WO1990002206A1 - Detection of non-a, non-b hepatitis - Google Patents

Detection of non-a, non-b hepatitis Download PDF

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Publication number
WO1990002206A1
WO1990002206A1 PCT/US1989/003638 US8903638W WO9002206A1 WO 1990002206 A1 WO1990002206 A1 WO 1990002206A1 US 8903638 W US8903638 W US 8903638W WO 9002206 A1 WO9002206 A1 WO 9002206A1
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PCT/US1989/003638
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French (fr)
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Belinda Seto
William G. Coleman, Jr.
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Publication of WO1990002206A1 publication Critical patent/WO1990002206A1/en

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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
    • 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
    • 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
    • G01N33/576—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
    • 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

  • New clones bearing nucleotide sequences of non-A, non-B hepatitis provides a method for testing blood to detct the presence of non-A, non-B hepatitis materials.
  • the cloned DNA is used as a hybridization probe to screen serum samples or biopsied liver specimens for homologous DNA sequences.
  • Polypeptides expressed by the cloned DNA may be used as antigens in assays to determine whether antibodies to non-A, non-B hepatitis are present.
  • the causative agent of non-A, non-B hepatitis accounts for 90% of the posttransfusion hepatitis in the United States. Of those blood recipients who are infected by transfusion, 50% develop chronic hepatitis. Those chronically infected persons develop chronic hepatitis. Those chronically infected persons continue to be carriers of the infectious disease.
  • the existence of the transmissible agent has been demonstrated by infection of chimpanzees by inoculation with serum from a chronic non-A, non-B hepatitis patient and followed by serial passage of virus into other chimpanzees.
  • the deposited material will be maintained for at least five years after the most recent request for a sample; for a period of 30 years from date of deposit; or for the life of the patent, whichever period is longest. If the deposited sample should become contaminated or non-viable, the sample will be replaced.
  • Plasmid pBR322 and Escherichia coli DH5 are available commercially and were obtained from Bethesda Research Laboratory in Gaithersburg, Maryland. Plasmids pSP65, GEM37, and GEM47 are available from Promega. E. coli strain p678-54 was obtained by request from Dr. Barbara Bachmann, E. coli Genetic Stock Center, Yale University School of Medicine.
  • Figure 1 indicates that hybridization with the DNA insert from clone pSC22 occurred at only the acute phase of the disease in serum and liver (see insert) samples. Note the response at approximately 5 weeks.
  • Figure 2 shows evidence of hybridization of sequences in clone pSC22 with cultures of peripheral blood mononuclear cells isolated from a patient diagnosed as suffering from non-A, non-B hepatitis.
  • Figures 3 and 4 Cultures of mink lung cells superinfected with non-A, non-B hepatitis viral particles evidence sequences hybridizable with sequences from pSC22.
  • Figure 5 shows lack of hybridization of sequences in culture non-A, non-B hepatitis infected mink lung cells (as used in Figures 3 and 4) when vector pBR322 (lacking insert DNA of pSC22) is used.
  • Figure 6 shows the sequence of the DNA insert of clone pSC22 and a sequence of the protein encoded by that DNA.
  • Figure 7 shows the sequence of the DNA insert of clone pSN31 and the protein sequence encoded by the DNA.
  • Figure 8 shows the bands of immunoreactive protein from the 764 DNA-insert clones.
  • Figure 9 is a diagramatic presentation of clones pSN31 and pSN32.
  • the proteins expressed by the clones may be used as immunogens to elicit production of antibodies against the non-A, non-B hepatitis antigens.
  • the clones pSC22 pLC30, pLC50, pSN30, pSN31, and pSN32 disclosed herein can be used to propagate non-A, non-B sequences for use as probes which will hybridize with a complementary strand DNA in infected test samples.
  • the DNA sequences may be purified by means known in the art. Purification by gel electrophoresis is exemplified. The probe may be radiolabeled by nick translation. Standard procedures such as Southern blot and slot blot tests are appropriate.
  • compositions of the invention may be provided in test kits for use as diagnostic tools.
  • Compositions wherein antigens or antibodies to non-A, non-B viral antigens have been attached to a solid support are particularly useful for such purposes.
  • the clone (pSC22) described in the following examples is constructed by ligating the dephosphorylated EcoRI-restricted pBR322 with the EcoRI digest of liver DNA isolated from a chimpanzee during acute non-A, non-B hepatitis. Competent E. coli strain P678-54 were prepared and transformed by known procedures. Tet, amp transformants were selected on L-broth containing 20 ug/ml of ampicillin and tetracyline. Mini-lysates of tet- and amp-resistant transformants were screened for DNA inserts into pBR322 by the relative migration of plasmid DNA on 0.7% agarose gel. The DNA insert present in clone pSC22 is 764 bp.
  • a single needle biopsied chimpanzee liver speciman (chimpanzee No . CA6 ) was suspended in 1 ml ice- cold TE buffer, pH 8.0 (10 mM Tris-HCl, ImM EDTA) in a Wheaton, Dounce type tissue grinder. The specimen was homogenized with the glass pestle 10 times. One ml of STE, pH 8.0 (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA) containing 4 mg/ml protease K (Boehringer-Mannheim) and 0.2% sodium lauryl sulfate was added to the homogenate and incubated for 2 hours at 37°C.
  • the mixture was extracted once with 2 ml phenol.
  • the aqueous phase was extracted with 2 ml phenol-chloroform (1:1 v/v).
  • the chloroform contains a 24:1 v/v ratio of isoamylalcohol.
  • the aqueous phase was treated with NaCl to a final concentration of 0.1 M.
