes present in a chimpanzee's infectious plasma with NANBH were isolated using purified human polyclonal anti-HCV antibodies that were attached to polystyrene microspheres.

polyclonal cells were pu from a human with encoded in the clone described in Section

Anti-NANB ^ antibodies raised from serum from NANBH using the SOD-HCV polypeptide

5-1-1. The method for purification was the

IV.E.

The purified anti-NANB ^ antibodies were attached to polystyrene beads (1/4 in diameter, specular finish, Precision Plastic Bali Co., Chicago, Illinois) by incubating each at room temperature overnight with 1 ml of antibodies (l microgram / ml in borate buffered brine, pH 8.5). Following overnight incubation, the beads were washed once with TBST ^ / JO mM Tris HCl, pH 8.0, 150 mM NaCl, 0.05% (v / v) Tween 20 ^ and then with buffered brine phosphate (PBS) containing 10 mg / ml BSA,

Control spheres were prepared in an identical manner, except that the purified anti-NANB ^ antibodies were replaced with total human immunoglobulin,

HCV capture from NANBH - infected chimpanzee plasma using anti - NANB antibodies _e . _Ί

5-1-1 linked to beads was performed as follows. The plasma from a chimpanzee with NANBH used is described in Section IV, A, 1 .. An aliquot (1 ml) of the chimpanzee plasma infected with NANBV was incubated for 3 hours at 37 ° C with each of the 5 spheres coated with either anti-NANB4 antibodies or control immunoglobulins. The spheres were washed 3 times with TBST,

IV, F «2. Hybridization of Nucleic Acid in Captured Particles to NANBC-cDNA nucleic acid component released from particles captured with anti-NANB4 antibodies was analyzed for hybridization to HCV cDNA derived from clone 81.

HCV particles were captured from NANBH-infected chimpanzee plasma, as described in IV.F.l. To release the nucleic acids from particles, the washed beads were incubated for 60 minutes at 37 ° C with 0.2 ml per sphere of a solution containing protein. nase 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 precipitated nucleic acid was pooled by centrifugation, dried, and dissolved in 50 mM Hepes, pH 7.5 · Parts duplicate aliquots of soluble nucleic acids from samples obtained from beads coated with anti-NANB antibodies and with control beads containing total human immunoglobulin were filtered over nitrocellulose filters. The filters were hybridized with an incision-translated probe, classified p, manufactured from the purified HCV cDNA fragment in clone 81. Methods for preparing the probe and for hybridization are described in Section IV.C.l ..

Autoradiographies of a probed filter containing nucleic acids from particles captured by spheres containing anti-NANB antibodies are shown in

Fig, 40. The extract obtained using the anti-NANB ^! (A ^, Ag) gave clear signs of hybridization to the control antibody extract (A ^, A ^) and the standard RNA control yeast consisting of lpg, 5pg 'and 10 pg of the purified 81 cDNA clone fragment, are reprjs seated at Cl-3, respectively.

These results demonstrate that the particles captured from the NANBH plasma by anti-NANB antibodies ^ _ ^ _ ^ contain nucleic acids that hybridize with HCV cDNA in clone 81, and thus provide further evidence that the cDNAs in these clones are derived from the etiologic agent for NANBH.

IV.G. Immunological Reactivity of C100-3 with Anti-NANB ^ Antibodies. . Purified p-l — 1 ......

The immunological reactivity of the C100-3 fusion polypeptide with anti-NANB ^ antibodies was determined by a radioimmunoassay, in which the antigens that were bound to a solid phase were competed with purified anti-NANB antibodies, and the complex antigen-antibody tectated with sheep anti-human antibodies classified · »p K 'ΐ. The immunological activity of the C100-3 polypeptide was compared to that of the SOD-NANB. .

C100-3 fusion polypeptide was synthesized and purified as described in Section IV, B, 5. and in Section IV, B, 6, respectively, the SOD-NANB4 fusion polypeptide was synthesized and purified as described in Section IV.B.l in Section IV, D, 1, respectively. Anti-NANB ^^^ antibodies were obtained as described in Section IV, E.

A hundred microliter aliquots containing varying amounts of C100-3 antigen purified in O, 125M Na borate buffer, pH 8.3, O, O75M NaCl (BBS) were added to each well of a microtiter plate ”(Dynatech Immulon 2 Removawell Strips). The plate was incubated at 4 ° C overnight in a humid chamber, after which the protein solution was removed and the wells washed 3 times with BBS containing 0.02% Triton X-100 (BBST), to avoid On non-specific binding, the reservoirs were coated with BSA by adding 100 microliters of a 5 mg / ml solution of BSA in BBS followed by incubation at room temperature for 1 hour, after which the excess BSA solution was removed. moved. Polypeptides in the coated wells were reacted with anti-NANB antibodies ^ _ ^ _ ^ by adding 1 microgram of antibody / well, and incubating the samples for 1 hour at 37 ° C · After incubation, the excess solution was removed by suction, and the reservoirs were washed 5 times with BBST. The binding of the anti-NANB4 to the fusion polypeptides was determined by the binding of the classified anti-human F '(ab) 2 IgG ³ I to the coated reservoirs. 100 microliter aliquots of the classified probe (specific activity 5-20 microcuries / microgram) were added to each reservoir, and the plates were incubated at 37 ° C for 1 hour, followed by removal of the excess probe by aspiration, and 5 washes with BBST, to the amount of radioactivity. The connected power in each reservoir was determined by counting on a counter that detects gamma radiation.

The results of the immunological reactivity of C100 with purified anti-NANB ₅ _ ₁ _ ₁ , when compared with those of NANB ^ · ^ with the purified antibodies are shown in Table 3.

TABLE 3

Immunological Reactivity of C100-3 compared with NANB ^ _ _{1 1} by Radioimmunoassay

RIA (cpm / test)

AG (ng) 400 320 240 l60 60 0 nanb ₅ _i-i 7332 6732 4954 4050 . 3051 57 C100-3 7450 6985 5920 5593 4096 67

The results in Table 3 show that the anti-ΝΑΝΒ ^ ι i recognizes an epitope (s) in the C100 half of the C100-3 polypeptide, thus the NANB ^ and C100 share a common epitope (s) ), The results suggest that the cDNA sequence encoding these NANBV epitope (s) is one that is present in both clone 5-1-1 and clone 81,

150 IV.Η, HCV characterization

IV.Η · 1. Characterization of Gflnoma HCV Fibrosity HCV genome was characterized in relation to its fibrosity by isolating the fraction of nucleic acid from particles captured on polystyrene beads coated with the anti-NANB antibody ^ p ^θ determining if the nucleic acid hybridized with more and / or less HCV cDNA fibers,

The particles were captured from an HCV-infected chimpanzee plate using polystyrene beads coated with the anti-NANB antibody „. _n immunopurified as described in Section IV.Fl, the nucleic acid component of the particles was released using the method described in Section IV, F, 2. Aliquots of the genomic nucleic acid isolated from the equivalent of 3 ^m l ^s of the high titer plasma were poured onto nitrocellulose filters. As controls, aliquots of denatured HCV cDNA from clone 81 (2 picograms) were also poured over the same filters. The filters were probed with a mixture of more or a mixture of less fibers classified p of single fiber DNA cloned from HCV cDNAs; cDNAs were excised from clones 40b, 81, and 25c.

Single fiber probes were obtained by removing HCV cDNAs from clones 81, 40b, and 25c with EcoR1, and cloning the cDNA fragments into M13, mpl8 and mpl9 / Messing vectors (1983J.7 · M13 clones were sequenced for determine whether they contained more or less fibers

151 was from DNA coming from, HCV cDNAs. The sequencing performed by the dideoxy chain termination method of Sanger et al. (1977).

Each of a duplicate set of filters containing aliquots of the isolated HCV genome from the captured particles was hybridized with either fiber probes. more _f or less fibers derived from HCV cDNAs.

Fig. 41 shows the autoradiographies. 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 were hybridized with the fibara plus probe mixture. The samples in panel II were probed by hybridization with the fiber probe mixture minus. The composition of the samples in the immunoblot panels are shown in table 4.

Table 4

A B

HCV Genome Passage *

------- x

X cDNA 81

-------- cDNA 81 x is a sample not described.

As can be seen from the results shown in Fig, 4l, only the least hybrid fiber DNA probe with the 0 genus

- 152 -

isolated HCV. This result, in combination with the result showing that the genome is sensitive to RNase and not DNase (See Section IV.C.2,), suggests that the NANBV genome is positive fibrated RNA.

