DD298526A5 - METHOD FOR DETECTING HCV NUCLEINE ACIDS - Google Patents

Abstract

; Hepatitis C virus Non-A non-B hepatitis; cDNA sequences; flaviviruses; flaviviruses; polynucleotides; polynucleotide; polypeptides; HCV epitopes; Antibody; HCV antigens; HCV nucleic acid; polynucleotide duplex; Detection of HCV nucleic acids

Description

For this 63 drawings

Method for the detection of HCV nucluric acid Technical area

The invention relates to materials and methods for controlling the spread of infection by the non-A non-B hepatitis virus (NANBV). More particularly, it relates to diagnostic DNA fragments, diagnostic proteins, diagnostic antibodies and protective antigens, and antibodies to a pathogen of NANB hepatitis, i. the hepatitis C virus.

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Quoted Paient no.

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Background to the invention

Non-A-non-B hepatitis (NANBH) is a transmissible disease or family of diseases believed to be induced by a virus and other forms of virus-associated liver disease, including those caused by the virus known hepatitis viruses, ie Hepatitis A virus (HAV), hepatitis B virus (HBV) and delta hepatitis virus (HDV), as well as those caused by cytomegalovirus (CMV) or Epstein-Barr virus (EBV ) differentiates hepatitis. NANBH was first identified in people with blood transfusions. Transmission from humans to chimpanzees and serial passages in chimpanzees has provided evidence that NANBH is due to transmissible infectious agent (s). However, NANBH's transmission agent is still unidentified and the number of pathogens causing this disease is unknown. The epidemiological evidence suggests that there may be three types of NANBH: the epidemic type transmitted by water, the type of blood or needle, and the sporadically occurring (community-acquired) type. However, the number of NANBH-causing pathogens is unknown

 The clinical diagnosis and detection of NANBH has so far mainly been done by excluding other viral markers. To

The methods used to detect putative NANBV antigens or antibodies include agar gel diffusion, migration electrophoresis, immunofluorescence microscopy, immunoelectron microscopy, radioimmunoassay and enzyme immunoassay. However, none of these assays has been found to be sufficiently sensitive, specific and reproducible to be useful as a diagnostic test for NANBH.

So far, there is neither clarity nor consistency in the identity or specificity of the antigen-antibody systems related to the NANBH pathogens. This is due, at least in part, to a prior or simultaneous HBV infection in individuals with NANBV and to the known complexity of soluble and finely divided antigens associated with HBV, as well as to the integration of HBV DNA into the genome of liver cells. Furthermore, there is the possibility that NANBH is caused by more than one infectious agent, and there could also be a misdiagnosis of NANBH. It is also unclear what serological assays in the serum of patients with NANBH detect. It has been postulated that agar gel diffusion and the migration electrophoresis assay detect autoimmune reactions or nonspecific protein interactions, sometimes occurring between serum samples, and that they do not represent specific NANBV antigen-antibody reactions. Immunofluorescence, entnn-immunoassay and radioimmunoassay appear to be low levels of a rheumatoid factor-like material commonly found in serine. on patients with NANBH and also on patients with other hepatitis and non-hepatitis diseases . Part of the observed reactivity may be an antibody to host-determined cytoplasmic antigens. There are a number of NANBV candidates. See, for example, For example, the summaries of Prince (1983), Feinstone and Hoofnagle (1984), Overby (1985, 1986, 1987), and the articles by Iwarson (1987). However, there is no evidence that any of these candidates is the etiological agent of NANBH.

The demand for sensitive, specific screening and detection methods for NANBV carriers and for NANBV-contaminated blood or blood products is of great importance. Post-transfusion hepatitis (PTH) occurs in almost 10% of transfusion patients, with NANBH responsible for up to 90% of these cases. The main problem with this disease is the frequent aggravation up to chronic Leborschädigung (25-55%).

Patient treatment, as well as the prevention of the transmission of NANBH through blood and blood products, or through close personal contacts, requires reliable diagnostic and prognostic means to detect nucleic acids, antigens, and antibodies associated with NANBV. In addition, there is an urgent need for effective vaccines and immunotherapeutic agents for the prevention and / or treatment of the disease.

Disclosure of the invention

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 a family of cDNA copies of portions of the HCV genome. These cDNA copies were isolated by a technique comprising: a novel step of screening expression production from cDNA libraries created from a finely divided pathogen in infected tissue with sera from patients with NANBH to obtain newly synthesized antigens derived from the Genome of the hitherto unisolated and uncharacterized virus pathogen were derived, and the selection of clones producing products that reacted immunologically only with sera from infected individuals compared to uninfected individuals. Studies of the nature of the HCV genome using probes developed from the HCV cDNA, such as the sequence information contained in the HCV cDNA, indicate that the HCV is a flavivirus or a flavivirus.

Portions of the cDNA sequences derived from the HCV are useful as probes to detect the presence of the virus in samples and to isolate naturally occurring variants of the virus. These cDNAs also provide polypeptide sequences of HCV antigens encoded in the HCV genome (s) and allow the production of polypeptides that are used as standards or as reagents in diagnostic and / or as components of Vaccines are useful. Antibodies, both polyclonal and monoclonal, directed against the HCV epitopes contained in these polypeptide sequences are also useful in diagnostic assays and therapeutic agents for the screening of antiviral agents and for the isolation of the NANBV pathogen. from which these cDNAs are derived are useful. In addition, by using probes derived from these cDNAs, it becomes possible to isolate and sequence other portions of the HCV genome to yield additional probes and polypeptides useful for the diagnosis and / or prophylactic as well as therapeutic treatment of NANOH.

Accordingly, with respect to the polynucleotides, some aspects of the invention relate to: a purified HCV polynucleotide; a recombinant HCV polynucleotide; a recombinant polynucleotide containing a sequence derived from an HCV genome or HCV cDNA; a recombinant polynucleotide encoding an epitope of HCV; a recombinant vector containing any of the above recombinant polynucleotides, and a host cell transformed with any of these vectors. Other aspects of the invention are: a recombinant expression system comprising an open reading frame (ORF) of DNA derived from an HCV genome or hCV cDNA, the ORF operably linked to a control sequence that is compatible with a desired host; a cell transformed with the recombinant expression system; and a polypeptide produced by the transformed cell. Further aspects of the invention are: purified HCV, a preparation of polypeptides from the purified HCV; a purified HCV polypeptide; a purified polypeptide containing an epitope that is immunologically identifiable with an epitope contained in HCV.

Included in the aspects of the invention are: a recombinant HCV polypeptide; a recombinant polypeptide consisting of a sequence derived from an HCV genome or HCV cDNA; a recombinant polypeptide consisting of an HCV epitope; and a fusion polypeptide consisting of an HCV polypeptide. The invention also includes: a monoclonal antibody directed against an HCV epitope; and a purified preparation of polyclonal antibodies directed against an HCV epitope. Another aspect of the invention is a particle which is immunogenic against HCV infection and contains a non-HCV polypeptide having an amino acid sequence capable of forming a particle when this sequence is generated in a eukaryotic host and an HCV epitope.

Another aspect of the invention is a polynucleotide probe for HCV. Aspects of the invention pertaining to analysis kits are those for: Analyzing samples for the presence of polynucleotides derived from the HCV by means of a polynucleotide probe containing a nucleotide sequence from the HCV of about 8 or more nucleotides in a suitable container. English container); Analysis of samples for the presence of an HCV antigen by means of an antibody directed against the HCV antigen to be detected in a suitable container; Analysis of samples for the presence of an antibody directed against an HCV antigen by means of a polypeptide containing an HCV epitope present in the HCV antigen in a suitable container.

Other aspects of the invention are: a polypeptide consisting of an HCV epitope linked to a solid substrate; and an antibody to an HCV epitope linked to a solid substrate.

Further aspects of the invention are: a method of generating a polypeptide having an HCV epitope by incubating host cells transformed with an expression vector, the vector containing a sequence encoding a polypeptide containing an HCV epitope, under condition t ; which allow the expression of the polypeptide; and a polypeptide containing an HCV epitope generated by this method.

The invention also encompasses a method of detecting HCV nucleic acids in a sample, wherein the nucleic acids of the sample react with a probe for an HCV polynucleotide under conditions which permit the formation of a polynucleotide duplex between the probe and the HCV nucleic acid from the sample allow; and for detecting a polynucleotide duplex containing the probe.

Immunoassays are also included in the invention. These include an immunoassay to detect an HCV antigen, wherein a sample suspected of containing an HCV antigen is incubated with a probe antibody directed against the HCV antigen to be detected under conditions which prevent the formation of an antigenic antigen. Allow antibody complex; and for detection of an atitigen-antibody complex containing the probe antibody. An immunoassay for detecting antibodies directed against an HCV antigen, wherein a sample suspected of containing anti-HCV antibodies is incubated with a sondsn polypeptide containing an epitope of the HCV under conditions which prevent the formation of an antibody Allow antigen complex; and for detection of the antibody-antigen complex containing the probe antigen.

Also included in the invention are vaccines for the treatment of HCV infection which comprise an immunogenic peptide containing an HCV epitope or an inactivated HCV preparation or an attenuated HCV preparation. Another aspect of the invention is a tissue culture infected with HCV.

Yet another aspect of the invention is a method of generating antibodies to HCV by administering to an individual an isolated immunogenic polypeptide containing an HCV epitope in an amount sufficient to elicit an immune response.

Yet another aspect of the invention is a method for isolating cDNA derived from the genome of an unidentified infectious agent by (a) providing host cells transformed with expression vectors containing a cDNA library prepared from nucleic acids which were isolated from the pathogen-infected tissue and allowed to grow these host cells under conditions permitting the expression of polypeptide (s) encoded in the cDNA; (b) there is an interaction of the expression products of the cDNA with an antibody-containing body component of an individual infected with the infectious agent under conditions which permit an immune reaction and the antibody-antigen complexes formed as a result of the interaction are detected; (c) culturing host cells expressing polypeptides that form antibody-antigen complexes according to step (b) under conditions that permit their growth as individual clones and that isolate these clones; (d) growing cells from the clones according to (c) under conditions which allow the expression of polypeptide (s) encoded in the cDNA, and the expression products with antibody-containing body components other than those mentioned in step (a) Individuals that are infected with the infectious agent and interact with control individuals that are not infected with the agent, and that the antibody-antigen complexes formed as a result of this interaction are detected; (e) culturing host cells that express polypeptides that form antibody-antigen complexes with antibody-containing body components of infected individuals and individuals that are suspected of being infected, rather than the components of control individuals, under conditions that indicate their growth as an individual Allow clones, and these clones are isolated; and (f) isolating the cDNA from the host cell clones of step (a).

Brief description of the drawings Fig. 1: shows the double-stranded nucleotide sequence of the HCV cDNA insert in clone 5-1-1 and the presumptive Amino acid sequence of the polypeptide encoded therein.

Fig. 2: shows the homologies of overlapping HCV cDNA sequences in clones 5-1-1,81,1-2 and 91. Fig. 3: shows a composite sequence of HCV cDNA derived from overlapping clones 81 , 1 -2 and 91 derived

and the amino acid sequence encoded therein

 Fig. 4: shows the double-stranded nucleotide sequence of the HCV cDNA insert in clone 81 and the presumptive one

Amino acid sequence of the polypeptide encoded therein

 Fig. 5: shows the HCV cDNA sequence in clone 36, the segment that overlaps the NANBV cDNA of clone 81, and those in FIG

Clone 36 encoded polypeptide sequence

 Fig. 6: shows the combined open reading frames (ORF) of HCV cDNAs in clones 36 and 81 and the one encoded therein

Polypeptide.

Figure 7: shows the HCV cDNA sequence in clone 32, the segment that overlaps clone 81, and the polypeptide encoded therein. Figure 8: shows the HCV cDNA sequence in clone 35, the segment that overlaps clone 36 and the polypeptide encoded therein. Figure 9: shows the combined open reading frames (ORF) of HCV cDNAs in clones 35, 36, 81 and 32 and the amino acid coded polypeptide.

Figure 10: shows the HCV cDNA sequence in clone 37b, the segment that overlaps clone 35, and the polypeptide encoded therein. Fig. 11: shows the HCV cDNA sequence in clone 33b, the segment that overlaps clone 32 and the polypeptide encoded therein. Figure 12: shows the HCV cDNA sequence in clone 40b, the segment that overlaps clone 37b, and the polypeptide encoded therein. Fig. 13: shows the HCV cDNA sequence in clone 25c, the segment that overlaps clone 33b, and the polypeptide encoded therein.

Fig. 14: shows the nucleotide sequence and the open reading frame (ORF) polypeptide encoded therein by the

HCV cDNAs in clones 40b, 37b, 35, 36, 81, 32, 33b and 25c. Fig. 15: shows the HCV cDNA sequence in clone 33c, the segment that overlaps clones 40b and 33c and the amino acids encoded in it.

Figure 16: shows the HCV cDNA sequence in clone 8h, the segment that overlaps clone 33c and the amino acids encoded therein. Figure 17: shows the HCV cDNA sequence in clone 7e, the segment that overlaps clone 8h and the amino acids encoded therein. Fig. 18: shows the HCV cDNA sequence in clone 14c, the segment that overlaps clone 25c and encodes it

Amino acids.

Figure 19: shows the HCV cDNA sequence in clone 8f, the segment that overlaps clone 14c and the amino acids encoded therein. Fig. 20: shows the HCV cDNA sequence in clone 33f, the segment that overlaps clone 8f and the amino acids encoded therein. Fig. 21: shows the HCV cDNA sequence in clone 33g, the segment that overlaps clone 33f and the amino acids encoded therein. Flg. Figure 22: shows the HCV cDNA sequence in clone 7f, the segment that overlaps and encodes the sequence in clone 7e

amino acids

 Fig. 23: shows the HCV cDNA sequence in clone 11b, the segment that overlaps and encodes the sequence in clone 7f

amino acids

 Figure 24: shows the HCV cDNA sequence in clone 14 i, the segment that overlaps and encodes the sequence in clone 11b

amino acids

 Fig. 25: shows the HCV cDNA sequence in clone 39c, the segment that overlaps the sequence in clone 33g and that in it

coded amino acids

 Figure 26: shows a composite HCV cDNA sequence derived from aligned cDNAs in clones 14i, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32 , 33b, 25c, 14c, 8f, 33f and 33g were derived; continues in the

extended ORF shown in the deduced sequence encoded amino acid sequence of the polypeptide. Figure 27: shows the sequence of the HCV cDNA in clone 12f, the segment overlaps the clone 14i and encodes it

amino acids

 Fig. 28: shows the sequence of the HCV cDNA in clone 35f, the segment that overlaps clone 39c and encodes it

amino acids

 Fig. 29: shows the sequence of HCV cDNA in clone 19g, the segment that overlaps Cfon 35f and encodes it

Amino acids.

Fig. 30: shows the sequence of clone 26g, the segment that overlaps clone 19g and the amino acids encoded therein. Fig. 31: shows the sequence of clone 15e, the segment that overlaps clone 26g and the amino acids encoded therein. Fig. 32: shows the sequence in a composite cDNA obtained by aligning clones 12f to 15e in FIG

derived from the 5'-3 'direction and the amino acids encoded in the continuous ORF. Fig. 33: shows a photograph of Western blots of a fusion protein, SOD-NAND ₅ -II. with chimpanzee serum out with BB-NANB,

HAV and HBV infected chimpanzees

Fig. 34: shows a photograph of Western blots of a fusion protein, SOD-NANB ₅ -I, with serum from NANBV, HAV and HBV -infected humans and from controls.

Fig. 35: shows in a map the significant features of the vector pAB24. Fig. 36: shows the putative amino acid sequence of the carboxy terminus of the fusion polypeptide C100-3 and the nucleotide sequence coding for it.

Figure 37 A: is a photograph of a Coomassie blue-stained polyacrylamide gel that identifies C100-3 expressed in yeast. Figure 37B: shows a Western blot of C100-3 with serum from a human infected with NANBV. Fig. 38: shows an autoradiogram of a Northern blot of RNA infected from the liver of one with BB-NANBV

Chimpanzee and probed with BB-NANBV cDNA from clone 81. Fig. 39: shows an autoradiogram of NANBV nucleic acid treated with RNase A or DNase I and with

BB-NANBV cDNA from clone 81 was probed

Fig. 40: shows an autoradiogram of nucleic acids extracted from NAN particles which are annealed with AnU-NANB _5. ,

infected plasma and labeled with ³² P-labeled NANBV-cDN /. from clone 81 were probed. Figure 41: shows autoradiograms of filters containing isolated NANBV nucleic acids labeled with ³² P-labeled plus

and minus-stranded DNA probes derived from NANBV cDNA in clone 81 were probed. Fig. 42: shows the homologies between a polypeptide encoded in HCV cDNA and an NS protein from the

Dengue flavivirus

 Figure 43: shows a histogram of the distribution of HCV infection in samples as determined by

ELISA screening

 Fig. 44: shows a histogram of the distribution of HCV infection in samples by means of two configurations of

Immunoglobulin-enzyme conjugate in an ELISA assay

 Fig. 45: shows the sequences in a starter mixture derived from a conserved sequence in NSI of flaviviruses.

Ways to Erftndungsgemsßen execution I. Definitions The term hepatitis C virus has been used by scientists working in this field for a previously unknown Pathogens reserved by NANBH. Accordingly, the term hepatitis C virus (HCV) refers to as used herein Meaning to a pathogen causing NANBH formerly known as NANBV and / or BB-NANBV. The terms HCV, NANBV and BB-NANBV are used interchangeably here. As an extension of this terminology is that by the HCV

disease, formerly known as NANB Hepatitis (NANBH), now called Hepatitis C. The terms NANBH and

Hepatitis C can be used interchangeably here. The term "HCV" as used herein denotes a virus species that causes NANBH, as well as debilitated Strains or incomplete, interfering (defective interfering) particles derived therefrom. As shown below

the HCV genome consists of RNA. It is known that RNA-containing viruses have relatively high, spontaneous mutation rates in the range

from 10 ~ ³ to 10 ~ ⁴ per nucleotide (Fields & Knlpe {1986]). There are therefore a variety of strains within the HCV species described below. The compositions and methods described herein allow for propagation, identification. Detection and isolation of the various related strains. In addition, they also allow for the diagnosis and production of vaccines for the various strains, and are useful for pharmacological antiviral miVHn screening assays by inhibiting the replication of HCV. The information provided herein, although derived from an HCV strain, hereafter referred to as CDC / HCV1, is sufficient for a virus taxonist to identify other strains belonging to these species. As described herein, we have discovered that the HCV is a flavivirus or flavivirus. Morphology and composition of flaviviral particles are known and discussed in Brinton (1986). In terms of morphology, the flaviviruses, generally speaking, contain in the center a nucleocapsid surrounded by a lipid-containing bilayer. The virions are spherical and have a diameter of about 40-50 nm. Their nuclei have a diameter of about 25-30nm. On the outside of the virion shell are projections with a length of about 5-1 nm with central knobs of about 2 nm in diameter.

The HCV encodes an epitope which is immunologically identifiable with an epitope in the HCV genome from which the cDNAs described herein have been derived, which preferably encodes epitope fst in a cDNA described herein. Compared to other known flaviviruses, the epitope is unique to HCV. This uniqueness of the epitope can be determined by its immunological reactivity with the HCV and its lack of immunological reactivity with other flavivirus species. Methods for determining immunological reactivity are known in the art, for example by radioimmunoassay, by ELISA, by hemagglutination, several examples of suitable techniques for assays being presented herein.

In addition to the above, the following parameters, either alone or in combination, are applicable to the identification of a strain as HCV. Since HCV strains are evolutionarily related, it is expected that the total homology of genomes at nucleotide level will be at least about 40%, preferably about 60% or more, and more preferably about 80% or more. In addition, the corresponding contiguous sequences are expected to be at least about 13 nucleotides in length. The correspondence between the putative genomic sequence of the HCV strain and the CDC / CH1 HCV cDNA sequence can be determined by techniques known in the art. For example, they can be determined by a direct comparison of the sequence information of the polynucleotide from the putative HCV and the HCV cDNA sequence (s) described herein. For example, they may also be determined by hybridizing the polynucleotides under conditions where stable duplexes are formed between homologous regions (e.g., those that would be used prior to SpDigestion), then digesting with single-stranded (n) specific nuclease (s) and then sizing the digested fragments.

Because of the evolutionary relatedness of the HCV strains, the putative HCV strains are identifiable by their homology at the polypeptide level. Generally speaking, over 40%, preferably over about 60%, and more preferably over about 80% of the HCV strains are homologous at the polypeptide level. The techniques for determining amino acid sequence homology are known in the art. The amino acid sequence can for example be determined directly and compared with the sequences presented here. For example, the nucleotide sequence of the genome of the putative HCV may also be determined (usually by a cDNA intermediate); the amino acid sequence encoded therein can be determined and the corresponding regions can be compared.

As used herein, a polynucleotide "derived from" a designated sequence, e.g., the HCV cDNA, particularly those shown in Figures 1-32, or from an HCV genome, refers to a polynucleotide sequence which is composed of a sequence of at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and most preferably at least about 15-20 nucleotides corresponding to, ie homologous or complementary to, a region of the designated nucleotide sequence Preferably, the sequence of the region from which the polynucleotide is derived is homologous or complementary to a sequence unique to an HCV genome. Whether a sequence is unique to the HCV genome or not can be understood by the For example, the sequence may be compared to sequences in databases, such as Genebank, to determine if it is present in the uninfected host or in other organisms. The sequence may also be linked to known sequences of other viral agents, including those known to induce hepatitis, such as those described in U.S. Pat. As HAV, HBV and HDV, and compared with other members of Flaviviridae. The correspondence or disagreement of the deduced sequence with other sequences can also be detected by hybridization under appropriate severe conditions. Hybridization techniques for determining the complementarity of the nucleic acid sequences are known in the art and are discussed below. See, for example, Maniatis and co-workers (1982). Furthermore, malformations of duplex polynucleotides resulting from hybridization may be induced by known techniques, such as. B. digestion with a nuclease such as S1, which digests specifically single-stranded regions in duplex polynucleotides. Regions from which typical DNA sequences can be "derived" include, but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and / or untranslated regions.

The derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but may be expressed in various ways, including, but not limited to, e.g. Chemical synthesis or DNA replication or reverse transcription or transcription based on the information given by the base sequence in the region (s) from which the polynucleotide is derived. Furthermore, combinations of regions corresponding to those of the designated sequence may be modified in a manner known in the art to suit their intended purpose. Likewise, a polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence, e.g., the sequences shown in Figures 1-32, or from an HCV genome, refers to a polypeptide having an amino acid sequence which is identical to that of a polypeptide encoded in the sequence or to a part thereof, the part consisting of at least 3 to 5 amino acids, preferably at least 8 to 10 amino acids and more preferably at least 11 to 15 amino acids, or which is identifiable immunologically with a polypeptide encoded in the sequence.

A recombinant or derived peptide is not necessarily of a designated nucleic acid sequence, e.g. the sequences shown in Figures 1-32, or translated from an HCV genome, it can be generated in a variety of ways,

including e.g. chemical synthesis or expression of a recombinant expression system, or isolation from mutant HCV.

The term "recombinant polynucleotide" as used herein refers to a pelynucleotide of genomic, cDNA, semisynthetic or synthetic origin which, by virtue of its origin or manipulation (1), is not linked to all or part of the polynucleotide with which it is linked is linked in nature or in the form of a library, and / or (2) is linked to a different polynucleotide than it would be in nature.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, which refers only to the primary structure of the molecule, and thus includes both double and single stranded DNA such as It also includes, for example, by methylation and / or capping modified and unmodified forms of the polynucleotides.

As used herein, the term "HCV containing a sequence corresponding to a cDNA" means that the HCV contains a polynucleotide sequence that is homologous or complementary to a sequence in the designated DNA, the degree of homology or complementarity to the cDNA being approximately The corresponding sequences will be at least about 70 nucleotides in length, preferably at least about 80 nucleotides, and more preferably at least 90 nucleotides in length The HCV sequence and cDNA can be determined by techniques known in the art, such as by direct comparison of the sequenced material with the described cDNAs or by hybridization and digestion with single-stranded nucleases and subsequent determination of the digested fragments The term "purified from virus nucleotide "refers to one HCV genome or fragment thereof that is substantially free of, i. that is, it contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of polypeptides to which the viral polynucleotide is naturally linked. The techniques for purifying the viral polynucleotides from the viral particles are known in the art and include e.g. disruption entails particles with a chaotropic agent and separation of the polynucleotide (s) and polypeptide (s) by ion exchange chromatography, affinity chromatography, and sedimentation by density. The term "purified virus polypeptide" refers to a HCV polypeptide or fragment thereof that is substantially free of, ie, contains less than 50%, preferably less than about 70%, and more preferably less than about 90%, of cellular components, The techniques for purifying the virus polypeptides are well known in the art, and examples of these techniques are discussed below: "Recombinant host cells", "host zohan", "ΙΙβΙΙθη", "cell lines", "cell cords" and other such terms, which refer to microorganisms or higher eukaryotic cell lines taken as unicellular units in culture, refer to cells which may be or have been used as recipients for a recombinant vector or other transfer DNA and include also the progeny of the original cell that was transfected. It is understood that the progeny of a single pea cell need not necessarily be identical in morphology or as genome or total DNA complement due to a random or intended mutation with the original parental cell. Progeny of the parental cell, which is sufficiently similar to the parental cell and characterized by relevant characteristics, such as the presence of a nucleotide sequence encoding a desired peptide, is included in the progeny as defined by this definition and is encompassed by the above terms. A "replicon" is any genetic element, ie, a plasmid, a chromosome, a virus that behaves as an autonomous unit of polynucleotide replication within a cell, ie that it is capable of replication under its own control. A "vector" is a replicon to which another polynucleotide segment is linked so as to effect the replication and / or expression of the attached segment.

"Control sequence" refers to polynucleotide sequences necessary to effect the expression of the coding sequences to which they are ligated The nature of these control sequences differs depending on the host organism; in prokaryotes, these control sequences are generally promoters, ribosome menthols and terminators; In eukaryotes, these control sequences are promoters, terminators, and in some instances enhancers The term "control sequences" is intended to include at least all components whose presence is necessary for expression, and may also include additional components whose presence is beneficial, such as e.g. B. Leadersequences <.en. "Operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting their function in the intended manner. A control sequence "operably linked" to a coding sequence is on ligated such that expression of the coding sequence is achieved under conditions that are compatible with the control sequences. An "open reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide, which region may be part of a coding sequence or a complete coding sequence.

A "coding sequence" is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when under the control of appropriate regulatory sequences The boundaries of the coding sequence are determined by a translation start codon at the 5 'terminus and a translation stop codon at the A coding sequence may include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. "Immunologically identifiable with / as" refers to the presence of epitope (s) and polypeptide (polypeptides), those present in the designated polypeptide (s), usually HCV proteins, are unique and unique to them. Immunological identity can be determined by antibody binding and / or binding competition; these techniques are well known to those skilled in the art and are also illustrated below. The uniqueness of an epitope can also by computer research in well-known databases, ι. Genebank, for the polynucleotide sequences encoding the epitope, as well as by amino acid sequence comparisons with other known proteins. As used herein, "epitope" refers to an antigenic determinant of a polypeptide: an epitope could comprise 3 amino acids in a spatial arrangement unique to the epitope, generally an epitope consisting of at least 5 such amino acids, and more generally, at least 8-10 such amino acids The methods for determining the spatial arrangement of the amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to the fact that the antibody recognizes a specific epitope contained in the polypeptide. The immunological reactivity can be determined by the antibody binding, in particular by the antibody Kinetics of antibody binding and / or by

Binding competition by targeting a known polypeptide or polypeptide (s) containing an epitope

the antibody (s) being targeted is used as competitor (s). The techniques for determining if a

Polypeptide immunologically reactive with an antibody are known in the art. As used herein, the term "HCV-containing immunogenic polypeptide" includes a natural one

occurring HCV polypeptides or fragments thereof as well as polypeptides made by other means, such as chemical synthesis, or expression of the polypeptide in a recombinant organism.

