HRP940493A2 - Nanbv diagnostics and vaccines - Google Patents

Description

The field of technology

The invention is from the field of biomolecular engineering.

Technical problem

The invention relates to materials and methodologies for managing the spread of non-A and non-B hepatitis virus (NANBV) infection. More specifically, the invention relates to diagnostic DNA fragments, diagnostic proteins, diagnostic antibodies and protective antigens and antibodies for the etiological agent of NANB hepatitis, e.g. hepatitis C virus.

State of the art

Non-A, Non-B hepatitis (NANBH) is a transmissible disease or family of diseases believed to be caused by a virus, and is distinct from other forms of viral liver disease, including those caused by known hepatitis viruses, eg hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis delta virus (HDV), as well as hepatitis caused by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). NANBH was first identified in transfusion recipients. Human-to-chimpanzee transmission, and serial transmission in chimpanzees, provides evidence that NANBH is caused by a transmissible infectious agent or agents.

However, the transmissible agent responsible for NANBH is still unidentified and the number of agents that cause the disease is unknown.

Epidemiological evidence suggests that there are perhaps three types of NANBH: the blood-resistant epidemic type; blood or needle-connected type; and sporadically represented type (commonly supplied type). However, the number of agents that can cause NANBH is unknown.

Clinical diagnosis and identification of NANBH was achieved primarily by excluding other viral markers. Among the methods used to detect the putative NANBH antigen and antibody are agar-gel diffusion, counterimmunoelectrophoresis, immunofluorescence microscopy, immunoelectron microscopy, radioimmunoassays, and enzyme-linked immunosorbent assay. However, none of these assays are sufficiently sensitive, specific, and reproducible to be used as a diagnostic test for NANBH.

So far, it is not clear or agreed upon how to identify or specify antigen-antibody systems that are associated with NANBH agents. This is caused, at least in part, by previous HBV or simultaneous infection of individuals with NANBH and integration of HBV DNA into the liver cell genome. Additionally, there is the possibility that NANBH is caused by more than one infectious agent, as well as the possibility that NANBH may go undiagnosed. In addition, it is unclear what serological tests detect in the serum of a patient with NANBH. Agar-gel diffusion and anti-immunoelectrophoresis assays have been hypothesized to detect autoimmune responses or non-specific protein interactions that sometimes exist between serum species, and which do not represent specific NANBV antigen-antibody reactions. Immunofluorescent and enzyme-linked immunosorbent assays and radioimmunoassays are used to detect low levels of rheumatoid factor-like material that is often present in the serum of patients with NANBH as well as in patients with other hepatic and nonhepatic diseases. Some of the detected reactivities may represent antibodies against cytoplasmic antigens of a specific host.

There are numerous NANBV candidates. See, for example, the works of Princ (1983). Feinston and Hoofnagle (1984) and Overby (1985, 1986, 1987) and articles by Iwarson (1987). However, there is no evidence that any of these candidates represent the etiological agent of NANBH.

The demand for sensitive, specific methods to test and identify carriers of NANBV and blood or blood products contaminated with NANBV is significant. Post-transfusion hepatitis (PTH) occurs in about 10% of patients and NANBH is estimated to account for 90% of these cases. The main problem of this disease is often progressive, even chronic liver damage (25%-55%).

Patient care as well as prevention of transmission of NANBH through blood and blood products or close personal contact requires reliable diagnostic and prognostic methods for detecting nucleic acids, antigens and antibodies against NANBV. Additionally, there is also a need for effective vaccines and immunotherapeutic agents for disease prevention and/or treatment.

Description of the invention with examples

The invention relates to the isolation and characterization of the newly discovered etiologic agent of NANBH, hepatitis C virus (HCV). More specifically, the invention provides a family of cDNA replicates of portions of the HCV genome. These cDNA replicates were isolated by a technique involving a novel step of testing expression products from a cDNA library generated from a specific agent in infected tissue with serum from patients with NANBH to detect a newly synthesized antigen derived from the genome of a previously unisolated and uncharacterized viral agent, and from selecting clones that produce products that they react immunologically only with serum from an infected person compared to uninfected persons.

Studies of the nature of the HCV genome, using probes derived from HCV cDNA, as well as sequence information contained in HCV cDNA, suggest that HCV is a flavivirus or a virus similar to it.

Portions of cDNA sequences derived from HCV are useful as probes for diagnosing the presence of the virus in samples, and for isolating naturally occurring variants of the virus. These cDNAs also make available the polypeptide sequences of the HCV antigens encoded in the HCV genome(s) and allow the production of polypeptides that are useful as standards or reagents in diagnostic tests and/or as vaccine components. Antibodies, polyclonal and monoclonal, directed against the HCV epitope contained in these polypeptide sequences are also useful for diagnostic tests, as therapeutic agents, for testing antiviral agents, and for isolating NANBV agents from which these cDNAs are derived. Additionally, using probes derived from these cDNAs, it is possible to isolate the sequences of other parts of the HCV genome, thus providing an increase in additional probes and polypeptides that are useful in the diagnosis and treatment, prophylactic and therapeutic NANBH.

Therefore, with respect to polynucleotides, some aspects of the invention are: purified HCV polynucleoid; recombinant HCV polynucleotide; a recombinant polynucleotide comprising a sequence derived from the HCV genome or from HCV cDNA; a recombinant polynucleotide encoding an HCV epitope; a recombinant vector encoding any of the above recombinant polynucleotides and a host cell transformed with any of these vectors.

Other aspects of the invention are: a recombinant expression system comprising an open reading frame (ORF) of DNA derived from the HCV genome or HCV cDNA where the ORF is operably linked to a control sequence compatible with the desired host, a cell transformed with the recombinant expression system, and a polypeptide produced by the transformed cell.

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

Included in aspects of the invention are recombinant HCV polypeptide; a recombinant polypeptide comprising a sequence derived from the HCV genome or from HCV cDNA; a recombinant polypeptide comprising the HCV epitope; and a fusion polypeptide comprising the HCV polypeptide.

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

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.

A still further aspect of the invention is a polynucleotide probe for HCV. Aspects of the invention relating to the equipment are those for: analyzing samples for the presence of polynucleotides derived from HCV comprising a polynucleotide probe containing an HCV nucleotide sequence of about 8 or more nucleotides, in a suitable container; to analyze samples for the presence of HCV antigen, which is detected in a suitable container; analyzing the samples for the presence of antibodies directed against the HCV antigen comprising a polypeptide containing an epitope present in the 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 the HCV epitope, coupled to a solid substrate.

Still further aspects of the invention are: a method for the production of a polypeptide containing an HCV epitope comprising incubating host cells with an expression vector containing a sequence encoding a polypeptide containing the epitope under conditions that allow the expression of said polypeptide; and an HCV epitope-containing polypeptide produced by the process.

The invention also includes a method for detecting HCV nucleic acids in a sample comprising reacting the nucleic acids with a probe for HCV polynucleotide under conditions that experience the formation of a polynucleotide duplex between the probe and HCV nucleic acid from the sample; and detecting the polynucleotide duplex containing the probe.

Immunoassays are also included in the invention. These include immunoassays for the detection of HCV antigen which involve incubating a sample suspected of containing HCV antigen with an antibody probe directed against the detected HCV antigen under conditions that allow the formation of an antigen-antibody complex containing the antibody probe. Immunoassay is the detection of antibodies directed against the HCV antigen, which includes incubating a sample suspected of containing anti-HCV antibodies with a polypeptide probe containing the HCV epitope, under conditions that allow the formation of an antigen-antibody complex; and detecting the antigen-antibody complex containing the antigen probe.

Also included in the invention are vaccines for the treatment of HCV infection comprising an immunogenic peptide containing an HCV epitope or a reactive preparation of HCV or a diluted preparation of HCV. Another aspect of the invention is tissue culture of cultured cells infected with HCV.

A still further aspect of the invention is a method for the production of antibodies to HCV comprising introducing into an individual an isolated immunogenic polypeptide containing an HCV epitope in an amount sufficient to produce an immune response.

A still further aspect of the invention is a method for isolating cDNA derived from the genome of an unidentified infectious agent, comprising a) providing transformed host cells with expression vectors containing a cDNA library obtained from nucleic acids isolated from tissue infected with the agent and culturing the host cells under conditions that enable expression polypeptide encoded in cDNA; b) interaction of cDNA expression products with an antibody containing a body component of an individual infected with said infectious agent under conditions that allow an immunoreaction, and detection of antibody-antigen complexes created as a result of the interaction; c) growth of host cells expressing polypeptides that form antibody-antigen complexes in step b) under conditions that allow their growth as individual clones and isolation of said clones; d) growth of cells from clones c) under conditions that allow the expression of polypeptides encoded in cDNA, and interaction of the expression products with an antibody containing body components of individuals other than individuals from stage a) infected with an infectious agent and with control individuals infected with the agent, and detecting antibody-antigen complexes formed as a result of the interaction; e) growth of host cells that express polypeptides that create antibody-antigen complexes with an antibody containing body components of infected persons and persons suspected of being infected and not with said components of control individuals under conditions that allow their growth as individual clones and isolation of said clones; and f) isolation of cDNA from host cells of clone e).

Short description of the pictures

Figure 1 shows the double nucleotide sequence of the HCV cDNA insert in clone 5-1-1, and

the putative amino acid sequence of the encoded polypeptide.

Figure 2 shows the overlapping homologues of the HCV cDNA sequences in clones 5-1-1, 81, 1-2 and 91.

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

Figure 4 shows the double nucleotide sequence of the HCV insert in column 81, and the putative amino acid sequence of the encoded polypeptide.

Figure 5 shows the HCV cDNA sequence in clone 36, the segment overlapping the NANBV cDNA of clone 81, and the polypeptide sequence encoded in clone 36.

Figure 6 shows the combined ORF of HCV cDNA in clones 36 or 81, and the encoded polypeptide.

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

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

Figure 9 shows the combined ORF of HCV cDNA in clones 35, 36, 81 and 32 and the encoded polypeptide.

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

Figure 11 shows the HCV cDNA sequence in clone 33b, the segment overlapping clone 32, and the encoded polypeptide.

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

Figure 13 shows the HCV cDNA sequence in clone 25c, and the encoded polypeptide.

Figure 14 shows the nucleotide sequence and encoded polypeptide of the ORF spanning the HCV cDNA in clones 40b, 37b, 35, 36, 81, 32, 33b and 25c.

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

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

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

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

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

Figure 20 shows the HCV cDNA sequence in clone 33f, the segment overlapping clone 8f, and the encoded amino acids.

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

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

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

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

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

Figure 26 shows the schematic of HCV cDNA sequences derived from the linear cDNAs in clones 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f and 33g; also show the amino acid sequence of the polypeptide encoded in the extended ORF in the deduced sequence.

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

Figure 28 shows the sequence of clone 35f, the segment overlapping clone 39c, and the encoded amino acids.

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

Figure 30 shows the sequence of clone 15e, the segment overlapping clone 26g, and the encoded amino acids.

Figure 31 shows the sequence in the cDNA system which was derived by linking clones 12f to 15e in 5' to 3; also shows the encoded amino acids in the continuous ORF.

Figure 33 shows a photograph of a Western blot of the fusion protein, SOD-NANB5-1-1 with chimpanzee serum from chimpanzees infected with BB-N ANB, HAV and HBV.

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

Figure 35 is a map showing the essential characteristics of vector pAB24.

Figure 36 shows the putative amino acid sequence of the achrono-terminus of the C100-3 fusion polypeptide and the encoded nucleotide sequence.

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

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

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

Figure 39 shows an autoradiograph of NANBV nucleic acid treated with RNase or DNase 1 and probed with BB-NANBV cDNA clone 81.

Figure 40 shows an autoradiograph of nucleic acids extracted from NANBV particles taken from infected plasma with anti-NANB5-1-1, and probed with 32P-labeled NANBV cDNA from clone 81.

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

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

Figure 43 shows a histogram of the distribution of HCV infection in random samples, as determined by ELISA testing.

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

Figure 45 shows the sequences in the primer mixture, derived from the conserved sequence in flavivirus NS1.

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

Figure 47 shows the sequence in the composition of the cDNA, which was derived by connecting clones k9-1 to 15e in the 5' to 3' direction; amino acids encoded in the ORF are also shown.

And Definitions

The term "hepatitis C virus" is reserved by experts in the field for the hitherto unknown etiological agent of NANBH. Therefore, as used herein, “hepatitis C virus” (HCV) refers to the agent caused by NANBH, formerly designated NANBV and/or BB-NANBV. The terms HCV, NANBV and BB-NANBV are used interchangeably here. As an extension of this terminology, the disease caused by HCV, formerly called NANB hepatitis (NANBH) is referred to as hepatitis C. The terms NANBH and hepatitis C may be used interchangeably herein.

The term “HCV”, as used herein, refers to viral species that cause NANBH, and rare species or defective interfering particles derived therefrom. As noted below, the HCV genome comprises RNA. The RNA contained in viruses is known to have a relatively high rate of spontaneous mutation, eg reportedly of the order of 10-3 to 10-4 per nucleotide (Fields & Knipe (1986)). Therefore, there are multiples in the HCV species described below. The compositions and procedures described herein provide for the advancement, identification, detection and isolation of various related professions. However, they also allow obtaining diagnostics and vaccines for various professions and have applications in testing procedures for anti-viral agents for pharmacological use in which they inhibit HCV replication.

The information provided here, although derived from a single HCV, then designated as CDC/HCV1, is sufficient to allow viral taxonomy to identify other species that fall within the species. As described herein, we have discovered that HCV is a flavivirus or a virus similar to it. The morphology and composition of the flavivirus particle are known and discussed by Brinton (1986). Regarding the morphology in general, the flavivirus contains a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 20-50 nm. Their core is about 25-30 nm in diameter. Between the outer surface of the virion envelope are projections that are about 5-10 nm long, with end nodes about 2 nm in diameter.

HCV encodes an epitope that is immunologically detectable with the epitope in the HCV genome from which the described cDNA is derived; preferably the epitope is encoded in the cDNA described herein. The epitope is unique to HCV when compared to other known flaviviruses. The uniqueness of the epitope can be determined by its immunological reactivity with HCV and lack of immunological reactivity with other flavivirus species. Methods for determining immunological reactivity are known in the art, for example, by radioimmunoassay, ELISA, hemagglutination, and a number of techniques suitable for testing that are provided herein.

In addition to the above, the following parameters are applicable, either alone or in combination, in identifying the species as HCV. Although HCV species are evolutionarily related, full genome homology at the nucleotide level is expected to be 40% or more, preferably about 60% or more and even preferably about 80% or more and will additionally correspond to contiguous sequences of at least about 13 nucleotides. The correspondence between the putative HCV species 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 direct comparison of the polynucleotide sequence information from the putative HCV, and the HCV cDNA sequence described under conditions that create stable duplexes between homologous regions (for example, those to be used before S1 digestion), followed by digestion with a single-specific nuclease ( nucleases) and then determining the size of the broken fragments.

Due to the evolutionary relatedness of HCV species, the putative HCV domains are more than 40% homologous, preferably above 60% and even more preferably above 80% at the polypeptide level. Techniques for determining amino acid sequence homology are known in the art. For example, the amino acid sequence can be determined directly and compared to the sequences provided herein. For example, the nucleotide sequence of the putative HCV genomic material can be determined (usually via a cDNA intermediate); the amino acid sequence encoded here can be determined and the corresponding regions can be compared.

As used herein, a polynucleotide "derived from" a designated sequence, for example HCV cDNA, particularly shown in Figures 1-32 or from the HCV genome, means a sequence that is composed of about 6 nucleotides, preferably at least 8 nucleotides and most preferably at least about 10 -12 nucleotides, and even more preferably at least about 15-20 corresponding nucleotides, for example homologous or complementary regions of the indicated nucleotide sequence. Preferably, the sequence of the region from which the polynucleotide is derived is homologous or complementary to a sequence that is unique to the HCV gene. Whether or not the sequence is unique to the HCV genome can be determined by techniques known to those skilled in the art, for example, the sequence can be compared to sequences in data banks, eg Genebank, to determine whether or not it is also present in an unidentified host or other organisms. The sequence may also be compared to known sequences of other viral agents, including those known to cause hepatitis, eg HAV, HSV, HDV and other members of the flaviviruses.

Matching or non-matching of the derived sequence to other sequences can also be determined by hybridization under suitable stringency 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). In addition, the dominance of one of the duplex polynucleotides created by hybridization can be determined by known techniques, including for example, digestion with a nuclease such as S1 that specifically cleaves single-stranded regions in duplex polynucleotides. Regions from which typical DNA sequences may be "derived" include, but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and/or non-translatable regions.

A derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but may be generated by some means, including for example, chemical synthesis or DNA replication or reverse transcription or transcription that is based on information provided by the base sequence from which the polynucleotide is derived. Additionally, combinations of regions corresponding to that of the indicated sequence may be modified in a manner known in the art to suit the purpose.

Similarly, a polypeptide or amino acid sequence “derived from” an indicated nucleic acid sequence, for example, the sequences of Figures 1-32 or from the HCV genome, means a polypeptide having an amino acid sequence identical to that of the polypeptide encoded by the sequence or part thereof, where the portion contains at least 3- 5 amino acids, and more preferably at least 8-10 amino acids and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with the polypeptide encoded in the sequence.

The recombinant or derived polypeptide need not be translated from the generated nucleic acid sequence, for example, the sequence in Figures 1-32 or from the HCV genome; it can be generated, for example, by chemical synthesis or expression of a recombinant expression system or by isolation from mutated HCV.

The term "recombinant polynucleotide" as used herein means a polynucleotide of genomic cDNA, of semi-synthetic or synthetic origin by virtue of its origin or handling:

1) is not associated with all or part of the polynucleotide with which it is associated in nature or in the form of a library: and/or

2) is bound to a polynucleotide other than the one to which it is bound in nature.

The term "polynucleotide" as used herein refers to the polymeric form of a nucleotide of either ribonucleotide or deoxyribonucleotide length. This term denotes only the primary structure of the molecule. Thus, this term includes double-stranded or single-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modification, for example by methylation and/or capping, and unmodified forms of polynucleotides.

As used herein, the term “HCV containing a sequence corresponding to cDNA” means that HCV contains a polynucleotide sequence that is homologous or complementary to a sequence in the resulting DNA; the degree of homology or complementarity to the cDNA will be about 50% or more, preferably at least about 70% and even more preferably at least about 90%. Matching 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. Correspondence between the HCV sequence and cDNA can be determined by techniques known in the art, including, for example, direct comparison of the sequenced material with cDNA described or by hybridization and digestion with single nucleases, which is further followed by determination of the destruction of the resulting fragments.

The term "purified viral polynucleotide" means an HCV genome or a fragment thereof that is mostly free, i.e. contains about 50%, preferably below about 70% and even more preferably below 90% of the polypeptide with which the viral polypeptide is naturally associated. Techniques for purifying viral polynucleotides from viral particles are known in the art, and include, for example, fractionation of the particles with a chaotropic agent and separation of polynucleotides and polypeptides by ion-exchange chromatography, affinity chromatography, and density sedimentation.

The term "purified viral pilipeptide" means an HCV polypeptide or fragment thereof that is mostly free, i.e. contains less than about 50%, preferably less than about 70% and even more preferably less than about 90% of the cellular component 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”, “cell lines”, “cell culture” and other such terms mean microorganisms or multi-eukaryotic cell lines cultured as single-celled organisms from cells that will be, or have been used as recipients for, a recombinant vector or the second DNA transfer, and includes the progeny of the original cell that may be transferred. It is understood that the progeny of one parent cell does not have to be completely identical in morphology or genomic or total DNA complement as the original parent due to accidental or deliberate mutation. A progen of a parental cell that is sufficiently similar to the parent characterized by a suitable feature, such as the presence of a nucleotide sequence encoding the desired peptide, is included in the progen covered by this definition, and is covered by the above terms.

A "replicon" is a genetic element, eg a plasmid, a chromosome, a virus that behaves autonomously, and a unit of polynucleotide replication in a cell; eg capable of replicating under its own control.

A "vector" is a replicon into which another polynucleotide segment is attached, so that it carries the replication and/or expression of the joined segment.

"Control sequence" means the polynucleotide sequences that are necessary to effect the expression of the coding sequences in which they are bound. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include the promoter, ribosome binding site, and terminators; in eukaryotes, generally such control sequences include promoters, terminators and in some cases, enhancers. The term “control sequence” is intended to include at a minimum all components whose presence is required for expression and may also include additional components whose presence is convenient, for example, leader sequences.

"Operably linked" means an assembly where the related components are described in such a way as to allow them to function in the desired manner. A control sequence "operably linked" to the 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 may represent part of the coding sequence or the entire coding sequence.

"Coding sequence" is a polynucleotide sequence that is transcribed into mRNA and/or translated into a code polypeptide under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by translation of the start codon to the 5'-terminus and translation of the end codon to the 3'-terminus. Coding sequences may include, but are not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

"Immunologically identifiable with/as" means the presence of epitope(s) and polypeptides that are also present and unique to the indicated polypeptides, usually HCV proteins. Immunological identity can be determined by the antibody that binds and/or participates in binding; these techniques are known to those skilled in the art and are illustrated below. Epitope uniqueness can also be determined by computer searching known data banks, eg Genebank, for polynucleotide sequences encoding the epitope and comparing the amino acid sequence to other known proteins.

As used herein, “epitope” refers to an antigenic determinant of a polypeptide; an epitope can encompass 3 amino acids in a spatial conformation that is unique to the epitope, usually an epitope contains at least 5 such amino acids and usually contains at least 8-10 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 two-dimensional nuclear magnetic resonance.

A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody because the antibody recognizes a specific epitope contained in the polypeptide. The immunological reactivity can be determined by the binding antibody, more precisely by the antibody binding kinetics and/or participation in the binding using the known polypeptide e) as a competitor, which contains the epitope against which the antibody is directed. Techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

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

The term "polypeptide" refers to the molecular chain of amino acids and does not refer to the specific length of the product; thus, the definition of polypeptide includes peptides, oligopeptides and proteins. The term also refers to post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like.

"Transformation" as used herein refers to the introduction of an exogenous polynucleotide into a host cell, with respect to the method used for introduction, for example, by direct uptake, transduction, or f-matting. An exogenous polynucleotide can be maintained as a non-integrated vector, for example, a plasmid, or alternatively can be integrated into a host gene.

"Treatment" as used herein means prophylaxis and/or therapy.

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

As used herein, the “plus arm” of a nucleic acid comprises a sequence that encodes a polypeptide. "Minus waist" contains a sequence that is complementary to that of "plus waist".

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

They also include the Flaviviridae, which were once classified as Togaviridae. See Fields & Knipe (1986).

As used herein “antibody-containing body component” refers to the individual body component that is the source of the antibody of interest. Body components that contain the antibody are known in the art and include, but are not limited to, for example, external parts of the respiratory, interstitial, and genitourinary tracts, tears, saliva, milk, white blood cells, and myeloma.

As used herein, “purified HCV” means a preparation of HCV that has been isolated from cellular substances with which the virus is normally associated, and from other types of virus that may be present in infected tissue. Techniques for isolating viruses are known to those skilled in the art and include, for example, centrifugation and affinity chromatography; the procedure for obtaining purified HCV is discussed below.

The implementation of this invention is carried out, unless otherwise indicated, by conventional techniques of molecular biology, microbiology, recombinant DNA and immunology known to those skilled in the art. Such techniques are fully explained in the literature. See, eg, Manitis, Fitsch & Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL (1982); DNA CLONING, VOLUMES I AND II (D.N. Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed. 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); series, A METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN CALLS (J.H.Miller and M.P.calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymolgy 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, N.Y.) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-VI (D.M. Weir and C.C. Blackwell eds. 1986).

All patents, patent applications and publications mentioned hereinabove and below are hereby incorporated by reference.