These data, and data from other laboratories concerning the physicochemical properties of a putative NANBV (s), are compatible with the possibility that HCV is a member of the Flaviviridae. However, the possibility that HCV represents a new class of viral agent has not been eliminated.

IV, H.2. Detection of Sequences in Captured Particles That

When Magnified by PCR Hybridize to HCV cDNA Derived from Clone 81

RNA in the captured particles was obtained as described in Section IV.H.l. The analysis for the sequences that hybridize to the HCV cDNA derived from clone 81 was performed using the amplification process. PCR, as described in Section IV, C.3, except that the hybridization probe was a kinetized oligonucleotide derived from the clone 81 cDNA sequence. The results showed that the extended sequences that hybridized to q clone 81 were derived from the HCV cDNA probe.

IV.H.3. Homology between Dengue Flavivirus Non-Structural Protein (MNVWVDl) and HCV Polypeptides Encoded by the combined ORF of clones 14i to 39c

The HCV cDNAs combined from clones 14 through 39c contain a continuous ORF, as shown in Fig, 26.0 polypeptide

153 encoded therein was analyzed for sequence homology with the region of the non-structural polypeptide (s) in Dengue Flavivirus (MNWVDl). The analysis used the database of the Dayhoff protein, and was performed on a computer . The results are shown in Fig. 42, where the symbol (s) indicates an exact homology, and the symbol (.) Indicates a subs. conservative substitution in the sequence; the dashed lines indicate spaces inserted in the sequence to make larger homologies. As seen in the figure, there is significant homology. captive between the sequence encoded in the HCV cDNA and the non-structural polypeptide (s) of the Dengue virus. In addition to the homology depicted in Fig. 42, analysis of the polypeptide segment encoded in a region towards the 3 'end of the cDNA also contained sequences that are homologous to the sequences in the Dengue polymerase. As a consequence it appears that the canonical sequence Gly-Asp-Asp (GDD), although essential for RNA, RNA-dependent polymerases is contained in the HCV cDNA encoded polypeptide, in a location that is compatible with that of the Dengue virus 2 (Data not shown).

IV.H.4. 0 HCV-DNA is not detectable in tissue infected with

NANBH

Two types of studies have yielded results suggesting that HCV-DNA is not detectable in tissue from an individual with NANBH. These results, together with those described in IV.C and IV.H.l. and IV.H, 2 provide evidence that HCV is not a virus containing DNA, and that this replication does not involve cDNA,

- 154 -

XV.Η.4.a. Southern Spots Production Process

In order to determine if the chimpanzee liver infected with NANBH contains detectable HCV-DNA (or HCV-cDNA), DNA restriction enzyme fragments isolated from this source have been spotted in the south, and the stains probed with HCV cDNA classified p. The results showed that the sified HCV cDNA clajs did not hybridize to the stained DNA from the infected chimpanzee liver. Nor did it hybridize to control the stained DNA from the liver of a normal chimpanzee. In contrast, in a positive control, a classified beta-interferon gene hybridized strongly to patches south of the macerated human placental DNA restriction enzyme. These systems were designed to detect a single copy of the gene that was to be detected with the classified probe.

The DNAs were isolated from the livers of two chimpanzees with NANBH. Control DNAs were isolated from the uninfected chimpanzee liver, and from human placentas. The process for extracting DNA was essentially according to Maniatis et al. (1982), and the DNA samples were treated with RNAse during the isolation process.

Each DNA sample was treated with either EcoRI, Mbol, or HincII (12 micrograms), according to indd. manufacturer's instructions. The macerated DNAs were electrophoresed on 1 # neutral agarose gels, stained in the south on nitrocellulose, and the stained material hybridized with the appropriate cDNA probe translated by incision (3x10 ⁶ cpm / ml of hybridization mixture) ), The DNA from infected chimpanzee liver and normal liver were hybridized with HCV cDNA classified ^J p, from clones 36 plus 81; the DNA from the human placenta was hybridized with DNA classi32 ^J p, from the beta-interferon gene. After hybridization, the spots were washed under strict conditions, that is, with a solution containing 0.1 x SSC, 0, 1 $ SDS at 65 ° C.

beta-interferon DNA gene was prepared as described by Houghton et al. (ΐ9θΐ).

IV, Η, 4.b, Magnification by PCR Technique

In order to determine whether HCV-DNA can be detected in the liver from chimpanzees with NANBH, the DNA was isolated from the tissue, and subjected to the amplification-detection POR technique using probe primers and polynucleotides derived from HCV cDNA from clone 81. The negative controls were DNA samples isolated from cells whose. of uninfected HepG2 tissue, and presumably from uninfected human placenta. The positive controls were samples of the negative control DNAs to which a relatively small known amount (250 molecules) of the HCV cDNA supplement from clone 81 was added.

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

In the studies, DNAs were isolated by the procedure

- 156 are described in Section IV.H.4.a, and the RNAs were extracted essentially as described by Chirgwin et al. (ΐ9θΐ).

DNA samples were isolated from 2 infected chimpanzee livers, from uninfected HepQ2 cells, and from human placenta. One microgram of each DNA was macerated with HindIII, according to the manufacturer's instructions. The macerated samples were subjected to PCR amplification and detection for expanded HCV cDNA essentially as described in Section IV.C.3, except that the reverse trans cryptase phase was omitted. The PCR primers and probe came from HCV cDNA clone 8l, and are described in Section IV.C, 3 .. Before amplification, for positive controls, a microgram sample of each DNA was bristled ”by the addition of 250 molecules of HCV cDNA supplement isolated from clone 81.

In order to determine whether HCV sequences were present in RNA isolated from chimpanzee livers with NANBH, samples containing 0.4 micrograms of total RNA were subjected to the amplification process essentially as described in Section IV.C, 3., Except in which the transcriptase in versa was omitted from some of the samples as a negative control. The PCR primers and probe were from HCV cDNA clone 81, as described above.

The results showed that the amplified sequences of the complementary HCV cDNA probe were not detectable in the DNA from the infected chimpanzee liver, but were detectable in the negative controls. In contrast, when

157 samples, including DNA from infected chimpanzee liver, were bristled ”with HCV cDNA prior to amplification, clone 81 sequences were detected in all positive control samples. In addition, in RNA studies, the extended sequences of HCV cDNA clone 81 were detected only when reverse transcriptase was used, strongly suggesting that the results were not due to DNA contamination.

These results show that hepatocytes from chimpanzees with NANBH do not, or only contain, undetectable levels of HCV DNA. Based on the study of bristling, if HCV DNA is present, it is at a level well below 0.06 copies per hepatocyte. In contrast, HCV sequences in total RNA from the same liver samples were easily detected with the PCR technique.

IV.X. ELISA Determinations for HCV Infection Using HCV clOO-3 as Test Antigen

All samples were tested using the

HCV cl00-3 ELISA. This assay uses the HCV antigen cl00-3 (which was synthesized and purified as described in Section IV, B, 5), and a horseradish peroxidase (HRP) conjugated to mouse monoclonal anti-human IgG.

Plates coated with the HCV cl00-3 antigen were prepared as follows, 'A solution containing coating buffer (50 mM Na borate, pH 9.0), 21 ml / plate, BSA (25 micrograms / ml), cl00-3 (2.50 micrograms / ml) was prepared

V, from just before the addition to the Removeawell Immulon I plates (Dynatech Corp.). After mixing for 5 minutes, 0.2 ml / solution reservoir was added to the plates, the plates were covered and incubated for 2 hours at 37 ° C, after which the solution was removed by aspiration. The reservoirs were washed once with 400 microliters of Wash Buffer (wash buffer) (100 mM sodium phosphate, pH 7.4, 140 mM sodium chloride, 0.1% (w / v) casein, 1 % (w / v) of Triton x-100, 0.01% (w / v) of Thimerosal), After removing the washing solution, 200 microliters / reservoir of Postcoat solution (10 mM sodium phosphate, pH 7.2,

150 mM sodium chloride, 0.1% (w / v) casein and 2 mM phenylmethylsulfonylfluoride (PMSF) were added, the plates were covered freely to prevent evaporation, and left to stand at room temperature for 30 minutes . The reservoirs were then aspirated to remove the solution, and lyophilized dry overnight, without shelf heating. The prepared plates can be stored at 2-8 ° C in sealed aluminum pouches,

In order to perform ELISA determination, 20 microliters of serum sample or control sample were added to a reservoir containing 200 microliters of diluent sample (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 two hours, after which the solution was removed by aspiration, and the reservoirs were washed with 4-00 microliters of washed buffer (buffered phosphate brine (PBS) containing 0 , 5% Tween 20). The washed reservoirs were treated with 200 microliters of mouse anti-human IgG-HRP conjugate contained in a diluent Ortho conjugate solution (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 5θ $ (v / v) d® fetal bovine serum, 1 $ (v / v) heat treated horse serum, 1 mM K ^ Fe (CN) ^,

0.05% (w / v) of Tween 20, 0.2% (w / v) of Thimerosal). The treatment was carried out for 1 hour at 37 ° C, the solution was removed by aspiration, and the reservoirs were washed with wash buffer which was also removed by aspiration. To determine the amount of binding enzyme conjugate,

200 microliters of the substrate solution (10 mg of 0-phenylenediamine dihydrochloride per 5 ml of Developer solution) were added. The Developer solution contains 50 mM sodium citrate adjusted to pH 5.1 with phosphoric acid, and 0.6 microliters / ml of H ₂ 0 ₂ to 30% · The plates containing the substrate solution were incubated in the dark for 30 minutes at room temperature, reactions were stopped by adding 50 microliters / ml of 4N sulfuric acid, and the ODs determined.

