WO1989012462A1 - Agent viral d'hepatite non-a/non-b transmis par voie enterique - Google Patents

Agent viral d'hepatite non-a/non-b transmis par voie enterique Download PDF

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WO1989012462A1
WO1989012462A1 PCT/US1989/002648 US8902648W WO8912462A1 WO 1989012462 A1 WO1989012462 A1 WO 1989012462A1 US 8902648 W US8902648 W US 8902648W WO 8912462 A1 WO8912462 A1 WO 8912462A1
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dna
derived
sequence
viral
nanb
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Gregory R. Reyes
Daniel W. Bradley
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Genelabs Incorporated
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Priority to KR1019900700347A priority Critical patent/KR0180530B1/ko
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • 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 to recombinant proteins, genes, and gene probes and more specifically to such proteins and probes derived from an enterically transmitted nonA/nonB hepatitis viral agent, and to diagnostic methods and vaccine applications which employ the proteins and probes.
  • ET-NANB Enterically transmitted non-A/non-B hepatitis viral agent is the reported cause of hepatitis in several epidemics and sporadic cases in Asia, Africa, and the Indian subcontinent. Infection is usually by water contaminated with feces, although the virus may also spread by close physical contact. The virus does not seem to cause chronic infection.
  • the viral etiology in ET-NANB has been demonstrated by infection of volunteers with pooled fecal isolates; Immune electron microscopy (IEM) studies have shown virus particles with 27-34 nm diameters in stools from infected individuals.
  • the virus particles reacted with antibodies in serum from infected individuals from geographically distinct regions, suggesting that a single viral agent or class is responsible for the majority of ET-NANB hepatitis seen worldwide. No antibody reaction was seen in serum from individuals infected with blood-transmitted NANB virus, indicating a different specificity between the two NANB types.
  • ET-NANB is characteristically an acute infection, often associated with fever and arthralgia, and with portal inflammation and associated bile stasis in liver biopsy specimens (Arankalle) . Symptoms are usually resolved within six weeks. Blood-transmitted NANB, by contrast, produces a chronic infection in about 50% of the cases. Fever and arthralgia are rarely seen, and inflammation has a predominantly parenchymal distribution (Khuroo, 1980).
  • the two viral agents can also be distinguished on the basis of primate host susceptibility. ET-NANB, but not the blood-transmitted agent, can be transmitted to cynomolgus monkeys.
  • the blood-transmitted agent is more readily transmitted to chimpanzees than is ET-NANB (Bradley, 1987).
  • ET-NANB Brain-NANB
  • One goal of this effort, requiring virus-specific genomic sequences, is to identify and characterize the nature of the virus and its protein products.
  • Another goal is to produce recombinant viral proteins which can be used in antibody-based diagnostic procedures and for a vaccine.
  • viral sequences associated with ET-NANB hepatitis have not been successfully identified or cloned heretofore, nor have any virus-specific proteins been identified or produced.
  • compositions as well as methods of preparation and use of the compositions are provided, where the compositions comprise viral proteins and fragments thereof derived from the viral agent for ET- NANB.
  • Methods for preparation of ET-NANB viral proteins include isolating ET-NANB genomic sequences which are then cloned and expressed in a host cell. The resultant recombinant viral proteins find use as diagnostic agents and as vaccines. The genomic sequences and fragments thereof find use in preparing ET-NANB viral proteins and as probes for virus detection.
  • Figure 1 shows vector constructions and manipulations used in obtaining and sequencing cloned ET-NANB fragment
  • Figures 2A-2B are representations of Southern blots in which a radiolabeled ET-NANB probe was hybridized with amplified cDNA fragments prepared from RNA isolated from infected (I) and non-infected (N) bile sources (2A), and from infected (I) and non- infected (N) stool-sample sources (2B).
  • compositions comprising generic sequences and fragments thereof derived from the viral agent for ET-NANB are provided, together with recombinant viral proteins produced using the genomic sequences and methods of using these compositions.
  • the genome of the ET-NANB viral agent is identified as containing a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KF1 (ET1.1) carried in E. coli strain BB4 and having ATCC deposit no. 67717.
  • Initial studies sequenced the two terminal regions of the insert and an intermediate region. The 5'-end region of this insert contains the sequence:
  • An intermediate region has the sequence:
  • CAG GCC CCG AAG GAG TCT CTG CGA GGG TTT TGG AAG AAA CAC
  • the 3'-end region contains the sequence:
  • the complimentary strand referred to here as the "reverse sequence,” is set forth below in the same manner as the forward sequence set forth above.
  • Non-A Non-B ET Burmese strain
  • Non-A Non-B ET Mexican Strain
  • hepatitis viral agent means a virus, virus type, or virus class which (1) causes water-borne, infectious hepatitis, (ii) is transmissible in cynomolgus monkeys, (i ⁇ ) is serologically distinct from hepatitis A virus (HAV) and hepatitis B virus (HAB), and (iv) includes a genomic region which is homologous to the 1.33 kb cDNA insert in plasmid pTZ-KFl(ETl.l) carried in E. coli strain BB4 identified by ATCC deposit number 67717.
  • Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions in which hybridized strands contain at most about 25-30% basepair mismatches.
  • two single-strand nucleic acid species will be homologous if they hybridize under the conditions described in Maniatis et al. , op. cit., pp. 320-323, but using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature - twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C - once, 30 minutes; then 2 x SCC, room temperature - twice, 10 minutes each.
  • homologous nucleic acid strands contain 15- 25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material), as is well known in the art.
  • a DNA fragment is "derived from” an ET-NANB viral agent if it has the same or substantially the same basepair sequence as a region of the viral agent genome. 4.
  • a protein is "derived from” an ET-NANB viral agent if it is encoded by an open reading frame of a DNA or RNA fragment derived from an ET-NANB viral agent.