  • Nucleic acids were precipitated twice with absolute alcohol and kept at -20°C for 30 minutes. The precipitate was centrifuged at 8,000 x g for 10 minutes and resuspended in 1 ml TE buffer, pH 8.0.
  • the nucleic acids were digested with 100 g/ml ribonuclease A at 37°C for 1 hour.
  • the phenol and phenol-chloroform extractions as described above were repeated.
  • the DNA is then treated with 0.1 M NaCl ethanol-precipitated, and centrifuged.
  • the restriction process was repeated using pBR322, 15 liters (1 ⁇ g/ul), instead of the liver DNA.
  • the EcoRI restricted CA6 liver DNA was electrophoresed in 0.7% low-melting agarose (Bethesda Research Laboratories) at 100 V in 100 mM Tris base, 100 mM anhydrous boric acid, and 2 mM Na2 EDTA H2O. At the end of the electrophoresis, the gel was divided into 3 seg- ments: top 4.5 cm, middle 3.5 cm, and bottom 2.3 cm. The DNA in each segment was recovered with the Bethesda Research Laboratory Pre-pac columns according to the manufacturer' s procedure.
  • pBR322 (1 mg/ml. New England BioLab) was EcoRI restricted and dephosphorylated with alkaline phosphatase from calf intestine (Boehringer-Mannheim). The treated vector, pBR322 (20 ⁇ l ) was incubated with 8 ul CA6 liver DNA from each of 3 segments separated by electrophoresis, 16 units of T4 DNA ligase (International Biotechnologies, Inc.), and 4 ⁇ l ligase buffer at 16°C for 2-3 minutes. LB broth (1 ml) was added, mixed gently, and incubated for 5-6 hours at 37°C.
  • the mixture was added to 5 ml soft LB agar containing 50 ug/ml amplicillin and 20 ⁇ g/ml tetracycline.
  • the soft agar mixture was immediately poured on top of 2 LB agar plates containing the same antibiotics and incubated overnight at 37°C.
  • pellets were resuspended in 12 ml of cold (sterile) 30 mM CaCl 2 and were allowed to stand on ice for 20 minutes. This incubation step was followed by centrifugation at 4,000 ⁇ g for 5 minutes.
  • the cell pellet was gently resuspended in 2.5 ml of ice-cold 30 mM CaCl 2 plus 15% glycerol. Samples (0.2 ml) of the competent cell suspension were stored in 1.5 ml Eppendorf tubes.
  • Transformants were grown as described and transferred to a nitrocellulose filter.
  • the filter was incubated on LB agar plate at 37°C overnight until colonies were 0.3 mm in diameter. Colony hybridization was performed as described by Grunstein.
  • the hybridization probe was 32p-labeled normal chimpanzee liver DNA. The colonies which did not react with the probe were selected. These were considered possible candidates to contain non-A, non-B hepatitis sequences.
  • Normal chimpanzee liver DNA was radiolabeled by [ 32 P]ATP using the same procedure as described above except that 2 ⁇ g normal chimpanzee liver DNA was used.
  • Example (b) illustrates the specific association of the cloned DNA insert with acute non-A, non-B hepatitis and the suitability for use in chimpanzees.
  • a chimpanzee is considered a surrogate human, permitting controlled evaluation in a protected environment.
  • a chronic non-A, non-B hepatitis patient known to contain 10 infectious viral particles/ml serum based on transmission of infectivity to chimpanzees was found positive by hybridization to the non-A, non-B hepatitis sequence in clone pSC22. The negative control individuals did not show reaction.
  • Chimpanzee No. 51 was screened to assure the absence of Epstein-Barr virus, cytomegalovirus, hepatitis A virus infection, and hepatitis B virus infection.
  • the animal was inoculated intravenously with 1 ml serum from a patient documented to be a chronic carrier of non-A, non-B hepatitis. Weekly bleedings from the animal were tested for serum aminotransferases.
  • These serial serum samples were also tested in a blind study for hybridization with the DNA insert in clone pSC22. The results shown in Fig. 1 clearly indicate that hybridization occurred only in the acute phase serum and liver samples. No evidence of hybridizable DNA is detected at other intervals.
  • the DNA insert from clone pSC22 can be used to detect infectious non-A, non-B hepatitis samples.
  • the non-A, non-B hepatitis sequence in clone pSC22 was found to hybridize to lijaer biopsied specimens of three non-A, non-B hepatitis infected chimpanzees, Nos. 1290, 1291, and 1279. Reaction was observed between 6 to 16 weeks post-inoculation. In contrast, no hybridization was observed in pre-inoculation specimens. Further, none of the liver specimens from two hepatitis B chimpanzees (Nos. 1196 and 59) showed hybridization (Table 1).
  • Chimpanzee liver cell cultures infected with non-A, non-B hepatitis viral particles from two patients contained sequences which hybridized with sequences from clone pSC22. The uninfected cultures showed no reaction.
  • Clones pLC30 and pLC50 were constructed in the same manner as the clone pSC22, using vector pSP65 instead of pBR322.
  • Clone pSC22 contains the DNA insert of Fig. 6, while pLC30 and pLC50 are complementary strands of DNA, one of which is the DNA insert of Fig. 6.
  • Clones pLC30 and pLC50 are effective for expression of polypeptides.
  • Plasmid pSC22 was subjected to EcoRI restriction to excise the 764 bp DNA insert.
  • the 764 bp DNA segment was then ligated into vector pGEM3Z to provide clone pSN30.
  • the pSN30 plasmid was transfected into E. coli DH50.