The useful materials and methods of this invention are made possible by providing a family of closely homologous nucleic acid nucleotide sequences isolated from a cDNA library derived from nucleic acid sequences present in the plasma of HCV infected chimpanzees.

The utility of this family of cDNAs shown in Figures 1-32 allows the creation of DNA probes and polypeptides useful in diagnosing NANBH caused by HCV infection and in testing blood donors as well as donated blood and blood products for infection. For example, it is possible to synthesize from the sequences DNA oligomers of about 8-10 nucleotides or more that are useful as hybridization probes for detecting the presence of the putative genomic genome in, for example, the serum of subjects suspected of harboring the virus or for testing donated blood for viral load. The family of cDNA sequences also allows the generation and production of HCV-specific polypeptides that are useful as diagnostic reagents for the presence of antibodies generated during NANBH. Antibodies to purified cDNA-derived polypeptides can also be used to detect putative antigens in infected individuals and in blood.

Knowledge of these cDNA sequences also ensures the creation and production of polypeptides that can be used as vaccines against HCV and also for the production of antibodies, which can be used for disease protection and/or therapy of HCV infected individuals.

Moreover, the family of cDNA sequences provides further characterization of the HCV genome. Polynucleotide probes derived from these sequences can be used to screen cDNA libraries for additional overlapping cDNA sequences, and can be used to obtain more overlapping sequences. Although a genome is a segment and segments do not have common sequences, these techniques can be used to obtain the sequences of the entire genome. However, if the genome is not a segment, other segments of the genome can be obtained by repeating the lambda-gt11 serological testing procedure used to isolate the cDNA clones described herein or alternatively by isolating the genome from purified HCV particles.

A family of cDNA sequences and polypeptides derived from these sequences, as well as antibodies directed against these polypeptides, are also useful in the isolation and identification of BB-NANBV agent(s).

This family of nucleotide sequences is not of human or chimpanzee origin, therefore it does not hybridize in either human or chimpanzee genomic DNA of an uninfected individual, therefore nucleotides of this family of sequences are present only in the liver and plasma of HCV-infected chimpanzees, and therefore the sequence is not present in Genebank. Additionally, the sequence family does not show significant homology to sequences contained in the HBV genome.

The sequence of one member of the family, contained in clone 5-1-1, has one continuous open frame (ORF) encoding a polypeptide of about 50 amino acids, serum from HCV-infected people contains antibodies that bind to this polypeptide, whereas serum from uninfected people does not antibodies to this polypeptide, finally, since the serum of uninfected chimpanzees does not contain antibodies to this polypeptide, antibodies are induced in chimpanzees following acute NANBH infection. However, antibodies to this polypeptide were not detected in chimpanzees and humans infected with HAV and HBV. Using these criteria, the sequence is the cDNA in the putative sequence, where the virus causes or is associated with NANBH; this cDNA sequence is shown in Figure 1. As discussed below, the cDNA sequence in clone 5-1-1 differs from that of other isolated cDNAs in that it contains 28 base pair extracts.

The composition of the other identified cDNA family members, which was isolated using as a probe the synthetic equivalent sequence to the cDNA fragment in clone 5-1-1, is shown in Figure 3. The cDNA family member in clone 81 is shown in Figure 5, and the composition of this sequence with hereof, clone 81 is shown in Figure 6. Other members of the cDNA family, including those present in clones 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f , 33f, 33g and 39c are described in Chapter IV.A. The composition of the cDNA in these clones is described in Chapter IV.A.18 and is shown in Figure 26. The composition of the cDNA shows that it contains a continuous ORF and thus encodes a polyprotein. This data is consistent with the suggestion discussed below that HCV is a flavivirus or a virus similar to it.

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

The information obtained from the further sequencing of the HCV genome, as well as from the further characterization of the HCV antigen and the characterization of the genome, will enable the creation and synthesis of additional samples and polypeptides and antibodies that can be used for diagnosis, for the prevention and therapy of HCV caused by NANBH, and for testing for infected blood and blood products.

The availability of HCV probes, including antigens and antibodies and polynucleotides derived from the genome from which the cDNA family is derived, also allows the development of tissue culture systems for use in resolving HCV biology. This leads to the development of new treatment methods based on antiviral compounds that inhibit or infection by HCV.

The procedure used to identify and isolate the etiologic agent for NANBH is novel, and may be applicable to the identification and/or isolation of hitherto uncharacterized genome-containing agents associated with a variety of diseases, including those induced by viruses, viroids, bacteria, fungi, and parasites. In this procedure, a cDNA library is considered from nucleic acids present in infected tissue from infected individuals. The library is created in a vector that allows expression of the polypeptide encoded in the cDNA. Clones of host cells containing a vector, which expresses an immunologically reactive polypeptide fragment of the etiological agent with selection by immunoassay of the expression products of the library with a body component containing antibody from another individual previously infected with the putative agent.

Steps in the immunoassay technique include interacting the vector-containing cDNA expression products with an antibody-containing body component of another infected individual and detecting the formation of an antibody-antigen complex between the expression products and the antibody of the other infected individual.

Isolated clones are further tested immunologically by interacting their expression products with antibody-containing body components of other individuals infected with the putative agent, and by detecting the formation of antigen-antibody complexes from infected individuals; and vectors containing cDNAs encoding polypeptides that react immunologically with antibodies from infected individuals and individuals suspected of being infected with the agent, but not from control individuals, are isolated. The infected individuals used to create the cDNA library and for the purpose of immunological testing are not of the same species.

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

II.A. Obtaining the cDNA sequence

Pooled sera from chimpanzees with chronic HCV infection and containing a high virus titer, eg at least 106 chimp. infectious doses/ml (CID/ml) were used to isolate virus particles; nucleic acids isolated from these particles are used as templates in the creation of a cDNA library of the viral genome. Procedures for isolating putative HCV particles and for generating a cDNA library in lambda-gt11 are discussed in section IA.A.1. Lambda-gt11 is a vector that was specifically developed for the expression of insert cDNAs as united polypeptides with betagalactosidase and for testing a large number of recombinant phages with a specific antiserum against a defined antigen. A lambda-gt11 cDNA library generated from a cDNA source containing cDNA with an average size of about 200 base pairs was tested for encoded epitopes that should specifically bind to serum derived from patients previously suffering from NANB hepatitis. Huynh T.V. et al (1985).

About 106 phages were tested and five positive phages were identified, purified and tested for binding specificity to serum from early humans and chimpanzees previously infected with the HCV agent. One of the phages, 5-1-1 binds 5 to 8 human sera tested. This binding occurs specifically for serum derived from patients prior to NANB hepatitis infection, with 7 normal blood serum donors showing no such binding.

The cDNA sequence in recombinant phage 5-1-1 was determined and shown in Figure 1. The polypeptide encoded by this cloned cDNA, which is in the same translation frame as the N-terminal beta-galactosidase, is part of the united polypeptide in the forward shown nucleotide sequence. This translational ORF therefore encodes an epitope(s) specifically recognized by serum from patients with NANB hepatitis infection.

The availability of cDNA in recombinant phage 5-1- allows the isolation of other clones containing additional segments and/or alternative segments of cDNA in the viral genome. The lambda-gt11 cDNA library described above was screened using a synthetic polynucleotide derived from the cloned 5-1-1 cDNA sequence. This testing yielded three other clones, which were identified as 81, 1-2 and 91; The cDNA contained in all clones was sequenced. See chapters IV.A.3 and IV.A.4. homology between four independent clones is shown in Figure 2, where homologies are indicated by vertical lines. Nucleotide sequences present uniquely in clones 5-1-1, 81, and 91 are indicated in lowercase letters.

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

Another difference between the clones is that clone 5-1-1 contains 28 base pairs at its 5'-terminus that are not present in the other clones. The extra sequence may be a 5'-terminal cloning artifact; 5'-terminal cloning artifacts are generally considered in cDNA method products.

Synthetic sequences derived from the 5'-region of HCV cDNA in clone 81 were used to test and isolate cDNA from a lambda-gt11 NANBV cDNA library overlapping clone 81 cDNA (Chapter IV.A.5). the sequences of the obtained cDNAs, which are in clone 36 and clone 32, are shown in Figure 5 and Figure 7, respectively.

Similarly, a synthetic polynucleotide based on the 5'-region of 36 was used to test and isolate cDNA from a lambda-gt11 N AN BV cDNA library overlapping clone 36 cDNA (Chapter IV.A.8). the purified recombinant phage clone containing the cDNA that hybridized to the synthetic polynucleotide probe was called clone 35 and the NANBV cDNA sequence contained in this clone is shown in Figure 8.

Using overlapping cDNA sequence isolation techniques, clones containing additional downstream and upstream HCV cDNA sequences were obtained. Isolation of these clones is described below in Section IV.A.

Analysis of the nucleotide sequences of the HCV cDNA encoded in the isolated clones shows that the composition of the cDNA is one long continuous ORF. Figure 26 shows the sequence composition of the cDNA from these clones together with the cloned HCV polypeptide.

The description of procedures for recovering cDNA sequences is mainly of historical interest. The resulting sequences (and their complements) are provided herein and the sequences, or a portion thereof, should be added using synthetic sequence details using procedures similar to those described herein.

Lambda-gt11 replicates from the HCV cDNA library and from clones 5-1-1, 81, 1-2 and 91 have been deposited under the Budapest Treaty names with the Anerican Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Maryland 20852 and designated are the following associated numbers.

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Upon approval and issuance of this application as a US patent, all restrictions on the availability of these deposits will be irrevocably removed; and inspection of the awarded deposit will be available during the processing of the aforementioned application with the designated attorney under 37 CFR 1.14 and 25 USC 1.22. However, deposits will be maintained for a period of 30 years from the date of deposit or five years after the last deposit request; or validity of the US patent for however long. These deposits are for convenience only, and are not required for the practice of this invention from the perspective of this description. The HCV cDNA sequences in the deposited material are incorporated herein by reference.

II.B. Obtaining viral polypeptides and fragments

The availability of cDNA sequences, either those isolated using the cDNA sequences of Figures 1-26, as discussed below, as well as the cDNA sequences of these Figures, permits the construction of expression vectors encoding the antigenically antigenic regions of the polypeptide encoded by one or the other strand. These antigenically active regions can be derived from coated or enveloped antigens or from core antigens, including, for example, polynucleotide binding proteins, polynucleotide polymerases, and other viral proteins required for replication and/or viral porticle formation. Fragments encoding the desired polypeptides are derived from cDNA clones using conventional restriction digestion or synthetic methods and ligated into vectors which may, for example, contain portions of fused sequences such as beta-galactosidase or superoxide dismutase (SOD), preferably SOD.

Methods and vectors useful for the production of polypeptides containing fused SOD sequences are described in European Patent Office Publication no. 0196056, published on October 1, 1986. Vectors encoding the fusion of the SAD polypeptide and the HCV polypeptide, eg, NANB5-1-1, NANB81 and CIOO-3, which is encoded by the HCV cDNA, are described in Sections IV.B.1, IV.B.2 and IV. B.4, respectively. Any desired portion of the HCV cDNA containing the open reading frame, in any region, can be obtained as a recombinant polypeptide, such as a mature or fusion protein; alternatively, the polypeptide encoded in the cDNA can be provided by chemical syntheses.

DNA encoding the desired polypeptide, whether in a spliced or mature form and whether or not it contains a sequence to permit secretion, can be ligated into expression vectors suitable for a common host. Both eukaryotic and prokaryotic host systems are now used in the production of recombinant polypeptides and some of the common control systems and host cell lines are given in Chapter III.A., below. The polypeptide is then isolated from lysed cells or from the culture medium and purified for extended use. Purification can be performed by techniques known in the art, for example, salt fractionation, chromatography on ion-exchange resins, affinity chromatography, centrifugation, and the like.

See, for example, Methods in Enzymology for various protein purification methods. Such polypeptides can be used as diagnostics or those that give rise in neutralizing antibodies can be formulated into a vaccine. Antibodies raised against these polypeptides can also be used as diagnostics, or for passive immunotherapy. Additionally, as discussed in Chapter II.J. below, antibodies to these polypeptides are useful for the isolation and identification of HCV particles.

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

II.C. Preparation of antigenic polypeptides and conjugation with a carrier

The antigenic region of a polypeptide is generally relatively small—typically 8 to 10 amino acids or less in length. Fragments of 5 amino acids can characterize the antigenic region. These segments may correspond to regions of the HCV antigen. Therefore, using cDNA from HCV as a base, DNA encoding short segments of HCV polypeptides can be expressed recombinantly either as fused proteins or as isolated polypeptides. Additionally, short amino acid sequences can usually be obtained by chemical synthesis. In cases where the synthesized polypeptide is correctly configured to provide the correct epitope, but is too small to be immunogenic, the polypeptide can be attached to a suitable carrier.

Techniques for such linkage are known in the art, including the formation of disulfide bonds using N-succinidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carbosylate (SMCC) derived from Pierce Company, Rockford, Illinois (if the peptide does not have a sulfhydryl group, this can be provided by adding a cysteine residue). These reagents form a disulfide bond between themselves and peptide cysteine residues on one protein and an amide bond through an epsilon-amino on a lysine or other amino group in another. Variants of such disulfide/amide forming agents are known.

See, for example, Immun. Rev. (1982) 62:185. Other bifunctional binding agents form a thioether bond rather than a disulfide bond. Many of these are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimdomethyl)cyclohexane-1-carboxylic acid, and the like. Carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The list given above is not exclusive and modifications of the mentioned compounds can clearly be used.

Any carrier that does not induce the production of antibodies harmful to the host can be used. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins; polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose granules and the like; polymeric amino acids such as polyglutamic acid, polylysine and the like; copolymer amino acids; and inactive virus particles, see, for example, Chapter II.D. Particularly applicable protein substrates are serum albumins, keyhol limper hemocyanin, molecules of immunoglobulin, thyroglobulin, ovalbumin, tetanus toxoid and other proteins well known to scientists.

II.D. Obtaining hybriden particles of immunogen containing HCV epitopes

The immunogeneticity of an HCV epitope can also be enhanced by obtaining it in mammalian or yeast systems fused or fused to particle-forming proteins such as, for example, those associated with hepatitis B surface antigen. Constructs where the NANBV epitope binds directly to the particle-building protein encoding sequence produce hybrids that are immunogenic with respect to the HCV epitope. Additionally, all of the resulting vectors include HBV-specific epitopes that have varying degrees of immunogenicity, such as, for example, the pre-S peptide. Thus, particles constructed from particles that build a protein that includes the HCV sequence are immunogenic with respect to HCV and HBV.

Hepatitis surface antigen (HBSAg) has been shown to be generated and incorporated into particles in S. cerevisiae (Valenzuela et al (1982)) as well as, for example, in mammalian cells (Valenzuela et al (1982)). The concentration of such particles has been shown to enhance the immunogenicity of the monomeric subunit. The constructs can also include the immunodominant epitope of HBSAg, which comprises the 55 amino acid pre-surface (pre-S) region, Neurath et al (1984). Constructions of the pre-S-HBSAg particle expressible in yeast are described in EPO 174.44 published on 19 March 1986; hybrids incorporating heterologous viral sequences for yeast expression are described in EPO 175,261, published March 26, 1966. Both applications are marked as indicated and are incorporated herein by reference. These constructs can also be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells using the SV40-dihydrofolate reductase vector (Michelle et al (1984)).

Additionally, part of the particle-building protein encoding the sequence can be replaced with codons encoding the HCV epitope. In this replacement, regions not required to mediate unit aggregation in the formation of immunogenic particles in yeast or mammals can be deleted, thus eliminating additional HBV antigenic sites from competition with the HCV epitope.

II.E Preparing the vaccine

Vaccines can be obtained from one or more immunogenic polypeptides derived from HCV cDNA as well as from the cDNA sequences in Figures 1-22 or the HCV genome to which they correspond. The observed homology between HCV and flaviviruses provides information regarding the polypeptides most effective as vaccines as well as the regions of the genome in which they are encoded. The general structure of the flavivirus genome is discussed in Rice et al (1986). Flavirus genomic RNA is believed to be only virus-specific mRNA species, and translated into three viral structural proteins, eg C, M and E as well as two large nonstructural proteins, NV4 and NV5, and a complex set of smaller nonstructural proteins.

The major neutralizing epitopes for flaviviruses are known to lie in the E (envelope) protein (Roehrig (1986)). The corresponding HCV E gene and polypeptide coding region can be predicted, based on homology to flaviviruses. Thus, vaccines may comprise recombinant polypeptides containing epitopes of HCV E. These polypeptides may be expressed in bacteria, yeast or mammalian cells or alternatively may be isolated from viral preparations. It has also been noted that other structural proteins may also contain epitopes that confer a rise in protective anti-HCV antibodies. Thus, polypeptides containing epitopes E, C and M can also be used, either alone or in combination, in HCV vaccines.

In addition to the above, immunization with NS1 (nonstructural protein 1) has been shown to result in protection against yellow fever (Schesinger et al (1986)). This is true even when immunization does not produce a rise in neutralizing antibodies. Thus, especially when this protein appears to be highly conserved among flaviviruses, HCV NS1 will also be protected against HCV infection. Even more, it has also been shown that non-structural proteins can provide protection against viral pathogens, even if they do not induce the production of neutralizing antibodies.

In view of the above, multivalent HCV vaccines may comprise one or more non-structural proteins. These vaccines may comprise, for example, recombinant HCV polypeptides and/or polypeptides isolated from virions. Additionally, the use of inactive HCV in vaccines is possible; these can be obtained using viral lysate preparations or other means known in the art to condition flavivirus inactivity, for example, treatment with organic solvents or detergents, or treatment with formalin.

Even more, vaccines can also be obtained from diluted HCV stocks. Preparations of diluted HCV volumes are described below.

Some of the proteins in flaviviruses are known to contain highly conserved regions, so some immunological cross-reactivity is expected between HCV and other flaviviruses. It is possible that epitopes shared between flaviviruses and HCV will confer an increase in protective antibodies against one or more disorders induced by these pathogenic agents. Thus, it may be possible to create multi-purpose vaccines, based on this knowledge. The addition of vaccines containing immunogenic polypeptide(s) as an active substance is known to those skilled in the art. Typically, such vaccines are prepared as liquid solutions or suspensions for injection; solid forms suitable for dissolution or suspension or liquid for injection can also be prepared. Preparations can also be emulsifying, or protein encapsulated in liposomes. Active immunogenic substances are often mixed with additives that are pharmaceutically acceptable and compatible with the active substance. Suitable additives are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.

Additionally, if desired, the vaccine may contain small amounts of adjuvants such as wetting and emulsifying agents, buffers and/or additives that enhance the effectiveness of the vaccine. Examples of supplements that may be effective for this include, but are not limited to: aluminyl hydroxide, N-acetyl-murmyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-murtyl-L-alamyl- D-isoglutamine (CGP 11637, listed as nor-MDP), N-acetylmurmyl-L-alanyl-D-isoglutaminyl-L-alamine-2-(1'-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, designated as MTP-PE) and RIBI containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of adjuncts can be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing the HCV antigenic sequence obtained by introducing this polypeptide into vaccines that also included various adjuncts.

Vaccines are commonly administered parenterally, by injection, for example, either subcutaneously or intramuscularly, additional formulations suitable for other routes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may be included, for example polyalkaline glycols and triglycerides; such suppositories can be made from mixtures containing the active substance in the range of 0.5 to 10%, preferably 1-2%. Oral formulations include such commonly used additives as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These preparations can be in the form of solutions, suspensions, tablets, pills, capsules, support the release of formulations or powders and contain 10-95% of the active substance, preferably 25-70%.

Proteins can be formulated in the vaccine as neutral or in salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of peptides) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acid or with organic acids such as acetic, oxalic, tartaric, malic and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron (III) hydroxides and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

II.F. Dose and administration of the vaccine

Vaccines are administered in a manner compatible with the dose of the formulation, and in such an amount that will be prophylactically and/or therapeutically effective. The amount that is introduced, which is generally in the range of 5 µg to 250 µg of antigen per dose, depending on the subject being treated, the capacity of his immune system according to the synthesis of antibodies and the degree of desired protection. The precise amount of the active substance that is required for intake may depend on the assessment of the one applying it and may be adjusted for each subject.

The vaccine can be given in a single dose or preferably in multiple divided doses. In a multi-dose schedule, the primary course of vaccination may be 1-10 separate doses, followed by other doses given at time intervals necessary to maintain and/or strengthen the immune response, for example, at 1-4 months for the second dose and if necessary by further doses after several months. The method of dosing will also, at least partially, be determined by the needs of the individual and depends on the assessment of the one doing the dosing.

Additionally, a vaccine containing an immunogenic HCV antigen can be administered with other immunoregulatory agents, for example, immunoglobulins.

II.G. Obtaining antibodies against HCV epitopes

The immunogenic polypeptides obtained as described above are used for the production of polyclonal and monoclonal antibodies. If polyclonal antibodies are desired, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing the HCV epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If the serum containing polyclonal antibodies to the HCV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for the production and processing of polyclonal antiserum are known in the art, see for example Nayer and Walker (1987).

Alternatively, polyclonal antibodies can be isolated from a mammal previously infected with HCV. An example of a procedure for the purification of antibodies to HCV epitopes from the serum of infected individuals, based on affinity chromatography and using the combined polypeptide of SOD and the polypeptide encoded in cDNA clone 5-1-1 is given in chapter V.E. Monoclonal antibodies directed against HCV epitopes can also be readily produced by those skilled in the art. The general methodology for obtaining monoclonal antibodies using hybridomas is well known. Immortal antibody-producing cell lines can be generated by cell fusion and also by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, eg, M. Schreier et al. (1980); Maherling et al. (1091); Kenneth et al. (1980); see also US Patent No. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,446,917; 4,472,500; 4,491,632 and 4,493,890. Panels of monoclonal antibodies produced against HCV epitopes can be tested for different properties; eg for isotype, epitope, affinity etc.

Antibodies, monoclonal and polyclonal, that are directed against HCV epitopes are particularly useful in diagnostics, and that are neutralizing are useful in passive immunotherapy. Monoclonal antibodies, specifically, can be used to raise anti-idiotype antibodies.

Anti-idiotype antibodies are immunoglobulins that carry the "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, Nisonoff A. et al. (1981) and Dressman 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 can also be used to treat NANBH as well as to elucidate the immunogenic regions of the HCV antigen.

II.H. Diagnostic oligonucleotide probes and equipment

Using the described portions of isolated HCV cDNA as bases, including those in Figures 1-32, oligomers of about 8 nucleotides or more can be obtained, either by excision or synthetically, that hydrate with the HCV genome and are useful in identifying the viral agent, further characterizing the viral genome as well as virus detection of sick individuals. Probes with HCV polynucleotides (natural and derived) are of a length that allows detection of unique sequences by hydridization. Although 6-8 nucleotides can be a working length, sequences of 10-12 nucleotides are preferred and around 20 nucleotides appears to be the optimum. Preferably, these sequences will be derived from regions that have no heterogeneity. These probes can be obtained using routine procedures, including automated oligonucleotide synthetic procedures. Useful probes include, for example, clone 5-1-1 and additional clones described herein, as well as various oligomers useful in screening cDNA libraries, see below. A complement to some unique part of the HCV genome will be satisfactory. For use as a probe, complete complementarity is preferred, although it may be unnecessary if fragment length is increased.

For the use of such samples as diagnostics, biological samples such as blood or serum are analyzed, treated, if desired, in order to extract the nucleic acids they contain. The resulting nucleic acid from the sample can be subjected to gel electrophoresis or other separation techniques; alternatively, the nucleic acid sample can be spot-absorbed without size separation. The trials are then marked. Suitable labels and methods for labeling probes are known in the art and include, for example, radiolabels by incorporation by translational cleavage or kinases, biotin, fluorescent probes and chemiluminescent probes. Nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable binding.

