SI8812138A - Nanbv diagnostics and vaccines - Google Patents

Abstract

The present invention provides for a family cDNA sequences derived from hepatitis C virus (HCV). These sequences encode antigens that react immunologically with antibodies present in subjects with non-A and non-B hepatitis (NANBH), which are usually found in subjects infected with hepatitis A virus (HAV) or virus Hepatitis B (HBV) is absent and also absent in control subjects. Comparison of these cDNA sequences with sequences in Genebank and hepatitis sequences delta virus (HDV) and HBV show absence of essential homology. Comparison of coded amino acid sequences in the cDNA sequences of Flavivirus indicate that HCV Flavivirus or a virus like it. HCV sequences cDNAs are useful for the construction of polynucleotides probes and for the synthesis of a polypeptide that is useful for immunoassays. Both polynucleotide probes and polypeptides may be useful in the diagnosis of HCV-elicited NANBH and for testing types of blood banks and donations at HCV infection. In addition, cDNA sequences can be useful for the synthesis of immunogenic polypeptides that can be used in vaccines for treatment, prophylaxis and / or therapy for HCV infection. In the cDNA sequence encoded polypeptides are also useful for increasing numbers antibodies against HCV and for purification antibodies directed against HCV antigens. These antibodies should be useful in immunoassays for detection of antigens bound to NANBH subjects and in giving blood bank. These antibodies are just like that useful for treating NANBH subjects. Reagents, provided by the present invention also provide isolation of NANBH agents and progression of these agents in the tissue of the culture system. They further provide reagents that are useful for antiviral testing agents for HCV, especially in culture tissue or in animal model systems.

Description

NANBV DIAGNOSTICS AND VACCINES

The field of technology

The invention is in the field of biomolecular engineering.

A technical problem

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

The state of the art

Non-A and non-B hepatitis (NANBH) is a transmissible disease or family of diseases that are believed to be caused by this virus and are different from other liver diseases caused by viruses, including those caused by known hepatitis viruses, e.g. hepatitis A virus (HAV), hepatitis B virus (HBV) and delta hepatitis virus (HDV), as well as cytomegalovirus-induced hepatitis (CMV) or Epsten-Barr virus (EBV). NANBH was originally identified in persons who received the transfusion. Transmission from human to chimpanzee and serial transmission in chimpanzees proves that NANBH is caused by a transmissible infectious agent or agents. However, the transmission agent responsible for NANBH has not yet been identified, and the number of agents causing the disease is unknown.

Epidemiological evidence suggests that there may be three types of NANBH:

1. Blood-resistant epidemic type

2. Blood or needle-associated type

However, the number of agents that can cause NANBH remains unknown.

Clinical diagnosis and identification of NANBH is performed primarily by excluding other viral markers. Among the methods used to detect said NANBV antigen and antibody are agar-gel diffusion, anti-immunoelectrophoresis, immunofluorescence microscopy, immuno-electron microscopy, radioimmunoassay and enzyme-linked immunosorbent assay. However, none of the above methods is sensitive, specific and reproductive enough to be used as a diagnostic test for NANBH.

For now, it is not clear or unanimous how to identify or specify the antigen and antibody of systems associated with NANBH agents. This is at least partly caused by pre-HBV or concomitant infection of the individual with NANBH and the known complexity of HBV-related solution and partial antigens, as well as by the integration of HBV DNA into the liver cell genome. In addition, it is also possible that NANBH is caused by the action of more than one infectious agent, as well as the possibility that NANBH will not be diagnosed. In addition, it is unclear what serological studies in the serum of a patient with NANBH detect. Agar-gel diffusion and anti-immune electrophoresis studies have been hypothesized to detect autoimmune responses or non-specific protein interactions that sometimes exist between serum species and do not represent a specific NANBV antigen-antibody reaction.

Immunofluorescence and enzyme-linked immunosorbent and radioimmunoassays are commonly found in the detection of low levels of rheumatoid factor-like material present in the serum of patients with NANBH, as well as in patients with other hepatitic and non-hepatitic staining. Some of the reactivities detected may be antibodies to the cytoplasmic antigens of the respective host.

There are numerous NANBV candidates. See, for example, the magazines Prince (1983), Feinstone and Hoofnagle (1984), and Overby (1985, 1986, 1987) and Iwarson's (1987) article. However, there is no evidence that some of these candidates represent the etiological agent of NANBH.

There is a great need for more sensitive, more specific methods for testing and identifying NANBV carriers, blood or blood products infected with NANBV. Post-transfusion hepatitis (PTH) occurs in about 10% of patients, and NANBH is thought to be responsible for up to 90% of these diseases. The main problem with this disease is often progressive to chronic liver damage (25-55%).

Patient care as well as prevention of NANBH transmission through blood and blood products or through personal contact requires reliable prognostic methods for detecting nucleic acids, antibodies and antigens for NANBV. There is also a need for effective vaccines and immunotherapeutic therapeutic agents to prevent and / or treat the disease.

Description of the invention with examples

The invention relates to the isolation and characterization of a newly discovered etiological agent of NANBH, the hepatitis C virus (HCV). More specifically, the invention provides the cDNA family of replicates of the HCV genome. These cDNA replicates are isolated by techniques involving a new stage of testing expression products from a cDNA library made from a specific agent in infected tissue with serum from a patient with NANBH to detect a newly synthesized antigen derived from a genome not yet isolated and uncharacterized u · '' - <h \ viral agent, and from select clones that produce products that react immunologically only with serum from an infected individual compared to uninfected individuals. Studies of the nature of the HCV genome using HCV cDNA-derived probes as well as the sequence of information contained in the HCV cDNA suggest that HCV is a flavivirus or a virus similar to this. Parts of cDNA sequences derived from HCV are useful as probes for the diagnosis of the presence of the virus in the samples and for isolation of naturally occurring variants of the virus. These cDNAs also construct accessible polypeptide sequences of HCV antigens encoded in the HCV genome (calm) and allow the production of polypeptides that are useful as standards or reagents in diagnostic tests and / or as components of vaccines. Polyclonal and monoclonal antibodies directed against the HCV epitope contained in these polypeptide sequences are also useful for diagnostic assays, as therapeutic agents, for testing antiviral agents and for isolating NANBV agents from which these cDNAs are derived. In addition, sequences of other parts of the HCV genome can be isolated using probes derived from these cDNAs, giving rise to additional probes and polypeptides that are useful in the diagnosis and treatment of prophylactic and therapeutic NANBH.

Therefore, with respect to polynucleotides, some aspects of the invention are as follows:

1. purified HCV polynucleotide

2. recombinant HCV polynucleotide

3. recombinant polynucleotide comprising a sequence derived from the HCV genome or from HCV cDNA

4. recombinant polynucleotide encoding an HCV epitope

5. a recombinant vector encoding some of the above recombinant polynucleotides and

The 6th host cell transformed with one of the above vectors

Other aspects of the present invention are:

1. Recombinant expression system comprising open reading sites (ORFs) of DNA derived from the HCV genome or HCV cDNA where the ORF is operatively linked to a control sequence compatible with the desired host

2. cell transformed with a recombinant expression system and

3. a polypeptide produced by a transformed cell

Still further aspects of the present invention are:

1. purified HCV

2. polypeptide preparation from purified HCV

3. purified HCV polypeptide

4. a purified polypeptide comprising an epitope that can be immunologically identified with an epitope contained in HCV.

Included in aspects of the invention are:

1. recombinant HCV polypeptide

2. a recombinant polypeptide comprising a sequence derived from the HCV genome or from the HCV cDNA

3. a recombinant polypeptide comprising the HCV epitope

4. a pooled polypeptide comprising the HCV polypeptide

The invention also includes:

1. A monoclonal antibody directed against the HCV epitope

2. purified preparation of polyclonal antibodies directed against the HCV epitope

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

A further aspect of the invention is a polynucleotide probe for HCV. Aspects of the invention pertaining to the equipment are those for analyzing a sample for the presence of an HCV antigen comprising a polynucleotide probe containing a nucleotide sequence of HCV of about 8 or more nucleotides in a suitable container; for analyzing samples for the presence of an HCV antigen comprising an antibody directed against the HCV antigen detected in a suitable container; sample analysis directed to the presence of antibodies directed against HCV antigens, comprising a polypeptide containing the HCV epitope present in the antigen in a suitable container. Other aspects of the present invention are: a polypeptide comprising an HCV epitope fused to a solid substrate; antibody to the HCV epitope coupled to a solid substrate.

Still further aspects of the invention are: a method for making a polypeptide comprising an HCV epitope comprising incubating a host cell with an expression vector comprising a sequence encoding the polypeptide with the epitope under conditions permitting expression of said polypeptide; and a polypeptide containing the HCV epitope produced by this method.

The invention also includes a method for detecting HCV nucleic acids in a sample, which comprises reacting the sample nucleic acids with an HCV polynucleotide probe under conditions that permit the construction of a polynucleotide duplex between the probe and the HCV nucleic acid from the sample; and detecting a polynucleotide sample including the probe.

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

The invention also includes vaccines for the treatment of HCV infection. Vaccines include an immunogenic peptide containing an HCV epitope or an inactive HCV preparation or a dilute HCV preparation.

A further aspect of the present invention is also the tissue of culture of cultured cells infected with HCV.

A further aspect of the present invention is a method for producing antibodies to HCV, comprising administering an isolated immunogenic polypeptide containing the HCV epitope into a specimen in an amount sufficient to trigger an immune response.

A further aspect of the invention is a method for isolating a cDNA derived from the genome of an unidentified infectious agent, comprising:

a) providing transformed host cells with expression vectors containing a cDNA library derived from nucleic acids isolated from agent-infected tissue and culturing said host cells under conditions permitting expression of a cDNA encoded polypeptide

b) the interaction of the cDNA expression products with an antibody containing a body component of a subject infected with said infectious agent under conditions that permit an immune reaction and detection of the antigen-antibody complex resulting from the interaction

c) growth of host cells expressing polypeptides that construct antigen-antibody complexes in step (b) under conditions that allow them to grow as individual clones and isolate said clones

d) the growth of cells from the clones of (c) under conditions permitting the expression of polypeptides encoded in cDNA and the interreaction of expression products with an antibody containing components of the body of an individual other than the body of step (a) infected with the infectious agent; and with control specimens infected with the agent, and detection of antigen-antibody complexes resulting from the interaction

e) the growth of host cells expressing polypeptides that form antigen-antibody complexes with an antibody containing the components of the bodies of infected specimens and specimens of infected individuals, and not said components of control individuals, under conditions that allow them to grow as individual clones and isolate said clones and ...

f) isolation of cDNA from clone host cells (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 homologs of HCV cDNA sequence overlaps in clones 5-1-1, 81, 1-2, and 91.

Figure 3 shows the positive HCV cDNA sequence derived from the 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 clone 81 and the putative amino acid sequence of the encoded polypeptide.

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

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

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

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

Figure 9 shows the pooled 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 overlying clone 35 and the encoded polypeptide.

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

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

Figure 13 shows the HCV cDNA sequence in clone 25c, the segment overlying clone 33b and the encoded polypeptide.

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

Figure 15 shows the HCV cDNA sequence in clone 33c, a segment overlying clones 40b and 33c and the encoded amino acids. Figure 16 shows the HCV cDNA sequence in clone 8h, a segment overlying clone 33c and the encoded amino acids.

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

Figure 18 shows the HCV cDNA sequence of clone 14c, a segment overlying clone 25c and the encoded amino acids.

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

Figure 20 shows the HCV cDNA sequence of clone 33f, a segment overlying clone 8f and the encoded amino acids.

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

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

Figure 23 shows the HCV cDNA sequence in clone 11b, a segment that overlaps the sequence in clone 7f and the encoded amino acids. Figure 24 shows the HCV cDNA sequence in clone 14i, a segment that overlaps the sequence in clone 11b and the encoded amino acids. Figure 25 shows the HCV cDNA sequence in clone 39c, a segment that overlaps the sequence in clone 33g, and the encoded amino acids. Figure 26 shows the structure of the HCV cDNA sequence derived from straight cDNAs in clones 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32,

33b, 25c, 14c, 8f, 33f and 33g. Also shown is the amino acid sequence of the polypeptide encoded in the stretched ORF in the derived sequence.

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

Figure 28 illustrates the HCV cDNA sequence in clone 35f, a segment overlying clone 39c and the encoded amino acids.

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

Figure 30 shows the sequence of clone 26g, a segment overlying clone 19g and the encoded amino acids.

Figure 31 shows the sequence of clone 15e, a segment overlying clone 26g and the encoded amino acids.

Figure 32 shows the sequence in the cDNA composition that is performed by linking clones 12f to 15e in a 5 'vs. 3' direction; also shows encoded amino acids in continuous ORF. Figure 33 shows a photograph of Western blots of the pooled protein, SOD-NANBs-i-i, with the chimpanzee serum from chimpanzees infected with BB-NANB, HAV and HBV.

Figure 34 shows a photograph of Western blots of the combined protein, SOD-NANB ₅ _i-i, with the serum of humans infected with NANBV, HAV, HBV and from a control group.

Figure 35 is a diagram showing the basic features of the pAB24 vector.

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

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

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

Figure 38 shows a Northern RNA stain autoradiograph isolated from the liver of a BB-NANBV infected chimpanzee examined with BB-NANBV cDNA in clone 81.

Figure 39 shows the NANBV nucleic acid autoradiograph treated with RNAase A or DNAase I and examined with BB-NANBV cDNA clone 81.

Figure 40 shows the autoradiograph of nucleic acids extracted from NANBV particles taken from plasma infected with anti-NANBs-ii and examined with ³² p-labeled NANBV cDNA from clone 81.

Figure 41 shows the autoradiographs of filters containing isolated NANBV nucleic acids examined with ³² recorded plus and minus DNA probes derived from NANBV cDNA in clone 81.

Figure 42 shows homologs between a polypeptide encoded in HCV cDNA and an NS protein from Dengue flavirus.

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 random probes using two immunoglobulin-enzyme conjugate configurations in ELIA testing. X Figure 45 shows the sequences in the primary mixture derived from a conserved sequence in a vNSl flavivirus.

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

Fig. 47 shows the sequence in the cDNA composition performed by linking clones k9-l to 15e in a 5 'to 3' direction; the amino acids encoded in the ORF are also shown.

Definitions

The term hepatitis C virus has been reserved by experts for a hitherto unknown aetiological agent of NANBH. for this reason, the hepatitis C virus (HCV) agent designated herein is a NANBH-induced agent previously designated NANBV and / or BB-NANBV. The terms HCV, NANBV and BB-NANBV are used interchangeably here. Like the extension of this terminology, HCV-induced disease, previously referred to as NANB hepatitis (NANBH), is referred to herein as hepatitis C. The term NANBH and hepatitis C may be used interchangeably here.

The term HCV, as used throughout this document, refers to the virus species causing NANBH and the diluted species or defective interfering particles derived therefrom. As shown below, it covers the HCV RNA genome. RNAs contained in viruses are known to have a high rate of spontaneous mutations, presumably in the order of IO ^-3 to 10 ⁴ per nucleotide (Fields & Knipe (1986)), therefore, multiple in the HCV species described below. The ingredients and procedures described herein allow the progression, identification, detection and isolation of various related structures. In addition, they also provide diagnostics and vaccines for different groups and are useful in methods of testing anti-viral agents for pharmacological use in which they inhibit HCV replication.

The information provided here is sufficient to allow viral taxonomy to identify other species similar to these, regardless of whether it was derived from single HCV, which was then designated CDC / HCV1. As described here, we have discovered that HCV is a flavivirus or virus similar to this. The morphology and composition of the Flavivirus particle are known and discussed by Brinton (1986). In general, according to morphology, Flavivirus contains a central nucleoplastid surrounded by a lipid double sheath. The virions are spherical with a diameter of about 40-50 nm. Their nuclei are about 25-30 nm in diameter. Between the outer surface of the virion sheath, projections are about 5-10 Jun in length, with terminal nodes about 2 µm in diameter. HCV encodes an epitope that is immunologically identifiable with an epitope in the HCV genome from which the described cDNA is then derived; the epitope encoded in the cDNA described herein is desirable. The epitope is unique in terms of HCV when compared to other known Flaviviruses. The uniqueness of an epitope can be determined by its immune reactivity with HCV and by a lack of immune reactivity with other Flavivirus species. Methods for detecting immune reactivity are known in the art, for example, by radioimmunoassay, Elisa screening, hemagglutination, and a variety of screening techniques as required herein. In addition to the above, the following parameters are applicable, both individually and in combination in identifying the species as HCV. Although HCV species are evolutionarily related, complete genome homology at the nucleotide level of 40% or more, preferably about 60% or more, preferably about 80% or more, can be expected to additionally correspond to continuous 13 nucleotide or more sequences. The correspondence between the putative HCV genomic sequence and the CDC / CH1 HCV cDNA sequence can be established by techniques known in the art. Thus, this can be determined by directly comparing the polynucleotide sequence information of the suspected HCV, and the HCV cDNA sequence described herein. Homology can also be determined by hybridization of the polynucleotide under conditions that allow the construction of stable duplexes between homologous regions (for example, those to be used before Si degradation), which is then followed by single-specific nuclease degradation (nuclease) and then by determining the size of the decomposed fragments.

Due to the evolutionary association of HCV species, suspected HCV species are identified by their homology at the polypeptide level. Generally speaking, these at the polypeptide level are preferably homologous in about 40% or more, more preferably about 60% or more, and in the most preferred cases above 80%. Techniques for determining the homology of amino acid sequences are known. For example, the amino acid sequence can be determined directly and then compared with the sequences provided herein. As an example, the nucleotide sequence of the genomic material of putative HCV can be determined (usually via a cDNA intermediate); the amino acid sequence encoded here can thus be determined at which point the corresponding regions can be compared. As used herein, a polynucleotide derived from a labeled sequence, for example HCV cDNA, especially those shown in Figures 1-32 or from the HCV genome, is a sequence consisting of 6 nucleotides, preferably at least 8 corresponding nucleotides, and most preferably, if there are at least about 1012, but preferably around 15-20, e.g. homologous or complementary regions of the indicated nucleotide sequence. It is desirable that the sequence of the region from which the polynucleotide is derived be homologous or complementary to a sequence unique to the HCV genome. If this is or is not unique to the HCV genome, the sequence can be determined by techniques known to those skilled in the art. For example, a sequence can be compared with sequences in databases such as Genebank to determine whether it is present in an uninfected host or other organisms. The sequence can also be compared to known sequences of other viral agents, including those known to cause hepatitis such as HAV, HBV, HDV and other Flavivirus members. Matching or not matching a particular sequence with other sequences can also be established through hybridization under certain, strict conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art, and will be described below. See also, for example, Manitatis et al. (1982). As an addition, the superiority of one of the duplex polynucleotides constructed by hybridization can be established by known techniques, including, for example, degradation by a nuclease such as Sl, which specifically degrades single regions in duplex polynucleotides.

Regions from which typical DNA sequences can be derived include, but are not limited to, such as regions encoding specific epitopes as well as non-transcribed and / or non-translatable regions.

A derived polynucleotide may not necessarily be derived from the nucleotide sequence shown, but may in some way be generated, for example, by chemical synthesis or DNA replication or reverse transcription or information-based transcription enabled by the sequence of bases in the region from which the polynucleotide is derived. Additionally, combinations of regions corresponding to those of the indicated sequence may be modified in a manner known in the art for coordination with purpose.

Similarly, a polypeptide or amino acid sequence derived from a designated nucleic acid sequence, for example the sequence shown in Figures 1-32 or from the HCV genome, is a polypeptide having an amino acid sequence identical to that of the polypeptide encoded in the sequence or portion thereof, wherein the portion contains at least 3-5 amino acids, more preferably at least 8-10 amino acids, and preferably at least 11-15 amino acids, or immunologically identifiable by the polypeptide encoded in the sequence.

The recombinant or derived polypeptide need not be translated from the generated nucleic acid sequence, for example, the sequences in Figures 1-32 or from the HCV genome; it may be produced in a manner such as, for example, 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, with the efficacy of its origin or function:

1. Not related to all or part of the polynucleotide to which it is linked in nature or in the form of a library and / or

2. is bound to a polynucleotide other than that to which it is bound in nature.

The term polynucleotide, as used herein, denotes a polymeric nucleotide of some length both ribonucleotide and deoxyribonucleotide. This term denotes only the primary structure of the molecule, thus, this term includes single and double DNA as well as single and double RNA. It also includes modified polynucleotide forms, for example by methylation and / or closure, as well as unmodified polynucleotide forms.

As used herein, the term HCV containing a sequence corresponding to a cDNA indicates that it contains an HCV polynucleotide sequence that is complementary or homologous to the sequence in the DNA constructed; the cDNA homology or complementarity rate will be about 50% or greater, preferably at least 70%, or better still at least about 90%. The sequences that will correspond will be at least 70 nucleotides long, preferably at least about 80 nucleotides, and most preferably at least about 90 nucleotides. The correspondence between HCV sequence and cDNA can be established by techniques known in the art, including, for example, direct comparison of the sequenced material with cDNA, or by hybridization and degradation with single nucleases, which is further accompanied by determination by degradation of the resulting fragments.

The term purified viral polynucleotide "denotes the HCV genome or fragment thereof, which is substantially free, i.e. it contains below 50%, preferably below 70%, and most preferably below 90% of the polypeptides to which the viral polynucleotide is naturally associated. Techniques for the purification of viral polynucleotides from viral particles are known in the art and include, for example, disruption of particles with a chaotropic agent and further separation of polynucleotides and polypeptides by ion exchange chromatography, affinity chromatography and density sedimentation.

The term purified viral polypeptide refers to an HCV polypeptide or fragment thereof that is substantially free, i.e. contains less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the cellular component to which the viral polypeptide is naturally associated, techniques for purifying viral polypeptides are known in the art, and examples of these techniques will be described below. Host cell recombinant host cells, cell types, cell culture, and other similar terms refer to microorganisms or more eukaryotic cell species cultured as unicellular creatures relative to cells that will or have been used as recipients for recombinant vector or other DNA transfer and include the progeny of the original cells which can be downloaded. It is understood that the progeny of a parent cell need not be exactly identical in morphology or genomic or shared DNA complement to the original parent due to a random or deliberate mutation. A parent cell progeny sufficiently similar to a parent, characterized by an appropriate property such as the presence of a nucleotide sequence encoding the desired polypeptide, is included in the progeny covered by this definition and is covered by the above terms.

Replicon is some genetic element, e.g. plasmid, chromosome, a virus that is transmitted as an autonomous unit of replication in a cell; e.g. capable of replication under its own control. The vector is the replicon in which it is bound, the second polynucleotide segment, so that it carries replication and / or expression of the fused segment.

The control sequence indicates the polynucleotide sequences required to isolate 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 contain a promoter, a ribosome binding site, and terminators; in eukaryotes, these control sequences include generally promoters, terminators, and in some cases enhancers. The term control sequence is directed to include at least all components whose presence is required for expression and may also include additional components whose presence is advantageous, for example, control sequences.

Operationally bound ”means an installation that describes the components of a bond that allows them to function in the desired way. The control sequence operably linked in the coding sequence is coupled in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.

The open reading site (ORF) is a region of a polynucleotide sequence encoding a polypeptide; this region may be part of a coding sequence or a common coding sequence. The coding sequence is a polynucleotide sequence that is transcribed into an mRNA and / or translated into a polypeptide when placed under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by translating the start codon at the 5′-terminus and translating the end codon at the 3′-terminus. The coding sequence may include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. Immunologically identifiable by / as indicates the presence of epitopes (types) and polypeptides, which are also present and unique with respect to the indicated polypeptides, typically HCV proteins. The immunological identity can be determined by an antibody that binds and / or participates in binding; these techniques are known to those skilled in the art but are also shown below. The uniqueness of the epitope can also be established by computer research into known databases, such as Genebank, for polynucleotide sequences encoding the epitope, and by comparing the amino acid sequence with other known proteins.

As used herein, the epitope denotes an antigenic determinant of a polypeptide; an epitope may comprise 3 amino acids in a spatial conformation unique to the epitope. Generally, the epitope contains at least 5 such amino acids, but usually these are 8-10. Methods for determining the spatial conformation of amino acids are known in the art, 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. Immunological reactivity can be determined by an antibody that binds, more specifically by the kinetics of antibody binding and / or participation in binding, using a known polypeptide (s) containing the epitope against which the antibody is directed, as a competitor to the antibody. Techniques for determining whether a polypeptide is immunologically reactive are known in the art.

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

The term polypeptide refers to a molecular chain of amino acids and does not indicate a specific length of the product; both peptides, oligopeptides and proteins are included in the definition of a polypeptide. The term also does not refer to post-expression modification of a polypeptide, for example, glycosylation, acetylation, phosphorylation and the like.

Transformation as used herein denotes the insertion of exogenous polynucleotide into host cells, according to the method used for insertion, for example, by direct reception, transduction or f-maturation. The exogenous polynucleotide may be reflected as a non-integrated vector, for example, a plasmid, or may alternatively be integrated into the host genome.

Treatment or. Treatment refers to prophylaxis and / or therapy herein.

An individual or specimen as used herein designates vertebrates, especially members of mammalian species, and includes but is not limited to pets, sport animals, primates and humans.

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

As used herein, a positive strand of the virus genome is one in which either the DNA or RNA genome is united and encodes the putative polypeptide (s). Examples of positively RNA viruses include Togaviridae, Coronaviridae,

Retroviridae, Picornaviridae and Calciviridae. They also include Flaviviridae that were once classified as Togaviridae. See Fields & Knipe (1986).

As used herein, a component of the body containing the antibody denotes that component of the individual bodies that are the origin of the antibodies of interest. The components of the body containing the antibody are known in the art and include, but are not limited to, plasma, serum, spinal fluid, lymph fluid, external parts of the respiratory, intestinal and genitourinary tract, tears, saliva, milk, white blood cells and myeloma .