  • Plasmid pSN30 was restricted with PstI and religated with T4 DNA polymerase to generate a new recombinant plasmid, pSN31, which contains a 464 bp DNA insert.
  • a clone wherein the 464 bp had the opposite orientation was obtained by insertion of the segment into pGEM4Z to provide plasmid pSN32.
  • Clones pSN31 and pSN32 have been tested in the manner exemplified using pSC22 and have been shown to have similar characteristics. However, the smaller DNA insert provides greater specificity of reaction with nonA, non-B hepatitis virus, viral products, and antibodies to non-A, non-B hepatitis. Hence, the clones containing the 464 bp DNA inserts are preferred embodiments of the invention.
  • the 464 bp inserts may be further digested to provide small fragments of DNA which could be used as probes using hybridization techniques. However, clones from the smaller segments would not be competent for use in production of the full range of viral antigens provided by the clones containing the 464 bp insert. A segment of DNA from the 464 bp insert containing as few as 20 bp could be used as a probe using known hybridization techniques.
  • Antigens expressed by the clones are useful for detection of antibodies to non-A, non-B hepatitis antigens. Enzyme tests such as the ELISA test of RIA are preferred for this purpose.
  • the antigenic polypeptides may also be used as vaccines to elicit antibodies to the virus in at-risk populations.
  • Compositions containing the polypeptides may be formulated using adjuvants customary in the art.
  • Antigenic polypeptides may also be used to elicit antibodies for use in tests for detection of viral products by means known in the art.
  • the use of monoclonal antibodies to the peptides in radioimmunological tests is a preferred means of detection.

Abstract

The invention of clones bearing nucleotide sequences of non-A, non-B hepatitis provides a method for testing blood to detect the presence of non-A, non-B hepatitis materials. The clone DNA is used as a hybridization probe to screen serum samples or biopsied liver specimens for homologous sequences. Polypeptides expressed by the cloned DNA may be used as antigens in assays to determine whether antibodies to non-A, non-B hepatitis are present.

Description

DETECTION OF NON-A, NON-B HEPATITIS
Summary of the Invention
New clones bearing nucleotide sequences of non-A, non-B hepatitis provides a method for testing blood to detct the presence of non-A, non-B hepatitis materials. The cloned DNA is used as a hybridization probe to screen serum samples or biopsied liver specimens for homologous DNA sequences. Polypeptides expressed by the cloned DNA may be used as antigens in assays to determine whether antibodies to non-A, non-B hepatitis are present.
Background of the Invention
The causative agent of non-A, non-B hepatitis accounts for 90% of the posttransfusion hepatitis in the United States. Of those blood recipients who are infected by transfusion, 50% develop chronic hepatitis. Those chronically infected persons develop chronic hepatitis. Those chronically infected persons continue to be carriers of the infectious disease. The existence of the transmissible agent has been demonstrated by infection of chimpanzees by inoculation with serum from a chronic non-A, non-B hepatitis patient and followed by serial passage of virus into other chimpanzees.
The need for a screening test which would evidence with specificity the presence of non-A, non-B hepatitis infection has long been recognized. [See Deinhart et al.. Bull. WHO 60, 661-691 (1982) and Tabor, Lab. World 31, 25-27 (1980).]
The availability of several antigen-antibody tests to determine presence of non-A, non-B hepatitis has been reported in the literature. Tabor et al. (U.S. Patent 4,395,395); Suh et al. [The Lancet, pages 178-180 (January 1981)] and Shirachi et al. [The Lancet, pages 853-856 (October 1978)] all report such assays. However, all of the previous methods lack sufficient sensitivity and specificity needed to provide reliable means for protecting the blood supply. All previously reported means of detection present serious disadvantages because of uncertainty of viral or host origins of antigens or antibodies.
The lack of a sufficiently reliable laboratory assay for use in diagnosis of non-A, non-B hepatitis has forced clinicians to rely on negative seriological tests for hepatitis A, hepatitis B, cytomegalovirus, and
Epstein-Barr virus.
At the International Symposium on Viral Hepatitis held in May 1987 in London, it was suggested clones might be used in detection of non-A, non-B hepatitis. No clones for such purposes were described.
Deposits of Materials
The subject matter of the invention has been deposited under conditions to assure access to the subject matter will be available to the public during pendancy of the application in accord with requirements under 37 CFR 1.14 and 35 U.S.C. 122 in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., on August 31, 1988 under accession numbers ATCC #67776 and ATCC #67777.
The deposited material will be maintained for at least five years after the most recent request for a sample; for a period of 30 years from date of deposit; or for the life of the patent, whichever period is longest. If the deposited sample should become contaminated or non-viable, the sample will be replaced.
Plasmid pBR322 and Escherichia coli DH5 are available commercially and were obtained from Bethesda Research Laboratory in Gaithersburg, Maryland. Plasmids pSP65, GEM37, and GEM47 are available from Promega. E. coli strain p678-54 was obtained by request from Dr. Barbara Bachmann, E. coli Genetic Stock Center, Yale University School of Medicine.
Description of the Figures
Figure 1 indicates that hybridization with the DNA insert from clone pSC22 occurred at only the acute phase of the disease in serum and liver (see insert) samples. Note the response at approximately 5 weeks.
Figure 2 shows evidence of hybridization of sequences in clone pSC22 with cultures of peripheral blood mononuclear cells isolated from a patient diagnosed as suffering from non-A, non-B hepatitis.
Figures 3 and 4: Cultures of mink lung cells superinfected with non-A, non-B hepatitis viral particles evidence sequences hybridizable with sequences from pSC22.