Tests can be made completely complementary to the HCV genome. Therefore, usually high bindings are desirable in order to prevent false positives. However, high binding conditions should only be used if the probes are complementary to regions of the viral genome that lack heterogeneity. The binding of hybridization is determined by a number of factors during hybridization and during the washing process, including temperature, ionic strength, length of time, and concentration of formamide. These factors are highlighted, for example, in Maniatis T. (1982).

In general, HCV genomic sequences are expected to be present in the serum of infected individuals at relatively low levels, eg at about 102-103 sequences per ml. This level may require amplification techniques to be used in hydridization tests. Such techniques are known in the art, for example, the Enzo Biochemical Corporation "Bio-Bridge" system uses terminal deoxynucleotide transferase to add unmodified 3'-poly-dT-ends to a DNA probe. The poly dT-terminated probe is hydrogenated from the target nucleotide sequence and then to the biotin-modified poly-A. PCT application 84/03520 and EPA 124221 describe a DNA hybridization assay in which: (1) the analyte is cleaved to single-stranded DNA that is complementary to an enzyme-labeled oligonucleotide; and (2) the resulting terminal duplex is hydrogenated to an enzyme-labeled oligonucleotide. EPA 204510 describes a DNA hybridization assay in which a DNA analyte is contacted with a probe having a terminus, such as a poly-dT terminus, an amplified region having a sequence that hydrolyzes the ends of the probe, such as a poly-A sequence, and capable of binding multiple labeled regions.

A particularly preferred technique may first comprise amplifying the target HCV sequence in serum about 1000-fold, eg about 10 6 sequences/ml. This can be done, for example, by the technique of Saiki et al (1986). The amplified sequence can then be detected using a hybridization assay as described in copending US application Ser. No. 2300-0171 filed Oct. 15, 1987, which is incorporated herein by reference. This hybridization assay, which should detect sequences at the 106/ml level, uses nucleic acid multimers that bind to single nucleic acid analytes and which also bind to amplify single labeled oligonucleotides. Convenient testing of solution phase sandwiches and procedures for obtaining samples in EPO 225,807 published 6/16/1987, which is incorporated herein by reference.

Samples can be packaged in diagnostic equipment. Diagnostic equipment includes a DNA test that can be labeled; alternatively, the DNA sample may be unlabeled and labeling components may be included in the kit. The kit may also contain other conveniently packaged reagents and materials necessary for the hybridization assay, for example standards, as well as text-based instructions.

II.I. Immunoassays and diagnostic devices

Both polypeptides that react immunologically with serum containing HCV antibodies, e.g., those derived from or encoded in the clones described in Chapter IV.A., and compositions thereof (see Chapter IV.A.) and antibodies raised against HCV-specific epitopes in these polypeptides, see for example chapter IV.E., are useful in immunoassays to detect the presence of HCV antibodies or the presence of virus and/or viral antigens in biological samples, including for example, blood or serum samples. The immunoassay plan is the subject of a large number of variants, many of which are known in science. For example, immunoassays can use a single viral antigen, for example, a polypeptide derived from one of the clones containing HCV cDNA, described in Section IV.A. or from the cDNA composition derived from the cDNA in these clones, or from the HCV genome from which the cDNA in these clones is derived; alternatively, the immunoassay may use a combination of viral antigens derived from these sources.

For example, a monoclonal antibody directed against a single viral antigen, monoclonal antibodies directed against various viral antigens, polyclonal antibodies directed against a single viral antigen, or polyclonal antibodies directed against various viral antigens can be used. Assay flows can be based, for example, on competition or direct reaction or sandwich-type assays. For example, solid supports can also be used or it can be done by immunoprecipitation.

Most tests involve the use of a labeled antibody or polypeptide; labels can be, for example, fluorescent, chemiluminescent, radioactive or colored molecules. Tests that amplify signals from trials are also known; examples are assays using biotin and avidin and enzyme-labeled and mediated immunoassays, such as ELISA assays.

The flavivirus model for HCV allows prediction of the likely location of diagnostic epitopes for virion structural proteins. C, pre-M, M and E domains are all, probably due to containing epitopes, significant potentials for detecting viral antigens, and especially for diagnosis. Similarly, domains of nonstructural proteins are expected to contain important diagnostic epitopes (eg NS5 encoding a putative polymerase; and NS1 encoding a putative complement-binding antigen). Recombinant polypeptides, or viral polypeptides, that include epitopes from these specific domains may be useful for detecting viral antibodies in infectious blood donors and infected patients.

Additional antibodies directed against E and/or M proteins can be used in immunoassays to detect viral antigens in patients with HCV induced by NANBH, and in infectious blood donors. However, these antibodies will be extremely useful in donor detection and in the acute phase of the patient.

Devices suitable for immunodiagnosis and containing appropriately labeled reagents are constructed by packaging appropriate materials, including polypeptides of the invention containing HCV epitopes or antibodies directed against HCV epitopes, in appropriate containers together with other reagents and materials necessary to perform the test, as well as an appropriate set of instructions for performing the test.

II.J. Further characterization of the HCV genome, virions and viral antigens using cDNA-derived probes to the viral genome

The HCV cDNA sequence information in the clones described in Chapter IV.A., as shown in Figures 1-32, can be used to obtain further information on the HCV genome sequence, and to identify and isolate the HCV agent and thus aid in its 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 may lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and antibodies directed against HCV epitopes that should be useful in the diagnosis and/or treatment of HCV-induced NANBH.

The cDNA information in the aforementioned clones is useful for creating probes for isolating additional cDNA sequences derived from as yet undefined regions of the HCV genome whose cDNAs in the clones described in Chapter IV.A., were derived. For example, labeled probes containing a sequence of about 8 or more nucleotides, and preferably 20 or more nucleotides, derived from regions near the 5'-end or 3'-end of the family of HCV cDNA sequences shown in Figures 1, 3, 6, 9, 14 and 32, can be used to isolate overlapping cDNA sequences from HCV cDNA libraries. These sequences that overlap the cDNAs in the clones described above, but which also contain sequences derived from regions of the genome from which the cDNAs in the clones mentioned above are not derived, can then be used to synthesize probes to identify other overlapping fragments that do not need to overlap in the cDNA clones described in chapter IV.A.

Although the HCV genome is segmented and the segments do not contain common sequences, it is possible to sequence the entire viral genome using the technique of isolating overlapping cDNAs derived from the viral genome. If, however, the genome is segmented by a genome lacking common sequences, the genome sequence can be determined by serological testing of lambda-gt11 HCV cDNA libraries, as used to isolate 5-1-1. The sequencing of cDNA isolates and the use of isolated cDNAs for the isolation and sequencing of clones is described in Chapter IV.A. Alternatively, characterization of genomic segments should be from viral genomes isolated from purified HCV particles. Procedures for purifying HCV particles and for detecting them during the purification process are described below. Methods for isolating polynucleotide genomes from viral particles are known in the art and one method that can be used is shown in Example IV.A.1. The isolated genomic segments should then be cloned and sequenced. Thus, with the information provided here, it is possible to clone and sequence the entire HCV genome given its nature.

Procedures for generating cDNA libraries are known in the art and are discussed above and below; the procedure for generating the HCV cDNA library in labda-gt11 is discussed below in section IV.A. However, cDNA libraries useful for testing with nucleic acid probes can occur in other vectors known in the art, for example, lambda-gt10 (Huynh et al. (1985)). HCV-derived cDNA detected using probes derived from the cDNA in Figures 1-32, and from probes synthesized from polynucleotides derived from these cDNAs can be isolated from the clone by destroying the isolated polynucleotide with the appropriate restriction enzyme (enzyme) and sequenced.

See, for example, chapter IV.A.3. and IV.A.4. for the techniques used to isolate and sequence the HCV cDNA overlapping the HCV cDNA in clone 5-1-1, Chapters IV.A.5.-IV.A.7. for isolation and sequencing of HCV cDNA overlapping that of clone 81, and chapter IV.A.8. and IV.A.9. to isolate and sequence a clone overlapping another clone (clone 36) overlapping clone 81.

The sequence information derived from these overlapping HCV cDNAs is useful for determining regions of homology and heterogeneity in the viral genome(s), which should indicate the presence of different genome regions and/or particle-damaged populations. It is also useful for creating hydridization assays to detect HCV or HCV antigen or HCV nucleic acids in biological samples, and during HCV isolation (discussed below), techniques used are described in Section II.G. However, the overlapping cDNAs can be used to create expression vectors for polypeptides derived from the HCV genome which also encodes the polypeptides encoded in clones 5-1-, 36, 81, 91 and 1-2 and other clones described in section IV.A. Techniques for generating these HCV epitope-containing polypeptides and for their use are analogous to those described for polypeptides derived from the NANBV cDNA sequences contained in clones 5-1-1, 32, 35, 36, 1-2, 81, and 91, discussed above and lower.

Coded in the family of cDNA sequences contained in clones 5-1-1, 32, 35, 36, 81, 91, 1-2 and other clones described in chapter IV.A. are antigen(s) containing epitopes that appear to be unique to HCV; eg, antibodies directed against these antigens are absent from individuals infected with HAV or HBV and from individuals not infected with HCV (see serological data in Chapter IV.B.). However, comparison of the sequence information of these cDNAs with the sequences of HAV, HBV, HDV and with genomic sequences in Genebank shows that minimal homology exists between these cDNAs and the polynucleotide sequences of these sources. Thus, antibodies directed against antigens encoded in the cDNA of these clones can be used to identify BB-NANBV particles isolated from infected individuals. Additionally, they are useful for isolating NANBH agent(s).

HCV particles can be isolated from the serum of BB-NANBV infected individuals or from cell culture following techniques known in the art, including for example, size-indiscriminate techniques such as sedimentation or exclusion methods or density-based techniques such as density gradient ultracentrifugation or sedimentation with agents such as polyethylene glycol or chromatography on various materials such as anion or cation exchangers and hydrophobicity binding materials as well as affinity columns. During the isolation procedure, the presence of HCV can be detected by hybridization analysis of the extracted genome using probes derived from HCV cDNA described below or by immunoassay (see chapter II.1.) using as a probe antibodies directed against the HCV antigen encoded in the family of cDNA sequences shown in Figures 1-32 and also directed against the HCV antigen encoded by the overlapping HCV cDNA sequences discussed above. Antibodies can be monoclonal or polyclonal and purification may be desirable when using them in immunoassays. The purification procedure for polynucleon antibodies directed against the antigen encoded in clone 5-1-1 is described in Chapter IV.E.; an analogous purification procedure can be used for antibodies directed against other HCV antigens.

Antibodies directed against HCV antigens encoded in the family of cDNAs shown in Figures 1-32, as well as those encoded in overlapping HCV cDNAs, which are fixed to solid supports, are useful for isolating HCV by immunoaffinity chromatography. Techniques for this are known in the art and include techniques for fixing antibodies to solid supports so that they retain their immunoselective activity; techniques can be those in which the antibodies are adsorbed to a support (see, for example, Kurstak in ENZYME IMMUNODIAGNOSIS, pp. 31-37), as well as those in which the antibodies are covalently attached to the support. In general, the techniques are similar to those used for covalent attachment of antigen to a solid support, which are generally described in Chapter IV.C.; however, spacers may be included in bifunctional binding agents so that the antigen binding site of the antibody remains accessible.

During the purification process, the presence of HCV can be detected and/or verified by nucleic acid hybridization, using as a probe polynucleotide derived from the family of HCV cDNA sequences shown in Figures 1-32, as well as from overlapping HCV cDMA sequences, described above. In this case, the fractions are treated under conditions that should cause the rupture of viral particles, for example, with detergents in the presence of chelating agents and the presence of viral nucleic acid determined by the hybridization techniques described in chapter II.H. Further confirmation that the isolated particles are the causative agents of HCV can be obtained by infecting chimpanzees with the isolated viral particles and then determining whether NANBH symptoms are present as a result of the infection.

Viral particles of purified preparations can be further characterized. The genomic nucleic acid was purified. Based on its sensitivity to Rnase and not DNase I, the virus appears to be composed of an RNA genome. See example IV.C.2., below. Helplessness and circularity or non-circularity can be determined by techniques known in the art, including, for example, their observation by electron microscopy, their migration in density gradients, and their sedimentation characteristics. Based on the hybridization of the obtained HCV genome to the negative strands of HCV cDNA, it appears that HCV can encompass the positive strand of the RNA genome (see chapter IV.H.1.). Techniques such as these are described, for example, in METHODS IN ENZYMOLOGY. Additionally, the purified nucleic acid can be cloned and sequenced by known techniques, including backup transcription, even though the genomic material is RNA. See, for example, Maniatis (1982), and Glover (1985). The use of nucleic acid derived from viral particles allows the sequencing of the entire genome, whether it is segmented or not.

Homology testing of the polypeptide encoded in the contiguous ORF of the pooled clones 14i to 39c (see Figure 26) shows that the HCV polypeptide contains regions of homology to corresponding proteins in flavivirus conserved regions. An example of this is described in chapter IV.H.3. This conclusion has many significant ramifications. First, this indication, in conjunction with the results showing that HCV contains a positive-fold genome of about 10,000 nucleotides in size, is consistent with the suggestion that HCV is a flavivirus or a virus similar to it. In general, flavivirus virions and their genomes have a relatively consistent structure and organization that is known. See Rice et at. (1982), and Brinton MA. (1988). Thus, structural genes encoding polypeptides C, pre M, M and E can be located in the 5'-terminus of genes upstream of clone 14i. However, using comparisons with other flaviviruses, the statement as preciting the location of the sequences encoding these proteins can be given.

Isolation of sequences upstream of these in clone 14i can be accomplished in a number of ways, provided herein for information purposes, which should be apparent to those skilled in the art. For example, a genome “walking” technique can be used to isolate other sequences that are 5' to that of clone 14i but that overlap that clone; this leads to the isolation of additional sequences. This technique is demonstrated below in Chapter IV.A. for example, flaviviruses are also known to have conserved epitopes and regions of conserved nucleic acid sequences. Polynucleotides containing conserved sequences should be used as probes that bind to the HCV genome, allowing their isolation. Additionally, these conserved sequences relative to that derived from the HCV cDNA shown in Figure 22 can be used to create primers for use in systems that amplify genomic sequences upstream of those in clone 14i, using polymerase chain reaction technology. An example of this is described below.

The structure of HCV can also be determined and its components isolated. Morphology and sizes can be determined, for example, using an electron microscope. The identification and location of specific viral polypeptide antigens, such as coat or envelope antigens, or internal antigens, such as linked amino acid proteins, core antigens, and polynucleotide polymerases can also be determined by, for example, determining whether the antigens are present as major or minor viral antigens. components, as well as by using antibodies directed against specific antigens encoded in isolated cDNAs as probes. This information is useful in vaccine development; for example, it may be desirable to include an external antigen in a vaccine preparation. Multivalent vaccines may comprise, for example, a polypeptide derived from a genome encoding a structural protein, for example E, as well as a polypeptide from another part of the genome, for example, a non-structural or structural polypeptide.

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

The suggestion that HCV is a flavivirus or a virus similar to it also provides information on the mechanisms by which HCV grows. The term "similar" means that the virus shows a significant degree of homology to known conserved regions and that the bulk of the genome is a single ORF. Procedures for culturing flaviviruses are known to those skilled in the art (see, for example, reviews by Brinston (1986) and Stollar (1980)). In general, suitable cells or cell lines for culturing HCV may include those known to support flavivirus replication, for example: monkey kidney cell lines (eg, MK2, VERO), porcine kidney cell lines (eg, PS); baby hamster kidney cell lines (eg, BHK), murine macrophage cell lines (eg, p388D1, MK1, Mm1); human macrophage cell lines (eg, U-937); human peripheral blood leukocytes; human adherent monocytes; hepatocytes or hepatocyte cell lines (eg HUH7, HEPG2); embio or embryonic cells (e.g. chicken embryo fibroblasts), or cell lines derived from invertebrates, preferably from insects (e.g. Drosophila cell lines), or more preferably from arthropods, for example, mosquito cell lines (e.g. A. Albopictus, Aedes aegypti , Cutex tritaeniorhynchus) or tick cell lines (eg RML-14 Dermacentor parumpertus).

It is possible for primary hepatocytes to be cultured and then infected with HCV; or alternatively, hepatocyte cultures can be derived from the liver of infected individuals (eg, human or chimpanzee). The last case is an example of a cell that is infected in vivo and then translated in vitro. Additionally, various immortalization procedures can be used to obtain cell lines derived from hepatocyte cultures. For example, primary liver cultures (before and after enrichment of the hepatocyte population) can be pooled into various cells to maintain stability. For example, also, cultures can be infected with transformation viruses or transformed with transforming agents in order to generate permanent or semi-permanent cell lines. Additionally, for example, cells in liver culture can be pooled to obtain cell lines (eg, HePG2). Methods for cell fusion are known in the art and include, for example, the use of fusion agents such as polyethylene glycol, Sendai virus, and Epstein-barr virus.

As discussed above, HCV is a flavivirus or a virus similar to it. Therefore, it is likely that HCV infection of cell lines can be performed by techniques known in the art for infecting cells with viral preparations under conditions that permit viral entry into the cell. Additionally, it is possible to obtain viral production by transfecting cells with isolated viral polynucleotides. Togavirus and Flavivirus RNA are known to be infectious in various vertebrate cell lines (Feerkorn and Shapiro 91974)), and in a mosquito cell line (Peleg (1969)). Methods for transfecting cell culture tissue with RNA duplexes, positive-strand RNAs and DNA (including cDNA) are known in the art and include, for example, techniques using electroporation and precipitation with DEAE-Dextran or calcium phosphate. A rich source of HCV RNA can be obtained by performing in vitro transcription of HCV cDNA corresponding to the complete genome. Transfection with this material or with cloned HCV cDNA should result in viral replication and in vitro viral progression.

In addition to cultured cells, an animal model system can be used for viral replication; animal systems in which flaviviruses are known are known to those skilled in the art (see, for example, review by Monath (1986)). Thus, HCV replication can take place not only in chimpanzees, but also in, for example, marmosets or infant mice.

II.L. Testing for antiviral agents for HCV

The availability of cell culture and animal model systems for HCV also allows testing for antiviral agents that inhibit HCV replication, and particularly for those agents that primarily allow growth and replication while inhibiting viral replication. These test methods are known to those skilled in the art. In general, antiviral agents have been tested at various concentrations for their effect on preventing viral replication in cell culture systems that support viral replication, and then on inhibiting infectivity or viral pathogenicity (and low toxicity) in animal model systems.

The methods and compositions provided herein for detecting antigens and HCV polynucleotides are useful for testing antiviral agents where they provide an alternative and perhaps more sensitive means of detecting the agent's effect on viral replication than cell plaque assays or ID50 assays, for example, the HCV polynucleotide probes described herein can can be used to determine the amount of nucleic acid produced in cell culture. This can be done, for example, by hybridizing infected cell nucleic acids with a labeled HCV-polynucleotide probe. For example, anti-HCV antibodies can also be used to identify and quantify HCV antigen in a cell culture using the immunoassays described herein. Additionally, where it may be desirable to determine the amount of antigen in an infected cell using a competitive assay, the polypeptides encoded by the HCV cDNA described herein are useful in these competitive assays. In general, recombinant HCV polypeptide from HCV cDNA should be labeled and inhibition of binding of this labeled polypeptide to HCV by antigen produced in an allergic culture system should be noted. However, these techniques are practically useful in cases where HCV may be able to replicate in a cell line without causing cell death.

II.M. Getting weakened waists HCV

In addition to the above, the use of tissue culture systems and/or animal model systems may be possible to isolate attenuated HCV strains. These professions should be suitable for vaccines or for the isolation of viral antigens. Attenuated segments are isolable after multiple passages in a cell culture and/or animal model.

Detection of weakened structures in an infected cell or individual is achieved by techniques known in the art and can include, for example, using antibodies against one or more epitopes encoded in HCV as a probe or using a polynucleotide containing the HCV sequence of at least about 8 nucleotides as a probe .

Alternatively, or additionally, an attenuated strain can be generated using the HCV genomic information provided herein and using recombinant techniques. In general, deletion of a region of the genome encoding, for example, a polypeptide related to pathogenicity, or allowing viral replication, should be attempted.

The additional generation of the genome should allow the expression of an epitope that gives an increase in neutralizing antibodies for HCV. The modified gene should then be used to transform cells that allow HCV replication, and grow the cells under conditions that allow viral replication. Attenuated HCV strains are useful not only for vaccine purposes but also as a source for commercial production of viral antigens, hence the processing of these viruses should require less stringent protective measures for those employed in virus production.

III. General procedures

The general techniques used in extracting genomes from viruses, obtaining and testing cDNA libraries, sequencing clones, constructing expression factors, transforming cells, performing immunoassays such as immunoassays and ELISA assays for cell growth in culture and the like are known in science and laboratory manuals. are available to describe these techniques. However, as a general guide, the following is a set of resources constantly available for such procedures and for materials applicable to their execution.

III.A. Hosts and expression control sequences

Prokaryotic and eukaryotic host cells can be used to express the desired coding sequences when appropriate control sequences are used that are compatible with the designated host. Among prokaryotic hosts, E. coli is the most commonly used. Expression control sequences for prokaryotes include promoters, which optionally contain operator moieties, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are generally derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and teracyclin resistance, and various pUC vectors also containing sequences conferring antibiotic resistance markers. These markers can be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al (1977)), the tryptophan (trp) promoter system (Goeddel et al. (1980)) and the lambda-derived PL promoter and N gene ribosome binding site (Shimatake et al. (1981)) and a hybrid tac promoter (De Boer et al. (1983)) derived from the trp and lac UV5 promoter sequences. The above systems are particularly compatible with E. coli; if desired, other prokaryotic hosts such as Bacillus or Pseudomonas species with appropriate control sequences can be used.

Eukaryotic hosts include yeast and mammalian cells in culture systems. Saccharomyced cerevisiae and Saccharomyces carlsbergenesisi are the most commonly used yeast hosts and correspond to fungal hosts. Yeast compatibilizer vectors carry markers that allow selection of successful transformants by conferring prototrophy to auxotrophic mutants or heavy metal resistance to wild-type strains. Yeast-compatible vectors may use a 2 micron origin of replication (Broach et al. (1983)), a combination of CEN3 and ARS1, or other means of ensuring replication, such as sequences that will result in the incorporation of the appropriate fragment into the host cell genome. Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al. (1968)); Holland et al. (1978) which include promoters for 3-phosphoglycerate kinase (Hitzeman (1980)). Terminators such as those derived from the enolase gene may also be included (Holland (1981)). Particularly useful control systems are those comprising a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or an alcohol dehydrogenase (ADH) regulatory promoter, terminators also derived from GAPDH, and if secretion is desired, a leader sequence from a yeast alpha vector. Additionally, the transcriptional regulatory region and the transcriptional initiator region are operably linked and may be such that they are not naturally linked in the wild-type organism. These systems are described in detail in EPO 120,551, published on October 3, 1984; EPO 116.201, published on August 22, 1984; and EPO 164,556; published on 18.12.1985. which are incorporated herein by reference under these designations.

Mammalian cell lines available as expression hosts are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATTC), including HeLa cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells, and numerous others cell lines. Suitable promoters for mammalian cells are also known in the art and include viral promoters such as those from Simian Virus 40 (SV40) (Fiers (1978)), Rous arcoma virus (RSV), adenovirus (ADV) and bovine papilloma virus (BPV). Mammalian leg cells also require terminator sequences and Poly A accessory sequences; enhancer sequences that increase expression may also be included, and sequences that cause gene amplification may also be desirable. These sequences are known in science. Vectors suitable for replication in mammalian cells may include viral replicons or sequences that ensure integration of appropriate sequences encoding NANBV epitopes into the host genome.