  • a virus-specific DNA clone can be produced by (a) isolating RNA from the bile of a cynomolgus monkey having a known ET-NANB infection, (b) cloning the cDNA fragments to form a fragment library, and (c) screening the library by differential hybridization to radiolabeled cDNAs from infected and non-infected bile sources.
  • ET-NANB infection in cynomolgus monkeys is initiated by inoculating the animals intravenously with a 10% w/v suspension from human case stools positive for 27-34 nm ET-NANB particles (mean diameter 32 nm) .
  • An infected animal is monitored for elevated levels of alanine aminotransferase, indicating hepatitis infection.
  • ET-NANB infection is confirmed by immunospecific binding of seropositive antibodies to virus-like particles (VLPs), according to published methods (Gravelle).
  • a stool (or bile) specimen taken from the infected animal 3-4 weeks after infection is diluted 1:10 with phosphate-buffered saline, and the 10% suspension is clarified by low- speed centrifugation and filtration successively through 1.2 and 0.45 micron filters.
  • the material may be further purified by pelleting through a 30% sucrose cushion (Bradley).
  • the resulting preparation of VLPs is mixed with diluted serum from human patients with known ET-NANB infection. After incubation overnight, the mixture is centrifuged overnight to pellet immune aggregates, and these are stained and examined by electron microscopy for antibody binding to the VLPs.
  • ET-NANB infection can also be confirmed by seroconversion to VLP-positive serum.
  • the serum of the infected animal is mixed as above with 27-34 nm VLPs isolated form the stool specimens of infected human cases and examined by immune electron microscopy for antibody binding to the VLPs.
  • Bile can be collected from ET-NANB positive animals by either cannulating the bile duct and collecting the bile fluid or by draining the bile duct during necropsy.
  • Total RNA is extracted from the bile by hot phenol extraction, as outlined in Example 1A.
  • the RNA fragments are used to synthesize corresponding duplex cDNA fragments by random priming, also as referenced in Example 1A.
  • the cDNA fragments may be fractionated by gel electrophoresis or density gradient centrifugation to obtain a desired size class of fragments, e.g., 500-4,000 basepai fragments.
  • alternative sources of viral material such as VLPs obtained from stool samples (as described in Example 4)
  • the bile source is preferred.
  • it has been found that bile from ET-NANB-infected monkeys shows a greater number of intact viral particles than material obtained from stool samples, as evidenced by immune electron microscopy. Bile obtained from an ET-NANB infected human or cynomolgus monkey, for use as a source of ET- NANB viral protein or genomic material, or intact virus, forms part of the present invention.
  • the cDNA fragments from above are cloned into a suitable cloning vector to form a cDNA library. This may be done by equipping blunt-ended fragments with a suitable end linker, such as an EcoRI sequence, and inserting the fragments into a suitable insertion site of a cloning vector, such as at a unique EcoRI site. After initial cloning, the library may be recloned, if desired, to increase the percentage of vectors containing a fragment insert.
  • the library construction described in Example IB is illustrative. Here cDNA fragments were blunt-ended, equipped with EcoRI ends, and inserted into the EcoRI site of the lambda phage vector gtlO.
  • the library phage which showed less than 5% fragment inserts, was isolated, and the fragment inserts recloned into the lambda gtlO vector, yielding more than 95% insert-containing phage.
  • the cDNA library is screened for sequences specific for ET-NANB by differential hybridization to cDNA probes derived from infected and non-infected sources. cDNA fragments from infected and non-infected source bile or stool viral isolates can be prepared as above. Radiolabeling the fragments is by random labeling, nick translation, or end labeling, according to conventional methods (Maniatis, p. 109).
  • cDNA library from above is screened by transfer to duplicate nitrocellulose filters, and hybridization with both infected-source and non-infected-source (control) radiolabeled probes, as detailed in Example 2.
  • clones can be selected if they hybridize under the conditions described in Maniatis et al. , op. cit. , pp. 320-323, but using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature - twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C - once, 30 minutes; then 2 x SCC, room temperature - twice, 10 minutes each.
  • Plaques which show selective hybridization to the infected-source probes are preferably replated at low plating density and rescreened as above, to isolate single clones which are specific for ET-NANB sequences. As indicated in Example 2, sixteen clones which hybridized specifically with infected-source probes were indentified by these procedures.
  • the basepair sequence of cloned regions of the ET-NANB fragments from Part B are determined by standard sequencing methods.
  • the fragment insert from the selected cloning vector is excised, isolated by gel electrophoresis, and inserted into a cloning vector whose basepair sequence on either side of the insertion site is known.
  • the particular vector employed in Example 3 is a pTZ-KFl vector shown at the left in Figure 1.
  • the ET-NANB fragment from the gtlO- 1.1 phage was inserted at the unique EcoRI site of the pTZ-KFl plasmid. Recombinants carrying the desired insert were identified by hybridization with the isolated 1.33 kilobase fragment, as described in Example 3.
  • pTZ-KFl E. coli strain BB4 infected with the pTZ-KFl(ETl.l) plasmid has been deposited with the American Type Culture Collection, Rockville, MD, and is identified by ATCC deposit nubmer 67717.
  • the pTZ-KFl(ETl.l) plasmid is illustrated at the bottom in Figure 1.
  • the fragment insert has 5' and 3 1 end regions denoted at A and C, respectively, and an intermediate region, denoted at B. The sequences in these regions were determined by standard dideoxy sequencing and are set forth above.
  • the three short sequences (A, B, and C) are from the same insert strand.
  • the B-region sequence was actually determined from the opposite strand, so that the B-region sequence shown above represent the complement of the sequence in the sequenced strand.
  • the base numbers of the partial sequences are approximate.
  • the invention includes ET-NANB-specific fragments or probes which hybridize with ET-NANB genomic sequences or cDNA fragments derived therefrom.
  • the fragments may include full-length cDNA fragments such as described in Section II, or may be derived from shorter sequence regions within cloned cDNA fragments. Shorter fragments can be prepared by enzymatic digestion of full-length fragments under conditions which yield desired-sized fragments, as will be described in Section IV.