The term "polypeptide" refers to a molecular chain of amino acids and not to a specific length of the product,

so that peptides, oligopeptides and proteins are included in the definition of the polypeptide. The term also does not refer to the modifications of the polypeptide after expression, such as glycosylations, acetylations,

Phosphorylations and the like.

"Transformation" as used herein refers to the insertion of an exogenous polynucleotide into a

Host cell regardless of the method used for the insertion, such as direct uptake, transduction or

"F-mating." The exogenous polynucleotide may be retained as a non-integrated vector, such as a plasmid, or may be integrated into the host genome in some other manner.

"Treatment" in the sense used refers to prophylaxis and / or therapy.

An "individual" as used herein refers to vertebrates, particularly members of mammalian species and

includes, but is not limited to, pets, sport animals, primates and humans. In the used here

Meaning, the "plus strand" of a nucleic acid contains the sequence encoding the polypeptide. The "minus strand" contains a sequence that is complementary to that of the "plus strand". As used herein, a "positive-stranded genome" is a virus of one in which the genome, whether RNA or DNA,

is single stranded and codes for a virus polypeptide / virus polypeptide. Examples of positive-stranded RNA viruses are Togaviridao,

Coronaviridae, Reteroviridae, Picornaviridae and Caliciviradae. Also included are the Flaviviridae, formerly known as Togaviridae were classified. See Fields & Knipe (1986). As used herein, "antibody-containing body component" refers to a component of the body

of an individual who is a source of the antibodies of interest. Antibody-containing body components are known in the art and include e.g. Plasma, serum, cerebrospinal fluid, lymph, the outer sections of the respiratory,

Digestive and genitourinary tracts, tears, saliva, milk, white blood cells and myeloma cells. As used herein, "purified HCV preparation" refers to an HCV preparation derived from the cellular Ingredients with which the virus is normally associated and from other virus species present in the infected tissue

could occur, was isolated. The techniques for isolating the viruses are known to those skilled in the art and include e.g. Centrifugation and affinity chromatography; a process for producing purified HCV will be explained below.

II. Description of the invention

Unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are conventional in the art, are given for carrying out the present invention. These techniques are explained in detail in the literature. See, e.g. Maniatis, Fitsch & Sambrock, MOLECULAR CLONING; A LABORATORY MANUAL (Molecular Cloning, a Laboratory Manual) (1982); DNA CLONING, VOLUMES 1 AND II (DNA Cloning, Volumes I and II) (Editor D.N. GIover, 1985); OLiGONUCLEOTIDE SYNTHESIS (Oligonucleotide Synthesis) (Editor M.J. Gait, 1984); NUCLEIC ACID HYBRIDIZATION (Nucleic Acid Hybridization) (Editor B.D. Harnes & S.J. Higgins, 1984); TRANSCRIPTION AND TRANSLATION (Transcription and Translation) (Editors B.D. Harnes & S.J.Higgins, 1984); ANIMAL CELL CULTURE (Animal Cell Culture) (Editor R. I. Frishney, 1986); IMMOBILIZED CELLS AND ENZYMES (Immobilized Cells and Enzyme) (IRL Press, 1986); B. Perbai, A PRACTICAL GUIDE TO MOLECUUR CLONING (A Practical Guide to Molecular Cloning) (1984); the series: METHODS IN ENZYMOLOGY (Methods of Enzymology) (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMALIAN CELLS (mammalian cell gene transfer vectors) (Editor JH Miller and MPCalos, 1987, Coldspark Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Methods of Enzymology, Volume 154 and Volume 155 Wu and Grossman and Wu respectively), editors Mayer and Walker (1987), IMMUNOCHEMICAL METHODS IN CELLAND MOLECULAR BIOLOGY (Academic Press, London), Seiles (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE (Protein Purification: Principles and Practice), Second Edition, (Springer Verlag, NY) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES HV (Handbook of Experimental Immunology, Volume HV), (publisher DMWeir and CC Blackwell, 1986).

All patents, patent applications and publications mentioned hereinbefore and hereinafter are incorporated herein by reference.

The useful materials and processes of the present invention are made possible by providing a family of closely homologous nucleotide sequences isolated from a cDNA library derived from nucleic acid sequences present in the plasma of an HCV-infected chimpanzee. This family of nucleotide sequences does not originate in either humans or chimpanzees because it does not hybridize to human or chimpanzee genomic DNA from uninfected individuals, and nucleotides of this family of sequences hybridize only to the liver and plasma of chimpanzees with HCV infection occur and because the sequence is not present on the Genebank. In addition, this family of sequences shows no significant homology to sequences contained in the HBV genome.

The seqmnz of a member of the family contained in clone 5-1-1 has a continuous open reading frame (ORF), which codes for a polypeptide of about 50 amino acids. The sera from HCV-infected humans contain antibodies that bind to this polypeptide, while the sera from uninfected humans do not contain antibodies that bind to this polypeptide. Finally, while the sera from uninfected chimpanzees do not contain antibodies that bind to this polypeptide, the antibodies in chimpanzees are induced after acute NANBH infection. Moreover, antibodies that bind to this polypeptide are not found in chimpanzees and humans infected with HAV and HBV

demonstrated. According to these criteria, the sequence is a cDNA against a viral sequence, which virus causes or is linked to NANBH; this cDNA sequence is shown in FIG. As will be discussed below, the cDNA sequence in clone 5-1-1 differs from that of the other isolated cDNAs in that it contains 28 extra base pairs. A composition of other identified members of the cDNA family isolated by using as a probe a synthetic sequence corresponding to a fragment of the cDNA in clone 6-1-1 is shown in Fig. 3. E = A member of the cDNA family isolated by using a synthetic sequence derived from the cDNA in clone 81 is described in Flg. 15, and a composition of this sequence with that of clone 81 is shown in FIG. Other members of the cDNA family, including those described in clones 17f, 141, 11b, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f , 35f, 19g, 26g, 15c, 33g and 39c are described in Section IV.A. described. A composition of the cDNAs in these clones is described in Section IV.A.9. described and shown in Fig. 39. The composite cDNA shows that it contains a continuous open reading frame and thus encodes a polyprotein. These data are consistent with the presumption discussed below that HCV is a flavivirus or fiaviartiges virus.

The availability of this family of cDNAs shown in Figures 1-32 permits the construction of DNA probes and polypeptides useful for the diagnosis of NANBH as a result of HCV infection and screening assays in blood donors as well as donated blood and blood products an infection are useful. For example, it is possible to synthesize from the sequences DNA oligomers of about 8-10 nucleotides or more which are useful as hybridizing probes to detect the presence of the viral gene in, for example, sera from individuals who may harbor the virus, or the like are useful for screening spondial blood for the forerunner of the virus. The family of cDNA sequences also allow the design and generation of HCV-specific polypeptides useful as diagnostic agents for the presence of antibodies raised during NANBH. Antibodies to purified polypeptides derived from the cDNAs can also be used to detect viral antigens in infected individuals and in blood.

Knowledge of these cDNA sequences also permits the design and production of polypeptides useful as vaccines against HCV and also for the production of antibodies which, in turn, can be used to protect against this disease and / or for therapy with HCV infected individuals can be used. In addition, the family of cDNA sequences allows further characterization of the HCV genome. The polynucleotide probes derived from these sequences can be used to screen cDNA libraries for additional overlapping cDNA sequences, which in turn can be used to obtain even more overlapping sequences. Unless the genome is segmented and the segments lack common sequences, this technique can be used to obtain the sequence of the entire genome. However, if the genome is segmented, can other segments of the genome be replaced by repetition? Lambda gt11 serological screening method for isolating the cDNA clones described herein or alternatively by isolating the genome from purified HCV particles.

The family of cDNA sequences and the polypeptides derived from these sequences as well as the antibodies directed against these polypeptides are useful in the isolation and identification of the BB-NANBV pathogen (s). For example, antibodies directed against HCV epitopes contained in polypeptides derived from the cDNAs can be used in processes based on affinity chromatography to isolate the virus. Alternatively, the antibodies can be used to identify virus particles isolated by other techniques. The viral antigens and the genomic material within the isolated virus particles can then be further characterized. The information obtained from further sequencing of the HCV genome (s), as well as from further characterization of HCV antigens and characterization of the genome, allows the design and synthesis of additional probes and polypeptides and antibodies useful for diagnosis, Prevention and treatment of HCV-induced NANBH and screening of infected blood and blood-like products. The availability of probes for HCV, including antigens and antibodies, as well as polynucleotides derived from the genome from which the family of cDNAs was derived, also allow the development of tissue culture systems which are of great benefit in brightening the biology of HCV become. This, in turn, may lead to the development of new treatments based on antiviral components that preferentially inhibit the replication of or infection by the HCV.

The method used to identify and isolate the pathogen of NANBH is novel and can be applied to the identification and / or isolation of previously uncharacterised pathogens containing a genome and those associated with a variety of diseases, including those caused by viruses, Viroids, bacteria, fungi and parasites are induced. In this method, a cDNA library was created from the nucleic acids present in the infected tissue from an infected individual. The library was created in a vector that allowed expression of the polypeptides encoded in the cDNA. Clones from host cells containing the vector expressing an immunologically reactive fragment of a polypeptide of the pathogen were selected by immunological screening of the library's expression products with an antibody-containing body component from another individual previously infected with the putative pathogen. The steps of the immunological screening procedure included the interaction of the expression products of the cDNA-containing vectors with the antibody-containing body component of a second infected individual and the detection of the formation of antibody-antigen complexes between the expression product (s) and antibodies of the second infected one Individual. The isolated clones are further subjected to immunological screening procedures by interacting their expression products with the antibody-containing body components of the other suspected virus-infected individuals and with control individuals uninfected with the putative agent and the formation of antigen-antibody Complexes with antibodies from the infected individuals has been demonstrated; and the cDNA-containing vectors encoding polypeptides that are immunologically isolated with antibodies from infected individuals and from individuals suspected of being infected with the agent, but not with control individuals. The infected individuals used for construction of the cDNA library and for immunological screening need not be of the same species. The cDNAs isolated as a result of this method and their expression products and the antibodies directed against the expression products are useful for the characterization and / or theimplementation of the etiological agent. As described in more detail below, this method has been used successfully to isolate a family of cDNAs derived from the HCV genome.

HA. Preparation of the cDNA sequence Serum collected from a chimpanzee with chronic HCV infection that has a high virus titer, that is

at least one 10 * chimpanzee infectious dose / ml (CID / ml) was used to isolate viral particles; the nucleic acids isolated from these particles were used as templates in the construction of a cDNA library to the viral genome. The procedures for isolating putative HVC particles and for constructing the cDNA library in Lambdagt 11 are described in Section IV.A.1. discussed. Lambda gt 11 is a vector that has been specifically designed to generate inserted cDNAs

Fueionspolypeptide with beta-galactosidase to express and a large number of recombinant phage with specific Antisera may form a certain antigen. The lambda gt 11 cDNA library, made from a

cDNA pool containing approximately 200-base-cDNA approximately 200 basepairs of cDNA was screened for coded epitopes that could specifically bind sera derived from previously NANB-Hepatitta-affected patients. Huynh, T.V.

et al. (1985). There were about 10 ^е phage screened, and five positive phages were identified, purified and r.nschließond for specificity of binding to sera from different humans and chimpanzees with zuvof

KCV agent were tested. One of the phages, 5-1-1, bound 6 of the 8 human sera tested. These Binding appeared to be selective for sera from patients previously exposed to NANB hepatitis, as 7 normal Blood donor sera did not have such a binding. The sequence of the cDNA in the recombinant phage 5-1-1 was determined and shown in FIG. The cloned through this

cDNA encoded polypeptide that is in the same translational pattern as the N-terminal beta galactosidase component of the

Fusion polypeptide is shown above the nucleotide sequence. Therefore, this translational open reading frame encodes an epitope

or epitopes that are specifically recognized by the sera of patients with NANB hepatitis infections.

The availability of the cDNA in recombinant phage 5-1-1 allowed the isolation of other clones containing the additional segments

and / or alternative segments of the cDNA for the viral genome. The lambda gt 11 cDNA described above

Library was constructed using a synthetic polynucleotide derived from the sequence of clonalite 5-1-1 cDNA

screened. This screen revealed three other clones identified as 81,1-2 and 91; the cDNAs contained in these clones were sequenced. See section IV.A.3. and IV.A.4. The homologies between the four independent clones are shown in Figure 2, where the homologies are indicated by vertical lines. Nucleoiidsequenzen are exclusively in the

Clones 5-1-1,81 and 91 are present and are indicated by small letters. The cloned cDNAs present in the recombinant phages in clones 5-1-1,81,1-2 are highly homologous and

differ only in two regions. First, nucleotide number 67 in clone 1-2 is one thymidine, while the other three

Clones in this position contain a cytidine residue. However, this substitution does not change the nature of the coded Amino acid. The second difference between the clones is that clone 5-1-1 contains 28 base pairs at its 5 'terminus, which in

other clones are not present. This additional sequence may be a 5'-terminal cloned artifact; 5'-terminally cloned artifacts are generally observed in the products of cDNA methods.

Synthetic sequences derived from the 5 'region and З' region of the HCV cDNA in clone 81 were used for screening and Isolating cDNAs from the lambda gt 11-NANBV cDNA library using the clone 81 cDNA (Section IV.A.5.).

overlap. The sequences of the resulting cDNAs present in clone 36 and clone 32, respectively, are shown in Fig. 5 and Fig. 7.

A synthetic polynucleotide based on the 5 'region of clone 36 was also used to screen and isolate

cDNAs from the lambda gt 11 NANBV cDNA library overlapping the clone 36 cDNA (Section IV.A.8.). Purified clone of recombinant phage-containing cDNA that hybridized to the synthetic polynucleotide probe was designated clone 35 and the NANBV cDNA sequence contained in this clone is shown in FIG.

By using the method for isolating overlapping cDNA sequences, additional upstream and

down tram-HCV-cDNA sequences. The isolation of these clones will be discussed later in Section IV.A. described.

Analysis of the nucleotide sequences of HCV cDNAs encoded within the isolated clones show that the

composite cDNA contains a long continuous open reading frame. Figure 26 shows the sequence of the composite cDNA from these clones together with the putative HCV polypeptide encoded therein.

The description of the method for obtaining the cDNA sequences is mostly of historical interest. The

resulting sequences (and their complements) are provided herein, and the sequences, or any part thereof, may be synthesized using synthetic methods or by a combination of the synthetic methods

Partial sequence restoration using similar methods to those described herein. Lambda gt 11 strains replicated from the HCV cDNA library and from clones Б-1-1,81,1-2 and 91, respectively

according to the provisions of the Budapest Treaty at the American Type Culture Collection (ATCC), 12301 Parklawn

Dr., Rockville, Maryland 20852 and identified with the following accession numbers.

Lambda-gt 11 ATCC-No. deposit date HCV cDNA library 40394 Dec. 1, 1987 Clone 81 40388 Nov. 17, 1987 Clone 91 40389 Nov. 17, 1987 Clone 1-2 40390 Nov. 17, 1987 Clone 5-1-1 40391 I8.N0V 1987

In granting and issuing this application as a United States patent, all restrictions on the availability of the deposit are irrevocably removed; Access to the designated deposits will be made possible during the pendency of the above application to the one designated by the patent agent authorized to do so under 37 CRF 1.14 and 35 USC 1.22. In addition, the designated deposits will be held for a period of thirty (30) years from the date of deposit, or for five (5) years after the last filing request; or over the life of the US patent, whichever is longer. These deposits and other deposited materials referred to herein are intended for convenience only and are not required to practice the present invention as the description proceeds. The HCV cDNA sequences in all of the deposited materials are incorporated herein by reference.

The above description, which concerns the "walking" of a genome by isolating overlapping cDNA sequences from the HCV lambda gt 11 library, provides a method by which cDNAs corresponding to the entire HCV genome can be isolated. However, it will be understood by those skilled in the art that the information provided herein enables other methods for the isolation of these cDNAs Some of these methods are described in Section IV.A., infra.

II.B. Preparation of viral polypeptides and fragments

The availability of cDNA sequences, either those isolated by use of the cDNA sequences shown in Figures 1 to 26, as set forth below, as well as the cDNA sequences in these figures, permits the construction of the expression vectors which encode the active site regions of the polypeptides encoded in each strand. These antigenically active regions may be selected from the coating or envelope antigens or from core antigens including e.g. Polynucleotide binding proteins, polynucleotide polymerase (s) and other viral proteins required for the replication and / or assembly of the virus particles. The fragments encoding the desired polypeptides are derived from cDNA clones using conventional restriction digestion or synthetic methods and ligated into vectors, e.g. B. contain parts of fusion sequences such as beta-galactosidase or superoxide dismutase (SOD), preferably SOD. Methods and vectors useful for the production of polypeptides containing fusion sequences of SOD are described in EPO Pub. 0196056, published on

October 10, 1986 described. Vectors encoding fusion polypeptides of SOD and HCV polypeptides, ie NANB ₆ -M, NANBg ₁ and C100-3, encoded in a composition of HCV cDNAs are described in Sections IV.B.1, IV.B .2 and IV.B.4 described. Any desired portion of the HCV cDNA containing an open reading frame in each of the two coding strands may be expressed as a recombinant polypeptide, e.g. B. as mature or fusion protein, are obtained; alternatively, a polypeptide encoded in the cDNA can be prepared by chemical synthesis.

The cDNA encoding the desired polypeptide, whether fused or mature, and whether or not a signal rate that allows secretion, may be ligated into expression vectors appropriate for each host. Both eukaryotic and prokaryotic host systems are currently used in the formation of recombinant polypeptides, and a summary of some of the more common control systems and host cell lines is given in Section Ш.A., below. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent necessary for its intended use. The detergent can be prepared by methods known in the art, e.g. Salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, etc. For the variety of methods for the purification of Froteinen see z. Such polypeptides may be used as diagnoses, or those which cause neutralization of antibodies may be formulated into vaccines The antibodies to the polypeptides may also be used as diagnoses or for diagnosis In addition, as discussed later in Section IU, the antibodies to these polypeptides are useful for isolating and identifying the HCV particles.

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

11. C. Preparation of Antigenic Polypeptides and Conjugation with Carriers

An antigenic region of a polypeptide is generally relatively small, typically 8 to 10 amino acids or less in length. Fragments of only 5 amino acids can characterize an antigenic region. These segments may correspond to regions of the HCV antigen. Accordingly, using the cDNAs of HCV as a base, DIVAs encoding short segments of HCV polypeptides can be recombinantly expressed either as fusion proteins or as isolated polypeptides. In addition, short amino acid sequences can be conveniently obtained by chemical synthesis. For example, in the case where the synthesized polypeptide is correctly configured to provide the correct epitope but is too small to be immunogenic, it can be bound to a suitable carrier.

A number of techniques for obtaining such compounds are known to those skilled in the art including the formation of disulfide compounds using N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and succinimidyl 4- (N-maleimidomethylcyclohexane-i-carboxylate (U.S. SMCC) purchased from the Pierce Company, Rockford, Illinois. (If the peptide does not have a sulfhydryl group, this can be provided by addition of a cysteine residue.) These reagents cause a disulfide bond between them and the peptide cysteine residues on one Protein as well as an amide bond by the epsilon-amino to one lysine, or other free amino groups in others The diversity of such disulphide / amide-forming agents is known, see, for example, Imi. ^ And Rev. (1982) 62: 185 Bifunctional coupling agents tend to form a thioether rather than a disulfide compound, Many of these thioether-forming agents are available commercially and include, but are not limited to, the following cosame esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-im-maleimidomethyl) -cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combination with succinimide or i-hydroxyl-nitro-sulfonic acid sodium salt. The above list is not intended to be exhaustive, and modifications of said compounds may, of course, be used.

Any carrier that does not itself induce the generation of host-damaging antibodies can be used. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; Polysaccharides such as latex-functionalized sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine and the like; amino acid copolymers; and inactive virus particles, see e.g. B. Section II.D. Particularly useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, egg albumin, tetanus toxoid, and other proteins well known to those skilled in the art.

II.D. Preparation of HCV Epltope-containing Hybrldpartlkellmmunogenen

The immunogenicity of the epitopes of HCV may also be improved by producing them in mammalian or yeast systems which fuse and assemble with particle-forming proteins, e.g. that is associated with hepatitis B surface antigen. The constructs in which the NANBV epitope is directly linked to particle-forming protein coding sequences produce hybrids that are immunogenic with respect to the HCV epitope. In addition, all of the vectors produced contain HBV specific epitopes with different degrees of immunogenicity such. For example, the pre-S peptide. Thus are

Particle-forming protein particles comprising HCV sequences immunizing with respect to HCV and HBV. The hepatitis surface antigen (HBSAg) was expressed in S. cerevisiae (Valenzuela et al. [1982]), e.g. also formed and assembled in mammalian cells (Valenzuela, P. et al. [1984]). The formation of such particles revealed that the immunogenicity of the monomer subunit is enhanced. The constructs may also contain the immunodominant epitope of HBSAg comprising 55 amino acids of the presurface (pre-S) region, Neurath et al. (1985). Constructs of the pre-S-HBSAg particles expressible in yeast are disclosed in EPO 174444, published March 19, 1986; Hybrids including heterologous viral sequences for yeast expression are disclosed in EPO 175261, published March 26, 1966. Both applications are hereby assigned to said assignee and are incorporated herein by reference. These constructs can also be expressed in mammalian cells, such as in Chinese hamster ovary (CHO) cells, using an SV40 dihydrofolate reductase vector (Michelle et al. (1984)). In addition, portions of the particle-forming protein-encoding sequence may be exchanged for HCV epitope-encoding codons. In this replacement regions that are not required to mediate aggregation of the moieties to form immunizing particles in yeast or mammals can be eradicated, thereby eliminating additional HBV antigenic sites competing with the HCV epitope.

U.E. Production of vaccines

Vaccines may be prepared from one or more immunogenic polypeptides derived from an HCV cDNA as well as from the cDNA sequences shown in Figures 1 to 32 or from the HCV genome to which they correspond. The homology observed between HCV and flaviviruses provides information concerning the polypeptides, which are most likely to be most effective as vaccines, as well as the rogues of the genome in which they are encoded. The general structure of the flavivirus genome is described in Rice et al. (1986). It is believed that the flavivirus genomic RNA is the only virus-specific mRNA species and will translate into three viral structural proteins translation, i. C, M and E as well as two large non-structural proteins, NV4 and NV5, and a complex group of smaller non-structural proteins. It is known that the major neutralization epitopes for flaviviruses are in the E (envelope) protein (Roehring [1986]). The region encoding the corresponding HCV E gene and polypeptide can be predicted from homology to the flavivirus. Thus, recombinant polypeptides may contain extensive HCV-E vaccine epitopes. These polypeptides can be expressed in bacterial, yeast or mammalian cells, or optionally isolated from viral preparations. It is also believed that the other structural proteins also contain epitopes that produce protective anti-HCV antibodies. Thus, in HCV vaccines, polypeptides may also be used which either individually or in combination contain the epitopes E, C and M.

In addition to the above, it has been demonstrated that immunization with NS1 (non-structural protein 1) results in protection against yellow fever (Schlesinger et al., 1986) .This is true, although immunization did not induce neutralizing antibodies It is also likely, especially since this protein appears to be highly conserved among the flaviviruses, that HCV-NS1 also protects against HCV infection. In addition, it is also known that non-structural proteins provide protection against viral pathogenicity, even if they do not produce neutralizing antibodies cause.

In view of the above, multivalent vaccines against HCV may include one or more structural proteins, and / or one or more non-structural proteins. These vaccines may contain, for example, recombinant HCV polypeptides and / or polypeptides isolated from virions. In addition, it is possible to use inactivated HCV in vaccines; inactivation may be by the production of viral lysates or by other means known to those skilled in the art which will cause the inactivation of the flaviviruses, for example by treatment with organic solvents or detergents, or by treatment with formalin. In addition, vaccines can also be produced from attenuated HCV strains. The production of the attenuated HCV strains will be described later. It is known that some of the proteins present in flaviviruses contain highly conserved regions, so that some immunological cross-reactivity between HCV and other flaviviruses must be assumed. It is possible that epitopes shared between the flaviviruses and HCV produce protective antibodies against one or more diseases caused by these pathogenic agents. Therefore, it may be possible to develop multipurpose vaccines based on this knowledge.

The preparation of vaccines containing an immunizing polypeptide (s) as an active ingredient is known to those skilled in the art. Typical is the production of such vaccines as injectables, either as liquid solutions or suspensions; Solid forms suitable for solution or suspension in a liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein may be encapsulated in liposomes. The immunizing agents are often mixed with pharmaceutically acceptable and drug-compatible excipients. Suitable carriers are, for. Water, physiological saline, dextrose, glycerol, ethanol, etc., and combinations thereof. If desired, the vaccines may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and / or adjuvants that enhance the effectiveness of the vaccine. Examples of effective adjuvants include, but are not limited to, aluminum hydroxide, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MPD), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637 , referred to as non-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine ^ -d '^' - dipalmitoyl-sn-glycero-S-hydroxyphosphryloxy) ethylamine (GP 19835A, referred to as MTP-PE) and RIBI containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL 4-TDM + CWS) in 2% squalene / Tween-80 emulsion. The efficacy of an adjuvant can be assessed by measuring the amount of antibody directed against an immunizing polypeptide containing an HCV antigenic sequence resulting from the administration of this polypeptide in vaccines also consisting of various adjuvants.

The vaccines are usually administered parenterally, by injection, for example, subcutaneously or intramuscularly. Other formulations suitable for other forms of administration include suppositories and, in some cases, oral formulations. Suppositories may contain traditional binding and carrier agents, e.g. For example, polyalkylene glycols or triglycerides; Such suppositories may be formed from mixtures containing an active ingredient content in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally used excipients as e.g. Pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets,

Contain pills, capsules, long-acting formulations or powders and 10% to 95% of the active ingredient, preferably 25% to 70%.

The proteins can be formulated into the vaccine in neutral or saline form. Pharmaceutically acceptable salts include acid addition salts (which are formed with free amino groups of the peptide) and those with inorganic acids, e.g. As hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic acid, etc., are formed. The salts formed with the free carboxyl groups may also be of inorganic bases, eg. For example, sodium, potassium, ammonium, calcium or iron (III) hydroxides, or such organic bases as isopropylamine, trimethylamine, 2-ethylamirtoethanol, histidine, procaine and the like can be derived.

II.F. Dosage and administration of vaccines

The vaccines are administered in a manner compatible with the dosage formulation and in an amount which is prophylactically and / or therapeutically effective. The amount to be administered will generally range from 5 micrograms to 250 micrograms of antigen per dose and will depend on the subject to be treated, the ability of the subject's immune system to synthesize antibodies, and the desired level of protection. Exact amounts of the required active ingredient to be administered will depend on the judgment of the physician and may be different for each individual. The vaccine may be administered as a single dose or, preferably, as a multiple dose. A multi-dose regimen means that a main course of vaccination may consist of 1 to 10 individual doses followed by other doses required during subsequent time intervals required to maintain or re-establish the immunity response, eg in 1 to 4 month (s). a second dose and, if necessary, after several months another dose or several doses. The Dosieri'igsschema is also, ie at least partially, determined on the basis of the needs of the individual and depends on the assessment of the doctor. In addition, the vaccine containing the immunizing HCV antigen (s) may be used in conjunction with other immunoregulating agents, e.g. ß. Immunoglobulins ^ be administered.

