EP0380656A1 - Defiziente hepadna-viren, produktive zellinie für impfstoffe sowie behandlung von erkrankungen und störungen der leber - Google Patents

Defiziente hepadna-viren, produktive zellinie für impfstoffe sowie behandlung von erkrankungen und störungen der leber

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Publication number
EP0380656A1
EP0380656A1 EP89910500A EP89910500A EP0380656A1 EP 0380656 A1 EP0380656 A1 EP 0380656A1 EP 89910500 A EP89910500 A EP 89910500A EP 89910500 A EP89910500 A EP 89910500A EP 0380656 A1 EP0380656 A1 EP 0380656A1
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European Patent Office
Prior art keywords
dna
hepadnavirus
defective
sequence
dna sequence
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English (en)
French (fr)
Inventor
Arthur Louis Horwich
Jesse William Summers
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Yale University
Fox Chase Cancer Center
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Yale University
Fox Chase Cancer Center
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Publication of EP0380656A1 publication Critical patent/EP0380656A1/de
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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
    • 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
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to replication-defective hepadnaviruses.
  • the invention relates to defective hepadnaviruses that are incapable of replication by themselves, yet whose pregenomic RNA, in the presence of an appropriate helper virus function, can be packaged and reverse transcribed into DNA.
  • the genomic DNA of such viruses can have deletions in the env, pol, and/or core genes.
  • the invention also relates to hepadnavirus packaging genomes, whose encoded pregenomic RNA cannot itself be packaged and/or reverse-transcribed into a double stranded DNA genome, yet is capable of supplying functions required in trans for packaging.
  • the defective hepadnaviruses of the invention which express immunogenic epitopes may be formulated as vaccines, or used as
  • immunostimulatory agents for the production of an immune response against hepatitis virus antigens.
  • the present invention is also directed to
  • defective hepadnaviruses which contain a heterologous gene sequence.
  • these recombinant viruses may be used for gene therapy of an inherited deficiency of an hepatic enzyme or an enzyme whose deficiency can be replaced by hepatic production.
  • recombinant hepadnaviruses containing a heterologous gene sequence encoding an imunogenic epitope may be formulated as vaccines for protection against
  • the present invention is also directed to the generation and maintenance of permanent hepatic cell lines which are stably transfected with the defective hepadnaviruses of the invention and are capable of producing infectious defective hepadnavirus particles.
  • the defective hepadnaviruses of the invention may also be formulated as therapeutic interfering agents, preventing the propagation or maintenance of infection by wild-type virus.
  • HEPADNAVIRUSES Hepadnaviruses, which include human hepatitis B virus (HBV) (Barker et al., 1975, Am. J. Med. Sci.
  • DHBV duck hepatitis B virus
  • Hepadnavirus virions are approximately 42 nm in diameter and consist of an envelope and nucleocapsid
  • the envelope contains the hepatitis B surface antigen (HBsAg) as well as
  • the nucleocapsid contains a circular DNA (3.0-3.3 kb in length), a DNA polymerase, protein kinase activity, and hepatitis B core antigen
  • the hepadnavirus genome is a small, circular, partly double stranded DNA molecule (reviewed in Ganem and Varmus, supra and Tiollais et al., supra).
  • the minus strand is linear and of a fixed length, 3-3.2 kb.
  • the plus strand is of variable length, ranging from 50-100% of that of the minus strand.
  • DHBV contains S, C, and P ORFs (see figure 1).
  • ORF S which codes for HBsAg, is divided into the S gene, pre-S1 region, and pre-S2 region.
  • ORF C which codes for HBcAg, is divided into the C gene and pre-C region.
  • ORF P encodes the viral
  • ORF X can potentially encode a
  • the first step involves the conversion of the
  • the covalently closed circular DNA is then transcribed by RNA polymerase II to generate two RNA species, genomic and subgenomic.