The examples provided below show that the ELISA microtiter descreening plate using the HCV clOO-3 antigen has a high degree of specificity, as evidenced by the initial reactivity ratio of about 1%, with a reactive repeat ratio of about 0 , 5% on random donors. The assay is able to detect an immune response both in the post acute phase of the infection and during the chronic phase of the disease. In addition, the assay is able to detect some samples that count negatively in the substitute tests for NANBH; these samples come from individuals with a history of NANBH, or from donors involved in the transmission of NANBH,

In the examples described below, the following abbreviations are used:

ALT Amino Alanine Transferase Anti-HBc Antibody against HBc Anti-HBsAg Antibody against HBsAg HBc Hepatitis B core antigen ABsAg Hepati surface antigen te B IG G Immunoglobulin G IgM Immunoglobulin M IU / L International Units / Liter AT Not available NT Not tested N Sample size Neg Negative OD Epic density Pos Positive s / co Signal / cutoff SD Standard deviation X Average or medium WNL Within normal limits

IV.I.1. HCV infection in a random blood donor population

A group of I.O56 samples (fresh sera) from random blood donors were obtained from the Irwin Memorial Blood Bank, San Francisco, California.

The test results obtained with these samples are summarized in a histogram showing the distribution of OD values (Fig. 43). As shown in Fig. 43, 4 samples read> 3, 1 sample reads between 1 and 3 »5 samples read between 0.4 and 1, and the remaining 1,046 samples read <0.4 with more than 90 $ of these samples reading <0.1,

The results on the random reactive samples are shown in Table 5 «Using an eut-off value equal to half plus 5 standard deviations, ten samples out of 1.056 (0.95%) were initially reactive. Of these, five samples (0.47%) were repeated as reactive when they were tested a second time using ELISA. Ta Bela 5 also shows the ALT and Anti-HBd status of each of the reactive samples repeatedly. Of particular interest is the fact that all five repeated reactive samples are negative in both substitute tests for NANBH, as long as there is positive numbering in the HCV ELISA.

162

TABLE 5

RESULTS ON REACTIVE SAMPLES

N = 1051 x = 0.049 *

SD = + 0.074

Cut-off: x + 5SD = 0.419 (o, 4oo + Control Negative) Samples Reagents Initials OD Repeated Reagents OD JKK ALT (UI / L) Anti HBc *** (OD) 4227 0.462 0.084 AT AT 6292 0.569 0.294 AT AT 6188 0.699 0.326 AT AT 6157 0.735 0.187 AT AT 6277 0.883 0.152 AT AT 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

10/1056 = $ 0.95 5/1056 = $ 0.47 * Sample readings> 1.5 were not included in the calculation of Meane SD.

** ALT> 68 IU / L is above normal limits.

*** Anti-HBc <0.535 (competition test) is considered positive.

W WWW

WNL: Within normal limits

- 163 IV, I.2, Chimpanzee Serum Samples

Serum samples from eleven chimpanzees were tested with HCV cl00-3 ELISA. Four of these chimpanzees were infected with NANBH from a contaminated portion of Factor VIII (presumably Hutchinson strain), following a process established in collaboration with Dr. Daniel Bradley at the Centers for Disease Control. As controls, four other chimpanzees were infected with HAV and three with HBV. Serum samples were obtained at different times after infection.

The results, which are summarized in Table 6, show the sero-conversion of the antibody, documented in all chimpanzees infected with the Hutchinson strain of NANBH. Following the acute phase of infection (as evidenced by the significant rise and subsequent return to normal ALT levels), antibodies to HCV cl00-3 became detectable in the sera of chimpanzees infected with NANBH on 4/4. These samples had previously been shown, as discussed in Section IV.B, 3. as positive by Western analysis, and by an RIA. In contrast, none of the control chimpanzees that had been infected with HAV or HBV showed evidence of reactivity in the ELISA

TABLE 6

SAMPLES SERUM CHIMPANZEE OD S / CO Date of 1 Inoculation Date of 0 Sangria ALT (UI / L) Ino ass sides Control 0.001 Negative Control 1.504 Positive Cutoff ’'0.401 Chimp, 1 -0.007 0.00 5/24/84 5/24/84 9 NANB 0.003 0.01 08/07/84 71 7-3,000 77.48 9/18/84 19 > 3,000 -7 7.48 10/24/84 - Chimp, 2 - O7 / o6 / 84 - - NANB -0.003 0.00 5/31 / 84 5 -0.005 0.00 06/28/84 52 0.945 2.36 8/20/84 13 z3, ooo 77.48 10/24/84 - Chimp, 3 0.005 0.01 3/14/85 3/14/85 8 NANB 0.017 0.04 4/26/85 205 0.006 0.01 5/6/85 14 1,010 2.52 8/20/85 6 Chimp, 4-0.006 0.00 03/11/85 H / 03/85 11 NANB 0.003 0.01 5/9/85 132 0.523 1.31 06/06/85 - 1,574 3.93 OI / O8 / 85 - Chimp.5 -0.006 0.00 11/21/80 11/21/80 4 HAV

- 165 -

TABLE 6 (Cont.)

Date of Date of ALT Inocu OD s / co Inoculation Sangria bond Chimp. 5 0.001 0.00 12/16/80 147 0.003 0.01 3O / 12 / 8O 18 0.006 0.01 07/29-21 / 08/81 5 Chimp. 6 --- - 5/25/82 ' - - HAV -0.005 0.00 5/17/82 - 0.001 0.00 6/10/82 106 -0.004 0.00 07/06/82 10 0.290 0.72 10/1/82 - Chimp. 7 -0.008 0.00 5/25/82 5/25/82 7 HAV -0.004 0.00 6/17/82 83 -0.006 0.00 9/16/82 5 0.005 0.01 10/9/82 - Chimp. 8 -0.007 0.00 11/21/80 21 / H / 8O 15 HAV 0.000 0.00 12/16/80 130 0.004 0.01 2/3/81 8 0.000 0.00 O3 / O6-IO / O6 / 8I M Chimp. 9 --- - · * · 7/24/80 - - HBV 0.019 0.05 08/22/10/10/79 - - - II / 03 / 8I 57 0.015 o, o4 07/01/05 / 08/08 9 0.008 0.02 10/1/81 6 Chimp. 10 -— - 5/12/82 - - HBV 0.011 0.03 21 / 04-12 / 05/82 9

166 TABLE 6 (Cont'd)

OD ’S / co Date of Date Sangria inoculation ALT Inocu (IU / L) lation Chimp.10 0.015 0.04 01 / 09-08 / 09/82 126 0.008 0.02 12/02/82 9 0.010 0.02 1/6/83 13 Chimp.ll - - 5/12/82 --- HBV . 0.000 0.00 06 / 01-12 / 05/82 11 - - 6/23/82 100 -0.003 0.00 06/06/07 07/07 - -0.003 0.00 10/28/82 9 -0.003 0.00 12/20/82 10

167 -

IV.I.3. Panel 1: Proven Infectious Sera From

Chronic Human NANBH carriers

A coded panel consisted of 22 simple samples, each in duplicate, for a total of 44 samples. The samples came from infectious sera tested from chronic NANBH transporters, infectious sera from implicated donors, and infectious sera from patients with acute NANBH. In addition, the samples came from highly pure negative controls, and other disease controls. This panel was provided by Dr, H. Alter from the Department of Health and Human Services, National Institutes of Health, Bethesda, Marilândia. The panel was built by Dr. Alter several years ago, and has been used by Dr. Alter as a qualification panel for putative NANBH assays.

