DD298524A5 - ANALYSIS KIT FOR HCV POLYNUCLEOTIDES, HCV ANTIGENES AND ANTIBODIES TAKEN AGAINST HCV ANTIGENS - Google Patents

Abstract

A family of cDNA sequences derived from hepatitis C virus (HCV) is provided. These sequences encode antigens which immunologically react with antibodies present in individuals with non-A non-B hepatitis (NANBH), but which generally do not occur in individuals infected with hepatitis A virus (HAV ) or hepatitis B virus (HBV) are infected and are also absent in control individuals. Comparison of these cDNA sequences with the sequences on the gene bank and with the hepatitis delta virus (HDV) and HBV sequences shows a lack of substantial homology. A comparison of the sequence of amino acids encoded in the cDNA with the sequences of flaviviruses indicates that the HCV is a flavivirus or a flavivirus. The HCV cDNA sequences are useful in the design of polynucleotide probes and for the synthesis of polypeptides that can be used in immunoassays. Both the polynucleotide probes and the polynucleotides may be useful for the diagnosis of HCV-induced NANBH and for the screening of blood bank samples and donors after HCV infection. In addition, these cDNA sequences may be useful for the synthesis of immunogenic polypeptides which may be used in vaccines for the treatment, prophylaxis and / or therapy of HCV infection. The polypeptides encoded in the cDNA sequences can also be used to develop antibodies to HCV antigens and to purify antibodies directed against the HCV antigens. These antibodies may be useful in immunoassays for the detection of HCV antigens associated with NANBH in individuals as well as in blood banks. In addition, these antibodies can be used in the treatment of NANBH in individuals. The reagents provided according to the invention also enable the isolation of NANBH pathogen (s) and the reproduction of this pathogen (s) in tissue culture systems. Furthermore, they provide reagents useful in screening for anti-viral agents of HCV, particularly in tissue culture or animal model systems. {Hepatitis C virus; Non-A non-B hepatitis; cDNA sequences; flaviviruses; flaviviruses; polynucleotides; polynucleotide; polypeptides; HCV epitopes; Antibody; HCV antigens; Screening of blood bank samples; analysis kit}

Description

For this 63 drawings

Analysis kit for HCV polynucleotides, HCV antigens and antibodies directed against HCV antigone

Technical area

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

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Cited patent no.

U.S. Patent No. 4,341,761 U.S. Patent No. 4,399,121 U.S. Patent No. 4,427,783 U.S. Patent No. 4,444,887 U.S. Patent No. 4,466,917 U.S. Patent No. 4,472,500 U.S. Patent No. 4,491,632 U.S. Patent No. 4,493,890

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 other than other forms of viral-bgliding liver disease, including those caused by the known hepatitis viruses, ie Hepatitis A virus (HAV), hepatitis B virus (HBV) and delta hepatitis viruo (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. Methods used to detect putative NANBV antigens or antibodies include agar gel diffusion, over-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 no clarity or consistency in the identity or specificity of the antigenic 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 a possibility that NANBH caused by more than one infectious agent wild 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 immigration electrophoresis assay detect autoimmune reactions or non-specific protein interactions that sometimes occur between serum samples, and that they do not represent specific NANBV antigen-antibody reactions. Immunofluorescence, enzyme immunoassay and radioimmunoassay appear to detect low levels of a rheumatoid factor-like material that is commonly found in the serum of patients with NANBH and also patients with other non-hepatitis and hepatitis. 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 of NANBV carriers and for NANBV-contaminated blood or blood products is of great importance. Posttransfusion 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 to chronic liver damage (25-55%).

Patient treatment, as well as the prevention of NANBH transmission through blood and blood products, or through close personal contact, 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 newly synthesized antigens resulting from were derived from the genome of the hitherto unisolated and uncharacterised virus pathogen, and the production of clones producing products which 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 as well as the sequence information contained in the HCV cDNA indicate that the HCV is a flavirus or a flaviartic virus.

Portions of the cDNA sequences derived from the HCV are useful as probes to detect the presence of the virus in samples, and to naturally isolate the virus. These cDNAs also provide polypeptide sequences of HCV antigens encoded in the HCV genome (s) and permit the production of polypeptides that are used as standards or as reagents in diagnostic assays 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 for 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 NANBH.

Accordingly, with respect to the polynucleotides, some aspects of the invention relate to: a purified HCV polynucleotide; a recombinant HCV polynucleotide; a recombinant polynucleotide, as 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, wherein the ORF is linked to a

Operably linked to a control sequence that is compatible with a desired host; a cell that is 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 HCA cDNA; a recombinant polypeptide consisting of an HCV epitope; and a fusion polypeptide consisting of an HCV polypeptide. The invention further 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 that is immunogenic against HCV infection and contains a non-HCV polypeptide having an amino acid sequence that can form 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 . 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. τι suitable container; Analyzing 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 producing a polypeptide containing an HCV epitope by incubating host cells transformed with an expression vector, wherein the vector contains a sequence encoding a polypeptide containing an HCV epitope, under conditions the expression of the polypeptide e. bowers; and a polypeptide containing an HCV epitope generated by this method.

The invention also encompasses a method for 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 induce the formation of a polycucleotide duplex between the probe and the HCV nucleic acid allow the sample; and for detection of a polynucloloid 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 that prevent the formation of an antigen-antibody. Allow complex; and for detecting an antigen-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 probe polypeptide containing an epitope of 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.

A still further 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 immunoreactant.

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 with the pathogen 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 that express 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 individual Allow clones, and these clones are isolated; and (f) isolating the cDNA from the host cell clones of step (e).

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 putative amino acid sequence of the polypeptide encoded therein.

Fig. 2: shows the homologies of the 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, 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 polypeptides encoded therein

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

coded polypeptide sequence

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

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 polypeptide encoded therein.

Fiy. 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. Figure 13: shows the HCV cDNA sequence in clone 25 o, 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 40 b, 37 b, 35, 36, 81, 32, 33 b and 25 c is extended. Fig. 15: shows the HCV cDNA sequence in clone 33c, the segment that overlaps clones 40b and 33c, and the amino acids encoded therein.

Fig. 16: shows the HCV cDNA sequence in clone 8h, the segment licking clone 33c and the amino acids encoded therein. Fig. 17: shows the HCV cDNA sequence in clone 7 e, the segment that overlaps clone 8 h and the amino acids encoded therein. Fig. 18: shows the HCV cDNA sequence in clone 14c, the segment that overlaps clone 25c and the amino acids encoded therein. 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 33 g, the segment that overlaps clone 33 f and the amino acids encoded therein. Fig. 22: shows the HCV cDNA sequence in clone 7f, the segment containing the sequences? overlapped in clone 7 e and encoded therein

amino acids

 Figure 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 and encodes the sequence in clone 33g

amino acids

 Fig. 26: shows a composite HCV cDNA sequence obtained 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; will continue in the

extended ORF shown in the deduced sequence amino acid sequence of the polypeptide. Figure 27: shows the sequence of HCV cDNA in clone 12f, the segment overlaps 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 the HCV cDNA in clone 19g, the segment that overlaps clone 35f and the amino acids encoded therein.

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

5'-3 'direction and the amino acids encoded in the continuos ORF. Fig. 33: shows a photograph of Western blots of a fusion protein, SOD-NAND ^ ₁ . ₍ · With chimpanzee serum out with BB-NANB,

HAV and HBV chimpanzees

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

Fig. 35: shows in a map the significant features of the vector pAB 24. Fig. 36: shows the putative amino acid sequence of the carboxy terminus of the fusion polypeptide C100-3 and the nucleotide sequence coding therefor.

Figure 37 A: is a photograph of a Coomassie blue-stained polyacrylamide gel that identifies C100-3 expressed in yeast. Figure 378: 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 derived from the liver of one infected 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 containing anti-N ANB _5. ,

were probed and probed with ³² P-labeled NANBV cDNA from clone 81. 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 homology 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 eriindeungsgemSßen execution

I. Definitions

The term hepatitis C virus was reserved by scientists working in this field for a previously unknown pathogen of NANBH. Accordingly, the term "hepatitis C virus" (HCV), as used herein, refers to a pathogen causing NANBH, formerly known as NANBV and / or BB-NANBV, the terms HCV, NANBV and BB-NANBV As an extension of this terminology, the disease caused by HCV, formerly known as NANB hepatitis (NANBH), is now called hepatitis C. The terms NANBH and hepatitis C can be used interchangeably herein.

The term "HCV" as used herein refers to a virus species that causes NANBH, as well as debilitated strains or defective interfering particles derived therefrom As will be shown below, the HCV genome is RNA that RNA-containing viruses relatively hoha, spontaneous mutation rates, which should be in the range of 10 ~ ³ to 10 ^"4 per nucleotide (Fields & Knipe (1986)), have. Thus, there are many strains within the HCV species described below. The compositions and methods described herein allow 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 screening for antiviral agents for pharmacological purposes by inhibiting the replication of HCV. The information provided herein, although derived from an HCV strain, hereinafter referred to as CDC / HCV1, is sufficient for a virus taxonomist 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 rar. Their cores have a diameter of about 25-30 nm. On the outer surface of the virion shell are projections with a length of about 5-10nm with Uirminalon buttons (knobs) of about 2 nm in diameter.

The HCV encodes an epitope that is immunologically identifiable with an epitope in the HCV genome from which the cDNAs described herein were derived, the epitope is preferably encoded 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 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 hybridization of the polynucleotides under conditions where stable duplexes are formed between homologous regions (e.g., those that would be used prior to the S, digestion), then digestion with single-stranded ones (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 determined by the For example, the sequence may be compared to sequences in databases, such as Genebank, to determine if they are known to those skilled in the art. whether 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 hepatitis. As HAV, HBV and HDV, and compared with other members of Flaviviridae. The correspondence or non-conformity of the deduced sequence with other sequences can also be determined 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 coworkers (1982). Furthermore, malformations of duplex polynucleotides resulting from hybridization may occur by known techniques, such as. For example, digestion with a nuclease such as S1, which specifically digests single-stranded regions into duplex polynucleotides, may include but is not limited to regions from which typical DNA sequences can be "derived", for example, regions specific for epitopes encode as well as non-transcribed and / or untranslated regions.

The deduced polynucleotide is not necessarily physically recovered from the nucleotide sequence shown, but may be obtained in various ways, including 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, eg, 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, which part consists 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 is immunologically identifiable with a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily of a designated nucleic acid sequence, e.g. The sequences shown in Figs. 1-32, or translated from an HCV genome, it can be generated in a variety of ways, including e.g. B. chemical synthesis or expression of a recombinant expression system, or isolation from mutant HCV.

The term "recombinant polynucleotide" as used herein refers to a polynucleotide 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 present 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, methylated and / or "capped" modified and unmodified forms of the polynucleotide.

As used herein, the term "HCV containing a sequence corresponding to a cDNA" means that the HCV contains a polynucieotide 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 about at least 90 nucleotides in length The 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 on a H A CV genome or fragment thereof that is substantially free of, ie, 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, for example, U.S. Pat. For example, disruption of the particles with a chaoirope 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 known in the art and examples of these techniques are discussed below: "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures" and other such terms which designate microorganisms or higher eukaryotic cell lines taken as unicellular units in culture, refer to cells that can be used as recipients for another transfer DNA or that use it, and also include the progeny of the original cell that transfects was a. It is understood that the progeny of a single parental cell need not necessarily be identical in morphology or as genomic 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 promoter, ribosome tins 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. leader sequences. "Operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting their function in the intended voice. A control sequence "operably linked" to the 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 encoding 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 initiation 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 known to those skilled in the art and are also illustrated below. The uniqueness of an epitope can also d-n oh Computerforschungen in known databases, z. 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 antibody binding, especially by kinetics antibody binding, and / or by binding competition, by using a known polypeptide or known polypeptide (s) containing an epitope against the antibody (s) targeted as a competitor (s); whether a polypeptide is immunologically reactive with an antibody is known in the art.

As used herein, the term "an HCV-containing immunogenic polypeptide" includes naturally occurring HCV polypeptides or fragments thereof as well as polypeptides produced 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 such that peptides, oligopeptides and proteins are included in the definition of the polypeptide, nor does it 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-mr :: °". The exogenous polynucleotide can be retained as a non-integrated vector, such as. A piasmide, or may be integrated into the host genome in some other way.

"Treatment" as used herein 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, sports animals, primates, and humans. As used herein, the "plus strand" of a nucleic acid includes the sequence, which codes for 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 in which the genome, whether RNA or DNA, is single-stranded and encodes a viral polypeptide / virus polypeptide.Examples of positive-stranded RNA viruses are Togaviridae, Coronaviridae, Retroviridae, Also included are the Flaviviridae, formerly classified as Togaviridae, see Fields & Knipe (1986).

As used herein, "antibody-containing body component" refers to a component of the body of an individual that is a source of the antibodies of interest. Antibody-containing body components are known in the art and include, for example, plasma, serum, cerebrospinal fluid, Lymph, the outer sections of the respiratory, digestive and urogenital tract, tears, saliva, milk, white blood cells and myeloma cells.

As used herein, "purified HCV preparation" refers to an HCV preparation that has been isolated from the cellular components with which the virus is normally associated and from other virus species that may be present in the infected tissue Isolation of the viruses are known to those skilled in the art and include, for example, centrifugation and affinity chromatography, and a method of preparing purified HVC is 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, for example, Maniatis, Fitsch & Sambrook, MOLECULAR CLONING; A LABORATORY MANUAL (Molecular Cloning, a Laboratory Manual) (1982); DNA CLONING, VOLUMES I AND II (DNA Cloning, Volumes I and II) (Editor D.N. Glover, 1985); OUGONUCLEOTIDE SYNTHESIS (Oligonucleotide Synthesis) (Editor MJ Gait, 1984); NUCLEIC ACID HYBRIDIZATION (Nucleic Acid Hybridization) (Editor B.D.Hames & S J.Higgins, 1984); TRANSCRIPTION AND TRANSLATION (Transcription and Translation) (Editors B.D. Harnes & SJ.Higgins, 1984); ANIMAL CELL CULTURE (Animal Cell Culture) (Editor R. I. Frishney, 1986); IMMOBILIZED CELLS AND ENZYMES (Immobilized Cells and Enzymes) (IRL Press, 1986); B. Perbai, A PRACTICAL GUIDE TO MOLECULAR 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 (gene transfer vectors for mammalian cells) (Editor JH Miller and MP Calos, 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Methods of Enzymology, Vol. 154 and Volume 155) (Ed Wu and Grossman bzvv. Wu), editors Mayer and Walker (1987), IMMUNOLOGICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London), Scopes (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE (Protein Purification: Principles and Practice), Second Edition, (Springer-Verlag, NY) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES WV (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 shows

Sequenzfemilie no significant homology to sequences contained in the HBV genome.