III.B. Transformations

Transformations can be those of known methods for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus and transducing the host cells with the virus, and by directly taking up the polynucleotide. The transformation procedure used depends on the host being transformed. For example, transformation of E. coli host sites with lambda-gt11 containing BB-NANBV sequences is discussed in the examples section, below. Bacterial transformation by direct uptake generally uses treatment with calcium or rubidium chloride (Cohen (1972); Maniatis (1982)). Transformation of yeast by direct uptake can be performed using the procedure of Hinnes et al (1978). Mammalian transformations by direct uptake can be performed using the calcium phosphate precipitation method of Graham and Van der Eb (1978) or various modifications thereof.

III.C. Creating a vector

Vector creation uses techniques known in the art. DNA coding of specific sites is performed by treatment with suitable restriction enzymes under conditions generally specified by the manufacturers of these commercially available enzymes. Generally, about 1 microgram of plasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 microliters of buffer solution by incubating for 1-2 hours at 37ºC. After incubation with restriction enzyme, protein is removed by phenol/chloroform extraction and DNA is separated by ethanol precipitation. Cleaved fragments can be separated using polyacrylamide or gel electrophoresis techniques, according to general procedures found in Methods in Enzymology (1980) 65; 499-560.

Broken sticky end fragments can be opened using E. coli DNA polymerase I (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S1 nucleose can also be used resulting in the hydrolysis of some parts of the single-stranded DNA.

Ligation is performed using the standard buffer and temperature conditions used by T4 DNA ligase and ATP; ligation of the sticky end requires less ATP and less ligase than ligation of the open end. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or bovine intestinal alkaline phosphatase to remove the 5'-phosphate and thus prevent religation of the vector; alternatively, restriction enzyme destruction of unwanted fragments can be used to prevent association.

The ligation mixture is transformed in a suitable cloning host, such as E. coli, and successful transformants are selected by, for example, antibiotic resistance and testing for correct construction.

III.D. Creating the desired DNA sequences

Genetic oligonucleotides can be obtained using an automated oligonucleotide synthesizer as described by Warner (1984). If desired, synthetic domains can be labeled with 32 P treatment with polynucleotide kinase in the presence of 32 P-ATP, using standard reaction conditions.

DNA sequences, including those isolated from cDNA libraries, 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 into phage as a single-stranded sequence, and translated into double-stranded DNA using DNA polymerase as a primer, a synthetic oligonucleotide complementary to the portion of the DNA being modified and having the desired modification incorporated into its sequence. The obtained DNA is transformed into a phage carried by the host bacterium. Cultures of transformed bacteria containing replicates of each type of phage are placed in agar to form a plaque. Theoretically, 50% of the new plaques contain phage that has the mutated sequence, and the remaining 50% have the original sequence. Plaque replicates are hybridized to label the synthetic probe at temperatures and conditions that allow hybridization with the correct region, but not with the unmodified sequence. Sequences identified by hybridization are isolated and cloned.

III.E. Hybridization with probe

DNA libraries can be screened using the procedure of Grunstein and Hogness (1975). Briefly, in this procedure, the DNA to be examined is immobilized on nitrocellulose filters, denatured, and prehybridized with a buffer containing 0-5% formamide, 0.75 M NaCl, 75 mM Na-citrate, 0.02% (w/v ) each of bovine serum albumin, polyvinylpyrrolidone, and Ficol, 50 mM Na-phosphate (pH 6.5), 0.1% SDS, and 100 micrograms/ml denatured DNA carrier. The process of formamide in the buffer, as well as the time and temperature conditions of the stages of prehybridization and then hybridization, depending on the required stringency. Oligomeric probes that require lower stringency conditions are generally used with a lower percentage of formamide, lower temperatures, and longer hybridization times. Probes containing more than 30 or 40 nucleotides such as derived from cDNA or genomic sequences generally use higher temperatures, eg about 40-42ºC and a higher percentage of formamide, eg 50%. After hybridization, the 5'32P-labeled oligonucleotide probe is added to the buffer and the filters are incubated in this mixture under hybridization conditions. After washing, the treated filters are subjected to autoradiography to show the location of the hybridization probe; The DNA in the appropriate locations on the original agar plates is used as the source of the desired DNA.

III.F. Construction verification and sequencing

For routine vector constructions, ligation mixtures are transformed into E.coli strain HB101 or another suitable host, and successful transformants are selected by antibiotic resistance or other markers. Plasmids from etad transformants are prepared according to the method of Clewell et al 91969, usually followed by chloramphenicol amplification (Celwell (1972)). DNA is isolated and analyzed, usually by restriction enzyme analysis and/or sequencing. Sequencing can be by the dideoxy method of Sangen et al (1977) as further described by Nessing et al (1981) or by the method of Maxam et al (1980). Banding problems sometimes observed in GC-rich regions are overcome by using T-deauguanosine according to Barr et al (1986).

III.G. Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) can be used to measure either antigen or antibody concentration. This method depends on enzyme conjugation, either to antigen or antibody, and uses binding enzyme activity as a quantitative marker. For antibody measurement, a known antigen is fixed on a solid phase (eg microplate or plastic cover), incubated with diluted test sera, washed, incubated with enzyme-labeled anti-immunoglobulin and washed again. Enzymes suitable for labeling are known in the art and include, for example, horseradish peroxidase. The bound enzyme activity towards the solid phase is measured by adding a specific substrate and determining proproduct formation or substrate utilization colorimetrically. The bound enzyme activity is a direct function of the amount of bound antibody.

For antigen measurement, a known specific antibody is fixed on a solid phase, the tested material containing the antigen is added, after incubation the solid phase is washed and another enzyme-labeled antibody is added. After the test, the substrate is added and the enzyme activity is assessed colorimetrically and related to the antigen concentration.

Examples

Examples of the present invention are described below which are provided for illustrative purposes only and do not limit the scope of the present invention. For purposes of this disclosure, numerous reactions in the scope of protection will be apparent to those skilled in the art. Procedures given, for example, in Chapter IV.A. may, if desired, be repeated but must, as techniques available for the construction of desired nucleotide sequences based on information provided by the invention. Expression was demonstrated in E. coli; however, other systems that are available are given in Chapter III.A. Additional epitopes derived from the genetic structure can also be produced, and used to generate antibodies as described below.

IV.A. Obtaining, isolating and sequencing HCV cDNA

IV.A.1. Obtaining HCV cDNA

The source of the NANB agent was a plasma pool derived from a chimpanzee with chronic NANBH. A chimpanzee was experimentally infected with the blood of another chimpanzee with chronic NANBH acquired from HCV infection in a contaminated batch of factor 8 concentrate derived from a human serum reserve, the chimpanzee plasma reserve was rewarded by combining many individual plasma samples containing high levels of alanine aminotransferase activity; this activity results from hepatic damage induced by HCV infection, therefore, 1 ml of 10-6 diluted reserve serum was given i.v. causing NANBH in another chimpanzee, his CID was at least 106/ml, ie he had a high infectious titer of the virus.

A cDNA library from a high titer plasma pool was generated as follows. First, viral particles are isolated from plasma; A 90 ml aliquot is diluted with 310 ml of a solution containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl. Debris is removed by centrifugation for 20 minutes at 15,000 rg at 20ºC. Viral particles in the resulting supernatant are then pelleted by centrifugation in a Beckman SW rotor at 28,000 rpm for 5 hours. To release the viral genome, the particles are disrupted by suspending the tablet in 15 ml of a solution containing 1% sodium duodecyl sulfate (SDS), 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, also containing mg/ml protectkinase k, and then incubation at 45ºC for 90 minutes. Nucleic acids are isolated by adding 0.8 µg MS2 bacteriophage RNA carrier, and extracting the mixture four times with 1:1 phenol:chloroform (phenol saturated with 0.5 M tris-HCl, pH 5.5, 0.1% (v /v) beta-mercaptoethanol, 0.1% (v/v) hydroxyquinoline and then extracted twice with chloroform. The aqueous phase was concentrated with 1-butanol before precipitation with 2.5 volumes of absolute ethanol overnight at -20ºC. The nucleic acid was isolated by centrifugation in a Beckman SW41 rotor at 40,000 rpm for 90 min at 4ºC, and dissolved in water treated with 0.05% (v/v) diethylpyro-carbonate and autoclaved.

Nucleic acid obtained by the above procedure (less than 2 µg) is denatured with 17.5 mM CH3HgOH; cDNA was synthesized using this denatured nucleic acid as a template, and cloned into the EcoRI site of phage lambda-gt11 using the methods described by Huynh (1985), excluding the significant primer replaced by oligo(dT) 12-18 during synthesis of the first cDNA strand by reverse transcriptase ( Taylor et al (1976)). The resulting double cDNA was fractionated according to size on a Sepharose CL-4B column; Eluted material around an average of 400, 300, 200, and 100 base pairs was added to cDNA pools 1, 2, 3, and 4, respectively. A lambda-gt11 cDNA library was created from cDNA in pool 3.

A lambda-gt11 cDNA library generated from reserve 3 is screened for epitopes that should specifically bind to serum derived from a patient with prior NANBH. About 106 phages were tested with patient serum using the methods of Huynh et al (1985), except that bound human antibodies were detected with 125I. Five positive phages were identified and purified. Five positive phages were tested for binding specificity to the serum of 8 different people previously infected with the NANBH agent, using the same method. Four of the phages encode a polypeptide that reacts immunologically with one of the human sera, e.g., one that was used to primary screen the phage library. The fifth phage (5-1-1) encodes a polypeptide that reacts immunologically with 5 out of 8 tested sera. However, this polypeptide does not react immunologically with the serum of 7 normal blood donors. Therefore, it is clear that clone 5-1-1 encodes a polypeptide that is specifically recognized immunologically by serum from NANB patients.

IV.A.2. Sequences of HCV cDNA in recombinant phage 5-1-1 and polypeptide encoded in the sequence

cDNA in recombinant phage 5-1-1 was sequenced by the method of Sanger et al (1977). Essentially, the cDNA was digested with EcoRI, isolated by size fractionation using gel electrophoresis. EcolRI restriction fragments were subcloned into the M13 vector, mp18 and mp19 (Messing (1983)) and sequenced using the dideoxyquine termiNaCl method of Sanger et al (1977). The resulting sequence is shown in Figure 1.

The encoded polypeptide in Figure 1 that is encoded in the HCV cDNA is in the same translational frame as the N-terminal beta-Galactose of the fused portion, as shown in Chapter IV.A., translational open reading frame (ORF) 5-1 -1 encodes epitope(s) specifically recognized by patient and chimpanzee serum for NANBH infection.

IV.A.3. Isolation of overlapping HCV cDNA into cDNA in clone 5-1-1

The overlapping HCV cDNA in clone 5-1-1 was obtained by testing the same lambda-gt11 library as described in section IV.A.1., with a synthetic polynucleotide derived from the HCV cDNA sequence in clones 5-1-1, as shown in Figure 1. The polynucleotide sequence used for testing was:

5'-TCC CTT CGA TGT ACG GTA AGT GCD GAG AGC ACT CTT CCA TCT CAT CGA ATC CTC GGT AGA GGA CTT CCC TGT CAG GT-3'

The lambda-gt11 library was tested with this assay, using the method described by Huynh (1985). About 1 in 50,000 clones hybridized with the probe. The three cDNA-containing clones that hybridized with the synthetic probe were designated 81, 1-2, and 91.

IV.A.4. Nucleotide sequences of overlapping HCV 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 Chapter IV.A.2. The sequences of these clones according to the HCV cDNA sequence in phage 5-1-1 are shown in Figure 2, which shows the structure encoding the detected HCV epitope and where homologies in the nucleotide sequences are indicated by vertical lines between the sequences.

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

Another difference is that clone 5-1-1 contains 28 base pairs that are not present in the other three clones. These base pairs are located at the beginning of the cDNA sequence in 5-1-1, and are indicated by lowercase letters. Based on the radiological data discussed below in Section IV.D., it is possible that the HCV epitope may be encoded in this 28 bp region.

The absence of 28 base pairs 5-1-1 from clones 81, 1-2 and 91 may mean that the cDNA in these clones is derived from defective HCV genomes; alternatively, the 28 bp region should be the end addition in the 5-1-1 clone.

The lowercase sequences in the nucleotide sequence of clones 81 and 91 simply indicate that these sequences have not been found in other cDNAs because those overlapping these regions have not yet been isolated.

The composition of HCV and cDNA sequences from overlapping cDNAs in clones 5-1-1, 81, 1-2 and 91 is shown in Figure 3. However, in this figure the unique 28 base pairs of clone 5-1-1 are omitted. The figure also shows the sequence of the polypeptide encoded in the ORF composition of the HCV cDNA.

IV.A.5. Isolation of overlapping HCV cDNAs to cDNA in clone 81

Isolation of HCV cDNA sequences derived from and overlapping those of clone 81 cDNA is performed as follows. A lambda-gt11 cDNA library is obtained as described in section IV.A.1. It was tested by hybridization with a synthetic polynucleotide probe that is homologous to the 5'-terminal sequence of clone 81. The sequence of clone 81 is shown in Figure 4. The sequence of the synthetic polynucleotide used for testing was: 5' CTG TCA GGT ATG ATT GCC GGC TTC CCG GAC 3 '

Methods were essentially as described by Huynh (1985), except that library filters were washed twice under stringent conditions, eg washed 5 times with SSC, 0.1% SDS at 55ºC for 30 min each time. About 1 in about 50,000 clones hybridized with the probe. A positive recombinant phage containing cDNA that hybridized to the sequence was isolated and purified. This phage was designated clone 36.

Downstream cDNA sequences, which overlap the carboxyl terminus of the sequence in clone 81 cDNA, were isolated using a procedure similar to that for isolating upstream cDNA sequences, except that a synthetic oligonucleotide probe was obtained, which is homologous to the 3'-terminal sequence of clone 81. The sequence of the synthetic polynucleotide used for testing She was:

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

A positive recombinant phage, containing a cDNA that hybridized to this latter sequence was isolated and purified, and designated clone 32.

IV.A.6. Nucleotide sequence of HCV cDNA in clone 36

The nucleotide sequence of the cDNA in clone 36 was determined essentially as described in Section IV.A.2. The duplicate sequence of this cDNA, its region of overlap with the HCV cDNA in clone 81 and the polypeptide encoded by the ORF are shown in Figure 5.

The ORF in clone 36 is in the same translation frame as the HCV antigen encoded in clone 81. Thus, in combination, the ORFs in clones 36 and 81 encode a polypeptide that represents part of the large HCV antigen. The sequence of this putative HCV polypeptide and the double-stranded DNA sequence it encodes, which was derived from the combined ORFs of HCV cDNA clones 36 and 81, is shown in Figure 6.

IV.A.7. Nucleotide sequences of HCV cDNA in clone 32

The nucleotide sequence of the cDNA in clone 32 was determined essentially as described in Section IV.A.2. for clone sequence 5-1-1. Clone sequence data indicate that the cDNA in recombinant phage clone 32 is derived from two different sources. One cDNA fragment includes 418 nucleotides derived from the HCV genome; the second fragment comprises 172 nucleotides derived from the bacteriophage MS2 genome, which was used as a carrier during the preparation of the lambda-gt11 plasma cDNA library.

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

IV.A.8. Isolation of the overlapping HCV cDNA in clone 36 cDNA

Isolation of HCV cDNA sequences downstream of and overlapping those of clone 36 cDNA is performed as described in Section IV.A.5., for those overlapping clone 81 cDNA, except that the synthetic polynucleotide is based on the 5'-region of the clone 36. The sequence of the synthetic polynucleotide used for testing was:

5' AAG CCA CCG TGT GCG CTA GGG CTC AAG CCC 3'

about 1 in 50,000 clones hybridize with the probe. An isolated, purified recombinant phage clone containing a cDNA hybridizing to this sequence was named clone 35.

IV.A.9. Nucleotide sequences of HCV cDNA 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 are shown in Figure 8.

Clone 35 clearly contains one continuous ORF that encodes a polypeptide in the same translation frame as that encoded by clone 36, clone 81, and clone 32. Figure 9 shows the sequence of another continuous ORF spanning clones 35, 36, 81, and 32 along with the putative HCV cDNA. polypeptide is coded here. This concatenated sequence was confirmed using other independent cDNA clones derived from the same lambda-gt11 cDNA library.

IV.A.10. Isolation of overlapping HCV cDNA to cDNA in clone 35

Isolation of HCV cDNA sequences upstream of and overlapping those of clone 35 cDNA is performed as described in Section IV.A.8. for those overlapping the clone36 cDNA, except that the synthetic polynucleotide was based on the 5'-region of clone 35. The sequence of the synthetic polynucleotide used for testing was:

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

About 1 in 50,000 clones hybridized with the probe. An isolated, purified recombinant phage clone containing cDNA that hybridized to this sequence was designated clone 37b.

IV.A.11. Nucleotide sequence of HCV 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 herein are shown in Figure 10.

The 5'-terminal nucleotide of clone 35 is T, while the corresponding one in clone 37b is A. cDNA from three other independent clones that was isolated during the procedure in which clone 37b was isolated, which is described in Chapter IV.A.10., were also sequenced. cDNA from these clones also A at this position. Thus, the 5'-terminal T in clone 35 may be an addition to the cloning process. Additions often grow at the 5' end of the cDNA molecule.

Clone 37b clearly contains a single continuous ORF encoding a polypeptide that is a continuation of the polypeptide encoded in the ORF with an extension through overlapping clones 35, 36, 81, and 32.

IV.A.12. Isolation of overlapping HCV cDNA according to clone 32 cDNA

Isolation of HCV cDNA sequences downstream of clone 32 were isolated as follows. First, clone cla is isolated using a synthetic hybridization probe based on the nucleotide sequence of the HCV cDNA sequence in clone 32. The method is essentially that described in Chapter IV.A.5., except that the probe sequence was:

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

using the nucleotide sequence from the cla clone, a second synthetic clone was synthesized having the sequence:

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

Testing the lambda-gt11 library using the derived sequence from the cla clone as a probe yields about 1 in 50,000 positive clones. The isolated, purified clone that hybridized with this probe was named clone 33b.

IV.A.13.

The nucleic acid sequence of the cDNA in clone 33b was determined as described in Section IV.A.2. The sequence, its region of overlap with that of clone 32 cDNA, and the putative encoded polypeptide are shown in Figure 11.

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

IV.A.14. Isolation of overlapping HCV cDNAs to cDNA clone 37b to cDNA clone 33b

In order to isolate HCV cDNAs overlapping the cDNAs in clones 37b and 33b, the following synthetic oligonucleotide probe, derived from the cDNA in these clones, was used to test the lambda gt-11 library, using mainly the method described in chapter IV.A.3. Tests were used:

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

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

to detect colonies containing HCV cDNA sequences overlapping those of clones 37b and 33b. About 1 in 50,000 colonies were detected with each test. The clone containing the cDNA that was upstream of and overlapping the cDNA in clone 37b was named clone 40b. The clone that contained the cDNA that was upstream of, and overlapped with, the cDNA in clone 33b was named clone 25c.

IV.A.15. Nucleotide sequences of HCV cDNA in clone 40b and clone 25c

The nucleotide sequences of the cDNAs in clone 40b and clone 25c were determined essentially as described in Section IV.A.2. The sequences of 40 b and 25c, their regions overlapping with cDNA in clones 37b and 33b, and the putative encoded polypeptide, are shown in Figure 12 (clone 40b) and Figure 13 (clone 25c).

The 5'-terminal nucleotide of clone 40b is G. However, cDNA from five other independent clones that were isolated during the procedure in which clone 40b was isolated, described in section IV.A.14. They are also sequenced. The cDNAs from these clones also contain a T in this position. Thus, G may represent a cloning accessory (see appendix in Chapter IV.A.11).

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

Clones 40b and 25c each clearly contain an ORF that is an extension of the continuous ORF in the previously sequenced clones. The nucleotide sequence of the ORF provided by clones 40b, 37b, 35, 36, 81, 32, 33b and 25c and the amino acid sequence of the putative encoded polypeptide are shown in Figure 14. In the figure, possible cloning additions have been omitted from the sequence, and shown instead are the corresponding sequences in the non-5'-terminal regions of the multiple overlapping clones.

IV.A.16. Preparation of HCV cDNA from cDNA in clones 36, 81 and 32

The composition of HCV cDNA, C100 was obtained as follows. First, the cDNA from each clone was cloned individually into the EcoRI site of the vector pGEM3-blue (Promega Biotec). The resulting recombinant vectors containing cDNA from clones 36, 81, and 32 were named pGEM3-blue, pGEM3-blue/81, and pGEM3-blue/32. Appropriately oriented recombinant pGEM3-blue/81 was digested with NaeI and NarI and the large (2850 mp) fragment was purified and ligated to the small (570 bp) NaeI/NarI purified fragment from pGEM3-blue/36. This assembly of cDNA from clones 36 and 81 was used to create a second pGEM3-blue vector containing the contiguous HCV ORF contained in the overlapping cDNA in these clones. This new plasmid is then digested with PvuII and EcoRI to release fragments of about 600 bp, which are then ligated to a small (500 bp) PvuII/EcoRI fragment isolated from the appropriately oriented pGEM3-blue/32 plasmid, and the cDNA assembly from clones 36, 81 and 32 was ligated into the EcoRI linearized vector pSODcf1, which is described in chapter IV.A.1. and which was used to express clone 5-1-1 in bacteria. Recombinants containing about 1270 bp of the EcoRI fragment of the HCV cDNA (C100) were selected, and the cDNA from the plasmid was cut with EcoRI and purified.

IV.A.17. Isolation and nucleotide sequences of HCV cDNA in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c

HCV cDNAs in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f and 39c were isolated using the overlapping cDNA fragment isolation technique from the lambda-gt11 HCV cDNA library described in Chapter IV.A.3. except that the probes used were generated from the last nucleotide sequence of the isolated clones from the 5' and 3' end of the combined HCV sequence. The frequency of clones that hybridize with the probes described below is about 1 in about 50,000 in each case.

The nucleotide sequences of HCV cDNA in clones 14i, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 99c were determined essentially as described in section IV.A.2., except that the cDNA cut from these phages was substituted for 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 presented in Figure 12. The nucleotide sequence of the probe used for isolated 33c was:

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

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

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 of clone 33c, and the encoded amino acids, are shown in Figure 16.

Clone 7e was isolated using a probe based on the nucleotide sequence in clone 8h. The nucleotide sequence of the probe was:

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

the HCV cDNA sequence in clone 72, the overlap with clone 8h and the amino acids encoded here are shown in Figure 17.

Clone 14c was isolated with a probe based on the nucleotide sequence in clone 25c. The sequence of clone 25c is shown in Figure 13.

The trial in the isolation of clone 14c had the sequence

5' ACC TTC ATT AAT GCC TAC ACC ACG GGC 3'

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

Clone 18 was isolated using a probe based on the nucleotide sequence in clone 14c. The nucleotide sequence of the probe 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 here are shown 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 here are shown in Figure 20.

Clone 33g was isolated using a probe based on the nucleotide sequence in clone 33f. The nucleotide sequence of the probe 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 encoded amino acids are shown in Figure 21.

Clone 7f was isolated using a probe based on the nucleotide sequence in clone 7e. The 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 that of clone 7f, and the encoded amino acids are shown in Figure 22.

Clone 11b was isolated using a probe based on the sequence of clone 7f. The nucleotide sequence of the probe was:

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

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

Clone 14i was isolated using a probe based on the nucleotide sequence in clone 11b. The nucleotide sequence of the probe was:

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

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

Clone 39c was isolated using a probe based on the nucleotide sequence in clone 33g. The nucleotide sequence of the probe was:

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

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

IV.A.18. Composition of HCV cDNA sequence derived from isolated clones containing HCV cDNA

The HCV cDNA sequences in the isolated clones described above were linked to create a system of HCV cDNA sequences. Isolated clones linked 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. The composition of the HCV cDNA sequence derived from the isolated clones and the amino acid coded here is shown in Figure 26.