As used herein, a purified HCV preparation is an HCV preparation that has been isolated from the cellular constituents to which the virus is normally associated and from other viruses that may be present in the infected tissue. Virus isolation techniques are known to those skilled in the art and include, for example, centrifugation and affinity chromatography; the process of obtaining purified HCV will be described further. The implementation of the present 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, for example, Manitatis, Fitsch & Sambrook, Molecular cloning; A Labolatorz manual (1982); DNA clonong, Volumes I and II (D.N.Glover ed. 1985); Oligonucleotide synthesis (M.J. Gait ed. 1984); Nucleic acid hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and translation (B.D. Hames & S.J. Higgins eds. 1984); Animal Whole Culture (R.I. Freshney ed. 1986); Immobilized Celery and enzymes (IRL Press, 1986); B. Perbal,

A Practical Guide to Molecular Cloning (1984); series, Methods in enzymologz (Academic press, Inc.); Gene transfer vectors for mammalian cellulitis (J.H. Miller & M.P. calos eds. 1987, Cold Spring Harbor Laboratory), Methods in enzymology Vol. 154 and vol. 155 (Wu and Grossman and Wu eds., Respectively), Mayer and Walker eds. (1987), Immunochemical methods in whole and molecular biology (Academic press London), Scopes, (1987), Protein purification: Principles and practice, Second Edition (Springer-Verlag, Υ.Υ.) in Handbook of experimental immunology, Volumes I -IV (DM Weir and CC Blackwell eds 1986).

All patents, patent applications, and publications cited herein and before are incorporated herein by reference.

Useful materials and methods of the present invention are made possible by raising a family of tightly homologous nucleotide sequences isolated from a cDNA library derived from nucleic acid sequences present in the HCF plasma of infected chimpanzees. This family of nucleic sequences is of neither human nor chimpanzee origin, therefore it does not hybridize to either human or chimpanzee genomic DNA of an uninfected individual, therefore the nucleotides of this family are only present in the liver and plasma of HCF infected chimpanzees, thus the sequence is not present in Genebank. Additionally, the sequence family does not show significant homology with the sequences contained in the HBV genome. The sequence of one family member contained in clone 5-1-1 has one continuous open site (ORF) encoding a polypeptide about 50 amino acids long. Serum from HCF infected humans contains antibodies that bind to this polypeptide, since serum from uninfected humans does not contain antibodies to this polypeptide. Finally, since the serum of uninfected chimpanzees does not contain an antibody for this polypeptide, antibodies in the chimpanzee body are induced following acute NANBH infection. However, antibodies to this peptide were not detected in chimpanzees and humans infected with HAV and HBV. Using these criteria, the cDNA sequence is in the putative sequence where the virus causes or is associated with NANBH; this cDNA sequence is shown in Figure 1. As described below, the cDNA sequence in clone 5-1-1 differs from those isolated from other cDNAs in that it contains 28 additional base pairs. The composition of other identified members of the cDNA family that was isolated by the use of a synthetic equivalent sequence with respect to the cDNA fragment in clone 5-1-1, shown in Figure 3. The cDNA family member that was isolated by the use of a synthetic cDNA-derived sequence in clone 81 is shown in Figure 5, and the composition of this sequence with the composition of 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 Section IV.A.18 and shown in Figure 26. The cDNA composition of these clones is described in FIG. described in Section IV.A.18 and shown in Figure 26. The cDNA composition indicates that it contains a continuous ORF and thus encodes a polyprotein. This information is consistent with the suggestion described below, where HCV is a Flavivirus or a virus similar to this.

The usefulness of this cDNA family shown in Figures 1-32 allows the construction of DNA probes and polypeptides that are useful in diagnosing HCF infection-induced NANBH and in testing blood donors as well as donated blood and blood products for infection. For example, from sequences, DNA oligomers of 8-10 nucleotides or more in length may be synthesized, which are useful as hybridization probes for detecting the presence of a putative genome in, for example, the serum of subjects suspected of cultivating a virus or for testing donated blood for the presence of a virus. The cDNA family of sequences also enables the production and production of HCV specific polypeptides that are useful as diagnostic reagents for the presence of antibodies generated during NANBH. Antibodies in purified cDNA derived polypeptides can also be used to detect putative antigens in infected individuals and in the blood.

Knowledge of these cDNA sequences also enables the construction and production of polypeptides, which can then be used as HCV vaccines, and for the production of antibodies that can be used to protect against disease and / or for the treatment of HCV infected individuals.

Moreover, the cDNA sequence family allows further characterization of the HCV genome. Polynucleotide probes derived from these sequences can also be used to test the cDNA library for additional overlay of cDNA sequences and can be used to obtain sequences that no longer overlap. Although the genome is a segment and the segments do not have a common sequence, these techniques can be used to obtain the entire genome sequence. However, if the genome is a segment, other segments of the genome can be obtained by repeating the lambda-gtll serological testing procedure used to isolate the cDNAs described here, or alternatively, by isolating the genome from purified HCV particles.

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

For example, antibodies directed against HCV epitopes contained in cDNA-derived polypeptides can be used in affinity chromatography-based procedures for virus isolation. Alternatively, antibodies can be used to identify viral particles that have been isolated by other techniques. Viral antigens and genomic material in isolated viral particles can then be characterized further.

Information obtained from further sequencing of the HCV genome, as well as from further characterization of the HCV antigen and characterization of the genome, will allow the construction and synthesis of additional probes, polypeptides and antibodies that can be used to diagnose, prevent and treat HCV-induced NANBH and to test to infected blood and blood products.

The availability of HCV probes, including antigens, antibodies, and polynucleotides derived from the gene from which the cDNA family is derived, also allow the development of a tissue culture system whose main use will be in elucidating the biology of HCV. This leads to the development of new therapies based on antiviral associations that inhibit HCV replication or infection.

The process used to identify and isolate the etiologic agent for NANBV is novel and can be used to isolate and / or identify hitherto uncharacterized gene-binding agents associated with a variety of diseases, including those induced by viruses, viroids, bacteria, fungi and parasites. In this cDNA procedure, the library is based on nucleic acids present in infected tissue from infected individuals. The library is being constructed in a vector that permits the expression of a cDNA encoded polypeptide. Clones of host cells containing a vector expressing an immunologically reactive fragment of an etiologic agent polypeptide are selected by immunological testing of the library's expression products with a body component containing an antibody from another specimen previously infected with said agent. Degrees in immunological testing include the interaction of vector-containing cDNA expression products with a body component containing the antibody of another infected subject and detection of antibody-antigen complex formation between expression products and antibodies of another infected individual. Isolated clones are further tested by immunological interaction of their expression products with components of the body containing the antibody of other specimens infected with the suspected agent and control individuals not infected with that agent, and detection of antigen-antibody complex construction with antibodies 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 specimens are isolated. The infected individuals used to build the cDNA library and for immunological testing are not of the same species. cDNAs isolated as a result of this process, and their expression products and antibodies directed against expression products, are useful in characterizing and obtaining an etiologic agent. As will be described in more detail below, this process is successfully used to isolate the cDNA family derived from the HCV genome.

II.A. Obtaining a cDNA sequence

Combined chimpanzee sera with chronic HCV infection were used to isolate the viral particles and contained high virus titers, e.g. at least 10 ⁶ chimps. infectious doses / ml (CID / ml); nucleic acids isolated from these particles are used as templates in the construction of the cDNA library of the viral genome. The procedures for isolating putative HCV particles and for constructing a cDNA library in lambda-gtll are explained in Chapter IA.Al Lambda-gtll is a vector that has been specifically developed for the expression of inserted cDNAs as aggregated beta-galactosidase polypeptides and for testing a large number of recombinant phages specific antisera produced against a defined antigen. A lambda-gtll cDNA library formed from a cDNA source containing cDNAs of average lengths of about 200 base pairs was tested for coded epitopes that should specifically bind to serum derived from patients who previously suffered from NANBH hepatitis; Huynh TV et al. (1985).

About 10 ⁶ phages were tested and 5 positive phages were identified that were roasted and tested for specificity of binding to serum from different humans and chimpanzees pre-infected with HCV. One phage, 5-1-1 binds 5 to 8 human sera tested. This association occurs as specific for serum derived from patients prior to NANB hepatitis infection, with 7 normal donors of blood serum showing no such binding. The cDNA sequence in recombinant phage 5-1-1 is determined and shown in Figure 1. The polypeptide encoded by this cloned cDNA, which at the same translational site as the N-terminal beta-galactosidase part of the pooled polypeptide, shows the nucleotide sequence below. This translational ORF therefore encodes epitope (s) that are specifically identified with serum from patients with NANB hepatitis infections.

The availability of cDNAS in recombinant phage 5-1-1 permits the isolation of other clones containing additional segments and / or alternative cDNA segments in the viral genome. The Lambdagtll cDNA library described below is tested using a synthetic polynucleotide derived from a cloned sequence

5-1-1 cDNA. This testing yields three other clones identified as 81, 1-2, and 91; The cDNA contained in these clones is sequenced. See Chapters IV.A.3 and IV.A.4. The homology between the four independent clones is shown in Figure 2, where the homologies are indicated by vertical lines. Nucleotide sequences present only in clones 5-1-1, 81 and 91 are indicated by lowercase letters.

The cloned cDNAs present in the recombinant phages in clones 5-11, 81, 1-2 and 91 are highly homologous and differ in only two regions. First, the nucleotide at site 67 in clone 1-2 is thymidine, while containing the other three clones at that site is cytidine. However, this substitution does not change the nature of the encoded amino acid. Another difference between clones is that they clone

5-1-1 on its 5 * -terminus contains 28 base pairs that are not present in other clones. An extra sequence may be a 5′-terminal cloning artifact; 5′-terminal cloning artifacts are commonly described in cDNA method products. Synthetic sequences derived from 5′-regions and 3′-regions of HCV cDNA in clone 81 are used to test and isolate cDNAs from the lambda-gtll NANBV cDNA library that breaks clone 81 cDNA (Chapter IV.A.5). The sequences of the obtained cDNAs, which are in clones 36 and 32, are shown in Figures 5 and 7.

Similarly, a synthetic polynucleotide based on the 5′-region of clone 36 is used to test and isolate the cDNA from the lambda-gtll NANBV cDNA library that overlaps clone 36 of the cDNA (Chapter IV.A.8). The purified clone of a recombinant phage containing a cDNA that has been hybridized to a synthetic nucleotide probe is called clone 35, and the NANBV cDNA sequence in this clone is shown in Figure 8.

Using the technique of isolating overlapping cDNA sequences, clones containing additional HCV cDNA sequences and counter currents are obtained. The isolation of these clones is described below in Chapter IV.A.

Analysis of the nucleotide sequences of HCV cDNAs encoded in the isolated clones shows that the cDNA composition forms a single long continuous ORF. Figure 26 shows the sequential cDNA compositions of these clones together with the encoded HCV polypeptide.

The description of the process for retrieving cDNA sequences is mainly of historical importance. The resulting sequences (and their complements) are provided herein, and the sequences or portions thereof must be obtained by the use of synthetic procedures, or by a combination of a synthetic procedure by re-separating the individual sequences through procedures similar to those described herein.

Lambda-gtll species replicated from the HCV cDNA library and from clones 5-1-1, 81, 1-2, and 91 are deferred under the terms of the Budapest Agreement with the American Type Culture Collection (ATCC), 12301 Parklawn Dr. Rockville, Maryland 20852, and are tagged with the following associated numbers:

Lambda-gtll ATCC no. Date of storage HCV cDNA library 40394 12/1/1987 clone 81 40388 11/17/1987 clone 91 40389 11/17/1987 clone 1-2 40390 11/17/1987 clone 5-1-1 40391 11/18/1987

Upon approval and issue of this application as a US patent, any restrictions on the availability of stored things will be irrevocably lifted; also an insight into the deposit being made will be available during the decision-making process of the advance filing with a designated designee under 37 CFR 1.14 and 35 USC 1.22. However, deposits constructed will be maintained for 30 years from the date of deposit or five years after the last request for storage; or the validity of a U.S. patent regardless of its duration. These deposits are for opportunity only and are not required for the practice of the present invention from the point of view of this invention. The HCV cDNA sequences in the deposited material are included in the present invention.

II.B. Retrieval of viral polypeptides and fragments

The availability of cDNA sequences, either those isolated using the cDNA sequences in Figures 1-26 as described below, as well as the cDNA sequences in these Figures, allows the construction of expression vectors encoding the antigenically active regions of the polypeptide encoded in one or the other nor. These antigenically active regions can be derived from coated or enveloped antigens or from core antigens, including, for example, protein-binding polynucleotide, polynucleotide polymerases, and other viral proteins required for replication and / or viral particle assembly. The fragments that encode the desired polypeptides are derived from cDNA clones using conventional restriction degradation or by synthetic processes, and then bind to vectors that may, for example, contain portions of fused sequences such as beta-galactosidase or superoxide dimutase (SOD), desirable SOD. Methods and vectors useful for the production of polypeptides containing pooled SOD sequences are described in European Patent Office Publication no. 0196056, issued

I. 10.1986. Vectors encoding the assembly of an SOD polypeptide and an HCV polypeptide, e.g. NANB ₅ _i_i, NANB ₈ i, and C100-3, which is encoded in the HCV cDNA composition, are described in Chapters IV.Bl, IV.B.2, and IV.B.4, respectively. A particular desired portion of an HCV cDNA containing an open reading site, whatever strands, can be obtained as a recombinant polypeptide, ie as a mature or pooled protein; alternatively, the cDNA encoded polypeptide can be made possible by chemical synthesis.

DNA encoding the desired polypeptide, whether in aggregate or mature form, or whether it contains or may not have one sequence to allow secretion, may be bound in expression vectors favorable to the conventional host. Now, both prokaryotic and eukaryotic host systems are used in the construction of recombinant vectors, some of the common control systems and host cell types are given in Chapter III.A., below. The polypeptide is then isolated from the lysed cells or from the culture medium and purified because of the widespread need for its use. Purification can be carried out by techniques known in the art, for example, salt fractionation, ion-exchange resin chromatography, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for various methods of protein purification. Such polypeptides can be used as diagnostics and those that give rise to neutralizing antibodies can be formulated into vaccines. Antibodies produced against these polypeptides can also be used as diagnostics or for passive immunotherapy. In addition, as described in Sec

II. J. below, antibodies against 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 culture tissue or in an infected host.

II.C. Antigenic polypeptide production and carrier conjugation

The antigenic region of the polypeptide is generally very small; typically 8 to 10 amino acids or less in length. Fragments of 5 amino acids long can characterize the antigenic region. These segments may correspond to HCV antigen regions. Therefore, the use of HCV cDNA as the base of DNA encoding short HCV polypeptide segments can be expressed recombinantly as pooled proterins or as isolated polypeptides. Additionally, short amino acid sequences can usually be obtained by chemical synthesis. In cases where the synthesized polypeptide is specifically configured to allow a specific epitope but is too small to be immunogenic, the polypeptide can bind to a suitable carrier.

Many techniques for such bonding are known in the art, including the construction of disulfide bonds via the use of N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) by Pierce Company, Rockford, Illinois (if the peptide does not have a sulfhydryl group, the peptide may be supplied by the addition of a cysteine residue). These reagents create a disulfide bond between themselves and the cysteine residues of the peptide on one protein and the amide bond via epsilon-ammonium on lysine or another amino group in another protein. Variants of such disulfide / amide-building agents are known. See, for example, Immun. Rev. (1982) 62: 185. Other bifunctional binding agents build a thioether rather than a disulfide bond. Many of these are commercially available and include 6-maleylmidocoproic acid reactive esters, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or 132 hydroxy-2-nitro-4-sulfonic acid, a sodium salt.

The list below is optional and modifications to those associations may apply. Any carrier that does not induce the production of antibodies that harm the host may be used. Suitable carriers are usually 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 viral particles, see, for example, Chapter II.D. Particularly useful protein substrates are serum albumin, keihol limpet hemocyanin, immunoglobin molecules, thyroglobulin, ovalbumin, tetanus toxoid and other proteins well known to those skilled in the art.

II.D. Acquisition of an immunogen hybrid fragment containing HCV epitopes

The immunogenicity of the HCV epitope can also be enhanced by their production in mammals or yeast systems combined or coupled to proteins that build particles with, such as, for example, those bound to hepatitis B surface antigen. Constructions where the NANBV epitope binds directly to a protein that builds the sequence-encoding particles produce hybrids that are immunogenic with respect to the HCV epitope. Additionally, all of the vectors obtained include HBV-specific epitopes that have different levels of immunogenicity, such as a pre-S peptide. Thus, particles constructed from particles that build a protein that includes HCV sequences are immunogenic with respect to HCV and HBV.

Hepatitis surface antigen (HBSAg) has been shown to be able to be upgraded and aggregated into particles in S.cervisiae (Valenzuela et al (1982)) as well as, for example, in mammalian cells (Valenzuela et al (1982)). The construction of such particles has been shown to enhance the immunogenicity of the monomer subunit. Constructs may also include the immunodominant epitope of HBSAg spanning 55 amino acids of the pre-surface (pre-S) region, Neurath et al (1984). The constructs of the pre-SHBSAg particle expressive in yeast are described in EPO 174.44, issued 3/19/1986; hybrids incorporating heterogeneous viral sequences for yeast expression are described in EPO 175.261, issued 3/26/1966. Both applications have been marked as indicated and are referenced here. These constructs can be expressed in mammalian cells such as Chinese hamster ovary (CHO) cells using the SV40-dihydrofolate reductase vector (Michelle et al (1984)).

Further, the parts of the protein that build the sequence-encoding particles can be replaced by codons encoding the HCV epitope. In this replacement, regions not required for mediating the aggregation of immunogenic particles in yeasts or mammals can be deleted, eliminating additional HBV antigen sites from competition with the HCV epitope.

II.E. Preparation of vaccines

The 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 from the HCV genome to which they are correspondent. The observed homology between HCV and Flavivirus provides information relating to the polypeptides that are 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 described in Rice et al. (1986). Flavivirus genomic RNA is believed to be only virus-specific mRNA species and translated into three viral structural proteins, e.g. C, M and E as well as two large non-structural proteins, NV4 and NV5, and a complex set of smaller non-structural proteins. The major neutralizing epitopes for Flaviviruses are known to lie in the E protein (envelope) (Roehrig 1986). The corresponding HCV E gene and the region-coding polypeptide can be pedigree based on homology to Flaviviruses. Thus, vaccines may also encompass recombinant polypeptides containing HCV E epitopes. These polypeptides may be expressed in bacteria, yeasts or mammalian cells, or alternatively, may be isolated from viral preparations. It has also been observed that other structural polypeptides may also contain epitopes that give rise to protective anti-HCV antibodies. Thus, polypeptides containing the E, C and M epitopes, either individually or in a group, may also be utilized in the vaccines.

In addition, immunization with NS1 (non-structural protein 1) has been shown to result in protection against yellow fever (Schlesinger et al. (1986)). This is true even when immunization does not cause growth in neutralizing antibodies. Thus, especially when it occurs that this protein is highly conservative among Flaviviruses, HCV NS1 will also be protective against HCV infection. It has also been shown that non-structural proteins can provide protection against viral pathogens, even if they do not induce the production of neutral antibodies.

From the above viewpoint, multivalent HCV vaccines may comprise one or more structural proteins and / or one or more non-structural proteins. These vaccines may include, for example, recombinant HCV polypeptides and / or polypeptides isolated from virion. Additionally, inactive HCV can be used in vaccines; these may be obtained by the preparation of viral lysates or other agents known in the art to condition Flavivirus inactivity, for example, treatment with organic solvents or detergents, or treatment with formalin. Moreover, vaccines can also be obtained from dilute HCV structures. Preparations of dilute HCV structures are described below.

Some of the proteins in Flaviviruses are known to contain highly conserved regions such that some kind of immune cross-reactivity is expected between HCV and other Flaviviruses. It is possible that epitopes shared between Flaviviruses and HCV will give rise to protective antibodies against one or more of the disorders caused by these pathogenic agents. This makes it possible to build a multiple-use vaccine based on this knowledge. The preparation of vaccines containing immunogenic polypeptide (s) as active ingredients is well known to those skilled in the art. Typically, such vaccines are prepared as liquid solutions or suspensions for injection; solid forms favorable for dissolution or suspension / liquid for injection may also be prepared. The preparations may also be emulsifiable or protein encapsulated in liposomes. Active immunogenic ingredients are often mixed with additives that are pharmaceutically acceptable and compatible with the active ingredients. Advantageous 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 excipients such as wetting and emulsifying agents, buffers and / or additives that enhance the effectiveness of the vaccine. Examples of additives that may be effective for this include, but are not limited to: aluminum hydroxide, N-acetyl-murmyl-L-threonyl-disoglutamine (thr-MDP), N-acetyl-nor-murmyl-L-alanyl-dizoglutamine (CGP 11637, referred to as nor-MDP), Nacetylmurmyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'diapalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, designated as MTP-PE) and RIBI containing three bacterial extracted components, monophosphoryl lipid A, trehalose dimicolate and wall skeleton cell (MPL + TDM + CWS) in 2% squalene / Tween 80 emulsion. The efficacy of the additives can be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing the HCV antigen sequence obtained by administering this polypeptide into vaccines that also covered the administration of various additives.

Vaccines are usually administered parenterally, by injection by subcutaneous or intramuscular injection. Additional formulations that are advantageous for other routes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers, for example, polyalkalic glycols or triglycerides, may be included; such suppositories may be formulated 12 compositions containing the active ingredients in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such commonly used additives as, for example, pharmaceutical mannitol, lactose, starch, magnesium steart, saccharin sodium, cellulose, magnesium carbonate, etc. These preparations may be in the form of a solution, suspension, tablet, pill, capsule, support the release of formulations or powders and contain 1095% of the active ingredient, preferably about 25-70%.

Proteins can be formulated into the vaccine as neutral or in salt form. Pharmaceutically acceptable salts include addition salts of acids (rewarded with free amino groups of peptides) and those formed with inorganic acids such as hydrochloric or phosphoric acid, and with organic acids such as acetic, oxalic, tartaric salts built up with free carboxylic groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferro-hydroxides, and with such organic bases as isopropylamine, trimethylamine, 2 -ethylamino ethanol, histidine, procaine, etc.

II.F. Dose and administration of vaccines

Vaccines should be administered in a dose compatible formulation and in such quantities as will be prophylactically and / or therapeutically effective. The amount administered and usually in the range of 5µς to 250 µg of antigen per dose, depending on the treated subject, the capacity of the subject's immune system for antibody synthesis and the degree of protection desired. The exact amounts of the active ingredient required for administration may depend on the evaluation of who uses the vaccine and can be tailored to each subject.

The vaccine may be administered in a single dose or, more preferably, in a multiple dose distribution. In the latter, the primary course of vaccination may be 1-10 separate doses, accompanied by other doses given at intervals and necessary to maintain and / or enhance the immune response, for example, to 1-4 months between doses, and if is required with additional doses after several months. The dosage regimen will also depend at least in part on the individual's need and will also depend on the judgment of the person administering the dosage.

Additionally, a vaccine containing an immunogenic HCV antigen may be administered together with other immunoregulatory agents, such as immuno-globulins.

II.G. Acquisition of antibodies against HCV epitopes

The immunogenic polypeptides obtained in the manner described below are used to produce antibodies both polyclonal and monoclonal. If polyclonal antibodies are desired, the selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide carrying the HCV epitope (s). The serum from the immunized animal is then collected and treated by known methods. 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 producing and processing polyclonal antisera are well known in the art, see, for example, Mayer and Walker (1987). Alternatively, polyclonal antibodies can be isolated from mammals that have been previously infected with HCV. An example of a method for purification of antibodies against HCV epitopes from serum of infected individuals based on affinity chromatography using a combined SOD polypeptide and a polypeptide cloned in cDNA clone 5-1-1 is given in Chapter V.E. Experts may also produce monoclonal antibodies directed against HCV epitopes. The general methodology for producing hybrid monoclonal antibodies is well known. Immortal antibody-producing cell types can be generated by cellular coupling, as well as by other techniques such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, for example, M. Schreier et al. (1980); Hammerling et al. (1981); Kennett et al. (1980); see also USA Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,632 and 4,493,890. Panels of monoclonal antibodies produced against HCV epitopes can be easily tested for different properties: e.g. for isotope, epitope, affinity, etc.

Antibodies, monoclonal and polyclonal, that target HCV epitopes are particularly useful in diagnoses, and those that are neutral in passive immunotherapy. Monoclonal antibodies, more specifically, can be used to increase the anti-idiotype of antibodies.

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

Techniques for increasing anti-idiotype antibodies are known in the art. See, for example, Grzych (1985), Mac Namara et al.

(1984) and Uytdehaag et al (1985). These anti-idiotype antibodies can also be used to treat NANBH as well as to explain immunogenic regions of the HCV antigen.

II.H. Diagnostic oligonucleotide probes and equipment

By using the described sections of isolated HCV cDNA as a base, including those in Figures 1-32, oligomers of about 8 nucleotides or more, either by excision or synthetically hybridizing with the HCV genome, can be obtained and useful for viral agent identification , further characterization of the viral genome as well as in the detection of viruses of diseased individuals. HCV polynucleotides (natural or derived) are lengths that allow the detection of specific sequences by hybridization. Although 6-8 nucleotide lengths may be working lengths, sequences of 10-12 nucleotide lengths are desirable, and about 20 nucleotides occur as optimum. These sequences will preferably be derived from regions lacking heterogeneity. These probes can be obtained using routine procedures, including automated oligonucleotide synthetic procedures. Useful probes include, for example, clone 5-1-1 and the additional clones described herein, as well as various oligomers useful in exploring cDNA libraries (see below). Complement to some unique part of the HCV genome will be satisfying. To use complement as a probe, complementarity is desirable, although not necessarily required if the fragment family is enlarged.

In order to use such probes for diagnostics, a biological sample such as blood or serum is analyzed and treated if desired to extract the nucleic acids it contains. The nucleic acid obtained from the sample may be subjected to gel electrophoresis or other separation techniques; alternatively, the nucleic acid sample can be absorbed in spot without size separation. The probes are then marked. Advantageous markers and methods for labeling probes are well known in the art and include, for example, radioactive markers incorporating translational notch or kinase, biotin, fluorescent probes and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under conditions of hybridization with favorable obligations.