The sequence of a member of the family contained in clone 5-1-1 has a continuous open leno frame (ORP) coding 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. In addition, antibodies that bind to this polypeptide are not detected in chimpanzees and humans infected with HAV and HBV. 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 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 a synthetic sequence corresponding to a fragment of cDNA in clone 5-1-1 is shown in FIG. A member of the cDNA family isolated by using a synthetic sequence derived from the cDNA in clone 81 is shown in FIG. 5, 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, 14i, 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 faviartiges virus.

The availability of this family of cDNAs shown in Figures 1-32 permits the construction of DNA sorbed and polypeptides useful in the diagnosis of NANBH as a result of HCV infection and screening assays in blood donors as well as donor blood and blood products are useful for an infection. 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 genome in, for example, sera of individuals who may harbor the virus, or the like useful for screening donated blood for the presence 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 the therapy of 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, other segments of the genome can be recovered by repeating the Lambdagtll serological screening procedure to isolate the cDNA clones described herein or, alternatively, 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 virus. Alternatively, the antibodies may 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 for 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 agent of NANBH is novel and can be used in the identification and / or isolation of previously uncharacterised pathogens containing a genome and those 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 pathogen polypeptide were selected by immunological screening of the library's expression products with an antibody-containing body composition 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 between the expression product (s). and antibodies of the second infected 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 individual with the suspected pathogen and with control individuals not infected 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

which 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 and it.re expression products isolated as a result of this method and the antibodies directed against the expression products are useful for the characterization and / or capture of the etiological agent. As will be described more fully below, this method has been successfully used to isolate a family of cDNAs derived from the HCV genome.

U.A. Preparation of the cDNA Sequonz

Sera collected from a chimpanzee with chronic HCV infection containing a high virus titer, i.e., at least a 10 ° chimpanzee infectious dose / ml (CID / ml), has been 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 Lambdagtll are discussed in Section IV. A. 1. Lambda-gt 11 is a vector specifically designed to express inserted cDNAs as fusion peptides with beta-galactosidase and to produce a large number of recombinant phage with specific antisera to a particular antigen. The lambda gt 11 cDNA library, prepared from a cDNA library containing about 200 basepair average size cDNA, was screened for coded epitopes that could specifically bind to sera derived from previously NANB hepatitis patients , Huynh, TV et al. (1085). Approximately 10 ^{e of} phage were screened and five positive phage were identified, purified and then tested for specificity of binding to sera from various humans and chimpanzees previously infected with HCV agent. One of the phages, 5-1-1, had bound 5 of the 8 tested human sera. This binding appeared to be selective for sera from patients previously exposed to NANB hepatitis infection since 7 normal blood donor sera did not exhibit such binding.

The sequence of the cDNA in the recombinant phage 5-1-1 was determined and shown in FIG. The polypeptide encoded by this cloned cDNA, which 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 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 additional segments and / or alternative segments of the cDNA for the viral genome. The lambda gt 11 cDNA library described above was screened using a synthetic polynucleotide derived from the sequence of the cloned 5-1-1 cDNA. 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. Nucleotide sequences are present exclusively in clones 5-1-1,81 and 91 and are designated by small letters.

The cloned cDNAs present in the recombinant phage 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 a thymidine, while the other three clones contain a cytidine residue in this position. However, this substitution does not alter the nature of the encoded amino acid.

The second difference between the clones is that clone 5-1-1 contains 28 base pairs at its 5'-terminus which are not present in other clones. This additional sequence may be a 5'-terminal cloned artifact; 5'-terminal cloned artifacts are generally observed in the products of cDNA methods.

Synthetic sequences derived from the 5 'region and 3' region of the HCV cDNA in clone 81 were used to screen and isolate cDNAs from the lambda gt 11 NANBV cDNA library encoding 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.

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 containing the clone 36 cDNA (Section IV. A. 8. ) overlap. A 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 downstream HCV cDNA sequences were obtained. The isolation of these clones is described later in Section IV. A.

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 assembled 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, can be prepared using synthetic methods or by a combination of the partial sequence repair synthetic methods using procedures similar to those described herein.

Lambda gt 11 strains replicated from the HCV cDNA library and from clones 5-1-1,81,1-2 and 91 were in accordance with the provisions of the Budapest Treaty in 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 Nov. 18, 1987

In granting and issuing this application as a United States patent, all restrictions on the availability of such deposits will be irrevocably removed; Access to the designated deposits is made possible during the pendency of the above application to the one designated by the patent agent authorized to do so under 37 CFR 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 is concerned with "walking" a genome by isolating overlapping cDNA sequences from the HCV lambda gt 11 library, provides a method to isolate t'as cDNAs corresponding to the entire HCV genome 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., supra.

II. B. Preparation of Vectoric 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 antigenically active regions of the polypeptide encoded in each strand. These antigenically active regions may be derived from the overrigid or envelope antigens or from core antigens including e.g. Polynucleotide binding proteins, polynucleotide polymerases) 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 are raised in 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 Publication No. 0196056, published October 10, 1986. Vectors encoding fusion polypeptides of SOD and HCV polypeptides, ie NANB ₅ ,... ,, NANB ₈ , and C100-3 encoded in a composition of HCV cDNAs are described in Sections IV. B. 1, IV. B. 2 or 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 in fused or mature form, and whether or not a signal sequence that allows secretion, may be ligated into expression vectors suitable for each host. Both eukaryotic and prokaryotic host systems are currently used in the production of recombinant polypeptides, and a summary of some of the more common control systems and host lines is provided in Section III. A., given 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 purification can be carried out by methods known in the art, e.g. B. salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, etc. take place. For the variety of methods for the purification of proteins, see e.g. Such "polypeptides" may be used as diagnoses, or those which cause neutralization of antibodies may be formulated in vaccines. [Gegen Die] Antibodies directed against these polypeptides may also be used as diagnoses or for the purposes of the art In addition, as discussed herein later in Section II, J., 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.

II. C. Preparation of Generic Polypeptides and Conjugation with Carriers An antigenic region of a polypeptide is generally relatively small, typically from 8 to 10 amino acids or a shorter length. Fragments of only 5 amino acids can characterize an antigenic region. Disso segments may correspond to regions of the HCV antigen. Thus, using the cDNAs of HCV as a base, DNAs 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-maleimidomethyl) cyclohexane-i-carboxylate ( 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 a 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 disulfide / amici-forming agents is known, see, for example, Immun. Rev. (1982) 62: 185.

Other bifunctional coupling agents form ehor a thioether as a disulfide compound. Many of these thioether-forming agents are available commercially and include effective esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-acylacetic acid, 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid, and the like. The carboxyl groups can be activated by combination with succinimide or 1-hydroxyl-2-nitro-4-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 carrier sources 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 commonly known to those skilled in the art.

D. D. Preparation of HCV-Epltopo Containing Hybrldpartlkelimmogenogen

The immunogenicity of the epitopes of HCV can also be assessed by producing them in mammalian or yeast systems that fuse and assemble with particle-forming proteins, such as. As that associated with hepatitis B surface antigen, are enhanced. 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 particles constructed from particle-forming protein which include HCV sequences are immunogenic with respect to HCV and HBV. The hepatitis surface antigen (HBSAg) has been described in S. cerevlslae (Valenzuela et al. [1982]), e.g. also formed in mammalian cells (Valenzuela, P., et al. [19841]) and assemblies. 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. a. (1985). Constructs of 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 coding 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 become.

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 regions 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 it is translated into three viral structural proteins, i. H. 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, vaccines comprising recombinant polypeptides may contain epitopes of HCV-E. 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 retain epitopes that produce protective anti-HCV antibodies. Consequently, it is also possible to use in HCV vaccines polypeptides 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 the immunization did not cause the formation of neutralizing antibodies. Thus, particularly as this protein appears to be highly conserved among the flaviviruses, it is likely 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 cause the production of neutralizing antibodies.

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 HCVs may provide 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 can also be emulsified, or the protein can 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 , nomen-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine ^ -li''-dipalmitoyl-sn-glycero-S-hydroxyphosphryloxylethylamine (CGP 19835A, referred to as MTP-PE) and RIBI containing three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in 2% Squalone / 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. B. 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, pills, capsules, long-acting formulations or powders and contain from 10% to 95% of the active ingredient, preferably from 25% to 70%.

The proteins could be formulated in neutral or saline form into the vaccine. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the peptide) and those with inorganic acids, e.g. Hydrogen chloride 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-ethylaminoethanol, 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 being treated, the ability of the subject's immune system to synthesize antibodies and the desired degree 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 which may be required during subsequent time intervals required to maintain or re-establish the immune response, e.g. For example, a second dose may be administered in 1 to 4 months, and, if necessary, after several months, another dose or doses may be administered. The dosage regimen will also, i. at least in part, based on the needs of the individual and depends on the judgment of the doctor.

In addition, the vaccine containing the immunizing HCV antigen (s) may be used in conjunction with other immunoregulating agents, e.g. Immunoglobulins, administered.

II.G. Production of Antibodies to HCV Epitopes

The immunogenic polypeptides prepared as described above are used to produce antibodies, both polyclonal and 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) Immunized animals are collected and treated according to 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. An example of a method of purifying antibodies to HCV epitopes from serum of an infected individual based on affinity chromatography using a fusion polypeptide of SOD and a polypeptide encoded within cDNA clone 5-1-1 is described in Section V. E, shown.

Monoclonal antibodies directed against HCV epitopes can also be readily prepared by one skilled in the art. The general procedure for the production of monoclonal antibodies by hybridomas is well known.

Immortal antibody-producing cell lines can be generated by cell fusion and also by other techniques, such as direct transformation of B-lymphocytes with engrafting DNA or transfection with the Epstein-Barr virus. See, for example, B. M. Schreier u. a., (1980); Hammerling u. a. (1981); Kennett u. a. (1980); Also, see U.S. Patent Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,887, 4,466,917, 4,472,500, 4,491,632, and 4,493,890. Monoclonal antibodies produced against HCV epilepsies may be different in their properties, i. H. Isotypes, epitope affinity, etc. are screened by means of lists.

Antibodies, both monoclonal and polyclonal, directed against HCV epitopes are particularly useful in diagnosis, and those that have a neutralizing effect are useful in passive immunotherapy. Monoclonal antibodies can specifically be used to develop anti-idiotypic antibodies.

Anti-idiotypic antibodies are immunoglobulins that carry an "inner" image of the antigen of the infectious agent against which protection is desired, See, e.g., Nisonoff, A., et al., (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 Uytdehaag et al. (1985). These anti-idiotypic antibodies may also be useful for the treatment of NANBH as well as for the lightening of the immunizing regions of the HCV antigen.

II. H. Diagnostic Oligonucleotide Probes and Kits

Using the disclosed portions of the isolated HCV cDNAs ' As base, including those in Figures 1 to 32, oligomers of about 8 nucleotides or more, either by excision or synthetically, can be made to hybridize to the HCV genome and in the identification of the virus agent or virus agents, as well as in the characterization of virus 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 sufficient to detect unique virus 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 that lack heterogeneity. These probes can be made using routine methods, including automated oligonucleotide synthesis methods. Among the useful probes are z. Clone 5-1-1 and the additional clones disclosed herein, as well as the various oligomers useful in probing the cDNA libraries that will be reported 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 contained therein. The nucleic acid resulting from the sample may be subjected to gel electrophoresis or other size separation methods; otherwise, 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. For example, by nick translation or kinase, biotin, fluorescent probes and chemiluminescent probes inserted radioactive labels. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions which are subject 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 virus 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 e.g. 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 amplification 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 transferases to attach to a DNA probe unmodified 3'-poly-dT tails. "The poly-dT-tailed Probe hybridizes 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 by: (DAnalyt hybridizing to a single-stranded DNA probe which has been enzyme-labeled 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 sondo that has a tail, such as a poly-dT tail, an amplifier strand having a sequence that hybridizes to the tail of the probe, such as a poly A sequence, and is capable of a variety of labeled ones Tie strands. A particularly desirable technique may first involve amplifying the target HCV sequences in sera at about 10,000-fold, ie, at about 10 ⁶ sequences / ml. This can be z. B. by the technique of Saiki et al. (1986) can be achieved. The amplified sequence (s) may then be determined using a hybridization assay described in co-pending U.S. Application, Attorney Oocket no. 2300-0171, filed October 15, 1987, and assigned to the assignee hereof, and hereby incorporated by reference thereto. This hybridization assay, designed to detect sequences as high as 10Vml, 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 db methods for the preparation of probes are described in EPO 225307 (?), Published June 16, 1987, application no. No. 807,624, assigned to the assignee hereof 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 pol / peptides that react immunologically with HCV antibody-containing serum, e.g. Those derived from, or encoded by, the clones described in Section IV. A. and their compositions (see Section IV. A.) and the antibodies directed against the HCV-specific epitopes therein Polypeptides have been developed, see, for. Section IV. E. are useful in immunoassays for detecting the presence of HCV anti-fp particles or for the presence of the virus and / or viral antigens 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 HCV cDNA-containing clones, see Section IV. A., or from the composite cDNAs derived from the cDNAs in these clones, or from the HCV genome from which the cDNA were derived in these clones; otherwise, the immunoassay may use a combination of viral antigens derived from these sources. It can, for. For example, a monoclonal antibody directed against a virus apitope (s), a combination of monoclonal antibodies directed against a viral antigen, monoclonal antibodies directed against different viral antigens, polyclonal antibodies anti-antigenic to the same virus or polyclonal antibodies directed against different viral antigens. The logs can z. For example, based on competitive, or on direct reaction or sandwich-type assays. The protocols may also use solid documentation B. or may be by immunoprecipitation. Most assays involve the use of a labeled antibody or polypeptides; the markers can z. B. 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 compliant-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 corresponding labeled reagents are co-packaged with appropriate containers by packaging the appropriate materials, including the polypeptides of the invention, which have HCV epitopes or antibodies directed against HCV epitopes the remaining reagents and materials required to perform the assay, as well as a suitable set of assay protocols.