The following heterogeneous sequences were considered in creating the composition sequence. Clone 33c contains HCV cDNA of 800 bp, which overlaps the cDNA in clones 40b and 37c. In clone 33c, as in 5 other overlapping clones, nucleotide ≠789 is G. However, in clone 37b (see Chapter IV.A.11.), the corresponding nucleotide is A. This sequence difference creates an apparent heterogeneity in the amino acids encoded here, which they can be Cys or Tyr, for G or A. This heterogeneity can have significant ramifications at the level of protein folding. Therefore, this sequence heterogeneity is indicated in Figure 26 which shows the composition of HCV cDNA.

Nucleic acid residue ?2 in clone 8h HCV cDNA is T. However, as shown below, the corresponding residue in clone 7e is A; although A in this position is also found in 3 other isolated overlapping clones. Thus, the T residue in clone 8h may represent a cloning accessory. Therefore, in Figure 26 the residue in this position is marked as A.

The 3'-terminal nucleotide in clone 8f HCV cDNA is G. However, the corresponding residue in clone 33f and 2 other overlapping clones is T. Therefore, in Figure 26 the residue at this position is labeled T.

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

Nucleotide residue ≠4 in clone 33g HCV cDNA is T. However, in clone 33f and in two other overlapping clones the corresponding residue is A. Therefore, in Figure 26 the corresponding residue is labeled A.

The 3-terminus of clone 14i is AA, although the corresponding dinucleotide in clone 11b and three other clones is TA. Therefore, in Figure 26, the TA residue is presented.

Decomposition of other sequence heterogeneities is discussed further.

Examination of the composition of HCV cDNA shows that it contains one large ORF. This indicates that the viral genome has been translated into a large polypeptide whose process competes with or follows translation.

IV.A.19. Isolation and nucleotide sequences of HCV cDNA in clones 12f, 35f, 19g, 26g and 15e

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

Isolation of clone 12f, which contains a cDNA upstream of the HCV cDNA in Figure 26 is performed using hybridization of a probe based on the nucleotide sequence in clone 14i. The nucleotide sequence of the probe 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 encoded amino acids are shown in Figure 27.

Isolation of clone 35f, which contains the downstream HCV cDNA of Figure 26, is performed using a hybridization probe based on the nucleotide sequence in clone 39c. The nucleotide sequence of the probe was

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

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

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

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

The HCV cDNA sequence of clone 19g, its overlap with the sequence in clone 39f and the encoded amino acids are shown in Figure 29.

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

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

The HCV cDNA sequence of clone 26g, its overlap with the sequence in clone 19g and the encoded amino acids in it are shown in Figure 30.

Clone 15e was isolated using a hybridization probe based on the 3' sequence of clone 26g. The nucleotide sequence of the probe was:

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

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

The clones described in this chapter have been deposited with the ATCC under the terms and conditions described in chapter II.A., and designated by the following numbers.

[image]

The HCV cDNA sequences in the isolated clones described above were linked to create the HCV cDNA sequence composition. Isolated clones, linked in the 5' to 3' direction are: 12f, 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c. , 19g, 26g, and 15e.

The composition of the HCV cDNA sequence derived from isolated clones and the amino acids encoded in it are shown in Figure 32.

IV.A.20. An alternative method of isolating the cDNA sequence upstream of the HCV cDNA sequence in clone 12f

Based on the largest 5' HCV sequence in Figure 32, derived from HCV cDNA in clone 12f, small synthetic oligonucleotide reverse transcriptase primers were synthesized and used to bind to the corresponding sequence in HCV genomic RNA, for primary reverse transcription of upstream sequences. The primer sequences are close to the known 5'-terminal sequence of clone 12f, but sufficiently downstream to allow generation of probe sequences upstream of the primer sequences. Standard setup and cloning procedures are used. The resulting cDNA libraries are tested with sequences upstream of the primer site (as inferred from the sequence run in clone 12f). HCV genomic RNA is obtained from plasma or liver samples from chimpanzees with NANBH or from analogous samples from humans with NANBH.

IV.A.21. An alternative method of using supply to isolate sequences from the 5'-terminal region of the HCV genome

In order to isolate the 5'-terminal sequences of the HCV RNA genome, the cDNA product of the first round of reverse transcription, which is duplexed with the RNA template, is supplied with oligo C. This is performed by incubating the product with terminal transferase in the presence of CTP. The second round of cDNA synthesis, which complements the first strand of cDNA, is performed using oligo G as a primer for the reverse transcriptase reaction. Sources of genomic HCV cRNA are as described in Section IV.A.20. Procedures for terminal transferase supply and for reverse transcriptase reactions are as in Mantials et al. (1982). The cDNA products were then cloned, tested and sequenced.

IV.A.22. An alternative method of using supply to isolate sequences from the 3'-terminal region of the HCV genome

This method is based on previously used methods for flavivirus RNA cDNA cloning. In this method, RNA is subjected to denaturing conditions to remove secondary structures at the 3'-terminus, and then enriched with poly A polymerase using rATP as a substrate. Reverse transcription of poly A-supplied RNA is catalyzed by reverse transcriptase, using oligo dT as a primer. Other strands of cDNA are synthesized, cDNA products are cloned, tested and sequenced.

IV.A.23. Generation of lambda-gr11 HCV cDNA libraries containing larger cDNA inserts

The method used to generate and test the lambda-gt-11 libraries was essentially as described in Section IV.A. except that the library was created from a pool of cDNA eluted from a Sepharose CL-48 column.

IV.A.24. Creation of HCV cDNA libraries using oligomers as primers

New HCV cDNA libraries were obtained from RNA derived from the infectious reserve plasma of chimpanzees described in chapter IV.A.1., and from the poly A+RNA fraction derived from the liver of these infected animals. cDNA was generated as described by Gubler and Hoffman (1983), except that the primers for the first strike cDNAs were two synthetic oligomers based on the HCV genome sequence described above. The primers based on the sequence of clones 11b and 7e were:

5' CTG GCT TGA AGA ATC 3' i

5' AGT TAG GCT GGT GAT TAT GC 3'

The obtained cDNAs were cloned into lambda bacteriophage vectors and were tested with various other synthetic oligomers, the sequences of which are based on the HCV sequence in Figure 32.

IV.B. Expression of polypeptide encoded in HCV cDNA and identification of expression products as HCV induced genes

IV.B.1. Expression of the polypeptide encoded in clone 5-1-1

The HCV polypeptide encoded by clone 5-1-1 (see section IV.A.2., above) is expressed as a fused polypeptide with superoxide dismutase (SOD). This is done by subcloning the clone 5-1-1 cDNA insert into the expression vector pSODcf1 (Steimer et al. (1986)), as follows.

First, DNA isolated from pSODcf1 is treated with bamH1 and EcoR1, and the following binder is ligated to the linear DNA generated by 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 insert is isolated.

The plasmid containing the insert was digested with EcoR1 The HCV cDNA insert in clone 5-1-1 was cut with EcoR1 and ligated into this EcoR1 linearized plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al. (1980)). Recombinants with 5-1-1 cDNA in the correct orientation for expression of the ORF shown in Figure 1 were identified by restriction mapping and nucleotide sequencing.

A recombinant battery from a single clone induced expression of the SOD-NANB5-1-1 polypeptide by bacterial growth in the presence of IPTG.

IV.B.2. Expression of the polypeptide encoded in clone 81

HCV cDNA contained in clone 81 is expressed as a SOD-NANB81 fusion polypeptide. The method for obtaining the vector encoding this fusion polypeptide is analogous to that used to generate the vector encoding SOD-NANB5-1-1, except that the source of the HCV cDNA was clone 81, which was isolated as described in Chapter IV.A .3., and for which the cDNA sequence was determined as described in Chapter IV.A.4. The nucleotide sequence of the HCV cDNA in 81 and the putative amino acid sequence of the polypeptide encoded herein is shown in Figure 4.

The HCV cDNA insert in clone 81 was cut at EcoR1 and ligated into pSODcf1 containing the linker (see IV.B.1.) and linearized by treatment with EcoR1. The DNA mixture was used to transform E. coli strain D1210. Recombinants from clone 81 HCV cDNA in the correct orientation for expression of the ORF shown in Figure 4 were identified by fragment mapping and nucleotide sequencing.

Recombinant bacteria from a single clone were induced to express the SOD-NANB81 polypeptide by growing the bacteria in the presence of IPTG.

IV.B.3. Identification of polypeptide encoded in clone 5-1-1 as HCV and NANBH bound antigen

The polypeptide encoded by HCV cDNA clone 5-1-1 was identified as a NANBH-related antigen by showing that sera from chimpanzees and humans infected with NANBH reacted immunologically with a fusion polypeptide, SOD-NANB5-1-1, which comprises superoxide dismutase at its N-terminus and in frame 5-1-1 antigen at its C-terminus. This was performed using Western staining (Towbin et al. (1979)) as follows.

A recombinant bacterial strain transformed with an expression vector encoding the SOD-NANB5-1-1 polypeptide, described in Section IV.B.1., was induced to express the fusion polypeptide by growth in the presence of IPTG. The total bacterial lysate is subjected to electrophoresis through polyacrylamide gels in the presence of SDS according to Laemmla (1970). Separated polypeptides were transferred to nitrocellulose filters (Towbin et al. (1979)). The filters were then cut into strips and the strips were incubated individually with various chimpanzee and human sera. Bound antibodies were detected by further incubation with 125I-labeled sheep Ig anti-serum, as described in section IV.A.1.

Characterization of the chimpanzee serum used for Western staining and the results shown in photographs of autoradiographed strips are shown in Figure 33. Nitrocellulose strips containing polypeptides were incubated with serum derived from chimpanzees at various times during acute NANBH (Hutchinson strain) infections (path 1-16), hepatitis A infections (path 17-24 and 26-33), and hepatitis B infections (path 34-44). Lanes 25 and 45 show positive controls in which immunoblots were incubated with serum from the patient used to identify recombinant clone 5-1-1 in the original lambda-gt11 library assay (see section IV.A.1.).

The band visible in the control lanes, 25 and 45 in Figure 23, shows the binding of the antibody to the NANB5-1-1 portion of the SOD fusion polypeptide. These antibodies do not show binding to SOD itself, so this was excluded as a negative control in these samples and should be seen as a band that migrates significantly faster than the SAD-NANB5-1-1 fusion polypeptide.

Lanes 1-16 in Figure 27 show antibody binding in serum samples from 4 chimpanzees; serum was obtained immediately before infection with NANBH and sequentially during acute infection as seen in the figure, although antibodies immunoreactive with SOD-NANB5-1-1 polypeptide were absent in serum samples obtained before introduction of infectious HCV inoculums and during the early acute phase infection, all four animals finally induce circulating antibodies to this polypeptide during the latter part or after the acute phase.

The additional bands observed on the immunoblots in the case of chimpanzees 3 and 4 were caused by binding to proteins of the host bacteria.

In contrast to the results obtained with serum from NANBH-infected chimpanzees, the development of antibodies to the NANB5-1-1 portion of the fusion polypeptide was not observed in 4 chimpanzees infected with HAV or 3 chimpanzees infected with HBV. The binding itself in these cases was based on the binding to proteins of the host bacteria, which also occurs in HCV-infected samples.

Characterization of the human serum used for Western blots and the results shown in the photograph of the autoradiographed strips is presented in Figure 34. The nitrocellulose strips containing the polypeptides were incubated with serum taken from humans at various times during infection with NANBH (lanes 1-21), HAV ( lanes 33-40) and HBV (lanes 41-49). Lanes 25 and 50 show positive controls in which immunoblots were incubated with serum from the patient used in the original lambda-gt11 library assay, described above. Lanes 22-24 and 26-32 show “uninfected” controls containing serum from “normal” blood donors.

As seen in Figure 34, serum from 9 NANBH patients, including the serum used to test the lambda-gt11 library, contained antibodies to the NANB5-1-1 pool of the fusion polypeptide. Serum from three patients with NANBH contains these antibodies. It is possible that anti-NANB5-1-1 antibodies will develop in the future in these patients. It is also possible that this lack of reaction results from various NANBH agents causing disease in individuals from whom inappropriate serum was obtained.

Figure 34 also shows that serum from many patients infected with HAV and HBV does not contain anti-NANB5-1-1 antibodies and that these antibodies are also not present in the serum of "normal" controls. Although one patient (lane 36) is reported to contain anti-NANB5-1-1 antibodies, it is possible that this patient was previously infected with NANBH, so NANBH involvement is very high and often subclinical.

These serological studies show that the cDNA in clone 5-1-1 encodes epitopes that are specifically recognized by the serum of patients and animals infected with BB-NANBV. Additionally, cDNA does not appear to be derived from primate genomes. A hybridization assay performed with clone 5-1-1 or with clone 81 does not hybridize in Southern blots of control human and chimpanzee genomic DNA from uninfected individuals under conditions where unique single-copy genes are detectable. These probes also do not hybridize in Southern blots to control bovine genomic DNA.

IV.B.4. Expression of the polypeptide encoded by the HCV cDNA system in clones 36, 81 and 32

The HCV polypeptide encoded by the ORF spanning clones 36, 81, and 32 was expressed as a fusion polypeptide with SOD. This is done by inserting the cDNA composition, C100, into an expression cassette containing the human superoxide dismutase gene, inserting the expression cassette into a yeast expression vector, and expressing the polypeptide in yeast.

An expression cassette containing the C100 cDNA assembly derived from clones 36, 81 and 32 was created by inserting an approximately 1270 bp EcoRI fragment into the EcoRI site of vector pS3-56 (also called pS356), yielding plasmid pS3-56C100. The creation of C100 is described in Chapter IV.A.16. forward.

Vector pS3-56, which is a derivative of pBR322, contains an expression cassette comprising the ADH2/GAPDH hybrid yeast promoter of the upstream superoxide dismutase gene and the downstream GAPDH transcriptional terminator. A similar cassette, containing these control elements and the superoxide dismutase gene, was described by Cousens et al. (1987) and associated EPO 196,056 published Nov. 1, 1986, commonly cited under this designation. The cassette in pS3-56, however, differs from that in Cousens et al. (1987) in that the heterologous proinsulin gene and immunoglobulin were deleted, and gln154 superoxide dismutase was replaced by an adapter sequence containing an EcoRI site. The adapter sequence is:

5' AAT TTG GGA ATT CCATAA TGA G 3'

AC CCT TAA GGT ATT ACT ACG CT

The EcoRI site allows the insertion of heterologous sequences that, when expressed from a vector containing the cassette, yield polypeptides that bind to superoxide dismutase via an oligopeptide linker containing the amino acid sequence:

-asn-leu-gly-ile-arg-

specimen pS356 was deposited on April 29, 1988 under the name Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rovkville, Maryland 20853, and is designated as no. 67683. The terms and conditions for the availability of this deposit and for maintaining the deposit are the same as those specified in Chapter II.A., for species containing NANBV-cDNA. This deposit is for convenience only and is not required to practice the present invention from the point of view of the description. The deposited material is further incorporated herein by reference.

After isolation of recombinants containing the C100 cDNA insert in the correct orientation, the expression cassette containing the C100 cDNA was excised from pS3-56C100 with BamHI and the 3400bp fragment containing the cassette was isolated and purified. This fragment was inserted into BamHI instead of the yeast vector pAB24.

Plasmid pAB24, whose significant forms are shown in the figure, is a yeast-based vector containing a set of two-micron replication sequences /Broach (1981)/ and pBR322 sequences, also containing the yeast URA3 gene derived from plasmid Yep24 /Botstein et al. (1979)/ and yeast LEU2d derived from plasmid pCI/1, EPO publication no. 116.201. Plasmid pAB24 was generated by cutting Yep24 with EcoRI and religating the vector to remove partial 2 micron sequences. The resulting plasmid, pCBou, was then digested with Xbai and an 8605 bp fragment of the vector was gel isolated. This isolated Xbai fragment was linked to a 4460 bp XBai fragment containing the LEU2d gene isolated from pCI/1; the orientation of the LEU2d gene is in the same direction as the URA3 gene. The expression insertion was in the unique MamHI site of the pBR322 sequence, thus disrupting the bacterial tetracycline resistance gene.

A recombinant plasmid containing the SOD C100 expression cassette, pAB24C100-3, was transformed into the yeast strain JSC 308 as well as other yeast strains. Cells were transformed as described by Hinnen et al. (1978) and applied to a uraselective plate. Colonies were inoculated in leu-selective medium and grown to saturation. The culture was induced to express the SOD-C100 polypeptide (named C100-3) by growth in YEP containing 1% glucose.

The JSC strain is of the MAT, leu 2, ura3(del)DM15 (GAP/ADRI) genotype integrated at the ADRI locus. In JSC 308, over expression of the positive activator gene product, ADRI, results in hyperrepression (relative to wild-type ADRI control) and significantly higher yields of expressed heterologous proteins when such proteins are synthesized via the ADR UAS regulatory system. The creation of yeast strain JSC 308 is described in co-sponsored US application ser. no. (responsible representative) no. 2300-0229 completed simultaneously and incorporated herein by reference. Sample JSC 208 was deposited on May 5, 1988 with the ATTC under the terms of the Budapest Treaty, and is marked with the designation no. 20879. The terms and conditions for deposit availability and maintenance are the same as those specified in Chapter II.A., for species containing HCV cDNA.

The complete integration of the C100-3 polypeptide encoded in pAB24C100 should contain the 154 amino acids of human SOD at the amino terminus, 5 amino acid residues derived from a synthetic adapter containing an EcoRI site. 363 amino acid residues derived from C100 DNA and 5 carboxy-terminal amino acids derived from the MS2 nucleotide sequence of the adjacent HCV cDNA sequence in clone 32 (see chapter IV.A.7.). The putative amino acid sequence of the carboxy-terminus of this polypeptide begins at the penultimate Ala residue of SOD and is shown in Figure 36; the nucleotide sequence encoding this part of the polypeptide is also shown.

IV.B.5. Identification of the polypeptide encoded in C100 as a NANBH-bound antigen

The C100-3 fusion polypeptide expressed from plasmid pAB24C100-3 in yeast JSC was characterized for size and the polypeptide encoded by C100 was identified as a NANBH-associated antigen by its immunoreactivity with serum from a human with chronic NANBH.

The C100-3 polypeptide, which was expressed as described in section IV.B.4., was analyzed as follows. JSC 308 yeast cells were transformed with pAB24 or with pAB24C100 and induced to express the heterologous plasmid encoding the polypeptide. Induced yeast cells in 1 ml culture (OD650 ml about 20 minutes) were tabulated by centrifugation at 10000 rpm for 1 minute and lysed by vigorous mixing (10x1 min) with 2 volumes of solution and 1 volume of glass beads (0.2 millimicron diameter). The solution contained 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram/ml pepstatin. Undissolved, lysate material including C100-3 polypeptide was collected by centrifugation (10,000 rpm for 5 minutes) and solubilized by boiling for 5 minutes in Laemmli SDS sample buffer (see Laemmli (1970)).

An amount of polypeptide equivalent to that in 0.3 ml of an induced yeast culture was electrophoresed through a 10% polyacrylamide gel in the presence of SDS according to Laemmla (1970). The standard protein was co-electrophoresed on the gel. Gels containing expressed polypeptides are stained with Coomassie brilliant blue or subjected to Western staining as described in section IV.B.2. using serum from a patient with chronic NANBH to determine the immunoreactivity of polypeptides expressed from pAB24 and from pAB24C100-3.

The results are shown in Figure 37. In Figure 37a, the polypeptides are stained with Coomassie Brilliant Blue. The insoluble polypeptide from JSC 308 transformed with pAB24 and from two different JVC clones transformed with pAB24C100 are shown in lane 1 (pAB24), and lanes 2 and 3. Comparisons of lanes 2 and 3 with and lane 1 show induced expression of a polypeptide corresponding to a molecular weight of about 54,000 daltons from JSC 308 transformed with pAB24C100, which was not induced in JSC 308 transformed with pAB24. This polypeptide is indicated by an arrow.

Figure 37b shows the results of "Western" staining of insoluble polypeptides expressed in JSC 308, JSC 308 transformed with pAB24 (lane 1) or with pAB24C100-3 (lane 2). Polypeptides expressed from pAB24 are not immunoreactive with NANBH human serum. However, as indicated by the arrow, JSC 308 transformed with pAB24C100-3 expressed an approximately 54,000 dalton molecular weight polypeptide that did not immunoreact with human NANBH serum. Other immunologically reactive polypeptides in lane 2 may be degraded and/or aggregated to produce this polypeptide of about 54,000 daltons.

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

The combined polypeptide, C100-3, comprises SOD at the N-terminus and within the C100 HCV-polypeptide was purified by differential extraction of the insoluble fraction of extracted yeast cells expressing the polypeptide.

The fusion polypeptide, C100-3 was expressed in yeast strain JSC 308 transformed with pAB24C100, as described in section IV.A.2. The yeast cells were then lysed by homogenization, the insoluble material in the lysate was extracted at pH 12.0 and the C100-3 in the remaining insoluble fraction was solidified in buffer containing SDS.

Yeast lazate was obtained mainly according to Nagahuma et al. (1984). A yeast cell suspension was prepared to contain 33% cells suspended in a solution (buffer A) containing 20 mM Tris-Hc1, pH 8.0, 1 mM dititreitol and 1 mM phenylmethylsulfonyl fluoride (PMSF). An aliquot of the suspension (15 ml) was mixed with an equal volume of glass beads (0.45-0.50 mm diameter) and the mixture was mixed at high speed on a Super Mixer (Lab Line Instruments, Inc.) for 8 minutes. The homogenate and glass beads were separated and the glass beads were evaporated three times with the same volume of buffer A as the originally packed cells. After combining the wash water and the homogenate, the insoluble material in the lysate was obtained by centrifuging the homogenate at 7000 g for 15 minutes at 4ºC, the tablets were resuspended in buffer A equal to twice the volume of packed cells and pelleted again by centrifugation at 7000 g for 15 minutes. This washing procedure is repeated three times.

Insoluble material from the lysate was extracted at pH 12.0 as follows. The tablet is suspended in a buffer containing 0.5 M NaCl, 1 mM EDTA where the suspended volume is 1.8 times the original packed cells. The pH of the suspension was adjusted by adding 0.2 volumes of 0.4 M Na-phosphate buffer, pH 12.0. After mixing, the suspension was centrifuged at 7000 g for 15 minutes at 4ºC and the supernatant was removed. The extraction was repeated 2 times. The extracted tablets were washed by suspending in 0.5 M NaCl, 1 mM EDTA, the volume of suspension used was equal to twice the volume of the original packed cells and then followed by centrifugation at 7000 g for 15 minutes at 4ºC.

The C100-3 polypeptide in the extracted tablet was dissolved by treatment with SDS. Tablets were suspended in buffer A equal to 0.9 volume of originally packed cells, to which 0.1 volume of 2% SDS was added. After mixing the suspension, it is centrifuged at 7000 g for 15 minutes at 4ºC. The resulting tablet is extracted with 3 times more SDS. The obtained supernatants containing C100-3 are collected.

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

The purified preparation of the combined polypeptide is analyzed using polyacrylamide electrophoresis according to Laemmil (1970). Based on this analysis, the polypeptide was greater than 80% pure and had an apparent molecular weight of 54,000 daltons.

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

IV.C.1. Identification of chimpanzee liver RNA with NANBH that hybridizes to HCV cDNA

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

RNA was isolated from chimpanzee liver biopsies from which high titer plasma was derived (see Chapter IV.A.1.) using the techniques described in Maniatis et al. (1982) to isolate total RNA from mammalian cells and to separate them into poly A+ and poly A- fractions. These RNA fractions were subjected to formaldehyde/agarose gel electrophoresis (1% w/v) and transferred to nitrocellulose (Maniatis et al. (1982)). Nitrocellulose filters were hybridized with radiolabeled HCV cDNA from clone 81 (see Figure 4 for nucleotide sequence insert). In preparation of the radiolabeled probe, the HCV cDNA insert isolated from clone 81 was radiolabeled with 32P translational cleavage using DNA polymerase I (Manniatis et al. (1982)).