The total panel was tested twice with the ELISA assay, and the results were sent to Dr, Alter to be noted. The results are shown in Table 7.

Although the Table refers to results from only one set of duplicates, the same values were obtained for each of the samples in duplicate.

As shown in Table 7, 6 sera that proved to be infectious in a chimpanzee model were strongly positive. The seventh infectious serum corresponded to a sample for a case of acute NANBH, and was not reactive in the ELISA. A sample from a donor involved both as a normal ALT and with equivocal results in chimpanzee studies

168 was non-reactive in the assay. Three other serial samples from an individual with acute NANBH were also non-reactive. All samples that came from highly pure negative controls, obtained from donors who had at least 10 blood donations with no implication of hepatitis, were non-reactive in the ELISA. Finally, four of the tested samples had previously been marked as positive in putative NANBH tests developed by others, but these assays were not confirmed. These four samples were negatively marked with the HCV ELISA.

- 169 TABLE 7

PANEL 1 of H, ALTER:

Panel 19 Result tado 22 Resul tado 1) Chimpanzee Transmission Infectious Testing A, Chronic NANB: Feet-Tx JF + + EB + + PG + + B, High ALT Implicated Donors BC + + JJ + + BB + + C, NANB Treble Feet-Tx WII * 2) Mistakenly infectious by Chimp Transmission A, Normal ALT Implicated Donor CC 3) NANB Treble Feet -Tx JL 1 Week «» JL 2 Weeks - - JL 3 Weeks - - Disease Controls A, Primary Biliary Cirrhosis EK B, Recovering Alcoholic Hepatitis IIB - 5) Registered Negative Controls DM A.D - - LV - - ML - - AII - - 6) Potential NANB antigens JS-80-01T-0 (Ishida) Asterix (Trepo) - - Zurtz (Arnold) - - Becassdine (Trepo) - -

170

O

IV.I.4. Panel 2: NANBH Donor / Receptor coded panel consisted of 10 unambiguous donor / recipient transfusions of associated NANBH, with a total of 188 samples. Each case consisted of samples from some or all donors for the recipient and samples in series (taken 3 'and 12 months after the transfusion) came from the recipient. Also included was a prior bleeding, taken from the recipient before The coded panel was provided by Dr. H, Alter, from NXH, and the results were sent to him for marking.

The results, which are summarized in Table 8, show that ELISA detected antibody seroconversion in 10 transfusion cases associated with NANBH. Samples from case 4 (in which seroconversion was not detected) reacted poorly in the ELISA. Two of the recipient samples were reactive 3 months after transfusion. At six months, 8 receptor samples were reactive; and at 12 months, with the exception of case 4, all samples were reactive. In addition, at least one positive antibody donor was found in 7 out of 10 cases, with 10 having two positive donors. Also, in case 10, the pre-bleeding of receptors was positive for HCV antibodies. Bleeding for one month from this receptor was dripped to borderline reaction levels, while it was raised to 4 and 10 month bleeding positive. Generally, an S / CO of 0.4 is considered positive. Thus, this case may represent a previous infection of the individual with HCV.

171 Samples of ALT and HBc status for all reagents, i.e. positive, are summarized in Table 9. As seen in the Table, 1/8 donor samples were negative for the substitute and reactive markers in the HCV antibody ELISA. On the other hand, recipient samples (followed up to 12 months after transfusion) had either elevated ALT, Anti-HBc positive, or both.

172 TABLE 8

DONOR PANEL / NANB CONTAINER

PANEL H, ALTER DONOR / NANB CONTAINER

CONTAINER · POS-TX

DONOR PRE-SANGRIA CASE 3 MONTHS 6 MONTHS 12 MONTHS

JHL SZC £ L JU SZ £ Q JL. , M2 _2SL_ · 00 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 .034 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. > 5,000 > 6.96 .061 0.14 : .078 0.18 2,262 5.28 > 3,000 > 6.96 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 .517 ’ 0.74 > 3,000 " > 6.96 > 3,000 ·· ’ > 6.96

> 5,000> 6.96 * 1 Month, Months, *** 10 Months

173

TABLE 9

ALT AND HBc STATUS FOR, REACTIVE SAMPLES IN

PANEL 1 BY H. ALTER

Samples_Anti-ALT * HBc **

Donors

Case 3 Normal Negative Case 5 High Positive Case 6 High Positive Case 7 Not available Negative Case 8 Normal Positive Case 9 High Not available Case 10 Normal Positive Case 10 Normal Positive

Containers

Case 1 12 6 mo High High Not tested Positive Case 2 12 mo 6 mo High High Not tested Negative Case 3 12 mo 6 mo High Normal Not tested j *** Not tested Case 5 12 mo 6 mo High High Not tested Not tested Case 6 6 mo 12 mo 3 mo High High High Negative Not tested Negative Case 7 6 mo High Negative 12 mo High Negative Case 8 12 mo 6 mo High Normal Not tested Positive Case 9 12 mo High Not tested Case 10 10 mo 4 mo High High Not tested Not tested ALT> 45 IU / l is above 'the limits normal.

Anti-HBc <50 $ (competition test) is considered positive Pre-Sangria and 3 samples were negative for HBc.

174

IV, I, 5. Determination of HCV Infection in Group Samples

High risk

Samples from high-risk groups were controlled using ELISA to determine reactivity to HCV antigen cl00-3 · These samples were obtained from Dr, Gary Tegtmeier, Community Blood Bank, Kan sas City, The results are summarized in the Table 10.

As shown in the table, the samples with ^one more high reactivity are obtained from hemophiliacs (76 $). In addition, samples from individuals with high ALT and positive for Anti-HBc, labeled 51% reactive, a value that is compatible with the value expected from clinical data and NANBH prevalence in this group, The incidence of antibody to HCV was also higher in blood donors with high ALT only, positive blood donors for antibodies to Hepatitis B nucleus only, and in blood donors rejected for reasons other than high ALT or anti-core antibody when compared to voluntary random donors.

5 TABLE 10

HIGH RISK GROUP SAMPLES WITH NANBH

ORUPO N Distribution N OD in Reagent High ALT 35 3 > 3, ooo 11.4% 1 0.728 Anti-HBc 24 5 > 3,000 20.8% High ALT, Anti-HBc 33 12 > 3,000 51.5% 1 2,768 1 . 2,324 1 '0.939 1 o, 95l 1 0.906 Accepted Donors 25 5 -> 3,000 20.0% Donors with History in 150 19 > 3,000 14.7% Hepatitis • 1 0.837 1 0.714 1 0.469

Hemophiliacs

31> 3,000 76.0%

2,568

2,483

2,000

1,979

1,495 ·

1.209

0.819

- 176

IV.I.6 Comparative Studies Using Monoclonal Antibodies

Anti-IgG or Anti-IgM, or Polyclonal Antibodies as a Second Antibody in HCV clOO-3 ELISA

The sensitivity of the ELISA determination using the anti-IgG monoclonal conjugate was compared to that obtained by using either the anti-IgM monoclonal conjugate, or replacing both with a polyclonal antiserum reported to be both heavy and light chain specific. The following studies were carried out.

IV, I.6, a, Serial Samples from Seroconverters

Serial samples from three cases of NANB seroconverters were studied in the HCV clOO-3 ELISA assay using either the anti-IgG monoclonal in the enzyme conjugate alone, or in combination with an anti-IgM monoclonal, or using a polyclonal antiserum. The samples were provided by Dr, Cladd Stevens, N.Y. Blood Center, N.Y.G., N.Y .. The sample stories are presented in Table 11.

The results obtained using an anti-IgG antibody-monoclonal enzyme conjugate are shown in Table

12. The data shows that a strong reactivity is initially 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 e.s conjugate as shown in Table 13. Three different ratios of

177 anti-IgG to anti-IgM were tested; the 1: 10,000 dilution of anti-IgG was constant across all. The dilutions tested for the anti-IgM monoclonal conjugate were 1: 30,000, 1: 60,000, and 1: 120,000. The data show that, according to studies with anti-IgG alone, a strong initial reactivity is detected in the samples 1 -4, 2-8, and 3-5 ·

The results obtained with the ELISA using the anti-IgG monoclonal conjugate (1: 10,000 dilution), or Tago polyclonal conjugate (1: 8θ dilution, θθθ), or Jackson polyclonal conjugate (1: 80,000 dilution) are shown in Table 14. The data indicates that strong initial reactivity is detected in samples 1-4, 2-8, and 3-5 using all three configurations; polyclonal antibodies Tago gave the lowest signals.