  • the genomic RNA (3.5 kb) contains the full complement of viral genetic
  • RNAs 2.1 and 2.4 kb in length, are most likely mRNAs for the pre-S1 (2.4 kb transcript) and pre S2 and S
  • Minus strand DNA is then synthesized by reverse transcription of pregenomic RNA using the viral
  • DNA is accomplished by copying the minus strand
  • DR1 and DR2 play important roles in this step (further detail is provided in Ganem and Varmus, supra; Seeger et al.,
  • RNA primer is apparently employed, derived from the 5' portion of the pregenomic RNA. This primer contains the DR1 sequence which has been proposed to anneal to the DR2 sequence present in the minus strand (Madson et al.,
  • the plus strand synthesis proceeds for variable distances, often terminating before complete copying of the minus strand.
  • HBsAg 22 nm surface antigen particles were shown to be excreted into the cell culture medium.
  • Patent Publication No. 0020251 (published December 10,
  • HBV infection has been observed to be highly polymorphic, ranging from inapparent forms in which individuals experience mild or no liver injury to acute hepatitis B, a moderately severe illness
  • the virus itself appears not to be cytotoxic. Variation in host immune response to virus infected cells appears to be a major determinant in the degree of severity of liver damage in individuals. Both humoral and cellular immune responses to HBcAg and HBsAg has generally been observed during acute and chronic HBV infection (reviewed in Robinson, 1986, In Fundamental Virology, Fields, B.N. and Knipe, D.M.
  • HBV DNA has been detected in such nonhepatic tissues as kidney, pancreas, and skin (Robinson, supra). Free viral DNA has also been detected in the kidney and pancreas of infected Peking ducks. HBV DNA has also been
  • hepatocytes in peripheral blood leukocytes and the bone marrow cells.
  • Hepadnavirus DNA in hepatocytes can exist as either free DNA or integrated into the host cellular chromosome (Tiollais et al., 1985, Nature (London) 317:489-495). Free HBV DNA is detected during acute and some chronic stages of HBV infection and usually represents intermediate forms of replication. In contrast, integrated sequences are mostly observed during chronic virus infection and hepatocellular carcinoma.
  • hepatocytes of hepatoma patients have been found to contain HBV DNA and HBsAg. Hepatoma has also been observed in animals chronically infected with hepadnaviruses. Furthermore, hepatoma was able to be induced experimentally in woodchucks by inoculation with WHBV at birth.
  • Virol. 62:861-862 hepatocellular carcinoma has been studied by Southern blot hybridization. It has generally been observed that viral DNA is integrated into the host chromosomal DNA at 1 to 12 sites. The virus-host chromosome junctions have generally been located in the vicinity of the cohesive end region between DR1 and DR2.
  • vidarabine phosphate can be administered by rapid intravenous infusion or intramuscularly. Even though vidarabine phosphate therapy resulted in the clearance of serum hepatitis B virus DNA, it did not lead to a sustained improvement in the accompanying liver disease.
  • Other antivirals that have been tested for their effectiveness in treating HBV infection include acyclovir and suramin.
  • U.S. Patent No. 4,741,901 discloses a vaccine comprising a 22 nm polypeptide particle made of mature hepatitis B surface antigen.
  • RNA viruses as vesicular stomatitis virus (VSV), parainfluenza virus, influenza viruses, alphaviruses and reoviruses
  • DI defective interfering
  • DI particles are not capable of self-replication in host cells, but do replicate when they coinfect a cell with a homotypic infectious virus (helper virus) that supplies the missing gene products. Replication of helper virus is greatly suppressed (autoinferference) since DI
  • VSV DI particles have been found to play a role in both acute and chronic infections. It has been shown that VSV DI particles modulate virulence in mice by initiating a cyclic pattern of VSV growth in vivo
  • DI particles may lie in aberrant replication events (Lazzarini et al., 1981, Cell
  • Defective retroviruses primarily differ from other RNA defective viruses in that defective
  • retroviruses generally do not modulate infection of full length retrovirus particles. However, defective retroviruses like other defective RNA viruses can only replicate in the presence of helper virus.
  • the helper virus and the defective virus need not be the same retrovirus; for example, a leukemia virus can act as helper virus to a defective sarcoma virus.