Probes can easily be made completely complementary to the HCV genome. This makes desirable, usually high, commitments to prevent false positivity. However, high-obligation conditions should only be used if probes are complementary to regions of the viral genome that lack heterogeneity. The obligation to hybridize is determined by a number of factors during the hybridization process and during the washing process, including temperature, ionic strength, long time, and formamide concentration. These factors are indicated, for example, in Manitatis T. (1982).

HCV genomic sequences are generally expected to be present in the serum of infected specimens at relatively low levels, e.g. in about 10 ² -10 ³ sequences per ml. This level may require the use of amplification techniques in hybridization studies. 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 hybridized to the target nucleotide sequence and then to biotin-modified poly-A, PCT Application 84/03520 and EPA124221 describe a DNA hybridization assay in which:

l.analyte is digested to single DNA complementary to the enzyme-labeled oligonucleotide and

The resulting final duplex is hybridized to the enzyme-labeled oligonucleotide.

EPA 204510 describes a DNA hybridization assay in which a DNA analyte contacts a probe having an end such as a poly-dT end, a reinforced structure having a sequence that hydrolyzes the termination of a probe such as poly-A sequences and which is able to bind a plurality of labeled threads . A particularly desirable technique may first capture the amplification of a target HCV sequence in serum about 10,000 times, e.g. about 10 ⁶ sequences / ml. This can be done, for example, with the technique of Saiki et al (1986). The amplified sequence can then be detected using the hybridization assay described in the associate US application under agent number 2300-0171 of October 15, 1987, which is incorporated herein by reference. This hybridization assay, which has to detect sequences at 10 ⁶ / ml, uses nucleic acid multimers that bind to the single nucleic acid analyte and which also binds to reproduce single labeled oligonucleotides. A favorable sandwich phase investigation of the solution phase and probe acquisition procedures are described in European Patent Publication 255.807 of 16.6.1987 and is used herein as a reference.

Probes can be installed in diagnostic equipment. Diagnostic equipment includes a DNA probe that can be labeled; alternatively, the DNA probe may be non-labeling components and may be present in the equipment. The equipment may also contain other conveniently stored reagents and materials required for the particular hybridization, such as standards as well as instructions for performing the test.

II.I. Intuno-examination and diagnostic devices

Both polypeptides that react immunologically with serum containing HCV antibodies e.g. those derived or encoded in the clones described in Chapter IV.A., and their composition (see Chapter IV.A) and antibodies generated against HCV specific epitopes in these polypeptides, see, for example, Chapter IV.E. they are useful in immunoassay for detecting 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 design is the subject of a large number of variants, many of which are known in the art. For example, immunoassay may use a single vir antigen, for example, a polypeptide derived from one of the clones containing HCV cDNA, as described in Chapter IV.A. or from a cDNA composition derived from the cDNA of these clones, or the HCV genome from which the cDNA in these clones is derived; alternatively, immunoassays may use a combination of viral antigens derived from these sources. For example, a monoclonal antibody directed against a viral epitope (epitopes), a combination of monoclonal antibodies directed against a single viral antigen, monoclonal antibodies directed against different viral antigens, polyclonal antibodies directed against the same viral antigen, or polyclonal anti-viral antibodies may be used. . Test runs may be based, for example, on comparisons or on a sandwich type of test. They can also be used, for example, solid supports, but can also be done by immunoassay. Most investigations involve the use of a labeled antibody or polypeptide; the markers may be, for example, fluorescent, chemiluminescent, radioactive or colored molecules. Also known are studies that amplify probe signals;

Examples of this are screening using biotin and avidin, as well as enzyme-tagged and indirect immunoassays such as ELISA.

The elavivirus model for HCV allows the prediction of probable locations of diagnostic epitopes for virion structural proteins. The C, pre-M, M, and E domains, all probably due to their epitope content, have significant potential for detection of viral antigens and specifically for diagnoses. Similarly, non-structural protein domains are expected to contain important diagnostic epitopes (e.g., NS5 encoding putative polymerase and NS1 encoding putative complement-binding antigen).

Recombinant polypeptides or viral polypeptides that include epitopes from these specific domains may be useful for detection of viral antibodies in infected blood donors or in infected patients.

Additionally, antibodies directed against E and / or M proteins can be used in immunoassay for the detection of viral antigens in patients with HCV-induced NANBH and in infected blood donors. However, these antibodies will be most useful in acute-phase donor detection. Apparatus suitable for immunodiagnosis and containing appropriate labeled agents shall be constructed by packing appropriate materials, including polypeptides of the invention containing HCV epitopes or antibodies directed against HCV epitopes, into appropriate containers, together with other reagents and materials required for testing, as also the appropriate set of instructions for conducting the investigation.

II.J. Further characterization of the HCV genome, Virion and viral antigens using a cDNA probe relative to the viral genome

HCV cDNA sequence information in the clones described in Chapter IV.A., as shown in Figures 1-32, can be used to generate further information on the HCV genome sequence and to identify and isolate HCV agents, thereby assisting in its characterization including the nature of the genome, the structure of the viral particle, and the nature of the antigen it consists of. This information may lead to additional polynucleotide probes, polypeptides derived from the HCV genome, and antibodies directed against the HCV epitope that could be useful in the diagnosis and / or treatment with HCV-induced NANBH. cDNA information in the clones mentioned above is useful for constructing probes to isolate additional cDNA sequences derived from as yet undefined regions of the HCV genome, whose cDNA in the clones is described in Chapter IV.A. are implemented. For example, labeled probes embodying a sequence of about 8 nucleotides in length or more, preferably 20 or more nucleotides, derived from regions related to the 5'-end or 3'-end of the HCV family of cDNA sequences shown in Figures 1, 3 , 6, 9, 14, 32 can be used to isolate overlapping cDNA sequences from HCV cDNA libraries. These cDNA overlapping sequences in the clones described below, and including sequences derived from the region of the genome from which the cDNAs in the clones mentioned below were not derived, can then be used to synthesize probes to identify other overlapping fragments that are not required, that they overlap in the cDNA clones described in Chapter IV.A. Although the HCV genome is segmented and the segments do not contain a common sequence, it is possible to sequence the entire viral genome using the technique of isolating overlapping cDNAs derived from the viral genome. But if the viral genome is a segmented genome that lacks common sequences, the genome sequence can be ordered by serologically testing lambda-gtll HCV cDNA libraries as used to isolate clone 5-1-1, sequencing cDNA isolates, and using isolated cDNAs for isolation overlapping fragments, this technique is described in Chapter IV.A. Alternatively, there must be characterization of genomic segments from viral genomes that have been isolated from purified HCV particles. Procedures for the purification of HCV particles and for their detection during the course of the purification process are described below. Methods for isolating polynucleotide genomes from viral particles are known in the art, and one of the methods that is angular for use is illustrated in Example IV.A.1. The isolated genomic segments must then be cloned and sequenced. Thus, with the information given here, it is possible to clone and sequence the entire HCV genome, given its nature.

The procedures for constructing cDNA libraries are known in the art, and will be described below; the procedure for constructing the HCV cDNA library in lambda-gtll is discussed below, in Chapter IV.A. However, cDNA libraries useful for this assay with nucleic acid probes can be constructed in other vectors such as lambda-gtlO (Huyunh et al. 1985). HCV-derived cDNA detected by the probes performed in Figures 1-32, and from probes synthesized from polynucleotides derived from these cDNAs can be isolated from the clone by degradation of the polynucleotides by a repressing restriction enzyme (or enzymes), and is then sequenced. See, for example, Chapter IV.A.3. and IV.A.4, where the techniques described are used for isolation and sequencing of HCV cDNA overlapping HCV cDNA in clone 5-1-1, Chapter IV.A.5-IV.A: 7 for isolation and sequencing of HCV cDNA, which covers that of clone 81 and Chapters IV.A.8 and IV.A.9. for isolating and sequencing a clone overlapping another clone (clone 36) overlying clone 81.

The sequence of information derived from these overlapping HCV cDNAs is useful for identifying regions in the viral genome that are homologous or heterogeneous and which must indicate the presence of different genome strains and / or populations of defective particles. It is also used for the construction of hybridization probes for the detection of HCV, HCV antigen or HCV nucleic acids in biological samples, and for HCV isolation (see below), the techniques used will be discussed in Chapter II.G. Overlapping cDNAs can also be used to generate expression vectors for polypeptides derived from the HCV genome, which also encodes polypeptides encoded in clones 5-1-1, 36, 81, 91, 1-2 and in other clones described in Sec.

IV.A. The techniques for constructing these polypeptides containing HCV epitopes and for antibodies directed against HCV epitopes contained therein, as well as for their use, are analogous to those described for polypeptides derived from NANBV cDNA sequences contained in clones 5-1 -1 32 32 35 36

1-2, 81, 91 as described below.

Encoded in the cDNA family of sequences contained in clones 5-1-1, 32, 35, 36, 81, 91, 1-2 and other clones described in Chapter IV.A. are antigens containing epitopes that appear against HCV as unique; e.g. antibodies directed against these antigens are absent in persons infected with HAV or HBV, and from individuals not infected with HCV (see serological data present in section IV.B).

However, comparing the sequence of information of these cDNAs with sequences from HAV, HBV, HCV and with genomic sequences in Genebank shows that there is minimal homology between these cDNAs and polynucleotide sequences of this origin. Thus, bodies targeting antigens encoded in the cDNAs of these clones can be used to identify BB_NANBV particles isolated from infected individuals. Additionally, these are useful for isolating the NANBH agent (or agents).

HCV particles can be isolated from the serum of BB-NANBV infected individuals or from cell culture by a method known in the art, for example, techniques based on differentiating the size of such sedimentation or exclusion methods, or density based techniques such as ultracentrifugation in density gradients or precipitation with agents such as polyethylene glycol or chromatography on various materials such as anionic or cation exchangeable materials and materials that bind between hydrophobicity as well as affinity clones. During the isolation process, the presence of HCV can be detected by hybridization analysis of the extracted genome, using a probe derived from HCV cDNA described below, or by immunoassay (see Chapter II.I.), where antibodies directed against HCV antigen are used as probes. is encoded in the family of cDNA sequences shown in Fig. 132 and also directed against the HCV antigen encoded in the overlapping HCV cDNA sequences discussed below. The antibodies can be monoclonal or polyclonal, and it is desirable to purify them before use in immunoassay. A process for purifying polyclonal bodies directed against antigen encoded in clone 5-1-1 is described in Chapter IV.E; an analogous purification process can be used for antibodies directed against other HCV antigens.

Antibodies directed against HCV antigens encoded in the cDNA family shown in Figures 1-32, as well as those encoded in overlapping HCV cDNAs and attached to pure carriers, are useful for HCV isolation by immunoaffinity chromatography. Techniques for this are known and include techniques for fixing antibodies to solid carriers so that they retain their immunoselective activity; techniques may also be those in which antibodies are adsorbed on a carrier (see, for example, Kurstak v ΝΖΥΜΝΖΥΜΕ

IMMUNODIAGNOSIS, p. 31-37) as well as those in which antibodies bind covalently to the vehicle. In general, the techniques are similar to those used for the covalent binding of the antigen to the solid support described in Chapter II.C .; however, spatial groups may be incorporated into bifunctional binding agents such 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 polynucleotides derived from the HCv family of cDNA sequences shown in Figures 1-32, as well as from overlapping HCV cDNA sequences as probes, as described below. In this case, the fractions are treated under conditions that should cause the viral particles to disintegrate, 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 of the fact that isolated particles are HCV-causing agents can be obtained by chimpanzee infection with isolated viral particles and then determining whether there are NANBH symptoms as a result of the infection. The viral particles from the purified preparations may then be characterized further. The genomic nucleic acid was purified. Based on its sensitivity to RNAase rather than DNAase I, we find that the virus is composed of the RNA genome. See, for example, IV.C.2, below. Dysfunctionality and circularity or non-circularity can be determined by techniques known in the art, including, for example, their observation under an electron microscope, their migration into density gradients, and their sedimentation characteristics. Based on the hybridization of the HCV genome obtained with respect to the negative HCV cDNA structures, it is reported that HCV can positively encompass the RNA genome (see section IV.H.1). Techniques like these are described in, e.g., METHODS IN

ENZYMOLOGY. Additionally, purified nucleic acid can be cloned and sequenced by known techniques, including reverse transcription, although it is an RNA genetic material.

See, for example, Manitatis (1982) and Glover (1985). The use of nucleic acid derived from viral particles enables sequencing of the entire genome, whether that is or is not segmented. Investigation of the homology of the polypeptide encoded in the unbroken ORF of pooled clones 14i to 39c (see Figure 26) shows that the HCV polypeptide contains regions of homology with the corresponding proteins in the conserved regions of Flavivirus.

An example of this is described in Chapter IV.H.3. This conclusion has several important meanings. First, this indication, with respect to the results showing that d HCV contains a positive-stranded genome of about 10,000 nucleotides, is consistent with the suggestion that HCV is a flavivirus or a virus similar to this. Generally speaking, Flavivirus virions and their genomes have a relatively consistent structure and organization known. See Rice et al., (1982) and Brinton M.A. (1988).

Such structural genes encoding C, pre-M / M and E polypeptides can be found at the 5'-terminus of the genome upstream of clone 14i. But through the use of comparisons with other flaviviruses, we can provide a report in the form of specifying the location of the sequences encoding these proteins.

Isolation of sequences upstream of those in clone 14i can be accomplished in a number of ways, which are given here for information only and should be clear to the skilled person. For example, genome-wide-ranging technique may be used to isolate other sequences that are 5 'relative to that of clone 14i but overlap with that clone; this leads to the isolation of additional sequences. This technique is demonstrated below in Chapter IV.A. Flaviviruses are also known to contain ODR.ANE epitopes and regions of conserved nucleic acid sequences. Polynucleotides containing conserved sequences should be used as probes that bind the HCV genome, allowing them to be isolated. Additionally, these sequences, in relation to those derived from HCV cDNA (shown in Figure 22), can be used to construct a starter, which is then used in systems that amplify genomic sequences located higher than those in clone 14i, with the application of polymerase chain reaction technology. An example of this is described below. The structure of HCV can also be identified and the isolation of its components is possible. Morphology and size can be ordered, for example, by an electron microscope. The identification and localization of specific viral polypeptide antigens, such as coated, enveloped or intrinsic antigens, such as protein-coupled amino acids, core antigens, and polynucleotide polymerases, can also be determined by, for example, determining whether antigens are present as major or minor viral components, as well as using antibodies directed against specific antigens encoded in isolated cDNAs as probes. This information is useful in the construction of vaccines; for example, when incorporating an external antigen into the vaccine preparation. Multivalent vaccines may, for example, comprise a polypeptide derived from a genome encoding a structural protein (such as E), as well as a polypeptide from another part of the genome, for example, a non-structural or structural polypeptide.

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

The suggestion that HCV is a Flavivirus or a virus similar to it also provides information on how to raise HCV. A term similar to it means that the virus shows a degree of homology with respect to known conservative regions and that the majority of the genome is one ORF. Cultivation procedures

Flaviviruses are known to those skilled in the art (see, for example, the journals of Brinton (1986) and Stollar (1980). Generally speaking, cells or cell types favorable for HCV cultivation may include those known, but may include replication of Flavivirus, for example: Monkey kidney cell types (eg MK ₂ , VERO); Swine kidney cell types (eg PS); Hamster pup kidney cell types (eg BHK); Murine macrophage cell species (e.g., p388Dl, MK1, Mml); Human macrophage cell types (e.g., U-937); human peripheral blood leukocytes; human monocytes; hepatiocytic or hepatocyte cell types (e.g., HUH7, HEPG2); embryos or embryonic cells (eg, chicken embryonic fibrioblasts); or cell types derived from invertebrates, desirable from insects (eg, cell type drosophila), also desirable from arthropods, for example, cell mosquito species (eg A. albopictus, Aedes aegypti, Cutex tritaeniorhynchus) or tick cell types (eg RML-14 Dermacentor parump It is possible that primary hepatocytes are first cultured and then infected with HCV; or alternatively, culture hepatocytes may be derived from the liver of infected individuals (e.g., human or chimpanzee). The last example is an example of a cell that is infected in vivo and subsequently brought in vitro. In addition, various non-sacrificial procedures can be used to obtain cell types derived from hepatocyte cultures. For example, primary liver cultures (before and after enrichment of the hepatocyte population) may be pooled into different cells to maintain stability. Cultures can also be infected with transformation viruses or transformed with transforming agents to produce permanent or semi-permanent cell types. Additionally, cells in liver culture may be pooled to produce cell types (e.g., HepG2). Methods for combining cells are known in the art and include, for example, the use of clustering agents such as polyethylene glycol, Sendai virus and Epstein-Barr virus.

As described below, HCV Flavivirus or virus is similar to this. For this reason, it is likely that HCV infection of cell types can be carried out by techniques known in the art as techniques used to infect Flaviviruses. These include, for example, incubation of cells with viral preparations under conditions that allow the virus to enter the cell. Viral production can also be obtained by transfection of cells with isolated viral polynucleotides. Togavirus and Flavivirus RNA are known to be infectious in various vertebral cell types (Pferkorn and Shapiro (1974)) and in mosquito cell types (Peleg (1969)). methods for transfection of cell culture tissue with RNA duplexes, RNA and DNA positivity (including cDNA) are known in the art and include techniques such as techniques that use electroporation and precipitation with DEAE-Dextran or calcium phosphate. Abundant origin of HCV RNA can be obtained by performing in vitro transcription of HCV cDNA corresponding to the entire genome. Transfection with this material or with cloned HCV cDNA should result in viral replication and in vitro progression of the virus.

In addition to cultured cells, the animal model of the system can also be used for viral replication; animated systems containing Flavivirus are known to those skilled in the art (see, for example, the journal Monath (1986)). Thus, HCV replication can take place not only in chimpanzees, but also in, for example, marmosets (long-tailed American monkey) or young mice.

II.L. Testing for anti-viral agents for HCV

The availability of cell culture and the animal model of the HCV system also allows testing for viral replication inhibiting anti-viral agents, and in particular for those agents that primarily allow growth and multiplication while inhibiting viral replication. These testing methods are known to those skilled in the art. In general, anti-viral agents are tested by observing at different concentrations their effect on the prevention of viral replication in a cell culture system that maintains viral replication, and by observing the inhibition of infectivity or viral pathogenicity (and low levels). toxicity) in the animal model of the system.

The methods and components for detecting antigens and HCV polynucleotides provided herein are useful for testing anti-viral agents, in which they provide alternative and possibly more sensitive agents for detecting the agent's effect on viral replication than cell-plate screening or ID ₅₀ screening. For example, the HCVpolynucleotide probes described herein may be used to determine the amount of nucleic acid produced in a cell culture. This can be accomplished by hybridization or competitive hybridization of infected cell nucleic acids with a labeled HCV polynucleotide probe. For example, anti-HCV antibodies may also be used to identify and quantify HCV antigen in the cell culture using the immunoassays described herein. In addition, it is desirable to determine the amount of antigen in the infected cell by means of competitive screening. In these competing assays, useful polypeptides are encoded in the HCV cDNA described herein. In general, recombinant HCV polypeptide from HCV cDNA should be labeled, and inhibition of binding of this labeled polypeptide to HCV polypeptide due to antigen produced in the cell culture system should be noted. However, these techniques are practically useful in cases where HCV may be able to replicate in the cell line without causing cell death.

II.M. Acquisition of attenuated HCV filaments

In addition to the above, the use of culture system and / or animal model systems may be possible due to the isolation of weakened HCV strands. These threads are useful for vaccines or for isolating viral antigens. The weakened filaments are isolated after multiple transitions in the culture cell and / or animal model. Detection of weakened strands in an infected cell or individual is achieved by techniques known in the art, which may include, for example, the use of antibodies to one or more HCV-encoded epitopes to replace a probe, or the use of a polynucleotide containing HCV sequence at least 8 nucleotides long as probes.

Alternatively, or additionally, the impaired species can be generated using the genomic HCV information provided here and using recombinant techniques. In general, deletion of regions of the coding genome, for example, of a pathogenicity-bound polypeptide that permits viral replication should be attempted. In addition, genome-wide assembly must be able to express an epitope that produces an increased amount of neutralizing antibodies to HCV. The modified genome is then used to transform cells that allow HCV replication and cell growth under conditions that allow viral replication. Impaired HCV structures are useful not only for vaccines but also as sources for the commercial production of viral antigens, and hence the processing of these viruses should require lesser protective criteria for employees in the production of the virus.

III. General procedures

Common techniques used in genome extraction from viruses, cDNA library acquisition and testing, clone sequencing, expression vector construction, cell transformation, immunoassays such as radioimmunoassays and ELISAs for cell growth in culture and the like are known and known in the art also described in lab manuals. However, as a general guide, a set of sources is constantly followed here, which is constantly available for such processes and for materials that are useful in their implementation.

III.A. Hosts and expression control sequences

The prokaryotic and eukaryotic host cells may be used to express the desired coding sequences when appropriate control sequences compatible with the designated host are used. Among prokaryotic hosts, E. coli is most commonly used. Expression control sequences for prokaryotes include promoters that optionally contain operator moieties and ribosome-binding sites. Prokaryotic host compatible transfer vectors are generally derived from, for example, a pBR322 plasmid containing operons that confer ampilicin and tetracycline resistance, and various pUC vectors that also contain sequences that confer antibiotic resistance markers. These markers can be used for selection of successful transformers by selection. Commonly used prokaryotic sequences include beta-lactamase (penicillinase) and lactose promoter systems (Chang et al. (1977)), tryptophan (trp) promoter system (Goeddel et al (1980)), and the lambda-derived P _L 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; other prokaryotic hosts, such as Bacillus or Pseudomonas species with appropriate control sequences, may be used if desired. Eukaryotic hosts include in the culture systems, yeasts and mammalian cells. Saccharomyces cerevisiae and Saccharomzces carlsbergenesis are the most commonly used yeast hosts and correspond to fungal hosts. Yeast compatible vectors carry markers that allow selection of successful transformants by administering prototrophy to auxotrophic mutants or resistance to wild-type species against heavy metals. Yeast compatible vectors can utilize a 2 micron start of replicate (Broach et al (1983)), a combination of CEN3 and ARS1, or other means of providing replication, such as sequences, that will result in the incorporation of a suitable fragment into the host cell genome. The control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al (1968); Holand et al (1987)) that include a promoter for 3 phosphoglycerate kinase (Hitzeman (1980)). Terminators such as those derived from gene enolase (Holland (1981)) may also be involved. Particularly useful are control systems comprising glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatory promoter, also terminators derived from GAPDH and, if desired, a control sequence from alpha yeast vectors. Additionally, the transcriptional regulatory region and the transcriptionally initiating region are operably linked and may not be naturally bound in the wild-type organism. These systems are detailed in EPO 120.551 issued

10/3/1984; EPO 116.201, issued 22.8.1984 and EPO 164.556, issued 18.12.1985, which are referred to in the present invention, are also referred to herein.

Mammalian cells available as expression hosts are known in the art and include many immortal cell types accessible from the American Type Culture Collection (ATCC), including HeLa cells, Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells and many other cell types. Suitable mammalian cell promoters are also known and include viral promoters such as those from Simian Virus 40 (SV40) (Fiers (1987), Rous arcoma virus (RSV), adenovirus (ADV) and bovine papilloma virus (BPV). may also require terminator sequences and poly A sequences; expression enhancer sequences may also be involved as well as amplification-inducing sequences. These sequences are known in the art. Vectors suitable for replication in mammalian cells may include viral replicons or sequences that provide integration of the corresponding sequences encoding NANBV epitopes in the host genome.

III.B. Transformations

Transformations can be performed by known methods for introducing polynucleotides into host cells, including, for example, packaging of polynucleotides into a virus and transduction of a host cell by virus and direct uptake of polynucleotides. The transformation process used depends on the host being transformed. For example, transforming E.coli cells from lambda-gtll containing BB-NANBV sequences will be described in the example in the section below. Bacterial transformation with direct uptake is generally used by treatment with calcium or rubidium chloride (Cohen (1972); Manitatis (1982)). Transformation of yeast by direct uptake can be performed using the procedure of Hinnen et al (1987). Mammalian transformations with direct uptake can be performed using the calcium phosphate precipitation method of Graham and Van der Eb (1978) or with various modifications of this.

III.C. Construction of vectors

Vector-building uses techniques known in the art. DNA digestion of specific sites is performed by treatment with appropriate 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 with 1 unit of enzyme in about 20 microliters of buffer solution, incubated at 37 ° C for 1-2 hours. After incubation with the restriction enzyme, the protein is removed by phenol / chloroform extraction and DNA is eliminated by ethanol precipitation. The cleaved fragments can be separated using polyacrylamide or gel electrophoresis techniques according to the general methods found in Methods and Enzymology (1980) 65: 449-560. The broken ends of the adhesive fragments can be opened using E.coli DNA polymerase I (Klenow) in the presence of the corresponding deoxynucleotide triphosphate (dNTPs) present in the mixture. Treatment with Sl nuclease can also be used, resulting in the hydrolysis of certain parts of single DNA.

Connections are made using standard buffer and temperature conditions used by T4DNA ligase and ATP; bonding of the adhesive end requires less ATP and less ligase than the binding of the open ends. When vector fragments are used as part of a coupling mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or bovine intestinal alkaline phosphatase to remove 5'-phosphate, thereby preventing vector re-binding; alternatively, restriction enzymatic degradation of unwanted fragments can be used to prevent binding.

The coupling mixture is transformed into a favorable cloning host such as E. coli, and successful transformants are selected by, for example, antibiotic resistance and testing for proper construction.

III.D. Construction of desired DNA sequences

Synthetic oligonucleotides can be obtained using the automatic synthesis of oligonucleotides as described by Warner (1984). If desired, synthetic structures can be characterized by ³² P treatment with a polynucleotide kinase in the presence of ³² P-ATP, under 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). The modified DNA is packaged in the phage as a simple sequence and then translated into double DNA using DNA polymerase as a primary synthetic oligonucleotide complementary to the portion of the DNA being modified and having the desired modification included in its sequence. The resulting double DNA is transformed into a phage carried by the host bacterium. The culture of transformed bacteria containing replicants of each strand of the phage is placed in agar to obtain plates. Theoretically, it contains 50% of the new mutant sequence phage plates and the other 50% has the original sequence. Plate replicates hybridize due to the labeling of the synthetic probe at temperatures and conditions that permit hybridization with a specific thread rather than an unmodified sequence. Sequences identified by hybridization are eliminated and cloned.