The results presented above show that to. the three configurations detect reactive samples at the same time after the acute phase of the disease (as evidenced by the ALT elevation). Furthermore, the results indicate that the sensitivity of HCV clOO-3 ELISA using the monoclonal anti-IgG enzyme conjugate is the same, or better than that obtained using the other tested configurations for the enzyme conjugate.

- 178 -

TABLE 11

DESCRIPTION OF SAMPLES FROM THE CLADD PANEL

STEVENS

Data HBsAg Anti-HBsAnti-HBc ALT Bilirubin

Case 1 1-1 5/8/81 1.0 91.7 12.9 4th, 0 -1.0 1-2 2/9/81 1.0 121.0 15.1 274.0 ιΛ 1-3 10/7/81 1.0 64.0 1 23.8 261.0 0.9 1-4 11/19/81 1.0 67.3 33.8 75.0 0.9 1-5 12/15/81 1.0 50.5 27.6 71.0 1.0 Case 2 2-1 10/19/81 1.0 1.0 116.2 17.0 -1.0 2-2 11/17/81 1.0 0.8 89.5 46.0 1.1 2-3 12/02/81 1.0 1.2 78.3 63.0 1.4 2-4 12/14/81 1.0 0.9 90.6 152.0 13 2-5 12/23/81 1.0 0.8 93.6 624.0 1.7 2-6 1/20/82 1.0 0.8 92.9 66, 0 1.5 2-7 2/15/82 1.0 0.8 86.7 70.0 1.3 2-8 3/17/82 1.0 0.9 69.8 24.0 -1.0 2-9 4/21/82 1.0 0.9 67.1 53.0 1.5 2-10 5/19/82 1.0 0.5 74.8 95.0 1.6 2-11 6/14/82 1.0 0.8 82.9 37.0 -1.0 Case 3 3-1 4/7/81 1.0 1.2 88.4 13.0 -1.0 3-2 5/12/81 1.0 1.1 126.2 236.0 0.4 3-3 5/30/81 1.0 0.7 99.9 471.0 0.2 3-4 6/9/81 1.0 1.2 110.8 315.0 0.4 3-5 7/7/81 1.0 1.1 89.9 273.0 0.4 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 10/14/81 1.0 0.9 102.5 180.0 0.5 3-9 II / II / 8I 1.0 1.0 84.6 154.0 o, 3

179

TABLE 12

ELISA RESULTS OBTAINED USING A CONJUGATE ΑΝΓΙ-Ϊ G

MONOCLONAL

Sample Date ALT OD s / o Negative Control 0.076 Cutoff 0.476 PC (1: 128) 1,390 Case # 1 1-1 5/8/81 40.0 0.178 0.37 1-2 9/2/81 274.0 0.154 0.32 1-3 10/7/81 261.0 0.129 0.27 1-4 11/19/81 75.0 0.937 1.97 1-5 12/15/81 71.0 > 3,000 > 6.30 Case Ψ 2 2-1 10/19/81 17.0 0.058 0.12 2-2 11/17/81 46.0 0.050 0.11 2-3 12/02/81 63.0 0.047 0.10 2-4 12/14/81 152.0 0.059 0.12 2-5 12/23/81 624.0 0.070 0.15 2-6 1/20/82 66.0 0.051 0, LI 2-7 02/15/82 70.0 0.139 0.29 2-8 3/17/82 24.0 1,867 3.92 2-9 ' 4/21/82 53.0 > 3,000 > 6.30 2-10 5/19/82 95.0 > 3,000 > 6.30 2-11 6/14/82 37.0 > 3,000 > 6.30 Case = H = 3 3-1 4/7/81 13.0 0.090 0.19 3-2 5/12/81 236.0 0.064 0.13 3-3 05/30/81 471.0 0.079 0.17 3-4 6/9/81 315.0 0.211 0.44 3-5 07/06 / 8J. 273.0 1.707 3.59 3-6 08/10/81 158.0 > 3,000 > 6.30 3-7 9/8/81 84.0 > 3,000 > 6.30 3-8 10/14/81 180.0 > 3,000 > 6.30 3-9 11/11/81 154.0 > 3,000 > 6.30

- 180 TABLE 13

ELISA RESULTS OBTAINED USING CONJUGATES and MONOCLONAL ANTI-IgG and ANTI-IGM

ELISAS NANB

Monoclonal Monoclonal Monoclonal IgG lílOK IgG lílOK IgG lílOK IgM 1: 3OK IgM ljóOK IgM l: 120K

Sample Date ALT OD S / CO OD S / CO OD S / CO With trolley 0.100 0.080 0.079 Negative CUTOFF PC (1: 128) 1,083 1,328 1,197 Case 41 = 1 1-1 08/05/81 40 0.173 0.162 0.070 1-2 9/2/81 27 ^ 0.194 o, i4i 0.079 1-3 10/7/81 26l 0.162 0.129 0.063 1-4 I9 / II / 8I 75 0.812 0.85 0.709 1-5 12/15/81 71 > 3.00 > 3, oo > 3.00 Case 44 2 2-1 10/19/81 17 0.442 0.045 0.085 2-2 11/17/81 46 0.102 0.029 0.030 2-3 12/02/81 63 0.059 0.036 0.027 2-4 12/14/81 152 0.065 0.041 0.025 2-5 12/23/81 624 0.082 0.033 0.032 2-6 1/20/82 66 0.102 0.042 0.027 2-7 02/15/82 70 0.188 0.068 0.096 2-8 3/17/82 24 1,728 1,668 1,541 2-9 4/21/82 53 > 3.00 2,443 > 3.00 2-10 5/19/82 95 > 3.00 > 3.00. > 3.00 2-11 6/14/82 37 > 3.00 > 3.00 > 3.00 Case 41 = 3 3-1 4/7/81 13 0.193 0.076 0.049 3-2 5/12/81 236 0.201 0.051 0.038 3-3 05/30/81 471 0.132 0.067 0.052 3-4 6/9/81 315 0.175 0.155 0.140 3-5 07/06/81 273 1,335 1,238 1,260 3-6 08/10/81 158 > 3.00 > 3.00 > 3.00 3-7 9/8/81 84 > 3.00 > 3.00 > 3.00 3-8 10/14/81 180 > 3; 00 > 3.00 > 3.00 3-9 H / ll / 81 154 > 3.00 > 3.00 > 3.00

181

TABLE l4

ELISA RESULTS OBTAINED USING POLICIAL CONJUGATES

ELISAS NAxNB

MONOCLONAL TAGO JACKSON 1: 1OK 1: 8OK libOK

SAMPLE DATE ALT OD s / o OD s / co OD s / co With trolley 0.076 0.045 0.154 Negative Cutoff 0.476 0.545 0.654 PC (1: 128) OxlSi .X5 · ¹ ' Case 44 1 1-1 08/05/81 40 0.178 0.37 0.067 0.12 0.153 0.23 1-2 9/2/81 274 0.154 0.32 0.097 0.18 0.225 0.34 1-3 O7 / IO / 81 261 0.129 0.27 0.026 0.05 0.167 0.26 1-4 11/19/81 75 0.937 1.97 0.324 0.60 0.793 1.21 1-5 12/15/81 71 7 * 3.00 > 6.30 1,778 3.27 > 3.00 > 4.59 Case 44 2 2-1 10/19/81 17 0.058 0.12 o, .o? 3 0.04 0.052 0.08 2-2 I7 / H / 8I 46 0.050 0.11 0.018 0.03 0.058 0.09 2-3 12/02/81 63 0.047 0.10 0.020 o, o4 0.060 0.09 2-4 12/14/81 152 0.059 0.12 0.025 0.05 0.054 0.08 2-5 12/23/81 624 0.070 0.15 0.026 0.05 0.074 0.11 2-6 1/20/82 66 0.051 0.11 0.018 0.03 0.058 0.09 2-7 02/15/82 70 0.139 0.29 0.037 0.07 the i46 0.22 2-8 3/17/82 24 1,867 3.92 0.355 0.65 1,429 2.19 2-9 4/21/82 53 73.00 > 6.30 0.748 1.37 > 3.00 > 4.59 2-10 5/19/82 95 > 3, oo > 6.30 1,025 1.88 > 3.00 ^ 4.59 2-11 6/14/82 37 ₇ 3, oo > 6.30 0.917 1.68 > 3.00 > 4.59 Case 44 3 3-1 4/7/81 13 0.090 0.19 0.049 0.09 0.138 0.21 3-2 5/12/81 236 0.064 0.13 o, o4o 0.07 0.094 0, i4 3-3 05/30/81 471 0.079 0.17 0.045 0.08 the, i44 0.22 3-4 6/9/81 315 0.211 0.44 0.085 0.16 0.275 0.42 3-5 07/06/81 273 1.707 3.59 0.272 0.50 1,773 2.71 3-6 08/10/81 158 73.00 > 6.30 1,347 2.47 > 3.00 > 4.59 3-7 9/8/81 84 > 3.00 > 6.30 2,294 4.21 > 3.00 > 4.59 3-8 10/14/81 180 > 3.00 > 6.30 > 3.00 > 5.50 > 3.00 > 4.59 3-9 n / ll / 81 154 > 3.00 > 6.30 > 3.00 > 5.50 > 3.00 > 4.59