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

The HCV cDNA sequence information in the clones described in Section IV. A. and shown in Figures 1-32 is directed against HCV epitopes used in the diagnosis and / or treatment of HCV-induced NANBH would be useful. 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, labeled probes containing a sequence of about 8 or more nucleotides, and preferably 20 or more nucleotides derived from regions near the 5 'term or 3' term of the family of HCV cDNA sequences may be derived 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 that do not necessarily overlap the cDNAs in the clones described in Section IV. A. may be 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 segmented 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 isolation and 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 organism (s) isolated from the purified HCV particles. Methods for purifying the HCV particles ur, d for their detection during the purification process are described herein below. 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 herein, it is possible to clone and sequence the entire HCV genome (s), disregarding its or their 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. 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 obtained from Clor. isolated and sequenced by digesting the isolated polynucleotide with the appropriate restriction enzyme (s). Please refer -. B. Section IV.A. 3rd 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 the isolation and sequencing of HCV cDNA overlapping those in clone 81 and section 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 while isolating HCV (discussed below) to use the techniques described in Section II.1G. In addition, the overlapping cDNAs can be used to generate expression vectors for polypeptides derived from the HCV genome (s), including those described in clones 5-1-1, 36, 81, 91 and 1-2 and encode polypeptides encoded in the other clones described 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 contain antigens that have epitopes that appear unique to HCV, i. Antibodies directed against these antigens are absent in individuals infected with HAV or HBV and in non-HCV infected individuals (see serological data in Section IV. B.). In addition, a comparison of the sequence information of these cDNAs with the sequences of HAV, HBV, HDV and with the genomic shows

Sequences in the library, that there is minimal homology between these cDNAs and the polynucleotide sequences 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 isolated from the sera of BB-NANBV-infected individuals or from cell cultures by methods known in the art, including e.g. 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 nycol or chromatography on a variety of materials such as anionic or cationic exchange materials, and hydrophobicity-binding materials such as also affinity columns are isolated. During the isolation procedure, the presence of HCV can be determined by hybridization analysis of the extracted genome using probes derived from HCV cDNAs described above or by immunoassay (see Section II. I.) using as probes against HCV Antigenic antibodies encoded within the family of cDNA sequences shown in Figures 1 to 32, and 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 procedure for polyclonal antibodies directed against antigen (s) encoded within clone 5-1-1 is described in Section IV, E.; 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 adsorbed to the support (see, e.g., Kurstak in "Enzyme Immunodiagnosis," 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 and 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 assayed 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 described in U.S. Pat derived from overlapping HCV cDNA sequences and described in the text above. In this case, the fractions would be treated under conditions causing disruption of the virus particles, e.g. With detergents in the presence of celts and in the presence of viral nucleic acids as described in section H.H. Further confirmation that the isolated particles are the drugs that induce HCV can be obtained 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 looks like the virus is composed of an RNA genome. See Example IV.C. 2nd, below. Strandability and circularity or non-circularity can be determined by techniques known in the art, including e.g. 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 reserver 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 polypeptides encoded within the continuous open reading frame of combined clones 141 to 390 (see Figure 26) shows that the HCV polypeptide contains homology regions at the corresponding proteins in the conserved regions of the flaviviruses. An example of this is given in Section IIV. 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 flavirirus or a flavivirus. In general, flavivirus virions and their genomes have a relatively consistent structure and organization that are known. See Rice et al. (1988) and Brinton, M.A. (1988). Thus, the structural genes encoding polypeptides C, pre-M / M and E can be located in the 5 'terminus of the genome upstream of clone 14i. 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 14i can be accomplished in a variety of ways, and the information provided herein will be obvious 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 flaviviruses have conserved epitopes and regions of conserved nucleic acid sequences, and polynucleotides containing the conserved sequences can be used as probes that bind the HCV genome, and thus In addition, these conserved sequences, in conjunction with those derived from the HCV cDNAs, see Fig. 22, can be used to design starters for use in systems, lie genome sequences upstream of those in clone 14i using polymerase chain reaction technology An example of this will be described below.

The structure of 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,

Core antigens and polynucleotide polymerase (s) can also be detected by, e.g. B. Determining whether the antigens are present as the largest or smallest virus components, as well as by using the antibodies directed against the specific antigens encoded within isolated cDNAs as probes. This information is useful for the design of vaccines, e.g. For example, it may be beneficial to include an external antigen in a vaccine preparation. Multivalent vaccines may, for. One from the genome encoding a structural protein, e.g. E, derived polypeptide as well as a polypeptide from another part of the genome, e.g. A non-structural or structural polypeptide.

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

The assumption that HCV is a flavivirus or a flavivirus also provides information on methods for growing HCV. The term "flavivoidal" 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 Methods for the production of flaviviruses are known to those skilled in the art (see, e.g. For example, the reviews of Brinton (1986) Slollar, V. [1080]). In general, suitable cells or cell lines for the growth of HCV may include those known to support flavivirus replication, eg, the following: monkey kidney Cell lines (eg, HK ₃ , VERQ); porcine kidney cell lines (eg, PS); baby hamster kidney cell lines (eg, BHK); mouse macrophage cell lines (eg, P38801, MK1, MmI); human macrophage cell lines (eg U-937); peripheral human blood leucocytes, adherent human monocytes; hepatocytes or hepatocyte cell lines (e.g., HUH7, HEPC2); embryo or embryonic cells (e.g., chick embryo fibroblasts or cell lines derived from invertebrates , preferably insects (e.g. Drosophila cell lines), or more preferably of arthropods, e.g. Mosquito cell lines (eg, Albopictus, Aedes aegypti, Cutex tritaeniohynchus) or tick cell lines (eg, RML-14, Dermacentor parumaportus).

It is possible that primary hepatocytes can be cultured and subsequently infected with HCV; or otherwise, the hepatocyte cultures could be recovered from the livers of the infected individuals (e.g., humans or chimpanzees).

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 recover cell lines derived from hepatocyte cultures. For example, primary liver cultures (before and after accumulation of the hepatocyte population) may fuse with a number of cells to maintain their stability. It can z. Also, for example, cultures may be infected with transforming viruses or transferred with transforming genes to produce permanent or semi-permanent cell lines. 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 such as polyethylene glycol, Sendai virus and Epstein-Barr virus.

As discussed earlier, HCV is a flavivirus or a flavivirus. Therefore, it is likely that HCV infection of cell lines can be performed by techniques known in the art for infecting the cells with flaviviruses. These include z. B. incubating the cells with virus preparations under conditions that allow virus entry into the cell. In addition, it is possible to achieve virus production by transfecting the cells with 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, for example, U.S. Pat. For example, techniques such as electroporation and precipitation with DEAE-dextran or calcium phosphate. An abundant source of HCV RNA can be obtained by performing the HCV-corresponding in vltro transcription of a dom-complete genome. Transfection with this material or with cloned HCV cDNA would have to result in virus replication and in vitro 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]). Thus, HCV replication can occur not only in chimpanzees, but also in marmosets and suckling mice, for example.

II. L. Screening for antiviral agents for HCV

The availability of cell cultures and animal model systems for HCV also allows 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 antiviral agents in that they provide an alternative and perhaps a more sensitive means of detecting the effect of the agent on viral replication the cell plaque assay or ID ₃₀ assay. The HCV polynucleotide probes described herein can be used, for example, to quantitate the amount of orphan viral nucleic acids in a cell culture. This could be z. By hybridization or competition hybridization of the infected cell nucleic acids with a labeled HCV polynucleotide probe, for example, anti-HCV antibodies may also be used to identify and quantitate 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 cDNAq described herein are useful in these competition assays In general, a recombinant HCV polypeptide derived from the HCV cDNA would be labeled and the inhibition of binding of this labeled polypeptide to an HCV polypeptide due to the antigen produced in the cell culture system would be monitored useful in cases where the HCV may be able to be in a cellini e to 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 were 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 involve the use of a poly nucleotide which will retain a HCV sequence of at least about 8 nucleotides as a probe. Alternatively, or in addition, an attenuated strain can be constructed using the genomic information provided by HCV herein and using recombinant techniques. In general, one would look 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 epitope that causes neutralization of antibodies to 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, as the processing of these viruses would require less stringent protective measures for the agents involved in viral production and / or production of viral products.

III. General methods

The general techniques used for extracting the genome from a virus, preparing and probing a cDNA library, sequencing the clones, constructing expression vectors, transforming the cells, 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 is a general guide that highlights some currently available sources of such procedures and materials useful in carrying them out.

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 the 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 obtain 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. [1980]) and the lambda-derived PL promoter and the 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 UVS promoters. The above systems are particularly compatible with E. coil; if desired, other prokaryotic hosts, such as strains of Bacillus or Pseudomonas, can be used with the appropriate control sequences. Eukaryotic hosts contain yeast and mammalian cells in the culture systems. Saccharomyces cerevislae and Saccharomyces carlsbergensis are the most commonly used yeast hosts and are also beneficial fungus swirlers. Yeast-compatible vectors carry markers that allow the selection of successful transformants by conferring prototropy on auxotrophic mutants or resistance to heavy metals on wild strains. Yeast-compatible vectors can use the

2 micron origin of replication (Broach et al. [1981]), the combination of CDN3 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 can also be included, such as those derived from the enolase gene (Holland [1981]). Particularly useful are control systems, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohide dehydrogenase (ADH) -regulatable promoter, also GAPDH-derived terminators, and if secretion is desired, include "leader" sequence from 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 available from 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 [1978]), 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. transformations

The transformation may be by any known method for the introduction of polynucleotides into a host cell, including *. B. packaging the polynucleotides into a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotides. The transformation procedure used depends on the host to be transformed. The transformation of E. coli host cells with lambda gt 11 containing BB-NANBV sequences, z. 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 recovered by precipitation 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 with cohesive ends may have blunt ends when using E. coli DNA polymerase I (Klenow) in the presence of appropriate deoxynucleoitide triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease, which results in the hydrolysis of any single-stranded DNA segments, can also be performed.

Ligation is 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-ligation 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. B. selected by antibiotic resistance and screened for correct construction.

III.D. Construction of desired DNA sequences

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

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 Is Starter, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and the like td containing 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 yield plaques. Theoretically, 50% of the new plaques contain phage with mutated sequence. The remaining 50% have the original sequence. Replicas of the plaques are hybridized to the labeled synthetic probe at temperatures and conditions that allow 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, 0.75M NaCl, 75 mM Na citrate, 0.02% (M / V) of Each contains bovine serum albumin, polyvinylpyrollidone and Ficoll, 50 mM Na phosphate (pH = 6.5), 0.1% SDS and 100 micrograms / ml carrier denatured DNA The percent formamide in the buffer as well as the time and temperature conditions Pre-hybridization 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, such as derived from cDNA or genomic sequences generally require higher temperatures, e.g., about 40 ° C to 42 ° C, and a high formamidide urgent, eg 50%. Subsequent to the pre-hybridization, 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.

III.F. Verification of construction and sequencing

For routine vector designs, ligation mixtures are transformed into E. coli strain HB 101 or other suitable host, and successful transformants are solubilized by antibiotic resistance or other markers. Plasmic / e from the transformants are then according to the method of Clewell u. a. (1969), usually prepared by monitoring chloramphenicol amplification (Clewell [1972]). The DNA is isolated and analyzed on the basis of restriction enzyme 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 as described by Barr et al. (1986).

III.G. Enzyme-linked immunosorbent assay

Enzyme-linked immunological Tist (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 (e.g., a microplate or plastic crucible), incubated with test serum dilutions, washed, incubated with anti-immunoglobulin labeled with an enzyme, and washed again. Enzymes suitable for labeling are known in the art and include e.g. Horseradish peroxidase. The enzyme activity bound to the solid phase is measured by the Zi 'of the specific substrate, and the determination of product formation or substrate utilization e follows colorimetrically. 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 present 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. In Section IV.A. however, it may be necessary to repeat these procedures, as techniques for constructing the desired nucleotide sequences are available based on the information provided by the invention. Expression is illustrated in E. coil, 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 hepatic injury due to HCV infection. Since 1 ml of a ~ ^e 10 induced dilution of this collected and intravenous serum in another chimpanzee NANBH, its CID (Zytomegaliesyndrom) was at least 10 ^U / ml, that is, it had a high infectious virus titer.

A cDNA library from the high titer plasma pool was generated as follows. First, viral particles were isolated from the plasma; A 90 ml aliquot was diluted with 310 ml of a 50 mM Tris-HCl, pH 8, C 1 mM EDTA, 10 mM NaCl-containing solution. Residues were removed by centrifugation at 15,000xg for 20 min. At 20 ° C. Viral particles in the resulting supernatant were then pelleted by centrifugation in a Beckman SW28 rotor at 28000 U / min for 5 hours at 2O ⁰ C. To release the viral genome, the particles were prepared by suspending the pellets in 15 ml solution containing 1 % Sodium dodecylsulfate (SDS), 10 mM EDTA, 10 mM Tris-HCl, pH 7, j, and also 2 mg / ml proteinase k, and then incubated at 45 ° C. for 90 min. The nucleic acids were isolated by adding 0.8 micrograms MS2 bacteriophage RNA as vehicle and the mixture was washed four times with a 1: 1 mixture of phenol-chloroform (phenol saturated with O, 5M Tris-HCl, pH 7.5, 0, 1 % (V / v) beta-mercaptoethanol, 0.1% (w / v) hydroxyquinoline, and then extracted twice with chloroform The aqueous phase was precipitated with 2.5 volumes of absolute ethanol and left overnight at -20 The nucleic acid was recovered by centrifugation in a Beckman SW41 rotor at 40,000 rpm for 90 minutes at 4 ° C and dissolved in water containing 0.05% (v / v) diethylpyrocarbonate treated and cooked in an autoclave.

The nucleic acid recovered by the above procedure (<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 gt11 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 replaced (Taylor et al. [1976]). The resulting double-stranded cDNAs were fractionated according to size on a Sepharose CL-4 B 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 et al. (1985), about 10 ^{s of} phage were screened with patient sera except that bound human antibodies could be detected with sheep anti-human IG antisera radiolabeled with ¹²⁶ I. 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 phages encoded

a polypeptide that immunologically reacted with only one human serum, i.e., one that was 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 encodes a polypeptide which is recognized immunologically specifically by sera from NANB patients.