Figure 5 shows lack of hybridization of sequences in culture non-A, non-B hepatitis infected mink lung cells (as used in Figures 3 and 4) when vector pBR322 (lacking insert DNA of pSC22) is used.
Figure 6 shows the sequence of the DNA insert of clone pSC22 and a sequence of the protein encoded by that DNA.
Figure 7 shows the sequence of the DNA insert of clone pSN31 and the protein sequence encoded by the DNA.
Figure 8 shows the bands of immunoreactive protein from the 764 DNA-insert clones.
Figure 9 is a diagramatic presentation of clones pSN31 and pSN32.
Description of the Invention.
It is the purpose of this invention to provide improved means for detecting non-A, non-B hepatitis virons, viral particles, and viral antigens.
It is a further purpose of this invention to provide improved means for detection of antibodies specifically reactive to non-A, non-B hepatitis antigens.
The proteins expressed by the clones may be used as immunogens to elicit production of antibodies against the non-A, non-B hepatitis antigens.
The clones pSC22 pLC30, pLC50, pSN30, pSN31, and pSN32 disclosed herein can be used to propagate non-A, non-B sequences for use as probes which will hybridize with a complementary strand DNA in infected test samples. The DNA sequences may be purified by means known in the art. Purification by gel electrophoresis is exemplified. The probe may be radiolabeled by nick translation. Standard procedures such as Southern blot and slot blot tests are appropriate.
Compositions of the invention may be provided in test kits for use as diagnostic tools. Compositions wherein antigens or antibodies to non-A, non-B viral antigens have been attached to a solid support are particularly useful for such purposes.
Preparation of Clone pSC22
The clone (pSC22) described in the following examples is constructed by ligating the dephosphorylated EcoRI-restricted pBR322 with the EcoRI digest of liver DNA isolated from a chimpanzee during acute non-A, non-B hepatitis. Competent E. coli strain P678-54 were prepared and transformed by known procedures. Tet, amp transformants were selected on L-broth containing 20 ug/ml of ampicillin and tetracyline. Mini-lysates of tet- and amp-resistant transformants were screened for DNA inserts into pBR322 by the relative migration of plasmid DNA on 0.7% agarose gel. The DNA insert present in clone pSC22 is 764 bp.
Example 1
Extraction of Liver DNA
A single needle biopsied chimpanzee liver speciman (chimpanzee No . CA6 ) was suspended in 1 ml ice- cold TE buffer, pH 8.0 (10 mM Tris-HCl, ImM EDTA) in a Wheaton, Dounce type tissue grinder. The specimen was homogenized with the glass pestle 10 times. One ml of STE, pH 8.0 (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA) containing 4 mg/ml protease K (Boehringer-Mannheim) and 0.2% sodium lauryl sulfate was added to the homogenate and incubated for 2 hours at 37°C. Following incubation, the mixture was extracted once with 2 ml phenol. The aqueous phase was extracted with 2 ml phenol-chloroform (1:1 v/v). The chloroform contains a 24:1 v/v ratio of isoamylalcohol. The aqueous phase was treated with NaCl to a final concentration of 0.1 M. Nucleic acids were precipitated twice with absolute alcohol and kept at -20°C for 30 minutes. The precipitate was centrifuged at 8,000 x g for 10 minutes and resuspended in 1 ml TE buffer, pH 8.0. The nucleic acids were digested with 100 g/ml ribonuclease A at 37°C for 1 hour. The phenol and phenol-chloroform extractions as described above were repeated. The DNA is then treated with 0.1 M NaCl ethanol-precipitated, and centrifuged.
EcoRI Restriction
CA6 liver DNA . . . . . 15 μl (2 jαg/^1)
10X Eco buffer . . . . . 2 μl (Bethesda Research Lab) Bovine serum albumin . . 1 μl (Bethesda Research Lab)
EcoRI . . . . . . . . . 2 μl (Bethesda Research Lab) incubated for 2 hours at 37°C.
The restriction process was repeated using pBR322, 15 liters (1μg/ul), instead of the liver DNA.
The EcoRI restricted CA6 liver DNA was electrophoresed in 0.7% low-melting agarose (Bethesda Research Laboratories) at 100 V in 100 mM Tris base, 100 mM anhydrous boric acid, and 2 mM Na2 EDTA H2O. At the end of the electrophoresis, the gel was divided into 3 seg- ments: top 4.5 cm, middle 3.5 cm, and bottom 2.3 cm. The DNA in each segment was recovered with the Bethesda Research Laboratory Pre-pac columns according to the manufacturer' s procedure.
Example 2
Cloning of CA6 Liver DNA
pBR322 (1 mg/ml. New England BioLab) was EcoRI restricted and dephosphorylated with alkaline phosphatase from calf intestine (Boehringer-Mannheim). The treated vector, pBR322 (20 μl ) was incubated with 8 ul CA6 liver DNA from each of 3 segments separated by electrophoresis, 16 units of T4 DNA ligase (International Biotechnologies, Inc.), and 4 μl ligase buffer at 16°C for 2-3 minutes. LB broth (1 ml) was added, mixed gently, and incubated for 5-6 hours at 37°C. The mixture was added to 5 ml soft LB agar containing 50 ug/ml amplicillin and 20 μg/ml tetracycline. The soft agar mixture was immediately poured on top of 2 LB agar plates containing the same antibiotics and incubated overnight at 37°C.
