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WO1994018217A1 - Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation - Google Patents

Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation Download PDF

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Publication number
WO1994018217A1
WO1994018217A1 PCT/US1993/000928 US9300928W WO9418217A1 WO 1994018217 A1 WO1994018217 A1 WO 1994018217A1 US 9300928 W US9300928 W US 9300928W WO 9418217 A1 WO9418217 A1 WO 9418217A1
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WIPO (PCT)
Prior art keywords
hgv
polypeptide
antibody
polynucleotide
hepatitis
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PCT/US1993/000928
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English (en)
Inventor
Nic C. Tassopoulos
Angelos E. Hatzakis
Hugo Troonen
Mary C. Kuhns
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Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to AU36061/93A priority Critical patent/AU3606193A/en
Priority to PCT/US1993/000928 priority patent/WO1994018217A1/fr
Publication of WO1994018217A1 publication Critical patent/WO1994018217A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to hepatitis-causing agents, and more particularly, relates to an acute hepatitis-causing agent which is non-A, non-B, non-C, non-D and non-E, and methods for its detection.
  • Acute non-A, non-B (NANB) hepatitis infection is considered to be a mild disease with an increased tendency to progress to a chronic state. Progression to chronic hepatitis has been reported in 32-65% of the patients with acute NANB hepatitis infection, regardless of the risk factor(s) involved. See, for example, H.J. Alter et al., in W. Szmuness et al., eds., Viral Hepatitis. Philadelphia, Franklin Institute Press 279-294 (1981); M. Berman et al., Ann Intern. Med. 91:1-6 (1979); R.E. Sampliner et al.. J. Med. Virol. 13:125-130 (1984); A.
  • a non-identifiable source of infection is implicated in approximately 30 to 50% of these cases, and this subset of acute NANB hepatitis has been designated either as community-acquired NANB (CA- NANB), sporadic NANB or cryptogenic NANB hepatitis.
  • CA- NANB community-acquired NANB
  • sporadic NANB sporadic NANB
  • cryptogenic NANB hepatitis Acute CA-NANB hepatitis is a poorly characterized entity, since its etiology is obscure and the diagnosis is rarely confirmed by histology.
  • HCV hepatitis C virus
  • HCV antibodies Although the detection of HCV antibodies eliminates 70 to 80% of NANBH infected blood from the blood supply system, the antibodies apparently are readily detected during the chronic state of the disease, while only 60% of the samples from the acute NANBH stage are HCV antibody positive. H. Alter et al., New Eng. J. Med. 321:1994-1500 (1989).
  • the prolonged interval between exposure to HCV and antibody detection and the lack of adequate information regarding the profile of immune response to various structural and non-structural proteins raises questions regarding not only the infectious state of the patient in the antibody negative phase during NANBH infection, but also whether HCV infection exists in these hosts. Therefore, there is a need for on-going research to determine whether other infectious agents may be responsible for "NANB" infections.
  • HCV infection is implicated in a minority of , acute CA-NANB cases in Greece, and have discovered the clinical, biochemical and histological differences between anti-HCV positive and anti-HCV negative acute CA-NANB hepatitis cases.
  • this acute non- A, non-B non-C, non-D and non-E hepatitis is a distinct clinicopathologic entity. Isolation of the entity is on-going, as is work to characterize the amino acid sequence of the entity. It is contemplated that reagents developed as a result of the nucleic acid sequence will be helpful for the diagnosis of the hepatitis illness associated with this distinct etiological agent, as well as useful for vaccine production and other uses which will be set forth hereinbelow.
  • This invention provides a purified HGV polynucleotide, a recombinant HGV polynucleotide, host cells and recombinant vectors.
  • the recombinant HGV polynucleotide comprises a sequence derived from an HGV genome.
  • the recombinant HGV polynucleotide comprises an epitope of HGV.
  • the invention also provides a recombinant expression system comprising an open reading frame of DNA or RNA derived from the HGV genome or cDNA, wherein the open reading frame is operably linked to a control sequence compatible with a desired host, cells transformed with said recombinant expression system and polypeptides produced by said cells.
  • the invention further provides purified HGV, which can comprise a preparation of HGV polypeptide. Additionally, the present invention provides a recombinant polypeptide comprising a sequence derived from an HGV genome or from HGV cDNA, and a recombinant polypeptide comprising an HGV epitope. Also provided is an antibody directed against at least one HGV epitope. The antibody is polyclonal or monoclonal. The invention further provides a fusion polypeptide comprising an HGV polypeptide.
  • the invention also provides a particle that is immunogenic against HGV infection, comprising a non-HGV polypeptide having an amino acid sequence capable of forming a particle when said sequence is produced in a eukaryotic or prokaryotic host, and an HGV epitope, a polynucleotide probe for HGV, and various test kits for performing various methods to detect either HGV antigen or HGV antibody.
  • the invention provides a method for producing a polypeptide containing an HGV epitope comprising incubating host cells transformed with an expression vector containing a sequence encoding a polypeptide containing an HGV epitope, under conditions and for a time which allows expression of said polypeptide.
  • a method for detecting HGV nucleic acids in a test sample suspected of containing HGV comprises reacting the test sample with a probe for an HGV polynucleotide under conditions and for a time which allows the formation of a complex between the probe and the HGV nucleic acid in the test sample; and detecting the complex which contains the probe.
  • An additional method for detecting HGV antigen in a test sample suspected of containing HGV comprises contacting a test sample with an antibody directed against HGV antigen to be detected for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and detecting said complex containing the antibody.
  • Still another method for detecting HGV antibodies in a test sample suspected of containing said antibodies comprises contacting the test sample with a probe polypeptide wherein said polypeptide contains an HGV epitope, for a time and under conditions sufficient to allow antigen antibody complexes to form; and detecting said complexes which contain the probe polypeptide.
  • a vaccine for treatment of HGV infection also is provided which comprises a pharmacologically effective dose of an immunogenic HGV polypeptide which contains an HGV epitope in a pharmaceutically acceptable excipient.
  • the vaccine for treatment of HGV infection also can comprise an inactivated or attenuated HGV in a pharmacologically effective dose in an pharmaceutically acceptable excipient.
  • the invention provides tissue culture grown cells infected with HGV and a method for producing antibodies to HGV comprising administering to an individual an isolated immunogenic polypeptide containing an HGV epitope in an amount sufficient to produce an immune response.
  • the present invention provides characterization of a newly ascertained etiological agent of non-A, non-B, non-C, non-D and non-E hepatitis-causing agent, so-termed "Hepatitis G Virus,” or "HGV.”
  • the present invention provides a method for determining the presence of the HGV etiological agent, methods for obtaining the nucleic acid library of this etiological agent created from a particulate agent in infected serum, plasma or liver homogenates from individuals, either humans or chimpanzees, with HGV to detect newly synthesized antigens derived from the genome of the heretofore unisolated and uncharacterized viral agent, and of selecting clones which produced products which reacted immunologically only with sera from infected individuals as compared to non-infected individuals.
  • Portions of the nucleic acid sequences derived from HGV are useful as probes to determine the presence of HGV in test samples, and to isolate naturally occurring variants of the virus. These sequences also make available polypeptide sequences of HGV antigens encoded within the HGV genome(s) and permits the production of polypeptides which are useful as standards or reagents in diagnostic tests and/or as components of vaccines. Monoclonal and polyclonal antibodies directed against at least one epitope contained within these polypeptide sequences, also are useful for diagnostic tests as well as therapeutic agents, for screening of antiviral agents, and for the isolation of the HGV agent from which these nucleic acid sequences are derived.
  • Isolation and sequencing of other portions of the HGV genome also can be accomplished by utilizing probes derived from these nucleic acid sequences, thus allowing additional probes and polypeptides of the HGV to be established, which will be useful in the diagnosis and/or treatment of HGV, both as a prophylactic and therapeutic agent.
  • purified HGV in another aspect of the invention there will be provided purified HGV; a preparation of polypeptides from the purified HGV; a purified HGV polypeptide; a purified polypeptide comprising an epitope which is immunologically identifiable with an epitope contained in HGV.
  • a recombinant expression system comprising an open reading frame (ORF) of DNA derived from an HGV genome or from HGV cDNA, wherein the ORF is operably linked to a control sequence compatible with a desired host, a cell transformed with the recombinant expression system, and a polypeptide produced by the transformed cell.
  • ORF open reading frame
  • Additional aspects of the present invention include a recombinant HCV polypeptide, a recombinant polypeptide comprised of a sequence derived from an HGV genome or from HGV cDNA; a recombinant polypeptide comprised of an HGV epitope and a fusion polypeptide comprised of an HGV polypeptide.
  • the present invention also provides methods for producing a monoclonal antibody which specifically binds to at least one epitope of HGV; a purified preparation of polyclonal antibodies which specifically bind to at least one HGV epitope; and methods for using these antibodies, which include diagnostic, prognostic and therapeutic uses.
  • a particle which is immunogenic against HGV infection comprising a non-HGV polypeptide having an amino acid sequence capable of forming a particle when said sequence is produced in a eukaryotic host, and an HGV epitope.
  • a polynucleotide probe for HGV also will be provided.
  • kits containing reagents which can be used for the detection of the presence and/or amount of polynucleotides derived from HGV, such reagents comprising a polynucleotide probe containing a nucleotide sequence from HGV of about 8 or more nucleotides in a suitable container; a reagent for detecting the presence and/or amount of an HGV antigen comprising an antibody directed against the HGV antigen to be detected in a suitable container; a reagent for detecting the presence and/or amount of antibodies directed against an HGV antigen comprising a polypeptide containing an HCV epitope present in the HGV antigen, provided in a suitable container.
  • kits for various assay formats also are provided by the present invention as described herein.
  • polypeptide comprising an HGV epitope attached to a solid phase and an antibody to an HGV epitope attached to a solid phase.
  • methods for producing a polypeptide containing an HGV epitope comprising incubating host cells transformed with an expression vector containing a sequence encoding a polypeptide containing an HGV epitope under conditions which allow expression of the polypeptide, and a polypeptide containing an HGV epitope produced by this method.
  • the present invention also provides assays which utilize the recombinant or synthetic polypeptides provided by the invention, as well as the antibodies described herein in various formats, any of which may employ a signal generating compound in the assay. Assays which do not utilize signal generating compounds to provide a means of detection also are provided. All of the assays described generally detect either antigen or antibody, or both, and include contacting a test sample with at least one reagent provided herein to form at least one antigen/antibody complex and detecting the presence of the complex. These assays are described in detail herein.
  • Vaccines for treatment of HGV infection comprising an immunogenic peptide containing an HGV epitope, or an inactivated preparation of HGV, or an attenuated preparation of HGV also are included in the present invention. Also included in the present invention is a method for producing antibodies to HGV comprising administering to an individual an isolated immunogenic polypeptide containing an HGV epitope in an amount sufficient to produce an immune response in the inoculated individual.
  • tissue culture grown cell infected with HGV is also provided by the present invention.