  • the fragments can be produced by oligonucleotide synthetic methods, using sequences derived from the cDNA fragments. Methods or services for producing selected-sequence oligonucleotide fragments are available.
  • the fragment can be shown to hybridize selectively with cDNA from infected sources.
  • the fragment was excised from the pTZ-KFl(ETl.l) plasmid, purified, and radiolabeled by random labeling. The radiolabeled fragment was hybridized with fractionated cDNAs from infected and non-infected sources to confirm that the probe reacts only with infected-source cDNAs.
  • Example 4 This method is illustrated in Example 4, where the above radiolabeled 1.33 kb fragment from pTZ-KFl(ETl.l) plasmid was examined for binding to cDNAs prepared from infected and non-infected sources.
  • the infected sources are (1) bile from a cynomolgus monkey infected with a strain of virus derived from stool samples from human patients from Burma with known ET-NANB infections and (2) a viral agent derived from the stool sample of a human ET-NANB patient from Mexico.
  • the cDNAs in each fragment mixture were first amplified by a linker/primer amplification method described in Example 4.
  • Fragment separation was on agarose gel, followed by Southern blotting and then hybridization to bind the radiolabeled 1.33 kb fragment to the fractionated cDNAs.
  • the lane containing cDNAs from the infected sources showed a smeared band of bound probe, as expected (cDNAs amplified by the linker/primer amplification method would be expected to have a broad range of sizes). No probe binding to the amplified cDNAs from the non-infected sources was observed. The results indicate that the 1.33 kb probe is specific for cDNA fragments associated with ET-NANB infection.
  • ET-NANB Africa sequence is derived from a common ET-NANB virus or virus class responsible for ET-NANB hepatitis infection worldwide.
  • probe binding to fractionated genomic fragments prepared from human or cynomolgus monkey genomic DNA was examined. No probe binding was observed to either genomic fraction, demonstrating that the ET-NANB fragment is not an endogenous human or cynomologus fragment.
  • ET-NANB-specific fragments One important use of the ET-NANB-specific fragments is for identifying ET-NANB-derived cDNAs which contain additional sequence information. The newly identified cDNAs, in turn, yield new fragment probes, allowing further iterations until the entire viral genome is identified and sequenced. Procedures for identifying additional ET-NANB library clones and generating new probes therefrom generally follow the cloning and selection procedures described in Section II.
  • the fragments are also useful as primers for a poly erase chain reaction method of detecting ET-NANB viral genomic material in a patient sample. This diagnostic method will be described in Section. V below.
  • ET-NANB proteins can be prepared by expressing open reading-frame coding regions in ET-NANB fragments.
  • the ET-NANB fragments used for protein expression are derived from cloned cDNAs which have been treated to produce desired-size fragments, and preferably random fragments with sizes predominantly between about 100 to about 300 base pairs.
  • Example 5 describes the preparation of such fragments by DNAs digestion. Because it is desired to obtain peptide antigens of between about 30 to about 100 amino acids, the digest fragments are preferably size fractionated, for example by gel electrophoresis, to select those in the approximately 100-300 basepair size range.
  • the ET-NANB fragments are inserted into a suitable expression vector.
  • One exemplary expression vector is lambda gtll, which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the beta-galactosidase gene.
  • the inserted sequence will be expressed as a beta-galactosidase fusion protein which contains the N-terminal portion of the beta-galactosidase gene, the heterologous peptide, and optionally the C-terminal region of the beta-galactosidase peptide (the C- terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon).
  • This vector also produces a temperature-sensitive repressor (cl857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C.
  • Advantages of this vector include: (1) highly efficient recombinant generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) high levels of recombinant fusion protein production.
  • phage containing a heterologous insert produces an inactive beta- galactosidase enzyme, phage with inserts can be readily identified by a beta-galactosidase colored-substrate reaction.
  • the viral digest fragments may be modified, if needed, to contain selected restriction-site linkers, such as
  • Example 1 illustrates methods for cloning the digest fragments into lambda gtll, which includes the steps of blunt-ending the fragments, ligating with EcoRI linkers, and introducing the fragments into EcoRI-cut lambda gtll.
  • the resulting viral genomic library may be checked to confirm that a relatively large
  • the viral genomic library formed above is screened for production of peptide antigen (expressed as a fusion protein) which is immunoreactive with antiserum from ET-NANB seropositive individuals.
  • host cells infected with phage library vectors are plated, as above, and the plate is blotted with a nitrocellulose filter to transfer recombinant protein antigens produced by the cells onto the filter.
  • the filter is then reacted with the ET-NANB antiserum, washed to remove unbound antibody, and reacted with reporter-labeled, anti-human antibody, which becomes bound to the filter, in sandwich fashion, through the anti-ET-NANB antibody.
  • phage plaques which are identified by virtue of their production of recombinant antigen of interest are re-examined at a relatively low density for production of antibody-reactive fusion protein.
  • Several recombinant phage clones which produced immunoreactive recombinant antigen were identified in the procedure.
  • the selected expression vectors may be used for scale-up production, for purposes of recombinant protein purification. Scale-up production is carried out using one of a variety of reported methods for (a) lysogenizing a suitable host, such as E.
  • a high-producer E. coli host BNN103
  • BNN103 high-producer E. coli host
  • One of the plates is grown at 32°C, at which viral lysogeny can occur, and the other at 42°C, at which the infecting phage is in a lytic stage and therefore prevents cell growth. Cells which grow at the lower but not the higher temperature are therefore assumed to be successfully lysogenized.
  • the lysogenized host cells are then grown under liquid culture conditions which favor high production of the fused protein containing the viral insert, and lysed by rapid freezing to release the desired fusion protein.
  • the recombinant peptide can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • a fused protein such as the beta-galactosidase fused protein prepared as above
  • the protein isolation techniques which are used can be adapted from those used in isolation of the native protein.