Hybridization was performed for 18 hours at 42ºC in a solution containing 10% (m/v) dextran sulfate, 50% (m/v) deionized formamide, 750 mM NaCl, 75 mM Na-citrate, 20 mM Na2HPO4, pH 6.5, 0.1% SDS, 0.02% (w/v) bovine serum albumin (BSA), 0.02% (w/v) Ficoll-400, 0.02% (w/v) polyvinylpyrrolidone, 100 micrograms/ml of salmon sperm DNA that had been fragmented by sonication and denaturation and 106 CFM/ml nick-translated cDNA probe.

An autoradiograph of the test filter is shown in Figure 38. Lane 1 contains 32P-labeled restriction fragment markers. Lanes 2-4 contain chimpanzee liver RNA as follows: lane 2 contains 30 micrograms of total RNA; lane 3 contains 30 micrograms of poly A-RNA; and lane 4 contains 20 micrograms of poly A+ RNA. As shown in Figure 32, chimpanzee livers with NANBH contain a heterogeneous population of bound poly A+ molecules that hydrolyze the HCV cDNA probe and range in size from about 5,000 to about 11,000 nucleotides. oVa RNA, which hydrolyzes towards HCV cDNA, may represent viral genomes and/or specific transcripts of the viral genome.

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

IV.C.2. Identification of HCV-derived RNA in the serum of an infected individual

Nucleic acids were extracted from particles isolated from high titer chimpanzee NANBH plasma as described in Section IV.A.1. An aliquot (equivalent to one ml of original plasma) of isolated nucleic acids was resuspended in 20 microliters of 50 mM Hepes, pH 7.5, 1 mM EDTA, and 16 micrograms/ml yeast soluble RNA. Samples were denatured by boiling for 5 min followed by flash freezing, 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 unit of DNase I in 10 mM MgCl2, 25 mM Hepes, pH 7.5); control samples were incubated without enzyme. Following incubation, 230 microliters of ice-cold 2xSSC containing 2 micrograms/ml yeast soluble RNA is added and samples are filtered on a nitrocellulose filter.

Filters are hybridized with a cDNA probe from clone 81, which has been 32 P-labeled by spot-translation. Figure 39 shows the autoradiograph of the filter. Hybridization signals were detected in the DNase-treated and control samples (lanes 2 and 1), but not in the RNase-treated sample (lane 3). Thus, RNase A treatment destroys the nucleic acid isolated from the particles and DNase I treatment has no effect, which strongly suggests that the HCV genome is composed of RNA.

IV.C.3. Detection of amplified HCV nucleic acid sequences derived from HCV nucleic acid sequences in liver and plasma species from chimpanzees with NANBH

HCV nucleic acids present in the liver and plasma of chimpanzees with NANBH and control chimpanzees are amplified using mainly the polymerase chain reaction PCR technique described by Saiki et al. (1986). Primary oligonucleotides were derived from the HCV cDNA sequence in clone 81 or clones 36 and 37. Amplified sequences were detected by gel electrophoresis and Southern staining, using as probes the corresponding cDNA polymerase with a sequence from the region between, but not including, the two primates.

RNA samples containing HCV sequences should be tested using an amplification system where they were isolated from a liver biopsy of a chimpanzee with NANBH and from two control chimpanzees. Isolation of the RNA fraction using the guadinium-thiocyanate procedure is described in Chapter IV.C.1.

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

Nucleic acids were extracted from plasma as follows. Either 0.1 ml or 0.1 ml of plasma was diluted to a final volume of 0.1 ml, with 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 polyadenylic acid, and incubated at 37ºC 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) saturated phenol. The phenolic phase was separated by centrifugation and re-extracted with TENB containing 0.1% SDS. The resulting aqueous phases from each extraction were collected and extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol /1:1(99:2)/, and then twice with an equal volume of a 99:1 mixture of chloroform/isoamyl alcohol. The phases are separated by centrifugation, the aqueous phase is brought to a final concentration of 0.2 M Na-acetate and the nucleic acids are precipitated by adding two volumes of alcohol. Complex nucleic acids are separated by ultracentrifugation in a SW 41 rotor at 38 K for 60 minutes at 4ºC.

In addition to the above, high titer chimpanzee plasma and control plasma pool were alternately extracted with 50 micrograms of poly A carrier by the method of Chomcyzski and Sacchi (1987). This procedure uses acid guanidinium thiocyanate extraction. RNA was isolated by centrifugation at 10,000 rpm for 10 minutes at 4ºC on an Eppendorf microfuge.

On two occasions, prior to cDNA synthesis in the PCR reaction, nucleic acids were extracted from plasma using the protein K/SDS/phenol method and further purified by binding and elution from S and S Eluitip-R clones. The procedure was followed according to the manufacturer's instructions.

The cDNA used as a template for the PCR reaction is derived from nucleic acids (either total nucleic acids or RNA) obtained as described above. Following ethanol precipitation, the precipitated nucleic acids are dried and resuspended in DEPC-treated distilled water.

Secondary structures in nucleic acids were disrupted by heating at 65ºC for 10 minutes and samples were immediately cooled on ice. cDNA was synthesized using 1 to 3 micrograms of total RNA from chimpanzee liver or from nucleic acids or RNA extracted from 10 to 100 microliters of plasma. Synthesis uses reverse transcriptase and is performed in a 25 microliter reaction using the procedure specified by the manufacturer, BRL. Primers for cDNA synthesis are those also used in the PCR reaction, described below. All reaction mixtures for cDNA synthesis contained 23 units of the RNase indicator, RNASIN (Fisher/Promega). After the cDNA synthesis, the reaction mixtures are diluted with water, boiled for 10 minutes and suddenly cooled on ice.

PCR reactions are performed mainly according to the manufacturer's instructions (Getus-Perkin-Elmer) except for the addition of 1 microgram of RNase A. Reactions are performed in a final volume of 100 microliters. PCR is performed for 35 cycles, using regimes of 37.72 and 94ºC.

Primers for cDNA synthesis for the PCR reaction are derived from HCV cDNA sequences in clone 81, clone 36 or clone 37. (HCV cDNA sequences of clones 81, 36 and 37b are shown in Figures 4, 5 and 10, respectively). The sequences of two 16-mer primers derived from clone 81 were:

5' CAA TCA TAC CTG ACa G 3'

and

5' GAT AAC CTC TGC CTG A 3'

The sequence of the primers from clone 36 was:

5' GCA TGT CAT GAT GTA T 3'

The sequence of the primers from clone 37b was:

5' ACA ATA CGT GTG TCA C 3'

In PCR reactions, primer pairs were composed of either two 16-mers derived from clone 81 or a 16-mer from clone 36 and a 16-mer from clone 37b. PCR reaction products were analyzed by product separation using alkaline gel electrophoresis, followed by Southern staining, and detection of amplified HCV cDNA sequences with a 32P-labeled internal oligonucleotide probe derived from the region of HCV cDNA that does not overlap the primers. PCR reaction mixtures are extracted with phenol/chloroform and nucleic acids from the aqueous phase with salt and ethanol. Precipitated nucleic acids are collected by centrifugation, dissolved in distilled water. Aliquots of samples are subjected to electrolysis on 1.8% alkaline agarose gels. Single-stranded DNA of 60, 108 and 161 nucleotides in length were electrophoresed together on gels as markers of molecular mass. After electrophoresis, the cDNA on the gel is translated into the "Biora Zeta probe" on paper. Prehybridization and hybridization and washing conditions are those specified by the manufacturer (Biorad).

Probes used for hybridization detection of amplified HCV cDNA sequences were as follows. When the PCR primer 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 primers. When a pair of PCR primers is derived from clones 36 and 37b, the probe is translationally cut, the HCV cDNA insert is derived from clone 35. The primers are derived from nucleotides 155-170 of clone 37b insert and insert 206-268 of clone 36, 3'-end of HCV The cDNA insert in clone 35 overlaps nucleotides 1-186 of the insert in clone 36; and the 5'-end of the clone 35 insert overlaps nucleotides 207-269 of the insert in clone 37b (compare Figures 5, 8 and 10). Thus, the cDNA insert in clone 35 encompasses part of the region between the clone 26 and 37b sequences of the derived primers and is useful as a probe for amplifying sequences involving these primers.

Analysis of RNA from liver species used both sets of primers and probes according to the above procedure. RNA from the liver of three chimpanzees with NANBH gave positive hybridization results for amplified sequences of the expected size (161 and 586 nucleotides for 81 and 36 and 37b), although control chimpanzees gave negative hybridization results. The same results are obtained when the experiment is repeated three times.

Analysis of nucleic acids and RNA from plasma was also performed according to the above procedure using primates and sample 81. Plasmas were from two chimpanzees with NANBH, from a control chimpanzee and plasma reserves from control chimpanzees. Both NANBH plasmas contained nucleic acids/RNAs that gave positive results in PCR amplification experiments, although both control plasmas gave negative results. These results have been repeated several times.

IV.D. Radioimmunoassay for the detection of HCV antibodies in the serum of infected individuals

Solid-phase radioassays for the detection of antibodies to HCV antigen were developed based on Tsu and Herzenberg (1980). Microtiter plates (Imulon 2, although removable strips) are coated with purified polypeptide containing HCV epitopes. Coated plates were incubated with human serum samples suspected of containing antibodies to HCV epitopes or appropriate controls. During incubation, the antibody, if present, binds immunologically to the solid phase antigen. After removing the unbound material and washing the microtiter vessels, human antibody-NANBV antigen complexes are detected by incubation with 125I-labeled sheep antihuman immunoglobulin. Unbound labeled antibody is removed by suction and the dishes are washed. The radioactivity in individual sources is determined; the amount of bound human anti-HCV antibody is proportional to the radioactivity of the source.

IV.D.1. Purification of the SOD-NANB5-1-1 fusion polypeptide

Purified SOD-NANB5-1-1 polypeptide expressed in recombinant bacteria as described in section IV.B.1. was purified from recombinant E. coli by differential extraction of cell extracts with urea 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 0.01 M Tris-HCl, pH 8.0, and 0.4 ml of 0.5 M EDTA, pH 8 was added. ,0. After 5 minutes at 0ºC, the mixture is centrifuged at 4000 g for 10 minutes. The resulting tablet was resuspended in 10 ml of 25% (w/v) sucrose containing 0.05 Tris-HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram/ml pepstatin A, which was then powdered by adding 0 .5 ml of lysozyme (10 mg/ml) and incubation at 0ºC for 10 minutes. After adding 10 ml of 1% (v/v) Triton X-100 in 0.05 M Tris-HCl, 1 mM EDTA, the mixture was incubated for an additional 10 minutes at 0ºC with occasional mixing. The obtained viscous solution is homogenized by passing it 6 times through a sterile 20-gauge hypodermic needle and centrifuged at 4000 g for 10 minutes.

Tablets containing SAD-NANB5-1-1 fusion protein were co-dissolved in 5 ml urea in 0.02 M Tris-HCl, pH 8.0, 1 mM dithiothreitol (buffer A) and applied to a Q-Sepharose fast-flow column equilibrated with buffer A The polypeptide was eluted with a linear gradient of 0.0 to 3.0 M NaCl in buffer A. After elution, fractions were analyzed by polyacrylamide gel electrophoresis in the presence of SDS to determine the content of SDS-NANB5-1-1. Fractions containing this polypeptide were collected and dialyzed against 6 M urea in 0.02 M sodium phosphate buffer, pH 6.0, 1 mM dithiothreitol (buffer B). The dialyzed sample was applied to a fast-flow S-Sepharose column equilibrated with buffer B and polypeptides were eluted with a linear gradient from 0.0 to 3.0 M NaCl in buffer B. Fractions were analyzed by polyacrylamide gel electrophoresis for the presence of SOD-NANB5-1- 1 and the corresponding fractions were collected.

The final product of the SOD-NANB5-1-1 polypeptide was examined by polyacrylamide gel electrophoresis in the presence of SDS. Based on this analysis, the preparation was more than 80% pure.

IV.D.2. Purification of the SOD-NANB81 fusion polypeptide

The SOD-NANB81 fusion polypeptide, expressed in a recombinant bacterium as described in section IV.B.2., was purified from recombinant E. coli by differential extraction of the cell extract with urea, followed by chromatography on anion and cation exchange columns using the procedure described for isolation of the combined SOD-NANB5-1-1 polypeptide (see chapter IV.D.1.).

The final SOD-NANB81 polypeptide preparation was electrophoresed on a polyacrylamide gel 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 diagnosed with NANBH were analyzed by radioimmunoassay (RIA) to determine antibodies to HCV epitopes present in the fused SOD-NANB5-1-1 and SOD-NANB81 polypeptides.

Microtiter plates were covered with SOD-NANB5-1-1 or SOD-NANB81, which were partially purified according to Sections IV.D.1. and IV.D.2. Washes were performed as follows.

100-microliter aliquots containing 0.5 micrograms of SOD-NANB5-1-1 or SOD-NANB81 in 0.125 M Na-borate buffer, pH 8.3, 0.075 M NaCl (BBS) were added to each well of a microtiter plate (Dynatech Immunol 2 Removewell Strips). The dish is incubated at 4ºC overnight in a humidified chamber, after which the protein solution is removed and the wells are washed three times with BBS containing 0.02% Triton X-100 (BBST).

To prevent nonspecific binding, wells are coated with bovine serum albumin (BSA) by adding 100 microliters of a 5 mg/ml solution of BSA in BBS and then incubating at room temperature for 1 hour; after this incubation, the BSA solution is removed. Polypeptides in coated wells are reacted with serum by adding 100 microliters of serum samples diluted 1:100 in 0.01 M Na-phosphate buffer, pH 7.2 0.15 M NaCl (PBS) containing 10 mg/ml BSA, and incubating the serum that contains springs for 1 hour at 37ºC. After incubation, the serum samples are removed by suction and the wells are washed 5 times with BBST.

Anti-NANB5-1-1 and anti-NANB81 bound to the pooled polypeptides were determined by binding of 125I-labeled F'(ab)2 sheep anti-human IgG to cover the source. Aliquots of 100 microliters of labeled sample (specific activity 5-20 microcuries/microgram) are added to each well and the dishes are incubated at 37ºC for 1 hour, excess sample is aspirated off and washed 5 times with BBST. The amount of radioactivity associated with each source is determined by counting on a counter that detects gamma radiation.

The results of anti-NANB5-1-1 and anti-NANB81 measurements in individuals with NANBH are given in Table 1.

Table 1

Detection of anti-5-1-1 and anti-81 in the serum of NANB, HAV and HAB hepatitis patients

[image] [image]

1 Sequential serum samples available from these patients

2 IVD intravenous drug use

3 AVH acute viral hepatitis

4 Post transfusion

As seen in Table 1, 19 of 32 sera from patients diagnosed as having NANBH were positive for antibodies directed against HCV epitopes present in SOD-NANB5-1-1 and SOD-NANB81.

However, serum samples that were positive were equally immunoreactive with SOD-NANB5-1-1 and NANB81. Serum samples of patient no. 1 were positive for SOD-NANB81 but not for SOD-NANB5-1-1. Serum samples of patients no. 10, 15 and 17 were positive for SOD-NANB5-1-1 but not for SOD-NANB81. Serum samples of patients no. 3, 8, 11 and 12 react equally with both combined polypeptides, while serum samples no. 2, 4, 7 and 9 were 2-3 times more reactive towards SOD-NANB5-1-1 than towards SOD-NANB81. These results suggest that NANB5-1-1 and NANB81 may contain at least three empty epitopes; eg, it is possible for each polypeptide to contain at least 1 single epitope and for two polypeptides to share at least 1 epitope.

IV.D.4. Specificity of solid phase RIA for NANBH

The specificity of the solid-phase RIA for NANBH was tested using sera from patients infected with HAV or HBV and sera from control individuals. Assays using partially purified SOD-NANB5-1-1 and SOD-NANB81 were generally performed as sera from patients previously diagnosed as having HAV or HBV or from blood banked individuals. Results for sera from HAV- or HBV-infected patients are shown in Table 1. The RIA was tested using 11 sera from HAV-infected patients and 20 sera from HBV-infected patients. As shown in Table 1, none of these sera gave a positive immune reaction with the pooled polypeptides containing the BB-NANBV epitopes.

RIA using NANB5-1-1 antigen was performed to determine the reactivity of serum from control individuals. Of 230 serum samples obtained from a population of normal blood donors, only 2 gave positive reactions in the RIA (data not shown). It is possible that the two blood donors to whom these serum samples belong belong to individuals previously exposed to HCV.

IV.D.5. NANB5-1-1 reactivity during NANBH infection

The presence of anti-NANB5-1-1 antibodies during NANBH infection of 2 patients and 4 chimpanzees was monitored by RIA, described in Section IV.D.3. Additionally, RIA was used to determine the presence or absence of anti-NANB5-1-1 antibodies during HAV and HBV infection in infected chimpanzees.

The results presented in Table 2 show that with chimpanzee and human anti-NANB5-1-1 antibodies, the following set of corner phases of NANBH infection was detected. Anti-NANB5-1-1 antibodies were not detected in serum samples from chimpanzees infected with HAV or HBV. Thus, anti-NANB5-1-1 antibodies serve as markers for individual exposure to HCV.

Table 2

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

[image]

T= day from initial sample

IV.E. Purification of polyclonal serum antibodies to NANB5-1-1

Based on the specific immunological reactivity of NANB5-1-1 polypeptide with antibodies in serum samples from patients with NANBH, a method was developed to purify serum antibodies that react immunologically with the epitope in NANB5-1-1 polypeptide (see chapter IV.D.1.) attached non-fusible carrier; coupling is such that the immobilized polypeptide retains its affinity for the antibody to NANB5-1-1, the antibody in serum samples is absorbed on the matrix-bound polypeptide. After testing to remove non-specifically bound materials and unbound materials, the bound bodies are released from the bound SOD-HCV polypeptide by changing pH and/or chaotropic reagents, eg, urea.

Nitrocellulose membranes containing bound NANB5-1-1 were prepared as follows. A nitrocellulose membrane, 2.1 cm Sartorius of 0.2 micron pores was washed three times for three minutes each with BBS, SOD-NANB5-1-1 was bound to the membrane by incubating the purified preparation in BBS at room temperature for 2 hours; alternatively by incubating at 4ºC overnight. The filter was washed three times with BBS, each time for three minutes. The remaining active sites on the membrane were blocked with BSA by incubation with 5 mg/ml BSA solution for 30 minutes. Excess BSA was removed by washing the membrane 5 times and three times with distilled water. The membrane containing viral antigen and BSA is then treated with 0.05 M glycine hydrochloride, pH 2.5, 0.10 M NaCl (GlyHCl) for 15 minutes, then washed with PBS for 3 minutes.

polyclonal anti-NANB5-1-1 antibodies were isolated by incubating membranes containing the fusion polypeptide with serum from individuals 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 elutions of GlyHCl, three minutes per elution. The pH of the eluent was adjusted to 8.0 by collecting each eluate in a tube containing 2.0 M Tris-HCl, pH 8.0. The extraction of anti-NANB5-1-1 antibodies after affinity chromatography is about 50%.

Nitrocellulose membranes containing bound viral antigen can be used repeatedly without appreciable loss of binding capacity. When reusing the membrane, after elution, the antibodies are washed three times with BBS for three minutes each. It is stored in BBS at 4ºC.

IV.F. Obtaining HCV particles from infected plasma using purified human polyclonal anti-HCV antibodies; Nucleic acid hybridization in the obtained particles according to HCV cDNA

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

Protein-nuclein coselin complexes present in the infectious plasma of chimpanzees with NANBH were isolated using purified human polyclonal anti-HCV antibodies bound to polystyrene beads.

Polyclonal anti-NANB5-1-1 antibodies were purified from human serum with NANBH using the SOD-HCV polypeptide encoded in 5-1-1. The purification method is described in Chapter IV.E.

Purified anti-NANB5-1-1 antibodies were bound to polystyrene balls (1/4" diameter, Precision Plastic Ball Co., Chicago, Illinois) by incubating each at room temperature overnight with 1 ml of antibody (1 microgram/ml in borate buffer, pH 8.5). After overnight incubation, the beads were washed once with TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.03% (v/v)) and then with phosphate buffered saline (PBS) containing 10 mg/ml BSA.

Control beads were prepared in an identical manner, except that purified anti-NANB5-1-1 antibodies were replaced with whole human immunoglobulin.

Recovery of HCV from NANBH-infected chimpanzee plasma using bead-bound anti-NANB5-1-1 antibodies is performed as follows. Chimpanzee plasma with NANBH was used as described in section IV.A.1. An aliquot (1 ml) of NANBV-infected chimpanzee plasma was incubated for 3 hours at 37ºC with five beads coated with anti-NANB5-1-1 antibodies or with control immunoglobulins. The beads were washed three times with TBST.

IV.F.2. Nucleic acid hybridization in the obtained particles according to NANBV-cDNA

The nucleic acid component released from particles obtained with anti-NANB5-1-1 antibodies was analyzed for hybridization to HCV cDNA derived from clone 81.

HCV particles were obtained from NANBH-infected chimpanzee plasma, as described in IV.F.1. To release nucleic acids from the particles, the washed beads are incubated for 60 minutes at 37ºC with 0.2 ml per bead of a solution containing proteinase K (1 mg/ml), 10 mM Trir-HCl, pH 7.5, 10 mM EDTA, 0, 25% (v/v) SDS, 10 µg/ml dissolved yeast RNA, and the supernatant solution was removed. The supernatant is extracted with phenol and chloroform and the nucleic acids are precipitated with ethanol overnight at -20ºC. The nucleic acid pellet was collected by centrifugation, dried and dissolved in 50 mM Hepes, pH 7.5. Duplicate aliquots of soluble nucleic acids from samples obtained from beads coated with anti-NANB5-1-1 antibodies and from control beads containing total human immunoglobulin were filtered onto nitrocellulose filters. Filters were hybridized with a 32P-labeled notch-translator probe made from a purified HCV cDNA fragment in clone 81. Methods for probe preparation and hybridization are described in Chapter IV.C.1.

Autoradiographs of test filters containing nucleic acids from particles obtained using beads containing anti-NANB5-1-1 antibodies are shown in Figure 34. The extract obtained using anti-NANB5-1-1 antibodies (A1, A2) gives clear hybridization signals relative to to the control antibody extract (A3, A4) and to the control yeast RNA (B1, B2). Standards containing lpg, 5 pg and 10 pg of the purified cDNA fragment of clone 81 are shown in CI-3.

These results show that particles obtained from NANBH plasma using anti-NANB5-1-1 antibodies contain nucleic acids that hybridize with HCV cDNA in clone 81 and thus provide further evidence that the cDNA in these clones is derived from the etiological agent for NANBH.

IV.G. Immunoreactivity of C100-3 with purified anti-NANB5-1-1 antibodies

The immunoreactivity of the C100-3 fused polypeptide with anti-NANB5-1-1 antibodies was performed by radioimmunoassay, in which antigens bound to the solid phase are halogenated with purified NANB5-1-1 antibodies and the antigen-antibody complex is detected with 125I-labeled sheep anti-human antibodies.

The immunological reactivity of the C100-3 polypeptide was compared with that of the SOD-NANB5-1-1 antigen.

The C100-3 fusion polypeptide was synthesized and purified as described in Section IV.B.5. and in chapter IV.B.6. The SOD-NANB5-1-1 fusion polypeptide was synthesized and purified as described in Section IV.B.1. and in chapter IV.D.1. Purified anti-NANB5-1-1 antibodies were obtained as described in section IV.E.