II.G. Production of antibodies against HCV-tpltope The immunogenic polypeptides prepared as described above are used to produce antibodies, both

of polyclonal as well as monoclonal. When polyclonal antibodies are desired, a selected mammal (e.g., a mouse, goat, rabbit, horse, etc.) is immunized with an immunity-producing polypeptide carrying one or more HCV epitope (s). The serum of the immunized animal is collected and treated according to the known procedures. When the polyclonal antibody to HCV epitope-containing serum contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography.

Techniques for the production and processing of polyclonal antisera are known in the art, see e.g. Mayer and Walker Alternatively, polyclonal antibodies can be isolated from a mammal that has previously been infected with HCV. On Example of a Purification Method of Antibodies to HCV Epsomes to Serum from an Infected Individual Affinity chromatogram using a fusion polypeptide of SOD and a cDNA clone 5-1-1

coded polypeptides is described in Section V.E. shown.

Monoclonal antibodies directed against HCV epitopes can also be readily prepared by one skilled in the art

become. The general procedure for the production of monoclonal antibodies by hybridomas is well known.

Immortal antibody-producing cell lines can be produced by cell fusion and other techniques, such as direct Transformation of B lymphocytes with tumorigenic DNA or transfection with Epstein-Barr virus

become. See, e.g. M. Schreier et al. (1980); Hammerling and others. (1981); Kennett et al. (1980); see also US patent no. 4,341,761.4,399,121.4,427,783,4,444,887,4,466,917,4,472,500,4,491,632 and 4,493,890. Monoclonal produced against HCV epitopes

Antibodies may differ in their different properties, i. H. Isotypes, epitope affinity, etc. by means of lists

be screened.

Antibodies, both monoclonal and polyclonal, directed against HCV epitopes are particularly useful in the art Diagnosis and those that have a neutralizing effect are useful in passive immunotherapy. Monoclonal antibodies can

specifically used to develop anti-idiotypic antibodies.

Anti-idiotypic antibodies are immunoglobulins that are an "internal counterpart" of the antigen of the infectious agent against which the antigen Protection is desired, wear. See, e.g. Nisonoff, A & a. (1981) and Dreesman et al. (1985). Techniques for the production of anti-idiotypic antibodies are well known in the art. See, for example, Grzych (1985), MacNamara et al. (1984) and Utdtdaag et al. (1985). Disso anti-idiotype antibodies can also be used for the treatment of NANBH as well as lightening the immunizing regions of the HCV antigen. H. H. Diagnostic oligonucleotide probes and kits

Using the disclosed portions of the isolated HCV cDNAs as a base, including those in Figs. 1 to 32, oligomers of about 8 nucleotides or more, either by excision or synthetically, can be made to hybridize to and hybridize with the HCV genome the identification of the virus agent or viral agents, as well as in the characterization of viral genomes as well as the detection of viruses in diseased people is useful. The probes for HCV polynucleotides (natural or derived) are of a length that allow the detection of unique viral sequences by hybridization. While 6 to 8 nucleotides represent a workable length, sequences of 10 to 12 nucleotides are preferred, and about 20 nucleotides appear to be of optimal length. These sequences are preferably derived from regions lacking in heterogeneity. These probes can be made using routine methods, including automated oligonucleotide synthesis methods. Among the useful probes are г. Clone 5-1-1 and the additional clones disclosed herein, as well as the various oligomers useful in probing the cDNA libraries disclosed hereinafter. A complement to any unique part of the HCV genome will be sufficient. For use as probes, complete complementarity is desirable, although this may be unnecessary as the length of the fragment increases. For use of such probes as diagnostic agents, the biological sample to be analyzed, such as blood or serum, is treated, if desired, to extract the nucleic acids ги contained therein. The nucleic acid resulting from the sample may be subjected to gel electrophoresis or other size separation methods; in the other case

For example, the nucleic acid samples may be spotted without size separation. Subsequently, the samples are marked. Suitable labels and methods for labeling the samples are known in the art and include e.g. B. by nick translation or kinase, biotin, fluorescent probes and chemiluminszente probes inserted radioactive labels. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions subjected to appropriate controls.

The probes can be made completely complementary to the HCV genome. Therefore, usually very strict control conditions are desirable to prevent false-positive results. However, the stringent control conditions should only be used if the probes are complementary to regions of the viral genome lacking heterogeneity.

The stringent control of hybridization is determined by a number of factors during hybridization and during the washing process, including temperature, ionic strength, time duration, and formamide concentration. These factors are z. As outlined by Maniatis, T. (1982).

In general, it is believed that the HCV genome sequences are present in the serum of infected individuals at relatively low levels, ie, from about 10 ² to 10 ³ sequences per ml. This magnitude may require that enhancement techniques be used in the hybridization assays. Such techniques are known in the art. For example, the "Bio-Bridge" system provided by Enzo Biochemical Corporation uses terminal deoxynucleotide transferase to add unmodified 3 "poly dT tails to a DNA probe. The poly-dT tailed probe is hybridized to the target nucleotide sequence and subsequently to a biotin-modified poly-A. PCT Application 84/03520 and EPA 124221 describe a DNA hybridization assay in which: (1) analyte is hybridized to a single-stranded DNA probe complementary to an enzyme-labeled oligonucleotide; and (2) the resulting tailed duplex is hybridized to an enzyme labeled oligonucleotide EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe having a tail, such as a poly dT-tail, an amplifier string having a sequence that hybridizes to the tail of the probe, such as a poly A sequence, and is capable of binding a variety of labeled strands A particularly desirable technique may be to first amplify the Target HCV sequences in sera may be about 100,000 fold, ie, about 10 ^e sequences / ml, which may be achieved, for example, by the din technique of Saiki et al., (1986) The amplified sequence (s) may or may then be assayed using a hybridization assay set forth in co-pending U.S. Application, Attorney Docket No. 2300-0171, filed October 15, 1987, and assigned to the assignee hereof, herewith incorporated by reference be included. This hybridization assay, designed to detect sequences as high as 10 ^e / ml, utilizes nucleic acid multimers that bind to the single-stranded analyte nucleic acid as well as to a variety of single-stranded labeled oligonucleotides. A suitable solution phase sandwich assay in which labeled polynucleotide probes can be used and the methods for preparing probes are described in EPO 225307 (?), Published June 16, 1987, Application Serial No. 807,624, which is assigned to U.S. Pat cited herein and incorporated herein by reference.

The probes can be packaged in diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; otherwise, the probe DNA may not be labeled and the additives for the label may be included in the kit. The kit may also contain other suitable packaged reagents and materials needed for the particular hybridization protocol, such as: Standards and also rules for the conduct of the test.

II.I. Immunoassay and diagnostic kits

Both polypeptides that react immunologically with HCV antibody-containing serum, e.g. Those derived from or encoded by the clones and described in Section IV.A. and their compositions (see Section IV.A.) and the antibodies that have been developed against the HCV-specific epitopes in these polypeptides, see e.g. Section IV.E., are in immunoassays for detecting the presence of HCV antibodies, or for the presence of the virus and / or viral antigons in biological samples, including e.g. For example, blood or serum samples are useful. The design of immunoassays is frequently altered and a variety of them are known in the art. The immunoassay can z. B. use a virus antigen, for. A polypeptide derived from any of the clones containing HCV cDNA, see Section IV.A., or from the composite cDNAs derived from the cDNAs in these clones, or from the HCV genome, of άβ · ν The cDNA was derived in these clones; otherwise, the immunoassay may use a combination of viral antigens derived from these sources. For example, a monoclonal antibody directed against a virus epitope (s), a combination of monoclonal antibodies directed against a viral antigen, monoclonal antibodies directed against different viral antigens, polyclonal antibodies raised against the same Virus antigens, or polyclonal antibodies directed against different viral antigens. The logs can z. For example, based on competitive, or direct reaction or sandwich type assays. The logs can z. B. also use solid documents, or can come about by immunoprecipitation. Most assays involve the use of a labeled antibody or polypeptide; For example, the labels can be made by fluorescent, chemiluminescent, radioactive or color molecules. Assays that amplify the signals from the probe are also known; Examples are assays that use biotin and avidin as well as enzyme-labeled and indirect immunoassays such as the ELISA assays.

The flavivirus model for HCV allows predictions regarding the likely location of diagnostic epitopes for virion structural proteins. The C, pre-M, M, and Ε domains likely contain all epitopes of significant potential for the detection of viral antigens, and especially for diagnostics. Likewise, it is believed that the domains of the non-structural proteins contain important diagnostic epitopes (e.g., NS5 encoding a putative polymerase and NS1 encoding a putative complement-binding antigen). Recombinant polypeptides, or viral polypeptides, that include epitopes from these specific domains may be useful for the detection of virus antibodies in infected blood donors and infected patients.

In addition, antibodies directed against B and / or M proteins can be used in immunoassays for the detection of viral antigens in patients with HCV-induced NANBH and in infected blood donors. In addition, these antibodies may be particularly useful in detecting donors and patients in the acute phase. Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are prepared by packaging the appropriate materials, including the polypeptides of the invention, the HCV epitopes, or

Antibodies directed against HCV epitopes, contained in appropriate containers, along with the remaining reagents and materials required to perform the assay, as well as a suitable set of assay constructs.

N.J. Further characterization of the HCV genome, virions and viral antigens using cDNA from Virus genome derived. probes

The HCV cDNA sequence information in the clones described in Section IV.A. 1 to 32 are directed against HCV epitopes which would be useful in the diagnosis and / or treatment of HCV-induced NANBH. The cDNA sequence information in the aforementioned clones is useful for the design of probes for the isolation of additional cDNA sequences derived from hitherto undefined regions of the HCV genome or genomes, those described in Section IV .A. described cDNAs were obtained in clones. For example, the labeled probes containing a sequence of about 8 or more nucleotides and preferably 20 or more nucleotides derived from regions near the 5'-termini or З'-termini of the family of HCV cDNA sequences and Figures 1, 3, 6, 9, 14 and 32 can be used to isolate overlapping cDNA sequences from HCV cDNA libraries. These sequences, which overlap the cDNAs in the aforementioned clones but which also contain sequences derived from the regions of the genome from which the cDNAs are not derived in the aforementioned clones, can then be used to synthesize probes for the identification of other overlapping fragments, not necessarily the cDNAs in the ІѴ.А. overlap described clones used. If the HCV genome is segmented and the segments do not share common sequences, it is possible to derive all or the entire virus genome (s) using the technique of isolating overlapping cDNAs derived from the virus genome (s) are to be sequenced. Although it is unlikely that if the genome is a segregated genome that has no common sequences, the sequence of the genome can be determined by serological screening of the lambda gt 11 HCV cDNA libraries, as in the isolation of clone 5-1 -1, by sequencing cDNA isolates and by using the isolated cDNAs to isolate overlapping fragments, using the methods described for the isolation and sequencing of the clones in Section IV.A. described technique has been applied. In the other case, the characterization of the genome segments could be derived from the viral genome (s) isolated from the purified HCV particles. Methods for purifying the HCV particles and for detecting them during the purification process have been described hereinbelow. Methods for the isolation of polynucleotide genomes from virus particles are known in the art, and an applicable method is described in Example IV.A.1. shown. The isolated genome segments could then be cloned and sequenced. Thus, with the information provided therein, it is possible to clone and sequence the entire HCV genome (s) regardless of its nature.

Methods for the construction of cDNA libraries are known in the art and are discussed above and below; a method for the construction of HCV cDNA libraries in lambda gt 11 is described in Section IV.A. discussed further below. The cDNA libraries useful for screening with nucleic acid probes can also be constructed in other vectors known in the art, e.g. B. Lambda-gt 10 (Huynh et al. [1985]). The HCV-derived cDNA detected by probes derived from the cDNAs in Figs. 1 to 32 and probes synthesized from the polynucleotides derived from these cDNAs can be prepared from the clone by digesting the isolated polynucleotide with the appropriate restriction enzyme (s). en) are isolated and sequenced. See, for example, B. Section IV.A.3. and IV.A.4. with respect to the techniques used for the isolation and sequencing of the HCV cDNA overlapping the HCV cDNA in clone 5-1-1; Sections IV.A.5. to IV.A.7. for isolation and sequencing of HCV cDNA overlapping those in clone 81 and sections IV.A.8 and IV.A.9 for the isolation and sequencing of a clone overlapping another clone (clone 36), the clone 81 overlaps.

The sequence information derived from these overlapping HCV cDNAs is useful for the determination of homology and heterogeneity regions within the virus genome (s), which in turn could indicate the presence of different genome strains, and / or populations of defective particles. They are also useful for the design of the hybridization probes to detect HCV or HCV antigens or HCV nucleic acids in biological samples, and during the isolation of HCV (discussed below) those described in Section II.G. to use the techniques described. In addition, the overlapping cDNAs can be used to generate expression vectors for polypeptides derived from the HCV genome (s), including those in clones 5-1-1,3G, 81,91 and 1-2 and encode encoded polypeptides in the other clones operated in Section IV.A. These techniques for the generation of these HCV epitope-containing polypeptides and for antibodies directed against the HCV epitopes they contain, as well as their uses, are described analogously to those for polypeptides derived from NANBV cDNA sequences and incorporated herein by reference Clones 5-1-1,32,35,36,1-2,81 and 91 are included and discussed in the text above and in the text below.

Encoded in the family of cDNA sequences, in clones 5-1-1,32,35,36,81,91,1-2 and in the others in Section IV.A. clones described are antigens or antigens which have epitopes which appear unique to HCV, ie antibodies directed against these antigens are absent in the individuals infected with HAV or HBV and in non-HCV infected individuals (see Section IV.B., serological data). Moreover, comparing the sequence information of these cDNAs with the sequences of HAV, HBV, HDV and genomic sequences in the library shows that there is minimal homology between these cDNAs and the polynucleotide sequence of these sources. Thus, antibodies directed against antigens encoded within the cDNAs of these clones can be used to identify BB-NANBV particles isolated from infected individuals. In addition, they are also useful for the isolation of NANBH active agent (s).

HCV particles can be obtained from the sera of BB-NANBV-infected individuals or from cell cultures by methods known in the art, including e.g. For example, techniques based on size discrimination, such as 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 a variety of materials such as anionic or cationic exchange materials, and materials that bind due to hydrophobicity as well as affinity columns are isolated. During the isolation procedure, the presence of HCV can be determined by the hybridization analysis of the extracted genome using probes derived from HCV cDNAs described above or by immunoassay (see section IM.) Using as probes against HCV antigens targeted antibodies are used,

which are encoded within the family of the cONA sequences shown in Figures 1 to 32, and are also directed against HCV antigens encoded within the overlapping HCV cDNA sequences discussed above. The antibodies may be monoclonal or polyclonal, and it may be desirable to purify the antibodies prior to use in the immunoassay. A purification ancestor for polyclonal antibodies directed against antigen (s) encoded within clone 5-1-1 is described in Section IV.E. described; Analogous purification methods can be used for antibodies directed against other HCV antigens.

Antibodies directed against HCV antigens encoded within the family of cDNAs shown in Figures 1 to 32, as well as those encoding within overlapping HCV cDNAs and attached to solid supports, are useful for isolating HCV useful by immunoaffinity chromatography. Techniques for immunoaffinity chromatography are known in the art, including techniques for attaching antibodies to solid supports so that they retain their immunoselective activity; the techniques may consist of those in which the antibodies are absorbed on the support (see, eg, Kurstak in "Enzyme Immunodiavisin", pages 31-37) as well as those in which the antibodies are covalently bound to the support In general, the techniques are similar to those used for the covalent attachment of antigens to a solid support, which are generally described in Section II.C. However, spacer groups can be included in the bifunctional coupling agents such that the antigen binding site of the Antibody remains accessible.

During the purification procedure, the presence of HCV can be detected and / or checked by nucleic acid hybridization using polynucleotides derived as probes from the family of HCV cDNA sequences shown in Figures 1 to 32 as well as those overlapping Derived HCV cDNA sequences and described in the above text. In this case, the fractions would be treated under conditions causing disruption of the virus particles, e.g. With detergents in the presence of chelating agents and in the presence of viral nucleic acids described in section H.H. described hybridization techniques were determined. Further confirmation that the isolated particles are the drugs that induce HCV can be gained by infecting chimpanzees with the isolated virus particles, then determining whether the NANBH symptoms result from the infection.

Viral particles from the purified preparations can then be further characterized. The genomic nucleic acid was purified. Based on their sensitivity to RNase, rather than DNase I, it appears that the virus is derived from an RNA genome z ¹ . is composed. See Example IV.C.2., Below. The stringency and circularity or non-circularity can be determined by techniques known in the art, including, for example, their visualization by electron microscopy, their migration in the density gradients, and their sedimentation characteristics. Based on the hybridization of the captured HCV genome to the negative strands of HCV cDNAs, it appears that the HCV consists of a positive-stranded RNA genome (see Section IV.H.1). Techniques like these are In addition, the purified nucleic acid can be cloned and sequenced by known techniques, including reverse transcriptase, since the genomic material is RNA, See, e.g., Maniatis (1982) and Glover (1985) Using the nucleic acid derived from the viral particles, it is possible to sequence the entire genome, depending on whether or not it is segmented.

Examination of the homology of the polypeptide encoded within the continuous open reading frame of combined clones 141 to 390 (see Figure 26) shows that the HCV polypeptide contains homologous regions at the corresponding proteins in the conserved regions of the flaviviruses. An example of this is given in Section IV.H.3. described. This finding has many important aspects. First, this evidence is related to the finding that the HCV contains a positive-stranded genome of approximately 10,000 nucleotides in size, consistent with the notion that the HCV is a flavivirus or a flavivirus. In general, flavivirus virions and their genomes have a relatively consistent structure and organization that are known. See Rice u. a. (1988) and Brinton, M.A. (1988). Thus, polypeptides C, pre-M / M and E encoding the structural genes can be localized upstream of clone 14І in the 5'-terminus of the genome. In addition, predictions can be made regarding the exact location of the sequences encoding these proteins, including comparison with other flaviviruses.

Isolation of the sequences upstream of those in clone 14 i can be accomplished in a variety of ways, and the information provided herein will be apparent to one skilled in the art. For example, the genomic "walking" technique can be used to isolate other sequences that are 5 'to those in clone 14i, but which overlap that clone, which in turn results in the isolation of additional sequences. For example, it is known that the flavivir have conserved epitopes and regions of conserved nucleic acid sequences, and polynucleotides containing the conserved sequences can be used as probes encoding the HCV. Bind genome and thus allow its isolation. In addition, these conserved sequences, in conjunction with those derived from the HCV cDNAs, see Figure 22, can be used to design starters for use in systems that amplify genome sequences upstream of those in clone 14i using the polymerase chain reaction technology. An example of this will be described below.

The structure of the HCV can also be determined and its components isolated. The morphology and size can be determined, for example, by electron microscopy. The identification and localization of specific virus polypeptide antigens such as coat or envelope antigens, or internal antigens such as nucleic acid binding proteins, nuclear antigeno and polynucleotide polymerase (s) may also be determined by, for example, determining whether the antigens are present as largest or smallest virus components, as well by the use of antibodies directed against the specific antigens encoded within isolated cDNAs as probes This information is useful in the design of vaccines, eg it may be beneficial to include an external antigen in a vaccine preparation Vaccines may, for example, contain a polypeptide derived from the genome encoding a structural protein, e.g., E, as well as a polypeptide from another filament of the genome, e.g., a non-structural or structural polypeptide.

II, K. Cell culture systems and animal model systems for HCV replication The assumption that HCV is a flavivirus or a flavivirus also provides information on methods for growing HCV. The term "flavi-like" means that the virus has a significant proportion of homology to the known conserved regions of the flaviviruses and that the major part of the genome is a single open reading frame B. The Reviews by Brinton [1986] Slollar, V.

[1980]). In general, suitable cells or cell lines for the growth of HCV may include those for that purpose

are known to support flavivirus replication, e.g. The following: monkey kidney cell lines (e.g., HK ₃ , VERQ);

Pork kidney tent lines (e.g., PS); Baby hamster kidney cell lines (e.g., BHK); Mouse macrophage cell lines (e.g., P38801, MK1, MMI); Human macrophage cell lines (e.g., U-937); peripheral human blood leucocytes, adherent human monocytes; hepatocytes

or hepatocyte cell lines (e.g., HUH 7, HEPC2); Embryo or embryonic cells (eg chick embryo fibroblasts or cells derived from invertebrates, preferably from insects (eg Drosophila cell lines), or more preferably from arthropods,

z. Mosquito cell lines (e.g., Albopictus, Aedes aegypti, Cutex tritaeniorhynchus) or tick cell lines, e.g. RML-14,

Dermacentor parumaportus) are derived. It is possible that primary hepatocytes can be cultured and subsequently infected with HCV; or in the other In this case, hepatocyte cultures could be obtained from the livers of infected individuals (e.g., humans or chimpanzees)

become.

In the latter case, an example is that of a cell that is infected in vivo and transferred in vitro. In addition, various immortalization methods can be used to derive from hepatocyte cultures To gain cell lines. For example, primary liver cultures (before and after enrichment of the hepatocyte population) may be associated with

a number of cells fuse to maintain their stability. It can z. B. cultures mittransformierenden viruses are infected, or be transferred with transforming genes to permanent or semi-permanent cell lines

produce. In addition, z. For example, cells in liver cultures may be fused to established cell lines (eg, HepG 2). Methods for cell fusion are known in the art and include e.g. B. the use of fusion agents as well

Polyothylene glycol, Sendai virus and Epstein-Barr virus. As discussed earlier, HCV is a flavivirus or a flavivirus. Therefore, it is likely that the HCV infection of Cell lines can be performed by techniques known in the art for the infection of cells with flaviviruses. These

include, for. B. incubating the cells with virus preparations under conditions that allow virus entry into the cell. Moreover, it is possible to achieve virus production by transferring the cells to isolated viral polynucleotides. It is known that togavirus and flavivirus RNAs are transmissible in a number of vertebrate cell lines (Pfefferkorn and Shapiro [1974]) and in a mosquito cell line (Peleg [1969]).

Methods for transfecting tissue culture cells with RNA duplexes, positive-stranded RNAs and DNAs (including

cDNAs) are known in the art and include e.g. Techniques such as electroporation and precipitation with DEAE

Dextran or calcium phosphate. A rich source of HCV RNA can be obtained by performing in vitro transcription

an HCV corresponding to the complete genome. Transfection with this material or with

cloned HCV cDNA would have to result in virus replication and in-vivo-propagation of the virus.

In addition to the cultured cells, animal model systems for viral replication can be used; animal Systems in which flaviviruses occur are known to those skilled in the art (see, for example, "Review" by Monath [1986]) HCV replication not only occur in chimpanzees, but z. Also in marmosets and suckling mice. II.L. Scri <rifng on anti-viral agents for HCV

The disclosure of cell cultures and animal model systems for HCV also allows for screening for antiviral agents that inhibit HCV replication, and particularly for those agents that preferentially allow cell growth and multiplication while inhibiting viral replication. These screening methods are known to those skilled in the art. In general, the antiviral agents become effective in a number of concentrations in their effect of preventing viral replication in cell culture systems that support viral replication and then inhibiting infectivity or viral pathogenicity (and a low level of toxicity) in an animal model system tested.

The methods and compositions provided herein for the detection of HCV antigens and HCV polynucleotides are useful for the screening of anti-viral agents in that they provide an alternative and perhaps a sensitive means for detecting the effect of the agent on viral replication the cell plaque assay or Юzo assay. The HCV polynucleotide probes described herein can be used, for example, to quantify the amount of viral nucleic acids produced in a cell culture. This could be z. By hybridization or competition of the 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 (s) in cell cultures Since it may also be desirable to quantify HCV antigens in the infected cell cultures by a competition assay, the polypeptides encoded in the cDNAs described herein are useful in these competition assays. In general, a recombinant HCV polypeptide derived from the HCV cDNA would be labeled, and inhibition of binding of this labeled polypeptide to an HCV polypeptide due to the antigen produced in the cell culture system would be monitored In addition, the techniques are particularly useful in cases where the HCV may be able to be in a cell replicate without causing cell death.

AROUND. Preparation of attenuated HCV strains

In addition to the above and using the tissue culture systems and / or model animal systems, it may be possible to isolate attenuated HCV strains. These strains would be suitable for vaccines or for isolating viral antigens. Attenuated strains are isolatable after multiple passages in cell cultures and / or an animal model. Detection of an attenuated strain in an infected calf or individual is accomplished by techniques known in the art, and could e.g. For example, the use of antibodies to one or more epitopes encoded in HCV as a probe may include the use of a polynucleotide that will retain an HCV sequence of at least about 8 nucleotides as a probe. Alternatively, or in addition, an attenuated strain may be constructed using the genomic information provided herein by HCV and using recombinant techniques. In general, one would try to erase a region of the genome, the z. B. encodes a polypeptide-related pathogenicity, but allows viral replication. In addition, genome construction would allow the expression of an Epi '-. M. which causes neutralization of antibodies to the HCV. The altered genome could then be used to transform cells that permit HCV replication, and the cells grown under these conditions allow for viral replication.

The attenuated strains of HCV are useful not only for vaccine purposes, but also as sources of commercial production of viral antigen, since processing these viruses would require less stringent protective measures for the agents involved in virus production and / or production of viral products.

III. General methods

The general techniques used for extracting the genome from a virus, for preparing and probing a cDNA library, sequencing the clones, constructing expression vectors, transforming the cells, for performing immunological tests such as radioimmunoassays and ELISA assays, for growing cells in cultures and the like are known in the art and laboratory manuals describing these techniques are available. The following text provides a general guide that shows some currently available sources of such procedures and materials thereof are useful in the practice.

III.A. Hosts and expression control sequences

Both prokaryotic and eukaryotic host cells may be used for the expression of the desired coding sequences if appropriate control sequences compatible with the labeled host are used. Among prokaryotic hosts E. coil is the most commonly used. Prokaryotic expression control sequences include promoters optionally containing operator portions and ribosome-forming sites. Transfer vectors that are compatible with the prokaryotic hosts are commonly used by e.g. Derived pBR322, an operon-containing plasmid conferring ampicillin and tetracycline resistance, and various pUC vectors which also contain sequences conferring antibiotic resistance markers. These markers can be used to gain successful transformants by selection. The commonly used prokaryotic control sequences include beta-lactamase (penicillinase) and lactose promoter systems (Chang, et al. [1977]), tryptophan (trp) promoter system (Goeddel et al. (1989)), and the lambda-derived P _L promoter and N-gene ribosome binding site (Shimatake et al., 1981) and the hybrid tac promoter (De Boer et al., 1983), derived from the sequences of the trp and lac UV5 promoters particularly compatible with E. coil, if desired, other prokaryotic hosts such as Bacillus or Pseudomonas strains can be used with the appropriate control sequences Eukaryotic hosts contain yeast and mammalian cells in the culture systems Saccharomyces cerevisiae and Saccharomyces carlsbergensls are the most widely used yeast hosts and yeast-compatible vectors bear markers that allow the selection of successful transformants by conferring prototropism on auxotrophs Allow mutants or resistance to heavy metals on wild strains. Yeast-competent vectors may employ the 2-micron origin of replication (Broach et al. [1983]), the combination of CDN 3 and ARS 1, or other means for ensuring replication, as well as sequences resulting in the incorporation of a suitable fragment into the host cell genome.

Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al., [1968], Holland et al., [1978]), including the promoter for 3 phosphoglycerate kinases (Hitzeman [1980]). Terminators may also be included, such as those derived from the enolasgene (Holland (198I).) Control systems, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohide dehydrogenase (ADH) -regulatable promoter, are also particularly useful terminators derived from GAPDH, and if secretion is desired, include "leader" sequence of yeast alpha factors.