  • a method for isolating cDNA derived from the genome of an unidentified infectious agent comprising (a) providing host cells transformed with expression vectors containing a cDNA library prepared from nucleic acids isolated from serum, plasma or liver tissue homogenates infected with the agent growing in said host cells under conditions and for a time which allow expression of polypeptide(s) encoded in the cDNA; (b) interacting the expression products of the cDNA with an antibody containing body component of an individual infected with said infectious agent under conditions and for a time to allow an immunoreaction (antigen antibody complex) to occur, and detecting the antigen/antibody complexes formed as a result of the interaction; (c) growing host cells which express polypeptides that form antigen-antibody complexes in step (b) under conditions and for a time which allows for their growth as individual clones and isolating these clones; (d) growing cells from the clones of (c) under conditions and for a
  • Hepatitis G Virus denotes a viral species which causes non-A, non-B, non-C, non-D, non-E, CA-hepatitis, and attenuated strains or defective interfering particles derived therefrom.
  • the methods as described herein will allow the identification of individuals who have acquired HGV.
  • the compositions and methods described herein will enable the propagation, identification, detection and isolation of the Hepatitis G Virus and its possible strains. Moreover, they also will allow the preparation of diagnostics and vaccines for the possible different strains of HGV, and will have utility in screening procedures for anti-viral agents. The information that will be will be sufficient to allow a viral taxonomist to identify other strains which fall within the species.
  • HGV encodes an epitope which is immunologically identifiable with an epitope in the HGV genome from which the nucleic acids which will be described herein are derived; preferably, the epitope is encoded in a sequence which will be described.
  • the epitope is unique to HGV when compared to other known Hepatitis-like viruses.
  • the uniqueness of the epitope may be determined by its immunological reactivity with HGV and lack of immunological reactivity with Hepatitis A, B, C, D and E viruses.
  • Methods for determining immunological reactivity are known in the art and include, for example, radioimmunoassay, ELISA, hemagglutination, and several examples of suitable techniques are described herein.
  • the following parameters are applicable, either alone or in combination, in identifying a strain of HGV. It is expected that the overall homology of the genomes at the nucleotide level will be about 40% or greater since it is now believed that the HGV strains may be genetically related, preferably about 60% or greater, and more preferably, about 80% or greater. In addition, there will be corresponding contiguous sequences of at least about 13 nucleotides.
  • the correspondence between the putative HGV strain genomic sequence and the sequence of HGV can be determined by a direct comparison of the sequence information of the polynucleotide from the putative HGV, and the HGV amino acid sequence(s) we are now in the process of ascertaining. Putative HGV strains are identifiable on the polypeptide level.
  • HGV strains are more than about 40% homologous, preferably more than about 60% homologous, and even more preferably, more than about 80% homologous at the polypeptide level.
  • the techniques for determining amino acid sequence homology include, for example, directly determining the amino acid sequence and comparing it to the sequences provided herein; determining the nucleotide sequence of the genomic material of the putative HGV (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing the corresponding regions.
  • a polypeptide "derived from" a designated sequence, for example, the
  • HGV cDNA refers to a polynucleotide sequence which is comprised of a sequence of approximately at least about 6 nucleotides, is preferably at least about 8 nucleotides, is more preferably at least about 10-12 nucleotides, and even more preferably is at least about 15-20 nucleotides corresponding, i.e., homologous to or complementary to, a region of the designated nucleotide sequence.
  • the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence which is unique to the HGV genome. Whether or not a sequence is complementary to or homologous to a sequence which is unique to an HGV genome can be determined by techniques known to those skilled in the art.
  • Comparisons to sequences in databanks can be used as a method to determine the uniqueness of a designated sequence.
  • Regions from which sequences may be derived include but are not limited to regions encoding specific epitopes, as well as non-translated and/or non-transcribed regions.
  • the derived polypeptide will not necessarily be derived physically from the nucleotide sequence of HGV, but may be generated in any manner, including but not limited to chemical synthesis, replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived.
  • combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
  • a polypeptide or amino acid sequence derived from a designated nucleic acid sequence or from the HGV genome refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably 15 to 20 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
  • a “recombinant protein” (“recombinant polynucleotide”) as used herein means at least a polypeptide of genomic, semisynthetic or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature or in the form of a library and/or is linked to a polynucleotide other than that to which it is linked in nature.
  • a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence of HGV or from an HGV genome. It also may be generated in any manner, including chemical synthesis or expression of a recombinant expression system, or isolation from mutated HGV.
  • polynucleotide as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, either by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • HGV containing a sequence corresponding to a cDNA means that the HGV contains a polynucleotide sequence which is homologous to or complementary to a sequence in the designated DNA.
  • the degree of homology or complementarity to the cDNA will be approximately 50% or greater, will preferably be at least about 70%, and even more preferably will be at least about 90%.
  • the sequence which correspond will be at least about 70 nucleotides, preferably at least about 80 nucleotides, and even more preferably at least about 90 nucleotides in length.
  • the correspondence between the HGV and the cDNA can be determined by methods known in the art, and include, for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
  • Purified viral polynucleotide refers to an HGV genome or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of polypeptides with which the viral polynucleotide is naturally associated.
  • Techniques for purifying viral polynucleotides include, for example, disruption of the particle with a chaotropic agent, and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density.
  • purified viral polypeptide means an HGV polypeptide or fragment thereof which is essentially free, that is., contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of of cellular components with which the viral polypeptide is naturally associated. Methods for purifying are known to the routineer.
  • Polypeptide indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term however is not intended to refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the original progeny of the original cell which has been transfected.
  • replicon means any genetic element, such as a plasmid, a chromosome, a virus, that behaves as an autonomous unit of polynucleotide replication within a cell. That is, it is capable of replication under its own control.
  • a “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers.
  • control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
  • “Operably linked” refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • the term "open reading frame” or “ORF” refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
  • a "coding sequence” is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' -terminus and a translation stop codon at the 3' -terminus.
  • a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polypeptide sequences.
  • immunologically identifiable with/as refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s), usually HGV proteins. Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein. The uniqueness of an epitope also can be determined by computer searches of known data banks, such as Genebank, for the polynucleotide sequences which encode the epitope, and by amino acid sequence comparisons with other known proteins.
  • epitope means an antigenic determinant of a polypeptide.
  • an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope.
  • an epitope consists of at least five such amino acids, and more usually, it consists of at least 8 to 10 amino acids.
  • Methods of examining spatial conformation 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 antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
  • immunogenic polypeptide containing an HGV epitope means naturally occurring HGV polypeptides or fragments thereof, as well as polypeptides prepared by other means, for example, chemical synthesis or the expression of the polypeptide in a recombinant organism.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction, or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Treatment refers to prophylaxis and/or therapy.
  • the term "individual” as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, primates and humans; more particularly the term refers to chimpanzees and humans.
  • plus strand denotes a nucleic acid that contains the sequence that encodes the polypeptide.
  • minus strand denotes a nucleic acid that contains a sequence that is complementary to that of the "plus” strand.
  • RNA or DNA Single-stranded and which encodes a viral polypeptide(s).
  • antibody containing body component refers to a component of an individual's body which is the source of the antibodies of interest. These components are well known in the art. These samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like, biological fluids such as cell culture supernatants, fixed tissue specimens and fixed cell specimens.
  • human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like, biological fluids such as cell culture supernatants, fixed tissue specimens and fixed cell specimens.
  • Purified HGV refers to a preparation of HGV which has been isolated from the cellular constituents with which the virus is normally associated, and from other types of viruses which may be present in the infected tissue.
  • the techniques for isolating viruses are known to those skilled in the art and include, for example, centrifugation and affinity chromatography. A method for the preparation of purified HGV is described herein.
  • the recombinant or synthetic peptides can be used to develop unique assays as described herein to detect either the presence of antigen or antibody to HGV.
  • These compositions also can be used to develop monoclonal and/or polyclonal antibodies with a specific recombinant protein or synthetic peptide which specifically bind to the immunological epitope of HGV which is desired by the routineer.
  • at least one polynucleotide of the invention can be used to develop vaccines by following methods known in the art.
  • the reagent employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, or a polypeptide (either recombinant or synthetic) employed in the assay.
  • Solid phases are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, and others.
  • the “solid phase” is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips and sheep red blood cells are all suitable examples.
  • Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a “solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well.
  • Such useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross- linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxyiic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sul
  • porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents including monoclonal antibodies.
  • Nylon also possesses similar characteristics and also is suitable.
  • porous solid supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm.
  • the pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.
  • the surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support. The irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
  • Suitable solid supports also are described in U.S. Patent Application Serial No. 227,272.
  • the “indicator reagent” “comprises a “signal generating compound” (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for HGV.
  • Specific binding member as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HGV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • labels include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds, radioactive elements, and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No.921,979 corresponding to EPO Publication No. 0 273,115.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi- automated systems wherein the solid phase comprises a microparticle.
  • Such systems include those described in pending U. S. Patent Applications 425,651 and 425,643, which correspond to published EPO applications Nos.
  • SPM scanning probe microscopy
  • the capture phase for example, at least one of the monoclonal antibodies of the invention
  • a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase.
  • scanning tunnelling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. Such a system is described in pending U. S. patent application Serial No.
  • one member of a specific binding partner is attached to a surface suitable for scanning.
  • the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
  • covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized.
  • Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner.
  • Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively.
  • Such activated surfaces can be used to link the binding partner directly (in the cases of amino or thiol) or the activated surface can be further reacted with linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-[N- maleimidomethyl] cyclohexane-1-carboxylate), SIAB (succinimidyl [4-iodoacetyl] aminobenzoate), and SMPB (succinimidyl 4-[l-maleimidophenyl] butyrate) to separate the binding partner from the surface.
  • linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-
  • the vinyl group can be oxidized to provide a means for covalent attachment. It also can be used as an anchor for the polymerization of various polymers such as poly acrylic acid, which can provide multiple attachment points for specific binding partners.
  • the amino surface can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capacity.
  • oxidizable dextrans examples include Dextran T-40 (molecular weight 40,000 daltons), Dextran T-l 10 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia), or Ficoll (molecular weight 70,000 daltons (available from Sigma Chemical Co., St. Louis, MO).
  • polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries described by pending U. S. Patent applications Serial No. 150,278, filed January 29, 1988, and Serial No.
  • the preferred method of attachment is by covalent means.
  • the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
  • the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
  • Various other assay formats may be used, including "sandwich" immunoassays and competitive probe assays.
  • the monoclonal antibodies of the present invention can be employed in various assay systems to determine the presence, if any, of HGV proteins in a test sample. Fragments of these monoclonal antibodies provided also may be used.
  • a polyclonal or monoclonal anti-HGV antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase is contacted with a test sample which may contain HGV proteins, to form a mixture.
  • a test sample which may contain HGV proteins
  • This mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes.
  • an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to an HGV region, or a combination of these antibodies, to which a signal generating compound has been attached, is contacted with the antigen/antibody complexes to form a second mixture.