III.B. Probe hybridization

DNA libraries can be searched using the procedure of Grunstein and Hogness (1975). In this process, the DNA under examination is immobilized on nitro-cellulose filters, denatured and re-hybridized with buffer containing 0-50% formamide, 0.75M NaCl, 75mM Na citrate, 0.02% (w / v) of each bovine serum albumin, polyvinyl pyrrolidone and Ficola, 50mM Na phosphate (pH 6.5), 0.1% SDS and 100 micrograms / ml of denatured DNA carrier. The percentage of formamide in the buffer as well as the time and temperature conditions of the rates of pre-hybridization and subsequent hybridization depend on the required rigor. Oligomer probes requiring a lower degree of rigor are generally used with a lower percentage of formamide, lower temperatures, and a longer hybridization time. Probes containing more than 30 or 40 nucleotides, such as those derived from cDNA or genomic sequences, generally use higher temperatures, e.g. about 40-42 ° C, and a higher percentage of formamide, e.g. about 50%. After pre-hybridization, a 5 '- ³² P-labeled probe is added to the buffer, and the filters are incubated in this mixture under hybridization conditions. After rinsing, the filters under consideration undergo autoradiography to indicate the location of the hybridization probe; DNA at appropriate locations on the original agar trays are used as the source of the desired DNA.

III.F. Construction verification and sequencing

For routine vector constructs, the coupling mixtures are transformed into E.coli type HB101 or another suitable host, and then successful transformants are selected by antibiotic resistance or other markers. Transformant plasmids are then prepared by the method of Clewell et al (1969), which usually follows chlorampheniclo amplification (Clewell (1972)). DNA is isolated and analyzed, usually by restriction enzyme analysis and / or sequencing. Sequencing can be done using the dideoxy method of Sanger et al (1977), as further described by Messing et al (1981), or with the method of Maxam et al (1980). The band thickening problems sometimes observed in GC-rich regions are resolved by the use of Tdeaoguanosine according to Barr (1986).

IZ 1.6. Binding enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) can be used to measure the concentration of an antigen or antibody. This method depends on the conjugation of the enzyme either against the antigen or the antibody, and uses the binding of the enzyme activity as a quantitative marker. For the purpose of measuring antibodies, the known antigen is fixed to a firm base (eg, micro tray or plastic cover), incubated with diluted test sera, washed, incubated with enzyme-labeled anti-immunoglobulin, and then washed again. Enzymes good for labeling are well known in the art and include, for example, horseradish peroxidase. The bound enzyme activity is measured by the solid phase by the addition of a specific substrate, and by determining the build of the product or the use of the substrate in a color-modifying manner. Bound enzyme activity is a direct function of the amount of bound antibody.

For the purpose of measuring the antigen, an individual specific antibody is bound to a solid base, then the test material containing the antigen is added, and after incubation, the solid phase is washed off and another enzyme labeled antibody is added. After washing, the substrate is added and the enzyme activity is colorimetrically checked and bound to the antigen concentration.

IV. Examples

Below are examples of the present invention that are for illustrative purposes only and do not limit the scope of the invention. In light of this discovery, a number of security developments will be apparent to those skilled in the art. The procedures are given, for example, in Chapter IV.A. may be repeated if desired, but not necessarily as techniques available to construct the desired nucleotide sequences based on information provided by the invention. Expression was shown on E. coli; other systems that are accessible are described in Chapter III.A. Additional epithets derived from the genomic structure can also easily be produced and used for antibody production as described below.

IV.A. HCV cDNA acquisition, isolation and sequencing

IV.A.l Acquisition of HCV cDNA

The source of the NANB agent was a plasma reserve derived from chimpanzees with chronic NANBH. The chimpanzee was experimentally infected with the blood of another chimpanzee with chronic NANBH obtained by HCV infection in a contaminated batch of factor 8 concentrate derived from human serum reserve. The plasma chimpanzee reserve was upgraded by combining many individual plasma samples containing high levels of alanine aminotransferase activity; this activity results from hepatic injury caused by HCV infection. So, lml of ICC ⁶ diluted reserve serum was given iv, which caused NANBH in another chimpanzee, and its CIDS was at least 10 ⁶ / ml, e.g. he had a high infection titer of the virus.

The cDNA library from the high titre plasma reserve was constructed as follows. First, viral particles were isolated from the plasma; The 90 ml aliquot is diluted with 310 ml of a solution containing 50mM Tris-HCl (pH 8.0), 1mM EDTA, 100mM NaCl. The waste is removed by centrifugation for 20 minutes at 15000 xg at 20 ° C. The viral particles in the resulting supernatant were then tabulated by centrifugation in a Beckman SW rotor at 28,000 rpm for 5 hours. To liberate the viral genome, the particles decompose by suspending the tablets in 15 ml of a solution containing 1% Na dodecyl sulfate (SDS), lOmM EDTA, lOmM Tris-HCl (pH 7.5) and also containing 2mg / ml proteinase k, then however, the whole mixture is incubated for 90 minutes at 45 ° C. Nucleic acids are isolated by adding 0.8 micrograms of MS2 bacteriophage RNA as carrier and extracting the mixture four times with a 1: 1 mixture of phenol: chloroform (phenol saturated with 0.5M Tris-HCl (pH 7.5),

0.1% (v / v) of beta-mercaptoethanol, 0.1% (v / v) of hydroxyquinoline, followed by two chloroform catheterations. The aqueous phase was concentrated with 1-butanol before precipitation with 2.5 volumes of absolute ethanol overnight at -20 ° C. Nucleic acid is eliminated by centrifugation in a Beckman SW41 rotor at 40,000 rpm for 90 minutes at 4 ° C and then dissolved in water treated with 0.05% (v / v) diethyl pyrocarbonate and then autoclaved. The nucleic acid obtained by the above procedure (less than 2 micrograms) is denatured with 17.5mM CH ₃ HgOH; cDNA is synthesized using this denatured nucleic acid for the template and cloned into the EcoRI site of phage lambda-gtll using the method described by Huynh (1985), with the exception of that random start replaced by oligo (dT) 12-18 during the synthesis of the first cDNA structures with reverse transcriptase (Taylor et al (1976)). The double cDNAs obtained are fractionated by size on a Sepharose CL-4B column; eluted material approximately 400, 300 in length,

200 and 100 bp are added to cDNA reserves 1,2,3 and 4. The lambdagtll cDNA library is upgraded from cDNA in reservoir 3. The lambda-gtll cDNA library made from reserve 3 is tested for epitopes that should specifically bind to serum derived from a patient who previously had NANBH. About 10 ⁶ phages are tested with the patient's serum using the method of Hyunha et al (1985), except that the bound human antibodies are detected with a sheep anti-human Ig antiserum that has been radiolabeled with ¹²⁵ J. It has been identified and purified 5 positive phages. Five positive phages were also tested for the specificity of binding to the serum of 8 different humans 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 for primary library test of the library. The fifth phage (5-1-1) encodes a polypeptide that reacts immunologically with 5 of the 8 sera tested. But this polypeptide does not react immunologically with the serum of 7 normal blood donors. For this reason, it is clear that it encodes a clone 5-1-1 polypeptide that is specifically recognized by immunologically with serum from NANB patients.

IV.A. 2. The HCV cDNA sequences in the recombinant phage 5-1-1 and the polypeptide encoded in the cDNA sequence in the recombinant phage 5-1-1 were sequenced by the method of Sanger et al (1977). As a central part of the method, DNA is cut with EcoRI, isolated by size fractionation using gel electrophoresis. EcoRI restriction fragments are encoded in vector M13, mpl8 and mpl9 (Messing (1983)) and sequenced using the dideoxychain termination method of Sanger et al (1977). The acquisition sequences are shown in Figure 1.

The encoded polypeptide of Fig. 1, which is encoded in the HCV cDNA, is in the same translation site as the N-terminal betagalactose moiety on the nucleus. As shown in Chapter IV.A., it encodes a translational open reading site (ORF) 5-1-1 epitope (s) that are specifically identified by the serum of patients and chimpanzees with NANBH infections.

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

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

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

The lambda-gtll library was tested with this probe using the procedure described by Huynh (1985). About 1 in 50,000 clones were hybridized with the probe. Three clones containing cDNAs that were hybridized with the synthetic probe were identified by numbers 81, 1-2, and 91, respectively.

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 mainly as in Chapter IV.A.2. The sequences of these clones are shown in FIG. 5-1-1 against the HCV cDNA sequence in Figure 2, which shows the structure encoding the detected HCV epitope, and homologies in nucleotide sequences are indicated by vertical lines between sequences.

The sequences of cloned HCV cDNAs are highly homologous in overlapping regions (see Figure 2). But, there are differences in the two regions. Nucleotide 67 in clone 1-2 is thymidine, while the other three clones at this site contain a citrine residue. It should also be noted that the same amino acid is encoded, regardless of whether C or T is present.

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 radioimmunological data that will be described below in Chapter IV.D, it is possible that the HCV epitope is encoded in this region 28 b.p.

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

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

The composition of the HCV cDNA sequence derived from the overlapping cDNAs in clones 5-1-1, 81, 1-2, and 91 is shown in Fig. 3. But no unique 28 b.p. clone 5-1-1. The figure also shows the sequence of the polypeptide encoded in the ORF of the HCV cDNA composition.

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

Isolation of HCV cDNA sequences upstream of and those overlapping in clone 81 c DNA is performed as follows. The Lambdagtll cDNA library is obtained as described in Chapter IV.A.1 and then tested by hybridization with a synthetic polynucleotide probe homologous to the 5'terminal sequence of clone 81. The sequence of this clone 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 ′

The methods were essentially as described in Huynh (1985), except that the library filters were washed twice under strict conditions, e.g. they were washed in 5 X SSC, 0.1% SDS at 55 ° C for 30 minutes each time. About 1 in every 50,000 clones was hybridized with the probe. A positive recombinant phage containing the sequence-hybridized cDNA was isolated and purified. This phage was designated clone 36.

Lower located cDNA sequences that overlay the carboxyl-terminus of the sequence in clone 81 of the cDNA were isolated by a procedure similar to that for isolating higher-lying cDNA sequences, except that a synthetic oligonucleotide probe homologous to the 3'- clone terminal sequences 81. The sequence of the synthetic polynucleotide used as the test probe was:

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

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

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

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

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

IV.A. 7 HCV cDNA nucleotide sequences in clone 32

The nucleotide sequence of cDNA in clone 32 was determined mainly as described in Chapter IV.A.2 for the clone sequence 5-1-1. Clone sequence data indicate that cDNA in clone 32 of the recombinant phage is derived from two different sources. One cDNA fragment comprises 418 nucleotides derived from the HCV genome; the second fragment comprises 172 nucleotides and was derived from the genome of the bacteriophage MS2, which is used as a carrier during the acquisition of the lambda-gtll 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 ORF encoded polypeptide are also indicated in the figure. This sequence contains one continuous ORF encoded by clone 81 at the same translation site as the HCV antigen.

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

Isolation of HCV cDNA sequences lower than those overlapping in clone 36 together with those overlapping in clone 36 is performed as described in Chapter IV.A.5 for those overlapping clone 81 with the exception of that the synthetic polynucleotide is based on the 5 * region of 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 was hybridized with this probe. An isolated and purified clone of a recombinant phage containing a cDNA that hybridizes this sequence was named clone 35.

IV.A. 9 Nucleotide sequence of HCV cDNA in clone 35

The nucleotide sequence of cDNA in clone 35 was determined mainly as described in Chapter IV.A.2. The sequence, its region of overlap with that of the cDNA in clone 36, and the putative encoded polypeptide are shown in Fig. 8. Clone 35 clearly contains one continuous ORF encoding the polypeptide at the same translation site as encoded by clone 36, clone 81 and clone 32. Figure 9 shows the sequence of a long continuous ORF that extends into clones 35, 36, 81, and 32 together with the putative HCV polypeptide encoded herein. This pooled sequence is confirmed using other independent cDNA clones derived from the same lambda-gtll cDNA library.

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

Isolation of HCV cDNA sequences located above and together with sequences overlapping those in clone 35 of the cDNA shall be performed as described in Chapter IV.A.8 for those overlapping with clone 36 of the 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 as follows:

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

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

IV.A. 11 HCV nucleotide sequence in clone 37b

The cDNA nucleotide sequence in clone 27b was determined mainly as described in Chapter IV.A.2. The sequence, its region of overlap with that of the cDNA in clone 35, and the putative polypeptide encoded here are shown in FIG. 10. The 5'-terminal nucleotide of clone 35 is T, while the corresponding nucleotide in clone 37b is A. cDNA from three other independent clones that were isolated during the process in which clone 37b was isolated (the procedure is described in Sec. IV.A.10) were also sequenced. cDNAs from these clones also contain A in this position. Thus, the 5′-terminal T in clone 35 may be an adjunct to the cloning process. Additions often grow at the 5'-end of the cDNA molecule.

Clone 37b contains clearly one continuous ORF encoding a polypeptide, which is a continuation of a polypeptide encoded in the ORF by stretching through the overlapping clones 35, 36, 81 and 32.

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

Isolation of HCV cDNA sequences downstream of clone 32 is performed as follows. First, the clone clone 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 synthetic probe sequence was as follows:

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

Using a nucleotide sequence from a clone of cl, a different synthetic nucleotide was synthesized that had the sequence:

5′-TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3 ′ Testing a lambda-gtll library using a sequence derived from a cla clone as a probe yields about 1 in 50,000 positive clones. The isolated and purified clone hybridized with this probe was named clone 33b.

IV.A. 13 Nucleotide sequence of cDNA in clone 33b

This has been determined as described in Chapter IV.A.2. The sequence, its overlap region with that of cDNA clone 32, and the putative encoded polypeptide are shown in Figure 11.

Clone 33b clearly contains a single continuous ORF, which is an extension of the ORFs of the overlapping clones 37b, 35, 36, 81 and 32. The polypeptide encoded in clone 33b is in the same translational site as that encoded in the extended ORFs of the overlapping clones.

IV.A. 14 Isolation of overlapping HCV cDNAs relative to clone 37b cDNAs against cDNAs in clone 33b

For the purpose of isolating HCV cDNAs overlapping cDNAs in clones 37b and 33b, the following synthetic oligonucleotide probe derived from cDNAs in these clones was used to test the lambda-gtll library using mainly the methods described in Chapter IV.A.3 . The probes used were:

5'-CAG GAT GCT GTC TCC CGC ACT CAA CGT C 3 'and 5'-TCC TGA GGC GAC TGC ACC AGT GGA TAA GCT 3' due to the detection of colonies containing HCV cDNA sequences overlapping those in clones 37b and 33b respectively . About 1 in 50,000 colonies were detected with each probe. The clone containing the cDNA that was higher than the cDNA overlapping in clone 37b was named clone 40b. A clone that contained a lower-lying cDNA and that overlapped the cDNA in clone 33b was named clone 25c.

IV.A. 15 HCV cDNA nucleotide sequences in clone 4Ob and clone 25c

The cDNA nucleotide sequences in clone 40b and clone 25c were determined essentially as described in section IV.A.2. The sequences 40b and 25c, their cDNA overlapping regions 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. But cDNAs from five other independent clones that were not isolated during the process in which clone 40b described in Chapter IV.A.14 was secreted were also sequenced. cDNAs from these clones also contain T in this position. That easy

G represents a cloning additive (see Appendix in Chapter IV.A.11).

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

Of the clones 40b and 25c, each clearly contains an ORF, which is an extension of the unbroken ORF in previously sequenced clones. The nucleotide sequence of the ORF, which extends beyond 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. No cloning additives are shown in the figure. Instead, the corresponding sequences in non-5′-terminal regions of multiple overlapping clones are shown.

IV.A. 16 Obtaining HCV cDNA Composition From cDNA in Clones 36,

In 32

The composition of HCV cDNA, C100 was upgraded as follows. First, cDNAs from clones 36, 81, and 32 are cut using EcoRI. The EcoRi fragment from each clone is then cloned individually in the EcoRi site of the pGEM3-blue vector (Promega Biotec).

The resulting recombinant vectors containing the cDNAs from clones 36, 81 and 32 are named pGEM3-blue / 36, pGEM3-blue / 81 and pGEM3-blue / 32, respectively. The appropriately oriented recombinant pGEM3-blue / 81 is degraded with Nael and Narl, after which the large (2850 bp) fragment is purified and linked to the small (570 bp) Nael / Narl purified fragment from pGEM3-blue / 36. This cDNA composition from clones 36 and 81 is used to generate another pGEM3-blue vector that contains continuous HCV ORFs contained in the overlapping cDNA in these clones. This new plasmid is then degraded with PvuII and EcoRi to release a fragment of about 680 bp in size, which then binds to a small (580 bp) PvuII / EcoRI fragment isolated from the appropriately oriented pGEM3-blue / 32 plasmid, thereby making the cDNA composition clones 36, 81 and 32 linked to the EcoRI linearized vector pSODcfl described in Chapter IV.Bl and used for expression of clone 5-1-1 in the bacterium. Recombinants containing about 1270 bp of EcoRI fragment of HCV cDNA composition (C100) are selected, and plasmid cDNA is digested 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 are isolated using the technique of isolating overlapping cDNA fragments from the lambda-gtll HCV cDNA library described in Chapter IV.A.3, with the exception of that the probes used were upgraded from the nucleotide sequences of the last isolated clones from the 5 'and 3' ends of the fused HCV sequence. The frequency of clones that have been hybridized with the probes described below is about 1 to about 50,000 in each case.

The HCV cDNA nucleotide sequences in clones 14i, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g, 39c are determined mainly as described in Chapter IV.A.2, except that the cDNA has been excised from these phages 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 isolation 33c was:

5′-ATC AGG ACC GGG GTC AGA ACA ATT ACC ACT 3 ′ The HCV cDNA sequence in clone 33c and switching to that of clone 40b is shown in Figure 15, which also shows the encoded amino acids.

Clone 8h was isolated using a nucleotide sequence based probe 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 switching to 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 CTG GTC ACG AGG CAC 3 ′ The HCV cDNA sequence in clone 71, clone 8h switching, 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 probe used for clone isolation had the following sequence:

5′-ACC TTC CCC ATT AAT GCC TAC ACC ACG GGC 3 ′ The HCV cDNA sequence in clone 14c, its switch with that in clone 25c, and the encoded amino acid 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 sequence of HCV cDNA c to clone 8f, its switching to 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 sequence of HCV cDNA in clone 33f, its switching with that in 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 CCT CTG AGC CCG 3 ′

The HCV cDNA sequence in clone 33g, its switching to 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 switching with clone 7e, 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 as follows:

5′-CAC CTA TGT TTA TAA CCA TCT CAC TCC TCT 3 ′ The HCV cDNA sequence in clone llb, its switching to 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 as follows:

5'-CTC TGT CAC CAT ATT ACA AGC GCT ATA TCA 3 'The HCV cDNA sequence in clone 14i, its switching with llb, and the amino acids encoded here are shown in Figure 24.

Clone 39c was isolated using a nucleotide sequence based probe 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 switch with clone 33g, and the amino acids encoded here are shown in Figure 25.

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

The HCV cDNA sequences in the isolated clones described below are linked to create the composition of the HCV cDNA sequence. Isolated clones connected in 5 'to 3' directions 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 isolated clones and the amino acids encoded here are shown in Figure 26.

In creating the composition sequence, the following heterogeneous sequences were examined in greater detail. Clone 33c contains an HCV cDNA of about 800 bp in length, which overlaps the cDNA in clones 40b and 37c. In clone 33c as well as in 5 other overlapping clones, nucleotide no. 789 G. But, in clone 37b (see Chapter IV.A.11), the corresponding nucleotide A. This sequence difference creates apparent heterogeneity in the amino acids encoded here, which may be CYS or TYR for G or A, respectively.

This heterogeneity can cause significant branching at the level of protein folding. Therefore, this sequence heterogeneity is indicated in Figure 26, which shows the composition of the HCV cDNA.

Nucleotide residue no. 2 in clone 8f of the HCV cDNA is G. However, the corresponding residue in clone 33f and 2h of the other overlapping clones is T. Therefore, in Fig. 26, the residue at this position is designated as T.

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

Nucleotide residue no. 4 in clone 33g of the HCV cDNA is T. However, in clone 33f and two other overlapping clones, this residue is A. Therefore, in Fig. 26, the corresponding residue is designated A.

The 3′ terminus of clone 14i is AA, although the corresponding dinucleotide in clone 11b and three other TA clones. As a result, the residual TA is presented in Figure 26.

Other sequence heterogeneities are described below. Examination of the HCV cDNA composition indicates that it contains one large ORF. This suggests that the viral genome is translated into one large polypeptide, the process of which is competitive or followed by translation.

IV.λ. 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 are isolated mainly by the technique described in Chapter IV.A.17, except that the probes were as indicated below.

The frequency of hybridized clones was about 1 in 50,000 in each case. The nucleotide sequences of Hcv cDNAs in these clones were determined essentially as described in Chapter IV.A.2, except that the cDNAs of the indicated clones were substituted with the cDNAs of clone 5-1-1.

Isolation of clone 12f containing a higher-lying cDNA than the HCV cDNA in Figure 26 is performed using a hybridization 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 folding with clone 14i, and the encoded amino acids are shown in Figure 27.

Isolation of clone 35f containing the lower-lying HCV cDNA in 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 GTC AAA AGT GAA GGC TAA CTT 3 ′

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

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

5′-TTG TCG TAT GAT ACC CGC TGC TTT GAC TCC 3 ′

The HCV cDNA sequence of clone 19g, its switching 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 of the probe was:

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

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

Clone 15e was isolated using a hybridization probe based on a 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 switching with the sequence in clone 26g, and the amino acids encoded therein are shown in Figure 31.

The clones described in this section have been stored by the ATCC under the terms and conditions described in Chapter II.A., and are identified by the following numbers:

lambda-gtll ATCC no. Saved date clone 12f 40514 10.nov.1988 clone 35f 40511 10.nov.1988 clone 15e 40513 10.nov.1988 clone K9-1 40512 10.nov.1988

The HCV cDNA sequences in the isolated clones, which will be described below, are linked in order to construct the composition of the HCV cDNA sequence. The isolated clones connected in 5 'to 3' directions 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 HCV cDNA sequence assembly derived from isolated clones and the amino acids encoded therein are shown in Figure 32.

IV.A.20 Alternative methods for isolating cDNA sequences located higher than the HCV cDNA sequence in clone 12f.

Based on the largest 5 'HCV sequence in Figure 32, which is derived from HCV cDNA in clone 12f, small synthetic reverse transcriptase oligonucleotide primers are synthesized and used to bind to the corresponding sequence in the HCV genomic RNA, due to the primary reverse transcription of higher-order sequences. The primary sequences are related to the known 5'-terminal sequences of clone 12f, but sufficiently lower enough to allow the construction of probe sequences in a higher position than the primary sequences lie. Standard naming and cloning procedures are used. The resulting cDNA libraries are tested with sequences that are located higher than the primary site (as concluded from performing the sequence in clone 12f). HCV genomic RNA is obtained from plasma or liver samples from chimpanzees with NANBH or from analog samples from humans with NANBH.

IV.A.21 Alternative methods of utilizing supply for isolation of sequences from the 5′-terminal region of the HCV genome

In order to isolate the final 5′-terminal sequences of the HCV RNA genome, the cDNA product of the first round of transcription duplicated by RNA template is supplied with oligo C. This is done 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 reverse transcriptase reaction. The origins of genomic HCV cRNA have been described in Chapter IV.A.20. The procedures for terminal transferase delivery and reverse transcriptase reactions were the same as in Manitatis et al. (1982). cDNA products are then cloned, tested and sequenced.

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

This method was based on previously used methods for cloning Flavivirus RNA cDNA. In this method, RNA is subjected to denaturation conditions, thereby removing secondary structures at the 3′-terminus and then supplying the structure with poly-A polymerase using rATP as a substrate. The reverse transcription of polyA-supplied RNA is catalyzed by reverse transcriptase, using oligo dT as a primer. Other cDNA strands are synthesized, cDNA products are cloned, tested and sequenced.

IV.A.23 Construction of lambda-gtll HCV cDNA libraries containing larger cDNA inserts.

The method used to construct and test lambda-gtll libraries was essentially the same as that described in Chapter IV.A except that the library was created from a cDNA reserve eluted from Sepharose CL-4B column.

IV.A. 24 Construction of HCV cDNA Libraries Using Synthetic Oligomers as References

Novel HCV cDNA libraries were derived from RNA derived from the chimpanzee infectious reserve of plasma described in Chapter IV.A. 1, and from the poly A ⁺ RNA fraction derived from the liver of these infected animals. cDNA was upgraded as described by Gubler and Hoffman (1983), except that the primers for the synthesis of the first cDNA strand were two synthetic oligomers based on the HCV genome sequence described below. Primers based on the sequence of clone ilb and 7e were respectively:

5′-CTG GCT TGA AGA ATC 3 ′ in

5'-AGT TAG GCT GGT GAT TAT GC 3 '

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

IV.B. Expression of HCV cDNA-encoded polypeptide and identification of expression products as HCV-induced gene

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

The HCV polypeptide encoded in clone 5-1-1 (see Chapter IV.A.2, below) is expressed as a superoxide dimutase (SOD) coupled polypeptide. This is done by subcloning the clone 5-1-1 cDNA insert into the expression vector pSODcfl (Steimer et al (1986)) as follows.