Example 3
Preparation of Competent E. coli p678-54
An overnight Lurin broth (LB) culture of E. coli p678-54 was used to inoculate (1:100 dilution) a 100 ml L3 culture. The new culture was vigorously shaken at 37°C until an OD590 of 0.685 was reached. The entire culture was placed in a 50°C water bath for 35 minutes with intermittent shaking. After the 35-minute incubat-ion at 50°C, the cultures were quickly chilled in a water-ice bath and placed on ice for 10 minutes. 25 ml of the chilled culture was placed in-»βterile 50 ml Sepco tubes and centrifuged at 12,000 x g for 5 minutes at 4°C. The pellets were resuspended in 12 ml of cold (sterile) 30 mM CaCl2 and were allowed to stand on ice for 20 minutes. This incubation step was followed by centrifugation at 4,000 × g for 5 minutes.
The cell pellet was gently resuspended in 2.5 ml of ice-cold 30 mM CaCl2 plus 15% glycerol. Samples (0.2 ml) of the competent cell suspension were stored in 1.5 ml Eppendorf tubes.
Example 4
Screening of Transformant for Specificity for
Non-A, Non-B Hepatitis by Colony Hybridization
Transformants were grown as described and transferred to a nitrocellulose filter. The filter was incubated on LB agar plate at 37°C overnight until colonies were 0.3 mm in diameter. Colony hybridization was performed as described by Grunstein. The hybridization probe was 32p-labeled normal chimpanzee liver DNA. The colonies which did not react with the probe were selected. These were considered possible candidates to contain non-A, non-B hepatitis sequences.
Example 5
Radiolabeling of Hybridization Probes by Nick Translation
Nick translation kit (New England Nuclear) [35S]dATP (4 μl) was dried by Savant speed centrifuge. The following were added to the dried nucleotide: 5 μl 5X buffer, dCTP, dGTP, dTTP (4 μl total), 500 ng (3 μl ) 764 bp non-A, non-B hepatitis DNA, 8 μl H2O, 2 μl DNA polymerase, and 2 μl deoxyribonuclease. The mixture was incubated at 14°C for 2-3 hours. The reaction was stopped by adding 0.19 M EDTA and incubated at 65°C for 10 minutes. The reaction mixture was desalted by a spun column of Sephadex G100.
Normal chimpanzee liver DNA was radiolabeled by [32P]ATP using the same procedure as described above except that 2 μg normal chimpanzee liver DNA was used.
Supplies for use in hybridization were obtained from Schleicher and Schuell.
After hybridization was complete, filters containing hybridization product were autoradiographed under x-ray film for identification of hybridization materials. Examples of Results:
Example (b) illustrates the specific association of the cloned DNA insert with acute non-A, non-B hepatitis and the suitability for use in chimpanzees. For the purposes of this invention, a chimpanzee is considered a surrogate human, permitting controlled evaluation in a protected environment.
Example (a )
A chronic non-A, non-B hepatitis patient (E.B.) known to contain 10 infectious viral particles/ml serum based on transmission of infectivity to chimpanzees was found positive by hybridization to the non-A, non-B hepatitis sequence in clone pSC22. The negative control individuals did not show reaction.
Example (b)
Chimpanzee No. 51 was screened to assure the absence of Epstein-Barr virus, cytomegalovirus, hepatitis A virus infection, and hepatitis B virus infection. The animal was inoculated intravenously with 1 ml serum from a patient documented to be a chronic carrier of non-A, non-B hepatitis. Weekly bleedings from the animal were tested for serum aminotransferases. These serial serum samples were also tested in a blind study for hybridization with the DNA insert in clone pSC22. The results shown in Fig. 1 clearly indicate that hybridization occurred only in the acute phase serum and liver samples. No evidence of hybridizable DNA is detected at other intervals. Thus, the DNA insert from clone pSC22 can be used to detect infectious non-A, non-B hepatitis samples.
Example (c)
Using the in situ hybridization technique, the non-A, non-B hepatitis sequence in clone pSC22 was found to hybridize to lijaer biopsied specimens of three non-A, non-B hepatitis infected chimpanzees, Nos. 1290, 1291, and 1279. Reaction was observed between 6 to 16 weeks post-inoculation. In contrast, no hybridization was observed in pre-inoculation specimens. Further, none of the liver specimens from two hepatitis B chimpanzees (Nos. 1196 and 59) showed hybridization (Table 1).
Example (d)
Cultures of peripheral blood mononuclear cells isolated from a non-A, non-B hepatitis patient (R.F.) were found to hybridize to sequences in clone pSC22 (Fig. 2).
Example (e)
Chimpanzee liver cell cultures infected with non-A, non-B hepatitis viral particles from two patients (E.B. and W.R. ) contained sequences which hybridized with sequences from clone pSC22. The uninfected cultures showed no reaction.
Example (f)
Cultures of mink lung cells superinfected with non-A, non-B hepatitis viral particles ( from 15 patients ) showed hybridizable sequences to clone pSC22 (Figs . 3 and 4 ) . A control experiment using only the vector , pBR322 , as probe showed no hybridization (Fig . 5 ) .
Table 1
In Situ Detection of NANB-Associated Nucleic Acid Sequences Using Radiolabeled Probes
Chimpanzees [35S]pBR322 [35S]HBV-DNA [35S]NANEH-DNA
No. 1290 (NANBH)
pre - - - post 4 w. - - -
6 w. - - +-
8 w. - - +
12 w. - - +
No. 1292 (NANBH)
pre - - - post 4 w. - - -
6 w. - - +
8 w. - - +
12 w. - - +
No. 1279 (NANBH)
post 5 w. - - +
14 w. - - +
16 w. - - +
No. 1196 (HBV)
post 6 w. - + -
12 w. - + -
No. 59 (HBV)
6 w. - + -
12 w. - + -
Clones pLC30 and pLC50 were constructed in the same manner as the clone pSC22, using vector pSP65 instead of pBR322. Clone pSC22 contains the DNA insert of Fig. 6, while pLC30 and pLC50 are complementary strands of DNA, one of which is the DNA insert of Fig. 6. Clones pLC30 and pLC50 are effective for expression of polypeptides.