In addition, the transcriptional regulatory region and the transcriptional initiation region that are operably linked to each other may be of the nature that they are not naturally associated in the wild-type organism. These systems are described in detail in EPO 120551, published October 3, 1984; EPO 1162Oi, published August 22, 1984, and EPO 164446, published December 18, 1985, all of which are assigned to the assignee hereof and are hereby incorporated by reference.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines provided by the American Type Culture Collection (ATCC) including HeLa cells, Chinese hamster, ovary (CHO ) Cells, baby hamster kidney (BHK) cells and a number of other cell lines. Suitable promoters for mammalian cells are known in the art and include viral promoters such as simian virus 40 (SV40) (Fiers P 978]), rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus ( BPV). Mammalian cells may also require terminator sequences and poly (A) addition sequences; Enhancer sequences that increase expression may also be included, as well as sequences that effect the amplification of the genes may also be desirable. These sequences are known in the art. Vectors suitable for replication in mammalian cells may contain viral replicons or sequences that guarantee the integration of appropriate sequences encoding NANBV epitopes into the host genome.

III.B. transformations

The transformation may be by any known method for the introduction of polynucleotides into a host cell, including e.g. B. packaging the polynucleotides into a virus and transducing a host cell with dwn virus, and by direct uptake of the polynucleotides. The transformation procedure used depends on the host to be transformed. Transformation of E. coli host cells with lambda gt11 containing BB-NANBV sequences is e.g. The bacterial transformation by direct uptake generally involves treatment with calcium or rubidium chloride (Cohen (1972); Maniatis [1982]). Yeast transformation by direct uptake can be performed using the method of Hinnen et al., (1978) The mammalian cell transformation by direct uptake can be performed using the calcium phosphate precipitation method of Graham and Van der Eb (1978) or the various known modifications thereof.

III.C. vector construction

The vector construction is accomplished by techniques known in the art. Sequence-specific cleavage is performed by treatment with appropriate restriction enzymes under conditions generally specified by the manufacturer of these commercially available enzymes. In general, about 1 microgram of plasmid or DNA sequence is cut through 1 unit of enzyme in about 20 microliters of buffer solution by incubation for 1 to 2 hours at 37 ^° C. After incubation with the restriction enzyme, the protein is removed by phenol / chloroform extraction and the DNA is passed through

Precipitation recovered with ethanol. The cut fragments can be separated using polyacrylamide or agarose gel electrophoresis techniques according to the general procedures outlined in "Methods in Enzymology" (1980) 65: 499-560.

Cut fragments having cohesive ends may have blunt ends using E. coli DNA polymerase I (Klenow) in the presence of appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. The treatment with SI nuclease resulting in the hydrolysis of any single-stranded DNA segments may also be performed.

Ligations are performed using standard buffers and temperature conditions using T4 DNA ligase and ATP; Ligation of cohesive ends requires less ATP and less ligase than blunt end ligations. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5'-phosphate and thus prevent re-regulation of the vector; otherwise, restriction enzyme digestion of unwanted fragments can be used to prevent ligation. Ligation mixtures are transformed into suitable cloning hosts such as E. coli and successful transformants are e.g. selected by antibiotic resistance and screened for correct construction.

III.D. Construction of desired DNA sequences

Synthetic oligonucleotides can be prepared using automated oligonucleotide synthesis agents as described by Warner (1984). If desired, the synthetic strands with ³² P by treatment with polynucleotide kinase in the presence of "p-ATP, be labeled using standard conditions for the reaction can.

DNA sequences, including those isolated from the cDNA libraries, can be modified by known techniques, including, for example, directed mutagenesis as described by Zoller (1982). In short, the DNA to be modified is packaged into a phage as a single-stranded sequence and converted to a double-stranded DNA with DNA polymerase using as a starter a synthetic oligonucleotide complementary to the portion of the DNA to be modified and which contains the desired modification in its own sequence. The resulting double-stranded DNA is transformed into a phage that supports the host bacterium. The cultures of transformed bacteria containing replications of each strand of phage are plated in agar to obtain plaques. Theoretically, 50% of the new mutant phage plaques were present. The remaining 50% have the original sequence. Replicas of the plaques are hybridized to the labeled synthetic probe at temperatures and conditions that permit hybridization with the correct strand, but not with the unaltered sequence. The sequences identified by hybridization are recovered and cloned.

III.E. Hybridization with probes

DNA libraries can be probed using the method of Grunstein and Hogness (1975). Briefly, in this method, the DNA to be probed is immobilized on nitrocellulose filters, denatured and pre-hybridized with a buffer containing 0 to 50% formamide, 0.7SM NaCl, 75 mM Na citrate, 0.02% (M / V). each containing bovine serum albumin, polyvinylpyrollidone and Ficoll, 50 mM Na phosphate (pH = 6.5), 0.1% SDS and 100 micrograms / ml "denatured" DNA, the percentage of formamide in the buffer as well as the and pre-hybridization temperature conditions and subsequent hybridization steps depend on the stringency required Oligomeric probes, which require less stringent conditions, are generally employed at lower percentages of formamide, lower temperatures and longer hybridization times Probes greater than 30 to 40 nucleotides as containing derived from cDNA or genomic sequences, for example, generally require higher temperatures, eg. about 40 ° C to 42 ⁰ C, and a raised Formami danteil, eg 50%. Subsequent to the pre-Hybridisi6rung the 5'- ³² p-labeled oligonucleotide probe is added to the buffer, and the filters are incubated under hybridizing conditions in this. After washing, the treated filters are autoradiographed to show the location of the hybridized probe; the DNA in appropriate locations on the original agar plates is used as the source of the desired DNA.

HIF. Verification of construction and sequencing

In routine routine designs, ligation mixtures are transformed into E. coli strain HB101 or other suitable host, and successful transformants are selected by antibiotic resistance or other markers. Plasmids from the T'ansformanten are then according to the method of Clswell et al. (1Э69), usually prepared by monitoring chloramphenic H amplification (Clewell [1972]). The DNA is isolated and analyzed on the basis of repression analysis and / or sequencing. Sequencing can be performed by the dideoxy method of Sanger et al. (1977), which continue to be used by Messing et al. (1981) or by the method of Maxam et al. (1980), problems with band compression sometimes observed in the GC-rich regions were identified by the use of T-deazo-guanosine according to Barru.a. (1986).

III.G. Enzyme-linked immunosorbent assay

The enzyme-linked immunological assay (ELISA) can be used to measure either antigen or antibody levels. This method depends on the conjugation of an enzyme to either an antigen or antibody and uses the bound enzyme activity as a quantitative label. To measure the antibody, the known antigen is fixed to a solid phase (eg, a microplate or plastic crucible), incubated with test serum dilutions, washed, incubated with anti-immunoglobulin labeled with an enzyme, and washed again. Suitable enzymes for the labeling are known in the art and include, for. B. horseradish peroxidase. The type of solid phase-bound enzyme activity is measured by the addition of the specific substrate, and the determination of product formation or substrate utilization is colorimetric. The bound enzyme activity is a direct function of the amount of bound antibody.

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

IV. Examples

In the following examples according to the invention are described, which serve only for illustration and do not limit the scope of the laying invention. In view of the present invention, numerous embodiments within the scope of the claims are apparent to those skilled in the art.

The z. 8. in Section IV.A. however, may or may not be repeated, as techniques for constructing the desired nucleotide sequences are available based on the information provided by the invention. Expression is illustrated in E. coli, but other systems are available that are described in Section III.A. be described in more detail. Additional epitopes derived from the genomic structure can also be produced and used to generate antibodies as described below.

IV.A. Preparation, isolation and sequencing of HCV cDNA IV.A.1. Preparation of HCV cDNA

The source of the NANB agent was a plasma pool derived from a chimpanzee with chronic NANBH. The chimpanzee was experimentally infected with blood from another chimpanzee with chronic NANBH, which in turn resulted from infection with HCV in a contaminated concentration level with factor 8 derived from pooled human serum. The chimpanzee plasma pool was prepared by combining many individual plasma samples having a high degree of alanine aminotransferase activity, this activity resulting from the hepatic injury due to the HCV infection. Since 1 ml of a 10 ^-8 dilution of this collected and intravenously administered serum in other chimpanzees caused NANBH, its CID (cytomegalovirus syndrome) was at least 10 ⁶ / ml, ie, it had a high infectious virus titer.

A cDNA library from the high titer plasma pool was generated as follows. First, virate particles were isolated from the plasma; A 90 ml aliquot was diluted with 310 ml of a 50 ml Tris-HCl, pH8.0, 1 mM EDTA, 10 mM NaCl-containing solution. Residues were by S '.' Min centrifugation at 1500Ox g at 2O ⁰ C removed. Viral particles in the resulting supernatant were anschließet ή pelleted by centrifugation in a Beckman SW-28 rotor at 28000 U / min for 5 hours at 20 ⁰ C. were To release the viral genome, the particles by suspending the pellets in 15 ml solution the 1% sodium dodecyl sulfate (SDS), 1OmM EDTA, 1OmM Tris-HCl, pH7.6, and also 2 mg / ml proteinase к broken and then incubated at 45 ⁰ C for 90 min. The nucleic acids were isolated by the addition of 0.8 micrograms MS 2 bacteriophage RNA as vehicle and the mixture was washed four times with a 1: 1 mixture of phenol-chloroform (phenol saturated with 0.5M Tris-HCl), pH7.5.0 , 1% (v / v) beta-mercaptoethanol, 0.1% (w / v) hydroxyquinoline, and then extracted twice with chloroform. The aqueous phase was concentrated before precipitation with 2.5 volumes of absolute ethanol and allowed to stand overnight at -20 ° C with 1-butanol. Nucloic acid was recovered by centrifugation in a Beckman SW-41 rotor at 40,000rpm for 90min at 4 ° C and dissolved in water which was treated with 0.05% (v / v) diethylpyrocarbonate and autoclaved.

The nucleic acid recovered by the above method was <2 micrograms) was denatured with 17.5 mM CH ₃ HgOH; cDNA was synthesized using this denatured nucleic acid as a template and cloned into the EcoRI site of phage lambda gt 11 using methods described by Huynh (1985), except that the random starters oligo (dT) -12-18 were synthesized during synthesis of the first cDNA strand by reverse transcriptase (Taylor et al. [1976]). The resulting double-stranded cDNAs were fractionated according to size on a Sepharose CL-4 3 column; Approximately 400,300,200 and 100 base pair eluted material were pooled in cDNA pools 1,2, 3 and 4, respectively. The lambda gt 11 cDNA library was generated from the cDNA in pool 3.

The lambda gt 11 cDNA library generated from pool 3 was screened for epitopes that can specifically bind serum derived from a patient previously on NANBH. Using the methods of Huynh, etc. (1985) about 10 ^е phage were screened with patient sera, except that bound human antibody with sheep anti-human Ig antisera that were radiolabeled with ¹²⁵ I could be detected. Five positive phages were identified and purified. The five positive phage were then tested for specificity of binding to sera from 8 different humans previously infected with the NANBH agent using the same method. Four of the phage encoded a polypeptide immunologically reactive with only one human serum, Me, ie, the one used for primary screening of the phage library. The fifth phage (5-1-1) encoded a polypeptide that immunologically reacted with 5 of the 8 sera tested. In addition, this polypeptide did not react immunologically with sera from 7 normal blood donors. Therefore, it appears that clone 5-1-1 produces a polypeptide that is immunologically recognized specifically by sera from NANB patients.

IV. A.2 HCV cDNA Sequences In Recombinant Phage 5-1-1 and In-Sequence Encoded Polypeptide The cDNA in recombinant phage 5-1-1 was analyzed by a method of Sanger et al. (1977). The cDNA was excised substantially with EcoRI and isolated by gel fractionation using gel electrophoresis. The EcoRI restriction fragments were subcloned into the M13 vectors mp18 and mp19 (Messing [1983]) and using the dideoxy chain termination method of Sanger et al. (1977). The obtained sequence is shown in FIG. The polypeptide encoded in Figure 1, encoded in the HCV cDNA, is in the same translational framework as the N-terminal beta-galactosidase component to which it is fused. As described in Section IV.A. For example, the translational open reading frame (ORF) of 5-1-1 encodes an epitope or epitopes specifically recognized by sera from NANBH-infected patients and chimpanzees.

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

The HCV cDNA overlapping the cDNA into chl 5-1-1 was screened by screening the same Lampda-gt 11 library as described in Section IV.A. 1, with a synthetic polynucleotide derived from the sequence of the HCV cDNA in clones 5-1-1 as shown in FIG. 1. The sequence of the polynucleotide used for the screening was:

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

The lambda gt11 library was screened with this probe using the method described by Huynh (1985). About 1 in F, 0000 clones hybridized with this probe. Three clones containing cDNA hybridized to the synthetic probe numbered 81.1-2 and 91.

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

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

The sequences of the cloned HCV cDNAs are highly homologous in the overlapping regions (see Figure 2). In two regions, however, there are differences. Nucleotide 67 in clone 1-2 is a thymidine, whereas the other three clones contain a cytidine residue at the cJ'f ser site. However, it should be noted that the same amino acid is encoded when either C or T occupy this position.

The second difference is that clone 5-1-1 contains 28 base pairs that are not present in the other three clones. These base pairs start at the start of the cDNA sequence in 5-1-1 and are indicated by small letters. On the basis of the radioimmunoassay data, discussed below in Section IV. D., it is possible that an HCV epitope may be encoded in this 28 bp region.

The presence of the 28 base pairs of 5-1-1 from clones 81, 1-2 and 91 may indicate that the cDNA in these clones was derived from defective HCV genomes; alternatively, the 28 bp region could be a terminal artifact in clone 5-1-1. The small lettered sequences in the nucleotide sequence of clones 81 and 91 simply indicate that these sequences were not found in other cDNAs since cDNAs overlapping these regions have not been isolated yet.

A composite HCV cDNA sequence deduced from the overlapping cDNAs in clones 5-1-1,81,1-2 and 91 is shown in FIG. In this figure, however, the unique 28 base pairs of clone 5-1-1 have been omitted. The figure also shows the sequence of the polypoptide encoded in the ORF of the composite HCV cDNA.

IV. A.5. Isolation of HCV cDNAs Overlapping the cDNA in Clone 81

The isolation of the HCV cDNA sequences upstream from and overlapping those in clone 81 cDNA was performed as follows. The lambda gt 11 cDNA library prepared according to Section IV. A. 1. was screened by hybridization with a synthetic polynucleotide probe homologous to a 5'-terminal sequence of clone 81. The sequence of clone 81 is shown in FIG. The sequence of the synthetic polynucleotide used for the screening was:

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

The methods were essentially those described by Huynh (1985), except that the library filters were subjected to two washes under stringent conditions, ie, the washes were performed in 5 χ SSC, 0.1 % SDS at 55 ^° C and each for 30 min. About 1 in 50,000 clones hybridized to the probe. A positive recombinant phage containing cDNA that hybridized to the sequence was isolated and purified

 This phage was numbered 36.

Downstream cDNA sequences overlapping the carboxyl end sequences in clone 81 cDNA were isolated using a method similar to that for isolation of upstream cDNA sequences, except that a synthetic oligonucleotide probe, which is homologous to a З'-terminal sequence of clone 81. The sequence of the synthetic polynucleotide used for the screening was:

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

A positive recombinant phage containing cDNA that hybridized to this latter sequence was isolated and purified and numbered with clone 32.

IV. A.6 Nucleotide Sequence of HCV cDNA In Clone 36 The nucleotide sequence of the cDNA in clone 36 was determined essentially as described in Section IV. A. 2. The

double-stranded sequence of this cDNA, its overlap region with the HCV cDNA in clone 81 and that encoded by the ORF

Polypeptides are shown in FIG. The ORF in clone 36 is in the same translational frame as the HCV antigen encoded in clone 81. Thus

In combination, the ORFs in clones 36 and 81 encode a polypeptide representing part of a large HCV antigen. The sequence of this putative HCV polypeptide and the double-stranded DNA sequence encoding it derived from the combined ORFs of the HCV cDNAs of clones 36 and 81 are shown in FIG.

IV. A. 7. Nucleotide Sequences of HCV cDNA In Clone 32 The nucleotide sequence of the cDNA in clone 32 was prepared essentially according to the method described in Section IV Sequence of clone 5-1 -1 determined. The sequence data showed that the cDNA in clone 32 recombinant phage consists of two

was derived from different sources. One fragment of the cDNA contained 418 nucleotides derived from the HCV genome, the other fragment comprised 172 nucleotides derived from the bacteriophage MS 2 genome which was used as a support during the preparation of the lambda gt 11 plasma cDNA library.

The sequence of the cDNA in clone 32 corresponding to the HCV genome is shown in FIG. The region of the sequences of the Clone 81 overlaps and the polypeptide encoded by the ORF is also shown in this figure. Contains this sequence

a continuous ORF located in the same translational frame as the HCV antigen encoded by clone 81.

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

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

5 ¹ AAG CCA CCG TGT GCG СТА GGG CTC AAG CCC 3 '.

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

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

The nucleotide sequence of the cDNA in clone 35 was determined essentially as described in Section IV. A. 2.. The sequence, its region of overlap with that of the cDNA in clone 36, and the putative polypeptide encoded therein are shown in FIG. Clone 35 apparently contains a single continuous ORF encoding a polypeptide in the same translational frame as encoded by Clcn 36, clone 81 and clone 32. Figure 9 shows the sequence of the long continuous ORF which extends through clones 35, 36, 81 and 32 and is coded with the putative HCV polypeptide therein. This combined sequence has been confirmed using other independent cDNA clones derived from the same lambda gt 11 cDNA library.

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

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

5 'CAG GAT GCT GTG TCC CGC ACT CAA CGT 3'.

About 1 in 50,000 clones hybridizes to the probe. The isolated, purified clone of recombinant phage containing cDNA that hybridized to a sequsnz was named clone? 7b.

IV.A.11. Nucleotide sequence of HCV in clone 37b

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

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

Clone 37b apparently contains a continuous open reading frame (ORF) encoding a polypeptide which is a continuation of the polypeptide encoded in the open reading frame extending through the overlapping clones, 35, 36, 81 and 32.

IV.A.12. Isolation of HCV cDNA Overlapping cDNA in Clone 32

The isolation of HCV cDNA sequences "downstream" from clone 32 was performed as follows: First, clone was isolated using a synthetic hybridization probe based on the nucleotide sequence of the HCV cDNA sequence in clone 32. The method was equivalent essentially that described in Section IV.A.5., except that the sequence of the synthetic probe looked like this:

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

Using the nucleotide sequence of Clon Ria, another synthetic nucleotide was synthesized which had the following sequence:

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

Screening of the lambda gt 11 library using the clon da-derived sequence as a probe gave approximately 1 in 50,000 positive colonies. An isolated purified clone that hybridized to this probe was named clone 33b.

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

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

Clone 33b evidently contains a continuous open reading frame which is an extension of the open reading frames in the overlapping clones, 37b, 35, 36, 81, and 32. The polypeptide encoded in clone 33b is in the same framework as that encoded in the extended open reading frame of these overlapping clones.

IV.A.14. Isolation of HCV cDNAs Overlapping the cDNA in Clone 37b and cDNA in Clone 33b

To isolate HCV cDNAs overlapping the cDNAs in clone 37b and clone 33b, the following synthetic oligonucleotide probes derived from the cDNAs in those clones were used to "screen" the lambda gt 11 library, essentially using the method described in Section IV.A.3.

5 'CAG GAT GCT GTC TCC CGC ACT CAA CCT С 3'

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

were used to find colonies containing HCV cDNA sequences that overlap those in clones 37b and 3b, respectively. About 1 in 100,000 colonies were detected with each probe. A clone containing cDNA "upstream" from and overlapping the cDNA in clone 37b was named clone 40b, a clone containing cDNA "downstream" from cDNA in clone 33b and overlapping it Clone was named 25c.

IV.A.15. Nucleotide Sequences of HCV cDNA in Clone 40b and In Clone 25c

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

The 5'-terminal nucleotide of clone 40b is a G. However, the cDNAs of five other independent clones isolated during the procedure were isolated in clone 40b, description in Section IV.A.14. Also sequenced.

The cDNAs of these clones also contain a T in this position. Thus, G can represent a cloning artifact (see discussion in Section IV.A.11.).

The 5 "end of clone 25c is ACT but the sequence of this region is in clone da (sequence not shown) and in clone 33b int TCA.

This difference may also represent a cloning artifact such as the 28 extra 5 'terminal nucleotides in clone 5-1-1.

Clones 40b and 25c evidently each contain an open reading frame (ORF), which is an extension of the continuous open reading frame in the previously sequenced clones. The nucleotide sequence of the open reading frame passing through clones 40b, 37b, 35, 36, 81, 32, 33b and 25c and the amino acid sequence of the putative polypeptides encoded therein are shown in FIG. In the figure, the potential artifacts have been omitted from the sequence, and instead the corresponding sequences are shown in non-5'-terminal regions of multiple overlap clones.

IV.A.16. Preparation of a Composite HCV cDNA from the cDNAs In Clones 36, 81 and 32 The composite HCV cDNA, C100, was constructed as follows. First, the cDNAs from clones 36, 81 and 32 were excised with EcoRI. The EcoRI fragment of the cDNA from each clone was individually cloned into the EcoRI site of the vector pGEM3-blue (Promega Biotec). The resulting recombinant vectors containing the cDNAs of clones 36, 81 and 32 were named pGEM3-blue / 36, pGEM 3-blue / 81 and pGEM 3-blue / 3, respectively. The appropriately oriented pGEM 3-blue / 81 recombinant was digested with Nael and NarI and the large (~ 2850bp) fragment was purified and cloned with the small (~ 570 bp) Nael / Narl-purified restriction fragment of pGEM 3-blue / 36 ligated. This composition of the cDNAs of clones 36 and 81 was used to generate another pGEM 3 blue vector containing the continuous HCV ORF contained in the overlap cDNA within these clones. This new plasmid was then digested with Pvull and EcoRI to release a fragment of about 680bp, which was then ligated to the small (580bp) Pvull / EcoRI fragment isolated from the appropriately oriented pGEM 3 -blue / 32 plasmid and the composite cDNA from clones 36, 81 and 32 was ligated into the Eco RI-linearized vector pSODcf 1 described in Section IV.B.1. and was used to express Cion 5-1-1 in bacteria. Recombinants containing> 1270bp EcoRI fragment of composite RCV cDNA (C100) were selected and the cDNA from the plasmids was excised with EcoRI and purified.

IV.A.17. Isolation and nucleotide sequences of HCV cDNAs in clones 141, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g, and 39c The HCV cDNAs in clone 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g, and 39c were isolated by the technique of isolating overlapping cDNA fragments from the lambda gt11 library of HCV cDNAs described in Section IV.A.1. to be discribed. The technique used was essentially as described in Section IV.A.3, except that the probes used were designed from the nucleotide sequence of the last isolated clones of the 5 'and the З' ends of the combined HCV sequences. The frequency of the clones that hybridized with the probes described below was about 1 in every 50,000.

The nucleotide sequences of the HCV cDNAs in clones 141,7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were prepared essentially as described in Section IV.A.2. except that the cDNA excised from these phages was substituted for the cDNA isolated from clone 5-1-1.

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

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

The sequence of the HCV cDNA in clone 33c and the overlap with that in clone 40b are shown in Fiy. 15, ai.ch shows the amino acids encoded therein.

Clone 8h was isolated with a probe based on the sequence of nucleotides in clone 33c. The nucleotide sequence of the probe

5 'AGA GAC AAC CAT GAG GTC CCC GGT GTT C 3'.

The sequence of the HCV cDNA in clone 8h and the overlap with that in clone 33c and the amino acids encoded therein are shown in FIG.

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

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

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

shown.

Clone 14c was isolated with a probe based on the sequence of nucleotides in clone 25 с. The sequence of clone 25 с is

shown in Fig. 13. The probe had the sequence when isolating clone 14c

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

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

Clone 8f was isolated using a probe based on the sequence of nucleotides in clone 14c. The nucleotide sequence of the probe was

5 'TCC ATC TCT CAA GGC AAC TTG CAC CGC TAA 3 ¹ .

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

shown.

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

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

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

Clone 33g was isolated using a probe based on the sequence of nucleotides in clone 33f. The nucleotide sequence of the probe was

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

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

Clone 7f was isolated using a probe based on the sequence of nucleotides in clone 7e. The nucleotide sequence of the probe was

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

The sequence of HCV cDNA in clone 7f, its overlap with clone 73, and the amino acids encoded therein are shown in FIG

shown.

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

5 'CAC СТА TGT TTA TAA CCA TGT CAC TCC TCT 3'.

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

shown.

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

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

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

shown.

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

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

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

IV.A.18. The composite HCV cDNA sequence derived from isolated HCV cDNA-containing clones The HCV sequences in the isolated clones described above were aligned to form a composite To provide HCV cDNA sequence. The isolated clones arranged in the 5'-4 'direction are: 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35,

36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g and 39c. A composite HCV cDNA sequence deduced from the isolated clones and the amino acids encoded therein are shown in FIG.

In creating the composite sequence, the following sequence heterogeneities were considered. Clone 33c

contains an 800 base pair HCV cDNA that overlaps the cDNAs in clones 40b and 37c. In clone 33c, as well as in other overlapping clones, nucleotide 789 is G. In clone 37b (see Section IV.A.11.), However, the corresponding nucleotide is an A. This sequence difference creates apparent heterogeneity in the amino acids encoded therein

CYS or TYR for G and A, respectively. This heterogeneity may be important ramifications in protein folding.

to have.

Nucleotide residue 2 in clone 8h HCV cDNA is T. However, as shown below, the corresponding residue in clone 7e

an A. In addition, an A in this position is also found in 3 other isolated overlapping clones. Thus, the T residue in clone 8h may represent a cloning artifact.

Therefore, the remainder in this position in Fig. 26 is referred to as an A.

The 3'-terninal nucleotide in clone ef HCV cDNA is a G. However, the corresponding residue in clone 33f and i-wei other overlapping clones is T. Therefore, the remainder in this position in Fig. 26 is considered to be a T calculated.

The З'-terminala sequence in clone f-HCV cDNA is TTGC. However, the corresponding sequence in clone 33g and in two other overlapping clones is ATTC. That is why in Flg. 26 represents the corresponding region as ATTC.

Nucleotide residue 4 in clone SSg HCV cDNA is a T. In clone 33f and in two other overlapping clones, however, the corresponding residue is A. Therefore, the corresponding residue in Figure 26 is designated as A.

The З'-end of Cion 14i is AA, while the corresponding duncleotide is TAb in clone 11b and TA in three other clones. Therefore, the TA remainder is shown in FIG.

The resolution of the other sequence heterogeneities is discussed above.

Examination of the composite HCV cDNA reveals that it contains a large open reading frame. This suggests that the virus genome is translated into a large polypeptide which is processed simultaneously with or subsequent to translation.

IV.A.19. Isolation and nucleotide sequences of HCV cDNAs In clones 12f, 3Sf, 19g, 26g and 15e The HCV cDNAs in clones 12f, 35f, 19g, 26g and 15e were prepared essentially as described in Section IV.A.17. technique except that the probes were as indicated below. The frequency of clones that hybridized with the probes was in each case about 1 in 50,000. The nucleotide sequences of the HCV cDNAs in these clones were essentially as described in Section IV.A.2. determined, except that the cDNA from the input clones was used in place of the cDNA isolated from clone 5-1-1.

Isolation of clone 12f, which contains cDNA "upstream" from the HCV cDNA in Figure 26, was done using a hybridization probe based on the sequence of nucleotides in clone 14i The nucleotide sequence of the probe was

5 'TGC TTG TGG ATG ATG СТА CTC ATA TCC СТА З ¹ .