IV.A.2. Sequences of the HCV cDNA 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]) using the dideoxyk'termation method of Sanger et al. a. (1977). The recovered sequence is shown in FIG. The coJie te polypeptide coded 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 5-1-1 translational open reading frame (ÜRF) encodes an epitope or epitopes specifically recognized by sera from NANBH infection-challenged patients and chimpanzees.

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

HCV cDNA overlapping the cDNA in clone 5-1-1 was screened by screening the same lambda gt 11 library as described in Section IV.A.1, one from the sequence of the HCV cDNA in the clones 5-1-1, as shown in Fig. 1, derived derived synthetic polynucleotide. The sequence of the polynucleotides used for the screening was:

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

The I.ambda gt11 library was screened with this probe using the method described by Huynh (1985). About 1 in 50,000 clones hybridized to 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 prepared essentially as described in Section IV.A.2. described ermitte't. 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). However, there are differences in two regions. Nucl jotid 67 in clone 1-2 is a thymidine, whereas the other three clones contain a cytidine residue at this 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 appear at the start of the cDNA sequence in 5-1 -1 and are indicated by small letters. Based on the radioimmunoassay data set forth below in Section IV.D. It is possible that an HCV epitope may be encoded in this 28 bB region.

The presence of the 28 base pairs of 5-1-1 from clones 81, 1-2 and 91 can signify 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 polypeptide encoded in the ORF of the composite HCV cDNA.

IV.A.5. Isolation of HCV cDNAs that overlap 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 81 is shown in FIG. The sequence of the synthetic polynucleotides 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 5x SSC, 0.1% SDS at 55 ^° C. and over 30 minutes each. 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, the to a 3'-terminal sequence of clone 81 was prepared. The sequence of the synthetic polynucleotides 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 prepared essentially as described in Section IV.A.2. described determined. The double-stranded sequence of this cDNA, its overlap region with the HCV cDNA in clone 81, and the polypeptide encoded by the ORF are shown in FIG.

The ORF in clone 36 is in the same translational frame as the HCV antigen encoded in clone 81. Thus, the ORFs in clones 36 and 81 in combination 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 is 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 as described in Section IV.A.2. method described for the sequence of clone 5-1-1. The sequence data showed that the cDNA in clone 32 recombinant phage was derived from two different sources. A fragment of the cDNA contained 418 nucleotides derived from the HCV genome; the other fragment comprised 172 nucleotides derived from the bacteriophage MS2 genome which was used as a support during the preparation of the Labda-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 overlapping that of clone 81 and the polypeptide encoded by the ORF are also shown in this figure. This sequence contains a continuous ORF that resides in the same translational framework 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 was based on the 5 'region of clone 36. The sequence of the synthetic polynucleotides used for the screening was:

5 'AAG CCA CCG TGT GCG CTA 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 prepared essentially as described in Section IV.A.2. described determined. 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 clone 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

The isolation of HCV cDNA sequences upstream of and overlapping those in clone 35 cDNA was performed as described in Section IV.A.8. described for those overlapping clone 36 cDNA, except that the synthetic polynucleotide was based on the 5 'region of clone 35. The sequence of the synthetic polynucleotides 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 this sequence was named clone 37b.

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

The nucleotide sequence of the cDNA in clone 37b was prepared 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 A. The cDNAs from three other independent clones isolated during the procedure, in clone 37b, as described in Section IV.A.10. was isolated were also sequenced. The cDNAs from these clones also contain an A in this position. Thus, the 5'-terminal T in clone 35 may 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 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:

-23- 298 524 5 'AGT GCA GTG GAT GAA CCG GCT GAT AGC CTT 3'.

Using the nucleotide sequence of clone de, 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 with 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 prepared 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 translational framework as that encoded in the extended open reading frame of these overlapping clones.

IV.A.14. Isolation of HCV cDNAs Overlapping 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 C 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 50,000 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 prepared 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 in which clone 40b was isolated, described in Section IV.A.14., Were also sequenced.

The cDNAs of αίβεβη 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 in clone (sequence not shown) and clone 33b is TCA.

This difference can also create a cloning site such as the 20 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 pGEM 3-blue (Promega Biotec). The resulting recombinant vectors containing the cDNAs of clones 36, 81 and 32 were named pGEM 3-blue / 36, pGEM 3-blue / 81 and pGEM3-blue / 3, respectively. The appropriately oriented pGEM3-blue / 81 recombinant was digested with Nael and NarI and the large ("2850 bp) fragment was purified and ligated with the small (~ 570 bp) Nael / NarI purified restriction fragment of pGEM 3-blue / 36 This composition of the cDNAs of clones 36 and 81 was used to generate another pGEM3 blue vector containing the continuous HCV ORF contained in the overlap cDNA within these clones Pvull and EcoRI digested to release a fragment of about 680bp, which was then ligated to the small (580 bp) Pvull / EcoRI fragment isolated from the appropriately oriented pGEM 3 blue / 32 plasmid and the assembled cDWA from clones 36, 81 and 32 was ligated into the Eco RI-linearized vector pSODcf 1 described in Section IV.B.1., which was used to express clone 5-1-1 in bacteria. the ~ 1270bp EcoRI fragment nt of composite HCV cDNA (C 100) were selected and the cDNA from the plasmids was excised with ticoRI and purified.

IV.A.17. Isolation and nucleotide sequences of HCV cDNAs in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g, and 39c The HCV cDNAs in clones 14i, 11b, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were isolated by the technique of isolating overlapping cDNA fragments from the lambda gt 11 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 3' 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 14i, 7f, 7e, 8h, 33c, 14c, 8f, 33f, 33g and 39c were determined essentially as described in Section IV.A.2 Phage-cut cDNA in place of cDNA clone 5-1-1 was isolated.

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 33a and the overlap with that in clone 40b are shown in Figure 15, which also 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 ¹ 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 8 h. 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 7e, the overlap with clone 8h and the amino acids encoded therein are shown in FIG.

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

shown.

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'.

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.

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

5 'TCC ATG GCT GTC CGC TTC CAC 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.

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

5 'CAC CTA 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.

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

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

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

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

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. HCV cDNA-Assembled Cloned Composite HCV cDNA Sequence The HCV cDNA sequences in the isolated clones described above were aligned to create a composite HCV cDNA sequence , The isolated clones arranged in the 5'-3 'direction are: 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g and 39c. A composite HCV cDNA sequence deduced from the isolated clones and the amino acids encoded therein are shown in Figure 26;

In creating the composite sequence, the following sequence heterogeneities were considered. Clone 33c contains a '. 800 base pair iCV cDNA overlapping the cDNAs in clones 40b and 37c. In clone 33c as well as in 5 others. 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 or A. This heterogeneity may have important ramifications in protein folding.

Nucleotide residue 2 in clone eh HCV cDNA is T. However, as shown below, the corresponding residue in clone 7 e is an A. In addition, an A in this position is also found in 3 other isolated overlapping clones. Thus, the T residue in clone 8 h can represent a cloning artifact. Therefore, the remainder in this position is designated as an A in FIG.

the 3'-terminal nucleotide in clone 8f HCV cDNA is a G. However, the corresponding residue in clone 33f and in two other overlapping clones is T. Therefore, the remainder in this position is designated as T in FIG ,

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

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

The 3'-end of clone 14i is AA, while there: corresponding dinucleotide in clone 11 b and in three other clones TA. 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 indicates 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, 35f, 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 sonads were as indicated below. The frequency of clones that hybridized with the probes was in each case about 1 in 5000C. The nucleotide sequences of the HCV cDNAs in these clones were prepared essentially as described in Section IV.A.2. determined, except that the cDNA from the indicated clones was used in place of the cDNA isolated from clone 5-1-1.

Isolation of clone 12f containing cDNA "upstream" from the HCV cDNA in Fig. 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 CTA CTC ATA TCC CTA 3'.

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 3 'sequence of clone 35f. The nucleotide sequence of the probe was

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

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

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

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

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

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

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

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

The clones described in this section were subjected to the procedures described in section U.A. conditions described at the ATCC and received the following access numbers:

Lambda-gt11 ATCC no. deposit date

Clon12f 40514 10.Nov.1988

Clon35f 40511 Nov. 10, 1988

Clon15e 40513 Nov. 10, 1988

ClonK9-1 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, 14i, 7f, 7e, 8h, 33c, 40b, 37b, 35, 36, 81, 32, 33b, 25c, 14c, 8f, 33f, 33g, 39c, 35f, 1sg, 26g and 15e.

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 3 'HCV Sequence in Figure 32 Derived from the HCV cDNA in Cion 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 sequences "upstream" from the priming sites (as deduced from the illustrated sequence at clone 12f.) The HCV genomic RNA is either from plasma or liver samples from chimpanzees with NANBH or obtained from analogue samples of humans using NANBH.

IV.A.21. Alternative method using "Tallen" 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 d.:_: reverse transcription, which is duplexed with the template RNA, with "tailed" Oligo-C by incubating the product in the presence of CTP with terminal transferase The second round of cDNA synthesis, which provides the complement of the first cDNA strand, is carried out using oligo-G as a primer for the reserve transcriptase. Reaction The sources of 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 Utilizing "Tallen" for Isolation This method is based on previously used methods for cloning cDNAs of flavivirus RNA, where the RNA is subjected to denaturing conditions to remove secondary structures at the 3 'end and then becomes using tailed rATP as a substrate with poly-A polymerase. 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 ß 11-HCV cDNA blanks 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 eluted from the Sepharose CL-48 column.

IV.A.24. Creation of HCV cDNA bliaviothecans using synthetic oligomers as primers. 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. Expression of Polypeptides Encoded in HCV cDNAs and Identification of Exprimented Products as HCV-Infused Antigens

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

The clone 5-1-1 encoded HCV polypeptide (see Section IV.A.2., Supra) was expressed as a superoxide dismutase (SOD) fusion polypeptide. This was done by subcloning the clone 5-1-1 cDNA insert into the expression vector pSODcfi (Steimer et al., 1986) in the following manner.

First, DNA isolated from pSODcf 1 was treated with PamH1 and EcoRI, and the following linker was ligated into the linear DNA created by the restriction enzymes:

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

After cloning, the plasmid containing the insert was isolated.

The plasmid containing the insert was restricted with EcoRI. The HCV cDNA insert in clone F -1 -1 was excised with EcoRI and ligated into this EcoRI-linearized plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al. [198O]). 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 for the expression of SOD-NANB ₅ .,., - polypeptides.

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

Contained in clone 81 HCV cDNA wurdo expressed as a SOD-NANB ₈ i-fusion polypeptide. The method for preparing the VuKtor 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 the dio DNA sequence as described in Section IV.A.4.

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

The HCV cDNA insert in clone 81 was excised with EcoRI and ligated into pSODcM 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 for expression of the SOD-NANBei polypeptide.

IV.B.3. Identification of the polypaptide encoded in clone 5-1-1 as an HCV and NANBH associated antigen. In the HCV cDNA of Clor. 5-1-1 encoded polypeptide was identified as a NANBH-associated antigen by demonstrating that sera from NANBH-infected chimpanzees and humans were immunologically reactive with the fusion polypeptide, SOD-NANB ₆ , 10, which is composed of superoxide dismutase at its N-terminus and the in-frame 5-1-1 antigen at its C-terminus, by Western blotting (Towbin et al., 1979) in the following manner.

A recombinant bacterial strain transformed with an expression vector encoding the SOD-NANB ^ H polypeptide, as described in Section IV.B.I., was induced by culturing 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 steroids were incubated with sera from chimpanzees at different time points during the acute NANBH [Hutchinson strain] infections (lane 1-16), hepatitis A infections (lanes 17-24 and 26-33), and hepatitis B infections (lane 33-44) Figures 25 and 45 show positive controls in which the immunoblots were incubated with serum from the patient used to identify the recombinant clone 5-1-1 in the original screening of the lambda gt 11 cDNA library (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 ₅ .,., 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 appeared as a band migrating significantly faster than the SOD-NANB ₆ .,., Fusion polypeptide.

Lanes 1-16 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. The Figure imparts the following: While antibodies recovered immunologically with the SOD-NANB ^ .i polypeptide were absent in the serum samples collected prior to administration of the infective HCV inoculum and during the early acute phase of infection all 4 animals finally during the last part of the acute phase or subsequently circulating antibodies against this polypeptide. 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 ^ ₅ .,., Component of the fusion polypeptide was observed in 4 chimpanzees infected with HAV and 3 chimpanzees, respectively. not infected with HBV. 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. Nitrocellulose strips containing polypeptides

were incubated with sera recovered from humans at different times during 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 used in the initial screening of the lambda gt11 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, sera from nine NANBH patients, including the serum used to screen the Lambdagt11 library, contained antibodies to the NANB ₅ .,., Component of the fusion polypeptide. The sera from three patients with NANBH did not contain these antibodies. It is possible that the ArHi-NANB ₅ .,..., Antibodies will 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.

Figure 34 also shows that sera from many patients infected with HAV and HBV did not contain ArItI-NANB ₆ .,... Antibodies, and that these antibodies were not present in the sera of "normal" controls as well. Patient (lane 36) evidently has anti-NANBj.,., - antibody, is it possible that this patient was previously infected with HCV, since the incidence of NANBH is very high and because it 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.B.4. Expression of the polypeptides encoded in a composition of the HCV DNA in clone 36, 81 and un-J 32 The HCV polypeptide encoded in the open reading frame, which extends through clones 36, 81 and 32, was treated with SOD as a fusion polypeptide expressed. This was done by inserting the composite cDNA, C100, into an expression cassette containing the human superoxide dismutase gene, inserting the expression cassette into a yeast expression vector, and expressing the polypeptide in yeast.

An expression cassette containing the C100 cDNA derived from clones 36, 81 and 32 was constructed by inserting the ~1207bp EcoRI fragment into the EcoRI site of the pS3-56 vector (also called pS356), the Plasmid pS3-56 _{c100 was} obtained. 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 GP0196056, published October 1, 1986, which is the common 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 gin, 54 of the superoxide dismutase is followed by an adapter sequence containing an Eco RI site The sequence of the adapter is:

5'-AAT TTG GGA ATT CCA TAA TGA G -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.

Following the isolation of recombinants containing the C100 cDNA insert in the correct orientation, the expression cassette C 100 cDNA-containing BamHI from pS3-56 _c, oo was excised, and the cassette-containing fragment of ~ 3400bp was isolated and cleaned. This fragment was then inserted into the BamHI site of yeast vector ρ AB24.