  • This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
  • the presence of HGV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound.
  • the amount of HGV antigen present in the test sample is proportional to the signal generated.
  • a polyclonal or monoclonal anti-HGV antibody or fragment thereof, or a combination of these antibodies which is bound to a solid support the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which specifically binds to HGV antigen, or a combination of these antibodies to which a signal generating compound is attached, are contacted to form a mixture.
  • This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
  • the presence, if any, of HGV proteins present in the test sample and captured on the solid phase is determined by detecting the measurable signal generated by the signal generating compound.
  • the amount of HGV proteins present in the test sample is proportional to the signal generated.
  • one or a combination of one or more monoclonal antibodies of the invention can be employed as a competitive probe for the detection of antibodies to HGV protein.
  • HGV proteins either alone or in combination, can be coated on a solid phase.
  • a test sample suspected of containing antibody to HGV antigen then is incubated with an indicator reagent comprising a signal generating compound and at least one monoclonal antibody of the invention for a time and under conditions sufficient to form antigen antibody complexes of either the test sample and indicator reagent to the solid phase or the indicator reagent to the solid phase.
  • the reduction in binding of the monoclonal antibody to the solid phase can be quantitatively measured.
  • a measurable reduction in the signal compared to the signal generated from a confirmed negative NANB, non-C, non-D, non-E hepatitis test sample indicates the presence of anti- HGV antibody in the test sample.
  • each of the monoclonal antibodies of the present invention can be employed in the detection of HGV antigens in fixed tissue sections, as well as fixed cells by immunohistochemical analysis.
  • these monoclonal antibodies can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific HGV proteins from cell cultures, or biological tissues such as blood and liver.
  • the monoclonal antibodies of the invention can also be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
  • the monoclonal antibodies or fragments thereof can be provided individually to detect HGV antigens. Combinations of the monoclonal antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one anti-HGV antibody of the invention with antibodies to other HGV regions, each having different binding specificities. Thus, this cocktail can include the monoclonal antibodies of the invention which are directed to HGV proteins and other monoclonal antibodies to other antigenic determinants of the HGV genome.
  • the polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to a specific HGV region or other HGV proteins used in the assay.
  • the polyclonal antibody used preferably is of mammalian origin; human, goat, rabbit or sheep anti-HCV polyclonal antibody can be used. Most preferably, the polyclonal antibody is rabbit polyclonal anti-HGV antibody.
  • the polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different HGV specificity, they would be useful for diagnosis, evaluation and prognosis of HGV infection, as well as for studying HGV protein differentiation and specificity.
  • the presence of antibody and/or antigen to HCV can be detected in a simultaneous assay, as follows.
  • a test sample is simultaneously contacted with a capture reagent of a first analyte, wherein said capture reagent comprises a first binding member specific for a first analyte attached to a solid phase and a capture reagent for a second analyte, wherein said capture reagent comprises a first binding member for a second analyte attached to a second solid phase, to thereby form a mixture.
  • This mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes.
  • Such so-formed complexes then are contacted with an indicator reagent comprising a member of a binding pair specific for the first analyte labelled with a signal generating compound and an indicator reagent comprising a member of a binding pair specific for the second analyte labelled with a signal generating compound to form a second mixture.
  • This second mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator reagent complexes and capture reagent/second analyte/indicator reagent complexes.
  • the presence of one or more analytes is determined by detecting a signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence of one or more analytes in the test sample.
  • proteins derived from human expression systems may be utilized as well as monoclonal antibodies produced from the proteins derived from the mammalian expression systems as disclosed herein. Such assay systems are described in greater detail in pending U.S. Patent Application Serial No. 07/574,821 entitled Simultaneous Assay for Detecting One Or More Analytes, which corresponds to EP Publication No. 0473065.
  • recombinant proteins may be utilized to detect the presence of anti-HGV in test samples.
  • a test sample is incubated with a solid phase to which at least one recombinant protein has been attached. These are reacted for a time and under conditions sufficient to form antigen/antibody complexes. Following incubation, the antigen/antibody complex is detected. Indicator reagents may be used to facilitate detection, depending upon the assay system chosen.
  • a test sample is contacted with a solid phase to which a recombinant protein produced as described herein is attached and also is contacted with a monoclonal or polyclonal antibody specific for the protein, which preferably has been labelled with an indicator reagent.
  • the solid phase After incubation for a time and under conditions sufficient for antibody/antigen complexes to form, the solid phase is separated from the free phase, and the label is detected in either the solid or free phase as an indication of the presence of HGV antibody.
  • Other assay formats utilizing the proteins of the present invention are contemplated. These include contacting a test sample with a solid phase to which at least one recombinant protein produced in the mammalian expression system has been attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen/antibody complexes, and then contacting the solid phase with a labelled recombinant antigen. Assays such as this and others are described in pending U.S. Patent Application Serial No. 07/787,710.
  • the present invention discloses the preference for the use of solid phases, it is contemplated that the proteins of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention. While the present invention discloses the preference for the use of solid phases, it is contemplated that the peptides of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.
  • the reagents and methods of the present invention are made possible by the provision of a family of closely homologous nucleotide sequences isolated from a genomic or cDNA library derived from nucleic acid sequences present in the plasma, serum or liver homogenate of an HGV infected individual, either chimpanzee or human.
  • This family of nucleotide sequences is not of human or chimpanzee origin, since it will be shown that it hybridizes to neither human nor chimpanzee genomic DNA from uninfected individuals, since nucleotides of this family of sequences are present only in liver (or liver homogenates), plasma or serum of individuals infected with HGV, and since the sequence is not present in Genebank.
  • sequences contained within isolated clones which encode polypeptides.
  • Sera, plasma or liver homogenates from HGV infected humans contain antibodies which bind to this polypeptide
  • sera, plasma or liver homogenates from non-infected humans do not contain antibodies to this polypeptide.
  • the antibodies are induced in individuals following acute NANB, non-C, non-D and non-E infection.
  • nucleic acid sequences permit the construction of DNA probes and polypeptides useful in diagnosing NANB, non- C, non-E hepatitis due to HGV infections, and in screening blood donors , donated blood, blood products and individuals for infection.
  • DNA oligomers of about eight to ten nucleotides, or larger, which re useful as hybridization probes to detect the presence of the viral genome in, for example, sera of subjects suspected of harboring the virus, or for screening donated blood for the presence of the virus.
  • the family of nucleic acid sequences also allows the design and production of HGV specific polypeptides which are useful as diagnostic reagents for the presence of antibodies raised during infection with HGV.
  • Antibodies to purified polypeptides derived from the nucleic acid sequences may also be used to detect viral antigens in infected individuals and in blood. These nucleic acid sequences also enable the design and production of polypeptides which may be used as vaccines against HGV, and also for the production of antibodies, which then may be used for protection of the disease, and/or for therapy of HGV infected individuals.
  • the family of nucleic acid sequences also enables further characterization of the HGV genome.
  • Polynucleotide probes derived from these sequences may be used to screen genomic or cDNA libraries for additional overlapping nucleic acid sequences which then may be used to obtain more overlapping sequences. Unless the genome is segmented and the segments lack common sequences, this technique may be used to gain the sequence of the entire genome. However, if the genome is segmented, other segments of the genome can be obtained by repeating the lambda-gtl 1 serological screening procedure used to isolate the clones which will be described herein, or alternatively by isolating the genome from purified HGV particles.
  • the family of cDNA sequences and the polypeptides derived from these sequences, as well as antibodies directed against these polypeptides, also are useful in the isolation and identification of the HGV etiological agent(s).
  • antibodies directed against HGV epitopes contained in polypeptides derived from the nucleic acid sequences may be used in methods based upon affinity chromatography to isolate the virus.
  • the antibodies can be used to identify viral particles isolated by other techniques. The viral antigens and the genomic material within the isolated viral particles then may be further characterized.
  • the information obtained from further sequencing of the HGV genome(s), as well as from further characterization of the HGV antigens and characterization of the genome enables the design and synthesis of additional probes and polypeptides and antibodies which may be used for diagnosis, prevention and therapy of HGV induced NANB hepatitis, and for screening for infected blood and blood-related products.
  • probes for HGV including antigens, antibodies and polynucleotides derived from the genome from which the family of nucleic acid sequences is derived also allows for the development of tissue culture systems which will be of major use in elucidating the biology of HGV. Once this is known, it is contemplated that new treatment regimens may be developed based upon antiviral compounds which preferentially inhibit the replication of or infection by HGV.
  • a genomic or cDNA library is created from the nucleic acids present in infected serum, plasma or liver homogenates from an infected individual, preferably a chimpanzee or human.
  • the library is created in a vector which allows the expression of polypeptides encoded in the nucleic acid sequences.
  • Clones of host cells containing the vector, which has expressed an immunologically reactive fragment of a polypeptide of the etiological agent (HGV) are selected by immunological screening of the expression products of the library with an antibody containing body component from another individual previously infected with the putative agent.
  • the steps in the immunological screening technique include interacting the expression products of the cloned nucleic acid sequences containing vectors with the antibody containing body component of a second infected individual, and detecting the formation of antigen-antibody complexes between the expression product(s) and antibodies of the second infected individual.
  • the isolated clones are screened further immunologically by interacting their expression products with the antibody containing body component of other individuals infected with the putative agent and detecting the formation of antigen-antibody complexes with antibodies from the infected individuals, and the nucleic acid sequences containing vectors which encode polypeptides which react immunologically with antibodies from infected individuals and individuals suspected of being infected the agent, but not with control individuals, are isolated.
  • nucleic acid sequences isolated as a result of this method and their expression products, and antibodies directed against the expression products, are useful in characterizing and/or capturing the etiological agent. This method is taught in EP Patent Application Publication No.O 318 216. Preparation of the Nucleic Acid Sequences
  • the lambda-gtl 1 cDNA library generated from a cDNA pool containing cDNA is screened for encoded epitopes that can bind specifically with sera derived from individuals who previously had experienced non-A, non-B, non-c, non-d and non-E hepatitis. See V. Hunyh et al., in D. Glover, ed, DNA Cloning Techniques: A Practical Approach. IRL Press,Oxford, England, pp. 49-78 (1985). Approximately 10° - 10 ⁇ phages are screened, from which positive phages are identified, purified, and then tested for specificity of binding to sera from different individuals previously infected with the HGV agent.
  • Phages which selectively bind sera, plasma from patients meeting the criteria described hereinbelow and not in patients who did not meet these described criteria, are preferred for further study.
  • clones containing additional upstream and downstream HGV sequences are obtained. The isolation of these clones is described hereinbelow.
  • sequences (and their complements) retrieved from the HGV library of sequences are provided herein, and the sequences or any portion thereof, can be prepared using synthetic methods or by a combination of synthetic methods with retrieval of partial sequences using methods similar to those described herein. This description thus provides one method by which genomic or cDNA sequences corresponding to the entire HGV genome may be isolated. Other methods for isolating these sequences, however, will be obvious to those skilled in the art and are considered to be within the scope of the present invention.