  • the protein can be isolated readily by simple affinity chromatography, by passing the cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody.
  • the ET-NANB protein of the invention may also be derived directly from the ET-NANB viral agent.
  • VLPs isolated from a stool sample from an infected individual, as above, are one suitable source of viral protein material.
  • the VLPs isolated from the stool sample may be further purified by affinity chromatography prior to protein isolation (see below) .
  • the viral agent may also be raised in cell culture, which provides a convenient and potentially concentrated source of viral protein.
  • Co-owned U.S. Patent Application Serial No. 846,757, filed April 1, 1986 describes an immortalized trioma liver cell which supports NANB infection in cell culture.
  • the trioma cell line is prepared by fusing human liver cells with a mouse/human fusion partner selected for human chromosome stability.
  • Cells containing the desired NANB viral agent can be identified by immuno- fluorescence methods, employing anti-ET-NANB human antibodies.
  • the viral agent is disrupted, prior to protein isolation, by conventional methods, which can include sonication, high- or low-salt conditions, or use of detergents.
  • ET-NANB viral protein can be carried out by affinity chromatography, using a purified anti-ET-NANB antibody attached according to standard methods to a suitable solid support.
  • the antibody itself may be purified by affinity chromatography, where an immunoreactive recombinant ETNANB protein, such as described above, is attached to a solid support, for isolation of anti-ET-NANB antibodies from an immune serum source. The bound antibody is released from the support by standard methods.
  • the anti-ET-NANB antibody may be a monoclonal antibody (Mab) prepared by immunizing a mouse or other animal with recombinant ET-NANB protein, isolating lymphocytes from the animal and immortalizing the cells with a suitable fusion partner, and selecting successful fusion products which react with the recombinant protein immunogen. These in turn may be used in affinity purification procedures, described above, to obtain native ET-NANB antigen.
  • Mob monoclonal antibody
  • the particles and antigens of the invention, as well as the genetic material, can be used in diagnostic assays.
  • Methods for detecting the presence of ET-NANB hepatitis comprise analyzing a biological sample such as a blood sample, stool sample or liver biopsy specimen for the presence of an analyte associated with ET-NANB hepatitis virus.
  • the analyte can be a nucleotide sequence which hybridizes with a probe comprising a sequence of at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences).
  • the analyte can be RNA or cDNA.
  • the analyte is typically a virus particle suspected of being ET-NANB or a particle for which this classification is being ruled out.
  • the virus particle can be further characterized as having an RNA viral genome comprising a sequence at least about 80% homologous to a sequence of at least 12 consecutive nucleotides of the "forward" and "reverse” sequences given above, usually at least about 90% homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially homologous to the full-length sequences.
  • the probe may contain a detectable label.
  • the analyte can also comprise an antibody which recognizes an antigen, such as a cell surface antigen, on a ET-NANB virus particle.
  • the analyte can also be a ET-NANB viral antigen.
  • the analyte is an antibody or an antigen, either a labelled antigen or antibody, respectively, can be used to bind to the analyte to form an immunological complex, which can then be detected by means of the label.
  • Immunoassays can be conducted either to determine the presence of antibodies in the host that have arisen from infection by ET-NANB hepatitis virus or by assays that directly determine the presence of virus particles or antigens. Such techniques are well known and need not be described here in detail. Exam ⁇ ples include both heterogeneous and homogeneous immuno- assay techniques. Both techniques are based on the formation of an immunological complex between the virus particle or its antigen and a corresponding specific antibody. Heterogeneous assays for viral antigens typically use a specific monoclonal or polyclonal antibody bound to a solid surface.
  • Sandwich assays are becoming increasingly popular. Homogeneous assays, which are carried out in solution without the presence of a solid phase, can also be used, for example by determining the difference in enzyme activity brought on by binding of free antibody to an enzyme-antigen conjugate. A number of suitable assays are disclosed in U.S. Patent Nos. 3,817,837, 4,006,360, 3,996,345. When assaying for the presence of antibodies induced by ET-NANB viruses, the viruses and antigens of the invention can be used as specific binding agents to detect either IgG or IgM antibodies.
  • IgM anti ⁇ bodies are typically the first antibodies that appear during the course of an infection, when IgG synthesis may not yet have been initiated, specifically distin ⁇ guishing between IgM and IgG antibodies present in the blood stream of a host will enable a physician or other investigator to determine whether the infection is recent or chronic.
  • test serum is reacted with a solid phase reagent having surface-bound ET-NANB protein antigen.
  • the reagent After binding anti-ET-NANB antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-ET-NANB antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.
  • the solid surface reagent in the above assay prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activate carboxyl, hydroxyl, or aldehyde group.
  • homogeneous assay In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium.
  • Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • spin-labeled reporters where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks)
  • fluorescent reporters where binding is detected by a change in fluorescence efficiency
  • enzyme reporters where antibody binding effects enzyme/substrate interactions
  • liposome-bound reporters where binding leads to lipo
  • the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
  • the examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent phase), and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
  • kits for carrying out the assay method just described.
  • the kit generally includes a support with surface-bound recombinant protein antigen which is (a) immunoreactive with antibodies present in individuals infected with enterically transmitted nonA/nonB viral agent and (b) derived from a viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETl.l) carried in E. Coli strain BB4, and having ATCC deposit no. 67717.
  • a reporter-labeled anti-human antibody in the kit is used for detecting surface-bound anti-ET-NANB antibody.
  • the genetic material of the invention can it ⁇ self be used in numerous assays as probes for genetic material present in naturally occurring infections.
  • One method for amplification of target nucleic acids, for later analysis by hybridization assays, is known as the polymerase chain reaction or PCR technique.
  • the PCR technique can be applied to detecting virus particles of the invention in suspected pathological samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth above.
  • the primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt).
  • This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer.
  • the degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2 n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence.