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

The isolation of clone 35f containing cDNA "downstream" from the HCV cDNA in Figure 26 was performed using a hybridization probe based on the sequence of nucleotides in clone 39c

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

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

Clone 19g was isolated using a hybridization probe based on the З 'sequence of clone 35f. The nucleotide sequence of the probe was

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

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

Isolation of clone 26g was done using a hybridization probe based on the З '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 overlap with the sequence in clone 19g, and the amino acids encoded therein are shown in FIG.

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

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

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

The clones described in this section were used under the conditions described in section U.A. described. Conditions were deposited with the ATCC and received the following access numbers:

Lambda gt11 ATCC-No. deposit date Clone Ш 40514 Nov. 10, 1988 Clon35f 40511 Nov. 10, 1988 Clone 15e 40513 Nov. 10, 1988 Clone K 9-1 40512 Nov. 10, 1988

The HCV cDNA sequences in the isolated clones described above were aligned to create the composite HCV cDNA sequence. The isolated clones arranged in the 5'-3 'direction are:

12f, Иі.У ^ е.вІі.ЗЗМОЬ.ЗУЬ.Зб.Зб.ві.З ^ ЗЗЬ.гБс.Нсв ^ ЗЗ ^ ЗЗд.ЗЭсЗбт, 19g, 26g and 1Бѳ.

A composite HCV cDNA sequence deduced from the isolated clones and the amino acids encoded therein are shown in FIG.

IV.A.20. Alternative Method of Isolating cDNA Sequences "upstream" from the HCV cDNA Sequence in Clone 12f Based on the largest З'-HCV sequence in Figure 32 derived from the HCV cDNA in clone 12f. For example, small synthetic oligonucleotide primers of reverse transcriptase are synthesized and used to bind to the corresponding sequence in HCV genomic RNA to initiate reverse transcription of the upstream sequences. The primer sequences are proximal to the known 5'-terminal sequence of clone 12f but sufficiently "downstream" to allow the design of probe sequences "upstream" of the primer sequences.

Known standard startup and execution methods are used. The resulting cDNA libraries are screened with upstream sequences from the priming sites (as deduced from the illustrated sequence at clone 12f). HCV genomic RNA is either extracted from plasma or liver samples from chimpanzees with NANBH or from analog samples obtained from humans using NANBH.

IV.A.21. Alternative method using "parts" to isolate sequences from the 5'-terminal region of the

HCV genome

To isolate the extreme 5'-terminal sequences of the HCV RNA genome, the cDNA product of the first round of reverse transcription, which is duplexed with the template RNA, is "tailed" with oligo-C The second round of cDN A synthesis, which provides the complement of the first cDNA strand, is performed using oligo-G as the primer for the reverse transcriptase reaction for genomic HCV RNA are described in Section IV.A.20 .. The methods for "tailing" with terminal transferase are as described in Maniatis et al. (1982). The cDNA products are then cloned, screened and sequenced.

IV.A.22. Alternative method using "parts" for insulation

This method is based on previously used methods for cloning cDNAs of flavivirus RNA. In this method, the RNA is subjected to denaturing conditions to remove secondary structures at the З'-end, and is then "tailed" using rATP as a substrate with poly-A polymerase. The reverse transcription of the poly-A tailed "RNA is catalyzed by reverse transcriptase using oligo-dT as a primer. The second cDNA strands are synthesized, the cDNA products are cloned, screened and sequenced.

IV.A.23. Creation of lambda gtl 1 HCV cDNA blot libraries containing larger cDNA inserts. The method used to create and screen the lambda gt11 library is essentially as described in Section IV.A.1, except that the library is generated from a pool of larger cDNAs isolated from the Sepharose CL-4B library. Column were eluted.

IV.A.24. Creation of HCV cDNA Blots by Priming Synthetic Olfomers. New HCV cDNA libraries were prepared from the RNA described in Section IV.A.1. obtained from the infectious chimpanzee plasma pool and from the poly-A ⁺ fraction recovered from the liver of this infected animal. The cDNA was constructed essentially as described by Gubler and Hoffman (1983), except that the primers for the synthesis of the first cDNA strand were two synthetic oligomers based on the sequence of the HCV genome described above. Primers based on the sequence of clones 11b and 7e were

5 'CTG GCT TGA AGA ATC 3'

or

 5 'AGT TAG GCT GGT GAT TAT GC 3'.

The resulting cDNAs were cloned into lambda bacteriophage vectors and screened with various other synthetic oligomers whose sequence was based on the HCV sequence, Figure 32.

IV.B. Expressfon of polypeptides encoded in HCV cDNAs and identification of the expressed products as HCV-inducible antigens

IV.B.1. Expression of the polypeptides encoded in clone 5-1-1 The HCV polypeptide encoded in clone 5-1-1 (see Section IV.A.2., Supra) was used as a fusion polypeptide Superoxide dismutase (SOD) expressed. This was done by subcloning the clone 5-1-1 cDNA insert into the expression vector

pSODcf 1 (Steimer et al. [1986]) in the following manner.

First, DNA isolated from pSODcf 1 was treated with BamH 1 and EcoRI, and the following linker was inserted into the linear DNA

ligated created by the restriction enzymes:

5 'GAT CCT GGA ATT CFG ATA A 3' 3 'GA CCT TAA GAC TT TTT AA 5'.

After cloning, the plamid containing Jas insert was isolated.

The insert containing plaque was restricted with Eco RI (restricted). The HCV cDNA insert in clone 5-1-1 was excised with EcoRI and ligated into this FcoRI linearized plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al. [19EO]). Recombinants with the 5-1-1 cDNA in the correct orientation for the expression of the ORF, see Figure 1, were identified by restriction mapping and nucleotide sequencing. Recombinant bacteria from a clone were induced by culturing the bacteria in the presence of IPTG to express the SOD-NANBs M polypeptide.

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

The HCV cDNA contained in clone 81 was expressed as a SOD-NANB fusion polypeptide. The method for preparing the vector encoding this fusion polypeptide was analogous to the method used to create the SOD-NANB ₅ ... Vector, except that the source of the HCV cDNA was clone 81, which was prepared according to the

Description in section IV.A.3. and for which the DNA sequence described in Section IV.A.4.

was determined. The nucleotide sequence, HCV cDNA in clone 81 and the putative amino acid sequence of the polypeptides encoded therein are shown in FIG.

The HCV cDNA insert in clone 81 was excised with EcoRI and ligated into pSODcf 1 containing the linker (see IV.B.1.) And linearized by treatment with EcoRI. The DNA mixture was used to transform E. coli strain D1210. Recombinants with the clone 81 HCV cDNA in the correct orientation for the expression of the ORF (open reading frame) shown in Figure 4 were identified by restriction mapping and nucleotide sequencing.

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

IV.B.3. Identification of Polypeptides Encoded in Clone 5-1-1 as an HCV and NANBH Associated Antigen The polypeptide encoded in the HCV cDNA of clone 5-1-1 was identified as a NANBH-associated antigen by demonstrating that sera of NANBH infected chimpanzees and humans immunologically react with the fusion polypeptide, SOD-NANB ₈ .,. ,, which is composed of superoxide d 'mutase at its N-terminus and the in-frame 5-1-1 antigen its C-terminus, by Western blotting (Towbin et al., 1979J) in the following manner.

A recombinant bacterial strain transformed with an expression vector encoding the SOD-NANB ^ .r polypeptide, as described in Section IV.B.I., was induced by growth in the presence of IPTG to express the fusion polypeptide. All bacterial lysate was electrophoresed through polyacrylamide gels in the presence of SDS according to Laemmli (1970). The separated polypeptides were transferred to nitrocellulose filters (Towbin et al., (1979)). The filters were then cut into thin strips and the strips were individually incubated with the different chimpanzee and human sera. Bound antibodies were detected by further incubation with ''! -Labeled sheep anti-human IG, as described in Section IV.A.1.

The characterization of the chimpanzee sera used for the Western blots and the results shown in the photograph of the autoradiographically picked strips are shown in Figure 33. Polypeptide-containing nitrocellulose strips were incubated with sera from chimpanzees at different time points during the acute NANBH Iutchinson's strain infections (lane 1-16), hepatitis A infections (lane 17-24 and 26-3), and hepatitis B infections (lane 34-44) and Figure 45 shows positive controls in which immunoboots were incubated with serum from the patient used to identify recombinant clone 5-1-1 in the original lambda gt11 cDNA library screen (see Section IV.A.1.). was taken.

The band visible in control lanes 25 and 45 in Figure 23 reflects the binding of antibodies to the NANB ₆ .,., Component of the SOD fusion polypeptide. Not only did these antibodies bind to SOD, as this was also included as a negative control in these samples, they would have appeared as a band migrating significantly faster than the SOD-NANB ^ io fusion polypeptide.

Lanes 1-6 of Figure 33 show antibody binding in serum samples from 4 chimpanzees. The samples were obtained immediately before infection with NANBH and then during the acute infection. While the serum samples collected prior to administration of the infectious HCV inoculum and during the early acute phase of infection lacked antibodies that immunologically reacted with the SOD-NANBs.,., Polypeptide, FIG. Finally, all 4 animals induced antibodies to this polypeptide circulating during the final part of the acute phase or subsequently to it. Additional bands observed on chimpanzees Nos. 3 and 4 on the immunoblots were due to background binding to host bacterial proteins.

In contrast to the results obtained with sera from NANBH-infected chimpanzees, the development of antibodies against the NANB ^ M component of the fusion polypeptide was detected in 4 chimpanzees infected with HAV and 3 chimpanzees infected with HBV, respectively were not observed. The only binding in these cases was background binding to host bacterial proteins, which also occurred in the HCV-infected samples. The characterization of the human sera used for the "Western blots" and the results shown in the photograph of the autoradiographically picked strips are shown in Figure 34. Polypeptide-containing nitrocellulose strips were incubated with sera obtained from humans at different times during the period Infection with NANBH (lane 1-21), HAV (lane 33-40), and HBV (lane 41-49). Lanes 25 and 50 show positive controls in which the immunoblots were incubated with patient serum which was used in original screen of the lambda gt 11 library described above. Lanes 22-24 and 26-32 show "uninfected" controls in which the sera were from "normal" blood donors.

As can be seen in Figure 34, the sera from nine NANBH patients, including the serum used to screen the Lambdagt II library, contained antibodies against the NANB &, -, - component of the fusion polypeptide. The sera from three patients with NANBH did not contain these antibodies. It is possible that the anti-NANB ^ M antibodies develop in these patients at a later date. It is also possible that this lack of response resulted from a different NANBV agent that was causative for the disease in the individuals from whom the non-responsive serum was taken.

Fig. 34 also shows that sera from many patients infected with HAV and HBV did not contain anti-NANBs.,... Antibodies, and that these antibodies were not present in the sera of "normal" controls, although an HAV patient (Lane 36) apparently has anti-NANB antibodies, it is possible that this patient was previously infected with HCV, since the incidence of NANBH is very high and is often subclinical.

These serological studies show that the cDNA in clone 5-1-1 encodes epitopes specifically recognized by sera from BB-NANBV infected patients and animals. In addition, the cDNA apparently is not derived from the primate genome. A hybridization probe made by clone 5-1-1 or clone 81 did not hybridize to "Southern blots" of control human and chimpanzee genomic DNA under conditions where unique, single-copy genes were detectable infected individuals. These probes also did not hybridize to "Southern blots" of control bovine genomic DNA.

IV. "4. Expression of polypeptides encoded in a composition of HCV DNAs in clones 36, 81 and 32 The HCV polypeptide encoded in the open reading frame extending through clones 36, 81 and 32 was probed with SOD was expressed as a fusion polypeptide by inserting the composite cDNA, C100, into an expression cassette containing the human superoxide dismutase gene, inserting the expression cassette into a yeast expression vector, and expressing the polypeptide in yeast.

An expression cassette containing the C100 cDNA derived from clones 36, 81 and 32 was constructed by inserting the ~1270 bp EcoRI fragment into the EcoRI site of the vector pS3-56 (also called pS356), the plasmid pS3-56 _C ioo was won. The construction of C100 is in Section IV.A.16 above. described.

Vector pS3-56, which is a pBR322 derivative, contains an expression cassette composed of the ADH2 / GAPDH hybrid yeast promoter "upstream" of the human superoxide dismutase gene and a "downstream" GAPDH transcription terminator. A similar cassette containing these control elements and the superoxide dismutase gene is described in Cousens et al. (1987) and co-pending application GPO 196056, published October 1, 1986, which is the co-owned property of the assignee of this application. The cassette in pS3-56, however, differs from that in Cousens et al. (1987), where the heterologous proinsulin gene and the immunoglobulin "hinge" are deleted, and where at the _{end of} the superoxide dismutase there is an adapter sequence containing an EcoRI site The sequence of the adapter is:

5 '-AATTTGGGAATTCCATAATGAG -3'

AC CCT TAA GGT ATT ACT CAG CT

The EcoRI site allows the insertion of heterologous sequences which, when expressed by a vector containing the cassette, yield polypeptides having an amino acid sequence:

Asn-leu-gly-ile-ar.

containing oligopeptide linker are fused to superoxide dismutase.

A sample of pS356 was deposited on Apr. 29, 1988 under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Maryland 20853, and received accession number 67683. The availability and access conditions the stored material and the preservation of the deposited material are the same as in section HA specified for strains containing NANBV cDNAs. This deposit is intended to provide relief, but not the practical embodiment of the invention in light of the description given herein. The deposited material is incorporated herein by reference.

After isolation of recombinants containing the C100 cDNA insert in the correct orientation, the C100 cDNA-containing expression cassette was excised with BamHI from pS3-56cioo and a cassette containing ~~ 3400bp fragment was isolated and purified. This fragment was then inserted into the BamHI site of yeast vector pAB24. Plasmid ρ AB 24, whose significant features are shown in Figure 35, is a yeast "shuttle" vector containing the complete 2-micron sequence for replication (Broach (1981)) and pBR322 sequences of plasmid YEp24 (Bostein et al. [1979]) derived yeast URA3 gene and the derived plasmid pCI / 1 yeast ^{LEU? d} gene. EPO publication no. 116,201. plasmid AB ρ 24 was described in US- Application Serial No. 138,894 owned by the assignee of the present invention Plasmid pAB24 was constructed by digesting VEp24 with EcoRI and religating the vector to remove the 2 micron partial sequence The resulting plasmid, YEP24deltaRI, was linearized by digestion with CIaI and ligated with the complete 2 micron plasmid which had been linearized with CIaI The resulting plasmid, pCBou, was then digested with SbaI and the 8605 bp VekUir fragment was gel isolated This isolated XbaI fragment became with a 4460 bp SbaI fragment containing the pCI / 1-isolated LEU ^2d gene. The orientation of the LEU ^2d gene is in the same direction as the URAS gene. Insertion of the expression was at the unique EcoRI site of the pBR322 sequence (illegible site), disrupting the gene for bacterial resistance to tetracycline.

The recombinant plasmid containing the SOD-C 100 expression cassette, pAB24100-3, was transformed into the yeast strain JSC308 as well as into other yeast strains. The cells were prepared as described by Hinnen et al. (1978) and plated on ura-selective plates. Single colonies were inoculated into leu-selective media and grown to saturation. The culture was induced by growth in YEP containing 1% glucose for expression of the SOD polypeptide (called C100-3).

Strain JSC 308 is of the genotype MAT, leu 2, ura 3 (del) DM15 (GAP / ADR 1) integrated at the ADR1 locus. In JSC308, overexpression of the positive activator gene product ADR1 results in hyperderepression (relative to an ADR1 wild-type control) and significantly higher yields of expressed heterologous proteins when such proteins are synthesized via an ADH2-UAS regulatory system. The construction of yeast strain JSC308 is disclosed in co-pending application, US Patent Application Serial No. 2300-0229, filed concurrently herewith and incorporated herein by reference. A sample of JSC 308 was deposited with the ATCC under the terms of the Budapest Treaty on May 5, 1988 and received accession number 20879. The conditions of availability and access to the deposited material and conservation of deposit are the same as those described in Section H.A. specified for HCV cDNAs containing strains.

The complete C 100-3 fusion polypeptide encoded in pAB 24C100-3 should contain 154 amino acids of human SOD at the amino terminus, 5 amino acid residues derived from the EcoRI site-containing synthetic adapter, 363 amino acid residues derived from C 100 cDNA, and 5 carboxy residues. contain terminal amino acids derived from the MS2 nucleotide sequence adjacent to the HCV cDNA sequence in clone 32. (See Section IV. A. 7.) The putative amino acid sequence of the carboxy terminus of this polypeptide starting at the penultimate Ala residue of SOD is shown in FIG. Also shown is the nucleotide sequence encoding this portion of the polypeptide.

IV. 5. Identification of the C10O-encoded polypeptide as a NANBH-associated antigen

The C 100-3 fusion polypeptide expressed from plasmid pAB 24 C100-3 in yeast strain JSC 308 was characterized for size, and the '.100 C polypeptide was identified for its immunological responsiveness to serum from a human with chanical NANBH identified as a NANBH-associated antigen.

The C 100-3 polypeptide prepared as described in Section IV. B.4. was expressed as follows. Yeast JSC-303 cells were transformed with ρ AB 24 or pAB24C 100-3 and were induced to express the heterologous plasmid-encoded polypeptide. The induced yeast cells in 1 ml of culture (OD _86Onm ~20) were _pelleted by centrifugation at 10000 rpm for one minute and were lysed by vortexing vigorously (10x1 min) with 2 volumes of solution and 1 volume of 0.2 nanometer diameter glass beads , The solution contained 50 mM Tris-HCl, pH 8.0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin. Insoluble material in the lysate containing the C 100-3 polypeptide was collected by centrifugation (10000 rpm for 5 minutes) and dissolved in Laemmli SDS sample buffer for 5 minutes. (See Laemmli (197O)). A polypeptide equivalent to that in 0.3 ml of the induced yeast culture was electrophoresed through 10% polyacrylamide gels in the presence of SDS according to Laemmli (1970) Protein standards were co-electrophoresed on the gels. The gels containing expressed polypeptides were either stained with Coomassie Brilliant Blue or subjected to Western blotting as described in Section IV. B. 2. using serum from a patient with chronic NANBH to assess the immunological responsiveness of pAB24 and from The results are shown in Figure 37. In Figure 37A, the polypeptides were stained with Coomassie Brilliant Blue The insoluble polypeptides of JAB 308 transformed with pAB 24 and of two further colonies of pAB 24 C100 3 can be seen in lanes 1 (pAB 24) and 2 and 3. A comparison of lanes 2 and 3 with lane 1 shows the induced Expression of a 54,000 dalton molecular weight polypeptide from JSC 308 transformed with pAB 24 C100-3, which is not induced in pAB24-transformed JSC 308. This polypeptide is indicated by the arrow. Figure 37B shows the results of the Western blots of the insoluble polypeptides expressed in ρ AB 24 (lane 1) or pAB 24C100-3 (lane 2) yeast strain JSC 308. The polypeptides expressed from pAB24 were with human serum immunoreactive with NANBH, however, as indicated by the arrow, JSC 308 transformed with pAB24C 100-3 expressed a polypeptide with a molecular weight of "54,000 daltons that reacted with the human NANBH serum. The other immunologically reactive polypeptides in lane 2 may be degradation and / or aggregation products of this 54,000 dalton polypeptide.

IV. B. 6. Purification of Fuslonpolypepttd C100-3 The fusion polypeptide composed of SOD at the N-terminus and in-frame C 100 HCV polypeptide at the C-terminus C100-3 was obtained by differential extraction of the insoluble fraction of the extracted host yeast cells containing the polypeptide

was purified.

The fusion polypeptide C100-3 was purified as described in Section IV.B.4. described in yeast strain transformed with pAB24Ci00-3 JSC 308 expressed. The yeast cells were then lysed by homogenization, the insoluble material in the lysate was added

pH 12.0 was extracted and C100-3 in the remaining insoluble fraction was solubilized in SDS-containing buffer.

The yeast lysate was prepared essentially according to Nagahuma et al. (1984). A yeast cell suspension was prepared. It consisted of 33% cells (v / v) suspended in a solution (Buffer A) containing 20 mM Tris-HCl, pH 8.0.1 mM Dithiothreitol and 1 mM phenylmethylsulfonyl fluoride (PMSF). An aliquot of the suspension (15ml) was added

an equal volume of glass beads (0.45 to 0.50 mm in diameter) and the mixture was added

Maximum speed for 8 minutes in a Super Mixer (Lab Line Instruments, Inc.). Homogenate and glass beads

were separated and the glass beads were washed three times with the same volume of Buffer A as that initially used

Cells washed. After combining washings and homogenate, the insoluble material in the lysate became

by centrifuging 4 ⁰ C for 15 minutes, resuspending the pellets in twice the volume of buffer A as the cells initially used, and pelletizing the material by centrifuging at 7,000 ° C for 15 minutes. This washing was repeated three times.

The insoluble material from the lysate was extracted at pH 12.0 as follows. The pellet was dissolved in buffer containing 0.5 M NaCl, 1 mM EDTA contained, with the suspending volume corresponding to 1.8 times dom of the cells initially employed. The

pH of the suspension was adjusted by adding 0.2 volume of 0.4 M Na phosphate buffer, pH 12.0. After mixing, the suspension was centrifuged at 4 ° C for 15 minutes at 7000 g, and the supernatant was removed. The

Extraction was repeated twice. The extracted pellets were washed by placing them in 0.5 M NaCl, 1 mM EDTA,

were suspended, using a suspension volume that was twice that of the cells initially used. Damn followed 15 minutes centrifugation at 4 ⁰ C at 7000 g.

The C 100-3 polypeptide in the extracted pellet was solubilized by treatment with SDS. The pellets were in one Buffer A amount corresponding to 0.9 volume of the volume of cells initially charged, and 0.1 volume

2% SDS was added. After mixing the suspension, it was centrifuged at 4 ^° C. for 15 minutes at 7000 g.

The resulting pellet was extracted a further 3 times with SDS. The resulting supernatants containing C100-3 were

collected.

This procedure purifies C100-3 more than 10-fold from the insoluble fraction of the yeast homogenate, and the Recovery of the polypeptide is more than 50%, The purified fusion polypeptide preparation was analyzed by polyacrylamide electrophoresis according to Laemmli (1970). Based

In this analysis, the polypeptide was more than 80% pure and had an apparent molecular weight of 54,000 daltons.

IV. C. Identification of RNA hybridizing to HCV cDNA in infected individuals IV. C. 1. Identification of HCV cDNA hybridized RNA in a chimpanzee liver with NANBH It has been demonstrated that RNA from the liver of a chimpanzee that had NANBH contained an RNA species that binds to in Cbn 81 hybridized by Northern blotting in the following manner. RNA was isolated from chimpanzee liver biopsy material, of which the high titer plasma (see Section IV. A. 1.).

obtained using techniques described in Maniatis et al. (1982) for the isolation of total RNA

Mammalian cells and for their separation into poly A ⁺ and poly A 'fractions. These RNA fractions were Electrophoresed on a formaldehyde / agarose gel (1% w / v) and transferred to nitrocellulose. (Maniatis

et al. [1982]). The nitrocellulose filters were hybridized with radiolabelled HCV cDNA from clone 81 (see Figure 4 for the nucleotide sequence of the insert). To prepare the radiolabelled probe, the HCV cDNA insert isolated from clone 81 was radiolabeled by nick translation using DNA polymerase I (Maniatis et al., 1982) Hybridization was at 42 ° for 18 hours C in a solution containing 10% (mass / volume) dextran sulfate, 50% (mass / volume) deionized formamide, 75 mM NaCl, 75 mM Na citrate, 20 mM Na ₂ HPO ₄ , pH 6.5, 1% SDS, 0.02% (mass / volume) Bovine serum albumin (BSA), 0.02% (mass / volume) Ficoll-400.0.02% (mass / volume) Polyvinylpyrrolidone, 100 micrograms / ml of salmon sperm DNA sheared and denatured by sonication containing 10 ^{μe of} CPM / ml of the nick-translated cDNA probe.

An autoradiographic image of the transmitted filter is shown in FIG. Lane 1 contains ³² P-labeled restriction fragment markers. Lanes 2-4 contain chimpanzee liver RNA as follows: Lane 2 contains 30 micrograms of total RNA; Lane 3 contains 30 micrograms of poly A RNA; and lane 4 contains 20 micrograms of poly A ⁺ RNA. As shown in Figure 32, the chimpanzee liver with NANBH contains a heterogeneous population of related poly A ⁺ RNA molecules which hybridizes to the HCV DNA probe and which is apparently about 5,000 to 11,000 nucleotides in size. This RNA, which hybridizes to the HCV cDNA, could be viral genomes and / or specific transcripts of the virus genome. The experiment described in Section IV. C. 2. below confirms the suggestion that HCV contains an RNA genome.

IV. C. 2. Identification of HCV-Derived RNA in the Serum of Infected Individuals Nucleic acids were isolated from particles prepared from chimpanzee NANBH plasma as described in Section IV

high titer were isolated. Aliquots (equivalent to 1 ml of original plasma) of the isolated nucleic acids were resuspended in 20 microliters of 50 mM Hepes, pH 7.5.1 mM EDTA and 16 micrograms / ml yeast-soluble RNA. The samples were denatured by boiling for 5 minutes, followed by RNase A (5 microliters containing 0.1 mg / ml RNase A in 25 mM EDTA, 40 mM Hepes, pH 7.5) or DNase I (5 microliters containing 1 Unit DNase I in 10 mM

MgCl ₂ , 25mM Hepes, pH 7.5). Control samples were incubated without enzyme. After incubation were

230 microliters of ice-cold 2XSSC containing 2 micrograms / ml of yeast-soluble RNA were added and the samples were placed on a

Nitrocellulose filtered. The filters were hybridized with a cDNA probe of clone 81 by nick translation

32p-marked. Fig. 39 shows an autoradiographic image of the filter. Hybridization signals were in the

DNase-treated and control samples (lanes 2 and 1, respectively), but not in the RNase-treated sample

(Lane 3). Thus, because RNase A treatment destroys the nucleic acids isolated from the particles and corrupts DNase treatment

Had effect, it can be assumed that the HCV genome consists of RNA. IV.C.3. Detection of HCV Nucleic Acid Sequences Derived from NANBH liver and plasma samples from chimpanzees

amplified nucleic acid sequences

In the liver and plasma of chimpanzees with NANBH and in control chimpanzees existing HCV nucleic acids were essentially using the Saiki et al. (1986) PCR technique (PCR = polymerase chain reaction) amplified. The primer oligonucleotides were derived from the HCV cDNA sequences in clone 81 or clones 36 and 37. The amplified sequences were detected by gel electrophoresis and Southern blotting using as probes the appropriate cDNA oligomer having a sequence from the region between, but not including, the two primers.

Samples of RNA with HCV sequences to be examined by the amplification system were isolated from liver biopsy material from three chimpanzees with NANBH and from two control chimpanzees. Isolation of the RNA fraction was performed by the methods described in Section IV.C. 1. described Guanidiniumthiocyanatprozedur.

RNA samples to be assayed by the amplification system were also isolated from the plasma of two chimpanzees with NANBH and a control chimpanzee and from a pool of control chimpanzee plasmas. One infected chimpanzee had a CID / ml equal to or greater than 10 ^s , and the other infected chimpanzee had a CID / ml equal to or greater than 10 ^s .