  • Nondefective retroviruses contain a long terminal repeat (LTR) region at the 5' and 3' end of the LTR
  • gag, pol and env genes are reviewed in Watson et al., 1987, In Molecular Biology of the Gene, Vol. 2, Benjamin/Cummings Publishing Co., Menlo Park, CA and Hanafusa, 1977, In Comprehensive Virology,
  • Nondefective retroviruses have not contained oncogenes.
  • Nondefective retroviruses generally induce leukemias after fairly long latent periods (e.g., HTLV I, feline leukemia virus).
  • glycoprotein made by the non-defective virus is N-defective virus
  • Defective retroviruses have also been used in the process of gene transduction in which the defective virus carries foreign DNA sequencs. For example, the transduction of primary cultures of adult rat
  • hepatocytes by replication-defective retroviruses that constitutively express high levels of ⁇ -galactosidase has been reported (Wilson et al., 1988, Proc. Natl.
  • DI particles have been observed to be generated from such DNA viruses as papovaviruses, herpes
  • RNA viruses these DI particles of DNA viruses are generated after high titer infection with wild-type virus.
  • DI particles of many different structures are also generated from DNA viruses which contain deletions, substitutions, and duplications of the wild-type genome.
  • DI particles have also been observed to attenuate acute virus infection in papovaviruses (Brochman, 1977, Proc. Med. Virol. 23:69-85) and adenoviruses (Larsen, 1982, Virology, 116:573-580).
  • papovaviruses Brochman, 1977, Proc. Med. Virol. 23:69-85
  • adenoviruses Larsen, 1982, Virology, 116:573-580.
  • DI particles from DNA viruses are thought to be involved in persistent infection.
  • DI genomes replicate autonomously in cells transformed by BK virus , a human papovavirus (Yogo et al., 1980, Virology 103:241-244).
  • Attenuation refers to the production of virus strains which have essentially lost their disease producing ability.
  • One way to accomplish this is to subject the virus to unusual growth conditions and/or frequent passage in cell culture. Viral mutants are then selected which have lost virulence but yet are capable of eliciting an immune response.
  • Attenuated viruses generally make good immunogens as they actually replicate in the host cell and elicit long-lasting immunity.
  • subunit vaccines for example, see U.S. Patent No. 3,636,191 by Blumberg and Millman. This involves immunization only with those proteins which contain the relevant immunological material.
  • subunit vaccines One advantage of subunit vaccines is that the irrelevant viral material is excluded. For many enveloped viruses, the virally encoded glycoprotein contains those epitopes which are capable of eliciting neutralizing antibodies.
  • the HBV subunit vaccine contains the hepatitis B surface antigen (HBsAg) purified from the blood of chronically infected carriers (Krugman, 1982, J.A.M.A.
  • This vaccine has been shown to be effective and safe in high risk adult populations, e.g., drug addicts and homosexuals and in newborn infants.
  • the vaccine is very expensive due to the finite supply of serum, complex purification and invactivation process as well as the lengthy safety test in chimpanzees required for its production and certification.
  • Subunit vaccines may also be prepared using synthetic peptides representing immunologically imported domains of surface antigens. This approach has been attempted with a number of viruses, e.g., foot and mouth disease (Bittle et al., 1982, Nature
  • the peptide is coupled to a carrier (e.g., hemocyanin, Poly-DL-alanine) and administered in conjunction with an adjuvant.
  • a carrier e.g., hemocyanin, Poly-DL-alanine
  • adjuvant e.g., Freund's adjuvant, a carcinogen, to potentiate a strong immune response, since they are relatively poor antigens.
  • the use of recombinant DNA technology for the production of subunit vaccines involves the molecular cloning and expression in an appropriate vector of the viral genetic information coding for those proteins which can elicit a neutralizing response in the host animal.
  • HBV and other viruses are examples.
  • One example involves the transfection of yeast with a recombinant in which
  • HBsAg is inserted into a yeast expression vector downstream from an inducible promoter (reviewed in
  • Yeast however, must be disrupted to release HBsAg.