  • the PCR method is de ⁇ scribed in a number of publications, including Saiki e_t al..
  • the invention includes a specific diagnostic method for determination of ET-NANB viral agent, based on selective amplification of ET-NANB fragments.
  • This method employs a pair of single-strand primers derived from non-homologous regions of opposite strands of a DNA duplex fragment, which in turn is derived from an enterically transmitted viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ-
  • KFl(ETl.l) carried in E. coli strain BB4, and having ATCC deposit no. 67717.
  • These "primer fragments,” which form one aspect of the invention, are prepared from ET-NANB fragments such as described in Section III above. The method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S. Patent No. 4,683,202, as discussed above.
  • a preferred starting material for preparation of a vaccine is the particle antigen isolated from bile.
  • the antigens are preferably initially recovered as intact particles as described above. However, it is also possible to pre ⁇ pare a suitable vaccine from particles isolated from other sources or non-particle recombinant antigens.
  • non-particle antigens typically soluble antigens
  • proteins derived from the viral envelope or viral capsid are preferred for use in preparing vac ⁇ cines. These proteins can be purified by affinity chromatography, also described above.
  • the purified protein is not immunogenic per se, it can be bound to a carrier to make the protein immunogenic.
  • Carriers include bovine serum albumin, keyhole limpet hemocyanin and the like. It is desir ⁇ able, but not necessary, to purify antigens to be sub ⁇ stantially free of human protein. However, it is more important that the antigens be free of proteins, viruses, and other substances not of human origin that may have been introduced by way of, or contamination of, the nutrient medium, cell lines, tissues, or patho- logical fluids from which the virus is cultured or obtained.
  • Vaccination can be conducted in conventional fashion.
  • the antigen whether a viral particle or a protein
  • a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like.
  • the immunogen is administered using standard techniques for antibody induction, such as by subcutaneous ad inistra- tion of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens.
  • An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less.
  • a specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant protein or protein mixture derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZ- KFl(ETl.l) carried in E. coli strain BB4, and having ATCC deposit no. 67717.
  • the vaccine is administered at periodic intervals until a significant titer of anti- ET-NANB antibody is detected in the serum.
  • the vaccine is intended to protect against ET-NANB infection.
  • the composi ⁇ tions can be used to prepare antibodies to ET-NANB virus particles.
  • the antibodies can be used directly as antiviral agents.
  • a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines.
  • the host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle.
  • the gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art.
  • the antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.
  • the antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas.
  • the antibodies can also be used as a means of enhancing the immune response since antibody-virus complexes are recognized by macrophages.
  • the anti- bodies can be administered in amounts similar to those used for other therapeutic administrations of anti ⁇ body.
  • pooled gamma globulin is admini ⁇ stered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells.
  • antibodies reactive with the ET-NANB virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an ET-NANB virus to enhance the immune response and/or the effectiveness of an antiviral drug.
  • anti-ET-NANB-virus antibodies can be induced by administering anti-idiotype anti ⁇ bodies as immunogens.
  • a purified antiET- NANB-virus antibody preparation prepared as descibed above is used to induce anti-idiotype antibody in a host animal.
  • the composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody.
  • antibodies pro ⁇ **d by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed.
  • serum or plasma is removed to provide an antibody composition.
  • composition can be purified as described above for anti-ET-NANB- virus antibodies, or by affinity chromatography using anti-ET-NANB-virus antibodies bound to the affinity matrix.
  • anti-idiotype antibodies produced are similar in conformation to the authentic ET-NANB antigen and may be used to prepare an ET-NANB vaccine rather than using a ET-NANB particle antigen.
  • the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutane- ously or the like in an effective concentration in a physiologically suitable diluent with or without adju ⁇ vant.
  • One or more booster injections may be desirable.
  • the anti-idiotype method of induction of anti-ET-NANB- virus antibodies can alleviate problems which may be caused by passive administration of anti-ET-NANB-virus antibodies, such as an adverse immune response, and those associated with administration of purified blood components, such as infection with as yet undiscovered viruses.
  • the ET-NANB derived proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis.
  • an ET-NANB protein, or mixture of proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera.
  • Antibody response to the injected proteins is monitored, during a several- week period following immunization, by periodic serum sampling to detect the presence an anti- ET-NANB serum antibodies, as described in Section IIA above.
  • the antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection.
  • the anitserum is also useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.
  • Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a ET-NANB virus (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy.
  • Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas.
  • Primary i ⁇ vitro immunization with peptides can also be used in the generation of human monoclonal antibodies. - .
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete anti ⁇ bodies of the desired specificity. For monoclonal anti-virus particle antibodies, the antibodies must bind to ET-NANB virus particles. For monoclonal anti- idiotype antibodies, the antibodies must bind to anti- virus particle antibodies. Cells producing antibodies of the desired specificity are selected.
  • DNAse I and alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN); EcoRI, EcoRI methylase, DNA ligase, and DNA Polymerase I, from New England Biolabs (NEB,
  • EcoRI linkers were obtained from NEB; and nitro blue tetrazolium (NBT),
  • cynos Two cynomolgus monkeys (cynos) were intravenously injected with a 10% suspension of a stool pool obtained from a second-passage cyno (cyno #37) infected with a strain of ET-NANB virus isolated from Burma cases whose stools were positive for ET-NANB, as evidenced by binding of 27-34 nm virus-like particles (VLPs) in the stool to immune serum from a known ET ⁇ NANB patient.
  • VLPs virus-like particles
  • the animals developed elevated levels of alanine aminotransferase (ALT) between 24-36 days after innoculation, and one excreted 27-34 nm VLPs in its bile in the pre-acute phase of infection.
  • ALT alanine aminotransferase
  • duplex cDNA fragments were blunt-ended with T4 DNA polymerase under standard conditions (Maniatis, p. 118), then extracted with phenol/chloroform and precipitated with ethanol.