Plasmid pAB24, the significant features of which are shown in Figure 35, is a yeast "shuUie" vector containing the complete 2-micron sequence for replication (Broach [1981]) and pBR322 sequences, and also contains that of plasmid YEp24 (Bostein et al., 1979)) derived yeast URA3 gene and the yeast LEU ^2d gene derived from plasmid pCI / 1 EPO Publication No. 116,201 Plasmid pAB24 has been described in US Pat Plasmid pAB24 was constructed by digesting YEp24 with EcoRI and religating the vector to remove the partial 2 micron sequence The resulting plasmid, YEP24de1taRI, was linearized by digestion with CIaI and incubated with complete 2- The resultant plasmid, pCBou, was then digested with SbaI and the 8605bp vector fragment was gel-isolated This isolated fragment of XbaI was ligated with a plasmid, which had been linearized with CIal 4460pb SbaI fragment containing the pCI / 1 -isolated LEU ^2d gene.

The orientation of the LEU ^2d gene has the same direction as the URA3 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-CIOO expression cassette, pAB24100-3, was transformed into the yeast strain JSC 308 as well as other yeast strains. The cells were prepared as described by Hinnen et al. (1978) and

clad 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 JSC 308, 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 the yeast strain JSC308 is disclosed in co-pending application, U.S. Application Serial No. (Att.) No. 2300-0229, filed concurrently herewith and incorporated herein by reference A sample of JSC 308 was deposited with the ATCC on May 5, 1988 under the terms of the Budapest Treaty and was granted accession number 20879. The conditions of availability and access to the deposited material and preservation of deposit are the same as those contained in section HA for HCV cDNAs Strains specified.

The complete C100-3 fusion polypeptide encoded in pAB24C100-3 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 C100 cDNA-derived amino acid residues, and 5 carboxy-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.B.5. Identification of In C100 Encoded Polypeptides as a NANBH-Associated Antigen The CIOO-3 fusion polypeptide expressed from plasmid pAB24C100-3 in yeast strain JSC 308 was characterized for size, and the C100 encoded polypeptide was identified as having a immunological reactivity with serum from a People with chronic NANBH identified as a NANBH-associated antigen.

The C10O-3 polypeptide described in Section IV.B.4. was expressed as follows. Yeast JSC-308 cells were transformed with pAB24 or with pAB24C100-3 and were induced to express the heterologous plasmid-encoded polypeptide. The induced yeast cells in 1 ml of culture (On _e6Onm ~20) were _pelleted by centrifugation at 10,000 rpm for 1 minute and were lysed by vigorous (10x 1 min) with 2 volume solution and 1 volume glass beads (0.2 nanometer diameter). were swirled. 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 (10,000 rpm for 5 minutes) and dissolved in Laemmli SDS sample buffer for 5 minutes. (See Laemmle [1970]). A polypeptide amount equivalent to the 0.3 ml of the induced yeast culture was subjected to electrophoresis through 10% polyacrylamide gels in the presence of SDS according to Laemmli (1970). Protein residues 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., Taking serum from a patient with chronic NANBH to assess the immunological responsiveness of pAB24 and from The results are shown in Figure 37. The polypeptides were stained with Coomassie Brilliant Blue in Figure 37 A. The insoluble polypeptides of pAB24 transformed JSC 308 and two further colonies of pAB24C100-3 are described in U.S. Pat See lanes 1 (pAB24) and 2 and 3, respectively, and a comparison of Lanes 2 and 3 with lane 1 shows the induced expression of a polypeptide corresponding to a molecular weight of "54,000 daltons from JSC 308 transformed with pAB24C100-3 that is not in induced with pAB24 transformed JSC308. This polypeptide is indicated by the arrow. Figure 37B shows the results of the Western blots of the insoluble polypeptides expressed in pAB24 (lane 1) or pAB24C100-3 (lane 2) yeast strain JSC 308. The polypeptides expressed from pAB24 were immunologically immunized with human serum with NANBH however, as indicated by the arrow, JSC 308 transformed with pAB24C100-3 expressed a ~ 54,000 dalton molecular weight polypeptide that reacted with the human NANBH serum The other immunologically reactive polypeptides in lane 2 may be degradations and / or aggregate products of this ~ 54,000 dalton polypeptide.

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

The fusion polypeptide C100-3 composed of SOD at the N-terminus and in-frame C 100 HCV polypeptide at the C-terminus was purified by differential extraction of the insoluble fraction of the extracted host yeast cells in which the polypeptide was expressed.

The fusion polypeptide C100-3 became, where in Section IV.B.4. described expressed in pAB24C100-3 transformed yeast strain JSC308. The yeast cells were then lysed by homogenization, the insoluble material in the lysate was extracted at pH 12.0, and C100-3 in the remaining insoluble fraction was solubilized in 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 (15 ml) was mixed with an equal volume of glass beads (0.45 to 0.50 mm diameter) and the mixture was vortexed at 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 the cells initially used. After combining washings and homogenate, the insoluble material in the lysate was recovered by centrifuging the homogenate at 4 ^° C. for 15 minutes, resuspending the pellets in twice the volume of buffer A as the cells initially employed, and then incubating the material for 15 minutes 7000 xg was repelleted. This washing was repeated three times.

The insoluble material from the lysate was extracted at pH 12.0 as follows. The pellet was suspended in buffer containing 0.5M NaCl, 1mM EDTA, with the suspending volume equal to 1.8 times that of the initial cells. The pH of the suspension was adjusted by adding 0.2 volume of 0.4M Na phosphate buffer, pH 12.0. After mixing, the suspension was centrifuged at 4 ^° C. for 15 minutes at 7,000 g and the supernatant was removed. The extraction was repeated twice. The extracted pellets were washed by placing them in 0.5M NaCl, 1mM EDTA,

were suspended, using a suspension volume twice that of the cells initially used. Dar? Η started centrifuging at 4 ° C at 700Ox g for 15 minutes.

The C 100-3 polypeptide in the extracted pallet was solubilized by treatment with SDS. The pellets were in one

Buffer A amount equal to 0.9 volume of the volume of cells initially charged, and 0.1 volume of 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 greater than 50%.

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

IV.C. Identification of RNA hybridizing to HCV cDNA in infected individuals IV.C.1. Identification of RNA hybridizing to HCV cDNA in the chimpanzee liver with NANBH

It was demonstrated that RNA from the liver of a chimpanzee having NANBH contained an RNA species which hybridized to HCV cDNA contained in Cbn 81 by "Northern blotting" in the following manner.

RNA was isolated from chimpanzee liver biopsy material from which the high titer plasma (see Section IV.A.1.) Was recovered using techniques described in Maniatis et al. (1982) for the isolation of total RNA from mammalian cells and for their separation into poly A ⁺ and poly A ~ fractions. These RNA fractions were electrophoresed on a formaldehyde / agarose gel (1% w / v) and transferred to nitrocellulose. (Maniatis et al., 1982). The nitrocellulose filters were hybridized with 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 Cion 81 was radiolabeled by nick translation using DNA polymerase I (Maniatis et al., 19821). Hybridization was carried out at 42 ° C for 18 hours 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% (w / v) bovine serum albumin (BSA = bovine seruni albumin), 0.02% (w / v) Ficoll-400.0.02% (w / v) polyvinylpyrrolidone, 100 micrograms / ml salmon sperm DNA sheared and denatured by sonication containing 10 ^e CPM / ml of the nick-translated cDNA probe.

An autoradiographic image of the separate filter is shown in FIG. Lane 1 contains ^J2 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 following in section IV.C.2. described experiment confirms the assumption that HCV contains an RN Α genome.

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

Nucleic acids were isolated from particles which, as described in Section IV.A.1. isolated from high titer chimpanzee NANBH plasma. 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 with DNase I (5 microliters containing of 1 unit of DNase I in 10 mM MgCl ₂ , 25 mM HBs pH 7.5). Control samples were incubated without enzyme. After incubation, 230 microliters of ice-cold 2XSSC containing 2 micrograms / ml of yeast-soluble RNA were added and the samples were filtered on a nitrocellulose filter. The filters were hybridized to a cDNA probe of clone 81 which had been 32p-labeled by nick translation, Figure 39 shows autoradiographic uptake of the filter Hybridization signals were recorded in the DNase-treated and control samples (lanes 2 and 5, respectively) 1), but not in the RNase-treated sample (lane 3), so since RNase-A treatment destroyed the nucleic acids isolated from the particles and DNase treatment had no effect, it can be taken for granted that the HCV Genome consists of RNA.

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

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 reachon) 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. The isolation of the RNA fraction was performed by the methods described in Section IV.C.1. Guanidiniumthiocyanatprozedur described.

RNA samples to be tested by the amplification system were also isolated from the plasma of two chimpanzees with NANBH and a control chimpanzee, as well as a pool of control chimpanzee plasmas. One infected chimpanzee had a CID / ml equal to or greater than 10 ^e , and the other infected chimpanzee had a CID / ml equal to or greater than 10 ⁵ .

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, Vroteinase K / SDS solution (0.05 M Tris-HCl, pH 8.0, 0.001 M EDTA, 0.1 M NaCl, incubated for 1 mg / ml proteinase Kund 0.5% SDS) containing 10 micrograms / ml Polydenylsäure, and 60 minutes at 37 ⁰ C. Following 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

twice with an equal volume of phenol / chloroform / isoamyl alcohol (1: 1 (99: 2)) and then extracted 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% 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 at 10,000 rpm for 10 minutes 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 / Pheiiol method were further purified by binding to and from 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, andf 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 ™ (Fisher / 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 temperature regime of 37 ⁰ 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 'GAT AAC CTC TGC CTG A 3'.

The sequence of the primer of clone 36 was:

5 'GCA TGT CAT GAT GTA T 3'

 The sequence of the clone 37 b primer was:

5 'ACA ATA CGT GTG TCA C 3 ¹ .

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 Alkaligelelektrophorese, followed by "Southern blotting" and detection of the amplified HCV-cDNA sequences with ^a32 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 centrifugation and dissolved in distilled water Aliquots of the samples were spiked to 1 Electrophoresis of 8% alkaline agarose gels Single-stranded DNAs of length 60.108 and 161 nucleotides were co-electrophoresed on gels as "molecular weight markers". After electrophoresis, the DNAs in the gel were transferred to Biorad-Zeta-Probe ™ paper. Prehybridization and hybridization as well as washing conditions were in accordance with the manufacturer's specification (Biorad).

The probes used for hybridization / detection of the amplified HCV cDNA sequences were as follows. 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 3'-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. (See Figures 5, 8 and 10.) Thus, the cDNA insert in clone 35 spans a portion of the region between the sequences of clones derived from clone 36 and clone 37b 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 Kontioli 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 with the primers and the probe from 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. Radiolum Assay for the Detection of HCV Antibodies In the Serum of Infected Individuals Solid phase radioimmunoassays for the detection of antibodies to HCV antigens were developed on the basis of TSU and Herzenberg (1980). Microtiter plates (Immulon 2, Removaweil 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 appropriate 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 and human antibody NANBV antigen are detected by incubation with sheep labeled anti-human immunoglobulin. "Unbound labeled antibody is removed by aspiration and the plates are washed. The radioactivity in individual wells is determined and the amount of human anti-HCV antibody bound is proportional to the radioactivity in the well.

IV.D.1. Purification of Fuslonpolypeptld SOD-NANB ₆ .,.

The fusion polypeptide SOD-NANB ₆ .,. ,, 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 Tris-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 pullet was suspended in 10 ml of 25% (mass / volume) sucrose containing 0.05N Tris-HCl, pH 8.0.1mM phenylmethylsulfonyl fluoride (PMSF) and 1 microgram / ml pepstatin A, followed by Add 0.5 ml of lysozyme (10 mg / ml) and incubate at ⁰ ° C for 10 minutes. After addition of 10 ml of 1% (v / v) Triton X-100 in 0.05 M Tris-HCl, pH 8.0, 1 mM EDTA, the mixture was stirred at ⁰ ° C. for 10 minutes with occasional shaking incubated. The resulting viscous solution was homogenized by passing 6 times through a 20 gauge sterile hypodermic needle and centrifuge at 13,000 g for 25 minutes. The pelleted material was suspended in 5 ml of 0.01 M Tris-HCl, pH 8.0, and the suspension was centrifuged at 4000 g. G for 10 minutes. The pellet containing SOD-NANB ^ fusion protein was dissolved in 5 ml of 6 M urea in 0.02 M Tris-HCl, pH 8.0.1 mM dithiothreitol (buffer A) and loaded on a buffer A balanced column of Q-Sepharose fast flow given. 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 ₆ .,. Fractions containing this polypeptide were collected and dialyzed against 5 M urea in 0.02 M sodium phosphate buffer, pH 6.0.1 mM dithiothreitol (Buffer B). The dialyzed sample was loaded onto a buffer B equilibrated column of S-Spharose Fast Flow, and polypeptides were eluted with a linear gradient of 0.0 to 0.3 M NaCl in buffer B. The fractions were analyzed by polyacrylamide gel electrophoresis for the presence of SOD-NANB ₆ .,., And the appropriate fractions were pooled.

The final SOD-NANB ₅ .,.. 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. Purification of fusion polypeptide SOD-NANB ₁₁

The fusion polypeptide SOD-NANB ₈₁ expressed in recombinant bacteria as described in Section IV.B.2. was prepared from recombinant E. coli by differential extraction of the cell extracts with urea followed by chromatography on anion and cation exchange columns using the procedure described for the isolation of fusion polypeptide SOD-NANB _6. , (see Section IV.D .1.), Cleaned.

The final SOD-NANBei 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 to HCV epitopes by solid phase radioimmunoassay 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 ₈ , existing HCV epitopes were detectable.