The nucleic acids were extracted from the plasma as follows. Either 0.1 ml or 0.01 ml of plasma was diluted to a final volume of 1.0 ml with a TENB / proteinase K / SDS solution (0.05M Tris-HCl, pH 8.0, 0.01 M EDTA incubated 0.1 M NaCl, 1 mg / ml proteinase K and 0.5% SDS) containing 10 micrograms / ml Polydenylsäure, and 60 minutes at 37 ⁰ C. After this proteinase K digestion, resulting plasma fractions were deproteinized by extraction with TE (10.0 mM Tris-HCl, pH 8.0, 1 mM EDTA) -saturated phenol. The phenol phase was separated by centrifugation and reextracted with TENB containing 0.1% SDS. The resulting aqueous phases from each extraction were pooled and extracted twice with an equal volume of phenol / chloroform / isoamyl alcohol / 1: 1 (99: 2) / and then twice with an equal volume of a 99: 1 mixture of chloroform / isoamyl alcohol. After phase separation by centrifugation, the aqueous phase was brought to a final concentration of 0.2 M Na-acetate and the nucleic acids were precipitated by the addition of two volumes of ethanol. The precipitated nucleic acids were recovered by ultracentrifugation in an SW-41 rotor at 38K for 60 minutes at 4 ° C.

In addition, the high titer chimpanzee plasma and the collected control plasma were alternately extracted by the method of Chomcyzaki and Sacchi (1987) with 50 micrograms of poly A carrier. In this procedure, acid guanidinium thiocyanate extraction is used. RNA was recovered by centrifugation for 10 minutes at 10,000 rpm at 4 ° C in an Eppendorf microfuge.

In two cases, prior to the synthesis of cDNA in the PCR reaction, the nucleic acids extracted from the plasma by the proteinase K / SDS / phenol method were further purified by binding to and out of S and S-Elutip R columns be eluted. The procedure was carried out according to the manufacturer's guidelines.

The cDNA used as template for the PCR reaction was derived from the nucleic acids prepared as described above (either total nucleic acids or RNA). Following ethanol precipitation, the precipitated nucleic acid was dried and resuspended in DEPC-treated distilled water. Secondary structures in the nucleic acids were separated by lOminütiges holding at 65 ⁰ C, and the samples were immediately cooled on ice. cDNA was synthesized using 1 to 3 micrograms of total chimpanzee RNA from the liver or nucleic acids (or RNA) extracted from 10 to 100 microliters of plasma. Reverse transcriptase was used in the synthesis and was carried out in a 25 microliter reaction vessel according to the protocol specified by the manufacturer, BRL. The primers for cDNA synthesis were those also used in the PCR reaction described below.

All reaction mixtures for cDNA synthesis contained 23 units of the RNase inhibitor RNASIN ™ (Fishery Promega). Following the cDNA synthesis, the reaction mixtures were diluted with water, boiled for 10 minutes, and cooled rapidly on ice.

The PCR reactions were carried out essentially according to the manufacturer's guidelines (Cetus-Perkin-Elmer), with the exception of the addition of 1 microgram RNase A. The reactions were carried out in a final volume of 100 microliters. The PCR technique was carried out in 35 cycles with a Temperturregime of 37 "C, 72 ⁰ C and 94 ⁰ C.

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

5 'CAA TCA TAC CTG ACA G 3' and 5'GATAAC CTC TGCCTG A3 '.

The sequence of the primer of clone 36 was:

5 'GCA TGT CAT GAT GTA T 3'

 The sequence of the clone 37b primer was:

5 'ACA ATA CGT GTG TCA 3'.

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

The products of the PCR reaction were carried separation of the products by Alkaligelektrophorese, followed by "Southern blotting" and detection of the amplified HCV-cDNA sequences with einor ³² p-labeled internal oligonucleotide probe, the overlapping of a non-primer region of the HCV cDNA 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 were collected by Zen centrifugation and dissolved in distilled water Single-stranded DNAs of 60.108 and 161 nucleotides in length were co-electrophoresed on gels as "molecular weight" markers. After electrophoresis, the DNAs in the gel were transferred to Biorad zeta probes ^TH paper. Prehybridization and hybridization as well as washing conditions conformed to the manufacturer's specification (Biorad).

The probes used for hybridization / detection of the amplified HCV cDNA sequences were the following. When the PCR primer pair was recovered from clone 81, the probe was a 108 mer with a sequence corresponding to that in the region between the sequences of the two primers. When the PCR primer pair was derived from clone 36 and clone 37b, the probe was inserted into the nick-translated HCV cDNA derived from clone 35. The primers are from nucleotides 155-170 of the clone 37 b insert and The З'-end of the HCV cDNA insert in clone 35 overlaps nucleotides 1-186 of the insert in clone 36 and the 5'-end of the clone 35. Insets overlap nucleotides 207-269 of the insert in clone 37b (compare Figures 5, 8 and 10) Thus, the cDNA insert in clone 35 spans a portion of the kegion between the sequences of clones derived from clone 36 and clone 37b and is useful as a probe for the amplified sequences that include these primers.

Analysis of the RNA from the liver samples was done according to the above procedure using both primer and probe groups. RNA from the liver of the three chimpanzees with NANBH provided positive hybridization results for amplification sequences of the expected size (161 and 586 nucleotides for 81 and 36 and 37b, respectively) while the control chimpanzees gave negative hybridization results. The same results were obtained when the experiment was repeated three times.

The analysis of the nucleic acids and RNA from the plasma was also carried out according to the above procedure using primers and the probe of clone 81. The plasmas were from two chimpanzees with NANBH, one control chimpanzee and pooled plasma from control chimpanzees. Both NANBH plasmas contained nucleic acids / RNA, which gave positive results in the PCR-amplified assay, while both control plasmas gave negative results. These results were obtained several times in retest tests.

IV.D. Radioimmunoassay for Detecting HCV Antibodies in Serum of Infected Individuals Solid phase radioimmunoassays for detecting antibodies to HCV antigens have been developed based on TSU and Herzenberg (1980). Microtiter plates (Immulon 2, R "movawell strips) are coated with purified polypeptides containing HCV epitopes. The coated plates are incubated with human serum samples suspected of containing antibodies to the HCV epitopes or with matched controls. During incubation, the antibody, if present, is immunologically bound to the solid phase antigen. After removal of the unbound material and washing of the microtiter plates, complexes of human anti-mRNA NANBV antigen are detected by incubation with "'! -Labeled sheep anti-human immunoglobulin. Unbound labeled antibody is removed by aspiration and the plates are washed. The radioactivity in individual wells is determined; the amount of human anti-HCV antibody bound is proportional to the radioactivity in the well.

IV.D.1. Purification of fusion polypeptide SOD-NANBs-M

The fusion polypeptide SOD-NANB ₅ .,... Expressed in recombinant bacteria as described in Section IV.B.1. was purified from the recombinant E. coli by differential extraction of the cell extracts with urea and subsequent chromatography on anion and cation exchange columns as follows.

Thawed cells of 1 liter of culture were resuspended in 10 ml of 20% (mass / volume) sucrose containing 0.01 M Tric-HCl, pH 8.0, and 0.4 ml of 0.5M EDTA, pH 8.0 were added. After 5 minutes at ⁰ ° C, the mixture was centrifuged at 4000 xg for 10 minutes. The resulting pellet was suspended in 10ml of 2b% (mass / volume) sucrose containing 0.05N Tris-HCl, pH 8.0.1mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram / ml Pepstatin A, followed by

Addition of 0.5 ml of lysozyme (10 mg / ml) followed by incubation at ⁰ ° C. for 10 minutes. After addition of 10 ml of 1% (v / v) Triton X-100 in 0.05M Tris-HCl, pH 8.0, 1 mM EDTA, the mixture was incubated for a further 10 minutes at ⁰ ° C. with occasional shaking , The resulting viscous solution was homogenized by passing 6 times through a 20 gauge sterile hypodermic needle and centrifuging for 25 minutes at 13 ° C. The pelleted material was suspended in 5 ml of 0.01 M Tris-HCl, pH 8.0, and the suspension was centrifuged at 4000g g for 10 minutes. The pellet containing SOD-NANB ₆ .,., - fusion protein was dissolved in 5 ml of 6M urea in 0.02M Tric-HCl, pH 8.0, 1 mM dithiothreitol (Buffer A) and placed on a buffer A balanced column given by Q-Sepharose Fast Flow. Polypeptides were eluted with a linear gradient of 0.0 to 0.3 M NaCl in Buffer A. After elution, the fractions were analyzed by polyacrylamide gel electrophoresis in the presence of SDS to determine their level of SOD-NANB ₅ -M. Fractions containing this polypeptide were collected and dlalyzed against 5M urea in 0.02M sodium phosphate buffer, pH 6.0.1mM dithiothreitol (Buffer B). The dialyzed sample was loaded onto a B-balanced column of S-Sepharose Fast Flow, and polypeptides were eluted with a linear gradient of 0.0-0.3 M NaCl in buffer B. The fractions were analyzed by polyacrylamide gel electrophoresis for the presence of SOD-NANB ₅ .,., And the appropriate fractions were pooled.

The final SOD-NANBe-M polypeptide preparation 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. Cleaning of Fuslonpolypeptld SOD-NANB ₁₁

The fusion polypeptide SOD-NANB ₈₁ expressed in recombinant bacteria as described in Section IV.B.2. from recombinant E. coil duich, differential extraction of the cell extracts with urea followed by chromatography on anion and cation exchange columns using those described for the isolation of fusion polypeptide SOD-NANB _5. ,

Procedure (see section IV.D.1.), Cleaned.

The final SOD-NANB ₈₁ polypeptide preparation was examined by electrophoresis on polyacrylamide gels in the presence of SDS. Based on this analysis, the preparation was more than 50% pure.

IV.D.3. Detection of Antibodies Against HCV Epitopes by Solid-Phase Radloimmunoassay Serum samples from 32 patients diagnosed with NANBH were analyzed by radioimmunoassay (RIA) to determine whether antibodies to fusion polypeptides SOD-NANB ₅ .,., And SOD -NANB ₈₁ existing HCV epitopes were detectable.

Microtiter plates were coated with SOD-NANB ₅ .,., Or SOD-NANB ₈ ) prepared according to sections IV.D.1. or IV.D.2. had been partially cleaned. The assays were performed as follows. 100 milliliter aliquots containing 0.1 to 0.5 micrograms of SOD-NANB ₅ .,., Or SOD-NANB ₈₁ in 0.125 M Na borate buffer, pH 8.3.0.075 M NaCl (BBS) were analyzed in each well of a microtiter plate (Dynatech Immulon 2 Removawell strips). The plate was incubated overnight at 4 ° C in a humid chamber, after which the protein solution was removed and the wells washed three times with BBS containing 0.02% Triton X-100 (BBST). To prevent nonspecific binding, the wells were coated with bovine serum albumin (BSA) with the addition of 100 microliters of a 5 mg / ml solution of BSA in BBS, followed by incubation at room temperature over a period of one hour. After this incubation, the BSA solution was removed. The polypeptides in the coated wells were reacted with serum by adding 100 microliter of serum samples diluted 1: 100 in 0.01 M Na phosphate buffer pH 7.2, 15.15 M NaCl (PBS) containing 10 mg / ml BSA, and the wells were incubated with the serum for 1 hour at 37 ° C. After incubation, the serum samples were removed by aspiration and the wells were washed 5 times with BBST. AnJt-NANB ₆ -L ₁ and anti-NANB ₈ i bound to the fusion polypeptides were determined by the binding of ''! -Labeled F '(ab) _2- sheep anti-human IgG to the coated wells 100 microliters of the labeled probe (specific activity 5-20 microcuries / microgram) were added to each well, and the plates were incubated for one hour at 37 ⁰ C, followed by joined removal of excess probe by aspiration and 5 washing with BBST. the The amount of radioactivity bound in each well was determined by counting in a counter that detects gamma radiation

The results of the detection of AnU-NANB ₅ .,., And anti-NANB ₈₎ in individuals with NANBH are given in Table 1.

Table 1

Detection of anti-5-1-1 and anti-81 in sera from NANB, HAV and HBV hepatitis patients

Patient Diagnosis S / N

Reference number Anti-5-1-1 Anti-81

1. 28 'Chronic NANB, IVD ²¹

Chronic NANB, IVD Chronic NANB, IVD

2. 29 'AVH ³¹ , NANB, sporadic

Chronis-4 NANB Chronical, NANB

3. 30 'AVH, NANB, IVD

Chronic NANB, IVD Chronic NANB, IVD

4. 31 Chronic NANB, PT "

5. 32 'Late AVH NANB, IVD

Late AVH NANB, IVD

6. 33 'AVH, NANB, IVD

AVH, NANB, IVD

0.77 4.20 1.14 5.14 2.11 4.05 1.09 1.05 33.89 11.39 36.22 13.67 1.90 1.54 34.17 30.28 32,45 30.84 16.09 8.05 0.69 0.94 0.73 0.68 1.66 1.96 1.53 0.56

Table 1 (continued)

patients diagnosis S / N Anti-5-1-1 Anti-81 reference number 34.40 7.55 7. 34 ' Chronic NANB, PT 45.55 13.11 Chronic NANB, PT 41.58 13,45 Chronic NANB, PT 44,20 15,48 Chronic NANB, PT 31.92 31,95 8:35 ' AVH NANB, IVD 6.87 4.45 "Healed" recents NANB, AVH 11.84 5.79 9. 36 SpäteAVHNANBPT 6.52 1.33 10. 37 AVH NANB, IVD 39.44 39.18 11. 38 SpäteAVHNANB.PT 42.22 37.54 12. 39 Chronic NANB, PT 1.35 1.17 13. 40 AVH, NANB, PT 0.35 0.28 54. 41 Chronic NANB? PT 6.25 2.34 15. 42 AVH, NANB, IVD 0.74 0.61 16. 43 Chronic NANB, PT 5.40 1.83 17. 44 AVH, NANB, PT 0.52 0.32 18. 45 Chronically, NANB, PT 23.35 4.15 19. 46 AVH, NANB 1.60 1.35 20. 47 AVH, TypeA 1.30 0.66 21. 48 AVH, Type A 1.44 0.74 22. 49 AVH, type A. 0.48 0.56 23. 50 Dissolved (resolved) recent AVH, type A. 0.68 0.64 24. 51 AVH, type A. 0.80 0.65 Dissolved AVH, type A. 1.38 1.04 25. 52 Resolved recent AVH, type A. 0.80 0.65 Resolved recent AVH, type A. 1.85 1.16 26. 53 AVH, Type A 1.02 0.88 Resolved recent AVH, type A. 1.35 0.74 27. 54 AVH, type A. 0.58 0.55 28. 55 Late AVH, HBV 0.84 1.06 29. 56 Chronically, HBV 3.20 1.60 30. 57 Late AVH, HBV 0.47 0.46 31. 58 Chronically, HBV 0.73 0.60 32. 59 ' AVH, HBV 0.43 0.44 Healed AVH, HBV 1.06 0.92 33. 60 ' AVH, HBV 0.75 0.68 Healed AVH, HBV 1.66 0.61 34. 61 ' AVH, HBV 0.63 0.36 Healed AVH, HBV 1.02 0.73 35. 62 ' AVH ₁ HBV 0.41 0.42 Healed AVH, HBV 1.24 1.31 36. 63 ' AVH, HBV 1.55 0.45 Healed AVH, HBV 0.82 0.79 37. 64 ' AVH ₁ HBV 0.53 0.37 Healed AVH, HBV 0.95 0.92 38. 65 ' AVH, HBV 0.95 0.92 Healed AVH, HBV 0.70 0.50 Healed AVH, HBV 1.03 0.68 39. 66 ' AVH, HBV 1.71 1.39 Healed AVH, HBV

1 Of these patients, sequential serum samples are available.

2 IVD = intravenous drug user

3 AVH = acute viral hepatitis

4 PT = posttransfusion (after transfusion)

As can be seen from Table 1, 19 out of 32 sera from patients diagnosed with NANBH were antibody-directed against HCV epitopes present in SOD-NANB ₈ .!., And SOD-NANB ₈ I , positive. However, the positive serum samples were not equally immunologically reactive with SOD-NANB _6. ), And S0D-NANB _S |. Patient # 1 serum samples were positive for SOD-NAN ₈ , but not for SOD-NANB ₆ .!.,. Serum samples from patients # 10, 15 and 17 were positive for SOD-NANB ₆ .,. ,, but not SOD-NANB ₈ ,. Serum samples from patients Nos. 3, 8, 11, and 12 reacted equally with both fusion polypeptides, while serum samples from patients Nos. 2, 4, 7, and 9 had 2 to 3 times greater response to SOD-NANB ₆ -M than compared SOD-NANB ₈₁ . These results suggest that NANB _6. , And NANB ₈ may contain at least three different epitopes, ie, it is possible that each polypeptide contains at least one unique epitope and that the two polypeptides share at least one epitope.

IV.D.4. Specifically, the solid phase RIA refers to NANBH

The specificity of solid phase radioimmunoassays for NANBH was tested by performing the assay on the basis of serum from HAV or HBV infected patients and sera from control individuals. Assays using leepwise purified SOD-NANB ₆ ,., And SOD-NANB ₈₁ were performed essentially as described in Section IV.D.3. except that the sera were 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. The radioimmunoassay was tested using 11 serum samples from HAV-infected patients and 20 serum samples from HBV-infected patients.

As shown in Table 1, none of these sera showed a positive immunological reaction with the BB-NANBV epitope-containing fusion polypeptides.

The RIA using the NANB _b ,., Antigen was used to determine the immunological responsiveness of serum from control individuals. Of 230 serum samples from normal blood donors, only two showed positive responses in RIA (data not shown). It is possible that the two blood donors from which these serum samples originated were once exposed to HCV.

IV.D.5. Reactivity of NANBs.,., In the course of NANBH infection

The presence of anti-NANB &.,., Antibodies in the course of NANBH infection in two patients and four chimpanzees was assessed with RIA as described in Section IV.D.3. tracked. In addition, radioimmunoassay was used to determine the presence or absence of anti-NANBs.,... Antibodies in the course of HAV and HBV infection in infected chimpanzees.

The results presented in Table 2 show that in chimpanzees and in humans anti-NANBs.,... Antibodies were detected after onset of the acute phase of NANBH infection. In serum samples from chimpanzees infected with HAV or HBV no ArUi-NANB ₅ .!., - antibodies were detected. Thus, AnU-NANB ₅ .,... Antibodies serve as an indicator (marker) for HCV exposure of an individual.

Table 2

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

Patient/ Sample date (day) hepatitis Anti-5-5-1 OLD 8th _ Schin rumen (0 = vaccination day) virus (S / N) (Mu / mL) 205 Patient 29 T " NANB 1.09 1180 14 T + 180 33.89 425 6 T + 208 36.22 - 11 Patient 30 T NANB 1.90 1830 132 T + 307 34.17 290 - T + 799 32,45 276 - Schimpi 0 NANB 0.87 9 4 76 0.93 71 147 118 23.67 19 18 154 32.41 - 5 Schimp2 0 NANB 1.00 5 - 21 1.08 52 106 73 4.64 13 10 138 23,01 - - Schimp3 0 NANB 1.08 7 43 1.44 83 53 1.82 5 159 11.87 Schimp4 -3 NANB 1.12 55 1.25 83 6.60 140 17.51 Schimp5 0 HAV 1.50 25 2.39 40 1.92 268 1.53 Schimp6 -8th HAV 0.85 15 - 41 0.81 129 1.33 Schimp7 0 HAV 1.17 22 1.60 115 1.55 139 1.60

Table 2 (continued)

Patient / chimpanzee Sample date (day) (0 = vaccination day) Hepatitis viruses Anti-5-5-1 (S / N) ALT (mu / ml) Schimp8 0 26 74 205 HAV 0.77 1.98 1.77 1.27 15 130 8 5 Schimp9 -290 379 435 HBV 0.77 3.29 2.77 15 9 6 SchimpiO 0 111-118 (pool) 205 240 HBV 2.35 2.74 2.05 1.78 8 96-155 (pool) 9 13 Chimp 11 0 28-56 (pool) 169 223 HBV 1.82 1.26 1.00 0.52 11 8-100 (pool) 9 10

* Date of initial sampling

IV.E. Purification of polyclonal Sorumantlkörpern against NANBg.,.,

Based on the specific immunological reactivity of the SOD-NANB.,.,. Polypeptide with the antibodies in serum samples from patients with NANBH, a method was described for the purification of serum antibodies immunological with the epitope (s) in NANB ₆ -M react, developed.

In this method, the affinity chromatography is exploited. Purified SOD-NANB ^ M polypeptide (see Section IV.DI) was linked to an insoluble support, with the immobilized polypeptide retaining its affinity for the antibody to NANB ₅ .,. The antibody in serum samples is absorbed on the matrix-bound polypeptide. After washing to remove the nonspecifically bound materials and unbound materials, the bound antibody is altered by changing the pH and / or chaotropic reagents such. Urea, released from the bound SOD-HCV polypeptide.

Nitrocellulose membranes containing bound SOD-NANB _6. , Were prepared as follows. A nitrocellulose membrane, 2.1 cm Sartorius with a pore size of 0.2 μm, was washed three times with BBS for 3 minutes. SOD-NANB & .1., Was bound to the membrane by incubation of the purified preparation in BBS at room temperature for 2 hours; in an alternative way was overnight at 4 ° C іпкиЫѳгі. The solution containing the bound antigen was removed and the filter was washed three times with BBS for three minutes each. The remaining active sites on the membrane were blocked by incubation with a 5 mg / ml BSA solution for 30 minutes. Excess BSA was 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 further treated by three 3-minute washes with PBS. The polyclonal AnIi-NANB ₅ .,... Antibodies were isolated by incubating the fusion polypeptide-containing membranes with serum from one subject with NANBH for two hours. After incubation, the filters were washed five times with BBS and twice with distilled water. The bound antibodies were then removed from each filter by 5 elutions of Glyl HCI, with each elution taking 3 minutes. The pH of the eluates was adjusted to 8.0 by collecting each eluate in a test tube containing 2.0 M Tris HCl at pH 8.0. The recovery of anti-NANB5.,., - antibody after affinity chromatography was about 50%.

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

IV.F. The trapping of HCV particles from infected plasma by purified human polyclonalor anti-HCV antibodies; Hybridization of the nucleic acids to the captured particles on HCV cDNA

IV.F.1. Infection of HCV Particles from Infected Plasma Using Human Polyclonal-Antl-HCV Antibody Protein nucleic acid complexes present in the infectious plasma of a chimpanzee with NANBH were purified by purified human polyclonal anti-HCV antibodies bound to polystyrene beads isolated. Polyclonal anti-NANB ₆ .,. - Antibodies were purified from human serum with NANBH using the polypeptide SOD-HCV encoded in clone 5-1-1. For purification, the procedure described in Section IV.E. described method applied.

Purified anti-NANB ^ ii antibodies were ligated to polystyrene beads ( ^1- inch diameter, mirror-smooth surface, Precision Plastic Ball Co., Chicago, Illinois) by incubating each bead overnight with 1 ml of antibody (1 microgram / ml in Borate-buffered saline, pH 8.5) was incubated. After overnight incubation, the beads were washed once with TBST (50 mM Tris HCl, pH 8.0, 15 mM NaCl, 0.05% (v / v) Tween 20) and then with phosphate buffered saline (PBS) containing 10 mg / ml BSA contained, washed.

Control beads were prepared in a similar manner except that the purified anti-NANBs-i antibodies were replaced with whole human immunoglobulin.

The trapping of HCV from NANBH-infected chimpanzee plasma by means of the AnH-NANB ₆ .,.., Antibodies bound to the beads was as follows: The plasma used from a chimpanzee with NANBH is described in Section IV.A.1. described. A

aliquot (1 ml) of the NANBV infected chimpanzee plasma was incubated for 3 hours at 37 ⁰ C with 5 beads coated with either anti-NANB ^ M-antibodies or with Kontrollimmunoglobulinen. The beads were washed three times with TBST.

IV.F.2. Hybridization of Nuclofenic Acid In the Submitted Particles to NANBV cDNA The nucleic acid component released from the particles captured with anti-NANB antibody was analyzed for hybridization to HCV cDNA derived from clone 81.

HCV particles were prepared from NANBH-infected chimpanzee plasma as described in Section IV.F.1. captured captured. To release the nucleic acids from the particles, the washed beads were incubated for 60 minutes at 37 ^° C. with 0.2 ml solution per bead, using the solution proteinase к (1 mg / ml), 10 mM Tris HCl, pH 7.5, 10 mM EDTA, 0.25% (M / V) SDS containing 10 micrograms / ml of soluble yeast RNA, the supernatant was removed. The supernatant was extracted with phenol and chloroform, and the nucleic acids were precipitated overnight at -2O ⁰ C. The nucleic acid precipitate was collected by centrifugation, dried and dissolved in 50 mM Hepes, pH 7.5. Exactly equal amounts of the soluble nucleic acids from the samples derived from the beads coated with anti-NANB ^, i antibodies and control beads containing the total human immunoglobulin were filtered on the nitrocellulose filters. The filters were hybridized with a ³² P-labeled "nick" -translated probe prepared from the purified HCV cDNA fragment in clone 81. The procedures for probe preparation and hybridization are described in Section IV.C. .1 described.

Autoradiograms of a probed filter containing the nucleic acids from particles captured by the beads containing AnU-NANB _5. , Antibodies were shown in FIG. The extract recovered with the aid of the anti-NANB ^ ti antibody (Ai, А _г ) gave clear hybridization signals relative to the control antibody extract (A ₃ , A ₄ ) and to the control yeast RNA (B ,, B ₂ ) , The standards consisting of 1 pg, 5pg and 10pg of the purified cDNA fragment of clone Ы are shown in C 1-3.

These results indicate that the particles captured from NANBH plasma by anti-NANB ^ n antibodies contain nucleic acids that hybridize to HCV cDNA in clone 81 and thus provide further evidence that the cDNAs in these clones are from the etiological agent of NANBH were derived.

IV.Q. Immunological Reactivity of C 100-3 with Purified Antl-NANBn.rAntlkSrpern The immunological reactivity of the C 100-3 fusion polypeptide with anti-NANB ₅ .,., - antibodies wu.de determined by a Ftadioimmunassay, wherein bound to a solid phase Antigens were tested with purified anti-NANB _6. , I antibodies and the antigen-antibody complex was detected with ¹²⁶ l-labeled sheep anti-human antibodies. The immiotic reactivity of C 100-3 polypeptide was compared to that of SOD-NANB _S .,., Antigen. The Fusbn polypeptide C 100-3 was synthesized are purified as described in Section IV.B.5. or in Section IV.B.6. described. The fusion polypeptide SOD-NANB _6. )! was as described in Section IV.B.1. or in Section IV.D.1. described synthesized and purified. The purified anti-NANB _W. , Antibodies were purified as described in Section IV.E. described won. Aliquots of 100 microliters containing varying amounts of purified C 100-3 antigen in 0.125M Na borate buffer, pH 8.3.0.075 M NaCl (BBS), were added to each well of a microtiter plate (Dynatech Immolon 2 Rernovawe'.l strips ). The plate was incubated overnight in a humid chamber at 4 ° C, after which the protein solution was removed and the wells were washed three times with BBS containing 0.02% Triton X-100 (BBST). To avoid nonspecific binding, the wells are coated with BSA by adding 100 microliters of a 5 mg / ml solution of BSA in BBS and then incubating for 1 hour at room temperature, after which the excess BSA solution was removed. The polypeptides in the coated wells were reacted with purified anti-NANB ^ M-antibodies for the reaction by 1 iMikrogramm antibody / well was added and the samples were incubated for 1 hour at 37 ⁰ C. After incubation, the excess solution was removed by aspiration and the wells were washed five times with BBST. Anii-NANB ₅ .,., Bound to the fusion polypeptides was detected by binding of '' labeled F '(ab) i sheep anti-human IgG to aio coated wells, 100 microliter aliquots of labeled probe ( Fig . specific activity 5-20 micro Curies / microgram) were added to each well, the plates were incubated for 1 hour at 37 ⁰ C, and then the excess probe was removed by aspiration and washed five times with BBGV. the bound in each well radioactivity was determined by counting in a gamma counter.