  • nucleic acid sequences permits the construction of expression vectors encoding antigenically active regions of the polypeptide encoded in either strand.
  • antigenically active regions may be derived from envelope (coat) or core antigens, including, for example, polynucleotide binding proteins, polynucleotide polymerase(s), and other viral proteins necessary for replication and/or assembly of the viral particle.
  • Fragments encoding the desired polypeptides are derived from the genomic or cDNA clones using conventional restriction digestion or by synthetic methods, and are ligated into vectors which may, for example, contain portions of fusion sequences such as beta-galactosidase (B-gal) or superoxide dismutase (SOD) or CMP-KDO synthetase (CKS).
  • B-gal beta-galactosidase
  • SOD superoxide dismutase
  • CKS CMP-KDO synthetase
  • nucleic acid sequence containing an open reading frame, in either sense strand can be obtained as a recombinant protein, such as a mature or fusion protein; alternatively, a polypeptide encoded in the HGV genome or cDNA can be provided by chemical synthesis.
  • the nucleic acid sequence encoding the desired polypeptide may be ligated into expression vectors suitable for any convenient host. Both eucaryotic and prokaryotic host systems are used in the art to form recombinant proteins, and some of these are listed herein.
  • the polypeptide then is isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification can be performed by techniques known in the art, and include salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, among others. Such polypeptides may be used as diagnostic reagents, or for passive immunotherapy.
  • HGV antigens are useful for isolating and identifying HGV particles.
  • the HGV antigens also may be isolated from HGV virions. These virions can be grown in HGV infected cells in tissue culture, or in an infected individual. Preparation of Antigenic Polypeptides and Conjugation With Solid Phase
  • An antigenic region or fragment of a polypeptide generally is relatively small, usually about 8 to 10 amino acids or less in length. Fragments of as few as 5 amino acids may characterize an antigenic region. These segments may correspond to regions of HGV antigen.
  • nucleic acid sequences encoding short segments of HGV polypeptides can be expressed recombinantly either as fusion proteins or as isolated polypeptides. These short amino acid sequences also can be obtained by chemical synthesis.
  • the small chemically synthesized polypeptides may be linked to a suitable carrier molecule when the synthesized polypeptide provided is correctly configured to provide the correct epitope but too small to be antigenic.
  • Linking methods include but are not limited to using N- succinimidyl-3-(2-pyrdylthio)propionate (SPDP) and succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC).
  • SPDP N- succinimidyl-3-(2-pyrdylthio)propionate
  • SMCC succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate
  • Polypeptides lacking sulfhydryl groups can be modified by adding a cysteine residue. These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in the other.
  • a variety of such disulfide/amide-forming agents are known.
  • bifunctional coupling agents form a thioester rather than a disulfide linkage.
  • Many of these thio-ether-forming agents are commercially available and are known to those of ordinary skill in the art.
  • the carboxyl groups can be activated by combining them with succinimide or l-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
  • Any carrier which does not itself induce the production of antibodies harmful to the host can be used. Suitable carriers include proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, polymeric amino acids such as polyglutamic acid, polylysine , amino acid copolymers and inactive virus particles, among others.
  • protein substrates include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and yet other proteins known to those skilled in the art.
  • HGV epitopes also may be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle- forming proteins such as those associated with HB V surface antigen. Constructs wherein the HGV epitope is linked directly to the particle-forming protein coding sequences produce hybrids which are immunogenic with respect to the HGV epitope.
  • all of the vectors prepared include epitopes specific for HGV, having varying degrees of immunogenicity.
  • Particles constructed from particle forming protein which include HGV sequences are immunogenic with respect to HGV and HBV.
  • Hepatitis B surface antigen has been determined to be formed and assembled into particles in S. cerevisiae and mammalian cells; the formation of these particles has been reported to enhance the immunogenicity of the monomer subunit.
  • P. Valenzuela et al. Nature 298:334 (1982); P. Valenzuela et al., in I. Millman et al., eds., Hepatitis B. Plenum Press, pp. 225-236 (1984).
  • the constructs may include immunodominant epitopes of HBsAg. Such constructs have been reported expressible in yeast, and hybrids including heterologous viral sequences for yeast expression have been disclosed.
  • Vaccines may be prepared from one or more immunogenic polypeptides derived from HGV nucleic acid sequences or from the HGV genome to which they correspond. Vaccines may comprise recombinant polypeptides containing epitope(s) of HGV. These polypeptides may be expressed in bacteria, yeast or mammalian cells, or alternatively may be isolated from viral preparations. It also is anticipated that various structural proteins may contain epitopes of HGV which give rise to protective anti-HGV antibodies. Thus, polypeptides containing at least one epitope of HGV may be used, either singly or in combinations, in HGV vaccines. It also is contemplated that nonstructural proteins as well as structural proteins may provide protection against viral pathogenicity, even if they do not cause the production of neutralizing antibodies.
  • multivalent vaccines against HGV may comprise one or more structural proteins, and/or one or more nonstructural proteins. These vaccines may be comprised of, for example, recombinant HGV polypeptides and/or polypeptides isolated from the virions. Additionally, it may be possible to use inactivated HGV in vaccines. Such inactivation may be be preparation of viral lysates, or by other means known in the art to cause inactivation of hepatitis-like viruses, for example, treatment with organic solvents or detergents, or treatment with formalin. Attenuated HGV strain preparation also is disclosed in the present invention.
  • vaccines which contain at least one immunogenic peptide as an active ingredient is known to one skilled in the art.
  • vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared.
  • the preparation may be emulsified, or the protein may be encapsulated in liposomes.
  • the active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient.
  • Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used.
  • the vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and or adjuvants which enhance the effectiveness of the vaccine.
  • such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D- isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L- alanyl-D-isoglutaminyl-L-alanine-2-( 1 '2'-dipalmitoyl-sn-glycero-3- hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI (MPL + TDM+ CWS) in a 2% squalene/Tween-80® emulsion.
  • thr-DMP N-acetyl-nornuramyl-L-alanyl-D- isoglutamine
  • an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an HGV antigenic sequence resulting from administration of this polypeptide in vaccines which also are comprised of the various adjuvants.
  • the vaccines usually are administered by intraveneous or intramuscular injection.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably, about 1% to about 2%.
  • Oral formulation include such normally employed excipients as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
  • the proteins used in the vaccine may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts such as acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and others known to those skilled in the art.
  • Salts formed with the free carboxyl groups also may be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides and the like, and such organic bases such as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine procaine, and others known to those skilled in the art.
  • Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject.
  • the vaccine may be given in a single or multiple dose schedule.
  • a multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reenforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months.
  • the dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment It is contemplated that the vaccine containing the immunogenic HGV antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, with immune globulins.
  • the immunogenic peptides prepared as described herein are used to produce antibodies, either polyclonal or monoclonal.
  • a selected mammal for example, a mouse, rabbit, goat, horse and the like
  • an immunogenic polypeptide bearing at least one HGV epitope Serum from the immunized animal is collected after an appropriate incubation period and treated according to known procedures. If serum containing polyclonal antibodies to an HGV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by, for example, immunoaffinity chromatography.
  • Polyclonal antibodies also can be isolated. Polyclonal antibodies may be obtained from a mammal previously infected with HGV. An example of a • method for purifying antibodies to HGV epitopes from serum of an individual infected with HGV using affinity chromatography is provided herein.
  • Monoclonal antibodies directed against HGV epitopes also can produced by one skilled in the art.
  • the general methodology for producing such antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G.R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press Inc., Boco Raton, FL (1982), as well as that taught by L. T. Mimms et al., Virology 176:604-619 (1990).
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See also, M.
  • Monoclonal and polyclonal antibodies thus developed, directed against HGV epitopes, are useful in diagnostic and prognostic applications, and also, those which are neutralizing are useful in passive immunotherapy.
  • Monoclonal antibodies especially can be used to produce anti-idiotype antibodies. These anti- idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, A. Nisonoff et al., Clin. Immunol. Immunopath. 21:397-406 (1981), and Dreesman et al., J. Infect. Dis. 151 :761 (1985).
  • idiotype antibodies are known in the art and exemplified, for example, in Grych et al., Nature 316:74 (1985); MacNamara et al., Science 226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225 (1985).
  • These anti-idiotypic antibodies also may be useful for treatment of HGV infection, as well as for elucidation of the immunogenic regions of HGV antigens.
  • oligomers of approximately 8 nucleotides or more can be prepared, either by excision or synthetically, which hybridize with the HGV genome and are useful in identification of the viral agent(s)., further characterization of the viral genome, as • well as in detection of the virus(es) in diseased individuals.
  • the natural or derived probes for HGV polynucleotides are a length which allows the detection of unique viral sequences by hybridization. While 6 to 8 nucleotides may be a workable length, sequences of 10 to 12 nucleotides are preferred, and those of about 20 nucleotides may be most preferred. These sequences preferably will derive from regions which lack heterogeneity.
  • probes can be prepared using routine, standard methods including automated oligonucleotide synthetic methods. A complement of any unique portion of the HGV genome will be satisfactory. Complete complementarity is desirable for use as probes, although it may be unnecessary as the length of the fragment is increased.
  • the biological test sample to be analyzed such as blood or serum may be treated such as to extract the nucleic acids contained therein.
  • the resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; or, the nucleic acid sample may be dot-blotted without size separation.
  • the probes then are labelled. Suitable labels are methods for attaching labels to probes are known in the art, and include but are not limited to radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent and chemiluminescent probes. Examples of many of these labels are disclosed herein.
  • the nucleic acids extracted from the sample then are treated with the labelled probe under hybridization conditions of suitable stringencies.
  • the probes can be made completely complementary to the HGV genome.
  • Hybridization can be carried out by a number of various techniques, including, for example, by Ligase Chain Reaction (LCR), Polymerase Chain Reaction (PCR). These techniques are described herein. It is contemplated that the HGV genome sequences may be present in serum of infected individuals at relatively low levels, for example, approximately 10 ⁇ -10 ⁇ sequences per ml.
  • This level may require that amplification techniques be used in hybridization assays, such as the Ligase Chain Reaction or the Polymerase Chain Reaction.
  • amplification techniques are known in the art.
  • the "Bio- Bridge” system uses terminal deoxynucleotide transferase to add unmodified 3'- poly-dT-tails to a nucleic acid probe (Enzo Biochem. Corp.). The poly dt- tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly- A.
  • EP 124221 there is described a DNA hybridization assay wherein the analyte is annealed to a single-stranded PNA probe that is complementary to an enzyme-labelled oligonucleotide, and the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide.
  • EP 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dt-tail, an amplifier strand that has a sequence that hybridizes to to the tail of the probe, such as a poly-A sequence, and which is capable of binding a plurality of labelled strands.
  • the technique first may involve amplification of the target HGV sequences in sera to approximately 10 ⁇ sequences/ml. This may be accomplished by following the methods described by Saiki et al., Nature 324:163 (1986).