First, DNA isolated from pSODcfl is treated with BamHI and EcoRI, and the following binder binds to 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 is digested with EcoRI. The HCV cDNA insert in clone 5-1-1 is cut with EcoRI and linked to this EcoRI linearized plasmid DNA. The DNA mixture is used to transform E. coli type D1210 (Sadler et al. (1980)). Recombinants from 5-1-1 cDNA in the correct orientation for ORF expression shown in Figure 1 were identified by restriction mapping and nucleotide sequencing. Recombinant bacteria from one clone induced the 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-NANBei pooled polypeptide. The method for obtaining a vector encoding this pooled polypeptide is analogous to that used to create a vector encoding SOD-NANB ₅ -! - i, except that the HCV cDNA source 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 HCV cDNA in clone 81 and the putative sequence of amino acids encoding the polypeptide are shown in Figure 4.

The HCV cDNA insert in clone 81 was excised using EcoRI and then coupled to pSOD cfl containing the binder (see IV.B.l) and linearized upon treatment with EcoRI. The DNA mixture was used to transform E. coli type D1210. Recombinants from clone 81 of the HCV cDNA in the correct orientation for ORF expression shown in Figure 4 were identified by intermittent mapping and nucleotide sequencing.

One clone recombinant bacterium was induced due to the expression of SOD-NANB ₈ i polypeptide by bacterial growth in the presence of IPTG.

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

The HCV cDNA encoded polypeptide of clone 5-1-1 was identified as a NANBH-linked antigen such that sera of chimpanzees and NANBH-infected humans have been shown to react immunologically with the SOD-NANB ₅ -and-1 pooled polypeptide superoxide dimutase at its N-terminus and site 5-1-1 antigen at its C-terminus. This was performed using Western dyeing (described in Towbin et al. (1979)) as follows.

The recombinant bacterial species is transformed with an expression vector encoding the SOD-NANBs-χ-ι polypeptide described in Chapter IV.Bl and is induced by expression of the association of the polypeptide with growth in the presence of IPTG. The total bacterial lysate is then electrophoresed through polyacrylamide gels in the presence of SDS according to Laemmmli (1970). Separate polypeptides are transferred to nitrocellulose filters (Towbin et al. (1979)). The filters are then cut into strips, which are then individually incubated with different chimpanzee and human sera. The bound antibodies were detected by further incubation with ¹²⁵ J labeled sheep Ig anti-serum as described in Chapter IV.A.1.

The characterization of the chimpanzee serum used for Western staining and the results shown in the photograph of autoradiographic strips are shown in Figure 33. Nitrocellulose strips containing polypeptides are incubated with serum derived from chimpanzees at various times during acute NANBH (Hutchinson strain) infections (pathways 1- 16), hepatitis A infection (lanes 17-24 and 26-33), and hepatitis B infection (lanes 34-44). Lanes 25 and 45 show positive controls in which immune stains are incubated with patient serum used to identify recombinant clone 5-1-1 in the original lambda-gtll library assay (see Chapter IV.A.1).

The band seen in the control paths 25 and 45 in Figure 23 shows the binding of the antibody to the NANBs portion of the SOD of the pooled poiipeptide. These antibodies do not show binding to the SOD itself, so this was ruled out as a negative control in these samples and should be observed as a band that migrates significantly faster than the SOD-NANB ₅ _i-i pooled polypeptide.

Paths 1-16 in Figure 27 show antibody binding in serum samples of 4 chimpanzees; serum was obtained immediately prior to NANBH infection and sequentially during acute infection. As can be seen from the figure, although antibodies that react immunologically with the SOD-NANB5-1-1 polypeptide in serum of samples obtained prior to infectious HCV inoculum and are absent during the early acute phase of infection, all 4 animals finally induce antibody circulation into this polypeptide during the last part or after the acute phase.

Additional bands observed on immuno-stains were found in chimpanzee no. 3 and 4 elicited by binding to host bacterial proteins.

Contrary to the results obtained with serum from chimpanzees infected with NANBH, the development of antibodies against NANBs-t-! part of the pooled polypeptide was not observed in 4 HAV-infected chimpanzees or 3 HBV-infected chimpanzees. Only the binding in these cases was the basis for binding to host bacterial proteins, which also takes place in HCV infected samples.

Characterization of human serum used for Viestern stains and the results shown in the photograph of autoradiographic strips is present in Figure 34. Nitrocellulose strips containing polypeptides were incubated with serum derived from humans at various intervals during NANBH infection (pathways 1-21), HAV (lanes 3340) and HBV (lanes 41-49). Pathways 25 and 50 show positive controls in which immuno-stains were incubated with patient serum, which was also used in the original lambda-gtll library testing described below. Lanes 22-24 and 26-32 show non-infected controls containing serum from normal blood donors.

As can be seen from Figure 34, the serum from 9 NANBH patients, including the serum used to test the lambda-gtll library, contained antibodies against the NANB5-1-1 pooled polypeptide group. Serum from three NANBH patients did not contain these antibodies. It may be that anti-NANBs-II antibodies may develop in these patients in the future. It is also possible that this lack of reaction results from a variety of NANBH agents that cause disease in the individuals from which the non-responding serum was taken. Figure 34 also shows that the serum from many patients infected with HAV and HBV does not contain anti-NANBs-II antibodies, and that these antibodies are also not present in the serum of normal controls. Although one patient (pathway 36) has been reported to contain anti-NANB ₅ -ii antibodies, it is possible that this patient was pre-infected with NANBH, therefore NANBH uptake is very high and often subclinical.

These serological studies indicate that cDNA in clone 5-1-1 encodes epitopes that are specifically identifiable by the serum of patients and animals infected with BB-NANBV. Additionally, cDNA does not appear derived from the primate genome. A hybridization probe made with clone 5-1-1 or clone 81 does not hybridize to Southern patches of control human and chimpanzee genomic DNA from uninfected individuals under conditions where unique enecopies genes are capable of being detected. These probes also do not hybridize to Southern blots of control bovine genomic DNA.

IV.B.4 Expression of HCV cDNA-encoded polypeptide in clones 36, 81, 32

The HCV polypeptide encoded in the ORF that extends through clones 36, 81, 32 is expressed as a splicing polypeptide with SOD. This is accomplished by placing the cDNA composition of C100 in an expression cassette containing the human superoxide dismutase gene, inserting the expression cassette into the yeast expression vector, and expressing the yeast polypeptide.

An expression cassette containing the C100 cDNA composition derived from clones 36, 81, and 32 is upgraded to accommodate about 1270 bp. The EcoRI fragment to the EcoRI vector site is pS3-56 (also called pS356), thus yielding the plasmid pS3-56 _C i ₀₀ . The construction of the C100 is described in Chapter IV.B. 16 below.

The pS3-56 vector, which is a pBR322 derivative, contains an expression cassette comprising the ADH2 / GAPDH hybrid yeast promoter of the higher-lying human superoxide dismutase gene and the lower-lying GAPDH transcription terminator. A similar cassette containing these controls and the superoxide dismutase gene has been described by Cousens et al. (1987) and in associated EPO 196.056 issued 1.10.1986, which is commonly referred to under this designation. The cassette in pS3-56, however, differs from the one in Cousens et al (1987) in that the heterologous proinsulin gene and immunoglobulin were deleted and glni54 superoxide dismutase was accompanied by an adapter sequence containing the EcoRI site. The adapter sequence was:

5'-TKAT TTG GGA ATT CCA TAA TGA G -3 '

AC CCT TAA GGT ATT ACG CT.

The EcoRI site permits the insertion of heterologous sequences which, when expressed from a vector containing the cassette, yield superoxide dismutase-binding polypeptides via an oligopeptide binder containing the following amino acid sequence:

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

VSample pS356 was stored on April 29, 1988 under the terms of the Budapest Agreement with the American Type Culture Collection (ATCC), 12301 Parklawn Dr. Rockville, Maryland 20853 and was designated no. 67683. The terms and conditions for accessing and maintaining this deposit are the same as those specified in Chapter II.A. for species containing NANBV-cDNA. This deposit is for convention purposes only and is not required for use of the present invention from the point of view of description. The material deposited hereinafter is referred to with the indication.

After isolation of recombinants containing the C100 cDNA insert in the correct orientation, an expression cassette containing the C100 cDNA is excised from pS3-56Ci ₀₀ with BamHI and a fragment about 3400 bp in length containing the cassette is isolated and purified. This fragment is attached to the BamHI site of the yeast vector pAB24. The plasmid pAB24, whose characteristic features are shown in the figure, is a yeast-based vector containing a complement of two-micron replication sequences (Broach (1981)) and pBR322 sequences. It also contains the yeast URA3 gene derived from plasmid Zep24 (Botstein et al. (1979)) and the LEU ^2d yeast gene derived from plasmid pCl / 1. EPO Publication no. 116.201. Plasmid pAB24 was upgraded by degradation

YEp24 with EcoRI and by reconnecting the vector to remove partial two-micron sequences. The resulting plasmid, YEP24deltaRI, was linearized by digestion with Clal and coupled to a complete 2-micron plasmid, which was linearized with Clal. The resulting plasmid, pCBou, was then digested with Xbal and the 8605 bp long vector fragment was gel isolated. This isolated Xbal fragment was then coupled to a 4460 bp Xbal fragment containing the LEU ^2d gene isolated from pCl / 1; the orientation of the LEU ^2d gene is in the same direction as the orientation of the URA3 gene. Expression uptake was in the unique MamHI site of the pBR322 sequence, thus interrupting the bacterial resistance gene for tetracycline. The recombinant plasmid pAB24C100-3 containing the SOD C100 expression cassette was transformed into yeast JSC 308 as well as other yeast species. Cells were transformed as described in Hinnen et al. (1987) and applied to a clock-selective tray. The colonies were then inoculated in a leu-selective environment and then allowed to grow until saturation. The culture was induced by expression of a SOD-CIOO polypeptide (termed C100-3) by growth in ΥΕΡ containing 1% glucose.

Type JSC 308 is of the genotype MAT, leu 2, ura3 (del) DM15 (GAP / ADRI) integrated at the ADRI locus. In JSC 308, through the expression of a positive activator of a gene product, ADRI results in hyperdepression (relative to wild-type ADRI control) and with significantly higher contributions of expressed heterologous proteins when such proteins are synthesized through the ADH2 UAS regulatory system. The construction of yeast species of JSC 308 was described in an associate application USA ser. no. (Responsible Agent No. 2300-0229) completed at the same time and entered here as a reference. Sample JSC 308 was stored on May 5, 1988 by the ATCC under the terms of Budapest_Treaty and was designated no. 20879. The terms and conditions for making the deposit available, and for reflecting it, are the same as those specified in Chapter II.A., for HCV cDNA-containing species.

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

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

C100-3 is a pooled polypeptide expressed from plasmid pAB24C100-3 in yeast of the JSC type and is size-characterized and the polypeptide encoded in C100 has been identified as a NANBH-linked antigen by its immunological reactivity with human serum with chronic NANBH. The C100-3 polypeptide expressed as described in Chapter IV.B.4 was analyzed as follows. JSC 308 yeast cells were transformed with pAB24 or pAB24C100 and induced to express a heterologous plasmid of the encoded polypeptide. Induced yeast cells in 1ml culture (ODesonm about 20) were tabulated by centrifugation at 10,000 rpm for 1 minute, then lysed by wet stirring (10x1 min) with 2 volumes of solution and 1 volume of glass beads (0.2 millimeter diameter). The solution contained 50mM Tris-HCl (pH 8.0), 1mM EDTA, 1mN phenyl methyl sulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin. Insoluble material in the lysate comprising the C100-3 polypeptide was collected by centrifugation (10,000 rpm for 5 minutes) and then dissolved by boiling for 5 minutes in Laemmli SDS buffer sample (see Laemmli (1970)). An amount of polypeptide equivalent to that in 0.3 ml of induced yeast culture was electrophoresed through 10% polyacrylamide gels in the presence of SDS according to Laemmli (1970). Standard proteins were electrophoresed on elves together. Gels containing expressed polypeptides were stained with kumas in brilliant blue or subjected to Western staining as described in section IV.B.2, using serum of patients with chronic NANBH to determine the immunological reactivity of the polypeptides that were expressed from pAB24 and from pAB24C100-3.

The results are shown in Figure 37. In this figure, the polypeptides were stained with Kumasi brilliant blue.

The undissolved polypeptide from JSC 308 was transformed with pAB24 and from two different JSC clones transformed with pAB24C1000 shown in path 1 (pAB24) and pathways 2 and 3 respectively. Comparison of pathways 2 and 3 with pathway 1 induces the induced expression of a polypeptide corresponding to a molecular weight of 54,000 Daltons of JSC 308 transformed with pAB24C100, which was not induced in JSC 308 transformed with pAB24. This polypeptide was indicated by an arrow.

Figure 31B shows the results of Western staining of undissolved polypeptides expressed in JSC 308 transformed with pAB24 (path 1) or pAB24C100-3 (path 2). The polypeptides expressed from pAB24 are not immunologically reactive with human serum with NANBH. However, as indicated by the arrow, JSC 308 transformed with pAB24C100-3 expressed polypeptide of about 54,000 Daltons that does not react immunologically with human NANBH serum. Other immunologically reactive polypeptides in pathway 2 can be degraded and / or aggregation produces about 54,000 Dalton polypeptides.

IV.Β.6 Purification of C100-3 pooled polypeptide

The cl00-3 pooled polypeptide captures SOD at the N-terminus and at the C100 site. The HCV polypeptide is purified by differential extraction of an insoluble fraction of extracted yeast cells in which the polypeptide is expressed.

The pooled polypeptide, C100-3, is expressed in the yeast species JSC 308, which has been transformed with pAB24C100 as described in Chapter IV.B.4. The yeast cells were then lysed by homogenization, and the undissolved material in the lysate was extracted to pH 12 and C100-3 in the remaining undissolved fraction was solidified in SDS-containing buffer.

Yeast lysate was obtained mainly by the method of Nagahum et al. (1984). The yeast cell suspension was prepared to contain 33% of the cells suspended in solution (buffer A) containing 20mM Tris-HCl (pH 8), 1mM dithitreitol and 1mM phenyl methyl sulfonyl fluoride (PMSF). An aliquot of the suspension (15 ml) was mixed with the same volume of glass beads (0.45-0.5 mm in diameter) and the mixture was stirred at top speed at Super Mixer (Lab Line Instruments, Inc.) for 8 minutes. Homogeneous! and the glass beads were separated from each other, the beads were washed three times with the same volume of buffer A as the original packed cells. After pooling the water from the rinse and the homogenate, the insoluble in lysate was obtained by centrifuging the homogenate at 7,000xg for 15 minutes at 4 ° C, the tablets were resuspended in buffer A equal to double the volume of the original packaged cells, and then re-tabulation with by centrifugation at 7000xg for 15 minutes. This rinsing process is performed three times. The insoluble lysate material was extracted to pH 12 as follows. The tablet was first suspended in buffer containing 0.5M NaCl, 1mM EDTA, where the suspended volume was 1.8 times the original packaged cells. The pH of the suspension was adjusted by adding 0.2 volumes of 0.4M Na phosphate buffer at pH 12. After stirring, the suspension was centrifuged at 7000xg for 15 minutes at 4 ° C after which the supernatant was removed. The extraction was repeated twice. The extracted tablets were washed by suspension in 0.5M NaCl, 1mM EDTA, and the suspension volume used was equal to twice the volume of the originally packed cells. This was followed by centrifugation at 7000xg for 15 minutes at 4 ° C. The C100-3 polypeptide in the extracted tablet was dissolved upon treatment with SDS. The tablets were suspended in buffer A of volume 0.9 volume of originally packaged cells and then 0.1 volume of 2% SDS was added. After stirring the suspension, the mixture was centrifuged at 7000xg for 15 minutes at 4 ° C. The resulting tablet is extracted with 3 times the amount of SDS. The resulting supernatants containing C100-3 were then collected.

This process purifies C100-3 more than 10x from the undissolved fraction of the yeast homogenate and separates more than 50% from the polypeptide. The purified preparation of the pooled polypeptide was analyzed by polyacrylamide gel electrophoresis according to Laemmli (1970). Based on this analysis, the polypeptide was more than 80% pure and had an apparent molecular weight of 54,000 Daltons.

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

IV.C. 1 Identification of RNA in chimpanzee liver by NANBH hybridizing to HCV cDNA

RNA from the chimpanzee liver having NANBH was shown to contain RNA species that hybridize to HCV cDNA contained in clone 81 by Northern staining as follows.

RNA was isolated from the liver by chimpanzee biopsy from which high titre plasma was obtained (see section IV.A.1) using the technique described in Manitatis et al. (1982) to isolate total RNA from mammalian cells and to separate them in the poly A ⁺ and poly A fractions. These RNA fractions were subjected to formaldehyde / agarose gel electrophoresis (1% w / v) and converted to nitrocellulose (Manitatis et al. (1982)). Nitrocellulose filters were hybridized with radiolabeled HCV cDNA from clone 81 (see Figure 4 for nucleotide sequence of insert a). In the preparation of the radiolabeled probe, the HCV cDNA insert isolated from clone 81 was radiolabeled with ³² P translation notch using DNA polymerase I (Manitatis et al. (1982)). Hybridization was performed for 18 hours at 42 ° C in a solution containing 10% (w / v) dextran sulfate, 50% (w / v) deionized formamide, 750mM NaCl, 75mM Na citrate, 20mM Na ₂ HPO4 (pH 6.5 ), 0.1% SDS, 0.02% (w / v) bovine albumin serum (BSA), 0.02% (w / v) Ficcol-400, 0.02% (w / v) polyvinylpyrrolidone, 100 micrograms / ml salmon sperm DNA, which is was separated by sonication and denaturation and 10 ⁶ CFM / ml with the notch-translated cDNA probe. The autoradiograph of the probe filter is shown in Figure 38. Route 1 contains ³² P-labeled restriction fragment markers. Lanes 2-4 contain the chimpanzee liver RNA as follows: Route 2 contains 30 micrograms of total RNA; route 3 contains 30 micrograms of poly A 'RNA; and path 4 contains 20 micrograms of poly A ⁺ RNA. As shown in Figure 32, the chimpanzee liver of the NANBH contains a heterogeneous population of bound poly A ⁺ RNA molecule, which hydrolyzes with respect to the HCV cDNA probe and occurs in size from about 5000 to about 11000 nucleotides in size. This RNA hydrolyzing to HCV cDNA may represent viral genomes and / or specific viral genome transcripts.

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

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

Nucleic acids are extracted from particles isolated from the high titre chimpanzee NANBH chimpanzee as described in Chapter IV.Al Aliquots (equivalent to one ml of original plasma) of isolated nucleic acids were resuspended in 20 microliters of 50mM Hepes (pH 7.5), lmM EDTA and 16 micrograms / ml of yeast RNA dissolved. Samples were denatured by boiling for 5 minutes and then freezing immediately and then treated with RNAase A (5 microliters containing 0.1 mg / ml RNAze A in 25mM EDTA, 40mM Hepes (pH 7.5)) or DNAase I (5 microliters containing 1 unit of DNAase I in 10mM MgCl ₂ , 25mM Hepes (pH 7.5)); control samples were incubated without enzymes.

An accompanying incubation, 230 microliters of ice-cold 2xSSC containing 2 micrograms / ml of the yeast RNA dissolved, is added and the samples are then filtered on a nitrocellulose filter. The filters were hybridized with a cDNA probe of clone 81 that was radiolabeled with cross-translation. Figure 39 shows the filter autoradiograph. Hybridization signals were detected in DNA for the treated and control samples (Pathways 2 and 1, respectively), but were not detected in the sample treated with RNAase (Pathway 3).

Thus, RNA processing degrades nucleic acids isolated from particles, and DNA processing has no effect, strongly suggesting 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 the liver and plasma chimpanzee species with NANBH

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

RNA samples containing HCV sequences should be examined using a amplification system. These samples were obtained by liver biopsy of 3 chimpanzees with NANBH and from 2 control chimpanzees. Isolation of the RNA fraction by the guanidium thiocyanate process was 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 the control chimpanzee as well as from the spare plasma from the control chimpanzees. One infected chimpanzee had a CID / ml equal to or greater than 10 ⁶ and the other infected chimpanzee had a CID / ml equal to or greater than 10 ⁵ .

Nucleic acids were extracted from plasma as follows. Either 0.1 ml or 0.01 ml of plasma was diluted to a maximum volume of 1 ml with TENB / proteokinase with K / SDS solution (0.05M Tris-HCl (pH 8)), 0.001M EDTA, 0.1M NaCl, 1mg / ml proteinase K and 0.5% SDS) containing 10 micrograms / ml polyadenylic acid is then incubated at 37 ° C for 60 minutes. Following this proteinase K degradation, the resulting plasma fractions are deproteinized by extraction with TE (10mM Tris-HCl (pH 8), 1mM EDTA) saturated phenol. The phenolic phase was then separated by centrifugation and re-extracted with TENB containing 0.1% SDS. The resulting aqueous phases from each extraction are collected and extracted twice with the same volume of phenol / chloroform / isoamyl alcohol (1: 1 (99: 2)) and then twice with the same volume of 99: 1 chloroform / isoamyl alcohol mixture. Phase separation is followed by centrifugation, the aqueous phase is brought to an extreme concentration of 0.2M Na acetate, and the nucleic acids are then precipitated by the addition of two volumes of ethanol. The precipitated nucleic acids were separated by ultracentrifugation in a SW41 rotor at 38 K for 60 minutes at 4 ° C.

In addition to the above, the high titre plasma chimpanzee and the control plasma reserve were alternatively extracted with 50 micrograms of poly A carrier by the procedure of Chomcyzski and Sacchi (1987). This process uses acid guanidine thiocyanate extraction. RNA was eliminated by centrifugation at 10,000 rpm for 10 minutes at 4 ° C in an Eppendorf microfuge.

In two cases, prior to cDNA synthesis in the PCR reaction, nucleic acids were extracted from plasma by proteinase K / SDS / phenol method, and then further purified by binding and elution from the S and S Elutip-R clone. We followed the procedure in the manufacturer's instructions.

The cDNA used as the template for the PCR reaction was derived from nucleic acids (either total nucleic acids or RNA) obtained as described below. Ethanol precipitation followed by precipitated nucleic acids was dried and resuspended in DEPC treated with distilled water.

Secondary structures in nucleic acids were dissolved by heating to 65 ° C for 10 minutes, after which the samples were immediately cooled on ice. cDNA was synthesized using 1 to 3 micrograms of total RNA from chimpanzee livers or from nucleic acids (or RNA) extracted from 10 to 100 microlitres of plasma. The synthesis uses reverse transcriptase and is performed in a 25 microliter reaction using a manufacturer-specific procedure, BRL. The primers for cDNA synthesis are the same as those used in the PCR reaction described below. All reaction mixtures for cDNA synthesis contained 23 RNAase inhibitor units, RNASIN (Fischer / Promega). After cDNA synthesis, the reaction mixtures were diluted with water, boiled for 10 minutes and then rapidly cooled on ice.

PCR reactions are performed mainly according to the manufacturer's instructions (Getus-Perkin-Elmer) with the exception of the addition of 1 microgram of RNAase A. The reactions are performed over 35 cycles using a mode of 37.72 and 94 ° C.

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

5'-CAA TCA TAC CTG ACA G 3 'in

5'-GAT AAC CTC TGC CTG A 3 '

The primary sequence of clone 36 was:

5′-GCA TGT CAT GAT GTA T 3 ′

The primary sequence of clone 37b was:

5'-ACA ATA CGT GTG TCA C 3 '

In PCR reactions, the primary pairs were recovered from either deh 16-mer derived from clone 81 or 16-mer from clone 36 and 16-mer from clone 37. PCR reaction products were analyzed by separation of products by alkaline gel electrophoresis, which is was monitored by Southern staining and detection of amplified HCV cDNA sequences with a ³² P-labeled internal oligonucleotide probe derived from the non-priming HCV cDNA region. The PCR reaction mixtures were extracted with phenol / chloroform, and the nucleic acids were precipitated from the aqueous phase with salt and ethanol. The precipitated nucleic acids are collected by centrifugation and dissolved in distilled water. Aliquots of the sample are then subjected to electrophoresis on 1.8% alkaline agarose gels. Single DNA lengths 60108 and 161 nucleotides are together electrophoresed on gels as molecular weight markers. After electrophoresis, the gel cDNA is translated into Biora Zeta probe ”on paper. Hybridization and hybridization and washing conditions are those specified by the manufacturer (Biorad).

Probes are used to detect hybridization of amplified HCV cDNA sequences as follows. When a pair of PCR primers was derived from clone 81, the probe was 108-mer with a sequence corresponding to one 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 translationally cuts the HCV cDNA insert and is derived from clone 35. Primers derived from nucleotides 155-170 of clone 37b insert and 206-268 clone 36 insert. clone 35 overlaps the nucleotides 1-186 of the insert in clone 36; and the 5'-end of insertion of clone 35 overlaps nucleotides 207-269 of insertion in clone 37b. (Compare Figures 5, 8 and 10). Thus, the cDNA insert in clone 35 encompasses a portion of the region between the sequences of clone 36 and 37b of the derived primers and is useful as a probe for amplifying sequences involving these primers.

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

Plasma nucleic acid and RNA analysis was also performed according to the above procedure using a primer and probe from clone 81. Plasmas were from two chimpanzees with NANBH, from control and plasma reserves from control chimpanzees. Both NANBH plasmas contained nucleic acids / RNAs that gave positive results in PCR amplified assays, although both control plasmas gave negative results. These results were again obtained several times.

IV.D. Radioisunological examination for the detection of serum HCV antibodies from infected individuals

Solid phase radioimmunoassays for the detection of antibodies to HCV antigen were based on Tsu and Herzenberg (1980). Microtiter vessels (Imulon 2, easily removable bands) were coated with a purified polypeptide containing HCV epitopes. The coated plates were incubated with human serum specimens suspected to contain antibodies to HCV epitopes or appropriate controls. During incubation, the antibody, if present, was immunologically bound to the solid phase antigen. After removal of the unbound material and washing of the microtiter dishes, human antibody-NANBV antigen complexes were detected by incubation with ¹²⁵ J-labeled anti-human immunoglobulin sheep. The unbound labeled antibody was removed by suction and the dishes were then washed. Radioactivity in individual sources is determined; the amount of bound human anti-HCV antibody is proportional to the radioactivity at the origin.