Table 3 shows the results of the immunological reactivity of C 100 with purified AnH-NANB ₅ .,., Compared to that of NANB5.1-1 with the purified antibodies.

Tabeile 3

Immunological reactivity of C 100-3 compared to NANB _5. , Detected by radioimmunoassay (R / A)

AG (ng) Rl A (count per minute / assay)

400 320 240 160 60 0

NANB ₅ .,., 7332 6732 4954 4050 3051 57

C100-3 7450 6985 5920 5593 4096 67

The results in Table 3 demonstrate that anti-NAN B _5. , Recognizes an epitope (or epitopes) in the C 100 component of the C 100-3 polypeptide. Thus, NANB ₅ ,., And C 100 share a common epitope (epitope). The results indicate that the cDNA sequence encoding this NANBV epitope (s) is present in both clone 5-1-1 and clone 81.

IV.H. Characterization of HCV

IV H.1. Characterization of the Stringency of the HCV Genome

The HCV ganoma was characterized for its strandedness by isolating the nucleic acid fraction from the particles captured on the polystyrene beads coated with anti-NANB ^ .r antibody and determining whether the isolated nucleic acid was positive and / or positive. or Minuc strands of the HCV cDNA hybridized.

As in Section IV.F.1. Particles were described from the HCV-infected chimpanzee plasma using

captured with immunopurified anti-NANB ^ o antibody-coated polystyrene beads. The nucleic acid component of the particles was analyzed by means of the method described in Section IV.F.2. released process described. Aliquots of isolated genomic nucleic acid corresponding to 3 ml of the high titer plasma were transferred to nitrocellulose filters. As controls, aliquots of denatured HCV cDNA from clone 81 (2 picograms) were also transferred to the same filters. The filters were probed with a ³² P-labeled mixture of plus or a mixture of minus strands of single-stranded DNA cloned from HCV cDNAs; the cDNAs were excised from clones 40b, 81 and 25c.

The single-stranded probes were obtained by excising the HCV cDNAs from clones 81, 40b and?. 5c by EcoRI and cloning the cDNA fragments into M 13 vector, mp 18 and mp 19 (Messing (1983)). , The M13 clones were sequenced to determine if they contained the Fius or Minus strands of DNA derived from the don HCV cDNAs. The sequencing was carried out with the dideoxy chain termination method according to Sanger and co-workers (1977).

From a set of Dupukat filters containing aliquots of the HCV genome isolated from the captured particles, each filter was analyzed with either plus or minus strand sondans derived from the HCV cDNAs. hybridized. Figure 41 shows the autoradiograms obtained by probing the NANBV genome, with the probe mixture derived from clones 81, 40 b and 25c. This mixture was used to increase the sensitivity of the hybridization assay. The samples in List I were hybridized with the plus strand probe mixture. The samples in List II were hybridized by hybridization to the minus strand-probe mixture. The composition of the samples in the lists of immunoblots is shown in Table 4.

Table 4

Lane A B

1 HCV genome »

2 ··

3 · CDNA81

4 - CDNA81

'is a sample not described.

As can be seen from the results of Figure 41, only the minus strand DNA probe hybridizes to the isolated HCV genome. This result, coupled with the finding that the genome is sensitive to RNase and not to DNase (see Section IV.C.2.), Suggests that the genome of NANBV is a positive-stranded RNA. These data, as well as the data from the other laboratories regarding the physicochemical properties of a probable NANBV, are consistent with the possibility that the HCV belongs to the Flaviviridae. However, the possibility that HCV represents a new class of viral agent has not yet been eliminated.

IV.H.2. Detection of Sequences in Captured Particles Derived from Clone 81 by Ampllatkatlon PCR Hybridize HCV cDNA

The RNA in trapped particles was analyzed as described in Section IV.H. 1st described won. Analysis for sequences that hybridize to the cus clone 81-derived HCV cDNA was performed using the PCR amplification method as described in Section IV.C.3. with the exception that the hybridizing probe was a kinase oligonucleotide derived from the cDNA sequence of clone 81. The results showed that the amplified sequences hybridize with the clone 81-derived HCV cDNA probe.

IV.H.3. Homology between the nonstructural protein of dengue flavivirus (MNWVD 1) and the HCV polypeptides

are encoded by the combined ORF of clones 141 to 39 с

As shown in Figure 26, the combined HCV cDNAs of clones 14І to 39c contain a continuous ORF. The polypeptide encoded therein was analyzed for sequence homology with the region of the non-structural polypeptide (s) in dengue flavivirus (MNWVD 1). For the analysis performed on the computer, the Dayhoff protein database was used. The results are shown in Figure 47, in which the symbol (:) indicates exact homology and the symbol (.) Indicates a conservative replacement in the sequence; the dashes indicate spaces that have been inserted into the sequence to achieve the greatest homology. As can be seen from the figure, there is significant homology between the sequence encoded in the HCV cDNA and dengue virus non-structural polypeptide (s). In addition to the homology shown in Figure 42, analysis of the polypeptide segment encoded in a region towards the З'-end of the cDNA also contained sequences homologous to sequences in dengue polymerase. Also of consequence is the discovery that the regulatory Gly-Asp-Asp (GDD) sequence believed to be important for RNA-dependent RNA polymerases is contained in the polypeptide encoded in HCV cDNA, and although in a place that is consistent with the Dengue-2 virus. (This data is not shown).

ІѴ.Н.4. HCV DNA is not detectable in tissue infected with NANBH

Two types of studies provide results suggesting that HCV DNA is undetectable in tissue from an individual with NANBH. These results, together with those in Sections IV.C. and IV.H.2. evidence that the HCV is not a DNA-containing virus and that its replication proceeds without cDNA involvement.

IV.H.4.a. Southern blotting

To determine whether NANBH-infected chimpanzee liver contains detectable HCV DNA (or HCV cDNA), restriction enzyme fragments of DNA were Southern blotted from this source and the blots were probed with ³² P-labeled HCV cDNA. The results show that the labeled HCV cDNA did not hybridize to the DNA transferred from the infected chimpanzee liver. It also did not hybridize to normal for control purposes

Chimpanzee liver transmitted DNA. On the contrary, in a positive control, a labeled probe of the beta-interferon gene hybridized strongly to Southern blots of restriction enzyme-digested human placental DNA These systems were designed to detect a single copy of the gene labeled with the labeled probe DNAs were isolated from the liver of two chimpanzees with NANBH Control DNAs were isolated from uninfected chimpanzee liver and from human placentas The DNA extraction procedure was performed essentially according to Maniatis and coworkers (1988), and the samples Both DNA samples were treated with either EcoRI, Mbol or HincII (12 micrograms) according to the manufacturer's instructions The digested DNAs were then further analyzed by electrophoresis on 1% neutral agarose gels, by Southern blotting treated on nitrocellulose, and the transferred material hybridized with the appropriate corresponding "nick" -translated probe cDNA (3 x 10 ^е counts per minute / ml of hybridization mixture). The DNA from infected chimpanzee liver and normal liver was hybridized with ³² P-labeled HCV cDNA from clones 36 and 81; the human placenta DNA was hybridized with ³² P-labeled DNA from the beta interferon gene. After Hybric'isierung the "blots" writhe washed under stringent conditions, ie with a solution containing 0.1 χ SSC, 0.1% SDS unri at a temperature of 69 ⁰ C. The beta-interferon gene DNA was prepared as described by Houghton and coworkers (1981).

IV.H.4.b. Ampicostomy by the PCR Technlk To determine whether the HCV DNA in chimpanzee liver can be detected with NANBH, DNA was removed from the tissue

isolated and the PCR amplification detection method with prewires of primers and probe polynucleotides consisting of

HCV cDNA from clone 81 were subjected. As negative controls were DNA samples that did not

infected HepG2 tissue cells and isolated from presumably uninfected human placenta.

As positive controls, samples of negative control DNAs revealed a known relatively small amount

(250 molecules) of the HCV cDNA insert from clone 81 was added. In addition, to confirm that RNA fractions isolated from the same liver of chimpanzees with NANBH contained sequences complementary to the HCV cDNA probe, the PCR amplification detection system was also applied to the isolated RNA samples.

In these studies, the DNAs were prepared by the method described in Section IV.HAa. described method isolated and the RNAs

were extracted essentially as described by Cbirgwin and coworkers (1981).

DNA samples were isolated from 2 infected chimpanzee liver, from uninfected HepG2 cells and from Hurnan placenta. One microgram of each DNA was digested with HindIII according to the manufacturer's instructions. The digested samples were

was amplified by PCR and the detection of the amplified HCV cDNA was carried out essentially according to the description in

Section IV.C.3., With the exception that the reserve transcript level has been omitted. The PCR primer and the probe

were obtained from HCV cDNA clone 81 and are described in Section IV.C.3. described. Before the amplification was for positive

Control a 1 microgram sample of each DNA by adding 250 molecules of HCV cDNA isolated from clone 81. Insert "spiked." To determine whether HCV sequences in RNA, dio from the livers of chimpanzees have been isolated with NANBH

were present, samples containing 0.4 micrograms of total RNA were essentially as in

Section IV.C.3. subjected to the described amplification process, but with some samples as a negative control

the reverse transcriptase was omitted. The PCR primer and probe were recovered from HCV cDNA clone 81 as described previously.

The results showed that the amplified sequences complementary to the HCV cDNA probe were not present in the DNAs

from infected chimpanzee liver were still detectable in the negative controls. On the contrary, when the samples, including the DNA from infected chimpanzee liver, were "spiked" with the HCV cDNA prior to amplification, the

Sequences from clone 81 detected in all positive control samples. In addition, amplified HCV cDNA sequences from clone 81 were detected in the RNA studies only if Reverse transcriptase was used, strongly suggesting that the results were not due to DNA contamination

were brought about.

These results show that chimpanzee hepatocytes with NANBH have no or undetectable levels of HCV DNA

contain. Based on the "spiking" assay, if HCV DNA is present, then it can be said that it accounts for well below 0.06 copies per hepatocyte. On the contrary, the HCV sequences in the total RNA from the same liver samples are eliminated easy to detect with the PCR technique.

! IV.. ELISA tests for HCV infection with HCV-d 00-3 as test antigen

All samples were tested by ELISA using HCV-d 00-3. This assay utilizes the НСѴ-сЮО-3 antigen (synthesized and purified as described in Section IV.B.5.) And a horseradish peroxidase-IHRPl conjugate of mouse monoclonal anti-human IgG.

The plates coated with НСѴ-сіОО-Апидеп were prepared as follows. A solution of coating buffer (5 mM Na borate, pH 9.0), 21 ml / plate, BSA (25 micrograms / ml), с 100-3 (2.50 micrograms / ml) was added to the Removeawell Immulon immediately before addition. I plates (Dynatech Corp.). After mixing for 5 minutes, 0.2 ml / well of the solution was added to the plates, they were covered and incubated at 37 ^° C. for 2 hours, after which the solution was removed by suction. The wells were washed once with 400 microliters of wash buffer (10 mM sodium phosphate, pH 7.4, 14, 10 mM sodium chloride, 0.1% (M / Vj casein, 1% (M / V j Triton X-100, 0.01% (M / V, thimerosal). After removal of the wash solution, 200 microliters / well of post-coat solution (10 mM sodium phosphate, pH7.2, 15mM sodium chloride, 0.1% [M / V] casein and mM phenylmethylsulfonyl fluoride [PMSFj) were added, the plates were loosely capped to give to prevent evaporation and were incubated for 30 minutes at room temperature so allowed to stand. the wells were aspirated to remove the solution and dry lyophilized overnight without heating. the prepared plates may be stored in sealed aluminum containers at 2-8 ⁰ C. to perform In the ELISA assay, 20 microliters of serum sample or control were added to a well containing 200 microliter sample diluent (10 mM sodium phosphate, pH 7.4, 50 mM sodium chloride, 1 mM EDTA, 0.1% [M / V] casein , 0.015% [M / V] Therosal, 1% [M / V] Triton X-100, 100 micrograms / ml yeast extract). The plates were sealed and incubated at 37 ⁰ C zwsi hours, after which the solution was removed by aspiration. The wells were washed with 400 microliters of wash buffer (phosphate buffered saline [PBS] containing 0.05% Tween 20). The washed wells were washed with 200 microliters of mouse anti-Kuman IgG HRP conjugate dissolved in a solution of ortho conjugate diluent (10 mM sodium phosphate, pH 7.2, 125 mM sodium chloride, 50% fetal bovine serum, V / V). 1% | V / V]

heat-treated horse serum containing 1mM K ₃ Fe (CN) ₆ , 0.05% [M / V] Tѵѵѳѳp 20.0.02% [M / V] thimerosal). The treatment lasted for 1 hour at 37 ° C, after which the solution was removed by suction and the wells were washed with washing buffer, which was also removed again by suction. To determine the amount of bound enzyme conjugate, 200 microliters of the substrate solution (10 mg O-phenylenediamine dihydrochloride per 5 ml developer solution) was added. The developer solution contains 5 mM sodium citrate adjusted to pH 5.1 with phosphoric acid and 0.6 microliter / ml. ml of ₃ % H ₂ O ₂ . The plates with the substrate solution were incubated in the dark for 30 minutes at room temperature, the reactions were stopped by adding 50 micro-rollers of 4 N sulfuric acid and the absorbance values (OD) were determined.

The examples presented below demonstrate that the microtiter plate screening ELISA using HCV C100-3 antigen has a high degree of specificity, such as the initial rate of reactivity of about 1%, with a replication reactivity rate of about 0.5% on random donors is proved. The Asse in is able to detect the immune reaction both in the post-acute phase of the infection and during the chronic disease phase. In addition, this test can be used for detection on some samples that have been negatively evaluated in the surrogate tests for NANBH. These samples are from individuals with a NANBH history or from donors affected by NANBH transmission (narrow, donors implicated in NANBH transmission). In the examples below, the following abbreviations are used:

ALT alanine-amino-transferase

Anti-HBc Antibody to HBc Anti-HBsAg Antibody to HBsAg HBc Hepatitis B core antigen ABsAg Hepatitis B surface antigen IgG immunoglobulins IgM ImmunoglobulinM IU / L International units / liter N / A is not available NT was not tested N sample size Neg negative OD extinction Pos positive S / CO Signal / cut-off value (narrow), signal / cutoff) SD standard deviation X Average or mean WNL within normal limits

IV.1.1. HCV infection in a population of random blood donors

A panel of 1056 samples (fresh sera) from randomly selected blood donors was obtained from Irwin Memorial Blood Bank, San Francisco, California. The test results from these samples are summarized in a histogram showing the distribution of extinction values (Figure 37). As can be seen from FIG. 37, 4 samples are> 3, one sample between 1 and 3.5 samples between 0.4 and 1 and the remaining 1046 samples are <0.4, with over 90% of these samples <0, Show 1.

The results of the reactive samples are listed in Table 5. Using a truncation value corresponding to the standard deviations mean plus 5, then 10 samples of 1056 (0.95%) were initially reactive. Of these, five samples (0.47%) repeatedly proved to be reactive when tested a second time by ELISA. Table 5 also shows the ALT and anti-HBd status for each of the repeatedly reactive samples. Of particular interest is the fact that all five repeated reactive samples were negative for NANBH in the two surrogate assays, but positive in the HCV ELISA.

Table 5

Results on reactive samples

N = 1051 = 0.419 (0.400 + negative control) OLD"" Anti-Hbc · ** X = 0.049 » OD repeated (IU / L) (OD) SD = ± 0.074 reactive samples N / A N / A Cut off value: χ + 5SD 0.084 N / A N / A rehearse 0.294 N / A N / A 0.326 N / A N / A 4227 0,187 N / A N / A 6292 0,152 30.14 1,433 6188 1,392 46.48 1,057 6157 > 3,000 48.53 1,343 6277 > 3,000 60.53 1,165 6397 > 3,000 WNL · · · · negative 6019 3,000 6651 6669 4003 ODderanfangs reactive samples 0.462 0.569 0.699 0.735 0.883 1,567 > 3,000 > 3,000 > 3,000 > 3,000

10/1056 = 0.95% 5/1056 = 0.47%

* Sample readings> 1.5 were not included in the calculation of the mean and statistical deviation. * ALT <68 IU / L are above normal limits

 *** Anti-HBc SO.535 (competition assay) is considered positive

"··· WNL: within normal limits.

IV.1.2. Chimpanzee serum samples

Serum samples from 11 chimpanzees were tested by HCV-c 100-3 ELISA. 4 of these chimpanzees were treated according to an established procedure in collaboration with Dr. med. Daniel Bradley of Centers of Disease Control Infected with NANBH from a Contaminated Charge Factor VIII (Probably Hutchinson's Strain). As controls, 4 other chimpanzees were infected with HAV and 3 with HBV. The serum samples were taken at different times after infection. The results summarized in Table 6 show documented antibody erokonversion in all chimpanzees infected with the Hutchinson strain of NANBH. After the acute phase of infection (as evidenced by the significant increase and subsequent return to normal ALT levels), antibodies to HCV-c 100-3 were detected in the sera of 4/4 NANBH-infected chimpanzees. Diesa samples have recently been tested as described in Section IV.B.3. was found to be positive by Western analysis and a radioimmunoassay. In contrast, none of the control chimpanzees infected with HAV or HBV showed any sign of reactivity in ELISA.

Table 6 OD S / Cl Chimpanzee serum samples 1,504 0.401 positive control 0.001 shutdown 0,007 0.00 negative 0,003 0.01 control 3,000 7.48 Chimpanzee 1 3,000 7.48 - - 0,003 0.00 0.005 0.00 Schlmpansö2 0.945 2.36 3,000 7.48 0.005 0.01 0,017 0.04 0,006 0.01 Chimpanzee 3 1,010 2.52 0,006 0.00 0,003 0.01 0.523 1.31 Schimpanse4 1.574 3.93

Inoculation date

Blood date

ALT IIU / L)

transfusion

24/5/84

07/06/84

14.03.85

11/3/85

24/5/84 9 5 08/07/84 71 52 18/9/84 19 13 24/10/84 - - 31/5/84 8th 28/6/84 205 20/8/84 14 24/10/84 6 14.03.85 11 26/4/85 132 06/05/85 - 20/8/85 11/3/85 09/05/85 06/06/85 08/01/85

NANB

NANB

NANB

NANB

Continued Table β

OD

S / CO

Inoculation date

Blood date

OLD (IU / L)

transfusion

Chimpanzee 5

Chimpanzee β

Chimpanzee 7

Chimpanzee 8

Schlmpanse9

0,006 0.00 0.001 0.00 0,003 0.01 0,006 0.01 0.005 0.00 0.001 0.00 0,004 0.00 0.29C 0.72 0,008 0.00 0,004 0.00 0,006 0.00 0.005 0.01 0,007 0.00 0,000 0.00 0,004 0.01 0,000 0.00

0.0 * 9

0,000

0,003

0.003 0.003

21/11/70

25/5/82

25/5/82

21/11/80

24/7/80

0.05

0,015 0.04 0,008 0.02 Chimpanzee 10 0.011 0.03 0,015 0.04 0.008 0.010 0.02 0.02 Chimpanzee 11 _ _

12/5/82

12/5/82

0.00

0.00

0.00 0.00

21.11.80 16.12.80 30.12.Й0 29.07.- 21.08.81

17.05.82 10.06.82 06.07.82 01.10.82

25.05.82 17.06.82 16.09.82 09.10.82

21.11.80 16.12.80 03.02.81 03.06.- 10.06.81

22.08.- 10.10.79 11.03.81 01.07.- 05.08.81 01.10.81

21.04.- 12.05.82 01.09.- 08.09.82 02.12.82 06.01.83

06.01.- 12.05.82 23.06.82 09.06.- 07.07.82 28.10.82 20.12.82

147

18

106 10

83 5

15 130 8 4.5

57 9

9 126

9 13

11 100

& 10

HAV

HAV

HAV

HAV

HBV

HBV

HBV

IV.1.3. List 1: Detected Infectious sera from human carriers chronic NANBH An encrypted list consisted of 22 single samples, each as a duplicate, d. H. a total of 44 samples. The samples were from proven infectious sera from chronic NANBH carriers, infectious sera from affected donors and infectious sera from NANBH patients in the acute phase.

In addition, the samples came from long-tracked negative controls and other disease controls. This list was written by Dr. H.Old provided by Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland. The list was written by dr. Age set up several years ago and has since been used by him as a qualifying list for tests on presumptive NANBH. (See cited reference to Dr. Alters article.) The entire list was tested twice by the ELISA method, and the results were sent to Dr. Witt. Age sent. The results of this evaluation are shown in Table 7. Although the table contains the results of only one duplicate dl, the same values were achieved for each of the duplicate samples.

As shown in Table 7, 6 sera that had been proven infectious in a chimpanzee model were strongly positive. The seventh infectious serum corresponded to a sample in an acute NANBH case and was unreactive in this ELISA. A sample from an affected donor with normal ALT values and ambiguous results in chimpanzee abnormalities was not reactive in this test. Three other serial samples from an individual with acute NANBH were also unreactive. All samples derived from the long-tracked negative controls were obtained from donors who had donated blood at least 10 times without hepatitis, and were unreactive in the ELISA. Finally, 4 of the tested samples were previously considered positive in presumptive NANBH tests developed by other scientists, but these tests were not confirmed.

Table 7

List 1 by H.Alter

List I. Result 2. Result

1) Infectious by chimpanzee transmission

a. Chronic NANB; Post-Tx

JF + +

EB + +

PG + +

b. Affected donors with increased ALT

BC + +

JJ + +

BB + +

Acute NANB; Post-tx Wl!

2) Not clearly infectious by chimpanzee transmission

a. Affected donors with normal ALT

CC - -

3) Acute NANB; Post-TX

JL 1st week JL 2nd week JL 3rd week

4) Disease checks Primary biliary cirrhosis

EK - -

b. Alcohol Hepatitis in convalescence

HB - -

5) Negative controls with history

DH - -

OC - -

LV - -

HL - All

6) Potential NANB "Antigens"

JS-80-01T-O (ISHIDA) Asterix (TREPO) Zurtz (ARNOLD) Becassdine (TREPO)

IV.1.4. List 2. Donor / recipient NANBH

The encrypted list consisted of 10 unambiguous transfusion-related donor / recipient NANBH cases with a total of 188 samples. Each of these cases consisted of samples from some or all donors to the recipient and from serial samples (which were taken 3.6 and 12 mornings after transfusion) from the recipient. A blood sample taken from the recipient prior to transfusion was also included. The encrypted list was written by Dr. med. H.Olter set up by NIH, and the results were sent to him.-Jr evaluation.

The results summarized in Table 8 show that the ELISA method demonstrated antibody seroconversion in 9 out of 10 cases of transfusion-related NANBH. Samples from case 4 (where no seroconversion was detected) consistently reacted poorly in the ELISA. Two out of the ten recipient samples were reactive 3 months after transfusion.

At 6 months, 8 recipient samples were reactive and at 12 months all samples except Case 4 were reactive.

In addition, at least 1 antibody positive donor was found in 7 of 10 cases, with case 10 being 2 positive donors. In case 10, the recipient's pre-transfusion blood sample was also positive for HCV antibody. The blood sample of this recipient after one month fell below the limit of reactive values, while the blood samples rose to positive levels after 4 and 10 months.

In general, an S / CO of 0.4 is considered positive. This case may thus be an earlier infection of the individual with HCV.

The ALT and HBc status for all reactive d. H. positive, samples are summarized in Table 9. As can be seen in the table, the 1/8 donor samples were negative for the surrogate markers and reactive in the HCV antibody ELISA method. On the other hand, the recipient samples (12 months after transfusion) had either elevated ALT or positive anti-HBc or both.

Table 3

List of locker / receiver NANB after H.Alter

case Spendur _ s / co receiver S / CO 3 months S / CO Post-TX S / CO 12 months S / CO - - blood sample 0.07 0.26 6 months 6.96 > 6.96 0.403 - 0.14 0.12 3.90 > 6.96 OD - 0.94 0.11 OD 0.13 OD 6.96 OD > 6.96 1. > 3,000 - before transfusion 0.15 0.112 0.17 > 3,000 0.16 > 3,000 0.50 Second > 3,000 > 6.96 OD 0.08 0,050 0.22 1,681 6.96 > 3,000 > 6.96 a. > 3,000 > 6.96 0.032 0.13 0.057 3.44 > 3,000 6.96 > 3,000 > 6.96 4th > 3,000 > 6.96 0.059 0.08 0.073 0.13 0.067 6.96 > 0.217 > 6.96 5th > 3,000 > 6.96 0,049 0.14 0.096 0.18 > 3,000 5.28 > 3,000 > 6.96 6th > 3,000 > 6.96 0,065 0.19 1.475 0.30 > 3,000 0.13 > 3,000 > 6.96 7th > 3,000 > 6.96 0.034 > 6.96 0.056 0.74 > 3,000 6.96 > 3,000 > 6.96 8th. Month: ·· - > 6.96 0.056 0.078 2,262 > 3,000 9th 4 months: 0.034 0,127 0,055 > 3,000 10th 0,061 0.317 " > 3,000 · » > 3,000 ··· 0,080 • - 1 > 3,000 ··· = 10 months

Table 9

ALT and HBc status for reactive samples in List 1 by H.Alter

rehearse 12Mon. 6Mon. Anti-ALT * HBC " donor elevated Case 3 12Mon. 6Mon. normal negative Case 5 elevated elevated positive Case 6 12Mon. 6Mon. elevated positive Case 7 elevated not available negative Case 8 12Mon. 6Mon. normal positive FaK 9 elevated elevated not available Case 10 6Mon. 3mon. normal positive Case 10 12Mon. elevated normal positive receiver elevated case 1 12Mon. 6 Мол. elevated positive elevated not tested Case 2 12Mon. 6Mon. elevated negative elevated not tested Case 3 12Mon. normal not tested··· 10Mon. 4Mon. not tested··· Case 5 elevated elevated not tested not tested Case 6 elevated negative negative not tested Case 7 elevated negative negative Case 8 normal positive not tested Case 9 elevated not tested Case 10 elevated not tested not posted

• ALT & 45 IU / L is above the normal limit

 ** Anti-HBc £ 50% (competition assay) is considered positive

 *** Blood sample before transfusion and 3 months after that were HBc negative.

IV.I.5. Determination of HCV infection In samples of high-risk groups Samples of high-risk groups were monitored by ELISA to determine the reactivity to НСѴ-сЮО-3 antigen. These samples were provided by D.Gary Tegtmeier, Community Blood Bank, Kansas City. The results are summarized in Table 10.

As can be seen from the table, the samples with the highest reactivity of hemophilone were obtained (76%). Further, samples from individuals with elevated ALT and anti-HBc positive were found to be 51% reactive, a value consistent with the expected clinical data and NANBH prevalence in this group. The presence of antibodies to HCV was also higher in blood donors with elevated ALT alone, in blood donors who were positive only for antibodies to hepatitis B core, and in blood donors who had other reasons than high ALT or anti-nuclear antibodies were rejected in comparison to random voluntary blood donors.

Table 10 Samples from NANBH risky groups

group 1 N 35 distribution OD % reactive 0.728 N 3,000 11.4% Increased ALT 24 3 1 33 5 3,000 20.8% Anti-HBc 1 2,768 5 3,000 51.5% Elevated ALT, anti-HBc 1 2,324 12 1 2,939 1 0,951 0.906 25 1 150 3,000 20.0% Rejected donors 1 0.837 5 3,000 14.7% Donor with hepatitis 1 0.714 19 prehistory 0.469 1 50 1 2,568 3,000 76.0% hemophilic 1 2,483 31 1 2,000 1 1,979 1 1,495 1 1,209 0.809

IV.1.6. Comparative studies using anti-IgG or anti-IgM monoclonal antibody or polyclonal antibody

as the second antibody in the НСѴ-сЮО-3 ELISA

The sensitivity of the ELISA measurement using anti-IgG monoclonal conjugate was compared with that achieved when either an anti-IgM monoclonal conjugate is used or when both are replaced by a polyclonal antiserum , which should be both heavy and light chain specific. The following investigations were carried out.