  • the amplified sequence(s) then may be detected using a hybridization assay such as those known in the art.
  • the probes can be packaged in diagnostic kits which include the probe nucleic acid sequence which sequence may be labelled; alternatively, the probe may be unlabelled and the ingredients for labelling could be included with the kit.
  • the kit also may contain other suitably packaged reagents and materials needed or desirable for the particular hybridization protocol, for example, standards as well as instructions for performing the assay. Immunoassay and Diagnostic Kits Both the polypeptides which react immunologically with serum containing
  • HGV antibodies and composites thereof, and the antibodies raised against the HGV specific epitopes in these polypeptides are useful in immunoassays to detect the presence of HGV antibodies, or the presence of the virus and/or viral antigens in biological test samples.
  • the design of these immunoassays is subject to variation, and a variety of these are known in the art; a variety of these have been described herein.
  • the immunoassay may utilize one viral antigen, such as a polypeptide derived from any clone-containing HGV nucleic acid sequence, or from the composite nucleic acid sequences derived from the HGV nucleic acid sequences in these clones, or from the HGV genome from which the nucleic acid sequences in these clones is derived.
  • the immunoassay may use of combination of viral antigens derived from these sources. It may use, for example, a monoclonal antibody directed against the same viral antigen, or polyclonal antibodies directed against different viral antigens. Assays can include but are not limited to those based on competition, direct reaction or sandwich-type assays. Assays may use solid phases or may be performed by immunoprecipitation or any other methods which do not utilize solid phases. Examples of assays which utilize labels as the signal generating compound and those labels are described herein. Signals also may be amplified by using biotin and avidin, enzyme labels or biotin anti-biotin systems, such as that described in pending U.S. patent application Serial Nos.
  • kits suitable for immunodiagnosis and containing the appropriate reagents are constructed by packaging the appropriate materials, including the polypeptides of the invention containing HGV epitopes or antibodies directed against HGV epitopes in suitable containers, along with the remaining reagents and materials required for the conduct of the assay, as well as suitable assay instructions. Further Characterization of the HGV Genome. Virions. and Viral Antigens Using Probes
  • the HGV nucleic acid sequences may be used to gain further information on the sequence of the HGV genome, and for identification and isolation of the HGV agent. Thus, it is contemplated that this knowledge will aid in the characterization of HGV including the nature of the HGV genome, the structure of the viral particle, and the nature of the antigens of which it is composed. This information, in turn, can lead to additional polynucleotide probes, polypeptides derived from the HGV genome, and antibodies directed against HGV epitopes which would be useful for the diagnosis and/or treatment of HGV caused non-A, non-B, non-C, non-D and non-E hepatitis.
  • the nucleic acid sequence information is useful for the design of probes for the isolation of additional nucleic acid sequences which are derived from yet undefined regions of the HGV genome.
  • labelled probes containing a sequence of 8 or more nucleotides, and preferably 20 or more nucleotides, which are derived from regions close to the 5'-termini or 3'-termini of the family of HGV nucleic acid sequences may be used to isolate overlapping nucleic acid sequences from HGV genomic or cDNA libraries.
  • sequences which overlap the HGV nucleic acid sequences may then be used to synthesize probes for identification of other overlapping fragments which do not necessarily overlap the nucleic acid sequences in the clones.
  • HGV genome is segmented and the segments lack common sequences, it is possible to sequence the entire viral genome(s) utilizing the technique of isolation of overlapping nucleic acid sequences derived from the viral genome(s).
  • the sequence of the genome can be determined serologically by screening lambda-gtl 1 HGV genomic or cDNA libraries, sequencing HGV genomic or cDNA isolates, and using the isolated HGV nucleic acid sequences to isolate overlapping fragments, using the techniques described for the isolation and sequencing of clones. Characterization of the genomic segments alternatively could be from the viral genome(s) isolated from purified HGV particles. Methods for purifying HGV particles and for detecting them during the purification procedure are described herein. Procedures for isolating polynucleotide genomes from viral particles are well-known in the art. The isolated genomic segments then could be cloned and sequenced.
  • HGV genomic or cDNA libraries are known in the art, and vectors useful for this purpose are known in the art. These vectors include lambda-gtl 1, lambda-gtlO, and others.
  • the HGV derived nucleic acid sequence detected by the probes derived from the HGV genomic or cDNAs may be isolated from the clone by digestion of the isolated polynucleotide with the appropriate restriction enzyme(s), and sequenced.
  • sequence information derived from these overlapping HGV nucleic acid sequences is useful for determining areas of homology and heterogeneity within the viral genome(s), which could indicate the presence of different strains of the genome and. or of populations of defective particles. It is also useful for the design of hybridization probes to detect HGV or HGV antigens or HGV nucleic acids in biological samples, and during the isolation of HGV, utilizing the techniques described herein.
  • the overlapping nucleic acid sequences may be used to create expression vectors for polypeptides derived from the HGV genome(s).
  • antigen(s) containing epitopes which are contemplated to be unique to HGV, i.e., antibodies directed against these antigens are absent from individuals infected with HAV, HBV, HCV, and HEV, and with the genomic sequences in Genebank are contemplated to indicate that minimal homology exists between these nucleic acid sequences and the polynucleotide sequences of those sources.
  • antibodies directed against the antigens encoded with the HGV nucleic acid sequences may be used to identify the non-A, non-B, non-C, non-D and non-E particle isolated from infected individuals. In addition, they also are useful for the isolation of the HGV agent(s).
  • HGV particles may be isolated from the sera of infected individuals or from cell cultures by any of the methods known in the art, including, for example, techniques based on size discrimination such as sedimentation or exclusion methods, or techniques based on density such as ultracentrifugation in density gradients, or precipitation with agents such as polyethylene glycol (PEG), or chromatography on a variety of materials such as anionic or cationic exchange materials, and materials which bind due to hydrophobic interactions, as well as affinity columns.
  • PEG polyethylene glycol
  • chromatography on a variety of materials such as anionic or cationic exchange materials, and materials which bind due to hydrophobic interactions, as well as affinity columns.
  • HGV may be detected by hybridization analysis of the extracted genome, using probes derived from HGV nucleic acid sequences or by immunoassay which utilize as probes antibodies directed against HGV antigens encoded within the family of HGV nucleic acid sequences.
  • the antibodies may be polyclonal or monoclonal, and it may be desirable to purify the antibodies before their use in the immunoassay.
  • Such antibodies directed against HGV antigens which are affixed to solid phases are useful for the isolation of HGV by immunoaffinity chromatography.
  • Methods for immunoaffinity chromatography are known in the art, and include methods for affixing antibodies to solid phases so that they retain their immunoselective activity. These methods include adsorption, and covalent binding.
  • Spacer groups may be included in the bifunctional coupling agents such that the antigen binding site of the antibody remains accessible.
  • HGV HGV genomic or cDNA sequences
  • Fractions are treated under conditions which would cause the disruption of viral particles, such as by use of detergents in the presence of chelating agents, and the presence of viral nucleic acid determined by hybridization techniques.
  • the isolated particles are the agents which induce HGV infection may be obtained by infecting an individual which is preferably a chimpanzee with the isolated virus particles, followed by a determination of whether the symptoms of non-A, non-B, non-C, non-D and non- E hepatitis, as described herein, result from the infection.
  • Such viral particles obtained from the purified preparations then may be further characterized.
  • the genomic nucleic acid once purified, can be tested to determine its sensitivity to RNAse or DNAse I; based on these tests, the determination of HGV as a RNA genome or DNA genome may be made.
  • the strandedness and circularity or non-circularity can be determined by methods known in the art including its visualization by electron microscopy, its migration in density gradients and its sedimentation characteristics. From hybridization of the HGV genome, the negative or positive strandedness of the purified nucleic acid can be determined.
  • the purified nucleic acid can be cloned and sequenced by known techniques, including reverse transcriptase, if the genomic material is RNA. Utilizing the nucleic acid derived from the viral particles, it then is possible to sequence the entire genome, whether or not it is segmented.
  • Determination of polypeptides containing conserved sequences may be useful for selecting probes which bind the HGV genome, thus allowing its isolation.
  • conserved sequences in conjunction with those derived from the HGV nucleic acid sequences may be used to design primers for use in systems which amplify genomic sequences.
  • the structure of HGV also may be determined and its components isolated. The morphology and size may be determined by electron microscopy, for example.
  • the identification and localization of specific viral polypeptide antigens such as envelope (coat) antigens, or internal antigens such as nucleic acid binding proteins or core antigens, and polynucleotide polymerase(s) also may be determined by ascertaining whether the antigens are present in major or minor viral components, as well as by utilizing antibodies directed against the specific antigens encoded within isolated nucleic acid sequences as probes. This information may be useful for diagnostic and therapeutic applications. For example, it may be preferable to include an exterior antigen in a vaccine preparation, or perhaps multivalent vaccines may be comprised of a polypeptide derived from the genome encoding a structural protein as well as a polypeptide from another portion of the genome, such as a nonstructural polypeptide.
  • suitable cells or cell lines for culturing HGV may include the following: monkey kidney cells such as MK2 and VERO, porcine kidney cell lines such as PS, baby hamster kidney cell lines such as BHK, murine macrophage cell lines such as P388D1, MK1 and Mml, human macrophage cell lines such as U- 937, human peripheral blood leukocytes, human adherent monocytes, hepatocytes or hepatocytic cell lines such as HUH7 and HepG2, embryos or embryonic cell such as chick embryo fibroblasts or cell lines derived from invertebrates, preferably from insects such as drosophia cell lines or more preferably from arthropods such as mosquito cell tines or tick cell lines It also is possible that primary hepatocytes can be cultured and then infected with HGV.
  • monkey kidney cells such as MK2 and VERO
  • porcine kidney cell lines such as PS
  • baby hamster kidney cell lines such as BHK
  • the hepatocyte cultures could be derived from the livers of infected individuals (human or chimpanzee). That latter case is an example of a cell line which is infected in vivo being passaged in vitro.
  • various immortalization methods can be used to obtain cell lines derived from hepatocyte cultures. For example, primary hver cultures (before and after enrichment of the hepatocyte population) may be fused to a variety of cells to maintain stability. Also, cultures may be infected with transforming viruses, or transfected with transforming genes in order to create permanent or semipermanent cell lines.
  • cells in Hver cultures may be fused to established cell lines such as PehG2. Methods for cell fusion are well-known to the routineer, and include the use of fusion agents such as PEG, Sendai Virus and Epstein-Barr Virus, among others.
  • HGV infection of cell lines may be accomplished by techniques such as incubating the cells with viral preparations under conditions which allow viral entry into the cell. It also may be possible to obtain viral production by transfecting the cells with isolated viral polynucleotides. Methods for transfecting tissue culture cells are known in the art and include but are not limited to techniques which use electroporation and precipitation with DEAE- Dextran or calcium phosphate. Transfection with cloned HGV genomic or cDNA should result in viral replication and the in vitro propagation of the virus. In addition to cultured cells, animal model systems may be used for viral replication. HGV rephcation thus may occur in chimpanzees and also in, for example, marmosets and suckling mice. Screening for Anti- Viral Agents For HGV
  • HGV human genome virus
  • screening for anti- viral agents which inhibit HGV replication possible and particularly for those agents which preferentially allow cell growth and multiplication while inhibiting viral rephcation.