182

IV. 1.6, b. Samples from Random Blood Donors

Samples from random blood donors (See Section IV.Il) were sieved for HCV infection using the HCV cl00-3 ELISA, where the antibody-enzyme conjugate was either an anti-IgG monoclonal conjugate or a polyclonal conjugate . The total number of samples weighed was 1077 ® 1056, for the polyclonal conjugate and the monoclonal conjugate, respectively. A summary of the screening results is shown in Table 15, and the sample distributions in the histogram in Fig. 44.

calculation of the mean and standard deviation was performed excluding samples that gave a signal above 1.5, that is, values 1073 00 were used for calculations using the polyclonal conjugate, and 1051 for the anti-IgG monoclonal conjugate. As seen in Table 15, when the polyclonal conjugate was used, the mean was changed from 0.0493 to 0.0931, and the standard deviation was increased from 0.074 to 0.0933. In addition, the results also show that if the x + 5SD criterion is used to define the cut-off assay, the configuration of the polyclonal enzyme conjugate in the ELISA requires a high cut-off value. This indicates reduced test specificity compared to the monoclonal system. On the other hand, as shown in the histogram in Fig. 44, a greater separation of results between negative and positive distributions occurs when random blood donors are passed through the ELISA using the anti-IgG monoclonal conjugate as compared to the assay using a classification commercial polyclonal.

- 183 TABLE 15

COMPARISON OF TWO ELISA CONFIGURATIONS IN SAMPLES

OF TEST FROM BLOOD DONORS

ANTI-IgG POLYCLONAL MONOCLONAL CONJUGATE (jackson)

Number of samples 1073 1051 Average (*) 0.0931 0.04926 Detour default (su) 0.0933 0.07427 5 3D 0.4666 0.3714 CUT-OFF (5 SD + x) 0.5596 0.4206

184 IV.J. Detection of HCV Seroconversion in Patients with NANBH from a Variety of

Geographic Locations

Sera from patients who were suspected of having NANBH based on high ALT levels, and who were negative in HAV and HBV tests were sieved using RIA essentially as described in Section IV, D., Except that HCV antigen cl00-3 was used as the screening antigen on microtiter plates. As can be seen from the results presented in Table 16, the RIA detected positive samples in a high percentage of cases.

TABLE 16

Seroconversion Frequencies for Anti-clOO-3 Enter

Patients with NANBH in Different Countries Parents Netherlands Italy Japan At the. Examined 5 36 26 At the. 1 Positive 3 29 19 $ Positive 60 80 73 IV.K. HCV Seroconversion Detection in Patient,

acquired community ¹¹ NANBH

Sera that were obtained from 100 patients with NANBH, for whom there was no route of transmission qb via (ie, no transfusions, use of drugs via i.v ,, promiscuity, etc.) were identified as factors of

185 -

risk), were provided by Dr. M. Halter of the Center for Disease Control, and by Dr. J. Dienstang of Harvard University. These samples were sieved using an RIA essentially as described in Section XV, D ,, except that the HCV antigen clOO-3 was used as the screening antigen bound to the microtiter plates. The results showed that of the 100 serum samples, 55 contained antibodies that reacted immunologically with the HCV antigen clOO-3 »

The results described above suggest that the

Acquired Community NANBH is also caused by HCV. In addition, since it has been shown here that HCV is related to Flaviviruses, most of which are transmitted by arthropods, HCV transmission in Acquired Community cases is also thought to result from arthropod transmission.

IV. L. Comparison of Incidence of HCV Antibodies and Replaced Markers in Donors Implicated in NANBH Transmission _z

A prospective study was conducted to determine whether recipients of blood from suspected donors of positive NANBH, who developed NANBH, s _and seroconverted to positive anti-HCV antibody. Blood donors were tested for abnormalities of the substituted marker that are currently used as markers for NANBH infection, i.e., elevated ALT levels, and the presence of the anti-core antibody.

In addition, donors were also tested for the presence of anti-HCV antibodies.

186 anti-HCV antibodies were determined using a radioimmunoassay as described in Section IV, K. The results of the study are shown in Table 17, which shows the patient's number. s.

te (column 1) the presence of anti-HCV antibodies in the patient's serum (column 2); the number of donations received by the patient, with each donation coming from a different donor (column 3); the presence of anti-HCV antibodies in the donor serum (column 4), and the abnormality of the donor substitute (column 5) (NT or - means not tested) (ALT is elevated transaminase, and ANTI-HBc anti-core antibody) .

The results in Table 17 demonstrate that the HCV antibody test is more accurate in detecting infected blood donors than the replaced marker tests. Nine out of ten patients, who developed symptoms of NANBH, tested positive for anti-HCV antibody seroconversion. Of the 11 suspected donors, (patient 6 received donations from two different individuals suspected of having NANBH), 9 were positive for antiI antibodies

-HCV, and 1 was positive uncertain, and therefore equivocal (donor for patient 1). In contrast, using the elevated ALT test, the 10 donors tested negative, and using the anti-core-antibody test 5 of the 10 donors tested negative. Of greater consequence, however, in the three cases (donors for patients 8, 9 'and 10) the ALT test and the ANTI-HBc test gave inconsistent results.

- 187 -

TABLE 17

Patient DEVELOPMENT OF ΑΝΤΙ-HCV ANTIBODIES IN PATIENTS RECEIVING BLOOD FROM DONORS SUSPECTED OF BEING NANBH CARRIERS Abnormality Substitut Alt Anti-HB Seroconversion . in es / Donors Donor Anti-HCV Positive Anti-HCV in Patient Np Donations 1 Yes 18 equiv. no no 2 Yes 18 Yes NT Yes 3 Yes 13 Yes no no 4 no 18 no - - 5 Yes 16 Yes Yes Yes 6 Yes 11 Yes 2) no no 7 Yes 15 Yes NT no 8 Yes 20 Yes no Yes 9 Yes 5 Yes Yes no 10 Yes 15 Yes no Yes

188

IV, Μ, Magnification to Cion HCV cDNA Sequences Using PCR and Primers Derived from Conserved Regions of Flavivirus Genomic Sequences

The results presented above, which suggest that HCV is a flavivirus or flavi virus analog, allow a strategy for the cloning of uncharacterized HCV cDNA sequences using the PCR technique, and primers derived from regions encoding flavivirus conserved amino acid sequences. Generally, one of the primers is derived from a defined HCV genomic sequence, and the other primer that flanks a non-sequenced HCV polynucleotide region is derived from a conserved region of the flavivirus genera.

Flavivirus genomes are known to contain conserved sequences within NS1 θ E polypeptides, which are encoded in the 5 'region of the genorn flavivirus. Matched sequences encoding these regions are upstream from the HCV cDNA sequence represented in Fig, 26. Thus, to isolate cDNA sequences derived from this HCV genorn region, upstream primers are outlined which are derived from the conserved sequences within these polypeptides. flavivirus. The downstream primers are derived from an upstream end of the known portion of the HCV cDNA.

Due to the degeneration of the code, it is likely that there will be mismatches between the flavivirus probes and the corresponding HCV genomic sequence. Therefore, an approach that is similar to that described by Lee (1988) is used. Lee's process uses mis oligonucleotide primers

- I89 supplemented to the reverse translation products of an amino acid sequence; the sequences in the mixed primers take into account any codon degeneration for the conserved amino acid sequence.