Microtiter plates were coated with SOD-NANBc.,., 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. 10 o milliliter aliquots containing 0.1 to 0.5 micrograms of SOD-NANB ₆ .,., Or SOD-NANB ₈ , in 0.125 M Na borate buffer, pH 8.3.0.075 M NaCl (BBS) into each well of a microtiter plate (Dynatech Immulon 2 Removawell strip). 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. AnU-NANB ₅ .,., 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 of 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 washes with BBST. The amount of radioactivity bound in each well was determined by counting in a counter that detects gamma radiation

The results of detecting 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

patients diagnosis Anti-5-1-1 S / N Anti-81 reference Chronic NANB, IVD ²¹ 0.77 4.20 number Chronic NANB, IVD 1.14 5.14 1. 28 ' Chronic NANB, IVD 2.11 4.05 AVH ³¹ , NANB, sporadically 1.09 1.05 Chronically, NANB 33.89 11.39 2. 29 ' Chronically, NANB 36,2Ü 13.67 AVH, NANB, IVD 1.90 1.54 Chronic NANB, IVD 34.17 30.28 3. 30 ' Chronic NANB, IVD 32,45 30.45 Chronic NANB, PT ⁴¹ 16.09 8.05 Late AVH NANB, IVD 0.69 0.94 4. 31 Late AVH NANB, IVD 0.73 0.68 5. 32 ' AVH, NANB, IVD 1.66 1.96 AVH, NANB, IVD 1.53 0.56 6. 33 ' Chronic NANB, PT 34.40 7.55 Chronic NANB, PT 45.55 13.11 7. 34 ' ChronischoNANB, PT 41.58 13,45 Chronic NANB, PT 44,20 15,48 AVH NANB, IVD 31.92 31,95 "Healed" Recent NANB, AVH 6.87 4.45 8. 35 ' SpäteAVHNANBPT 11.84 5.79 AVH NANB, IVD 6.52 1.33 9. 36 SpäteAVHNANB, PT 39.44 39.18 10. 37 Chronic NANB, PT 42.22 37.54 11. 38 AVH, NANB, PT 1.35 1.17 12. 39 Chronic NANB, PT 12:35 0.28 13. 40 AVH, NANB, IVD 6.25 2.34 14. 41 Chronic NANB, PT 0.74 0.61 15. 42 AVH, NANB, PT 5.40 1.83 16. 43 Chronically, NANB, PT 0.52 0.32 17. 44 AVH, NANB 23.35 4.15 18. 45 AVH, Type A 1.60 1.35 19. 46 AVH, type A. 1.30 0.66 20. 47 AVH, type A. 1.44 0.74 21. 48 Resolved 0.48 0.56 22. 49 Recent AVH, Type A 23. 50 AVH, type A. 0.68 0.64 GelösteAVH.TypA 0.80 0.65 24. 51 Resolved recent AVH, type A. 1.38 1.04 Resolved recent AVH, type A. 0.80 0.65 25. 52 AVH, type A. 1.85 1.16 Resolved recent AVH, type A. 1.02 0.88 26. 53 AVH, type A. 1.35 0.74 Late AVH, HBV 0.58 0.55 27. 54 Chronically, HBV 0.84 1.06 28. 55 Late AVH, HBV 3.20 1.60 29. 56 Chronically, HBV 0.47 0.46 30. 57 AVH, HBV 0.73 0.60 31. 58 Healed AVH, HBV 0.43 0.44 32. 59 ' AVH, HBV 1.06 0.92 Healed AVH, HBV 0.75 0.68 33. 60 ' AVH, HBV 1.66 0.61 Healed AVH, HBV 0.63 0.36 34. 61 ' AVH, HBV 1.02 0.73 Healed AVH, HBV 0.41 0.42 35. 62 ' AVH, HBV 1.24 1.31 Healed AVH, HBV 1.55 0.45 36. 63 ' AVH, HBV 0.82 0.79 Healed AVH, HBV 0.53 0.37 37. 64 '

0.95 0.92 0.95 0.92 0.70 0.50 1.03 0.68 1.71 1.39

Continuation Table 1

Patient Reference S / N Number Diagnosis Anti-5-1-1 Anti-81

38. 65 'AVH, HBV

Healed AVH, HBV Healed AVH, HBV

39. 66 ¹ AVH, HBV

Healed AVH, HBV

1 Of these patients, sequential serum samples are available.

2 IVD = intravenous drug user

3 AVH = acute viral hepatitis

4 PT = post-transfusion (after transfusion).

As can be seen from Table 1, 19 out of 32 sera from patients diagnosed with NANBH were antibody-directed to HCV epitopes present in SOD-NANB ₆ .,., And SOD-NANB ₉ , positive. However, the positive serum samples were not equally immunologically reactive with SOD-NANB _6. , And SOD-NANB ₈ I serum samples from Patient No. 1 were positive to SOD-NAN ₈₁ but not to JOD-NANB ₅ .,. , Serum samples from patients Nos. 10, 15 and 17 were positive for SOD-NANB ₆ .,. ,, but not for SOD-NANB ₈ I serum samples from patients Nos. 3, _8, 11 and 12 reacted equally with both fusion polypeptides while serum samples to 3 times stronger response exhibited by the patients Nr.2,4,7 and 9 with respect to SOD-NANB _6.,., a 2 than to SOD-NANB _8. These results suggest that NANB _6. , And NANB _{81 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. Specificity of solid phase RIA with respect 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 partially purified SOD-NANB ₆ ,., And SOD-NANB ₀ 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 immunologic response with the fusion polypeptides containing BB-11ANBV epitopes. The RIA using the NANB ₆ .,., 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 came were once exposed to HCV.

IV.D.5. Reactivity of NANB _5. , During the course of the NANBH infection

The presence of anti-NANBe-M 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-NANB ^ .p antibodies in the course of HAV and HBV infection

Chimpanzees applied.

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. No anti-NANBs,,, antibodies were detected in serum samples of chimpanzees infected with HAV or HBV. Thus, anti-NANB antibodies are used as indicators (markers) for HCV exposure of an individual.

Table 2

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

Patent/ Sample date (day) hepatitis Anti-5-1-1 \ OLD - 5 8th 4 - - 7 - 15 chimp (0 = vaccination day) virus (S / N) (Mu / mL) 52 205 147 106 83 130 Patient 29 T * NANB 1.09 1180 13 14 18 10 5 8th T + 180 33.89 425 - 6 5 5 T + 208 36.22 - 11 15 Patient 30 T NANB 1.90 1830 132 9 T + 307 34.17 290 - 6 T + 799 32,45 276 - 8th Schimpi 0 NANB 0.87 9 96-155 (pool) 76 0.93 71 9 118 23.67 19 13 154 32.41 11 Schimp2 0 NANB 1.00 8-100 (pool) 21 1.08 9 73 4.64 10 138 23,01 Schimp3 0 NANB 1.08 43 1.44 53 1.82 159 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 Schimp8 0 HAV 0.77 26 1.98 74 1.77 205 1.27 Schimp9 -290 HBV 0.77 379 3.29 435 2.77 SchimpiO 0 HBV 2.35 111- 118 (pool) 2.74 205 2.05 240 1.78 SchirnpH 0 HBV 1.82 28-56 (pool) 1.26 169 1.00 223 0.52

* = Day of initial sampling

IV.E. Purification of polyclonal serum antibodies against NANB ₅ .,.,

Based on the specific immunological reactivity of the SOD-NANB ₅ .,., Polypeptide with the antibodies in serum samples from patients with NANBH, a procedure was followed for the purification of serum antibodies immunological with the epitope (s) in NANB5.1.1 react, developed.

In this method, the affinity chromatography is exploited. Purified SOD-NANB ₅ .!., Polypeptide (see Section IV.DI) was attached to an insoluble support, with the immobilized polypeptide retaining its affinity for the antibody to NANBe-M. The antibody in serum samples is absorbed on the matrix-bound polypoptide. After this

Washing to remove the non-specifically bound materials and unbound materials will cause the bound antibody to change pH and / or chaotropic reagents, such as. 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 _5. , Was bound to the membrane by incubation of the purified preparation in BBS at room temperature for 2 hours; in an alternative manner, it was incubated overnight at 4 ° C. 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 anti-NANB &.,., - 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, OM Tris HCl pH 8.0. The recovery of the anti-NANB ₅ .,., - 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 ingestion of HCV particles from infected plasma by purified human polyclonal anti-HCV antibodies; Hybridization of nucleic acid in the captured particles to HCV cDNA

IV.F.1. The capture tone of HCV infected human plasma HCV perticolins using human polyclonal anti-HCV antibody proteic 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 were, isolated. Polyclonal anti-NANBc,.,., Antibodies were purified from human serum with NANBH using d3 * in C! On 5-1-1 encoded polypeptide SOD-HCV. For purification, the procedure described in Section IV.E. described method applied. The purified anti-NANB6 antibodies were bound to polystyrene beads (diameter ¹ / inch, mirror-smooth surface, Precision Plastic Ball Co., Chicago, Illinois) by placing each bead overnight with 1 ml of antibody (1 microgram / ml in Porat-buffered saline, pH 8.5). After overnight incubation, the beads were washed once with TBST (50r.mM Tris HCl, pH 8.0, 15 mM NaCl, 0.05% (v / v) Tween 20) and then with phosphate buffered saline (PBS) 10mg / ml BSA, washed.

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

The trapping of HCV from NANBH-infected chimpanzee plasma by means of bead-bound anti-NANBs.,... Antibodies was as follows: The plasma used from a chimpanzee with NANBH is described in Section IV.A.1. described. An aliquot (1 ml) of the NANBV infected chimpanzee plasma was incubated for 3 hours at 37 ° C with 5 beads each coated with either anti-NANB5.,., Antibodies or control immunoglobulins. The beads were washed three times with TBST.

IV.F.2. Hybridization of Nuclilic Acid in the Captured Particles to NANBV cDNA The nucleic acid component released from the particles captured with AnH-NANB ₅ .,... Antibodies 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. In order 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, the solution being proteinase k (1 mg / ml), 10 mM Tris HCl, pH 7.5.1 mM EDTA, C., 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 -20 ° 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 antibody 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. Autoradiograms of a probed filter containing the nucleic acids from particles captured by the beads containing AnU-NANB ₅ .,..., Antibodies are shown in FIG. Extract recovered from anti-NANB ^ n antibody (A ,, A ₂ ) gave clear hybridization signals relative to the control antibody extract (A ₃ , A ₄ ) and control yeast RNA (B ₁ , B ₂ ) 1 pg, 5pg and 10 pg of the purified cUNA fragment of clone 81 are shown in C1-3.

These results show that those captured from NANBH plasma by AmJ-NANB ₅ .,..., Antibodies. "Nucleic acids that hybridize with HCV cDNA in clone 81 contain additional evidence that the cDNAs in these clones were derived from the aetiological agent of NANBH.

IV.G. Immunological Reactivity of C100-3 with Purified AnU-NANB ₆ .,., - Antibodies The immunological reactivity of the C 100-3 fusion polypeptide with anti-NANB ₅ .,... Antibodies was determined by radioimmunoassay using a solid state immunoassay Phase-bound antigens with purified anti-NANB ₅ .,., - antibodies were tested and the antigen-antibody complex was detected with ''! -Labeled sheep anti-human antibodies.

The immunological reactivity of C 100-3 polypeptide was compared to that of SOD-NANB ₅ .,, Antigens.

The fusion polypeptide C100-3 was synthesized and purified as described in Section IV.B.5. or in Section IV.B.6. described.

The fusion polypeptide SOD-NANB ₅ .!., As described in Section IV.B.1. or in Section IV.D.1. described synthesized and purified. The purified anti-NANB ^ i., 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 0.125 M Na borate buffer, pH 8.3.0.075M NaCl (BBS), were added to each well of a microtiter plate (Dynatech Immolon 2 Removawell 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 turns, the wells were 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 ^,., Antibodies by adding 1 microgram of antibody / well and the pores were incubated at 37 ⁰ C for 1 hour. After incubation, the excess solution was removed by aspiration and the wells were washed five times with BBST. Anu-NANB ₆ .,., Bound to the fusion polypeptides, was detected by binding of '' labeled F '(ab) ₂ sheep anti-human IgG to the coated wells, aliquots of 100 microliters of labeled probe (specific activity 5-20 micro Curies / microgram) were added to each well, the plates were incubated for 1 Stundfe at 37 ⁰ C, and then the excess probe was removed by aspiration and washed five times with BBST. the bound in each well radioactivity was Counting in a gamma ray counter determined.

Table 3 shows the results of immunological reactivity of C100 with purified AnU-NANB ₅ .,., As compared to that of NANB _6. ).) With the purified antibodies.

Table 3

Immunological reactivity of C100-3 compared to NANB _6. , Detected by radioimmunoassay (R / A)

AG (ng) RIA (count per minute / assay) 400 320 240 6732 6985 4954 5920 160 60 0 NANB ₅ .,., C100-3 7332 7450 4050 5 593 3051 4096 57 67

The results in Table 3 show that AmI-NANB ₅ .,., Recognizes an epitope (or epitopes) in the C100 component of the C 100-3 polypeptide. Thus, NANB ₅ ,., And C100 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 sterility of the HCV genome

The HCV genome was characterized for strandability by isolating the nucleic acid fraction from the particles captured on the polystyrene beads coated with anti-NANB6.,..., Antibodies and determining whether the isolated nucleic acid was positive and / or minus strands of the HCV cDNA hybridized.

As in Section IV.F.1. described particles were captured from the HCV-infected chimpanzee plasma using Polysirenporlen coated with immuno-purified anti-NANB ^ M antibody. 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 cleaving the HCV cDNAs from clones 81, 40b and 25c by EcoRI and cloning the cDNA fragments into M 13 vectors, mp 18 and mp 19 (Messing [1983]). The M13 clones were sequenced to determine if they contained the plus or minus strands of DNA derived from the HCV cDNAs. The sequencing was carried out with the dideoxy chain termination method according to Sanger and co-workers (1977).

From a set of duplicate filters containing aliquots of the HCV genome isolated from the captured particles, each filter was hybridized with either plus or minus strand probes derived from the HCV cDNAs , Figure 41 shows the autoradiograms obtained from probing the NANBV genome, with the probe mixture derived from clones 81, 40b 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 an unspecified sample.

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 which hybridize by PCR to amplicon derived from clone 81-derived HCV cDNA

The RNA in trapped particles was analyzed as described in Section IV.H.1. described won. Analysis for sequences which hybridize in the 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 Clon81. The results indicated that the amplified sequences hybridize with the clone 81-derived HCV cDNA probe.

IV.H.3 Homology between dengue flavivirus non-structural protein (MNWVD 1) and the HCV polypeptides encoded by the combined ORF of clones 141 to 39c

As shown in Figure 26, the combined HCV cDNAs of clones 14i to 39c contain a continuous ORF. The polypeptide encoded therein was analyzed for sequence homology with the region of the non-structural or non-structural dengue flavivirus peptides (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 3 '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 was believed to be important for RNA-dependent RNA polymerases. in the polypeptide encoded in HCV cDNA, at a location consistent with that in dengue 2 virus. (This data is not shown).