  • screening methods are known in the art.
  • the anti- viral agents are tested at a variety of concentrations, for their effect on preventing viral replication in cell culture systems which support viral rephcation, and then for an inhibition of infectivity or of viral pathogenicity, and a low level of toxicity, in an animal model system.
  • HGV antigens and HGV polynucleotides are useful for screening of anti- viral agents because they provide an alternative, and perhaps a more sensitive means, for detecting the agent's effect on viral rephcation than the cell plaque assay or ID5 0 assay.
  • the agent's effect on viral rephcation than the cell plaque assay or ID5 0 assay.
  • HGV polynucleotide probes described herein may be used to quantitate the amount of viral nucleic acid produced in a cell culture. This could be performed by hybridization or competition hybridization of the infected cell nucleic acids with a labelled HGV polynucleotide probe. Also, anti-HGV antibodies may be used to identify and quantitate HGV antigen(s) in the cell culture utilizing the immunoassays described herein. Also, since it may be desirable to quantitate HGV antigens in the infected cell culture by a competition assay, the polypeptides encoded within the HGV nucleic acid sequences described herein are useful for these assays.
  • a recombinant HGV polypeptide derived from the HGV genomic or cDNA would be labelled, and the inhibition of binding of this labelled polypeptide to an HGV polypeptide due to the antigen produced in the cell culture system would be monitored. These methods are especially useful in cases where the HGV may be able to replicate in a cell tines without causing cell death.
  • Preparation of Attenuated Strains of HGV It maybe possible to isolate attenuated strains of HGV by utilizing the tissue culture systems and/or animal models systems provided herein. These attenuated strains would be useful for vaccines, or for the isolation of viral antigens. Attenuated strains are isolatable after multiple passages in cell culture and/or an animal model.
  • an attenuated strain in an infected cell or individual is achievable by following methods known in the art and could include the use of antibodies to one or more epitopes encoded in HGV as a probe or the use of a polynucleotide containing an HGV sequence of at least about 8 nucleotides in length as a probe.
  • an attenuated strain may be constructed utilizing the genomic information of HGV provided herein, and utilizing recombinant techniques. Usually an attempt is made to delete a region of the genome encoding a polypeptide related to pathogenicity but not to viral replication. The genomic construction would allow the expression of an epitope which gives rise to neutralizing antibodies for HGV.
  • HGV strains are useful not only for vaccine purposes, but also as sources for the commercial production of viral antigens, since the processing of these viruses would require less stringent protection measures for the employees involved in viral production and/or the production of viral products.
  • procaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used.
  • E. coli is most frequently used.
  • Expression control sequences for prokaryotics include promoters, optionally containing operator portions, and ribosome binding sites.
  • Transfer vectors compatible with prokaryotic hosts are commonly derived from the plasmid pBR322 which contains operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection.
  • prokaryotic control sequences include the beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 198:1056 [1977]) the tryptophan promoter system (reported by Goeddel et al. , Nucleic Acid Res 8:4057 [1980]) and the lambda-derived PI promoter and N gene ribosome binding site (Shimatake et al., Nature 292:128 [1981]) and the hybrid Tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA 292:128 [1983]) derived from sequences of the trg and lac UV5 promoters.
  • E. coli particularly compatible with E. coli: however, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used if desired, with corresponding control sequences.
  • Eukaryotic hosts include yeast and mammalian cells in culture systems.
  • Saccharomyces cerevisiae and Saccharomyces carlsbergensis are the most commonly used yeast hosts, and are convenient fungal hosts.
  • Yeast compatible vectors carry markers which permit selection of successful transformants by conferring protrophy to auxotrophic mutants or resistance to heavy metals on wild- type strains.
  • Yeast compatible vectors may employ the 2 micron origin of replication (as described by Broach et al., Meth. Enz. 101:307 [1983]), the combination of CEN3 and ARS1 or other means for assuring rephcation, such as sequences which will result in incorporation of an appropriate fragment into the host cell genome.
  • Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes, including the promoter for 3 phosphophycerate kinase. See, for example, Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968), Holland et al., Biochemistry 17:4900 (1978) and Hitzeman I Biol. Chem. 255:2073 (1980). Terminators also may be included, such as those derived from the enolase gene as reported by Holland, J. Biol. Chem.256:1385 (1981).
  • particularly useful control systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, leader sequences from yeast alpha factor.
  • GPDH glyceraldehyde-3 phosphate dehydrogenase
  • ADH alcohol dehydrogenase
  • terminators also derived from GAPDH
  • leader sequences from yeast alpha factor if secretion is desired, leader sequences from yeast alpha factor.
  • the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines which are available from the American Type Culture Collection. These include HeLa cell, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others. Suitable promoters for mammalian cells also are known in the art and include viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV), cytomegalovirus (CMV).
  • Simian Virus 40 SV40
  • Rous sarcoma virus RSV
  • ADV adenovirus
  • BCV bovine papilloma virus
  • CMV cytomegalovirus
  • Mammalian cells also may require terminator sequences and poly A addition sequences; enhancer sequences which increase expression also may be included, and sequences which cause amplification of the gene also may be desirable. These sequences are known in the art.
  • Vectors suitable for rephcation in mammalian cells may include viral replicons, or sequences which insure integration of the appropriate sequences encoding non-A, non-B, non-C, non-E epitopes into the host genome.
  • An example of a mammalian expression system for HCV is described in U.S. Patent Application Serial No. 07/830,024, filed January 31, 1992. Transformations
  • Transformation may be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide.
  • the transformation procedures selected depends upon the host to be transformed.
  • Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride. Cohen, Proc. Natl. Acad. Sci. USA 69:2110 (1972).
  • Yeast transformation by direct uptake may be conducted using the calcium phosphate precipitation method of Graham et al., Virology 52:526 (1978), or modification thereof.
  • Vector Construction
  • Vector construction employs methods known in the art. Generally, site- specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. Usually, about 1 microgram ( ⁇ g) of plasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 ⁇ l of buffer solution by incubation at 37 °C for .1 to 2 hours. After incubation with the restriction enzyme, protein is removed by phenol/chloroform extraction and the DNA recovered by precipitation with ethanol. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis methods, according to methods known by the routineer.
  • Sticky end cleavage fragments may be blunt ended using E. coli DNA polymerase 1 (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with SI nuclease also may be used, resulting in the hydrolysis of any single stranded DNA portions. Ligations are performed using standard buffer and temperature conditions using T4 DNA ligase and ATP. Sticky end ligations require less ATP and less ligase than blunt end ligations.
  • vector fragments When vector fragments are used as part of a ligation mixture, the vector fragment often is treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5'- phosphate and thus prevent religation of the vector. Or, 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 selected by methods including antibiotic resistance, and then screened for the correct construction. Construction of Desired DNA Sequences
  • Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner, DNA 3:401 (1984). If desired, the synthetic strands may be labelled with 32 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 genomic or cDNA libraries, may be modified by known methods which include site directed mutagenesis as described by ZoUer, Nucleic Acids Res. 10:6487 (1982).
  • the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence.
  • Culture of the transformed bacteria, which contain replications of each strand of the phage are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50% have the original sequence.
  • Replicates of the plaques are hybridized to labelled synthetic probe at temperatures and conditions suitable for hybridization with the correct strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned. Hybridization With Probe
  • HGV genomic or DNA libraries may be probed using the procedure described by Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 73:3961 (1975). Briefly, the DNA to be probed is immobilized on nitrocellulose filters, denatured and prehybridized with a buffer which contains 0-50% formamide, 0.75 M NaCl, 75 mM Na citrate, 0.02% (w/v) each of bovine serum albumin (BSA), polyvinyl pyrollidone and Ficoll, 50 mM Na Phosphate (pH 6.5), 0.1% SDS and 100 ⁇ g/ml carrier denatured DNA.
  • BSA bovine serum albumin
  • PHSS polyvinyl pyrollidone
  • Ficoll 50 mM Na Phosphate
  • SDS 100 ⁇ g/ml carrier denatured DNA.
  • the percentage of formamide in the buffer, as well as the time and temperature conditions of the prehybridization and subsequent hybridization steps depends on the stringency required. Oligomeric probes which require lower stringency conditions are generally used with low percentages of formamide, lower temperatures, and longer hybridization times. Probes containing more than 30 or 40 nucleotides such as those derived from cDNA or genomic sequences generally employ higher temperatures, for example, about 40 to 42°C, and a high percentage, for example, 50% formamide. Following prehybridization, a 32 P-labelled oligonucleotide probe is added to the buffer, and the filters are incubated in this mixture under hybridization conditions. After washing, the treated filters are subjected to autoradiography to show the location of the hybridized probe. DNA in corresponding locations on the original agar plates is used as the source of the desired DNA. Verification of Construction and Sequencing
  • ligation mixtures are transformed into E. coli strain HB101 or other suitable host and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants then are prepared according to the method of Clewell et al., Proc. Natl. Acad. Sci. USA 62: 1159 (1969) usually following chloramphenicol amphfication as reported by Clewell et al., J. Bacteriol. 110:667 (1972). The DNA is isolated and analyzed usually by restriction enzyme analysis and.or sequencing. Sequencing may be by the well-known dideoxy method of Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977) as further described by Messing et al., Nucleic Acid Res. 9:309 (1981), or by the method reported by Maxam et al., Methods in Enzymology
  • Enzyme-Linked Immunosorbent assay Enzyme-Linked Immunosorbent assay
  • ELISA Enzyme-Linked Immunosorbent assay
  • This method depends upon conjugation of an enzyme label to either an antigen or antibody, and uses the bound enzyme activity (signal generated) as a quantitative label (measurable generated signal). Methods which utilize enzymes as labels are described herein, as are examples of such enzyme labels.
  • the source of the non-A, non-B,non-C, non-E agent is an individual or pooled plasma, serum or liver homogenate from a human or chimpanzee infected with the HGV virus meeting the clinical and laboratory criteria described herein.
  • a chimpanzee alternatively can be experimentally infected with blood from another individual with non-A, non-B,non-C, non-E hepatitis meeting the criteria described hereinbelow.
  • a pool can be made by combining many individual plasma, serum or liver homogenate samples containing high levels of alanine transferase activity; this activity results from hepatic injury due to HGV infection.
  • a nucleic acid library from plasma, serum or hver homogenate preferably but not necessarily high liter, is generated as follows. First viral particles are isolated from the plasma, serum or liver homogenate; then an aliquot is diluted in a buffered solution, such as one containing 50 mM Tris- HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl. Debris is removed by centrifugation, for example, for 20 minutes at 15,000 x g at 20°C.