IV.Dl Purification of the SOD-NANB Combined Polypeptide ₅ .ii

The purified SOD-NANB5-1-1 polypeptide expressed in the recombinant bacterium as described in Chapter IV.Bl was purified from recombinant E. coli by differential extraction of the cells of the extract with urea followed by chromatography on anionic and cation exchangeable columns as follows.

Thawed cells from 1 liter of culture were resuspended in 10 ml of 20% (w / v) sucrose containing 0.01M Tris-HCl (pH 8), 0.4 ml 0.5M EDTA (pH 8). After 5 minutes at 0 ° C, the mixture was centrifuged at 4000xg for 10 minutes. The resulting table was then resuspended in 10 acres of 25% (w / v) sucrose containing 0.05M Tris-HCl (pH 8), 1mM phenyl methyl sulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin A, which is was then monitored by the addition of 0.5 ml of lysozyme (10 mg / ml) and incubation at 0 ° C for 10 minutes. After the addition of 10 ml of 1% (w / v) Triton Χ-100 in 0.05M Tris-HCl (pH 8), lmM EDTA, the mixture was incubated for a further 10 minutes at 0 ° C with occasional stirring. The viscous solution obtained was homologated with Run 6 times through a sterile 20-gauge hypodermic needle and centrifuge at 13000xg for 25 minutes. The tabulated material was suspended in 5 ml of 0.01M Tris-HCl (pH 8), then the suspension was centrifuged at 4000xg for 10 minutes. Tablets containing SOD-NANB5-1-1 pooled protein were dissolved in 5 ml of urea in 0.02M Tris-HCl (pH 8), 1mM dithiothreitol (buffer A) and applied to a Q-Sepharore column of fast-acting buffer A. The polypeptide was eluted with a linear gradient of 0.0 to 0.3M NaCl in buffer A.

After elution, fractions were analyzed by polyacrylic gel electrophoresis in the presence of SDS to determine the content of S DS-NANB 5_ i-ι. The fractions containing this polypeptide were collected and dialyzed against 6M urea in 0.02M Sodium phosphate buffer (pH 6), 1mM dithiothreitol (buffer B). The dialyzed sample was applied on a S-Sepharose column of fast-flow equilibration with buffer B, after which the polypeptides were eluted with a linear gradient of 0.0 to 0.3M NaCl in buffer B. Fractions were analyzed by polyacrylamide gel electrophoresis for the presence of SOD-NANB5-1- 1, after which the corresponding fractions were combined.

The final product of SOD-NANB ₅ and polypeptide was examined by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 80% pure.

IV.D.2 Purification of the SOD-ΝΑΝΒβχ pooled polypeptide

The pooled S0D-NANB _ei polypeptide expressed in recombinant bacteria as described in Section IV.B.2 was purified from recombinant E.coli with differential

100 extraction of the cell extract with urea followed by chromatography on anionic and cation exchangeable columns using the procedure described for isolating the pooled poiipeptide (see Chapter IV.D.1). The final preparation of SOD-NANB ₈ i polypeptide was examined by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 50% pure.

IV. D. 3 Antibody detection of HCV epi tops by solid phase radio-immunoassay

Serum samples from 32 patients diagnosed with NANBH were analyzed by radioimmunoassay (RIA) for the determination of antibodies against HCV epitopes present in the combined polypeptides SOD-NANB5-1-1 and SOD-NANB ₈ i ·

The microtiter vessels were coated with SOD-NANB5-1-1 or SODNANB ₈ i, which were partially purified according to Chapters IV.D. 1 and IV.D.2 respectively. Investigations were carried out as follows:

100 microliter aliquots containing 0.5 micrograms of SOD-NANB5-1-1 or SOD-NANB ₈ iv 0.125M To each well of microtiter vessel (Dynatech Immulon 2 Removawell Strips) is added to borate buffer (pH 8.3), 0.075M NaCl (BBS). . The vessel was incubated overnight at 4 ° C in a humid room, after which the protein solution was removed and the sources washed three times with BBS containing 0.02% Triniton Χ-100 (BBST). To prevent nonspecific binding, the sources were coated with bovine albumin serum (BSA) while adding 100 microlitres of 5mG / ml BSA solution in BBS, followed by incubation at room temperature for 1 hour; after this incubation, the BSA solution is removed. Coated-source polypeptides react with serum by adding 100 microlitres of serum samples diluted 1: 100 in 0.01M In phosphate buffer (pH 7.2) 0.15M NaCl (PBS) containing 10 mg / ml BSA and incubating serum containing sources 1 hour at 37 ° C. Po

In incubation, serum samples are removed by suction and the sources are washed 5 times with BBST.

The anti-NANB ₅ _i-i and anti-NANB ₀ i bound to the pooled polypeptides were determined by binding of ¹²⁵ J-labeled F '(ab) ₂ sheep of anti-human IgG to coat the origin. Aliquots of the labeled probe, volumes of about 100 microlitres (specific activity of 5-20 microcircuits / microgram) are added to each source, and then the dishes are incubated at 37 ° C for 1 hour. The excess probe is then removed by suction and then rinsed with BBST for 5 minutes. The amount of radioactivity bound to each source is determined by counting on a counter that detects gamma radiation.

The results of detection of anti-NANBs-ii and NANB _0i in individuals with NANBH are given in Table 1.

Table 1: Detection of anti-5-1-1 and anti-81 in serum NANB, HAV and HAB hepatitis patients reference number diagnosis of Anti-5-1-1 Anti-81 patient

1.28 ¹ NANB Chronicle, IVD ² 0.77 4.20 NANB Chronicle, IVD 1.14 5.14 NANBV Chronicle, IVD 2.11 4.05 2.29 ¹ AVH ³ , NANB, Sporadic 1.09 1,05 NANB Chronicle 33.89 11,39 NANB Chronicle 36,22 13, 67 3. 30 ¹ AVH, NANB, IVD 1, 90 1, 54 NANB Chronicle, IVD 34.17 30,28 NANB Chronicle, IVD 32.45 30,84 4. 31 NANB Chronicle, PT ⁴ 16.09 8.05 5. 32 ¹ late AVH, NANB, IVD 0, 69 0, 94 late AVH, NANB, IVD 0.73 0, 68 33 ¹ AVH, NANB, IVD 1, 66 1, 96 AVH, NANB, IVD 1.53 0.56

102

7. 34 ¹ NANB Chronicle, PT 34, 40 7.55 NANB Chronicle, PT 45.55 13.11 NANB Chronicle, PT 41.58 13,45 NANB Chronicle, PT 44.20 15,48 8. 35 ^χ AVH, NANB, IVD 31,92 31,95 (recently healed) NANB, AVH 6.87 4,45 9. 36 late AVH, NANB, PT 11,84 5.79 10. 37 AVH, NANB, PT 6.52 1.33 11. 38 late AVH, NANB, PT 39.44 39.18 12. 39 NANB Chronicle, PT 42,22 37.54 13. 40 AVH, NANB, PT 1.35 1.17 14. 41 NANB Chronicle, PT 0.35 0.28 15. 42 AVH, NANB, IVD 6.25 2,34 16. 43 NANB Chronicle, PT 0.74 0.61 17. 44 AVH, NANB, PT 5,40 1.83 18. 45 Chronicle, NANB, PT 0.52 4,45 19. 46 AVH, NANB 23,35 4,45 20. 47 AVH, Type A 1.60 1.35 21. 48 AVH, Type A 1,30 0.66 22. 49 AVH, Type A 1.44 0.74 23. 50 recently resolved AVH, Type A 0.48 0.56 24. 51 AVH, Type A 0.68 0, 64 resolved AVH, Type A 0.80 0.65 25. 52 recently solved AVH, Tip A 1.38 1.04 recently solved AVH, Tip A 0.80 0, 65 26. 53 AVH, Type A 1.85 1.16 recently solved AVH, Tip A 1.02 0.88 27. 54 AVH, Type A 1, 35 0.74 28. 55 late AVH, HBV 0, 58 0, 55 29. 56 HBV Chronicle 0.84 1.06 30. 57 late AVH, HBV 3.20 1, 60 31. 58 HBV Chronicle 0, 47 0.46 32. 59 ¹ AVH, HBV 0.73 0, 60

103

healed AVH, HBV 0.43 0, 44 33. 60 ¹ AVH, HBV 1.06 0, 92 healed AVH, HBV 0.75 0, 68 34. 61 ¹ AVH, HBV 1, 66 0, 61 healed AVH, HBV 0, 63 0.36 35. 62 ¹ AVH, HBV 1.02 0.73 healed AVH, HBV 0.41 0.42 36. 63 ¹ AVH, HBV 1,24 1, 31 healed AVH, HBV 1.55 0.45 37. 64 ¹ AVH, HBV 0.82 0.79 healed AVH, HBV 0.53 0.37 38. 65 ¹ AVH, HBV 0, 95 0, 92 healed AVH, HBV 0.70 0.50 39. 66 ¹ AVH, HBV 1.03 0, 68 healed AVH, HBV 1,71 1.39

Legend: ¹ sequential serum samples available from these patients

2IVD intravenous administration ³ AVH acute viral hepatitis ⁴ PT Post transfusion

As can be seen in Table 1, of the 32 patients diagnosed with NANBH, 19 of these sera-positive antibodies were directed against HCV epitopes present in SOD-NANB5_i-i and SOD-NANBH _e i.

But, serum samples that were positive were not uniformly immunologically reactive with SOD-NANBs -! -! and SOD-NANB ₈ i. Patient serum samples No.1 were positive with respect to SODNANB ₈ and not with SOD-NANB ₅ -i-! . Patient serum samples No.10, 15 and 17 were positive for SODNANB5 -! -! but not according to SOD-NANB ₈ i. Patient serum samples no. 3, 8, 11 and 12 react equally with both

104 combined polypeptides, while serum samples from patients no. 2, 4, 7 and 9 2 to 3 times more reactive with SOD-NANB5-1-1 than with SOD-NANB ₈ i · These results suggest that SOD-NANB5-1-1 and SOD-ΝΑΝΒβι may contain at least three different epitopes; e.g. it is possible that each polypeptide contains at least 1 different epitope and that the two polypeptides share at least 1 epitope.

IV.D.4 RIA Solid Phase Specificity for NANB

The solid phase specificity of RIA for NANB is tested by testing on the sera of patients infected with HAV or HBV and on sera of control individuals. Investigations into the use of partially purified SOD-NANB ₅ _i_i and SOD-ΝΑΝΒθι are mainly performed as described in Chapter IV.D.3 except that the serum was from patients previously diagnosed with HAV or HBV , or from individuals who were blood donors. The results for sera from HAV and HBV infected patients are shown in Table 1.

RIA was tested with 11 serum types from HAV infected patients and 20 serum species from HBV infected patients. As shown in Table 1, none of the sera gave a positive immune reaction with pooled polypeptides containing BB-NANBV epitopes.

RIA use of NANBVs-i-i antigen was performed to determine serum reactivity from control specimens. Of the 230 serum samples obtained from the normal blood donor population, only 2 gave a positive reaction in the RIA (data not shown). It may be that the blood donors from which these serum samples were derived came from individuals previously in contact with HCV.

IV.D.5 Reactivity of NANB ₅ -ii during NANBH infection

The presence of anti-NANBs-i antibodies during NANBH infection of 2 patients and 4 chimpanzees was monitored by RIA, which was

105 described in Chapter IV.D.3. Additionally, RIA was also used to determine the presence or absence of anti-NANB ₅ and antibodies during chimpanzee infection with NAV or HBV. The results shown in Table 2 show that the following set of acute phases of NANBH infection is detected by chimpanzee and human anti-NANBs_ and antibodies. AntiNANB _5- i antibodies were not detected in serum samples from chimpanzees infected with HAV or HBV. Thus, antiNANB5-1-1 antibodies serve as markers for individual exposure to HCV.

Table 2: Seroconversion in sequential serum samples of hepatitis and chimpanzee patients using 5-1-1 antigen.

Patient / Sample Date (Days) Hepatitis Anti-5-1-1 ALT

chimpanzee 0. inoculation days viruses (S / N) (IU / L) The patient 29 T NANB 1.09 1180 T + 180 33.89 425 T + 208 36,22 - The patient 30 T NANB 1.90 1830 T + 307 34.17 290 T + 799 32.45 276 Chimpanzee 1 0 NANB 0.87 9 76 0.93 71 118 23, 67 19 154 32.41 - Chimpanzee 2 0 NANB 1.00 5 21 1.08 52 73 4, 64 13 138 25.01 - Chimpanzee 3 0 NANB 1.08 8 43 1, 44 205 53 1.82 14

106

159 11,87 6 Chimpanzee 4 -3 NANBV 1.12 11 55 1,25 132 83 6, 60 - 140 17.51 - Chimpanzee 5 0 HAV 1.50 4 25 2.39 147 40 1.92 18 268 1, 53 5 Chimpanzee 6 -8 HAV 0.85 - 15 - 106 41 0.81 10 129 1.33 - Chimpanzee 7 0 HAV 1.17 7 22 1, 60 83 115 1.55 5 139 1, 60 - Chimpanzee 8 0 HAV 0.77 15 26 1.98 130 74 1.77 8 205 1,27 5 Chimpanzee 9 -290 HBV 1.74 - 379 3.29 9 435 2.77 6 Chimpanzee 10 0 2, 35 8 111-118 (pool) HBV 2.74 96-156 (pool) 205 2.05 9 240 1.78 13 Chimpanzee 11 0 HBV 1, 82 11 28-56 (pool) 1,26 8-100 (pool) 169 - 9 223 0, 52 10

Legend: T is the day of the initial pattern

107

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

Based on the specific immunological reactivity of SOD-NANBs-i-i polypeptide with antibodies in serum samples from patients with NABH, a method of purification of serum antibodies immunologically reacting with the epitope in NANB5-1-1 was developed. This method uses affinity chromatography. Purified SODNANBs-i-! the polypeptide (see Chapter IV.D. 1) is attached to the undissolved support; attachment is such that the stationary polypeptide retains its affinity for the antibody against NANBs. The antibody in serum samples is adsorbed on the matrix-bound polypeptide. After washing to remove non-specific bound materials and unbound materials, the bound bodies are released from the bound SOD-HCV polypeptide by changing the pH and / or by chaotropic agents, e.g. with urea.

Nitrocellulose membranes containing bound NANBs-i are obtained as follows. Nitrocellulose membrane, 2.1 cm Sartorius with 0.2 micron pores was washed 3 times in 3 minutes with BBS. SOD-NANBs -! -! is bound to the membrane by incubation of the purified preparation in BBS at room temperature for 2 hours; or alternatively by incubation at 4 ° C overnight. The filter is then washed three times with BBS, every 3 minutes. The remaining active sites on the membrane were blocked by BBS incubation with 5mg / ml BSA solution for 30 minutes. Excess BSA is removed by washing the membrane five times with BBS and washing three times with distilled water. The membrane containing the viral antigen and BSA was then treated with 0.05M glycine hydrochloride (pH 2.5), 0.1M NaCl (GlyHCl) for 15 minutes and then washed with PBS for 3 minutes.

Polyclonal anti-NANB ₅ -ii antibodies were isolated by incubation of membranes containing the pooled polypeptide with serum from individuals with NANBH for 2 hours. After incubation

The 108 filters were washed 5 times with BBS and twice with distilled water. The bound antibodies were then eluted from each filter with five elution with GlyHCl, three minutes after elution. The pH of the eluate was then adjusted to 8 by collecting each eluate in a tube containing 2M Tris-HCl (pH 8). Separation of antiNANBs -! -! of antibodies by affinity chromatography is about 50%.

Nitrocellulose membranes containing bound viral antigen can be used several times without significant decrease in binding capacity. When reusing the membrane, the antibodies are washed with elution with BBS three times for 3 minutes after elution. These are stored in BBS at 4 ° C.

IV.F Extraction of HCV particles from infected plasma using purified human polyclonal anti-HCV antibodies / Nucleic acid hybridization in obtained anti-HCV cDNA particles

IV.F. 1 Extraction of HCV particles from infected avalanche using human polyclonal anti-HCV antibodies

Protein-nucleic acid complexes present in infected chimpanzee plasma with NANBH are isolated by purified human polyclonal anti-HCV antibodies bound to polystyrene beads.

Polyclonal anti-HCV antibodies are purified from human serum by NANBH using the SOD-HCV polypeptide encoded in 51-1. The purification method was described in Chapter IV.E.

Purified anti-NANB _5- ii antibodies were bound to polystyrene beads (1/4 in diameter, mirror prepared, Precision Plastic Bali Co., Chicago, Illinois) by incubating each bead at room temperature overnight with 1 ml of antibody (1 microgram / ml in buffer buffer (pH 8.5)). After overnight incubation, the beads are washed once

109 with TBST (50mM Tris-HCl (pH 8), 150mM NaCl, 0.05% (v / v) followed by phosphorous buffer (PBS) containing lOmg / ml BSA. The control beads are prepared in an identical manner except this that purified anti-NANBs -! -! antibodies are replaced with complete human immunoglobin.

Harvesting of HCV from a NANBH infected chimpanzee using bead-bound anti-NANBs-1 antibodies is performed as follows. Chimpanzee plasma with NANBH is used as described in Chapter IV.A.1. An aliquot (1 ml) of the NANBV-infected plasma chimpanzee was incubated for 3 hours at 37 ° C with five beads coated with anti-NANBs-1 antibodies or control immunoglobulins. The beads are washed three times with TBST.

IV.F.2 Nucleic acid hybridization in the resulting anti-NANBV-cDNA particles

A nucleic acid component released from particles obtained with anti-NANB5_ and antibodies is analyzed for hybridization against HCV cDNA derived from clone 81.

HCV particles are obtained from a NANBH-infected plasma chimpanzee as described in Chapter IV.F.1. In order to liberate nucleic acids from the particles, the washed beads are incubated for 60 minutes at 37 ° C with 0.2 ml of solution (this contains proteinase K (1 mg / ml), 10mM Tris-HCl (pH 7.5), 10mM EDTA, 0.25% (v / v) SDS, 10 μg / ml of yeast RNA dissolved) per bead, after which the supernatant solution is removed. The supernatant was extracted with phenol and chloroform, and the nucleic acids were precipitated with ethanol overnight at -20 ° C. The nucleic acid precipitate was collected by centrifugation, then dried and dissolved in 50mM Hepes (pH 7.5). Diluted aliquots of the dissolved nucleic acids from the sample obtained from beads coated with anti-NANBs-1 antibodies and control beads containing whole human immunoglobin are filtered on

110 nitrocellulose filters. These are hybridized with a ³² Recorded transection probe made from the purified HCV cDNA fragment in clone 81. Methods for probe acquisition and hybridization are described in Chapter IV.CI

Autoradiographs of probe filters containing nucleic acid particles from beads obtained by means of a bead containing anti-NANBs-II antibodies are shown in Figure 34. The extract obtained using anti-NANBs-II antibodies (Ai, A ₂ ) gives clear hybridization signals relative to the control extract of antibodies (A ₃ , AJ and against the yeast control RNA (Bi, B ₂ ). Standards containing the Ipg, 5pg, and lOpg purified cDNA fragment of clone 81 are shown in Cl-3, respectively.

These results indicate that particles derived from NANBH plasma by anti-NANBs-1 antibodies, nucleic acids that hybridize with HCV cDNA in clone 81, thus providing further evidence that the cDNA in these clones is derived from an etiological agent for NANBH.

IV.6. Immunological reactivity of C100-3 with purified antiNANBj -! -! antibodies

The immunological reactivity of the C100-3 fusion polypeptide with antiNANB ₅ antibodies is performed by radioimmunoassay in which the solid phase bound antigens are halogenated with purified NANB5-1-1 antibodies. The antigen-antibody complex is detected with ¹²⁵ J-labeled sheep anti-human antibodies.

The immunological reactivity of the C100-3 polypeptide is primed by the reactivity of the SOD-NANB ₅ and antigen.

The C100-3 pooled polypeptide is synthesized and purified as described in Chapter IV.B.5 and Chapter IV.B.6, respectively. The combined SOD-NANBs-i-i polypeptide is synthesized and purified as described in Chapter IV.B.l and

111

IV.D. 1, respectively. Purified anti-NANBs-i antibodies are obtained as described in section IV.E.

An aliquot of 100 μΐ containing different amounts of purified C100-3 antigen in 0.125M In borate buffer (pH 8.3), 0.075M NaCl (BBS) is added to each source on a microtiter vessel (Dynatech Immulon; bi. Mark; op. Trans) with two with breakaway straps. The vessel is then incubated at 4 ° C overnight in a humidified room. The protein solution was then removed and the sources washed three times with BBS containing 0.02% Triton Χ-100 (BBST). To prevent nonspecific binding, the sources are coated with BSA by adding 100 μΐ 5mg / ml BSA solution in BBS, then incubated at room temperature for 1 hour and then the excess BSA solution is removed. The polypeptides in the coated sources react with purified anti-NANBs-II antibodies after the addition of 1µg of antibody / sample and incubation of the sample for 1 hour at 37 ° C. After incubation, the excess solution is removed by suction and the sources are washed 5 times with BBST. AntiNANB5-1-1 bound to pooled polypeptides was determined by binding to ¹²⁵ J-labeled F '(ab) ₂ anti-human IgG sheep on labeled sources. Aliquots of 100 μΐ volumes of labeled probe (specific activity 5–20 microcircuits / microgram) are added to each source, after which the vessels are incubated for 1 hour at 37 ° C, and the excess probe is then removed by suction and washed 5 times with BBST. the amount of radioactivity bound to each source is determined by counting on a counter that detects gamma radiation. The results of the immunological reactivity of C100 with purified antiNANB5-1-1 compared with those of NANB5-1-1 with purified antibodies are shown in Table 3.

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Table 3: Immunological reactivity of C100-3 compared to ΝΆΝΒ5-1-1 radioassay.

RIA (cpm / test)

AG (ng) 400 320 240 160 60 0 NANB5-1-1 7732 6732 4954 4050 3051 57 C100-3 7450 6985 5920 5593 4096 67 Results in the table 3 show , Yes anti-NANBs and recognized

epitopes in the C100 portion of the C100-3 polypeptide. Thus, NANB5-1-1 and C100 share common epitopes. The results suggest that the cDNA sequence encoding this NANBV epitope is one that is present in both clones 5-1-1 and 81.

IV.H characterization of HCV

IV.H. 1 Characterization of HCV genome complexity

The HCV genome is characterized in terms of frequency by isolating the nucleic acid fraction from particles obtained on anti-NANBs -! -! antibodies labeled with polyester beads and determining whether the isolated nucleic acids hybridize with the plus and / or minus HCV cDNA strands.

The particles were obtained from HCV plasma of infected chimpanzees using a polyester bead coated with an immuno-purified anti-NANBs-i antibody as described in Chapter IV.F.1. The particle nucleic acid component is released using the method described in Chapter IV.F.2. Aliquots of isolated genomic nucleic acid equivalent to 3 ml of high plasma titer are applied to nitrocellulose filters. As controls, aliquots of denatured HCV cDNA from clone 81 (2 are also applied to these filters)

113 picograms). The filters are examined with a ³² P-labeled mixture of plus or minus single DNA strands cloned from HCV cDNA; cDNAs are excised from clones 40b, 81 and 25c.

Single probes are obtained by cutting HCV cDNAs from clones 81, 40b and 25c with EcoRI and cloning the cDNA fragment in M13 vectors, rap! 8 and mpl9 (Messing (1983)). M13 clones are sequenced to determine whether they contain plus or minus DNA strands derived from HCV cDNA. Sequencing using the dideoxygenase termination process of Sanger et al. (1977).

Each of the sets of duplicate filters containing aliquots of the HCV genome isolated from the resulting particles is hybridized with a plus or minus strand of HCV cDNA probe. Figure 35 shows the autoradiographs obtained from the NANBV genome scan with a mixture of probes derived from clones 81, 40b and 25c.

The mixture is used to increase the sensitivity of hybridization testing. The samples in panel I are hybridized with a plus-chain probe mixture. Samples in panel II are examined by hybridization with a minus the chain of probes. The composition of the samples in the panels that were immuno-stained is presented in Table 4.

Table 4:

pot

A

HCV genome cDNA 81 cDNA 81 * non-descriptive pattern

As can be seen from the result in Figure 35, only a minus strand of DNA probes hybridizes with the isolated HCV genome. This one

114 result, combined with a result showing genome sensitivity with respect to RNAase I but not gelde to DNAase (see Chapter IV.C.2) suggests that the NANBV genome is a positive RNA strand.

These data and data from other laboratories relating to the physicochemical properties of suspected NANBV are consistent with the hypothesis that HCV is a member of Flaviviridiae. However, the possibility that HCV represents a new type of viral agent has not been eliminated.

IV.H.2 Detection of sequences in obtained particles amplified by PCR hybridization against HCV cDNA from clone 81

RNA in the resulting particles was obtained as described in Chapter IV.H. The analysis for HCV cDNA hybridizing sequences derived from clone 81 was performed by PCR amplification as described in Chapter IV.C.3, except that the hybridization probe was a kinized oligonucleotide derived from a Sl cDNA sequence clone. The results indicate that the amplified sequences hybridized with clone 81 are derived from HCV cDNA probes.

IV.H.3 Homology between the non-structural protein of Dengue Flavivirus (MNWWVD1) and the HCV polypeptide encoded by the pooled ORF of clones 14i-39c

The pooled cDNAs from clones 14i to 39c contain a single continuous ORF, as shown in Figure 26. The encoded polypeptide was analyzed for homology to the non-structural polypeptide region in Dangue flavivirus (MNWVD1). The Dayhoff protein database was used in the analysis. The analysis was performed with a computer. The results are shown in Figure 36, where the symbol (:) indicates exact homology and the symbol (.) Indicates a conservative replacement in sequence; dashes indicate spaces placed in the sequence for maximum homology. as shown in the figure, it exists between the sequence encoded in HCV cDNA and non-structural

115 Dengue virus protein significant homology. In addition to the homology of Fig. 36, the analysis of the polypeptide segment encoded in the 3'-end region and the cDNA also comprise sequences homologous to the sequences in Dengue polymerase. As a consequence, we conclude that the canonical Gly-Asp-Asp (GDD) sequence must be crucial for the RNA polymerases contained in the HCV cDNA encoded polypeptide at a location consistent with that of the Dengue 2 virus (data not shown).