Three sets of primary mixtures are generated, based on the amino acid homologies found in various flaviviruses, including Dengue-2,4 (D-2,4), Japanese Encephalitis Virus (JEV), Yellow Fever (YF) and West Nile Virus ( WN) The primary mixture derived from the upstream (5'-l) sequence conserved 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- protein E of D-2, JEV, YF, and WN. The following primary mixture (5'-2) is based on a conserved sequence downstream of protein E, phe-asp-gly-asp-ser-tyr-ileu-phe-gly-asp-ser-tyr-ileu, and is derived from phe-gly-asp; the conserved sequence is present in D-2, JEV, YF, and WN. The third primary mixture (5'-3) '© 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, D-4, JEV, YF, and WN. The individual primers that form the 5'-3 mixture are shown in Fig. 45. In addition to the varied sequences derived from the conserved region, each primer in each mixture also contains a constant region at the 5 'end that contains a sequence encoding enzyme sites restriction, HindIII, Mbol, and EcoRl.

downstream primary, ssc5b2OA, is derived from a nucleotide sequence in clone 5h, which contains HCV cDNA

190 with sequences overlapping those of clones 14 and 11b. The sequence of ssc5h20A is

5 'GTA ATA TGG TGA CAG AGT CA 3'.

An alternative primer, ssc5h3 ^ A, can also be used. This primer is derived from a sequence in clone 5h, and furthermore contains nucleotides at the 5 'end that create an enzyme restriction site, thus facilitating cloning. The sequence of ssc5h3 ^ A is

5 ’GAT CTC TAG AGA AAT CAA TAT GGT GAC AGA GTC A 3’.

The PCR reaction, which was first described by Saiki et al. (1986), is performed essentially as described in Lee et al. (1988), except that the standard for cDNA is RNA isolated from HCV-infected chimpanzee liver, as described in Section IV.C.2, or from viral particles isolated from HCV-infected chimpanzee serum, as described in Section IV.A.1. In addition, the quenching conditions are less stringent in the first round of expansion (O, oh NaCl, and 25 ° C) since part of the primer will be tempered with the HCV sequence and only 9 nucleotides, and there may be more with combinations. Furthermore, if ssc5h34A is used, additional sequences not derived from the HCV genome tend to be dese. to stabilize the hybrid standard primer. After the first round of expansion, the quenching conditions may be more stringent (Ο, ΟόόΜ NaCl, and 32 ° C-37 ° C), since the extended sequences now contain regions that are complementary to, or twice as primary . In addition, the first 10 amplification cycles are treated with Klenow's enzyme I, under PCR conditions

- 191 suitable for that enzyme. After completing these cycles, the samples are extracted, and treated with Taq polymerase, according to the package instructions (kit), as provided by Cetus / Perkin-Elmer.

After enlargement, the extended HCV cDNA sequences are detected by hybridization using a probe derived from the clone. This probe is derived from sequences upstream from those used to derive the primer, and do not overlap with the sequences of the primers derived from clone 5h. The probe sequence 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 '.

IV.N.1 Creation of the HCV cDNA Libraria from a Chimpanzee's Liver with Infectious NANBH

An HCV cDNA book was created from a chimpanzee figure from which the HCV cDNA book in Section IV.A.l was created. The technique for creating the librarian was similar to that of Section IV, A.24, except for this different source of RNA, and because a primer based on the HCV cDNA sequence in clone 11b was used. The primary sequence was

5 'CTG GCT TGA AGA ATC 3'

IV.N.2, Isolation and Sequence of Nucleotide Overlapping HCV cDNA in Clone k9-l to cDNA in Clone 11b 'clone k9-l was isolated from the librarian HCV cDNA created from the liver of a chimpanzee infected with

192

NANBH, as described in Section IV, A, 25, The library was screened for clones that overlap the sequence in clone 11b, using a clone that overlaps clone 11b at terminal 5 ', clone lie. The sequence of clone 11b is shown in Figure 23. Positive clones were isolated at a frequency of 1 in 500,000. An isolated clone, k9-l, was subjected to further study. The overlapping nature of the HCV cDNA in the clone ne k9-l to the 5 'end of the HCV-cDNA sequence in Fig.

was confirmed by polling the clone with 0 Alex46; This last clone contains a 30 base pair HCV cDNA sequence that matches those - base pairs at the 5 'end of the HCV cDNA in clone l4i, described above,

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

The HCV cDNA sequence in clone k9-l was aligned with those of the clones described in Section IV, A.19. to create a composite HCV cDNA sequence, the k9-l sequence being placed upstream of the sequence shown in Fig, 32. The composite HCV cDNA that includes the k9-l sequence, and the amino acids encoded therein, are represented in Fig, 47 .

The sequence of the amino acids encoded in the 5 'region of the HCV cDNA represented in Fig. 47 was compared with the corresponding region of one of the strains of Dengue virus, described above, in relation to the profile of hydrophobicide regions.

193 de and hydrophilicity. This comparison showed that the HCV and Dengue polypeptides encoded in this region, which corresponds to the NS1 encoding region (or a portion thereof), has a similar hydrophobic / hydrophilic profile.

The information provided below allows the identification of HCV strains. Isolation and characterization of other HCV strains can be accomplished by isolating nucleic acids from body components that contain viral particles, creating librarian cDNAs using polynucleotide probes. It is based on the HCV cDNA probes described below, sieving the librarians for clones containing HCV cDNA sequences described below, and comparing the HCV cDNAs from the isolated isolates with the cDNAs described below. Polypeptides encoded in them, or in the viral genome, can be controlled for immunological cross-reactivity using the polypeptides and antibodies described above. Strains that fit within the HCV parameters, as described in the Definitions above, are easily identifiable. Other methods for identifying HCV strains are obvious to those of skill in the art, based on the information provided herein.

Description of Figures (Continued)

Fig. 46 shows the HCV cDNA sequence in clone k9-1, the segment that overlaps with the cDNA in Fig, 26, and the amino acids encoded therein.

Fig. 47 shows the sequence in a composite cDNA that was derived by aligning clones k9-l at 15e in the direction

5 'to 3', θ also shows the continuous encoded amino acids.

at

ORF

Industrial Applicability

The invention, in the various manifestations disclosed here, has several industrial uses, some of which are as follows. HCV cDNAs can be used to probe the probes for detecting HCV nuoleic acids in samples. Probes derived from cDNAs can be used to detect HCV nucleic acids in, for example, synthetic chemical reactions. They can also be used for screening programs for anti-viral agents, to determine the effect of agents on inhibition of viral replication in cell culture systems, and animal model systems. HCV polynucleotide probes are also useful for detecting viral nucleic acids in humans, and thus can serve as a basis for diagnosing HCV infections in humans.

In addition to the above, the cDNAs provided herein provide information and means for synthesizing polypeptides containing HCV epitopes. These polypeptides are useful in detecting antibodies to HCV antigens. A series of immunoassays for HCV infection, based on recombinant polypeptides containing HCV epitopes, is described here, and will find commercial use in the diagnosis of HCV-induced NANBH in the screening of blood bank donors for infectious hepatitis caused HCV, and also to detect contaminated blood from infectious blood donors.

195

'V

ο

Viral antigens will also be useful in controlling the effectiveness of anti-viral agents in animal model systems. In addition, the HCV-derived polypeptides cDNAs disclosed herein will find use as vaccines for treating HCV infections.

Polypeptides derived from HCV cDNAs, in addition to the uses indicated above, are also useful for raising anti-HCV antibodies. Therefore, they can be used in anti-HCV vaccines. However, antibodies produced as a result of immunization with HCV polypeptides are also very useful in detecting the presence of viral antigens in samples. Thus, they can be used to test the production of HCV polypeptides in chemical systems. The anti. anti-HCV bodies can also be used to control the effectiveness of anti-viral agents in screening programs in which these agents are tested in tissue culture systems. They can also be used for passive immunotherapy, and for diagnosis of NANBH caused by HCV allowing the detection of viral antigen (s) in both donors and blood recipients. Another important use for anti-HCV antibodies is a chromatographic affinity for the purification of viruses and viral polypeptides, purified viruses and viral polypeptide preparations can be used in vaccines ^ However, the purified virus can also be useful for the development of culture systems. cell in which HCV replicates.

Cell culture systems containing cells

- 196 infected with HCV may have several uses. They can be used for the production on a relatively large scale of HCV, which is normally a low-titer virus. These systems will also be useful for elucidating the bio. molecular virology, and lead to the development of anti-viral agents. Cell culture systems will also be useful in screening for the effectiveness of anti-agents. -virals. In addition, HCV permissive cell culture systems are useful for producing attenuated strains of HCV.