  • Viral particles in the resulting supernatant then are pelleted by centrifugation under appropriate conditions which can be determined routinely by one skilled in the art.
  • the particles are disrupted by suspending the pellets in an aliquot of an SDS suspension, for example, one containing 1% SDS, 120 mM EDTA, 10 mM Tris- HC1, pH 7.5, which also contains 2 mg/ml proteinase K, which is followed by incubation at appropriate conditions, for example, 45°C for 90 minutes.
  • Nucleic acids are isolated by adding, for example, 0.8 ⁇ g MS2 bacteriophage RNA as carrier, and extracting the mixture four times with a 1 : 1 mixture of phenolxhloroform (phenol saturated with 0.5M Tris-HCl, pH 7.5, 0.1 % (v/v) beta-mercaptoethanol, 0.1% (w/v) hydroxyquinolone, followed by extraction two times with chloroform.
  • the aqueous phase is concentrated with, for example, 1- butanol prior to precipitation with 2.5 volumes of absolute ethanol overnight at - 20°C.
  • Nucleic acids are recovered by centrifugation in, for example, a Beckman SW41 rotor at 40,000 rpm for 90 min at 4°C, and dissolved in water that Is treated with 0.05% (v/v) diethylpyrocarbonate and autoclaved.
  • Nucleic acid obtained by the above procedure is denatured with, for example, 17.5 mM CH 3 HgOH; cDNA then is synthesized using this denatured nucleic acid as template, and is cloned into the EcoRI site of phase lambda-gtl 1, for example, by using methods described by Huynh (1985) supra, except that random primers replace oligo(dT) 12-18 during the synthesis of the first nucleic acid strand by reverse transcriptase (see Taylor et al., [1976]). The resulting double stranded nucleic acid sequences are fractionated according to size on a Sepharose CL-4B column, for example.
  • Eluted material of approximate mean size 400, 300, 200 and 100 base-pairs are pooled into genomic pools.
  • the lambda- gtl 1 cDNA library is generated from the cDNA in at least one of the pools.
  • the etiological agent is a DNA virus, methods for cloning genomic DNA may be useful and are known to those skilled in the art.
  • the lambda-gtl 1 genomic library generated thusly is screened for epitopes that can bind specifically with serum, plasma or a hver homogenate from an individual who had previously experienced non-A, non-B, non-C, non-E hepatitis (one which meets the criteria as set forth hereinbelow).
  • About lO O 7 phage are screened with sera, plasma, or liver homogenates using the methods of Huyng et al. (supra).
  • Bound human antibody can be detected with sheep anti-human Ig antisera that is radio-labeled with 125 I or other suitable reporter molecules including HRPO, alkaline phosphatase and others. Positive phages are identified and purified.
  • the phages then are tested for specificity of binding to sera from a pre-determined number of different humans previously infected with the HGV agent , using the same method.
  • the phage will encode a polypeptide that reacts with all or a majority of the sera, plasma or liver homogenates that are tested, and will not react with sera, plasma or Hver homogenates from individuals who are determined to be "negative” according to the criteria set forth herein for the HGV agent as weU as hepatitis A, B, C, and E.
  • ALT serum alanine aminotransferase
  • Hepatitis was characterized as icteric (bilirubin >2.0 mg/dL) or anicteric (bilirubin ⁇ 2.0 mg/dL).
  • Relapse of acute hepatitis was diagnosed when acute icteric or anicteric hepatitis or abrupt elevation of ALT activity (about five times the upper limit of normal) occurred within the first six months.
  • Acute NANBH was defined by the absence of serological markers for acute hepatitis A (IgM anti-HAV negative) and acute EB V infection (IgM antibody to EBV capsid antigen negative).
  • IgM anti-HAV negative acute hepatitis A
  • IgM antibody to EBV capsid antigen negative The absence of antibody titers against nuclear, mitochondrial, microsomal or smooth muscle antigens excluded the diagnosis of autoimmune chronic active liver disease. The first day when symptoms and/or dark colored urine appeared was considered the onset of illness.
  • Progression to chronic hepatitis was characterized by elevated ALT activity six months after the onset of acute hepatitis and/or the presence of histological changes compatible with chronic Hver disease. Also, cases which had normal liver enzymes at six months and subsequently developed (a) elevated values in two or more consecutive determinations, each obtained at least at one-month intervals, or (b) fluctuating values, were considered to be chronic cases. Laboratory Determinations
  • CommerciaUy available enzyme immunoassays were used for the detection of HBsAg (available from Abbott Laboratories, Abbott Park, IL), IgM anti-HBc (CORZYME®-M available from Abbott Laboratories, Abbott Park, IL) and IgM anti-HAV (HAVAB® available from Abbott Laboratories, Abbott Park, IL).
  • IgM antibody to the viral capsid antigen of Epstein-Barr Virus (EBV) was determined by indirect immunofluorescence, following the protocol and using the reagents of the commerciaUy available kit Gull IFA (available from GuU Labs, Midvale, Utah 84047).
  • HCV assay Five-hundred and twenty serum specimens were tested for antibodies to HCV by a second-generation enzyme immunoassay (available from Abbott GmbH Diagnostika, Weisbaden, Germany). This HCV assay is based on recombinant HCV non-structural antigen derived from the NS4 (so-called "clOO”) and NS3 (so-called “c33”) regions and on a structural antigen derived from the 5' region of the HCV genome (so-called "putative core”).
  • ASA Abbott's supplemental assay
  • Assay for anti-HEV was performed using a peptide or recombinant protein from a cloned sequence of HEV which was obtained from Genelabs, Redwood City, California. This assay is being developed by Abbott Laboratories.
  • HCV RT-PCR was performed as foUows on serial samples from seven patients negative for antibodies for HCV (54 total serum samples).
  • 25 ⁇ l of serum was diluted 10-fold in digestion buffer.
  • the final concentration in the diluted sample was 50.5 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 0.1 M NaCl, 0.5% SDS, 1 mg/ml Proteinase K and 20.2 ⁇ g/ml tRNA.
  • the diluted sample was incubated at 37°C for 60 minutes. Proteins were removed by one extraction with phenol, one with phenol/chloroform, and one with chloroform alone.
  • RNA was resuspended in 5 ⁇ l of DEPC treated water water containing 1 unit/ ⁇ l RNasin and ImM Dithiothreitol. The resuspended RNA was heated at 37°C for 30 minutes, 65 °C for 3 minutes and then cooled on ice.
  • Otigonucleotide primers were commercially synthesized for the RT-PCR. The primer pair produced a 293 bp sequence from the 5' non- coding region of the HCV genome.
  • the anti-sense and sense primers were as described in Wang et al., Lancet 337:48 (1991). These primers are presented as SEQ. ID NO. 1 (anti-sense primer) and SEQ. ID NO. 2 (sense prirner).
  • the first strand reaction mix contained 5 ⁇ l of sample or control RNA, 100 mM Tris (pH 8.3), 76 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM dATP, dGTP, dTTP, 0.5 mM dCTP, 4 mM each primer, 5 units of AMV-RT and 25 units of RNasin in a final volume of 25 ⁇ l.
  • Synthesis was performed at 37°C for 60 minutes. Then, 50 ⁇ l of water was added to the cDNA reaction. The dtiuted reaction was boiled for 10 minutes and cooled on ice. Amplification reactions were a final volume of 100 ⁇ l containing 32.5 mM Tris (pH 8.3), 56.5 mM KCl, 3.6 mM MgCl2, 0.34 mM dATP, dGTP, dTTP, 0.22 mM dCTP, 1 mM each primer and 5 units of Taq polymerase. AH samples were overlaid with mineral oU.
  • Hybridization of the blot was at 1- 1.5 x 10 6 cpm ml in 10% dextran sulfate and 6x SSC at 65°C for 1 to 1.5 hours.
  • the blots were twice washed at room temperature in 2x SSC, 0.1% SDS for five minutes and then twice for 15 minutes at 65°C in O.lx SSC, 0.1% SDS.
  • the blots were autoradiographed at room temperature with intensifying screens for 3 to 7 days. The results were scored under code. The results of this assay, the anti- HEV and others are summarized in Table 1.
  • Hver biopsy specimens were obtained from 51 patients giving informed consent. Of the 62 tissue specimens, 46 were obtained 5 ⁇ 3 weeks (range from two to 17 weeks) after the onset of acute hepatitis (acute-phase) and the remaining 16 were obtained from 14 patients 12.7 ⁇ 5.5 (range of six to 32) months after the onset of acute hepatitis (chronic phase). Histological lesions were interpreted in accordance with internationaUy accepted criteria and Mindly to clinical and serological information as described by L. Bianchi et al., Lancet ti:914-919 (1977). The severity of acute hepatitis was classified as ⁇ tild, moderate and severe according to the extent and the type of Hver cell necrosis.
  • Mild acute hepatitis was typified by few to moderate focal necrosis, moderate hepatitis by multiple focal and/or small confluent necrosis in zone 3 and severe hepatitis by large confluent necrosis in zone 3 and bridging necrosis with or without fibrosis. Extensive confluent and bridging necrosis suggested possible transition to cirrhosis.
  • Statistical analysis was carried out by using the Fisher's exact and chi- square tests for 2x2 and 2xK comparisons, respectively, and one way analysis of variance. Histological Findings
  • HBcAg and HBsAg were investigated in paraffin sections of serum formalin fixed Hver biopsies of seven patients negative for aU HCV and HEV markers (see Table 1).
  • the PAP method of immunoperoxidase known to those of ordinary skiU in the art was applied using the polyclonal anti- HBc and anti-HBs antibodies avaUable from DAKO in dUution of 1:200 and 1:1000, respectively. Liver biopsies expressing the viral markers served as positive controls. HBcAg and HBsAg were not detected in any of the seven examined cases. TABLE 2
  • the clinical presentation of acute CA-NANB hepatitis was fulminant in one patient (1.5%), protracted with impaired regeneration (ascites, prolonged prothrombin time > 5 seconds, albumin ⁇ 3g/dL) in seven patients (10.3%) and benign in the remaining 60 patients (88.2%).
  • the patient who survived fulminant hepatitis had three consecutive episodes of acute icteric hepatitis at six, 10 and 12 months later; this patient died of chronic Hver fahure (postnecrotic inactive cirrhosis) 13 months after the first episode of acute hepatitis.
  • Peak g-globulin (x ⁇ SD) g/dL 1.83 ⁇ .74 1.49 ⁇ 0.34 1.59+0.34 0.149
  • a denotes possible transition to chronic hepatitis and/or cirrhosis
  • chronic hepatitis developed significantly more frequently in severe than in non-severe cases (5/8 or 62.5% versus 10/49 or 20.4%, P 0.024).
  • HCV also was found to be the major etiological agent of acute parenterally- transmitted NANB hepatitis in Greece.N. Tassopoulos et al., Gastroenterology 102(3): in press (1992). Moreover, none of the histologicaUy and/or clinicaUy severe CA-NANB hepatitis was anti-HCV positive.