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

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

IV.H.4.a Southern staining process

In order to determine whether the liver of a NANBH-infected chimpanzee contains detectable HCV-DNA (or HCV-cDNA), restriction enzyme DNA fragments isolated from this Southern origin are stained, and stains are examined with ³² Recorded HCV cDNA. The results show that the labeled HCV cDNA does not hybridize with the smeared DNA from the liver of the infected chimpanzee. Nor does it hybridize with the DNA stain of a normal chimpanzee's liver. On the contrary, in the positive control, the labeled probe of the beta-interferon gene strongly hybridizes with Southern spots with the restriction enzyme of degraded human placental DNA. These systems are rewarded for detecting a single copy of the gene to be detected by the labeled probe.

DNAs were isolated from the livers of two chimpanzees with NANBH. Control DNAs were isolated from the liver of an uninfected chimpanzee and from a human placenta. Extraction process

116

DNA is mainly that of Manitatis et al. (1982), and DNA samples are treated with RNAase during the isolation process. Each DNA sample is treated with EcoRI, Mbol or HincII (12 micrograms) according to the manufacturer's instructions. The digested DNA was electrophoresed on 1% neutral agarose gels, then Southern stained on nitrocellulose, and then this material was hybridized with a suitable translator-notched cDNA probe (3x10 ⁶ cpm / ml hybridization mixture). DNA from the liver of infected chimpanzees and from normal liver is hybridized with ³² P-labeled HCV cDNA from clones 36 plus 81; Human placental DNA was hybridized with ³² P-labeled DNA from the betaine interferon gene. After hybridization, the stains were washed under strict conditions, e.g. with a solution containing 0.1xSSC, 0.1% SDS at 65 ° C.

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

IV.H.4.b Amplification by PCR technique

In order to determine whether HCV-DNA could be detected in the chimpanzees' livers with NANBH, DNA was isolated from the tissue and subjected to PCR amplification detection using primers and probe polynucleotides derived from HCV DNA from clone 81. Negative controls were samples isolated from tissue cells of culture of uninfected HepG2, and probably from uninfected human placenta. Positive controls were samples of negative DNA control to which a known small amount (250 molecules) of HCV cDNA inserts from clones 81 was added. In addition, to confirm the assertion that RNA fractions from the same NANBH chimpanzee chimneys containing complementary HCV sequences cDNA probes, PCR amplification-detection system also used on isolated RNA samples.

When examined, DNAs were isolated by the procedure described in Chapter IV.H.4.a, and RNAs were extracted mainly in the manner described by Chirgwin et al. (1981).

117

DNA samples were isolated from the liver of 2 infected chimpanzees, from uninfected HepG2 cells and from a 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 and detection for amplified HCV cDNA mainly as described in Chapter IV.C.3, except that the stage was omitted. reverse transcriptases.

The PCR primers and probe were from HCV cDNA from clone 81 and are described in Chapter IV.C.3. Prior to amplification, one microgram of a sample of each DNA was spiked by adding 250 HCV cDNA molecules of the insert isolated from clone 81 for positive controls. DNA subjected to the amplification process mainly according to the instructions in Chapter IV.C.3 except that reverse transcriptase was omitted as a negative control for some reason. The PCR primers and probe were from HCV cDNA from clone 81 as described below.

The results indicate that the sequence amplification of the complementary HCV cDNA probe was neither detectable in DNA from the chimpanzee infected liver, nor in the negative controls. Conversely, if samples including DNA from the liver of an infected chimpanzee were spiked with HCV cDNA prior to amplification, clone 81 sequences were detected in all positive control samples. Additionally, in RNA amplification, HCV amplified cDNA sequences are only detected when reverse transcriptase is used, strongly suggesting that the results are not caused by DNA contamination.

These results indicate that hepatocytes from chimpanzees with NANBH do not contain, or contain undetectable, HCV cDNA levels. Based on the aforementioned study, if HCV cDNA is present, it is significantly below 0.06 copies per hepatocyte at the level.

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In contrast, HCV sequences in total RNA from the same liver samples can be detected by PCR technique.

IV. I. ELISA for HCV Infection Using HCV C100-3 as an Antigen Test

All samples were examined using HCV C100-3 ELISA. This assay uses an HCV C100-3 antigen that has been synthesized and purified as described in Chapter IV.B.5 and horseradish peroxidase (HRP) conjugate of mouse monoclonal anti-human IgG.

Containers coated with HCV C100-3 antigen were obtained as follows. Solution containing coating buffer (50mM Na-borate (pH 9), 21 ml / vessel, BSA (25pg / ml), C100-3 (2.5 pg / ml) prepared immediately prior to addition to Removeawell Immulon I (trademark After 5 minutes of stirring, 0.2 ml / well of the solution was added to the dishes, which were then covered and incubated for 2 hours at 37 ° C, after which the solution was removed by suction. were washed once with 400 μΐ wash buffer (lOOmM Na-phosphate (pH 7.4), 140mM NaCl, 0.1% (w / v) casein, 1% (w / v) Trinitone Χ-100, 0.01% (w / v) After removal of the solution after rinsing, 200 μΐ / source of Post-spill solution (lOmM Na-phosphate (pH 7.2), 150mM NaCl, 0.1% (w / v) casein and 2mM phenyl methyl sulfonyl fluoride (PMSF) was added )), the vessels were loosely covered to prevent evaporation, and allowed to stand at room temperature for 30 minutes. The springs were then sucked in to remove the solution and lyophilized overnight to dry, dry. The resulting containers can be stored at 2-8 ° C in submerged aluminum containers.

In order to perform ELISA determination, a source containing 200 pl of the diluted sample (100mM Na-phosphate (pH 7.4), 500mM NaCl, 1mM EDTA, 0.1% (w / v) casein, 0.015 (w / v) therosal,

119

1% (w / v Triton Χ-100, lOOgg / ml yeast extract), add 20 μΐ of serum or control sample.

The dishes were then warmed and incubated at 37 ° C for two hours, then the solution was removed by suction and the sources were washed with 400μ1 buffer (phosphate buffered solution (PBS) containing 0.5% Tween 20). The washed sources were treated with 200 μΐ of mouse anti-human IgG-HRP conjugate contained in a solution of Ortho conjugate diluent (lOmM Na-phosphate (pH 7.2), 150mM NaCl, 50% (v / v) fetal bovine serum, 1% heat treated horse serum, lmM K ₃ Fe (CN) ₆ ) for 1 hour at 37 ° C, after which the solution was removed by suction and the sources were washed with buffer, which was also removed by suction. To determine the amount of bound enzyme conjugate, 200 μΐ of the substrate solution (10 mg of 0-phenylenediamindiclor per 5 ml of developing solution) was added. The developing solution contained 50mM Na-citrate adjusted to pH 5.1 with phosphoric acid and 0.6 μΐ / ml 30% H ₂ O ₂ . The containers containing the substrate solution were incubated in the dark for 30 minutes at room temperature, and the reactions were then stopped by the addition of 50 μΐ / ml sulfuric acid, after which Ods was determined.

The examples given below show that a microtiter ELISA test vessel using HCV C100-3 antigen has a high degree of specificity, as evidenced by an initial reactivity rate of about 1% with a repeated reactive rate of magnitude of about 0.5% of random donors. The test is capable of detecting immuno-responses in the post-acute phase of infection as well as during the chronic phase of the disease. In addition, it is capable of detecting some samples that were negative in other NANBH tests; these samples come from specimens with a history of NANBH or donors covered by the NANBH transmission.

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The following codes were used in the examples below:

ALT

Anti-HBc

Anti-HBsAg

Hbc

AbsAg

IgG

IgM

IU / L

NA

NT

N

Neg

OD

Pos

S / CO

SD x

WN L alanine amino transferase Hbc antibody HBsAg antibody hepatitis B core antigen hepatitis B surface antigen immunoglobulin G immunoglobulin M international unit / liter not available tested sample size negative optical density positive signal / section standard deviation medium or average within normal limits

IV.I.l HCV infection in a population of random donors

A pool of 1056 serum samples from random blood donors obtained from Irwin Memorial Blood Bank, San Francisco, California. The test results obtained with these samples were collected in a histogram showing the distribution of OD values (Figure 37). As can be seen from Figure 37, 4 samples count above 3, 1 sample counts between 1 and 3, 5 samples count between 0.4 and 1 and the remaining samples count below 0.4, with over 90% of these samples counting below 0.1.

The results with non-reactive random samples are given in Table 5. Using the cut-off values of equals

121 mean values plus 5 standard deviations, 10 samples out of 1056 (0.95%) were initially reactive. Of these, 5 samples (0.47%) were repeated as reactive when tested for ELISA. Table 5 also shows ALT and anti-HBd status for each replicate reactive sample. Of particular interest is the fact that all 5 replicate reactive samples are negative with both other NANBH assays, although they are positive by HCV ELISA.

Table 5: Reactivity results of random samples

section: Patterns N = 1051 x = 0.049 * control) SD = ± 0.074 x + SSD = 0.419 The initial ones reactivity OD (0.4 + negative Reactivity at the beginning OD ALT ** (UI / L) Anti HBC *** OD 4227 0,462 th most common 0,084 NA NA 6292 0,569 th most common 0.294 NA NA 6188 0, 699 0, 326 NA NA 6157 0.735 0,187 NA NA 6277 0.883 0,152 NA NA 6397 1,567 th most common 1,392 th most common 30.14 1,433 th most common 6019 > 3,000 > 3,000 46, 48 1,057 th most common 6651 > 3,000 > 3,000 48.53 1,343 th most common 6669 > 3,000 > 3,000 60, 53 1,165 th most common 4003 > 3,000 > 3,000 WNL **** Negative 10/1056 = 0. 95% 5/1056 = 0.47%

* samples counting above 1.5 and not included in the mean and Sd

122 ** ALT above 68 IU / L is the upper limit of normal *** Anti-HBc is below 0.535 (competitiveness test) considered positive **** WN I: within normal range

IV. 1.2 Chinpanzes serum samples

Serum samples from 11 chimpanzees were tested by HCV C100-3 ELISA. Four of these chimpanzees were infected with NANBH from infected Factors VIII (mainly Hutchinson species). The following is a description of the procedure with Dr. Daniel Bradley at the Centers for Disease Control. As a control, the other 4 chimpanzees were infected with HAV and three were infected with HBV. Serum samples were obtained at different times after infection.

The results are shown in Table 6 and show documented antibody seroconversion in all chimpanzees infected with the Hutchinson species NANBH. Following the acute phase of infection (as recorded with significant growth and subsequent return to normal ALT levels), antibodies against HCV C1003 become detectable in the serum of 4/4 NANBH infected chimpanzees. These samples, as described in Chapter IV.B.3, are positive by Western analysis and RIA. In contrast, none of the control chimpanzees that were infected with HAV or HBV show evident reactivity in the ELISA.

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Table 6: Chimpanzee serum samples

OD S / Co Date Date ALT transfusion inoculum. bloody. (UI / L)

Neg. contr. 0.001 Pos. contr. 1,504 section 0.401

Chimpanzee 1 -0,007 0.003 0.00 24.5.84 0.01 > 7.48 > 7.48 24.05.84 07.08.84 18.09.84 10/24/84 9 71 19 NANB > 3,000 > 3,000 Chimpanzee 2 - - 07.06.84 - - NANB -0,003 0.00 05/31/84 5 -0.05 0.00 28.06.84 52 0.945 2,36 08/20/84 13 > 3,000 > 7.48 10/24/84 - Chimpanzee 3 0,005 0.01 14.03.85 14.03.85 8 NANB 0.017 0.04 04/26/85 205 0.006 0.01 06.05.85 14 1,010 th most common 2.52 08/20/85 6 Chimpanzee 4 -0,006 0.00 11.03.85 11.03.85 11 NANB 0.003 0.01 09.05.85 132 0,523 th most common 1.31 06.06.85 - 1,574 th most common 3, 93 01.08.85 - Chimpanzee 5 -0,006 0.00 11/21/80 11/21/80 4 HAV 0.001 0.00 12/16/80 147 0, 003 0.01 12/30/80 18 0, 006 0.01 29.07-21. 08.80 5 Chimpanzee 6 - - 25.05.82 - - HAV -0,005 0.00 17.05.82 - 0.001 0.00 10.06.82 106 -0,004 0.00 06.07.82 10

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0.290 0.72 01.10.82 - - Chimpanzee 7 -0,008 0.00 25.05 .82 25.05.82 7 HAV -0,004 0.00 17.06.82 83 -0,006 0.00 16.09.82 5 0,005 0.01 09.10.82 - Chimpanzee 8 -0,007 0.00 21.11 .80 11/21/80 15 HAV 0.000 0.00 12/15/80 130 0.004 0.01 03.02.81 8 0.000 0.00 03 .06-10.06.81 4.5 Chimpanzee 9 - - 27.07 .80 - - HBV 0.019 0.05 22 .08-10.10.79 - - - 11 .03.81 57 0.015 0.04 01.07-05.08.81 9 0.008 0.02 01.10.81 6 Chimpanzee 10 - - 12.05 .82 - - HBV 0.011 0.03 21. 04-12.05.82 9 0.015 0.04 01. 09-08.09.82 126 0.008 0.02 02.12.82 9 0.010 0.02 06.01.83 13 Chimpanzee 11 - - 05.12 .82 - - HBV 0.000 0.00 06. 01-12.05.82 11 - - 23.06.82 100 -0,003 0.00 09. 06-07.07.82 - -0,003 0.00 10/28/82 9 -0,003 0.00 12/20/82 10

ZV. 1.3. Panel I: Verification of infectious sera from chronic human NANBH carriers

A panel consisting of 22 unique samples, each in duplicate, for a total of 44 samples was coded. The samples were from verified infectious serum from a chronic NANBH carrier, infectious serum from retracted donors, and an infectious serum from an acute phase NANBH patient.

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In addition, samples were from highly pedigree negative controls and other disease controls. We got this panel from Dr. H. Alter Department of Health, Bethseda, Maryland.

The panel was designed by Dr. Alter several years earlier, and used it as a qualification panel to investigate the alleged NANBH.

The whole panel was examined twice by ELISA and the results were sent to Dr. Alter for confirmation. The validation results are shown in Table 7. Although this only shows the results of one set of duplicates, some values were obtained for each of the duplicate samples.

As Table 7 also shows, the 6 sera tested infectious in the chimpanzee model were strictly positive. The seventh infectious sample matched the sample for the acute NANBH case and was not reactive in this ELISA. Samples from covered donors with normal ALT levels and with uncertain results in chimpanzee studies were not reactive in the reaction.

Three other serial samples from one individual with acute NANBH were also non-reactive. All samples coming from highly pedigree controls obtained from donors who had at least 10 hepatitis-free blood donations were non-reactive in the ELISA. Finally, 4 of the samples were previously tested positive for NANBH investigations to others, but were not confirmed in these investigations. These four samples were negative by HCV ELISA.

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Table 7: H. Alter panel I:

Panel

1st result 2nd result

1.) proven infection by chimpanzee transmission A. NANB Chronicle: Post-Tx JF + + EB + + PG + + B. donors with elevated ALT involved BC + + JJ + + BB + + C. Acute NANB: Post-Tx WH - - 2.) suspected infection with chimpanzee transmission A. donor involved with normal ALT CC - - 3.) Acute NANB: Post-TX JL Week 1 - - JL Week 2 - - JL Week 3 - - 4.) Disease controls A. Primary biliary cirrhosis EC - - B. Alcoholic hepatitis in detection HB - - 5.) Pedigree negative controls DM - - DC - -

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LV

ML

AH

6.) Potential NANB Antigens

JS-80-OLT-0 (Ishida) star (Trepo) zurtz (Arnold)

Becasidin

IV. 1.4 Panel 2: NANBH donor / receptor

The coded panel consisted of 10 uncertain NANBH transfusion-related donor receptor cases, with a total of 188 samples. Each case consists of several or all donors according to the receptor, as well as a series of samples (taken 3, 6 and 12 months after transfusion) and receptors. Blood leaks from the receptor before transfusion are also included. The coded panel is the work of Dr. H. Alter from the NIH, and the results were sent to him for verification.

The results collected in Table 8 show ELISA detected antibody conversion in 9 out of 10 transfusion-bound NANBH cases. Samples from 4 cases (where there was no seroconversion) consistently react poorly in ELISA. Two of the 10 receptor samples were reactive 3 months after transfusion. There were 6 receptors after 6 months; after 12 months, with the exception of Case 4, all samples were reactive. In addition, at least 1 positive donor antibody is found in 7 out of 10 cases, with 10 having two positive donors. Likewise, in Example 10, the receptor that previously donated blood was positive for HCV antibodies. After one month, the bleeding of this receptor fell to the threshold line of reactive levels, while taking blood at 4 and 10 months was positive. In general, a S / CO of 0.4 is considered

128 positive. Thus, this case may represent pre-infection of an individual with HCV.

ALT and Hbc for reactive e.g. positive samples are summarized in Table 9. As can be seen from the table, 1/8 of donor samples were negative for other markers and reactive in HCV ELISA antibodies. On the other hand, the sample receptor (monitored 12 months after transfusion) was raised to ALT, positive Anti-HBc, or both.

Table 8: Donor / receptor: NANB panel H. Alter donor / receptor NANB panel

Receptor before

Donor bleeding for 3 months

Ex. OD S / CO OD S / CO OD S / CO 1. - - 0.032 0.07 0,112 0.26 2. - - 0,059 0.14 0.050 0.12 3. 0,403 0.94 0.49 0.11 0,057 0.13 4. - - 0.065 0.15 0,073 0.17 5. > 3,000 > 6, 96 0.034 0.08 0.096 0.22 6. > 3,000 > 6.96 0,056 0.13 1,475 th most common 3, 44 7. > 3,000 > 6.96 0.034 0.08 0,056 0.13 8. > 3,000 > 6.96 0.061 0.14 0,078 0.18 9. > 3,000 > 6, 96 0.080 0.19 0.127 0, 30 10. > 3,000 > 6, 96 > 3,000 > 6, 96 0,317 * 0.74 > 3,000 > 6, 96

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Table 8 (continued)

Post-TX

6 months 12 months Ex. OD S / CO OD S / CO 1.> 3,000 > 6.96 > 3,000 > 6.96 2. 1,681 3, 90 > 3,000 > 6.96 3.> 3,000 > 6.96 > 3,000 > 6.96 4. 0.067 0.16 0,217 th most common 0.50 5.> 3,000 > 6.96 > 3,000 > 6.96 6.> 3,000 > 6, 96 > 3,000 > 6.96 7.> 3,000 > 6.96 > 3,000 > 6, 96 8.> 2,262 > 5.28 > 3,000 > 6, 96 9.> 0.055 0.13 > 3,000 > 6.96 10.> 3,000 ** > 6.96 > 3,000 ***> 6.96 Legend: * 1 Month, ** 4 months, *** 10 months Table 9: ALT And Hbc status for reactive samples in H panels 1 Patterns Anti-ALT * Hbc ** DONORS Example 3 normal negative Example 5 elevated positive Example 6 elevated positive Example 7 not accessible negative Example 8 normal positive Example 9 elevated not accessible Example 10 normal positive Example 10 normal positive

Al ter

130

RECEPTORS Example 1 6me elevated positive 12me elevated not investigated Example 2 6me elevated negative 12me elevated not investigated Example 3 6me normal not tested *** 12me elevated not explored *** Example 5 6me elevated not tested 12me elevated not investigated Example 6 3me elevated negative 6me elevated negative 12me elevated not investigated Example 7 6me elevated negative 12me elevated negative Example 8 6me normal positive 12me not investigated positive Example 9 12me elevated not investigated Example 1C i 4me elevated not investigated Oh elevated

Legend: * ALT 45 IU / L is above the upper limit ** anti-HBc 50% (competitiveness study) is considered positive *** before bleeding and 3 months the samples were negative for Hbc.

IV. 1.5 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 shown in Table 10.

As shown in the table, the samples with the highest reactivity were obtained from hemophiliacs (76%). Additionally, samples from individuals with elevated ALT and positive for Anti-HBc were 51% reactive, a value consistent with the value expected from clinical data

131 and NANBH dominates this group. HCV antibody levels were also higher in the blood of donors with elevated ALT, blood donors that were positive for antibodies to Hepatitis B nuclei and in blood obtained for reasons other than high ALT or anti-nuclear antibodies, if administered. with random volunteer donors.

Table 10: NANBH HIGH RISK SAMPLES GROUP

Group Distribution% reactivity

N N OD elevated ALT 35 3 > 3,000 11/4 1 0.728 Anti-HBc 24 5 > 3,000 20,8 elevated ALT, Anti-HBc 33 12 > 3,000 51.5 1 2,768 th most common 1 2,324 th most common 1 0.939 1 0.951 1 0, 906 rejected donors 25 5 > 3,000 20,0 donors with history of hepatitis 150 19 > 3,000 14.7 1 0.837 1 0.714 1 0,469 th most common hemophiliacs 50 31 > 3,000 76,0 1 2,568 th most common 1 2,483 th most common 1 2,000 1 1, 979 1 1,495 th most common 1 1,209 th most common 1 0.819

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IV. 1.6. Comparison of screening using Anti-IgG or AntiIgM monoclonal antibodies or polyclonal antibodies as another antibody in HCV C100-3 ELISA

The sensitivity of ELISAs using anti-IgG monoclonal conjugates was compared with the results obtained, either anti-IgM monoclonal conjugates or by replacing them with polyclonal antisera that were said to be specific for light and heavy chains. The following investigations were carried out.

IV.1.6.a Serial samples from a seroconverter

Serial samples from three NANBH seroconverter cases were examined in HCV C100-3 ELISA by screening using either anti-IgG monoclonal alone or in combination with anti-IgM monoclonal or using a polyclonal antiserum in the enzyme conjugate. Samples were obtained from Dr. Cladd Stevens, Ν.Υ. Blood Center, Ν.Υ. 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 indicate that strong reactivity can be detected in samples 1, 4, 2-8 and 3-5 of 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 conjugates and anti-IgM conjugate are displayed in Table 13. Three different anti-IgG anti-IgM ratios 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 indicate that, in agreement with the study of anti-IgG alone, strong reactivity was shown in samples 1-4, 2-8 and 3-5.

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Results obtained by ELISA using anti-IgG monoclonal conjugate (diluted 1: 100000) or Tago polyclonal conjugate (diluted 1: 80000) or Jackson polyclonal horse. (1: 80000) are summarized in Table 14. The data indicate that strong initial reactivity was detected in samples 1-4, 2-8, and 3-5 using all three configurations; Tago polyclonal antibodies give the lowest signals.

The results shown further show that all three configurations detect reactive patterns at the same time after the acute phase of the disease (as evidenced by increased ALT). But the results also show that the sensitivity of the HCV C100-3 ELISA using anti-IgG monoclonal enzyme conjugate is equal to or even better than that obtained when using other tested configurations for enzyme conjugates.