Preparation of Clones pSN30, pSN31, and pSN32
Plasmid pSC22 was subjected to EcoRI restriction to excise the 764 bp DNA insert. The 764 bp DNA segment was then ligated into vector pGEM3Z to provide clone pSN30. The pSN30 plasmid was transfected into E. coli DH50.
Plasmid pSN30 was restricted with PstI and religated with T4 DNA polymerase to generate a new recombinant plasmid, pSN31, which contains a 464 bp DNA insert. A clone wherein the 464 bp had the opposite orientation was obtained by insertion of the segment into pGEM4Z to provide plasmid pSN32.
Clones pSN31 and pSN32 have been tested in the manner exemplified using pSC22 and have been shown to have similar characteristics. However, the smaller DNA insert provides greater specificity of reaction with nonA, non-B hepatitis virus, viral products, and antibodies to non-A, non-B hepatitis. Hence, the clones containing the 464 bp DNA inserts are preferred embodiments of the invention.
The 464 bp inserts may be further digested to provide small fragments of DNA which could be used as probes using hybridization techniques. However, clones from the smaller segments would not be competent for use in production of the full range of viral antigens provided by the clones containing the 464 bp insert. A segment of DNA from the 464 bp insert containing as few as 20 bp could be used as a probe using known hybridization techniques.
Antigens expressed by the clones are useful for detection of antibodies to non-A, non-B hepatitis antigens. Enzyme tests such as the ELISA test of RIA are preferred for this purpose.
The antigenic polypeptides may also be used as vaccines to elicit antibodies to the virus in at-risk populations. Compositions containing the polypeptides may be formulated using adjuvants customary in the art.
Antigenic polypeptides may also be used to elicit antibodies for use in tests for detection of viral products by means known in the art. The use of monoclonal antibodies to the peptides in radioimmunological tests is a preferred means of detection.

Claims

1 . A clone or vector containing a DNA insert which encodes a protein having in whole or in part the amino acid sequence
Met Thr Tyr Ala Gln Asp Arg Ala Leu Arg Glu Glu Val Tyr
Ala Ala Tyr Cys Thr Arg Ala Ser Asp Gln Gly Pro Asn Ala
Gly Gln Asn Asp Asn Gly Pro Val Met Glu Gln lie Leu Asp
Leu Arg Gln Glu Leu Ala Gln Leu Leu Gly Tyr Ala Ser Phe
Ser Glu Leu Ser Leu Ala Thr Lys Met Ala Glu Ser Ser Asp Gln Val Leu Ser Phe Leu Arg Asp Leu Ala Lys Arg Ser Lys
Pro Phe Ala Ala Gln Asp Leu Gln Gln Leu Lys Ala Tyr Ala
Ala Glu Gln Glv Cys Ala Asp Leu Gln Ser Trp Asp Ser Gly
Phe Tyr Gly Glu Lys Leu Arg Glu Gln Arg Tyr Ser Val Ser Gln Glu Ala Leu Arg Ala Tyr Phe Pro lie Asp Lys Val Leu Gly Gly Leu Phe Ala lie Val Gln Arg Leu Tyr Gly lie Glu lie Ala Glu Leu Lys Gly Phe Asp Thr Trp His Pro Asp Val
Arg Leu Phe Glu lie Lys Glu Asn Gly Glu His Val Gly Arg
Phe Phe Phe Asp Leu Tyr Ala Arg Ala Asn Lys Arg Ala Val
Pro Gly Trp Met Ala Pro Val Thr Ala Ala Val Pro Leu Met Ala Cys Cys Lys Ala Pro Ser Pro Thr Trp Cys Ala Thr Ser
Leu Arg Pro Thr Ala Ala Ser Leu Pro Cys
or a DNA sequence complementary thereto .
2 . A clone or vector of claim 1 wherein the DNA insert contains the sequence
1 GAA TTCCCCA GCTACTACGC CGTCATGACC TAOGCCCAGG ACCGTGCCCT
61 GTSEAOGCCG CCTACTGCAC CCGTGCCTCG GACCAAGGCC OGAATGCCGG TCAGAACGAT 121 AACGGCCCGG TGADGGAACA GATCCTOGAC CTGOGTCAGG AACDGGCCCA ATTGCTGGGT 181 TATGOGTCGT TCTCGGSGCT GAGCCTGGCC ACCAAGATGG COGAGTCCAG OGACCSGGTG 241 CTCAGCTTCC TGOGTGACCT GGCCAAGOGC AGCAAGCOGT TTGCOGCCCA GGACCEGCSG 301 CAACTCAAGG CCTATGCCGC OGAGCAAGGC TGOGCCGATC TGCAAAGCTG GGACAGCGGT 361 TTCTAOGGCG AAAAACTGCG TGAGCAGOGC TACAGOSTGT CCCAGGAAGC GCTAGGGGCC 421 TACTTCCCCA TOGACAAAGT GCTEGGCGGC CTGTCTGCCA TTGTGCAGOG CCTGTAOGGC 481 ATOGAGATOG COGAGCTTAA AGGCTTOGAC A CCTGGCACC CGGATGTGOG CTTGTTOGAG 541 ATCAAGGSGA AOGGCGAGCA OGTCGGAOGT TTLTTLTTCG ACCTGTACGC COGOGCCAAC 601 AAGOGTGOGG TGCCDGGATG GATGGOGCCC GTGACOGCCG COGTACCGTT GATGGOGTGC 661 TGCAAAGCCC CGTOGCCAAC CTGGTGTGCA ACTTCACTCC GGCOGACAGC GGCAAGCCTS 721 CCCTGCIGAC CTACGAIGAA GTCACCACCC TGTTCCAOGA ATTC or a complementary sequence.