  • the blunt-ended material was ligated with EcoRI linkers under standard conditions (Maniatis, pp. 396-397) and digested with EcoRI to remove redundant linker ends. Non-ligated linkers were removed by sequential isopropanol precipitation.
  • Lambda gtlO phage vector (Huynh) was obtained from Promega Biotec (Madison, WI). This cloning vector has a unique EcoRI cloning site in the phage cl repressor gene.
  • the cDNA fragments from above were introduced into the EcoRI site by mixing 0.5 - 1.0 ug EcoRI-cleaved gtlO, 0.5-3 ⁇ l of the above duplex fragments, 0.5 ⁇ l 10X ligation buffer, 0.5 ⁇ l ligase (200 units), and distilled water to 5 ⁇ l. The mixture was incubated overnight at 14°C, followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).
  • the packaged phage were used to infect an E. coli hfl strain, such as strain HG415.
  • E. coli, strain C600 hfl, avialable from Promega Biotec, Madison, WI could be used.
  • EcoRI-ended fragments was less than 5% by analysis of 20 random plaques.
  • the resultant cDNA library was plated and phage were eluted from the selection plates by addition of elution buffer. After DNA extraction from the phage, the DNA was digested with EcoRI to release the heterogeneous insert population, and the DNA fragments were fractionated on agarose to remove phage fragments. The 500-4,000 basepair inserts were isolated and recloned into lambda gtlO as above, and the packaged phage was used to infect E. coli strain HG415. The percentage of successful recombinants was greater than 95%. The phage library was plated on E. coli strain HG415, at about 5,000 plaques/plate, on a total of 8 plates.
  • Duplex cDNA fragments from noninfected and ET- NANB-infected cynomolgus monkeys were prepared as in Example 1.
  • the cDNA fragments were radiolabeled by random priming, using a random-priming labeling kit obtained from Boehringer-Mannheim (Indianapolis, IN).
  • the plated cDNA library from Example 1 was transferred to each of two nitrocellulose filters, and the phage DNA was fixed on the filters by baking, according to standard methods (Maniatis, pp. 320- 323).
  • the duplicate filters were hybridized with either,infected-source or control cDNA probes from above. Autoradiographs of the filters were examined to identify library clones which hybridized with radiolabeled cDNA probes from infected source only, i.e., did not hybridize with cDNA probes from the non ⁇ infected source. Sixteen such clones, out of a total of about 40,000 clones examined, were identified by this subtraction selection method.
  • Each of the sixteen clones was picked and replated at low concentration on an agar plate.
  • the clones on each plate were transferred to two nitro ⁇ cellulose as duplicate lifts, and examined for hybrid ⁇ ization to radiolabeled cDNA probes from infected and noninfected sources, as above.
  • Clones were selected which showed selective binding for infected-source probes (i.e., binding with infected-source probes and substantially no binding with non-infected-source probes).
  • One of the clones which bound selectively to probe from infected source was isolated for further study.
  • the selected vector was identified as lambda gtlO-1.1, indicated in Figure 1.
  • Example 3 ET-NANB Sequence Clone lambda gtlO-1.1 from Example 2 was digested with EcoRI to release the heterologous insert, which was separated from the vector fragments by gel electrophoresis. The electrophoretic mobility of the fragment was consistent with a 1.33 kb fragment. This fragment, which contained EcoRI ends, was inserted into the EcoRI site of a pTZ-KFl vector, whose construction and properties are described in co-owned U.S. patent application for "Cloning Vector System and Method for Rare Clone Identification", Serial No. 125, 650, filed November 25, 1987.
  • this plasmid contains a unique EcoRI site adjacent a T7 polymerase promoter site, and plasmid and phage origins of replication. The sequence immediately adjacent each side of the EcoRI site is known.
  • E. coli BB4 bacteria obtained from Stratagene (La Jolla, CA, were transformed with the plasmid.
  • Radiolabeled ET-NANB probe was prepared by excising the 1.33 kb insert from the lambda gtlO-1.1 phage in Example 2, separating the fragment by gel electrophoresis, and randomly labeling as above.
  • Bacteria transfected with the above pTZ-KFl and containing the desired ET-NANB insert were selected by replica lift and hybridization with the radiolabeled ET-NANB probe, according to methods outlined in Example 2.
  • Example 4 Detecting ET-NANB Sequences cDNA fragment mixtures from the bile of noninfected and ET-NANB-infected cynomolgus monkeys were prepared as above.
  • the cDNA fragments obtained from human stool samples were prepared as follows. Thirty ml of a 10% stool suspension obtained from an individual from Mexico diagnosed as infected with ET ⁇ NANB as a result of an ET-NANB outbreak, and a similar volume of stool from a healthy, non-infected individual, were layered over a 30% sucrose density gradient cushion, and centrifuged at 25,000 xg for 6 hr in an SW27 rotor, at 15°C.
  • the pelleted material from the infected-source stool contained 27-34 nm VLP particles characteristic of ET-NANB infection in the infected-stool sample.
  • RNA was isolated from the sucrose-gradient pellets in both the infected and non ⁇ infected samples, and the isolated RNA was used to produce cDNA fragments as described in Example 1.
  • the cDNA fragment mixtures from infected and non-infected bile source, and from infected and non ⁇ infected human-stool source were each amplified by a novel linker/primer replication method described in co- owned patent application serial number 07/208,512 for "DNA Amplification and Subtraction Technique," filed June 17, 1988. Briefly, the fragments in each sample were blunt-ended with DNA Pol I then extracted with phenol/chloroform and precipitated with ethanol. The blunt-ended material was ligated with linkers having the following sequence: 5 '-GGAATTCGCGGCCGCTCG-3 ' 3 '-TTCCTTAAGCGCCGGCGAGC-5 '
  • duplex fragements were digested with Nrul to remove linker dimers, mixed with a primer having the sequence 5 ' -GGAATTCGCGGCCGCTCG-3 ' , and then heat denatured and cooled to room temperature to form single-strand DNA/primer complexes.