IV.H.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, provide evidence that the HCV is not a DNA-containing virus and that its replication proceeds without inclusion of cDNA.

IV.H.4.a. Southern-blottlng method

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 p-labeled HCV cDNA Results showed that the labeled HCV cDNA did not hybridize to the DNA transferred from the infected smectite liver and did not hybridize to the DNA transferred to the control of normal chimpanzee liver, to the contrary, in a positive control, a labeled probe of beta interferon hybridized. Gene strong on Southern blots of restriction enzyme-digested human placental DNA. These systems were designed to detect a single copy of the gene that was to be detected 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 procedure for extraction of DNA was performed essentially according to Maniatis and coworkers (1988), and the samples were treated with RNase during the isolation process. 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 treated by electrophoresis on 1% neutral agarose gels, by Southern blotting on nitrocellulose, and the transferred material hybridized with the corresponding nick-translated probe cDNA (3x10 ⁶ counts per minute / ml of hybridization mixture ). The DNA from infected chimpanzee liver and normal liver was hybridized with ³ 'p-labeled HCV cDNA from clones 36 and 81; the DNA from human placenta was hybridized with ³² P-labeled DNA from the beta-interferon gene. After hybridization, the blots were washed under stringent conditions, ie with a solution containing 0.1% SSC, 0.1% SDS and at a temperature of 69 ^° C. The beta interferon gene DNA was analyzed as manufactured by Houghton and co-workers (1981).

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

To determine if the HCV DNA in chimpanzee liver was to be detected with NANBH, DNA was isolated from the tissue and subjected to the PCR ampicculation detection method using primers and probe polynucleotides derived from clone 81 HCV cDNA , As negative controls, DNA samples isolated from uninfected HepG2 tissue culture cells and from suspected uninfected human placenta were used.

As positive controls, samples of negative control DNAs to which a known relatively small amount (250 molecules) of the HCV cDNA insert from clone 81 had been added were found. 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 also became isolated on the RNA samples applied.

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

DNA samples were isolated from 2 infected chimpanzee livers, from uninfected HepG2 cells and from human placenta.

One microgram of each DNA was digested with Hind HI according to the manufacturer's instructions. The digested samples were amplified by PCR and the detection of the amplified HCV cDNA was performed essentially as described in Section IV. C. 3. except that the reverse transcriptase step was omitted. The PCR primers and the probe were recovered from HCV cDNA clone 81 and are described in Section IV. C. 3. *** " Before the amplification was for positive

Controls a 1-microgram piobe of each DNA by adding "spiked" 250 molecules of HCV cDNA insert isolated from clone 81. To determine if HCV-C levels have been isolated in RNA derived from the livers of chimpanzees using NANBH were present, samples containing 0.4 micrograms of total RNA were subjected to an amplification process essentially as described in Section IV. C.3., but with some samples as a negative control, the reverse transcriptase was omitted PCR primer and probe were recovered from HCV cDNA clone 81 as described previously.

The results showed that the amplified sequences that are complementary to the HCV cDNA probe were not detectable in the infected chimpanzee liver DNAs or in the negative controls. On the contrary, if the samples, including the DNA from infected chimpanzee liver, were spiked with the HCV cDNA prior to amplification, the sequences from clone 81 were detected in all positive control samples and amplified HCV cDNAs were also amplified in the RNA assays. Clone 81 sequences were detected only when reverse transcriptase was used, strongly suggesting that the results were not due to DNA contamination.

These results demonstrate that chimpanzee hepatocytes with NANBH contain no or undetectable levels of HCV DNA. Based on the "spiking" study, it can be said that if HCV DNA is present then 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 were easily detected by the PCR technique.

IV. I. ELISA tests for HCV infection with HCV-c 100-3 as test antigen

All samples were tested by ELISA using HCV-c 100-3. This assay uses the HCV c 100-3 antigen (synthesized and purified as described in Section IV. B. 5.) and a horseradish peroxidase (HRP) conjugate of mouse monoclonal anti-human IgG.

The plates coated with the HCV c 100-3 antigen were prepared as follows. A solution of coating buffer (50 mM Na borate, pH 9.0), 21 ml / plate, BSA (25 microg / ml), c 100-3 (2.50 micrograms / ml) was added immediately before removal of Immealon-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 mM sodium chloride, 0.1% M / V casein, 1% (M / V) Triton X-100.0.01%. Vl thimerosal). After removal of the wash solution, 200 microliters / well of postcoat solution (10 mM sodium phosphate, pH 7.2, 15 mM sodium chloride, 0.1% [M / V] casein and mM phenylmethylsulfonyl fluoride [PMSF] were added, the plates were loosely capped to give a And allowed to stand for 30 minutes at room temperature, then the wells were leached to remove the solution and lyophilized dry overnight without heating The prepared plates can be stored at 2-8 ° C in sealed aluminum blisters ELISA assays were added to 20 microliters of serum sample or control in a well containing 200 microliters of sample diluent (10 mM sodium phosphate, pH 7.4, 50 mM sodium chloride, 1 mM EDTA, 0.1% M / V casein, 0.015% M / ml. V] Therosal, 1% [w / v] Triton X-100,100 micrograms / ml yeast extract) was prepared. the plates were sealed and incubated for two hours at 37 ⁰ C, after which the solution by suction, was removed. 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-human IgG-HRP conjugate dissolved in a solution of ortho-conjugate diluent (10 mM sodium phosphate, pH7.2, 15 mM sodium chloride, 50% [V / V] ^p otal bovine serum, 1% [V / V] of heat-treated horse serum containing 1 mM K ₃ Fe (CN) ₆ , 0.05% [M / V] Tween 20.0.02% [M / V] Thimerosai). The treatment lasts for 1 hour at 37 ⁰ C, then 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 of O-phenylenediamine dihydrochloride per 5 ml of 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 of 30% 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 microliters of 4N sulfuric acid and the absorbance values (OD) were determined.

The examples presented below demonstrate that the microliter plate screening ELISA using HCV-C100 antigen has a high degree of specificity, as evidenced by the initial rate of reactivity of about 1% with a replication reactivity rate of about 0.5% on random donors , The assay is capable of detecting the immune response both during 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 implicated in NANBH transmission (donors implicated in NANBH transmission). In the examples below, the following abbreviations are used:

OLD Alanine aminotransferase Anti-HBc Antibody to HBc Anti-HBsAg Antigens against HBsAg HBc Hepatitis B Kernaniigen ABsAg Hepatitis B surface antigen IgG Immunoglobulin G 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 / cutoff value

SD

WNL

Standard deviation average or mean 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 * ODderwiedorholt (IU / L) IOD) 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 4 227 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 OD the beginning 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

 *** AnIi-HBc s0.535 (competition assay) is considered positive

 ** ·· WNL: within normal limits.

IV.1.2. Chimpanzee serum samples

Serum samples from 11 chimpanzees were tested by HCV-dOC-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 seroconversion in all chimpanzees infected with the Hutchinson strain of NANBH. After the acute phase of the infection (as evidenced by the significant increase and subsequent return to normal ALT levels), antibodies to HCV-c100-3 could be detected in the sera of 4/4 NANBH infected chimpanzees. These samples have recently been analyzed 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 Chimpanzee serum samples

OD

S / CO

Inoculation date

Blood date

OLD (IU / L)

transfusion

positive 1,504 0.00 24/5/84 24/5/84 9 5 - control 0.401 0.01 08/07/84 71 52 15 _ 9 shutdown 7.48 18/9/84 19 13 130 negative 0.001 7Ί8 24/10/84 - - 8th 57 126 control 0,007 - 07/06/84 - - 8th 4.5 9 Chimpanzee 1 0,003 0.00 31/5/84 205 9 3,000 0.00 28/6/84 14 6 13 3,000 2.36 20/8/84 6 - 7.48 24/10/84 11 Chimpanzee 2 0,003 0.01 14.03.85 14.03.85 132 0.005 0.04 26/4/85 - 0.945 0.01 06/05/85 - 3,000 2.52 20/8/85 4 0.005 0.00 11/3/85 11/3/85 147 Chimpanzee 3 0,017 0.01 09/05/85 18 0,006 1.31 06/06/85 5 1,010 3.93 08/01/85 0,006 0.00 21/11/70 21/11/80 Schimpanse4 0,003 0.00 16/12/80 _ 0.523 0.01 30/12/80 106 1.574 0.01 29.07.- 10 0,006 21/8/81 Chimpanzee 5 0.001 - 25/5/82 _ 7 0,003 0.00 17/5/82 83 0,006 0.00 10/6/82 5 0.00 07/06/82 - 0.72 10/1/82 Chimpanzee 6 0.005 0.00 25/5/82 25/5/82 0.001 0.00 17/6/82 0,004 0.00 16/9/82 0,290 0.01 10/9/82 0,008 0.00 21/11/80 21/11/80 Chimpanzee 7 0,004 0.00 16/12/80 0,006 0.01 03/02/81 0.005 0.00 03.06.- 0,007 10/6/81 Chimpanzee 8 0,000 _ 24/7/80 _ 0,004 0.05 22.08.- 0,000 10/10/79 - 11/3/81 - 0.04 01.07.- Chimpanzee 9 0.019 08/05/81 0.02 10/1/81 - 12/5/82 0,015 0.03 21.04.- 12/5/82 0,008 0.04 01.09.- _ 09/08/82 Chimpanzee 10 0.011 0.02 12/2/82 0.02 06/01/83 0,015 0,008 0,010

NANB

NANB

NANB

NANB

HAV

HAV

HAV

HAV

HBV

HBV

Continuation Table 6

OD S / CO

blood OLD 9 -42- 298 524 inoculation date (IU / L) 10 trans date _ _ fusion 12/5/82 06.01.- 11 HBV 12/5/82 23/6/82 100 09.06.- _ 07/07/82 28/10/82 20/12/82

Chimpanzees - -

0.000 0.00

0.003 0.00

0.003 0.00

0.003 0.00

IV.1.3. List 1: Detected infectious sera from human carriers of chronic NANBH An encrypted list consisted of 22 single samples, each as a duplicate, i. 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. Alter of 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 quoted Bezuyiu.) · · · TifDr. Age article.)

The whole Lisiu. was tested twice by the ELISA method and the results were sent to Dr. med. Age sent. The results of this evaluation are shown in Table 7. Although the table contains the results of only one duplicate set, 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 highly 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 the chimpanzee studies 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 + +

c. Acute NANB; Post-tx

WII

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 liver zircon

EK -

b. Alcohol Hepatitis in convalescence

HB -

5) Negative controls with history

DH

OC - -

LV -

HL - -

Alles

Continuation Table 7

I.Ergebnis

2.Ergebnis

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 (taken 3.6 and 12 months after transfusion) from the recipient. Also included was a blood sample taken from the recipient of the transfusion. The encrypted list was written by Dr. med. H. NIH and the results were sent to him for evaluation.

The results summarized in Table 8 show that the ELISA method detected 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 out of 10 cases, with case 10 having 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 after 4 and

10 months to positive values.

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

Be HCV.

The ALT and HBc status for all reactive, i. 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 8

List of donor / recipient NANB after H.Alter

case donor - S / CO receiver 0.07 3 months S / CO Post-TX S / CO 12 months S / CO - - Blood sample 0.14 0.26 6 months 6.96 > 6.96 0.403 - transfusion 0.11 0.12 3.90 > 6.96 OD - 0.94 0.15 OD 0.13 OD 6.96 OD > 6.96 1. > 3,000 - 0.08 0.112 0.17 > 3,000 0.16 > 3,000 0.50 Second > 3,000 > 6.96 0.13 0,050 0.22 1,681 6.96 > 3,000 > 6.96 Third > 3,000 > 6.96 OD S / CO 0.08 0.057 3.44 > 3,000 6.96 > 3,000 > 6.96 4th > 3,000 > 6.96 0.032 0.14 0.073 0.13 0.067 6.96 > 0.217 > 6.96 5th > 3,000 > 6.96 0.059 0.19 0.096 0.18 > 3,000 5.28 > 3,000 > 6.96 6th > 3,000 > G, 96 0,049 6.96 1.475 0.30 > 3,000 0.13 > 3,000 > 6.96 7th > 3,000 > 6.96 0,065 0.056 0.74 > 3,000 6.96 > 3,000 > 6.96 8th. 1 month: "- 4 > 6.96 0.034 0.078 2,262 > 3,000 9th Months: »·· - 0.056 0,127 0,055 > 3,000 10th 0.034 0.317 * > 3,000 »» > 3,000 *** 0,061 • _ 0,080 > 3,000> 10 months.

Table 9

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

rehearse

Anti-ALT "

HBC "

Dispenser Case Case Case Case Case 8 Case 9 Case 10 Case 10

normal negative elevated positive elevated positive not available negative normal positive elevated not available normal positive normal positive

Continuation Table 9

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

rehearse 1 12Mon. 6Mon. Anti-ALT " HBC " receiver elevated case 2 12Mon. 6Mon. elevated positive elevated not tested case 3 12Mon. 6Mon. elevated negative elevated not tested case 5 12Mon. 6Mon. normal not tested · * ¹¹ elevated not tested*" case 6 6Mon. 3mon. elevated nichi tested 12Mon. elevated not tested case elevated elevated negative 7 12Mon. 6Mon. negative elevated not tested case 8th 12Mon. 6Mon. elevated negative elevated negative case 9 12Mon. normal positive 10 10Mon. 4Mon. not tested case elevated elevated not tested case elevated not tested not tested

* ALT> 45IU / L is above the normal limit

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

 "* Blood sample before transfusion and 3 months after that were llβc-negative.

IV.1.5. Determination of HCV infection In samples of high-risk groups Samples of high-risk groups were monitored by ELISA to determine reactivity to HCV c100- 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 hemophiles 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 levels of ALT or anti-nuclear antibodies Compared to random voluntary blood donors were rejected.

Table 10

Samples from NANBH risky groups

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

IV.1.6. Comparative studies using anti-IgG or anti-IgM monoclonal antibody or polyclonal antibody as second antibody for HCV ciOO-3 ELISA

The sensitivity of the ELISA measurement using anti-IgG monoclonal conjugate was compared to 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.