For convenience, anti-HCV antibodies and HCV polypeptides, whether natural or recombinant, can be packaged in kits.

method used to isolate HCV cDNA, which consists of the preparation of a cDNA library derived from an individual's infected tissue, in an expression vector, and selected clones that produce the expression products, which react immunologically with antibodies, in body components containing antibody, from infected individuals and not from uninfected individuals, may also be applicable to the isolation of cDNAs derived from other disease-associated agents, hitherto uncharacterized, which are made up of a genomic component. This, in turn, can lead to the isolation and characterization of these agents, and to diagnostic reagents and vaccines for these other associated disease agents.

Claims (43)

1 - A method for producing an HCV polypeptide, characterized in that it comprises the incubation of transformed host cells with an expression vector, 'which contains a sequence encoding an HCV polypeptide, under conditions that allow the polypeptide to be expressed. The method as claimed in claim 1, characterized in that the sequence encoding the HCV polypeptide comes from an HCV genome. The method as claimed in claim 1, characterized in that the sequence encoding the HCV polypeptide is derived from HCV cDNA. The co-form method claimed in claim 3, characterized in that the sequence encoding the HCV polypeptide is derived from the HCV sequence. cDNA in Figure 32. The method as claimed in claim 3, characterized in that the DNA sequence is contained in clone 5-1-1. I 6-0 method as claimed in claim 3, characterized in that the DNA sequence is contained in clone 81. Method as claimed in claim 3, characterized in that the DNA sequence is contained in clone 100. 8 - A method for producing a polypeptide comprising an epitope immunologically identifiable with an epitope contained in HCV, characterized by the Tact of understanding the incubation of host cells transformed with an expression vector, containing a sequence that encodes _{: x} the polypeptide under conditions that allow expression of the polypeptide. . 9 - A method of producing a fusion polypeptide containing an HCV polypeptide, characterized by the fact that it comprises the incubation of transformed host cells with an expression vector, containing a sequence that encodes the fusion polypeptide, under conditions that allow the expression of the polypeptide . 10-0 method as claimed in claim 9, characterized in that the polypeptide contains a superoxide dismutase polypeptide fused to an HCV polypeptide. 11 - A method for producing an HCV polypeptide, characterized by the fact that an HCV preparation is obtained, and isolating the said 199 antigen of said HCV preparation. 12 - A method for producing an HCV polypeptide, characterized by the fact that it comprises the binding of amino acids, under conditions that allow the formation of the peptide junction, in which the polypeptide sequence comes from the HCV genome. 13. The method as claimed in claim 12, characterized in that the polypeptide is derived from HCV cDNA, 14. The method as claimed in claim 13, characterized in that the polypeptide is from HCV cDNA, in Figure 32. 15 - A method of isolating HGV, characterized by the fact that it comprises incubating a preparation containing HCV, with anti-HCV antibodies, 'under conditions that allow the formation of an anti-HCV antibody-HCV complex, and isolating the complex. 16 - A method of preparing purified polyclonal antibodies intended to combat an HCV antigen, characterized by the fact that it comprises obtaining an antibody containing the body component of an individual who has reacted immunologically to the antigen; the interaction of the antibody containing body component with HCV antigen, under conditions that allow the formation of an HCV antigen-antibody complex, isolating said complex, and isolating said antibody from said complex. 17 - A method for preparing monoclonal antibodies directed against an HCV antigen, characterized by the fact that it comprises obtaining a hybridoma from antibody-producing cells from an individual immunized with the HCV antigen, and incubating the hybridoma under conditions that allow the expression of the anti-body. 18 - A method of preparing an HCV polynucleotide, characterized by the fact that it comprises obtaining a host cell transformed with a vector containing the HCV polynucleotide, and developing the host cells under conditions that allow the polynucleotide to replicate. 19 - A method of preparing an HCV polynucleotide, characterized by the fact that it comprises obtaining an HCV preparation, and isolating the HCV polynucleotide. 20 - A method of preparing an HCV polynucleotide, characterized by the fact that it comprises the binding of nucleotides under conditions that result in the formation of phosphodiester bonds between the nucleotides, and in which the polynucleotide sequence comes from a genome HCV. 21 - A method of preparing an HCV polynucleotide, characterized by the fact that it comprises nucleotide bonding, under conditions that result in the formation of phosphodiester bonds, between nucleotides, and in which the polynucleotide sequence comes from HCV cDNA. 22-0 method as claimed in claim 21, characterized in that the polynucleotide sequence comes from HCV cDNA, in Figure 32. 23 - An HCV production method, characterized by the fact that it comprises obtaining an HCV-infected cell, and developing the cell in culture, under conditions that allow 201 viral replication. 24 - A method of preparing an immobilized HCV polypeptide, characterized by the fact that it comprises the joining of the HCV polypeptide to a solid substrate. 25 - A method of preparing an immobilized polypeptide containing an HCV epitope, characterized by the fact that it comprises the joining of the polypeptide, containing the HCV polypeptide, to a solid substrate. 26 - A method of preparing an immobilized anti-HCV antibody, characterized by the fact that it comprises the joining of the antibody to a solid substrate. 27 - A method of preparing an immobilized antibody directed against an epitope that is immunologically identifiable with an HCV epitope, characterized in that it comprises the joining of the antibody to a solid substrate. 28 - A method for detecting HCV nucleic acids in a sample, characterized by the fact that it comprises: a) the reaction of the sample nucleic acids with a probe, for an HCV polynucleotide, under conditions that allow the formation of a polynucleotide duplex between the probe and the HCV nucleic acid of the sample; ,and b) detecting a polynucleotide duplex that contains the probe; A method as claimed in claim 28, characterized in that the reaction is carried out with a poly202 β probe nucleotide from · HCV cDNA. 30 - A method for detecting an HCV antigen by immunoassay, characterized by the fact that it comprises: a) incubating a sample suspected of containing an HCV antigen with an antibody probe directed against an epitope on an antigen to be detected under conditions that allow the formation of an HCV antigen-antibody probe complex; and b) the detection of the HCV antigen-antibody probe complex. 31. The method of claim 30, wherein the HCV antigen is encoded in the HCV cDNA. 32. The method as claimed in claim 30, characterized in that the antigen, against which the antibody is directed, is encoded in clone 5-1-1. 33. The method as claimed in claim 30, characterized in that the antigen, against which the antibody is directed, is encoded in Figure 32. 34 - A method for detecting antibodies directed against an HCV antigen, by means of an immunoassay, characterized by the fact that it comprises: a) the incubation of a sample that is suspected to contain anti-HCV antibodies, with a probe polypeptide containing an HCV epitope under conditions that allow the formation of an anti-HCV antibody - probe polypeptide complex; and b) detection of the anti-HCV antibody - probe polypeptide complex. The 35-0 method as claimed in claim 34, characterized in that the probe polypeptide comes from an HCV genome. 36. The method as claimed in claim 34, characterized in that the probe polypeptide is derived from HCV cDNA. 37 - The method as claimed in claim 36, characterized in that the probe polypeptide is derived from clone 5-1-1 38. The method as claimed in claim 36, characterized in that the probe polypeptide comes from clone 81 39. The method of claim 36, wherein the probe polypeptide is from clone C100. 40. The method as claimed in claim 37, characterized in that the probe polypeptide is NANBg _ ^ _ p ' 41 - The method as claimed in claim 38, characterized in that the probe polypeptide is ΝΑΝΒθρ 42. The method as claimed in claim 39, characterized in that the probe polypeptide is C100-3. 43 - Method for isolating cDNA derived from the genome of an unidentified infectious agent, characterized by the fact that it comprises: a) supply of host cells transformed with expression vectors that contain a cDNA heritage prepared from nucleic acids isolated from tissue infected with the agent and development of said host cells under conditions that allow the expression of coded polypeptide (s) • 204 of the cDNA. b) interaction of the cDNA expression products with an antibody that contains the body component of an individual infected by said infectious agent, under conditions that allow an immunoreaction, and detection of the antibody-antigen complexes, formed as a result of the interaction; c) development of host cells that express polypeptides that form antibody-antigen complexes in step (b), under conditions that allow their development as individual clones and isolation of said clones; I d) development of cells from clones of (c), under conditions that allow the expression of encoded polypeptide (s) within cDNA, and interaction of expression products with an antibody containing body components, from different individuals the individual from stage (a), who is infected with the infectious agent, and with control subjects infected with the agent, and detection of antibody-antigen complexes formed as a result of the interaction; e) development of host cells that express polypeptides that form antibody-antigen complexes, containing the antibody body components of infected individuals and individuals suspected of being infected, and not with the said components of control individuals, under conditions that allow their 'development as individual clones and isolation of said clones; and f) isolation of cDNA from the clones of the host cell of (e). Lisbon, November 18, 1988