Aliquots of 100 µl containing varying amounts of purified C100-3 antigen in 0.125 M Na-borate buffer, pH 8.3, 0.075 M NaCl (BBS) are added to each well of a microtiter plate (Dynatech Immulon 2 Removewell Strips). The plate was incubated at 4ºC overnight in a humidified chamber, after which the protein solution was removed and the wells were washed three times with BBS containing 0.02% Triton X-100 (BBST). To prevent non-specific binding, the wells were coated with BSA by adding 10 µl of a 5 mg/ml solution of BSA in BBS and then incubated at room temperature for 1 hour, after which the excess BSA solution was removed. Polypeptides in coated wells are reacted with purified anti-NANB5-1-1 antibodies by adding 1 µg of antibody per well, and incubating the samples for 1 hour at 37ºC. After incubation, the excess solution was removed by suction and the wells were evaporated 5 times with BBST. Anti-NANB5-1-1 bound to the pooled polypeptides was determined by binding of 125I-labeled F'(ab)2 sheep anti-human IgG to coated wells. Aliquots of 100 µl of labeled sample (specific activities 5-20 microcuri/microgram) were added to each well and the dishes were incubated for 1 hour at 37ºC and then the excess was removed by suction, which was further followed by a quintuplicate assay with BBST. The amount of radioactivity associated with each source is determined by counting on a counter that detects gamma radiation.

The results of the immunoreactivity of C100 with purified anti-NANB5-1-1 compared to those for NANB5-1-1 with purified antibodies are shown in Table 3.

Table 3

Immunoreactivity of C100-3 compared to NANB5-1-1 by radioimmunoassay

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The results in Table 3 show that anti-NANB5-1-1 recognizes the epitope(s) in the C100 portion of the C100-3 polypeptide. Thus, NANB5-1-1 and C100 share common epitopes. The results indicate that the cDNA sequence encoding this NANBV epitope is the one present in both clone 5-1-1 and clone 81.

IV.H. Characterization of HCV

IV.H.1. Characterization of the composition of the HCV genome

The HCV genome was characterized with respect to the strand by isolating the nucleic acid fraction from particles obtained on anti-NANB5-1-1 antibody coated polystyrene beads and determining whether the isolated nucleic acids hybridize with the plus and/or minus strands of HCV cDNA.

Particles were obtained from plasma of HCV-infected chimpanzees using polystyrene beads coated with immunopurified anti-NANB5-1-1 antibody as described in Section IV.F.1. The nucleic acid component of the particle is released using the method described in Section IV.F.2. Aliquots of isolated genomic nucleic acid equivalent to 3m1s of high titer plasma were spotted onto nitrocellulose filters. As a control, aliquots of denatured HCV cDNA from clone 81 (2 picograms) were also applied to the same filters. Filters were probed with a 32P-moistened mixture of plus or minus a strand of single-stranded DNA cloned from HCV cDNA; cDNAs were cut from clones 40b, 81 and 25c.

Single probes were obtained by cutting HCV cDNA from clones 81, 40b and 25c with EcoRI and cloned cDNA fragments in M13 vectors, mp18 and mp19 /Messing (1983)/. M13 clones were sequenced to determine whether they contained plus or minus strands of DNA derived from HCV cDNA. Sequencing was done using the dideoxy chain termination method of Sanger et al. (1977).

Each of the sets of duplicate filters containing aliquots of the HCV genome isolated from the obtained particles is hydrolyzed with a plus or minus strand of the probe derived from HCV cDNA. Figure 35 shows autoradiographs obtained from the NANBV genome assay with a mixture of probes derived from clones 81, 40b and 25c. The mixture was used to increase the sensitivity of hybridization tests. Samples in panel I were hybridized with plus-length probe mix. The samples in panel II were tested by hybridization with the minus waist probe mixture. The composition of the samples in the immunostained panels is presented in Table 4.

Table 4

[image] * undescribed sample

As can be seen from the results in Figure 35, only the minus side of the DNA probe hybridizes with the isolated HCV genome. This result, in combination with the result showing the sensitivity of the genome to RNase and not to DNase (see chapter IV.C.2.), indicates that the NANBV genome is positive RNA.

These data and data from other laboratories regarding the physicochemical properties of putative NANBV are consistent with the possibility that HCV is a member of the Flaviviridae. However, the possibility that HCV represents a new class of viral agents has not been eliminated.

IV.H.2. Detection of sequences in the obtained particles which, when amplified by PCR hybridization to HCV cDNA, are derived from clone 81

The RNA in the obtained particles is as described in section IV.H. Analysis for sequences hybridizing to HCV cDNA derived from clone 81 was performed using PCP amplification, as described in Section IV.C.3., except that the hybridization probe was a quinized oligonucleotide derived from clone 81 cDNA sequence. The results show that the amplified sequences hybridized with clone 81 derived from the HCV cDNA probe.

IV.H.3. Homology between the Dengue Flavivirus non-structural protein (MNWWD1) and the HCV polypeptide encoded by the merged ORF clone 14i-39c

The pooled HCV cDNA clones 14i and 39c contain a single continuous ORF, as shown in Figure 26. The encoded polypeptide was analyzed for homology to the nonstructural polypeptide region of Dangue flavivirus (MNWWD1). The analysis used the Dayhoff protein database and was performed by computer. The results are shown in Figure 36, where the symbol (:) indicates homology and the symbol (.) indicates a conservative substitution in the sequence; dashes indicate spaces inserted into the sequence to achieve the highest homology. As can be seen from the figure, there is significant homology between the sequence encoded in the HCV cDNA and the non-structural polypeptide segment encoded in the region towards the 3'-end and the cDNAs also contain sequences that are homologous to the sequences in the Dengue polymerase. As a consequence, it is found that the canonical gly-asp-asp (GDD) sequence should be essential for RNA-dependent RNA polymerases contained in the polypeptide encoded in the HCV cDNA, in a location consistent with that of the Dengue 2 virus (data not shown).

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

Two types of studies provide results indicating that HCV-DNA is not detectable in tissue from individuals with NANBH. These results, together with those described in IV.C. and IV.H.1. and IV.H.2. prove that HCV is not a DNA-containing virus and that its replication does not involve cDNA.

IV.H.4.a. Southern smear procedure

In order to determine whether the NANBH of the liver of an infected chimpanzee contains detectable HCV-DNA (or HCV cDNA), restriction enzyme DNA fragments isolated from this source were Southern blotted and the smears were probed with 32P-labeled HCV cDNA. The results show that labeled HCV cDNA hydrolyzes with smeared DNA from the liver of an infected chimpanzee. It also does not hybridize with a DNA smear from the liver of a normal chimpanzee. In contrast, in the positive control the labeled beta-interferon gene probe hybridizes strongly with Southern blot of restriction enzyme-digested human placental DNA. These systems are designed to detect a single copy of a gene to be detected with a labeled probe.

DNA was isolated from the liver of two chimpanzees with NANBH. Control DNAs were isolated from the liver of an uninfected chimpanzee and from human placenta. The procedure for extracting DNA was mainly done according to Maniatis et al. (1982) and DNA samples were treated with RNase during the isolation procedure.

Each DNA sample was treated with EcoRI, MboI or HincII (12 micrograms), according to the manufacturer's instructions. Fragmented DNAs were electrophoresed on 1% neutral agarose gels, Southern smeared on nitrocellulose and the smeared material was hybridized with the corresponding translationally cleaved cDNA probe (3x106 cpm/ml of hybridization mixture). DNA from the liver of an infected chimpanzee and from the liver of a normal chimpanzee was hybridized with 32P-labeled HCV cDNA from clones 36 plus 81; DNA from human placenta hybridized with 32P-labeled DNA from beta-interferon gel. After hybridization, blots were printed under stringent conditions eg with a solution containing 0.1xSSC, 0.1% SDS at 65ºC.

Beta-interferon gene DNA was obtained as described by Hougton et al. (1981).

IV.H.4.b. Amplification using the PCR technique

In order to determine whether HCV-DNA could be detected in the liver of chimpanzees with NANBH, DNA was isolated from tissue, and subjected to the PCR detection technique by amplification using primers and probe polynucleotides derived from HCV cDNA from clone 81. Negative controls were samples isolated from cell culture tissues of uninfected HepG2 and from presumably uninfected human placenta. Positive controls were negative control DNA samples to which a known small amount (250 molecules) of HCV cDNA insert from clone 81 was added.

To further confirm that RNA fractions isolated from the same livers of chimpanzees with NANBH contained sequences complementary to the HCV cDNA probe, an amplification-detection PCR assay was also used on the isolated RNA samples.

In the study, DNA was isolated using the procedure described in section IV.H.4.a. and RNA was extracted essentially as described by Chirgwin et al. (1981).

DNA samples were isolated from the liver of 2 infected chimpanzees, from uninfected HepG2 cells and from human placenta. One microgram of each DNA was digested with HindIII according to the manufacturer's instructions. The digested samples were subjected to PCR amplification for the detection of amplified HCV cDNA essentially as described in Chapter IV.V.3., except that the reverse transcriptase step was omitted. PCR primers and probes were from HCV cDNA clone 81 and are described in Chapter IV.C.3. Before amplification, for positive controls, one microgram of each DNA sample was “spiked” with the addition of 250 molecules of HCV cDNA insert isolated from clone 81.

In order to determine whether HCV sequences were present in RNA isolated from the livers of chimpanzees with NANBH, samples containing 0.4 micrograms of total RNA were subjected to an amplification procedure essentially as described in Section IV.C.3., except that the reverse transcriptase from some samples as a negative control. PCR primers and probes were from HCV cDNA clone 81 as described above.

The results show that the amplified sequences of the complementary HCV cDNA probe are not detectable in the negative controls. In contrast, when samples include DNA from the liver of an infected chimpanzee, HCV cDNA amplifies rather “spiked,” clone 81 sequences are detected in all known control samples. Furthermore, in the RNA study, amplified HCV cDNA clone 81 sequences were detected only when reverse transcriptase was used, strongly indicating that the results were not induced by DNA contamination with NaCl.

These results indicate that chimpanzee hepatocytes with NANBH contain no or undetectable levels of HCV DNA. Based on the aforementioned study, if HCV DNA is present then it is at a level well below 0.6 copies per hepatocyte. In contrast, HCV sequences in total RNA from the same liver samples were easily determined by the PCR technique.

AND YOU. ELISA determination for HCV infection using HCV C100-3 as antigen test

All samples were tested using the HCV C100-3 ELISA. This assay uses HCV C100-3 antigen synthesized and purified as described in Section IV.B.5., and a horseradish peroxidase (HRP) conjugate of mouse monoclonal anti-human IgG.

Dishes coated with HCV C100-3 antigen were obtained as follows. Solution containing plating buffer (50 mM Na-boronate, pH 9.0), 21 ml/well, BSA (25 µg/ml), C100-3 (2.50 µg/ml) prepared immediately before addition to Removeawell Immunolon And containers (Dynatech Corp.). After mixing for 5 minutes, 0.2 ml/well of the solution was added to the dishes, they were covered and incubated for 2 hours at 37ºC, after which the solution was removed by suction. Samples were washed once with 400 µl of wash buffer (100 mM sodium phosphate, pH 7.4, 140 mM sodium chloride, 0.1% (w/v) casein, 1% (w/v) Triton X-100 , 0.01% (w/v) Thimerosal).After removing the assay solution, 200 µl/well of post-overflow solution (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 0.1% ( m/v) casein and 2 mM phenylmethylsulfonifluoride (PMSF), the dishes were loosely covered to prevent evaporation and allowed to stand at room temperature for 30 min. The wells were then aspirated to remove the solution and lyophilized overnight to dryness without heating.

The resulting containers can be stored at 2-8ºC in sealed aluminum bags.

To perform the ELISA determination, 20 microliters of serum sample or control sample is added to a well containing 200 microliters of diluted sample (100 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 1 mM EDTA, 0.1% (m /v) casein, 0.015 (m/v) Therosal, 1% (m/v) Triton X-100, 100 µl/ml yeast extract).

The dishes were closed and incubated at 37ºC for two hours, after which the solution was removed by suction and the wells were washed with 400 microliters of buffer (phosphate-buffered saline (PBS) containing 0.05% Tween 20). Washed wells were tested with 200 µl mouse anti-human IgG-HRP conjugate contained in a solution of otroconjugate diluent (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 50% (v/v) fetal bovine serum , 1% heat-treated horse serum, 1 mM K3Fe(CN)6 for 1 hour at 37ºC, the solution is removed by suction and the wells are washed with washing buffer which is also removed by suction.To determine the amount of bound enzyme conjugate, 200 µl of substrate is added solution (10 mg of phenyldiamine dichloride per 5 ml of developing solution). The developing solution contains 50 mM sodium citrate adjusted to pH 5.1 with phosphoric acid and 0.6 µl/ml of 30% H2O2. Dishes containing the substrate solution were incubated in the dark for 30 minutes at room temperature, and the reactions are stopped by the addition of 50 µl/ml 4N sulfuric acid and the ODS is determined.

The examples below show that microtiter plate ELISA testing using HCV C100-3 antigen has a high degree of specificity, as evident from an initial reactivity rate of about 1%, with a repeat reactive rate of about 0.5% of random donors. The test is able to detect the immune response in the sub-acute phase of the infection, as well as during the chronic phase of the disease. In addition, the test is capable of detecting some samples that are negative in other tests for NANBH; these samples come from individuals with a history of NANBH or donors affected by NANBH transmission.

In the following examples, the tags are used:

ALT alanine amino transferase

Anti-HBc antibody against HBc

Anti-HBsAg antibody against HBsAg

HBc hepatitis B core antigen

AbsAg hepatitis B surface antigen

IgG immunoglobulin G

IgM immunoglobulin M

IU/L interNaClonal units/liter

NA is not available

NT not tested

N sample size

Neg negative

OD optical density

Pos positive

S/CO signal/clip

SD standard deviation

x average or mean value

WNL in normal range

IV.I.1. HCV infection in the random donor population

A pool of 1056 samples (fresh serum) from random blood donors obtained from the Irwin Memorial Blood Bank, San Francisco, California. Test results obtained with these samples are summarized in a histogram showing the distribution of OD values (Figure 37). As seen in Figure 37, 4 samples read above 3, 1 sample read between 1 and 3, 5 samples read between 0.4 and 1, and the remaining samples read below 0.4, with over 90% of all samples read below 0.1.

The results for non-reactive random samples are given in Table 5. Using cut-off values equal to the mean plus 5 standard deviations, ten samples out of 1056 (0.95%) were initially reactive. Of these, five samples (0.47%) were repeatedly reactive when tested a second time by ELISA.

Table 5 also shows the ALT and anti-HBd status for each repeat reactive sample. Of particular interest is the fact that all five repeated reactive samples are negative in both NANBH replacement tests, although they are positive in the HCV ELISA.

Table 5

Results of reactivity of random samples

N=1051

ξ=0.049*

SD=+ 0.074

Trimmed: ξ + 5SD=0.419 (0.400+Negative Control)

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* samples that read above 1.5 are not included in the mean vr. and SD

** ALT above 68 IU/L is the upper normal limit

*** Anti-HBc below 0.535 (competition test) are considered positive

**** WN I: within normal limits

IV.I.2. Chimpanzee serum samples

Serum samples from 11 chimpanzees were tested with the HCV C100-3 ELISA. Four of these chimpanzees were infected with NANBH from infected Factor VIII (mostly Hutchinson species), following a description of the procedure with Dr. Daniel Brandey at the Centers for Disease Control. As a control, four other chimpanzees were infected with HAV and three with HBV. Serum samples were obtained at various times after infection.

The results summarized in Table 6 show documented antibody seroconversion in all chimpanzees infected with Hutchinson type NANBH. Following the acute phase of infection (as evidenced by a significant rise and subsequent return to normal ALT levels), antibodies to HCV C100-3 become detectable in the serum of 4/4 NANBH-infected chimpanzees. As previously shown, these samples were positive by Western analysis and RIA, as discussed in Section IV.B.3. In contrast, none of the control chimpanzees infected with HAV or HBV showed evident reactivity in the ELISA.

Table 6 Chimpanzee serum samples

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IV.I.3. Panel I: Screening of infectious sera from chronic human NANBH carriers

A panel consisting of 22 unique samples, each in duplicate, was coded, with a total of 44 samples. The samples were from verified infectious serum of chronic NANBH carriers, infectious serum of enrolled donors and infectious serum from acute phase NANBH patients. In addition, samples were from highly selected negative controls and other disease controls. This panel was obtained from Dr. H. Alter, Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland. The panel was constructed by Dr. Alter several years ago and used as a qualifying panel to examine alleged NANBH. The entire panel was tested twice with the ELISA experiment and the results were sent to Dr. Alter for confirmation. The validation results are shown in Table 7. Although the table shows the results of only one set of duplicates, some values were obtained for each of the duplicate samples.

As shown in Table 7, the 6 sera tested to be infectious in the chimpanzee model were strictly positive. The seventh infectious serum corresponds to the sample for the acute NANBH case and is not reactive in this ELISA test. A sample of affected donors with normal ALT levels and with uncertain results in experiments with chimpanzees are not reactive. Three other serial samples from a single individual with acute NANBH were also nonreactive. All samples coming from highly selected controls, obtained from donors who gave blood at least 10 times without hepatitis procedures, were ELISA non-reactive. Finally, four of the samples previously tested positive in alleged other NANBH trials were not confirmed here. These four samples were negative with HCV ELISA.

Table 7

H. Alters panel 1:

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IV.I.4. Panel 2: Donor/recipient NANBH

The coded panel was composed of 10 uncertain cases of donor-recipient transfusion-related NANBH, with a total of 188 samples. Each case is composed of some or all donors per recipient, and serial samples (including 3, 6, and 12 months post-transfusion) of recipients. Pre-transfusion recipient samples were also included. The coded panel was provided by Dr. H. Alter, of the NIH, and the results were sent to him for summary.

The results, summarized in Table 8, show ELISA antibody-detected superconversion in 9 out of 10 cases of transfused NANBH. Samples in 4 cases (where no superconversion was detected) consistently reacted weakly in ELISA. Two of the 10 recipient samples were reactive 3 months after transfusion. After six months, 8 recipient samples were reactive; and after 12 months, with the exception of case 4, all were reactive. In addition, at least one positive donor antibody is found in 7 out of 10 cases, with case 10 having two positive donors. Also, in case 10, the recipient who had previously donated blood was positive for HCV antibodies. One month after this, the recipient fell to a borderline reactive level, while when taking blood after 4 and 10 months, it was representative of the individual's previous infection with HCV.

ALT and HBc for reactive, eg positive samples, are summarized in Table 9. As can be seen from the table, 1/8 donor samples were negative for other markers and reactive for HCV antibody ELISA. On the other hand, the recipient of the samples (followed 12 months after transfusion) is elevated in ALT, positive anti-HBc or both.

Table 8

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* 1 month, ** 4 months, *** 10 months

Table 9

ALT and HBC status for reactive samples in H. Alter panel 1

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* ALT 45 IU/L is above the upper limit

** anti-HBc 50% (competitiveness test) is considered positive

*** before bleeding and 3 months samples were negative for HBc

IV.I.5. Determination of HCV infection in high-risk sample groups

Samples from high-risk groups were observed using ELISA to determine reactivity to HCV C100-3 antigen. The results are summarized in Table 10.

As shown in the table, the samples with the highest reactivity were obtained from hemophiliacs (76%). In addition, samples from individuals with elevated ALT and positive for anti-HBc are 51% reactive. A value consistent with the value expected from clinical data and NAMNH prevails in this group. Antibodies to HCV were also higher in the blood of donors with elevated ALT, hepatitis B core antibody-positive blood donors, and blood donors rejected for reasons other than high ALT or anti-core antibodies, when compared to random volunteers. .

Table 10

NANBH high-risk sample groups

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IV.I.6. Parallel studies using anti-IgG or anti-IgM monoclonal antibodies or polyclonal antibodies as second antibody in HCV C100-3 ELISA

The sensitivities of Elisa assays using an anti-IgG monoclonal conjugate were compared to those obtained using either an anti-IgM monoclonal conjugate or replacing both with a polyclonal antiserum said to be specific for light and heavy chains. The following tests were performed.

IV.I.6.a. Serial samples from seroconverters

Serial samples from three cases of NANB seroconverters were studied in the HCV C100-3 ELISA assay using an enzyme conjugate of either anti-IgG monoclonal alone or in combination with anti-IgM monoclonal or using polyclonal antiserum. Samples were obtained from Dr. Cladd Stevens, N.Y. Blood Center, N.Y. The sample histories are shown in Table 11.

The results obtained using the anti-IgG monoclonal antibody-enzyme conjugate are shown in Table 12. The data show that strong reactivity is detected in samples 1-4, 2-8 and 3-5, cases 1, 2 and 3, respectively.

The results obtained using the combination of anti-IgG monoclonal conjugate and anti-IgM conjugate are shown in table 13. Three different ratios of anti-IgG to anti-IgM were tested; 1:10000 dilution of anti-IgG was constant. The dilutions tested for the anti-IgM monoclonal conjugate were 1:30000, 1:60000 and 1:120000. The data show that in agreement with the study of the anti-IgG itself, initial strong reactivity was detected in samples 1-4, 2-8 and 3-5.

Results obtained with ELISA using anti-IgG monoclonal conjugate (1:10000 dilution) or Tago polyclonal conjugate (1:80000 dilution) or Jackson polyclonal conjugate (1:80000 dilution) are shown in Table 14.

The data show that initial strong reactivity was detected in samples 1-4, 2-8 and 3-5 using all three conjugations; Tago polyclonal antibodies give the lowest signals.

The results presented above show that all three configurations detect reactive samples at the same time after the acute phase of the disease (as evident by dedicated ALT). However, the result shows that the sensitivity of the HCV C100-3 ELISA using the anti-IgG monoclonal-enzyme conjugate is equal to or better than that obtained using other enzyme conjugate configurations tested.

Table 11

Description of samples from the Cladd Stevens panel

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Table 12

ELISA results using anti-InG monoclonal conjugate

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Table 13

ELISA results obtained using anti-IgG and anti-IgM monoclonal conjugate

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Table 14

ELISA results obtained using polyclonal conjugates

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IV.I.6.b. Random donor samples

Samples from random donors (see Chapter IV.I.) were tested for HCV infection using the C100-3 ELISA, in which the antibody-enzyme conjugate was an anti-IgG monoclonal conjugate or a polyclonal conjugate. The total number of tested samples was 1077 and 1056 for polyclonal and monoclonal conjugate, respectively. The summarized results are shown in table 15, and the distribution of the sample is shown in the histogram in figure 44.

The calculation of the average and standard deviation was performed by excluding the samples that gave a signal above 1.5, ie 1073 values were used for the calculation of the polyclonal conjugate and 1051 for the anti-IgG monoclonal conjugate. As seen in Table 15, when the polyclonal conjugate is used, the average shift is from 0.0493 to 0.0931 and the standard deviation is increased from 0.074 to 0.0933.

However, the results also show that if the x+5SD criterion is used to define assay cutoff, the polyclonal enzyme-conjugate configuration in ELISA requires a higher overlap value. This indicates a reduced specificity of the assay compared to the monoclonal system. Additionally, as can be seen from the histogram in Figure 44, a greater separation of results between the negative and positive distributions is obtained when random blood donors are tested in an ELISA using an anti-IgG monoclonal conjugate compared to an assay using a commercial polyclonal label.

Table 15

Comparison of two ELISA configurations in random blood donor sample testing

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IV.J. Detection of HCV seroconversion of NANBH patients from various genetic locations

Serum from patients suspected of having NANBH based on elevated ALT levels, and who were negative for HAV and HBV assays, was tested using RIA essentially as described in Chapter IV.D., except that the HCV C100-3 antigen was used as a test antigen in a microtiter dish. As can be seen from the results given in Table 16, the RIA detects positive samples in a high proportion of cases.