IV.I.6.8. Serial samples from seroconverters

Serial samples from three cases of NANB seroconverters were tested in the HCV c1OO ELISA assay using either the anti-IgG monoclonal antiserum alone or in combination with an anti-IGM monoclonal antiserum or a polyclonal antiserum in the enzyme conjugate Antiserum was used. The samples were taken by 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 anti-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 anti-IgG monoclonal conjugate and anti-IgM conjugate are shown in Table 13. Three different ratios of anti-IgG to anti-IgM were tested. The 1: 10000 dilution of anti-IgG was constant throughout the tests. The dilutions tested in the anti-IgM monoclonal conjugate were 1: 30000.1: 60,000 and 1: 120000. The data show that, consistent with the anti-IgG studies alone, the initial high reactivity was demonstrated in samples 1-4,2-8 and 3-5.

The results obtained with the ELISA test with anti-IgG monoclonal conjugate (1: 100000 dilution), or with Tagopolyclonal conjugate (1: 80000 dilution) or Jackson polyclonal conjugate (1: 80000 dilution) , 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 detect reactive samples at the same time after the acute disease phase (as indicated by the increase in ALT values). In addition, the results indicate that the sensitivity of the HCV ciOO-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

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, C 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 Case 3 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 12

ELISA test results obtained using Anti-If G monoclonal conjugate

date

OLD

OD

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 FAIH 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 Case 3 09/05/81 315.0 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

Table 13

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

ELISA TestsanNANB

date OLD Monoclo- Monoclo- Monoclo- dimensional dimensional dimensional IgG1: 10K IgG1: 10K IgG1: 10K lgM1: 30K lgM1: 60K 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 FAIH 19/11/81 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 0.059 0,036 0.027 2-4 0,065 0,041 0,025 2-5 0.082 0.033 0.032 2-6 0,102 0,042 0.027

Continued table

ELISA TestsanNANB

OLD Monoclo- Monoclo- Monoclo- 70 dimensional dimensional dimensional 24 IgG1: 10K IgG1: 10K IgG1: 10K 53 lgM1: 30K lgM1: 60K lgM1: 120K date 95 OD S / CO OD S / CO OD S / CO 15/2/82 37 0.188 0,068 0.096 17/3/82 13 1,728 1,668 1,541 21/4/82 236 > 3.00 2,443 > 3.00 19/5/82 471 > 3.00 > 3.00 > 3.00 14/6/82 315 > 3.00 > 3.00 > 3.00 07/04/81 273 0,193 0,076 0,049 12/5/81 158 0.201 0,051 0,038 30/5/81 84 0.132 0.067 0,052 09/06/81 180 0,175 0,155 0.140 07/06/81 154 1,335 1,238 1,260 10/8/81 > 3.00 > 3.00 > 3.00 09/08/81 > 3.00 > 3.00 > 3.00 14/10/81 > 3.00 > 3.00 > 3.00 11/11/81 > 3.00 > 3.00 > 3.00

Case 3

table

ELISA test results obtained using polyclonal conjugates

date OLD ELISA TestsanNANB S / CO TAGO S / CO OD Jackson monoclonal 1:80 K 0.154 1: 80K 1: 10K OD 0,654 S / CO sample OD 0,045 2,154 Neg. control 0,076 0.545 cut-off value 08/05/81 40 0,476 0.37 0.727 0.12 0.153 PC (1: 128) 09/02/81 274 1,390 0.32 0.18 0.225 FAIM 10/7/81 261 0.27 0.067 0.05 0.167 0.23 1-1 19/11/81 75 0,178 1.97 0.097 0.60 0.793 0.34 1-2 15.12.81 71 0.154 6.30 0.026 3.27 > 3.00 0.26 1-3 0,129 0,324 1.21 1-4 19/10/81 17 0.937 0.12 1.778 0.04 0,052 4.59 1-5 17/11/81 46 > 3.00 0.11 0.03 0.058 Case 2 12/2/81 63 0.10 0.023 0.04 0,060 0.08 2-1 14/12/81 152 0.058 0.12 0,018 0.05 0.054 0.09 2-2 23/12/81 624 0,050 0.15 0,020 0.05 0.074 0.09 2-3 20/1/82 66 0.047 0.11 0,025 0.03 0.058 0.08 2-4 15/2/82 70 0.059 0.29 0.026 0.07 0.146 0.11 2-5 17/3/82 24 0,070 3.92 0,018 0.65 1,429 0.09 2-6 21/4/82 53 0,051 > 6.30 0.037 1.37 > 3.00 0.22 2-7 19/5/82 95 0,139 > 6.30 0.355 1.88 > 3.00 2.19 2-8 14/6/82 37 1,867 > 6.30 0,748 1.68 > 3.00 > 4.59 2-9 > 3.00 1.025 > 4.59 2-10 07/04/81 13 > 3.00 0.19 0.917 0.09 0.138 > 4.59 2-11 12/5/81 236 > 3.00 0.13 0.07 0.094 Case 3 30/5/81 471 0.17 0,049 0.08 0.144 0.21 3-1 09/06/81 315 0,090 0.44 0,040 0.16 0,275 0.14 3-2 07/06/81 273 0.064 0.59 0,045 0.50 1,773 0.22 3-3 10/8/81 158 0.079 > 6.30 0.085 2.47 > 3.00 0.42 3-4 09/08/81 84 0.211 > 6.30 0.272 4.21 > 3.00 2.71 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 > 4.59 3-8 > 3.00 > 3.00 > 4.59 3-9 > 3.00

IV.I.e.b. Prcben of random blood donors

Samples from random blood donors (see section IV.1.1.) Were screened for HCV infection by HCV c100-3 ELISA, where the antibody / En'.ymkonjuyat was either an anti-IgG monoclonal conjugate or a polyclonal conjugate. The total number of scans was 1077 for polyclonal conjugate and 1056 for monoclonal conjugate, respectively. A summary ^{Λ Ίί ό} ^ irtwni.n -iiobnisse carried out in Table 15, and the sample distributions are shown in the histogram in Fig.44.

The calculation of the average and standard deviation was done excluding the samples giving a signal above 1.5, d. that is, 1073 OD values were used for the polyclonal conjugate calculations and 1051 for the anti-IgG monoclonal conjugate. As can be seen in Table 15, the mean 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 were used to define the assay cut-off value, the polyclonal / enzyme conjugate configurations required a higher cut-off value in the ELISA assay. This indicates a lower assay specificity compared to the monoclonal system. In addition, as can be seen from the histogram in Fig. 44, a larger separation of the results between negative and positive distribution occurs when using random blood donors in an ELISA using the anti-IgG monoclonal conjugate as compared to an assay a conventional polyclonal label.

Table 15

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

conjugate Polyclonal Arti-IgG monoclonal (Jackson) Number of samples 1073 1 051 Average (x) 0.0931 0.04926 Standard deviation (SD) 0.0933 0.07427 5SD .4666 .3714 cut-off value (5SO + x) .5596 .4206

IV.J. Evidence of HCV seroconversion in NANBH patents from many geographic areas

Sera from patients suspected of having elevated ALT levels of NANBH in which HAV and HBV tests 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

i.and Niederliinde Italy Nip Number of examined cases 5 36 26 Positive number 3 29 19 % positive 60 80 73

IV.K. HCV Seroconjugase Evidence in "Community Acquired" NANBH Serenans were provided by Dr. H. Alter of the Center of Disease Control and Dr. J. Tuesday of Harvard University, of 100 patients with NANBH No transfusions, no user of oral drugs, no promiscuity, etc., were identified as risk factors.) These samples were analyzed by radicimmunoassay essentially according to the procedure described in section IV.D. except that the HCV c100-3 antigen was used as a screening antigen on the microtiter plates, and the results showed that of the 100 serum samples, 55 contained antibodies that were immunologically reactive with the HCV c100- antigen The results described above indicate that NANBH "Community Acquired" is also caused by HCV.

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

IV.L. Comparison of the occurrence of HCV antibodies and surrogate markers in donors affected by NANBH transmission

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. described radioimmunoassays. The results of the examination are shown 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 donations the patient had received, each donation being 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 - 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) , On the other hand, using the test for elevated ALT values, 6 of the 10 donors tested are negative, and using the anther antibody test, 5 out 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 18 No - Yes 5 Yes 16 Yes Yes No 6 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 Yes 10 Yes 15 Yes No

The same donor as anti-NANBH positive.

IV.M. Amplification to clone HCV cDNA sequence by PCR and methods derived from conserved regions of Flavl virus genome sequences were derived

The results presented above, which suggest that the HCV is a flavivirus or a flavivirus, allow a strategy for cionating uncharacteristic HCV cDNA sequences by the PCR technique and with primers derived from the regions that have been identified 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 26. 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 "Dowi stream" primers are derived from an upstream end of the known part of the HCV cDNA.

Because of the degeneracy of the code, it is possible that mismatches will occur between the flaviviruses and the corresponding HCV genomic sequence. Therefore, a strategy similar to that described by Lee (1988) is used. The Leesche ancestor exploits mixed oligonucleotide primers that are complementary to the products of the reverse translation of an amino acid sequence, the sequences in the mixed primers take into account each codon generation of the conserved amino acid sequence. Three sets of primer mixtures were created, based on the amino acid homologies found in flour flaviviruses, including dengue 2,4 (D-2,4), Japan encephalitis virus (YEV), yellow fever (YF), and West Nile virus (WN), were found. The primer mixture derived from the most "upstream" conserved sequences (5'-1) is based on the amino acid sequence Gly-Trp-Gly, which is part of the conserved sequence Asp-Arg-Gly-Trp-Gly-AspN, which is located in the The next primer mix (5'-2) is based on a downstream conserved sequence in the Ε protein, Phe-Asp-Giy-Asp-Ser-Tyr-ileum -Phe-Gly-Asp-Ser-Tyr-Ileu and is derived from Phe-Gly-Asp; the conserved sequence is present in D-2, JEV, YF and WN. The third primer mixture (5'-3) is based on the amino acid sequence Arg-Ser-Cys, which is part of the conserved sequence Cys-Cys-Arg-Ser-Cys in 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 HindIII, Mbol and EcoRI.

The "downstream" primer, ssc5h20A, is derived from a nucleotide sequence in clone 5h containing HCV cDNA with sequences overlapping sequences in clonans 14i and 11b The sequence of ssc5h20A is

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

An alternative primer, ssc5h34A, 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 enabling 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, 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 has been described. In addition, the annealing conditions in the first round of amplification are less severe (0.6M NaCl and 25 "C) because the portion of the primer that anneals to the HCV sequence is only 9 nucleotides and it is could lead to mismatches. In addition, when ssc5h34A is used, the additional sequences that are not derived from the HCV genome tend to destabilize the primer-template hybrid. After the first round of amplification, the "annealing" conditions can be more stringent (0,066M NaCI and 32 ° C to 37 ⁰ C), as amplified sequences now contain regions which are complementary u? Primers or their duplicates. In addition, the 10 cycles of amplification were performed with the Klenow enzyme I under PCR conditions suitable for this enzyme After completion of these cycles, the samples are extracted and treated according to the kit instructions as provided by Cetus / Perkin-Elmer with Tag polymerase. After amplification, 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 the primer derived from clone 5h. The sequence of the probe is

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 the 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 Figure 23. Positive clones were isolated at a frequency of 1 in 500000. An isolated clone, K9-1, was subjected to further studies: 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 14i as described previously.

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 k9-1 was compared to that described in Section IV.A.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.

The sequence of the amino acids encoded in the 5 'region of the HCV cDNA shown in Fig. 47 was compared with the corresponding region in one of the strains of the dengue virus described above with respect to the profile of the regions for hydrophobicity and hydrophilicity. This comparison showed that the polypeptides encoded in this region from HCV and dengue virus, this region corresponding to the region encoding NS1 (or a part thereof), have 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 comparing the HCV cDNAs from the new isolates with the cDNAs described below. The polypeptides encoded therein or in the virus genome can be monitored for immunological cross-reactivity by utilizing the polypeptides and antibodies described above. 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 aligning clones k9-1 to 15e in the 5 'to 3' direction; it also shows the amino acids encoded in the continuous ORF.

-51- 298 524 Industrial Applicability

The invention presented herein in various disclosures has many industrial applications, some of which are presented below. The HCV cDNAs can be used to design probes for detection of HCV nucleic acids in samples. The derived from the cDNAs probes can be used for the detection of HCV nucleic acids z. B. be 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 cell culture systems and in animal model systems. The HCV polynucleotide probes are also useful in the detection of viral nucleic acids in humans and thus may 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 the detection of antibodies to HCV antigens. Described herein is a series of immunoassays for HCV infection based on recombinant polypeptides containing HCV epitopes, commercial application in the diagnosis of HCV-induced NANBH, screening of blood donors for HCV-induced infectious hepatitis, and also when 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 infections. 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 antiviral agents in screening programs in which these agents are tested in tissue culture systems. Furthermore, they can also be used for passive immunotherapy and to diagnose HCV-induced NANBH by detecting the virus antigen (s) both in the blood donor and in 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 can be used in vaccines. In addition, 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 usually 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.

Conveniently, 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, the can also be used in the isolation of cDNAs derived from other disease-associated agents, not heretofore characterized in the preceding, from antibodies that are immunologically reactive with antibody-containing body components from other infected individuals, but not with those from uninfected individuals a genomic component.

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 (8)

An analysis kit for analyzing samples for the presence of polynucleotides derived from the HCV characterized by a polynucleotide probe containing a nucleotide sequence from the HCV of <Hwa 8 or more nucleotides in a suitable container. 2. Analytical kit for the analysis of samples for the presence of an HCV antigen, characterized by an antibody which is directed against the HCV antigen to be detected, in a suitable container. An analysis kit for analyzing samples for the presence of antibodies directed against an HCV antigen characterized by a polypeptide containing an HCV epitope present in the HCV antigen in a suitable container. 4. Analysis kit according to claim 3, characterized in that the polypeptide is linked to a solid substrate. 5. Analysis kit according to claim 3 or 4, characterized in that the polypeptide comprises an HCV epitope, since in the HCV cDNA sequence shown in FIG. 47 is coded. 6. Analysis kit according to claim 3, characterized in that the polypeptide comprises an HCV epitope which is in an HCV cDNA sequence in ATCC no. 40394, ATCC no. 40388, ATCC no. 40389, ATCC no. 40390, ATCC no. 40391, ATCC no. 40514, ATCC no. 40511, ATCC no. 40512 or ATCC no. 40513 is coded. 7. Analysis kit according to claim 3, characterized in that the polypeptide comprises an epitope of C100-3. 8. Analysis kit according to claim 3, characterized in that the polypeptide comprises C100-3.