  • HCV is involved in a minority of CA-NANB hepatitis in Greece, clinicaUy and histologicaUy severe cases of acute hepatitis with a decreased rate of chronicity were observed in anti-HCV negative patients, and a non-A, non-B, non-C, non-D and non-E agent is responsible for the majority of CA-NANB hepatitis cases in Greece.
  • Inocula for transmission studies are obtained from the Greek patients previously described herein and are selected from the group including undUuted or concentrated serum or plasma obtained from individuals or pooled from several individuals, liver tissue or extracts of Hver tissue either from individuals or specimen extracts pooled from several individuals, and leukocyte preparations from individual patients or pooled from several patients. Animals are inoculated intraveneously or by direct injection into the liver. Appropriate.
  • control animals are inoculated with samples from healthy humans or from humans with Hver disease that is known to be non-viral in origin. Prior to inoculation, samples of serum or plasma, leukocytes and liver tissue are obtained for testing to establish basehne results for each animal. At least one baseline time point is included. FoUowing inoculation, serum or plasma, leukocyte, and liver tissue samples are obtained at weekly intervals for four months. The foUowing parameters are studied to determine whether development of hepatic disease occurs or has occurred: enzyme activities including alanine aminotransferse activity, gamma-glutamyltransferase activity, aspartate aminotransf erase, serologic tests for hepatitis B markers including HBsAg,
  • HBeAg anti-HBc, anti-HBs
  • serologic tests for hepatitis C including anti-HCV c 100-3, anti-HCV 33c, anti-HCV CORE, anti-HCV env, anti-HCV NS5, serologic tests for hepatitis A including anti-HAV and serologic tests for hepatitis E including anti-HEV.
  • Liver tissue is examined to evaluate histologic, Hght and electron microscopic, ultrastructural and immunohistologic alterations in the liver of aU animals in the study. For example, approximately 30 to 35% of the Greek patients described herein demonstrate periportal necrosis of Hver tissue during the acute phase of disease.
  • Periportal necrosis of liver tissue during the acute phase of hepatitis C infection has not been observed and thus, this marker serves to distinguish disease cause by the putative new agent HGV from disease caused by the hepatitis C virus.
  • Immunohistologic studies of the liver include staining for known antigens of the hepatitis B, C and E viruses using antibody preparations directed against viral epitopes. Nucleic acids from the serum, Hver and leukocyte preparations are examined for HB V DNA, HCV RNA by direct hybridization, polymerase chain reaction, and in situ hybridization in the case of Hver tissue and leukocytes. Elevations in the enzyme activities supra result from liver damage and are indicative of viral infection of the Hver. Markers for hepatitis B, C and E are foUowed as exclusionary criteria for Hver disease caused by the putative new agent, HGV. The development of acute or chronic liver disease in the test animal is followed.
  • Anti-HCV refers to c-100, 33c and core
  • NAME POREMBSKI, PRISCILLA E.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne des séquences d'acide nucléique du virus de l'hépatite G (VHG) utiles dans différentes applications thérapeutiques et diagnostiques, des kits d'utilisation des séquences d'acide nucléique du VHG, des particules immunogènes du VHG, et un procédé de production d'anticorps, polyclonaux ou monoclonaux, à partir des séquences d'acide nucléique du VHG, contre le VHG.
PCT/US1993/000928 1992-02-03 1993-02-03 Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation WO1994018217A1 (fr)

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AU36061/93A AU3606193A (en) 1992-02-03 1993-02-03 Non-a, non-b, non-c, non-d, non-e hepatitis reagents and methods for their use
PCT/US1993/000928 WO1994018217A1 (fr) 1993-02-03 1993-02-03 Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation

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WO1995032291A2 (fr) * 1994-05-20 1995-11-30 Genelabs Technologies, Inc. Virus de l'hepatite g et son clonage moleculaire
EP0736601A2 (fr) * 1995-04-06 1996-10-09 Abbott Laboratories Réactifs pour la détection associés avec l'hépatite non-A, non-B, non-C, non-D et non-E ainsi que procédés d'utilisation de ces derniers
WO1997007224A1 (fr) * 1995-08-14 1997-02-27 Abbott Laboratories Reactifs et procedes permettant de moduler la traduction de proteines de l'hepatite gbv
WO1997019195A1 (fr) * 1995-11-21 1997-05-29 Boehringer Mannheim Gmbh Amplification d'acides nucleiques et identification d'un nouveau virus d'hepatite non a- non b- non c- non d- non e
WO1997027333A1 (fr) * 1996-01-24 1997-07-31 Wabco B.V. Procede de detection de sequences de nucleotides de virus associes a l'hepatite non a-non e peptides et compositions
US5709997A (en) * 1995-08-14 1998-01-20 Abbott Laboratories Nucleic acid detection of hepatitis GB virus
EP0832901A1 (fr) * 1996-09-18 1998-04-01 Roche Diagnostics GmbH Anticorps contre le virus hépatite G, leur utilisation diagnostique pour la détection de HGV, et comme agent thérapeutique
US5766840A (en) * 1994-05-20 1998-06-16 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US5807670A (en) * 1995-08-14 1998-09-15 Abbott Laboratories Detection of hepatitis GB virus genotypes
US5843450A (en) * 1994-02-14 1998-12-01 Abbott Laboratories Hepatitis GB Virus synthetic peptides and uses thereof
US5859230A (en) * 1992-07-30 1999-01-12 Genelabs Technologies, Inc. Non-A/non-B/non-C/non-D/non-E hepatitis agents and molecular cloning thereof
US5874563A (en) * 1994-05-20 1999-02-23 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US6051374A (en) * 1994-02-14 2000-04-18 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6156495A (en) * 1994-02-14 2000-12-05 Abbott Laboratories Hepatitis GB virus recombinant proteins and uses thereof
US6451578B1 (en) 1994-02-14 2002-09-17 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6558898B1 (en) 1994-02-14 2003-05-06 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6586568B1 (en) 1994-02-14 2003-07-01 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6720166B2 (en) 1994-02-14 2004-04-13 Abbott Laboratories Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use
US20050118181A1 (en) * 2003-06-05 2005-06-02 Stapleton Jack T. GB virus C (hepatitis G virus) for the treatment of HIV
CN112592961A (zh) * 2021-01-07 2021-04-02 山东科硕生物技术有限公司 一种核酸样本保存液及其制备方法和应用

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US5077193A (en) * 1988-12-20 1991-12-31 Immuno Japan Inc. Non-a, non-b hepatitis virus genome rna, cdna and virus antigen protein

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US5032511A (en) * 1987-03-31 1991-07-16 Mitsubishi Kasei Corporation DNA fragments coding for antigens specific to non-A non-B hepatitis, expression vectors containing said DNA fragments, transformants and process for producing said antigens
US5077193A (en) * 1988-12-20 1991-12-31 Immuno Japan Inc. Non-a, non-b hepatitis virus genome rna, cdna and virus antigen protein

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Publication number Priority date Publication date Assignee Title
US5859230A (en) * 1992-07-30 1999-01-12 Genelabs Technologies, Inc. Non-A/non-B/non-C/non-D/non-E hepatitis agents and molecular cloning thereof
US5843450A (en) * 1994-02-14 1998-12-01 Abbott Laboratories Hepatitis GB Virus synthetic peptides and uses thereof
US6720166B2 (en) 1994-02-14 2004-04-13 Abbott Laboratories Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use
US6586568B1 (en) 1994-02-14 2003-07-01 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6558898B1 (en) 1994-02-14 2003-05-06 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6451578B1 (en) 1994-02-14 2002-09-17 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6156495A (en) * 1994-02-14 2000-12-05 Abbott Laboratories Hepatitis GB virus recombinant proteins and uses thereof
US6051374A (en) * 1994-02-14 2000-04-18 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US5981172A (en) * 1994-02-14 1999-11-09 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
US5856134A (en) * 1994-05-20 1999-01-05 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US5874563A (en) * 1994-05-20 1999-02-23 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US5824507A (en) * 1994-05-20 1998-10-20 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US5766840A (en) * 1994-05-20 1998-06-16 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
US5849532A (en) * 1994-05-20 1998-12-15 Genelabs Technologies, Inc. Hepatitis G virus and molecular cloning thereof
WO1995032291A2 (fr) * 1994-05-20 1995-11-30 Genelabs Technologies, Inc. Virus de l'hepatite g et son clonage moleculaire
WO1995032291A3 (fr) * 1994-05-20 1996-03-07 Genelabs Tech Inc Virus de l'hepatite g et son clonage moleculaire
CN1125877C (zh) * 1994-05-20 2003-10-29 基因实验室技术有限公司 庚型肝炎病毒及其分子克隆
EP0736601A3 (fr) * 1995-04-06 1997-10-22 Abbott Lab Réactifs pour la détection associés avec l'hépatite non-A, non-B, non-C, non-D et non-E ainsi que procédés d'utilisation de ces derniers
EP0736601A2 (fr) * 1995-04-06 1996-10-09 Abbott Laboratories Réactifs pour la détection associés avec l'hépatite non-A, non-B, non-C, non-D et non-E ainsi que procédés d'utilisation de ces derniers
US5955318A (en) * 1995-08-14 1999-09-21 Abbott Laboratories Reagents and methods useful for controlling the translation of hepatitis GBV proteins
US5709997A (en) * 1995-08-14 1998-01-20 Abbott Laboratories Nucleic acid detection of hepatitis GB virus
US5807670A (en) * 1995-08-14 1998-09-15 Abbott Laboratories Detection of hepatitis GB virus genotypes
WO1997007224A1 (fr) * 1995-08-14 1997-02-27 Abbott Laboratories Reactifs et procedes permettant de moduler la traduction de proteines de l'hepatite gbv
WO1997019195A1 (fr) * 1995-11-21 1997-05-29 Boehringer Mannheim Gmbh Amplification d'acides nucleiques et identification d'un nouveau virus d'hepatite non a- non b- non c- non d- non e
WO1997027333A1 (fr) * 1996-01-24 1997-07-31 Wabco B.V. Procede de detection de sequences de nucleotides de virus associes a l'hepatite non a-non e peptides et compositions
EP0832901A1 (fr) * 1996-09-18 1998-04-01 Roche Diagnostics GmbH Anticorps contre le virus hépatite G, leur utilisation diagnostique pour la détection de HGV, et comme agent thérapeutique
US20050118181A1 (en) * 2003-06-05 2005-06-02 Stapleton Jack T. GB virus C (hepatitis G virus) for the treatment of HIV
US8858946B2 (en) 2003-06-05 2014-10-14 The University Of Iowa Research Foundation Method for inhibiting HIV-1 replication utilizing anti-GBV-C E2 antibodies
US10034936B2 (en) 2003-06-05 2018-07-31 The University Of Iowa Research Foundation Method of inducing a cross-reactive HIV-1 immune response by administering a composition comprising the GBV-C E2 protein
CN112592961A (zh) * 2021-01-07 2021-04-02 山东科硕生物技术有限公司 一种核酸样本保存液及其制备方法和应用

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