Table 16

Seroconversion frequencies for anti-C100-3 among NANBH patients in various countries of the world

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IV.K. Detection of HCV seroconversion in patients with “newly acquired” NANBH

Serum that was obtained from 100 patients with NANBH, for whom the usual route of transmission of transfusions (eg, no transfusion, IV drug use, intercourse, etc. are declared as risk factors) was obtained from Dr. Alter of the Center for Disease Control et al. Diestag from Harvard University. These samples were tested using RIA essentially as described in Section IV.D., except that the HCV C100-3 antigen used as the antigen was plated on microtiter plates. The results show that out of 100 serum samples, 55 contain antibodies and react immunologically with the HCV C100-3 antigen.

The results described above indicate that “generally acquired” NANBH is also induced with HCV. However, as shown here, HCV is associated with the flavivirus most transmitted by arthropods, and it is suggested that HCV transmission in “generally acquired” cases also results in arthropod transmission.

IV.L. Comparison of HCV antibody uptake and surrogacy markers in donors affected by NANBH transmission

A prospective study was performed to determine whether a recipient of blood from a suspected NANBH donor who develops NANBH seroconverts to anti-HCV antibody positive. Blood donors were tested for surrogate markers of abnormalities commonly used as markers for NANBH infection, eg, elevated ALR levels and the presence of anti-nuclear antibodies. Additionally, donors were also screened for the presence of anti-HCV antibodies. Determination of the presence of anti-HCV antibodies was determined using an immunoassay as described in Chapter IV.K. The results of the study are presented in table 17, which shows: number of patients (column 1); the presence of anti-HCV antibodies in the patients' serum (column 2); number of doses received by the patient, with each dose being from a different donor (clone 3); the presence of anti-HCV antibodies in the donor's serum (column 4); and donor surrogate abnormality (column 5) (NT or - means not tested) (AT is elevated transaminase and ANTI-HBc is anti-core antibody).

The results in Table 17 show that the HCV antibody test is more accurate in detecting infected donor blood than surrogate labeling tests. None of the 10 patients who developed NANBH symptoms tested positive for anti-HCV antibody seroconversion. Of the 11 suspected donors (patient 6 received 6 donations from two different individuals suspected of being NANBH carriers), 9 were positive for anti-HCV antibodies and 1 was borderline positive and therefore equivocal (donor for patient 1) . In contrast, using an elevated ALT test, 6 of 10 donors tested were negative. It is noted that in three cases (providers to patients 8, 9 and 10), the ALT test and the anti-HBc test give inconsistent results.

Table 17

Development of anti-HCV antibodies in patients receiving blood from donors suspected of being carriers of NANBH

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IV.M. Amplification for cloned HCV cDNA sequences using PCR and primers derived from conserved regions of the Flavivirus genomic sequence

The results presented above indicate that the HCV flavivirus or a virus similar to it, allows a strategy for cloning uncharacterized HCV cDNA sequences using the PCT technique and primers derived from the coding region of amino acid sequences in flaviviruses. Generally, one of the primers is derived from a defined HCV genomic sequence, and other primers that bypass the region of the unsequenced HCV polynucleotide are derived from a conserved region of the flavivirus genome. Flavivirus genomes are known to contain conserved sequences in NS1 and Epolypeptides, which are iodinated in the 5'-region of the flavivirus genome. The corresponding sequences encoding these regions lie upstream of the HCV cDNA sequences derived from this region of the HCV genome, upstream primers derived from conserved sequences in these flaviviral polypeptides are indicated. Downstream primers were derived from the upstream end of a known portion of HCV cDNA.

Due to the degeneracy of the code, there are likely to be discrepancies between flavivirus probes and the corresponding HCV genomic sequence. Therefore, a strategy similar to that described by Lee (1988) was used.

This procedure uses mixed oligonucleotide primers complementary to the reverse translation products of the amino acid sequence; sequences in the mixed primers taken into account for each coding degeneracy for the conserved amino acid sequence.

Three sets of primers were created, based on amino acid homologues found in several flaviviruses, including Dengue-2.4 (D-2.4), Japanese encephalitis virus (JEV), yellow fever (YF), and West Nile virus (WN). The primer mixture derived from the most upstream conserved sequence (5'-1) is based on the amino acid sequence gly-trp-gly, which is partially conserved in the aps-arg-gly-trp-gly-aspN sequence reported in E protein D-2, JEV , YF and WN. The following primer mixture (5'-2) is based on the downstream conserved sequence in the E protein, phe-asp-gly-asp-ser-tvr-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 primer mixture (5'-3) is based on the amino acid sequence arg-ser-cys, which is part of the conserved sequence cys-cys-arg-ser-cys in NS1 protein D-2, D-4, JEV, YF and WN . The individual primers that create the mixture in 5'-3 are shown in Figure 45. In addition to the variable sequences derived from the conserved region, each example in each mixture also contains the sequence encoding the sites for the restriction enzymes, HinIII, MboI and EcoRI.

The downstream primer, ssc5h20A, is derived from the nucleotide sequence in clone 5h, which contains HCV cDNA with sequences overlapping those of clones 14i and 11b. the sequence of ssc5h20A is

5' GTA ATA TGG TGA CAG AGT CA 3'

the alternative primer ssc5h32A can also be used. This primer is derived from the sequence in clone 5h, and additionally contains a nucleotide at the 5'-end that creates a restriction enzyme site, which facilitates cloning. The sequence of ssc5h32A is

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

The PCR reaction, initially described by Saiki et al. (1986) was performed essentially as described by Lee et al. (1988), except that the template for cDNA and RNA is isolated from the liver of an HCV-infected chimpanzee, as described in Section IV.C.2. or from viral particles isolated from HCV-infected chimpanzee serum as described in section IV.A.1. Additionally, the cleavage conditions are less strict in the first round of application (0.6 M NaCl and 25ºC), although the part of the primer that will be cleaved to the HCV sequence is only 9 nucleotides and may be poorly joined. However, if ssc5h34A is used, additional sequences not derived from the HCV genome tend to stabilize the primer-template hybrid. After the first round of examples, the cleavage conditions may be more stringent (0.066 M NaCl and 32ºC-37ºC) although the applied sequences now contain regions that are complementary or duplicate primers. In addition, the first 10 application cycles are performed with Klenow enzyme I, under appropriate PCR conditions for that enzyme. After the completion of these cycles, the samples are extracted and treated with Taq polymerase, according to the instructions of the equipment provided by Cetus-Perkin-Elmer.

After administration, amplified HCV cDNA sequences are detected by hybridization using a probe derived from clone 5h. this probe is derived from sequences upstream of those used to derive the primer, and does not overlap the clone 5h sequence of the derived primer. The rehearsal 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 HCV cDNA library from chimpanzee liver with NANBH infection

The HCV library was created from the chimpanzee liver from which the HCV cDNA library was created in Chapter IV.A.1. The technique for creating the library is similar to that in Chapter IV.A.24. except that a different RNA and 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 nucleotide sequence of overlapping HCV cDNA in clone k9-1 to cDNA in clone 11b

Clone k9-1 was isolated from an HCV cDNA library generated from the liver of a NANBH-infected chimpanzee, as described in Section IV.A.25. The library was screened for clones overlapping the sequence in clone 11 b, using a clone overlapping clone 11 b at the 5'-terminus, clone 11e. The sequence of clone 11b is shown in Figure 23. Positive clones were isolated at a frequency of 1 in 500000. One isolated clone k9-1 was subjected to further study. The overlapping nature of the HCV cDNA in clone k9-1 at the 5' end of the HCV cDNA sequence in Figure 26 was confirmed by testing the clone with Alex46; this last clone contains an HCV cDNA sequence of 30 base pairs corresponding to those base pairs at the 5'-terminus of the HCV cDNA in clone 14i, described above.

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

The HCV cDNA sequence in clone k9-1 is related to that of the clones described in Section IV.A.19. To create a composition of the HCV cDNA sequence with the k9-1 sequence placed upstream of the sequence shown in Figure 32. The composition of the HCV cDNA including the k9-1 sequence and the amino acids encoded therein are shown in Figure 47.

The sequence of amino acids encoded in the 5'-region of HCV cDNA shown in Figure 47 is compared with the corresponding region of the Dengue virus waist, described above, with respect to the profile of the hydrophobicity and hydrophilicity regions. This comparison shows that the polypeptide from HCV and Dengue are encoded in this region, which corresponds to the region encoding NS1 (or part thereof), and have a similar hydrophobic/hydrophilic profile.

The information provided below allows for the identification of HCV professions. Isolation and characterization of other HCV lineages can be performed by isolating nucleic acids from body components containing viral particles, yielding cDNA libraries using polynucleotide probes based on the HCV cDNA probes described below, screening libraries for clones containing the HCV cDNA sequences described below, and comparing HCV cDNA from the new cDNA isolates described below. Polypeptides encoded herein or in the viral genome can be monitored by immunobiological cross-reactivity using the polypeptides and antibodies described above. Occupations that fall within the HCV parameters as described in the definitions section, ahead, are easily identified. Other procedures for identifying HCV strains will be apparent to those skilled in the art based on the information provided herein.

Citation of the best applicant known way of commercial use of the invention

The invention in various manifestations described here has many industrial examples, some of which follow. HCV cDNA can be used to create a probe to detect HCV nucleic acids in samples. Probes derived from cDNA can be used to detect HCV nucleic acids in, for example, chemical synthetic reactions. These can also be used in testing programs for antiviral agents to determine the effect of the agent in inhibiting viral replication in cell culture systems and animal model systems. HCV polynucleotides in humans can thus serve as a basis for the diagnosis of HCV infections in humans.

In addition, the cDNAs provided herein provide the information and means for the synthesis of polypeptides containing HCV epitopes. These polypeptides are useful in determining antibodies to HCV antigens. A series of tests for HCV infection, based on recombinant polypeptides containing HCV epitopes are described here and will find commercial application in the diagnosis of HCV causing NANBH in testing donor blood banks for HCV-induced hepatitis, and also for detecting contaminated blood from infectious donor blood. Viral antigens will also be used to monitor the efficacy of antiviral agents in animal model systems. In addition, the polypeptides derived from HCV cDNA described herein will have application as vaccines for the treatment of HCV infection.

Polypeptides derived from HCV cDNA, in addition to the examples mentioned above, are also applicable for raising anti-HCV antibodies. Thus, they can be used in anti-HCV vaccines. However, antibodies produced as a result of immunization with HCV polypeptides are also 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. Anti-HCV antibodies can also be used to monitor the efficacy of antiviral agents in testing programs where these agents are tested in tissue culture systems. They can also be used for passive immunotherapy, and diagnosis of HCV causing NANBH by allowing detection of viral antigens in blood donors and recipients. Another important application for anti-HCV antibodies is in affinity chromatography for the purification of viruses and viral polypeptides. Purified virus and viral polypeptide preparations can be used in vaccines. However, purified virus may be applicable for developing cell cultures in which HCV replicates.

Cell culture systems containing HCV infected cells will have many applications. They can be used for relatively large production of HCV which is normally a low titer virus. These systems will also be used for molecular biology of viruses and lead to the development of anti-viral agents. Cell culture systems will also be applicable in testing the efficacy of antiviral agents. Additionally, HCV permeabilized cell culture systems are useful for producing attenuated HCV strains. Conventionally, anti-HCV antibodies and HCV polypeptides, whether native or recombinant, can be packaged into a kit.

A method used to isolate HCV cDNA, which includes obtaining a cDNA library derived from an infected tissue of an individual in an expression vector iii selecting clones that produce expression products that react immunologically with antibodies in body components containing antibodies from another infected individual and not from non-infected individuals, it can also be used to isolate cDNA from other, so far uncharacterized disease-causing agents, which are composed of a genomic component. This can lead to the isolation and characterization of these agents and diagnostic reagents and vaccines for these other disease-causing agents.

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Claims (77)

1. Polypeptide, characterized by the fact that it is in a completely isolated form that contains an uninterrupted sequence of at least 10 amino acids that is encoded by the hepatitis C virus (HCV) and that contains an antigenic determinant, whereby HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. 2. Polypeptide according to claim 1, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous with the sequence of 859 amino acids, according to Figure 14. 3. Polypeptide according to claim 1 or 2, characterized in that it contains at least 15 amino acids. 4. Polypeptide according to any of the preceding claims, characterized in that it is obtained by expression of recombinant DNA. 5. Polypeptide according to any claim from 1 to 3, characterized in that it is obtained by chemical synthesis. 6. The polypeptide according to any one of claims 1 to 5, characterized in that the uninterrupted sequence of amino acids conforms to Figure 14. 7. The polypeptide according to any one of claims 1 to 5, characterized in that the uninterrupted sequence of amino acids conforms to Figure 47. 8. The polypeptide according to any one of claims 1 to 5, characterized in that the continuous sequence is encoded in the lambda-gtll cDNA library deposited in the American Type Culture Collection (ATCC) under accession number 40394. 9. The polypeptide according to any one of claims 1 to 8, characterized in that the continuous sequence is that of a non-structural viral protein. 10. The polypeptide according to any one of claims 1 to 5, 7 or 8, characterized in that the continuous sequence is that of a structural viral protein. 11. A polypeptide according to any of claims 1 to 8, characterized in that its sequence is shown in any of Figures 1, 3 to 32, 36, 46 and 47, or whose sequence is encoded in a polynucleotide that can selectively hybridize with a polynucleotide that is shown in any figure 1, 3 to 32, 36, 46 or 47. 12. The polypeptide according to any one of claims 1 to 11, characterized in that the polypeptide is bound to a solid phase. 13. Accessories for performing an immunological analysis, characterized in that it contains the polypeptide according to any of claims 1 to 12 in a suitable container. 14. A mixture, characterized in that it contains a completely isolated polypeptide according to any one of claims 1 to 11, which is mixed with a pharmaceutically acceptable excipient. 15. Vaccine, indicated by the fact that its composition complies with requirement 14. 16. Kit for immunological analysis intended for the detection of antibodies to the hepatitis C virus (HCV) (anti-HCV antibody), characterized by the fact that it includes: (a) obtaining a polypeptide comprising an antigenic determinant capable of binding to said anti-HCV antibody, wherein said antigenic determinant comprises a continuous amino acid sequence encoded by the HCV genome and wherein HCV is characterized by: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. (b) incubating the biological sample with said polypeptide under conditions that enable the formation of an antibody-antigen complex; and (c) determining whether an antigen-antibody complex containing said polypeptide has formed. 17. Immunological analysis according to claim 16, characterized in that the polypeptide is obtained by expression of recombinant DNA. 18. Immunological analysis according to claim 16, characterized in that the polypeptide is obtained by chemical synthesis. 19. Immunological analysis according to any one of claims 16 to 18, characterized in that said polypeptide is attached to a solid support. 20. Immunological analysis according to any of claims 16 to 19, characterized in that said antigen-antibody complexes are detected by incubating the complex with a labeled anti-human immunoglobulin antibody. 21. Immunological analysis according to claim 20, characterized in that said anti-human immunoglobulin antibody is labeled with an enzyme. 22. Immunological analysis according to any claim from 16 to 21, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous with the 859 amino acid sequence, according to Figure 14. 23. Immunological analysis according to any claim from 16 to 22, characterized in that the continuous sequence consists of at least 10 amino acids. 24. Immunological analysis according to any claim from 16 to 23, characterized in that the continuous sequence consists of at least 15 amino acids. 25. The immunoassay according to any one of claims 16 to 24, characterized in that the unbroken sequence is shown in Figure 14. 26. The immunoassay according to any one of claims 16 to 24, characterized in that the unbroken sequence is shown in Figure 47. 27. The immunoassay according to any of claims 16 to 24, characterized in that the continuous sequence shown in any of Figures 1, 3 to 32, 36, 46 and 47, or whose sequence is encoded in a polynucleotide that can selectively hybridize to the polynucleotide which is shown in any figure 1, 3 to 32, 36, 46 or 47. 28. The immunoassay according to any one of claims 16 to 27, characterized in that the continuous sequence is encoded in the lambda-gt11 cDNA library deposited in the American Type Culture Collection (ATCC) under accession number 40394. 29. An immunoassay according to any one of claims 16 to 28, characterized in that the continuous sequence is that of a non-structural viral protein. 30. An immunoassay according to any of claims 16 to 24 or 26 to 28, characterized in that the continuous sequence is that of a structural viral protein. 31. An immobilized polypeptide for use in an immunoassay according to any one of claims 16 to 30, characterized in that the polypeptide contains an antigenic determinant that can bind to an anti-HCV antibody as set forth in claim 16. 32. A polynucleotide that is in a completely isolated form, characterized by the fact that it contains an uninterrupted sequence of nucleotides, which can be selectively hybridized to the genome of the hepatitis C virus (HCV) or its complement, wherein HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. 33. Polynucleotide according to claim 32, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous to the sequence of 859 amino acids, according to Figure 14. 34. Polynucleotide according to claim 32 or 33, characterized in that the uninterrupted sequence consists of at least 10 nucleotides. 35. Polynucleotide according to claim 34, characterized in that the continuous sequence consists of at least 15 nucleotides. 36. Polynucleotide according to claim 35, characterized in that the continuous sequence consists of at least 20 nucleotides. 37. A polynucleotide according to any of claims 32 to 36, characterized in that said polynucleotide is a DNA polynucleotide. 38. A polynucleotide according to any one of claims 32 to 36, characterized in that said polynucleotide is an RNA polynucleotide. 39. The polynucleotide according to any one of claims 32 to 38, characterized in that it is bound to a solid phase. 40. A sample comprising a polynucleotide according to any one of claims 32 to 39, further comprising a detectable label. 41. An analysis kit, characterized in that it contains a polynucleotide probe according to any of claims 32 to 40, in a suitable container. 42. Polymerase chain reaction (PCR) kit, characterized in that it contains a pair of initiators capable of initiating cDNA synthesis in a PCR reaction, wherein each of said initiators is a polynucleotide according to any of claims 32 to 37. 43. PCR kit according to claim 42, characterized in that it further includes a polynucleotide probe that can selectively hybridize the region of the HCV genome between, wherein the HCV sequences from which the initiators are derived are not included. 44. Method for carrying out the polymerase chain reaction, characterized in that the initiators are a pair of polynucleotides in accordance with any of claims 32 to 37. 45. A method of analyzing a sample in the presence or absence of HCV polynucleotide, characterized in that it includes the following: (a) bringing the sample into mutual contact with a probe comprising a polynucleotide according to any one of claims 32 to 40, under conditions that enable said probe to hybridize to the HCV polynucleotide or its complement in the sample; and (b) determining whether polynucleotide duplicates comprising said probe have been formed. 46. DNA polynucleotide encoding a polypeptide, characterized in that the polypeptide contains an uninterrupted sequence of at least 10 amino acids encoded by the genome of the hepatitis C virus (HCV) and contains an antigenic determinant, whereby HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. 47. DNA nucleotide according to claim 46, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous with the sequence of 859 amino acids, according to Figure 14. 48. DNA polynucleotide according to claim 46 or 47, characterized in that said continuous sequence encodes at least 15 amino acids. 49. The DNA polynucleotide according to any one of claims 46 to 48, characterized in that the continuous sequence is shown in Figure 14. 50. The DNA polynucleotide according to any one of claims 46 to 48, characterized in that the continuous sequence is shown in Figure 47. 51. The DNA polynucleotide according to any one of claims 46 to 48, characterized in that the continuous sequence is encoded in the lambda-gtl 1 cDNA library deposited in the American Type Culture Collection (ATCC) under accession number 40394. 52. The DNA polynucleotide according to any of claims 46 to 48, characterized in that the continuous sequence shown in any of Figures 1, 3 to 32, 36, 46 and 47, or whose sequence is encoded in a polynucleotide that can selectively hybridize with the polynucleotide which is shown in any figure 1, 3 to 32, 36, 46 or 47. 53. The DNA polynucleotide according to any one of claims 46 to 52, characterized in that said continuous sequence is that of a non-structural viral protein. 54. The DNA polynucleotide according to any of claims 46 to 58 or 50 to 52, characterized in that said continuous sequence is that of a structural viral protein. 55. Recombinant vector, characterized in that it contains a coding sequence comprising a DNA polynucleotide according to any one of claims 46 to 54. 56. A host cell that has been transformed with a recombinant vector according to claim 55, characterized in that the coding sequence is operably linked to a control sequence that can enable the expression of the coding sequence of the host cell. 57. A method for obtaining a recombinant HCV polypeptide, characterized in that it includes incubating the host cell according to claim 56, under conditions that enable the expression of the coding sequence. 58. A mixture of anti-HCV antibodies, characterized in that it contains antibodies that bind said antigenic determinant of the polypeptide according to any of claims 1 to 12 which is (a) a purified preparation of polyclonal antibodies or (b) a mixture of monoclonal antibodies. 59. The mixture according to claim 58, characterized in that the anti-HCV antibodies are bound to the solid phase. 60. Kit for immunological analysis, characterized in that it contains an anti-HCV antibody according to claim 58 or 59 in a suitable container. 61. Method of immunological analysis for detecting HCV antigen in a sample, indicated by including: (a) obtaining an anti-HCV antibody according to claims 58 or 59; (b) incubating the sample with said mixture of anti-HCV antibodies under conditions that enable the formation of an antibody-antigen complex; and (c) determining whether an antibody-antigen complex comprising an anti-HCV antibody has been formed. 62. A polypeptide containing a continuous sequence of at least 10 amino acids encoded by the hepatitis C virus (HCV) genome and containing an antigenic determinant, indicated by the fact that said continuous sequence is combined with a non-HCV amino acid sequence, whereby HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. 63. Polypeptide according to claim 62, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous with the sequence of 859 amino acids, according to Figure 14. 64. Polypeptide according to claim 62 or 63, characterized in that the non-HCV amino acid sequence contains a signal sequence. 65. Polypeptide according to claim 62 or 63, characterized in that the non-HCV amino acid sequence contains the amino acid sequence from beta-galactosidase or superoxide dismutase. 66. The polypeptide according to claim 62 or 63, characterized in that the non-HCV amino acid sequence comprises a particle-forming protein. 67. The polypeptide according to claim 66, characterized in that the particle-forming protein comprises hepatitis B surface antigen. 68. The polypeptide according to any one of claims 1 to 12 or 62 to 67 for use in the production of an anti-HCV antibody, characterized in that it involves administering the polypeptide to a mammal in an amount sufficient to elicit an immune response. 69. A mixture containing a polypeptide according to any one of claims 62 to 67, characterized in that the polypeptide is mixed with a pharmaceutically acceptable excipient. 70. Vaccine, characterized by the fact that it corresponds to claim 69. 71. A method for the growth of hepatitis C virus (HCV), characterized in that it includes cells infected with HCV, and the growth of said cells in vitro, wherein said HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14. 72. The method according to claim 71, characterized in that said polyprotein contains an amino acid sequence that is at least 60% homologous with the sequence of 859 amino acids, according to Figure 14. 73. The method according to claim 71 or 72, characterized in that said cells contain primary cells. 74. The method according to claim 71 or 72, characterized in that said cells comprise a cell line. 75. The method according to claim 71 or 72, characterized in that said cells are hepatocytes or macrophages. 76. Hepatitis C virus (HCV) immunoassay antigen that is bound to a solid phase, characterized in that HCV is characterized by the following: (i) positively positioned RNA genome; (ii) said genome contains an open loading frame (ORF) encoding a polyprotein; and (iii) said polyprotein contains an amino acid sequence that is at least 40% homologous with the 859 amino acid sequence shown in Figure 14; said antigen contains an antigenic determinant that is immunologically reactive with an anti-HCV antibody, where (a) said HCV antibody immunologically reactive against the antigenic determinant (i) which is encoded by the HCV cDNA insert in the lambda-gl 1 library which is deposited in the American Type Culture Collection (ATTC) under accession number 40394 or (ii) which found in Figure 47; and (b) said reference antigenic determinant is immunologically reactive with the serum of humans infected with HCV. 77. HCV antigen according to claim 76, characterized in that said reference antigenic determinant is shown in Figure 14.