ІѴ.І.б.а. Serial samples from seroconverters

Serial samples from three cases of NANB seroconverters were tested in the HCV-dOO ELISA test using enzyme conjugate

either the anti-IgG monoclonal antiserum was used alone or in combination with an anti-IGM monoclonal antiserum, or a polyclonal antiserum was used. The samples were obtained from Dr. Cladd Stevens, N. Y. Blood

Center, N.Y.C., N.Y. The sample histories are listed in Table 11. The results obtained with an AnIII IgG monoclonal antibody / enzyme conjugate are shown in Table 12. The data

show that strong reactivity was initially detected in samples 1-4,2-8 and 3-5 of cases 1,2 and 3, respectively.

The results obtained by a combination of an anti-IgG monoclonal anti-conjugate and an anti-IgM conjugate

are listed in Table 13. Three different ratios of anti-IgG to anti-IgM were tested. The dilution 1: 10,000 of anti-IgG was constant throughout the tests. Those tested in the anti-IgM monoclonal conjugate

Dilutions were 1: 30000.1: 60000 and 1: 120000. The data show that in accordance with the investigations

with anti-IgG alone, the initial strong reactivity was demonstrated in samples 1-4,2-8 and 3-5.

The results obtained with the ELISA test with anti-IgG monoclonal conjugate (dilution 1: 100000), or with Tagopolyclonal conjugate (dilution 1: 80000) or with Jackson polyclonal conjugate (dilution 1: 80000)

are listed in Table 14. The data show that the initial high reactivity was detected in samples 1-4,2-8 and 3-5 in all three configurations. Tago polyclonal antibodies gave the lowest signals.

The results presented above show that all three configurations have reactive samples at the same time after the acute Demonstrate disease phase (as indicated by the increase in ALT levels). In addition, the results show

that the sensitivity of the HCV-c 100-3 ELISA assay by anti-IgG monoclonal enzyme conjugate is equal to or better than that obtained in the other configurations of the enzyme conjugate tested.

tables

Description of samples according to the list of Cladd Stevens

date

HBsAg

Anti-HBs

Anti-HBc

OLD

Table 12

ELISA test results obtained using anti-IgQ monoclonal conjugate

sample

date

OLD

S / CO

Neg. control 08/05/81 40.0 0,076 0.37 cut-off value 09/02/81 274.0 0,476 0.32 PC (1: 128) 10/7/81 261.0 1,390 0.27 FAIM 19/11/81 75.0 1.97 1-1 15.12.81 71.0 0,178 6.30 1-2 0.154 1-3 19/10/81 17.0 0,129 0.12 1-4 17/11/81 46.0 0.937 0.11 1-5 12/2/81 63.0 3,000 0.10 Case 2 14/12/81 152.0 0.12 2-1 23/12/81 624.0 0.058 0.15 2-2 20/1/82 66.0 0,050 0.11 2-3 15/2/82 70.0 0.047 0.29 2-4 17/3/82 24.0 0.059 3.92 2-5 21/4/82 53.0 0,070 6.30 2-6 19/5/82 95.0 0,051 6.30 2-7 14/6/82 37.0 0,139 6.30 2-8 1,867 2-9 07/04/81 13.0 3,000 0.19 2-10 12/5/81 236.0 3,000 0.13 2-11 30/5/81 471.0 3,000 0.17 Fall3 09/06/81 315.0 0.44 3-1 07/06/81 273.0 0,090 3.59 3-2 10/8/81 158.0 0.064 6.30 3-3 09/08/81 84.0 0.079 6.30 3-4 14/10/81 180.0 0.211 6.30 3-5 11/11/81 154.0 1,707 6.30 3-6 3,000 3-7 3,000 3-8 3,000 3-9 3,000

bilirubin

FAIM 08/05/81 1.0 91.7 12.9 40.0 -1.0 1-1 09/02/81 1.0 121.0 15.1 274.0 1.4 1-2 10/7/81 1.0 64.0 23.8 261.0 0.9 1-3 19/11/81 1.0 67.3 33.8 75.0 0.9 1-4 15.12.81 1.0 50.5 27.6 71.0 1.0 1-5 Case 2 19/10/81 1.0 1.0 116.2 17.0 -1.0 2-1 17/11/81 1.0 0.8 89.5 46.0 1.1 2-2 12/2/81 1.0 1.2 78.3 63.0 1.4 2-3 14/12/81 1.0 0.9 90.6 152.0 1.4 2-4 23/12/81 1.0 0.8 93.6 624.0 1.7 2-5 20/1/82 1.0 0.8 92.9 66.0 1.5 2-6 15/2/82 1.0 0.8 86.7 70.0 1.3 2-7 17/3/82 1.0 0.9 69.8 24.0 -1.0 2-8 21/4/82 1.0 0.9 67.1 53.0 1.5 2-9 19/5/82 1.0 0.5 74.8 95.0 1.6 2-10 14/6/82 1.0 0.8 82.9 37.0 -1.0 2-11 Fall3 07/04/81 1.0 1.2 88.4 13.0 -1.0 3-1 12/5/81 1.0 1.1 162.2 236.0 0.4 3-2 30/5/81 1.0 0.7 99.9 471.0 0.2 3-3 09/06/81 1.0 1.2 110.8 315.0 0.4 3-4 07/06/81 1.0 1.1 89.9 273.0 0.4 3-5 10/8/81 1.0 1.0 118.2 158.0 0.4 3-6 09/08/81 1.0 1.0 112.3 84.0 0.3 3-7 14/10/81 1.0 0.9 102.5 180.0 0.5 3-8 11/11/81 1.0 1.0 84.6 154.0 0.3 3-9

Table 13 ELISA test results obtained using anti-IgG and anti-IgM monoclonal conjugate ELISA TestsanNANB

date OLD Monoclo- Monoclo- Monoclo- nates dimensional dimensional IgG1: 10K IgG 1: 10K IgGIMOK lgM1: 30K IgM 1:60 K lgM1: 120K sample OD S / CO OD S / CO OD S / CO Neg. control 08/05/81 40 0,100 0,080 0.079 cut-off value 09/02/81 274 PC (1: 128) 10/7/81 261 1,083 1,328 1,197 FAIM 1S.11.81 75 1-1 15.12.81 71 0.173 0.162 0,070 1-2 0.194 0.141 0.079 1-3 19/10/81 17 0.162 0,129 0.063 1-4 17/11/81 46 0.812 0.85 0.709 1-5 12/2/81 63 > 3.00 > 3.00 > 3.00 Case 2 14/12/81 152 2-1 23/12/81 624 0,442 0,045 0.085 2-2 20/1/82 66 0,102 0,029 0,030 2-3 15/2/82 70 0.059 0,036 0.027 2-4 17/3/82 24 0,065 0,041 0,025 2-5 21/4/82 53 0.082 0.033 0.032 2-6 19/5/82 95 0,102 0,042 0.027 2-7 14/6/82 37 0.188 0,068 0.096 2-8 1,728 1,668 1,541 2-9 07/04/81 13 > 3.00 2,443 > 3.00 2-10 12/5/81 236 > 3.00 > 3.00 > 3.00 2-11 30/5/81 471 > 3.00 > 3.00 > 3.00 FaIIS 09/06/81 315 3-1 07/06/81 273 0,193 0,076 0,049 3-2 10/8/81 158 0.201 0,051 0,038 3-3 09/08/81 C4 0.132 0.067 0,052 3-4 14/10/81 180 0,175 0,155 0.140 3-5 11/11/81 154 1,335 1,238 1,260 3-6 > 3.00 > 3.00 > 3.00 3-7 > 3.00 > 3.00 > 3.00 3-8 > 3.00 > 3.00 > 3.00 3-9 > 3.00 > 3.00 > 3.00

Table 14 ELISA test results obtained using polyclonal conjugates

date OLD ELISA TestsanNANB S / CO TAGO S / CO Jackson S / CO monoclonal 1:80 K 1:80 K 1: 10K OD OD sample OD 0,045 0.154 Neg. control 0,076 0.545 0,654 cut-off value 08/05/81 40 0,476 0.37 0.727 0.12 2,154 0.23 PC (1: 128) 09/02/81 274 1,390 0.32 0.18 0.34 FAIM 10/7/81 261 0.27 0.067 0.05 0.153 0.26 1-1 19/11/81 75 0,178 1.97 0.097 0.60 0.225 1.21 1-2 15.12.81 71 0.154 6.30 0,025 3.27 0.167 4.59 1-3 0,129 0,324 0.793 1-4 19/10/81 17 0.937 0.12 1.778 0.04 > 3.00 0.08 1-5 17/11/81 46 > 3.00 0.11 0.03 0.09 Case 2 12/2/81 63 0.10 0.023 0.04 0,052 0.09 2-1 14/12/81 152 0.058 0.12 0,018 0.05 0.058 0.08 2-2 23/12/81 624 0,050 0.15 0,020 0.05 0,060 0.11 2-3 20/1/82 C6 0.047 0.11 0,025 0.03 0.054 0.09 2-4 0.059 0.026 0.074 2-5 0,070 0,018 0.058 2-6 0,051

Continuation Table 14

date OLD EUSA-TestsanNANB S / CO TAGO S / CO Jackson I S / CO 15/2/82 70 monoclonal 0.29 1:80 K 0.07 1:80 K 0.22 17/3/82 24 1: 10K 3.92 OD 0.65 OD 2.19 sample 21/4/82 53 OD > 6.30 0.037 1.37 0.146 > 4.59 2-7 19/5/82 95 0,139 > 6,30 0.355 1.88 1,429 > 4.59 2-8 14/6/82 37 1,867 > 6.30 0,748 1.68 > 3.00 > 4.59 2-9 > 3.00 1.025 > 3.00 2-Ю 7:04:01 13 > 3.00 0.19 0.917 0.09 > 3.00 0.21 2-11 12/5/81 236 > 3.00 0.13 0.07 0.14 Fall3 30/5/81 471 0.17 0,049 0.08 0.138 0.22 3-1 09/06/81 315 0,090 0.44 0,040 0.16 0.094 0.42 3-2 07/06/81 273 0.064 0.59 0,045 0.50 0.144 2.71 3-3 10/8/81 158 0.079 > 6.30 0.085 2.47 0,275 > 4.59 3-4 09/08/81 84 0.211 > 6.30 0.272 4.21 1,773 > 4, 'J9 3-5 14/10/81 180 1,707 > 6.30 1,347 > 5.50 > 3.00 > 4.59 3-6 11/11/81 154 > 3.00 > 6.30 2,294 > 5.50 > 3.00 > 4.59 3-7 > 3.00 > 3.00 > 3.00 3-8 > 3.00 > 3.00 > 3.00 3-9 > 3.00

ІѴ.І.б.Ь. Samples of random blood donors

Samples from random blood donors (see Section IV.1.1.) Were screened for HCV infection by HCV c 100-3 ELISA, where the antibody / enzyme conjugate was either an anti-IgG monoclonal conjugate or a polyclonal conjugate. The total number of samples screened was 1077 for polyclonal conjugate and 105S for monoclonal conjugate, respectively. A summary of the screening results is given in Table 16 and the sample distributions are shown in the histogram in FIG.

The calculation of the mean and standard deviation was made excluding the samples giving a signal above 1.5, i.e. 1073 OD values were used for the polyclonal conjugate calculations and 1051 for the anti-IgG monoclonaloma conjugate. As can be seen in Table 15, the average value shifted from 0.0493 to 0.0931 and the standard deviation increased from 0.074 to 0.0933 when the polyclonal conjugate was used. In addition, the results also show that when the criteria of χ + 5SD are used to define the assay cut value, the polyclonal / enzyme conjugate configuration in the ELISA test requires a higher cutoff value. This indicates a lower assay specificity compared to the monoclonal system. In addition, as can be seen from the histogram in Figure 44, a greater separation of the results between negative and positive distribution occurs when compared to random blood donors in an ELISA using the anti-IgG monoclonal conjugate in comparison Assay be screened using a conventional polyclonal label.

Table 15

Comparison of two ELISA configurations in test samples from random blood donors

conjugate Polyclonal Anti-IgG monoclonal (Jackson) Number of samples 1073 1051 Average (x) 0.0931 0.04926 Standard deviation (SD) 0.0933 0.07427 5SD 0,46U6 .3714 cut-off value (5SD + x) .5596 .4206

IV a. Detection of HCV Seroconversion in NANBH Patients from Many Geographic Areas Sera from patients suspected of having elevated ALT levels of NANBH in whom HAV and HBV tactics had been negative were analyzed by radioimmunoassay essentially as described in Section IV .D. However, the HCV c100-3 antigen was used as a screening antigen on the microtiter plates. As can be seen from the results shown in Table 16, radioimmunoassay detected positive samples in a high percentage of cases.

Table 16

Frequency of seroconversion in anti-c100-3 among NANBH patients from different countries

country Netherlands Italy Japan Number of examined cases 5 36 26 positive number 3 29 19 % positive 60 80 73

IV.K. HCV seroconversion clones In "Community Acquired" NANBH Serenes were provided by Dr. H. Alter of the Center of Disease Control and Dr. J. Tuesday of Harvard University, who are primarily VO patients with NANBH originated, where no obvious transmission could be seen (. ie no ^Ί 'π, -> ^ .. r.en k-iri users of intravenous drugs, no promiscuity, etc. were identified as risk factors.) This ^r'c.:. In general, the procedure was repeated by redox immunoassay according to the method described in section IV.D., except that the НСѴ-сЮО-З-Атідеп was used as a screening antigen on the microtiter plates in that, of the 100 serum samples, 55 contained antibodies which reacted immunologically with the HCV c 100-3 antigen The results described above indicate that the "Community Acquired" NANBH is also caused by the HCV ill.

Further, as it has been shown here that the HCV is related to the flaviruses, most of which are transmitted by arthropods, this suggests that the HCV transmission of the "community acquired" hall was also by arthropods.

IV.L. Comparison of the occurrence of HCV antibodies ur.d surrogate markers in donors receiving NANBH transmission

are affected

A prognostic study was conducted to determine whether seroconversion to anti-HCV antibody positive had occurred in recipients of blood from presumably NANBH-positive donors who had developed NANBH. Blood donors were tested for abnormal surrogate markers, which are now used as markers in NANBH infection, d. H. increased ALT levels and the presence of anti-nuclear antibodies. In addition, the donors were also tested for the presence of anti-HCV antibodies. The determination of whether anti-HCV antibodies are present was made by means of a procedure described in Section IV.K. Radloimmunassays described. The test results are listed in Table 17, which shows the patient number (column 1); the presence of anti-HCV antibodies in the patient serum (column 2); the number of blood donations that the patient received, each donation from another donor (column 3), the presence of anti-HCV antibodies in the donor serum (column 4), and the surrogate abnormality of the donor (column 5) ) (NT or - means not tested.) (ALT means increased transaminase and anti-HBc means anti-nuclear antibody.)

The results in Table 17 show that the HCV antibody test is more accurate in finding infected blood donors than the surrogate marker tests. Nine out of 10 patients who developed NANBH symptoms were positive for anti-HCV antibody seroconversion. Of the 11 suspected infected individuals (patient 6 received donations from two different donors who are believed to be NANBH carriers) 9 were positive for anti-HCV antibodies and 1 was just above the borderline positive and therefore ambiguous (donor from patient 1) , If, on the other hand, the test is used for elevated ALT values, then 6 of the 10 donors tested are negative, and if one uses the antibody antibody tost, 5 of the 10 donors tested are negative. Of greater impact, however, is the fact that the ALT test and the anti-HBc test did not yield consistent results in 3 cases (donors for patients 8,9 and 10).

Table 17 Development of anti-HCV antibodies in patients receiving blood from donors who are believed to be NANBH carriers

patient Anti-HCV seroconversion number of Anti-HCV positive Surrogatabnormalität Anti-HB in the patient Donations / donor donor OLD No 1 Yes 18 ambiguous No Yes 2 Yes 18 Yes NT No 3 Yes 13 Yes No - 4 No ιε No - Yes 5 Yes 16 Yes Yes No β Yes 11 Yes 2) No Yes Yes No 7 Yes 15 Yes NT Yes 8th Yes 20 Yes No No 9 Yes 5 Yes Yes ia 10 ia 15 ia nain

The same donor as anti-NANBH positive.

IV.M. Amplification for cionating HCV cDNA sequences by PCR and primers derived from conserved regions of Flavivlrus genome sequences were derived

The results presented above, suggesting that the HCV is a flavivirus or a flavivirus, allow a strategy for cloning of uncharacteristic HCV cDNA sequences by the PCR technique and with primers derived from the regions that code for conserved amino acid sequences in the flaviviruses. In general, one of the primers is derived from a defined HCV genomic sequence, and the other primer flanking a region of non-sequenced HCV polynucleotide is derived from a conserved region of the flavivirus genome. The flavivirus genomes are known to contain conserved sequences within the NS1 and E polypeptides encoded in the 5 'region of the flavivirus genome. The corresponding sequences encoding these regions are "upstream" of the HCV cDNA sequence, as shown in Figure 28. Thus, to isolate the cDNA sequences derived from this region of the HCV genome, "upstream" primers are designed derived from the conserved sequences within these flavivirus polypeptides. The "downstream" primers are derived from the "upstream" end of the known part of the HCV cDNA.

Because of the degeneracy of the coda, it is possible that mismatches will occur between the ravivirus probes and the corresponding HCV genome sequence. Therefore, a strategy similar to that described by Lee (1988) is used. The Leesche method utilizes mixed oligonucleotide primers that are complementary to the products of the reverse translation of an amino acid sequence, the sequences in the mixed primers carry each

Codon generation of the conserved amino acid sequence calculation. Three sets of primer mixtures were created, based on amino acid homologies present in several flaviviruses, including dengue 2.4 ID-2.4, Japan encephalitis virus (YEV), yellow fever (YF), and West Nile virus. Virus (WN), were found. The primer mixture derived from the most .upstream "conserved sequences (54) is based on the amino acid sequence Gly-Trp-Gly, which is part of the conserved sequence Asp-Arg-Gly-Trp-Gly-AspN found in the Ε protein of D-2, JEV, YF and WN The next primer mixture (5'-2) is based on a "downstream" conserved sequence in the Ε protein, Phe-Asp-Gly-Asp-Ser-Tyr-Ileu-Phe- Gly-Asp-Ser-Tyrlleu and is derived from Phe-Gly-Asp; the conserved sequence is present in D-2, JEV, YF and WN. The third primer mixture (5'-3) is based on the amino acid sequence Arg-Ser-Cys, which is part of the conserved sequence Cys-Cys-Arg-Ser-Cys in the NS1 protein of D-2, D-4, JEV, YF and WN is. The individual primers which form the mixture in 5'-3 are shown in FIG. In addition, to the different sequences derived from the conserved region, each primer in each mixture also contains a constant region at the 5 'end which contains a sequence coding for sites for the restriction enzymes Hind III, Mbol and EcoRI ,

The "downstream" primer, ssc5h20A, is derived from a nucleotide sequence in clone 5h containing HCV cDNA with sequences that overlap sequences in clones 14І and 11b. The sequence of ssc5h20A is

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

An alternative primer, ssc5 h34 A, can also be used. This primer is derived from a sequence in clone 5h and additionally contains nucleotides at the 5 'end which form a restriction enzyme site, thus allowing for cionation. The sequence of ssc5h34A is

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

The PCR reaction, first described by Saiki and coworkers (1986), is performed essentially as described by Lee and coworkers (1988), except that the template for the cDNA is RNA derived from HCV-infected chimpanzee liver isolated as described in Section IV.C.2. or derived from viral particles isolated from HCV-infected chimpanzee serum as described in Section IV.A.1. In addition, the "annealing" are conditions in the first round of Amplikation less severe (0.6 M NaCl and 25 ⁰ C), since the part of the primer which hybridizes to the HCV sequence is (anneal) only 9 nucleotides, and there In addition, when ssc5 h34 A is used, the extra sequences not derived from the HCV genome tend to destabilize the primer-template hybrid. "After the first round of amplification, annealing Conditions be more stringent (0.066M NaCl and 32 ⁰ C to 37 ⁰ C) since amplified sequences now contain regions that are complementary to the primers or their duplicates. In addition, the first 10 cycles of amplification with Klenow enzyme I are carried out under PCR conditions suitable for this enzyme. After completion of these cycles, the samples are extracted and treated with Taq polymerase according to the kit instructions provided by Cetus / Perkin-Elmer. After Arr olifikation the amplified HCV cDNA sequences are detected by hybridization using a probe derived from clone 5h. This probe is derived from sequences "upstream" from those from which the primer was derived, and does not overlap the sequences of clone 5h-derived primers

5 'CCC AGC GGC GTA CGC CGT GGA CAC GGA GGT GGC CGC GTC GTG TGG CGG TGT TGT TCT CGT CGG GTT GAT GGC GC 3'.

IV.N.1. Creation of chimpanzee liver HCV cDNA library with infectious NANBH A chimpanzee liver HCV cDNA library was prepared from the HCV cDNA library in Section IV.A.1. had been created. The technique for creating the library was similar to that of section IV.A.24 except that a different RNA source and a primer based on the sequence of HCV cDNA in clone 11b was used. The sequence of the primer is

5 'CTG GCT TGA AGA ATC 3'.

IV.N.2. Isolation and Nucleotide Sequence of Overlapping HCV cDNA In clone K9-1 to cDNA in clone 11b Clone K9-1 was isolated from the HCV cDNA library created from the liver of a NANBH-infected chimpanzee as described in Section IV. A.25. has been described. The library was screened for clones that overlap the sequence in clone 11b using a clone that overlaps clone 11b at the 5 'terminus, clone 11e. The sequence of clone 11b is shown in FIG. Positive clones were with frequency ', to 500,000 isolated. An isolated clone, K9-1, was subjected to further investigations. The overlapping nature of the HCV cDNA in clone k9-1 to the 5 'end of the HCV cDNA sequence in Figure 26 was confirmed by probing the clone with clone Alex46, this latter clone containing a HCV cDNA sequence of 30 Base pairs corresponding to the base pairs at the 5 'terminus of the HCV cDNA in clone 14 i as described above.

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

The HCV cDNA sequence in clone к9-1 was compared with those in section ІѴ.А.19. aligned clones to create a composite HCV cDNA sequence with the k9-1 sequence placed "upstream" of the sequence shown in Figure 32. The composite HCV cDNA encoding the k9-1 And the amino acids encoded therein is shown in FIG. 47.

The sequence of the amino acids encoded in the 5 'region of the HCV cDNA shown in Fig. 47 was identified with the corresponding region in one of the dengue virus strains described above with respect to the profile of the regions for hydrophobicity and hydrophilicity. This comparison showed that the polypeptides encoded in this region consist of HCV and

Dengue virus, which region corresponds to the region encoding NS1 (or a portion thereof), has a similar hydrophobic / hydrophilic profile.

The information provided below allows the identification of HCV strains. The isolation and characterization of other HCV strains can be accomplished by isolating the nucleic acids from body components containing viral particles by creating cDNA libraries using polynucleotide probes based on the HCV cDNA probes described below, by screening the libraries Clones containing the HCV cDNA sequences described below, and by comparing the HCV cDNAs from the new isolates with the cDNAs described below. The polypeptides encoded therein or in the viral genome can be monitored for utilization of the polypeptides described above and for immunological cross-reactivity. Strains that meet the parameters of HCV described in the Definitions section above are easily identifiable. Other methods of identifying HCV strains will be apparent to those skilled in the art based on the information provided herein.

Continuation of the description of the figures

Figure 36 shows the HCV cDNA sequence in clone k9-1, the segment that overlaps the cDNA in Figure 26 and the amino acids encoded therein. Fig. 47 shows the sequence in a composite cDNA derived by dressing clones к9-1 to 15e in the 5 'to З' direction; it also shows the amino acids encoded in the continuous ORF.

Industrial applicability

The invention presented here in various disclosures has many industrial applications, some of which are presented in the following. The HCV cDNAs can be used to design probes for detection of HCV nucleic acids in samples. The probes derived from the cDNAs can be used to detect HCV nucleic acids, e.g. used in chemical synthesis reactions. They may also be used in anti-viral agent screening programs, to determine the effect of viral replication inhibition agents in taring culture systems and in animal model systems. The HCV polynucleotide probes are also useful in detecting human Vlrus nucleic acids and thus can serve as a basis for the diagnosis of HCV infections in humans.

In addition to the above, the cDNAs provided herein provide information and provide a means of synthesizing polypeptides containing HCV epitopes. These polypeptides are useful in detecting antibodies to HCV antigens. Described herein is a series of HCV infection-based immunoassays based on recombinant polypeptides containing HCV epitopes, the commercial application in the diagnosis of HCV-induced NANBH, in the screening of blood donors for HCV-induced infectious hepatitis and also in detecting contaminated blood from infectious blood donors. The viral antigens will also be useful in monitoring the efficacy of anti-viral agents in animal models. In addition, the polypeptides derived from the HCV cDNAs disclosed herein will be useful as vaccines for the treatment of HCV infection.

The polypeptides derived from the HCV cDNAs are also useful for developing anti-HCV antibodies, besides the uses mentioned above. They can therefore be used in anti-HCV vaccines. The antibodies produced as a result of immunization with the HCV polypeptides are also useful in detecting the presence of viral antigen in samples. They can thus be used to test the production of HCV polypeptides in chemical systems. The anti-HCV antibodies can also be used to monitor the efficacy of anti-viral agents in screening programs in which these agents are tested in tissue culture systems. Furthermore, they may also be used for passive immunotherapy and for the diagnosis of HCV-induced NANBH by detecting the virus antigen (s) in both the blood donor and the recipient. Another important use of anti-HCV antibodies is in affinity chromatography for the purification of virus and viral polypeptides. The purified virus and viral polypeptide preparations may be terminated in vaccines. In addition, the purified virus may also be useful in the development of cell culture systems in which HCV replicates.

Cell culture systems containing HCV-infected cells can be used in a variety of ways. They can be used on a relatively large scale for production of HCV, which is typically a low titer virus. These systems will also be useful in elucidating the molecular biology of the virus and leading to the development of antiviral agents. The cell culture systems will also be useful in screening for the efficacy of the antiviral agents. In addition, HCV-permissive cell culture systems are useful in the production of attenuated HCV strains.

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

The method used to isolate HCV cDNA and from the production of a cDNA library derived from the infected tissue of an individual in an expression vector, and in the selection of clones producing the expression products immunologically with Antibodies in antibody-containing body components from other infected individuals, but not with those of uninfected individuals, may also be used in the isolation of cDNAs derived from other, uncharacterized, disease-accompanying agents described hereinbefore from a genomic Component exist.

This, in turn, could lead to the isolation and characterization of these agents as well as diagnostic reagents and vaccines for these other disease-accompanying agents.

Claims (3)

1. A method for the detection of HCV nucleic acids in a sample, characterized in that a) nucleic acids of the sample react with one another for a HCV polynucleotide probe under conditions permitting the formation of a polynucleotide duplex between the probe and the HCV nucleic acid from the sample; and b) a polynucleotide duplex containing the probe is detected. 2. The method according to claim 1, characterized in that the HCV polynucleotide is contained in the HCV cDNA sequence according to Fig.47. 3. The method according to claim 1, characterized in that the HCV polynucleotide in an HCV cDNA sequence in ATCC no. 40388, ATCC no. 40389, ATCC no. 40390, ATCC no. 40391, ATCC No. 40S94, ATCC No. 40511, ATCC No. 40512, ATCC No. 40513 or ATCC No. 40514.