  • the complexes were replicated to form duplex fragments by addition of Thermus aquaticus (Taq) polymerase and all four deoxynucleotides.
  • the replication procedures involving successive strand denaturation, formation of strand/primer complexes, and replication, was repeated 25 times.
  • the amplified cDNA sequences were fractionated by agarose gel electrophoresis, using a 2% agarose matrix.
  • the filters were hybridized to a random-labeled 3 P probe prepared by (i) treating the pTZ-KFl(ETl.l) plasmid from above with EcoRI, (ii) isolating the released 1.33 kb ET-NANB fragment, and (iii) randomly labeling the isolated fragment.
  • the probe hybridization was performed by conventional Southern blotting methods (Maniatis, pp. 382-389).
  • Figure 2 shows the hybridization pattern obtained with cDNAs from infected (I) and non-infected (N) bile sources (2A) and from infected (I) and non ⁇ infected (N) human stool sources (2B).
  • the ET-NANB probe hybridized with fragments obtained from both of the infected sources, but was non-homologous to sequences obtained from either of the non-infected sources, thus confirming the specificity of derived sequence.
  • the pTZ-KFl(ETl.l) plasmid from Example 2 was digested with EcoRI to release the 1.33 kb ET-NANB insert which was purified from the linearized plasmid by gel electrophoresis.
  • the purified fragment was suspended in a standard digest buffer (0.5M Tris HCl, pH 7.5; 1 mg/ml BSA; lOmM MnC ⁇ ) to a concentration of about 1 mg/ml and digested with DNAse I at room temperature for about 5 minutes.
  • a standard digest buffer 0.5M Tris HCl, pH 7.5; 1 mg/ml BSA; lOmM MnC ⁇
  • the fragments in the digest mixture were blunt- ended and ligated with EcoRI linkers as in Example 1.
  • the resultant fragments were analyzed by electrophoresis (5-10V/cm) on 1.2% agarose gel, using PhiX17 /HaeIII and lambda/HindiII size markers.
  • the 100-300 bp fraction was eluted onto NA45 strips (Schleicher and Schuell), which were then placed into 1.5 ml microtubes with eluting solution (1 M NaCl, 50 mM arginine, pH 9.0), and incubated at 67°C for 30-60 minutes.
  • the eluted DNA was phenol/chloroform extracted and then precipitated with two volumes of ethanol.
  • the pellet was resuspended in 20 ⁇ l TE (0.01 M Tris HCl, pH 7.5, 0.001 M EDTA) .
  • Lambda gtll phage vector (Huynh) was obtained from Promega Biotec (Madison, WI) .
  • This cloning vector has a unique EcoRI cloning site 53 base pairs upstream from the beta-galactosidase translation termination codon.
  • the genomic fragments from above were introduced into the EcoRI site by mixing 0.5-1.0 ⁇ g EcoRI-cleaved gtll, 0.3-3 ⁇ l of the above sized fragments, 0.5 ⁇ l 10X ligation buffer (above), 0.5 ⁇ l ligase (200 units), and distilled water to 5 ⁇ l. The mixture was incubated overnight at 14°C, followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).
  • the packaged phage were used to infect E. coli strain KM392, obtained from Dr. Kevin Moore, DNAX (Palo Alto, CA) .
  • E. Coli strain Y1090 available from the American Type Culture Collection (ATCC #37197), could be used.
  • the infected bacteria were plated and the resultant colonies were checked for loss of beta-galactosidase activity-(clear plaques) in the presence of X-gal using a standard X-gal substrate plaque assay method (Maniatis). About 50% of the phage plaques showed loss of beta-galactosidase enzyme activity (recombinants) .
  • ET-NANB convalescent .antiserum was obtained from patients infected during documented ET-NANB outbreaks in Mexico, Borneo, Pakistan, Somalia, and Burma. The sera were immunoreactive with VLPs in stool specimens from each of several other patients with ET ⁇ NANB hepatitis.
  • a lawn of E. coli KM392 cells infected with about 10 ⁇ pfu of the phage stock from above was prepared on a 150 mm plate and incubated, inverted, for 5-8 hours at 37°C.
  • the lawn was overlaid with a nitrocellulose sheet, causing transfer of expressed ET- NANB recombinant protein from the plaques to the paper.
  • the plate and filter were indexed for matching corresponding plate and filter positions.
  • the filter was washed twice in TBST buffer (10 mM Tris, pH 8.0, 105 mM NaCl, 0.05% Tween 20), blocked with AIB (TBST buffer with 1% gelatin), washed again in TBST, and incubated overnight after addition of antiserum (diluted to 1:50 in AIB, 12-15 ml/plate).
  • the sheet was washed twice in TBST and then contacted with enzyme-labeled anti-human antibody to attach the labeled antibody at filter sites containing antigen recognized by the antiserum.
  • the filter was developed in a substrate medium containing 33 ⁇ l NBT (50 mg/ml stock solution maintained at 5 ⁇ C) mixed with 16 ⁇ l BCIP (50 mg/ml stock solution maintained at 5°C) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgCl 2 ). Purple color appeared at points of antigen production, as recognized by the antiserum.
  • the areas of antigen production determined in the previous step were replated at about 100-200 pfu on an 82 mm plate.
  • the identified plaques were picked and eluted in phage buffer (Maniatis, p. 443).
  • a series of subclones derived from the original pTZ-KFl (ET1.1) plasmid from Example 2 were isolated using the same techniques described above. Each of these five subclones were immunoreactive with a pool of anti-ET antisera noted in C.
  • the subclones contained short sequences from the "reverse" sequence set forth previously. The beginning and ending points of the sequences in the subclones (relative to the full "reverse” sequence), are identified in the table below.
  • the coding system for this epitope falls between nucleotide 2 (5'-end) and 101 P'-end). Genetic sequences related to this short sequence are therefore also preferred, as are peptides produced using this coding region.