3. A clone of claim 1 having designation pSC22, pLC30, or pLC50.
4. A composition of matter containing at least one peptide expressed by a clone of claim 1, its salt, ester, or amide.
5. A composition of matter comprising a DNA sequence which encodes a protein having an amino acid sequence
Met Thr Tyr Ala Gln Asp Arg Ala Leu Arg Glu Glu Val Tyr Ala Ala Tyr Cys Thr Arg Ala Ser Asp Gln Gly Pro Asn Ala Gly Gln Asn Asp Asn Gly Pro Val Met Glu Gln lle Leu Asp Leu Arg Gln Glu Leu Ala Gln Leu Leu Gly Tyr Ala Ser Pne Ser Glu Leu Ser Leu Ala Thr Lys Met Ala Glu Ser Ser Asp Gln Val Leu Ser Phe Leu Arg Asp Leu Ala Lys Arg Ser Lys Pro Phe Ala Ala Gln Asp Leu Gln Gln Leu Lys Ala Tyr Ala Ala Glu Gln Glv Cvs Ala Asp Leu Gln Ser Trp Asp Ser Gly Phe Tys Gly Glu Lys Leu Arg Glu Gln Arg Tyr Ser Val Ser Gln Glu Ala Leu Arg Ala Tyr Phe Pro lle Asp Lys Val Leu Gly Gly Leu Phe Ala lle Val Gln Arg Leu Tyr Gly lle Glu lle Ala Glu Leu Lys Gly Phe Asp Thr Trp His Pro Asp Val Arg Leu Phe Glu lle Lys Glu Asn Gly Glu His Val Gly Arg Phe Phe Phe Asp Leu Tyr Ala Arg Ala Asn Lys Arg Ala Val Pro Gly Trp Met Ala Pro Val Thr Ala Ala Val Pro Leu Met Ala Cys Cys Lys Ala Pro Ser Pro Thr Trp Cys Ala Thr Ser
Leu Arg Pro Thr Ala Ala Ser Leu Pro Cys
or a fragment thereof.
6. A composition of claim 4 in a pharmaceutically acceptable carrier.
7. A vector of claim 1 which is pLC30, pLC50, pSC22, or pSN30 containing the designated DNA insert.
8. A composition of claim 6 which contains an adjuvant.
9. A composition of matter which comprises a polypeptide produced by a clone of claim 1 which has been labeled .
10. A composition of matter comprising at least one polyclonal or monoclonal reactive to an antigen produced by the clones of claim 1 in a carrier.
11. A composition of matter comprising cell growth media from cell cultures transfected with a clone of claim 1.
12. A composition of claim 5 having as a component whole blood or blood serum.
13. A method for detecting presence of non-A, non-B hepatitis DNA, DNA fragments, or RNA produced therefrom comprising the steps of:
(1) isolating DNA from growth media containing a clone of claim 1;
(2) mixing test serum of cells from biopsy with an isolate obtained in step (1) under hybri- dizing conditions; and
(3) observing samples to identify presence or absence of hybridization.
14. A method of claim 13 wherein the test used is a Southern blot, Northern blot, or slot blot test.
15. A method of claim 13 wherein auto- radiography is the method used to identify presence of hybridization.
16. A method of identifying., presence of antibodies to non-A, non-B hepatitis in a sample by contacting the sample with an antigenig peptide composition of claim 4 and observing evidence of antigen-antibody interaction.
17. A method of claim 16 wherein the test is an ELISA test.
18. A clone or vector containing a DNA insert which encodes a protein having in whole or in part the amino acid sequence
Sequence of 464
1 GAATTOGTGG AACAGGGTGG TGACTTCATC GTGGGTCAGC AGGGCSGGCT TGCOSCTGTC 61 GGCCGGAGTG AAGTTGCACA CCAGGTTGGC GAQGGGGCTT TGCAGCAOGC CATCAACGGT
121 AOGGCGGOGG TCAOGGGCGC CATCCATCCA GGCACOGCAC GCTTGTTGGC GOGGGOGTAC 181 AGGTOGAAGA AGAAAOGTCC GAOGTGCTOG COGTTCTCCT TSATCTCGAA CAAGOGCACA 241 TCCGGGTGCC AGGTGTOGAA GCCTITAAGC TCGGOGATCT CGATGCOGTA CAGGCGCTGC 301 ACAATGGCAA ACAGGCCGCC CAGCACTTTG TGGATGGGGA AEΠAGGCGCG TAGOGCTTCC 361 TGGGACACGC TGTAGCGCTG CTCAOGCAGT TT TTCGCCGT AGAAACCGCT GTCECAGCTT 421 TGCAGATOGG CGCAGCCTTG CTOGGCGGCA TAGGCCTTGA GTTG
Sequence displayed from position 1 to end ( position
464 )
Sequence numbered from position 1
or a DNA sequence complementary thereof .