Table. 11: Description of samples From the Clado-Stevens panel date HBsAg Anti-HBs Anti HBc ALT Biliru

Example 1-1 1 5. 8.81 1.0 91,7 12.9 40,0 -1.0 1-2 2. 9.81 1.0 121,0 15.1 274,0 1.4 1-3 7. 10.81 1.0 64,0 23,8 261,0 0.9 1-4 19. 11.81 1.0 67.3 33,8 75,0 0.9 1-5 15. 12.81 1.0 50.5 27, 6 71,0 1.0 Example 2-1 2 19 .10.81 1.0 1.0 116,2 17,0 -1.0 2-2 17 .11.81 1.0 0.8 89.5 46.0 1.1 2-3 02 .12.81 1.0 1,2 78, 3 63,0 1.4 2-4 14 .12.81 1.0 0.9 90.6 152,0 1.4 2-5 23 .12.81 1.0 0, 8 93, 6 624,0 1.7 2-6 20 .01.82 1.0 0.8 92, 9 66,0 1.5

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2-7 02/15/82 1.0 0.8 86,7 70,0 1.3 2-8 17.03.82 1.0 0.9 69,8 24,0 -ι, ο 2-9 04/21/82 1.0 0.9 67.1 53,0 1.5 2-10 19.05.82 1.0 0.5 74,8 95,0 1.6 2-11 14.06.82 1.0 0.8 82,9 37,0 -ι, ο Example 3 3-1 07.04.81 1.0 1,2 88, 4 13,0 -ι, ο 3-2 12.05.81 1.0 1.1 126,2 236,0, 0.4 3-3 30.05.81 1.0 0.7 99, 9 471,0 0.2 3-4 09.06.81 1.0 1,2 110,8 315,0 0.4 3-5 06.07.81 1.0 1.1 89,9 273,0 0.4 3-6 10.08.81 1.0 1.0 118.2 158.0 0.4 3-7 08.09.81 1.0 1.0 112,3 84,0 0.3 3-8 10/14/81 1.0 0.9 102,5 180,0 0.5 3-9 11.11.81 1.0 1.0 84.6 154.0 0.3

Table 12: ELISA results using ANTI-IgG monoclonal conjugate ALT OD S / CO The pattern date neg. control 0,076 Cut 0,476 th most common PC (1: 128) 1, 390 Example 1 1-1 05.08.81 40,0 0,178 0.37 1-2 02.09.81 274,0 0,154 0.32 1-3 07.10.81 261,0 0,129 0.27 1-4 11/19/81 75,0 0.937 1, 97 1-5 12/15/81 71,0 > 3,000 > 6, 30

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Example 2

2-1 10/19/81 2-2 17.11.81 2-3 02.12.81 2-4 12/14/81 2-5 23.12.81 2-6 20.02.82 2-7 02/15/82 2-8 17.03.82 2-9 04/21/82 2-10 19.05.82 2-11 14.06.82 Example 3 3-1 07.04.81 3-2 12.05.81 3-3 30.05.81 3-4 09.06.81 3-5 06.07.81 3-6 10.08.81 3-7 08.09.81 3-8 10/14/81 3-9 11.11.81

, 0 0.058 0.12, 0 0.050 0.11, 0 0.047 0.10, 0 0.059 0.12, 0 0.070 0.15, 0 0.051 0.11, 0 0.139 0.29, 0 1.867 3, 92, 0 > 3.000> 6.30, 0> 3.000> 6.30, 0> 3.000> 6.30, 0 0.090 0.19, 0 0.064 0.13, 0 0.079 0.17, 0 0.211 0.44, 0 1.707 3 , 59, 0> 3.000> 6.30, 0> 3.000> 6.30, 0> 3.000> 6.30, 0> 3.000> 6.30

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Table 13: ELISA results obtained using ANTI-IgG and ANTI-IgM monoclonal conjugates

NANB ELISAs

Sample Date ALT monoclonal IgG l: 10K IgM l: 30K FROM S / CO monoclonal IgG l: 10K IgM l: 60K FROM S / CO monoclonal IgG l: 10K IgM l: 120K FROM S / CO Neg control 0.100 0.080 0,079 Cut PC ( 1: 128) 1,083 th most common 1,328 th most common 1,197 th most common Example 1 1-1 05.08.81 40 0,173 0,162 th most common 0.070 1-2 02.09.81 274 0,194 0,141 0,079 1-3 07.10.81 261 0,162 th most common 0,129 0,063 1-4 11/19/81 75 0.812 0.85 0.709 1-5 12/15/81 71 > 3,000 > 3,000 > 3,000 Prixner2 2-1 10/19/81 17 0,442 th most common 0.045 0,085 2-2 17.11.81 46 0.102 0.029 0.030 2-3 02.12.81 63 0,059 0.036 0.027 2-4 12/14/81 152 0.065 0.041 0.025 2-5 23.12.81 624 0,082 0.033 0.32 2-6 01/20/82 66 0.102 0.042 0.027 2-7 02/15/82 70 0,188 0.068 0.096 2-8 17.03.82 24 1,728 th most common 1,668 th most common 1, 541 2-9 04/21/82 53 > 3.00 2,443 th most common > 3.00 2-10 19.05.82 95 > 3.00 > 3.00 > 3.00 2-11 14.06.82 37 > 3.00 > 3.00 > 3.00

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Example 3

3-1 07.04.81 13 0.193 0,076 0.049 3-2 12.05.81 236 0.201 0,051 0.038 3-3 30.05.81 471 0,132 0.067 0,052 3-4 09.06.81 315 0.175 0,155 0,140 3-5 06.07.81 273 1,335 th most common 1,238 th most common 1,260 th most common 3-6 10.08.81 158 > 3.00 > 3.00 > 3.00 3-7 08.09.81 84 > 3.00 > 3.00 > 3.00 3-8 10/14/81 180 > 3.00 > 3.00 > 3.00 3-9 11.11.81 154 > 3.00 > 3.00 > 3.00

Table 14: ELISA results obtained using polyclonal conjugates

NAB ELISA

Sample Date ALT Monoclonal l: 10K Tago l: 80K Jackson l: 80K OD i S / CO OD S / CO OD S / CO Neg control 0.07 0.045 0,154 Cut 0,476 th most common 0.545 0.654 PC (1: 128) 1,390 th most common 0.727 2,154 th most common Example 1 1-1 05.08.81 40 0,178 0.37 0.067 0.12 0.153 0.23 1-2 02.09.81 274 0,154 0.32 0,097 0.18 0.225 0, 34 1-3 07.10.81 261 0,129 0.27 0,026 th most common 0.05 0.167 0.26 1-4 11/19/81 75 0, 937 1, 97 0, 324 0, 60 0.793 1.21 1-5 12/15/81 71 > 3,00: > 6.30 1,778 th most common 3.27 > 3.00 > 4,59 Example 2 2-1 10/19/81 17 0,058 0.12 0,023 th most common 0.04 0,052 0.08 2-2 11/17/81 46 0.050 0.11 0.018 0.03 0,058 0.09 2-3 02.12.81 63 0.047 0.10 0.020 0.04 0.060 0.09 2-4 12/14/81 152 0,059 0.12 0.025 0.05 0,054 0.08

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2-5 23.12.81 624 0.070 0.15 0,026 th most common 0.05 0,074 0.11 2-6 01/20/82 66 0,051 0.11 0.018 0.03 0,058 0.09 2-7 02/15/82 70 0,139 0.29 0.037 0.07 0,146 0.22 2-8 17.03.82 24 1,867 th most common 3, 92 0,355 th most common 0.65 1,429 th most common 2.19 2-9 04/21/82 53 > 3.00 > 6.30 0.748 1.37 > 3.00 > 4,59 2-10 19.05.82 95 > 3.00 > 6.30 1,025 th most common 1.88 > 3.00 > 4,59 2-11 14.06.82 37 > 3.00 > 6.30 0, 917 1.68 > 3.00 > 4,59

Example 3

3-1 07.04.81 13 0.090 0.19 0.049 0.09 0,138 0.21 3-2 12.05.81 236 0.064 0.13 0.040 0.07 0,094 0.14 3-3 30.05.81 471 0,079 0.17 0.045 0.08 0,144 0.22 3-4 09.06.81 315 0.211 0.44 0,085 0.16 0.275 0.42 3-5 06.07.81 273 1,707 th most common 3.59 0.272 0.50 1,773 th most common 2.71 3-6 10.08.81 158 > 3.00 > 6.30 1,347 th most common 2.47 > 3.00 > 4,59 3-7 08.09.81 84 > 3.00 > 6, 30 2,294 th most common 4.21 > 3.00 > 4,59 3-8 10/14/81 180 > 3.00 > 6, 30 > 3.00 > 5.50 > 3.30 > 4,59 3-9 11.11.81 154 > 3.00 > 6.30 > 3.00 > 5.50 > 3.00 > 4,59 IV. I .6.b Samples from random donors

Random donor samples (see section IV.I) are tested for HCV infection using a C100-3 ELISA in which the antibody-enzyme conjugate was an anti-IgG monoclonal conjugate or a polyclonal conjugate. The total number of samples tested was 1077 and 1056 for the polyclonal conjugate and the monoclonal conjugate, respectively. The collected results are shown in Table 15, and the distribution of the sample is shown in the histogram in Figure 44.

Average and standard deviation computation was performed by excluding samples giving a signal above 1.5 i.e.. 1073 OD values were used to calculate the use of the polyclonal conjugate and 1051 for the anti-IgG monoclonal conjugate. As shown in Table 15, when a polyclonal conjugate is used, the average osd measurement is 0.0493 to

139

0.0931 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 study termination, the polyclonal -enzyme conjugate configuration in the ELISA requires a greater value of termination. 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 result between negative and positive distributions is obtained when random blood donors are tested in ELISA using an anti-IgG monoclonal conjugate compared to studies using a commercial polyclonal label.

Table 15: Comparison of the two ELISA configurations in random blood donor sample testing

Polyclonal Anti-IgG Monoclonal Conjugate (Jackson)

no. of the sample 1073 1051 average (x) 0,0931 0,04926 stand. deviac. (SD) 0,0933 0,07427 5 SD 0,4666 0,3714 interruption (5 SD + x) 0,5596 0,4206

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

Serum from patients suspected of having NANBH based on elevated ALT levels and negative for HAV and HBV tests was tested with RIA mainly as described in Chapter IV.D., with the exception of this that the HCV C100-3 antigen was used as the test antigen in the microtiter vessel. As can be seen from the results given in Table 16, RIA detects positive samples in a high percentage of cases.

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Table 16: Seroconversion frequencies for Anti-C100-3 med

NANBH patients in different countries Japan Country The Netherlands Italy no. investigated 5 36 26 no. of the positive 3 29 19 % positive 60 80 73

IV.K. Detection of HCV seroconversion in patients with commonly obtained NANBH

Serum obtained from 100 NANBH patients for whom the usual route of transfusion (eg without transfusion, IV drug administration, pregnancy, etc., were declared risk factors) was obtained from Dr. Alter from the Centers for Disease Control and Dr. Harvard University Dienstag. These specimens were tested with the RIA mainly as described in Chapter IV.D., except that the HCV C100-3 antigen was used as the antigen for testing in microtiter vessels. The results showed that out of 100 serum samples, 55 contained antibodies and reacted immunologically with HCV C100-3 antigen. The results described further suggest that the commonly obtained NANBH is also HCV-induced. However, as shown here that HCV is associated with Flaviviruses most transmitted by arthropods, we can also conclude that HCV in these cases is transmitted through arthropod transmission.

IV.L. Comparison of HCV Antibody and Surrogate Margin Capture in NANBH Transfer Donors

A prospective study was conducted to determine whether a blood receptor from a suspected NANBH positive donor developing NANBH seroconverted to an anti-HCV antibody

141 positive. Blood donors have been tested for surrogate markers of abnormalities commonly used as markers for NANBH infection, insane. elevated ALT levels and for the presence of anti-core antibodies. In addition, donors were also tested for the presence of anti-HCV antibodies. Determination of the presence of anti-HCV antibodies was performed by immunoassay as described in section IV.K. The results of the study are shown in Table 17 showing: number of patients (column 1), presence of anti-HCV antibodies in the patient's serum (column 2), number of benefits the patient receives with each donation (column 3) ), the presence of anti-HCV antibodies in donor serum (column 4) and surrogate donor abnormality (column 5) (NT or - indicates untested area) (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 than the surrogate marker tests even in the detection of infected donor blood. 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 times from two different donors suspected to be NANBH carriers), 9 were positive for anti-HCV antibodies, 1 was at the limit of positivity and was therefore ambiguous ( donor for patient 1). In contrast, when using the elevated ALT test, 6 out of 10 donors tested were negative using the antinuclear antibody test, and 5 out of 10 donors were negative. In three cases (donors to patients 8, 9, and 10), the ALT test and the ANTI-HBc test were observed to produce consistent results.

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Table 17: Development of ANTI-HCV antibodies in patients receiving blood from donors suspected to be NANBH carriers

The patient anti-HCV superconvert, in patients no. give / donor Anti-HCV positive alternate abnormal anti-HB donor ALT 1 Yes 18 ambiguity. no no 2 Yes 18 Yes NT Yes 3 Yes 13 Yes no no 4 no 18 no - - 5 Yes 16 Yes Yes Yes 6 Yes 11 yes (2) no no 7 Yes 15 Yes NT no 8 Yes 20 Yes no Yes 9 Yes 5 Yes Yes no 10 Yes 15 Yes no Yes

IV.M. Amplification for cloning HCV cDNA sequences using PCR and primers derived from converted regions Flavivirus genomic sequences

The results shown below, suggesting that HCV is a Flavivirus or a similar virus, permit a strategy for cloning uncharacterized HCV cDNA sequences used by PCR and primers derived from the region encoding amino acid sequences in Flaviviruses. Generally speaking, one of the primers is derived from a defined HCV genomic sequence, and the other primers that bypass the region of the non-sequenced HCV polynucleotide are derived from conserved regions of the Flavivirus genome. Flavivirus genomes contain conserved sequences in NS1 and

Epolipeptides encoded in the 5'-region of the Flavivirus genome. The corresponding sequences encoding these regions lie higher than

143

The HCV cDNA sequence is shown in Figure 26. Thus, the isolation of cDNA sequences derived from this region of the HCV genome indicates higher-lying primers derived from conserved sequences in these flavivirus polypeptides.

The lower primers are derived from the higher end of the known portion of the HCV cDNA.

Due to code degeneration, there is likely to be a discrepancy between flavivirus probes and the corresponding HCV genomic sequence. For this reason, a strategy similar to that described by Lee (1988) is used. This process uses mixed oligonucleotide primers complementary to the reverse translational products of the amino acid sequence; sequences in mixed primers taken to account for each codon degeneration for a conservative amino acid sequence. Three sets of primary mixtures were made based on amino acid homologs found in several flaviviruses, including Dengue-2,4 (D-2,4), Japanese Encephalitis virus (JEV), Yellow fever (YF), and West Nile Virus (WN). The primary mixture derived from the highest-lying conserved sequence (5'-l) was based on the amino acid sequence of Gly-Trp-Gly, which was partially conserved in the Asp-Arg-Gly-Trp-Gly-AspN sequence found in E protein D-2, JEV, YF and WN. The following primary mixture (5'-2) was based on a lower-lying conserved sequence in the E protein, Phe-Asp-Gly-Asp-Ser-Tyr-Ile-Phe-Gly-Asp-Ser-TyrIle and derived from Phe- Gly-Asp; the conserved sequence was present in D-2, JEV, YF, and WN. The third primary mixture (5′-3) was based on the amino acid sequence of Arg-SerCys in NS1 protein D-2, D-4, JEV, YF and WN. The individual primers that construct the mixture in 5′-3 are shown in Figure 45. In addition to the variable sequences derived from the conserved region, each primer in each mixture also contains a sequence encoding restriction enzyme sites, Hinll1, Mbol, and EcoRI.

144

In the lower case, ssc5h20A is derived from a nucleotide sequence in clone 5h containing HCV cDNA with sequences of clones 14i and 11b. The sequence of ssc5h20A is:

5'-GTA ATA TTG TGA CAG AGT CA 3 '

An alternative example of ssc5h34A may also be used. This example is derived from a sequence in clone 5h and in the appendix contains nucleotides at the 5′-end, which form a restriction enzyme site, which facilitates cloning. The sequence of ssc5h34A is:

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

The PCR reaction originally described by Saiki et al. (1986) is implemented mainly as described in 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 Chapter IV.A.l. Additionally, the crossing conditions are less stringent in the first round of use (0.6M NaCl and 25 ° C), although the part that primes to cross to the HCV sequence is only 9 nucleotides long, making them poorly coupled. But when ssc5h34A is used, additional sequences that have not been derived from the HCV genome make it more difficult to stabilize the primary-template hybrid. After the first round of application, the crossover conditions should be stricter (0.066M NaCl and 32-37 ° C), although the sequences used now contain regions that are complementary or are duplicates of primers. In addition, the first 10 cycles of use are performed with Maple / Enzyme I, under the appropriate PCR conditions for that enzyme. After completion of these cycles, the samples are extracted and treated with Taq polymerase according to the directions of the equipment provided by CetusPerkin-Elmer.

After use, amplified HCV cDNA sequences are detected by hybridization of the probe derived from clone 5h. This probe is derived from sequences that are higher than those used for isolation of primers and does not overlap the sequence from clone 5h of the derived primer. The probe sequence is:

5 'CCC AGC GGC GTA CGC GCT GGA CAC GGA GGT GGC CGC GTC

GTG TGG CGG TGT TGT TCT CGT CGG GTT GAT GGC GC 3 ’.

145

IV.N.l Construction of HCV cDNA Library from Chinpanzes Liver with NANBH Infection

The HCV library was made from the liver of the chimpanzee from which the HCV cDNA library from Chapter IV.A.1 was created from the liver. The technique for constructing the library is similar to that in Chapter IV.A.24 except that a different RNA source was used and the primer was based on the HCV cDNA sequence in clone 11b. 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-l relative to cDNA in clone llb

The k9-l clone was isolated from an HCV cDNA library made from the liver of a NANBH infected chimpanzee as described in Chapter IV.A.25. The library was tested for clones that overlap the sequence in clone llb using a clone that overlaps clone llb at the 5'-terminus of clone lle. The sequential clone llb is shown in Figure 23. Positive clones were isolated at a frequency of 1 in 500000. One isolated clone k9-l was subjected to further study. The overlapping nature of HCV cDNA in the k9-l clone at the 5'-end of the HCV cDNA sequence in Fig. 26 was confirmed by examining the clone from Alex46; this latter clone contains the HCV cDNA sequence of 30 base pairs that correspond to those pairs at the 5'-terminus of the HCV cDNA in clone 14i described below.

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

The HCV cDNA sequence in clone k9-l is linked to that of the clones described in Chapter IV.A.19 for the purpose of constructing the composition of the HCV cDNA sequence with k9-l sequence positioned higher than the sequence

146 shown in Figure 32. The HCV cDNA composition comprising the k9-l sequence and the amino acids encoded therein are shown in Figure 47.

The amino acid sequence encoded in the 5′-region of the HCV cDNA shown in Figure 47 was compared with the corresponding Dengue virus region described below with respect to the hydrophobic and hydrophilic profile of the region. This comparison shows that the polypeptides from HCV and Dengue encode in this region corresponding to the region encoded by NS1 (or a portion thereof) and have a similar hydrophobic / hydrophilic profile.

The information provided below allows the identification of the HCV chain. Isolation and characterization of other HCV chains can be performed by isolating nucleic acids from body components containing viral particles that construct cDNA libraries with polynucleotide probes based on HCV cDNA probes, which will be described below. Testing libraries with clones containing HCV cDNA sequences described below and comparing HCV cDNA from new isolates with the cDNA described below. Polypeptides encoded here or in the viral genome can be monitored by immunobiological cross-reactivity using the polypeptides and antibodies described below. Chains that fall into HCV parameters as described in the definitions section are easy to identify. Other methods for identifying HCV chains will be readily apparent to those skilled in the art and will be based on the information provided in the present invention.

for

CHIRON CORPORATION Emeryville, California

USA

Representative:

IV,

INDUSTRIAL USE

The present invention, as described in various ways, has many industrial uses, some of which will be listed. HCV cDNA can be used to construct a probe for the detection of HCV nucleic acids in samples. CDNA probes can be used to detect HCV nucleic acids in, for example, chemical synthetic reactions. They can also be used in antiviral agent testing programs to determine the effects of agents in inhibiting viral replication in cell culture systems and animal models of the system. Human HCV polynucleotides can also be used as a basis for the diagnosis of human HCV infections.

In addition, the cDNAs provided by the present invention provide information and agents for the synthesis of polypeptides containing HCV epitopes. These polypeptides are useful in determining antibodies in HCV antigens. A series of tests for HCV infection based on recombinant polypeptides containing HCV epitopes are described, and the commercial utility is found in the diagnosis of HCV challenging NANBH in donor blood tests for HCV-induced hepatitis and also for the detection of contaminated blood from infectious blood donors. Viral antigens will also be used to monitor the performance of antiviral agents in animal models of the system. In addition, polypeptides derived from HCV cDNAs described in the present invention are useful as vaccines for the treatment of HCV infections.

HCV cDNA polypeptides, in addition to the areas listed above, are also useful for the growth of HCV antibodies. Thus, they can be used in anti-HCV vaccines. Anti-libraries resulting from immunization with HCV polypeptides are also useful in detecting the presence of viral <RTI ID = 0.0> i </RTI> agents in samples, but can also be used to investigate the production of HCV polypeptides in chemical systems. Anti-HCV antibodies are also useful in monitoring the usefulness of antiviral agents in research programs where these agents are tested in the tissue of a culture system. In addition, they can be used for passive immunotherapy and diagnosis of NANBH-inducing HCV by allowing the detection of viral antigens in donors and blood receptors. Another important utility for anti-HCV bodies is in affinity chromatography for purification of viral and viral polypeptides. Purified virus and viral polypeptide preparations can be used in vaccines. The purified virus can also be used to develop cell cultures where HCVs are replicates.

Cell culture systems containing HCV infected cells will have many uses. They can be used for a relatively large volume of HCV production, which is normally a low titre virus. These systems will also be useful in the molecular biology of viruses and enable the development of antiviral agents. Cell culture systems will be useful in testing the effectiveness of antiviral agents. In addition, HCV permeable cell culture systems are useful for the production of impaired HCV structures. Particularly appropriate and useful is the fact that anti-HCV antibodies and HCV polypeptides, natural or recombinant, can be incorporated into the equipment. The method used to isolate HCV cDNA, comprising obtaining a cDNA library derived from infected tissue of a subject in an expression vector and selecting clones that produce expression products that react immunologically with antibodies in components of bodies containing antibodies of another infected subject and not of non-infected subjects can also be used to isolate cDNAs of other, hitherto uncharacterized, agents that cause

Μ diseases that consist of a genomic component. This can lead to the isolation and characterization of these agents and diagnostic reagents and vaccines for other agents that cause the disease.

Claims (43)

PATENT REQUIREMENTS A recombinant polynucleotide encoding an HCV epitope. Recombinant polynucleotide according to claim 1, characterized in that said polynucleotide is DNA, and said HCV epitope is present within an adjacent sequence of at least 10 amino acids encoded by HCV cDNA from a cDNA library deposited at the American Type Culture Collection cultures; under number 40394. Recombinant polynucleotide according to claim 2, characterized in that said adjacent sequence comprises at least 15 amino acids. Recombinant polynucleotide according to claim 1, characterized in that said polynucleotide is DNA, and said HCV epitope is present within the adjacent sequence of at least 10 amino acids contained in the amino acid sequence shown in Figure 14. A recombinant polynucleotide according to claim 4, characterized in that said adjacent sequence comprises at least 15 amino acids. Recombinant polynucleotide according to claim 1, characterized in that said polynucleotide is DNA, and said HCV epitope is present within the adjacent sequence of at least 10 amino acids contained in the amino acid sequence shown in Figure 47. A recombinant polynucleotide according to claim 6, characterized in that said adjacent sequence comprises at least 15 amino acids. The recombinant polynucleotide according to claim 1, characterized in that said polynucleotide is DNA and said HCV epitope is present within the adjacent sequence of at least 15 amino acids contained in the HCV polyprotein. Μ, .. A vector comprising an ORF Opened reading frame, characterized in that it is operatively linked to a control sequence from a host cell, wherein said ORF is a recombinant polynucleotide according to any one of claims 2 to 8. 10. The host cell, characterized in that it is transformed with the vector of claim 9. A method of producing a polypeptide comprising an HCV epitope, characterized in that it comprises incubating the host cells according to claim 10 under conditions that permit the expression of said ORF. A purified polypeptide comprising an epitope that is immunologically recognizable by an epitope in HCV. A purified polypeptide according to claim 12, characterized in that said epitope lies within an adjacent sequence of at least 10 amino acids encoded by HCV cDNA from a cDNA library deposited at / deposited at / American Type Culture Collection, under number 40394. A purified polypeptide according to claim 13, characterized in that said adjacent sequence comprises at least 15 amino acids. A purified polypeptide according to claim 12, characterized in that said epitope lies within the adjacent sequence of at least 10 amino acids contained in the amino acid sequence shown in Figure 14. A purified polypeptide according to claim 15, characterized in that said adjacent sequence comprises at least 15 amino acids. A purified polypeptide according to claim 12, characterized in that said epitope lies within the adjacent sequence of at least 10 amino acids contained in the amino acid sequence shown in Figure 47. A purified polypeptide according to claim 17, characterized in that said adjacent sequence comprises at least 15 amino acids. dl .- -. A purified polypeptide according to claim 12, characterized in that said epitope lies within an adjacent sequence of at least 15 amino acids found in the HCV polyprotein. A purified polypeptide according to any one of claims 12 to 19, characterized in that the polypeptide is prepared by the expression of recombinant DNA or by chemical synthesis. 21. The purified polypeptide according to claim 20, characterized in that the polypeptide is prepared by expression of recombinant DNA. A purified polypeptide according to any one of claims 12 to 21, characterized in that the polypeptide is fixed to the solid phase. A purified polypeptide according to any one of claims 12 to 21, characterized in that the polypeptide is fixed to the wall of the microtiter vessel and said wall is coated with BSA (albumin serum) to prevent non-specific antibody binding. Apparatus for the analysis of biological samples for the presence of antibodies directed against the HCV antigen, characterized in that it comprises the polypeptide of any one of claims 12 to 23 in a suitable container. 25.Immunoassay for the detection of antibodies directed against HCV antigen, in biological samples, characterized in that they contain: (a) incubating the biological samples with the polypeptide of any one of claims 12 to 23 under conditions that permit the construction of an antibody-antigen complex; and (b) detecting said HCV-directed antibodies by detecting the presence of an antibody-antigen complex containing said polypeptide. A vaccine composition comprising a purified polypeptide according to any one of claims 13 to 21 mixed with a pharmaceutically acceptable recipient. The vaccine composition of claim 26, wherein said adjacent sequence is of the HCV protein structure. / 1¾ v '. - · · · · A monoclonal antibody, characterized in that it is directed against the HCV epitope. The monoclonal antibody of claim 29, wherein said HCV epitope is contained in an amino acid sequence encoded by HCV cDNA from a cDNA library deposited with American Type Culture Collection under number 40394. 30. Purified polyclonal antibody preparation, characterized in that it is directed against the HCV epitope. A purified polyclonal antibody preparation according to claim 30, characterized in that it contains antibodies directed against HCV epitopes contained within an amino acid sequence encoded by HCV cDNA from a cDNA library deposited with American Type Culture Collection under number 40394. 32. HCV probe, characterized in that it is a polynucleotide probe for HCV. The polynucleotide probe of claim 32, comprising at least 10 polynucleotides capable of selective hybridization in the HCV genome or complement thereof. The polynucleotide probe of claim 33, wherein said adjacent sequence contains at least 12 nucleotides. The polynucleotide probe of claim 33, wherein said adjacent sequence contains at least 20 nucleotides. A polynucleotide probe according to any one of claims 33 to 35, characterized in that said adjacent sequence is contained within the HCV cDNA from the cDNA library deposited at American Type Culture Collection under number 40394. A polynucleotide probe according to any one of claims 33 to 35, characterized in that said adjacent sequence is contained in Figure 14. A polynucleotide probe according to any one of claims 33 to 35, characterized in that said adjacent sequence is contained in Fig. 47. 39. Equipment for analyzing samples for the presence of HCV-derived polynucleotides, characterized in that it comprises a polynucleotide probe according to any one of claims 33 to 35 in a suitable container. 40. A method for detecting HCV nucleic acids in a sample, characterized in that it comprises: (a) incubating the nucleic acids from the sample with the polynucleotide probe of any one of claims 32 to 38, under conditions that permit the construction of a polynucleotide duplex between said polynucleotide probe and any HCV nucleic acid in said sample; and (b) detecting the presence of polynucleotide duplexes containing said polynucleotide probe. 41. Adult cell culture tissue, characterized in that it is infected with HCV. A HCV infected cell according to claim 41, characterized in that it is a cell of a human macrophage cell type or, a hepatocyte cell type. The HCV infected cell of claim 41, wherein the cell is a hepatocyte.