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Abstract

Protéines virales dérivées d'un agent de l'hépatite virale non-A/non-B transmis par voie entérique. Dans un mode de réalisation, la protéine est capable de réagir immunologiquement avec des anticorps présents dans des personnes infectées par l'agent de l'hépatite virale. Cette protéine est utile dans un procédé de diagnostic de l'infection par l'agent transmis par voie entérique. Des sondes d'ADN sont dérivées d'une séquence clonée de l'agent viral. Ces sondes sont utiles pour identifier et pour mettre en séquence l'ensemble de l'agent viral et pour dépister l'agent viral dans un échantillon infecté, au moyen de l'amplification, spécifique des sondes, de fragments d'ADN dérivés de virus.
PCT/US1989/002648 1988-06-17 1989-06-16 Agent viral d'hepatite non-a/non-b transmis par voie enterique WO1989012462A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0447984A1 (fr) * 1990-03-20 1991-09-25 Abbott Laboratories Globuline hyperimmune contre le virus de l'hépatite C et son procédé de préparation
WO1992004472A1 (fr) * 1990-09-07 1992-03-19 Oklahoma Medical Research Foundation Antigenes associes a la polymyosite et a la dermatomyosite
EP0476130A1 (fr) * 1990-04-05 1992-03-25 Genelabs Technologies, Inc. Agent viral d'hepatite non a/non b transmis par voie enterique et epitopes caracteristiques de cet agent
WO1993019780A1 (fr) * 1992-03-27 1993-10-14 Smithkline Beecham Biologicals (S.A.) Vaccins anti-hepatite contenant le lipide monophosphorylique 3-o-desacyle a
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
US5686239A (en) * 1988-06-17 1997-11-11 Genelabs Technologies, Inc. Hepatitis E virus peptides and methods
US5770689A (en) * 1988-06-17 1998-06-23 Genelabs Technologies, Inc. Hepatitis E virus ORF Z peptides
US6210675B1 (en) 1989-12-18 2001-04-03 Glaxo Wellcome Inc. PT-NANB hepatitis polypeptides
US6620414B2 (en) 1992-03-27 2003-09-16 Smithkline Beecham Biologicals (S.A.) Hepatitis vaccines containing 3-0-deacylated monophoshoryl lipid A
US7166287B1 (en) 1989-12-18 2007-01-23 Glaxo Wellcome Inc. Viral agent

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US5824649A (en) * 1988-06-17 1998-10-20 Genelabs Technologies, Inc. DNA sequences of enterically transmitted non-A/non-B hepatitis viral agent and characteristic epitopes thereof
US5741490A (en) * 1988-06-17 1998-04-21 Genelabs Technologies, Inc. Hepatitis E virus vaccine and method
AU3748489A (en) * 1988-06-17 1990-01-12 Genelabs Incorporated Enterically transmitted non-a/non-b hepatitis viral agent
JP2656995B2 (ja) * 1989-03-17 1997-09-24 カイロン コーポレイション Ｎａｎｂｖの診断用薬

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
US5770689A (en) * 1988-06-17 1998-06-23 Genelabs Technologies, Inc. Hepatitis E virus ORF Z peptides
US5686239A (en) * 1988-06-17 1997-11-11 Genelabs Technologies, Inc. Hepatitis E virus peptides and methods
US6210675B1 (en) 1989-12-18 2001-04-03 Glaxo Wellcome Inc. PT-NANB hepatitis polypeptides
US7166287B1 (en) 1989-12-18 2007-01-23 Glaxo Wellcome Inc. Viral agent
EP0447984A1 (fr) * 1990-03-20 1991-09-25 Abbott Laboratories Globuline hyperimmune contre le virus de l'hépatite C et son procédé de préparation
EP0476130A4 (en) * 1990-04-05 1993-05-19 Genelabs Inc Enterically transmitted non-a/non-b hepatitis viral agent and characteristic epitopes thereof
EP0476130A1 (fr) * 1990-04-05 1992-03-25 Genelabs Technologies, Inc. Agent viral d'hepatite non a/non b transmis par voie enterique et epitopes caracteristiques de cet agent
WO1992004472A1 (fr) * 1990-09-07 1992-03-19 Oklahoma Medical Research Foundation Antigenes associes a la polymyosite et a la dermatomyosite
US6160107A (en) * 1990-09-07 2000-12-12 Oklahoma Medical Research Foundation Nucleic acids encoding antigens associated with polymyositis and dermatomositis
WO1993019780A1 (fr) * 1992-03-27 1993-10-14 Smithkline Beecham Biologicals (S.A.) Vaccins anti-hepatite contenant le lipide monophosphorylique 3-o-desacyle a
US6620414B2 (en) 1992-03-27 2003-09-16 Smithkline Beecham Biologicals (S.A.) Hepatitis vaccines containing 3-0-deacylated monophoshoryl lipid A
US6893644B2 (en) 1992-03-27 2005-05-17 Smithkline Beecham Biologicals S.A. Hepatitis vaccines containing 3-O-deacylated monophoshoryl lipid A

Also Published As

Publication number Publication date
AU639209B2 (en) 1993-07-22
EP0419576A4 (en) 1991-11-27
JP4072604B2 (ja) 2008-04-09
JP2002097198A (ja) 2002-04-02
AU3857489A (en) 1990-01-12
JPH03506026A (ja) 1991-12-26
SG49225A1 (en) 1998-05-18
EP0419576A1 (fr) 1991-04-03
AU3748489A (en) 1990-01-12
KR0180530B1 (ko) 1999-03-20
KR900701317A (ko) 1990-12-01
JP2000184895A (ja) 2000-07-04
WO1989012641A1 (fr) 1989-12-28
JP2005053924A (ja) 2005-03-03
CA1341464C (fr) 2004-11-09
JP2007031440A (ja) 2007-02-08

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