19 . A clone of vector having a DNA insert containing the sequence
Sequence of 464
1 GAATTOGTGG AACAGGGTGG TGACTTCATC GTGGGTCAGC AGGGCAGGCT TGCOGCTGTC 61 GGCQGGAGTG AAGTTGCACA CCAGGTTGGC GAGGGGGCTT TGCAGCAOGC CATCAAOGGT 121 ACGGCGGGGG TCACGGGCGC CATCCATCCA GGCACOGCAC GCTTGTIGGC GCGGGCGTAC 181 AGGTCGAAGA AGAAACGTCC GAOCTGCTOG COGTTCTCCT TGATCTOGAA CAAGOGCACA 241 TCCGGGTGCC AGGTGTCGAA GCCTTTAAGC TOGGCGATCT OGATGCCGTA CAGGOGCTGC 301 ACAATGGCAA ACAGGCCGCC CAGCACTTTG TGGATGGGGA AGTAGGCGGG TAGGGCTTCC 361 TGGGACACGC TGTAGGGCTG CTCAOGCAGT TTTTCGCOGT AGAAACCGCT GTCfXAGCTT 421 TGCAGATOGG OGCAGCCTTG CTOGGCGGCA TAGGCCTTGA GTTG
20 . A DNA sequence of the structure
Sequence of 464
1 GAATTOGTGG AACAGGGTGG TGACTTCATC GTGGGTCAGC AGGGCAGGCT TGCCGCTGTC
61 GGCOGGAGTG AAGTTGCACA CCAGGTTGGC GAOGGGGCTT TGCAGCAOGC CATCAAOGGT
121 AOGGCGGOGG TCACGGGCGC CATCXATCCA GGCACGGCAC GCTTGTTGGC GOGGGOGTAC 181 AGGTOGAAGA AGAAACGTCC GAOGTGCTOG CCGTTCTCCT TGATCTOGAA CAAGCGCACA
241 TCOGGGTGCC AGGTGTCGAA GCCTTTAAGC TOGGCGATCT OGATGCOGTA CAGGCGCTGC
301 ACAATGGCAA ACAGGCCGCC CAGCACTTTG TOGATGGGGA AGTAGGCGGG TAGOGCTTCC
361 TGGGACACGC TGT AGCGCTG CTCAOGCAGT TTTTOGCOGT AGAAACCGCT GTCCCAGCTT
421 TGCAGATOGG OGCAGCCTTG CTOGGCGGCA TAGGCCTTGA G TTG
or a strand complementary thereto , or a fragment of either the designated sequence or a complementary strand .
21 . A DNA fragment of claim 20 containing at least 20 base pairs .
22 . A composition of matter comprising at least one peptide having an amino acid sequence expressed by a clone or vector of claim 18 .
23 . A composition of matter comprising the full complement of proteins expressed by a clone of claim 18.
24. A vector of claim 18 which is pSN31 or pSN32.
25. A composition of claim 23 formulated as an immunogen.
26. A polypeptide produced by a clone of claim 18 which has been labeled.
27. A composition of matter comprising at least one polyclonal or monoclonal antibody reactive with an antigen produced by a clone of claim 18.
28. A composition of matter comprising cell growth media from cell cultures transfected with a clone of claim 18.
29. A composition of matter comprising a DNA sequence of claim 18 and whole blood or blood serum.
30. A method of detecting presence of non-A, non-B hepatitis DNA, DNA fragments, or RNA produced therefrom comprising the steps of:
(1) mixing test serum or cells from biopsy with a DNA sequence of claim 18 under hybridizing conditions; and
(2) observing samples to identify the presence or absence of hybridization.
31. A method of claim 30 wherein the test used is a Southern blot. Northern blot, or slot blot test.
32. A method of claim 30 wherein auto- radiography is the method used to identify presence of hybridization.
33. A method of identifying the presence of antibodies to non-A, non-B hepatitis in a sample by contacting the sample with an antigenic peptide composition of claim 22 and observing for evidence of antigen-antibody interaction.
34. A method of claim 31 wherein the test is an ELISA test.
PCT/US1989/003638 1988-08-24 1989-08-24 Detection of non-a, non-b hepatitis WO1990002206A1 (en)

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US5106726A (en) * 1990-02-16 1992-04-21 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV
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US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain 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
US7166287B1 (en) 1989-12-18 2007-01-23 Glaxo Wellcome Inc. Viral agent
US6210675B1 (en) 1989-12-18 2001-04-03 Glaxo Wellcome Inc. PT-NANB hepatitis polypeptides
US5106726A (en) * 1990-02-16 1992-04-21 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV
US5436126A (en) * 1990-02-16 1995-07-25 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and prevention thereof as vaccines
US5747239A (en) * 1990-02-16 1998-05-05 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and preventions thereof as vaccines
US5714314A (en) * 1990-06-12 1998-02-03 National Institute Of Health Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method
US5734019A (en) * 1990-06-12 1998-03-31 National Institute Of Health Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method
US5998130A (en) * 1990-06-25 1999-12-07 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
US6217872B1 (en) 1990-06-25 2001-04-17 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
EP0544838A1 (en) * 1990-08-25 1993-06-09 New York Blood Center, Inc. Non-a, non-b hepatitis virus antigen, diagnostic methods and vaccines
EP0544838A4 (en) * 1990-08-25 1997-09-10 New York Blood Center Inc Non-a, non-b hepatitis virus antigen, diagnostic methods and vaccines
US5635346A (en) * 1991-03-26 1997-06-03 Dade International Inc. Assay for Non-A Non-B hepatitis
EP0531974A1 (en) * 1991-09-12 1993-03-17 Cedars-Sinai Medical Center Direct detection of hepatitis C virus RNA

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