  • the antigen is then purified by isopycnic and rate zonal centrifugation combined with immunoaffinity chromatography.
  • recombinant viruses Analogous approaches have been attempted in mammalian cells using recombinant viruses.
  • a virus is used as a vector to express foreign genes inserted into its genome.
  • the recombinant virus Upon introduction into host animals, the recombinant virus expresses the inserted foreign gene and may thereby elicit a host immune response to such gene product.
  • HBsAg as well as other surface antigens have been produced using recombinant vaccinia virus (Smith et al., 1983, Nature (London)
  • bovine papilloma virus (Statowa et al., supra, pp. 239-243), and adenoviruses (Morin et al.,
  • Gene therapy refers to the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of diseases or disorders.
  • the foreign gene is transferred into a cell that proliferates to spread the new gene throughout the cell population.
  • stem cells, or pluripotent progenitor cells are usually the target of gene transfer, since they are proliferative cells that produce various progeny lineages which will
  • DEAE dextran shows the capability of integrating transferred genes stably in a wide variety of cell types.
  • Recombinant retrovirus vectors have been widely used experimentally to transduce hematopoietic stem and progenitor cells.
  • Genes that have been successfully expressed in mice after transfer by retrovirus vectors include human hypoxanthine
  • Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance gene
  • Adenoassociated virus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance (Hermonat and Muzyczka,
  • the present invention is directed to replication defective hepadnaviruses.
  • the present invention is directed to replication defective hepadnaviruses.
  • invention relates to two types of defective
  • the first type (termed herein "particle-defective" hepadnavirus genomes) are
  • hepadnaviruses containing such a particle-defective genome can be defective in production of core antigen (HBcAg), viral polymerase, and/or surface antigen (HBsAg).
  • HBcAg core antigen
  • HBsAg surface antigen
  • the second type of defective hepadnavirus genomes according to the present invention are genomes which can be transcribed into pregenomic
  • mRNAs that encode protein(s) which are able to supply an in trans viral function necessary for packaging a second RNA into virions or for transcribing the second RNA into virion DNA.
  • packaging genomes include but are not limited to genomic DNA which contains a deletion in the direct repeat (DR) region, DR1.
  • DR direct repeat
  • Hepadnavirus virion particles containing a particle-defective genome can be produced by
  • helper hepadnavirus packaging genome(s).
  • virion can also be produced by coexpression of the particle-defective genome and nucleic acid vector (s)
  • plasmids e.g., plasmids
  • trans packaging functions e.g., reverse transcriptase, core, envelope functions
  • hepadnavirus containing a particle-defective genome
  • packaged particle-defective genome can then be used for the infection of a hepatocyte, to which the particle-defective DNA is delivered and in which it can be expressed, but which viral DNA cannot then bring about another round of hepadnavirus infection due to its defective nature.
  • the present invention also relates to therapeutic uses for both packaged particle-defective genomes and packaging genome products. Both packaged
  • hepadnavirus packaging genome products which express immunogenic epitopes (e.g., HBsAg)
  • immunogenic epitopes e.g., HBsAg
  • such viruses may be used as immunostimulatory agents for the treatment of hepadnavirus infection or its
  • the present invention is also directed to
  • packaged recombinant particle-defective genomes which comprise a heterologous gene sequence
  • various regions of the hepadnavirus genome which include but are not limited to those sequencs encoding the viral functions that can be complemented in trans by the packaging genomes can be replaced with a heterologous gene sequence which will be expressed under the control of viral or other regulatory sequences when introduced into a given organism.
  • Such recombinant viruses may be used for genetic therapy of enzyme def iciencies which can be treated by hepatic enzyme production (e.g. blood coagulation factors).
  • recombinant hepadnaviruses containing a heterologous gene may be formulated as vaccines for protection against infection by a pathogenic organism or for protection against conditions or disorders caused by the presence of an antigen.
  • packaged particle-defective genomes can be used as agents which interfere with propagation or maintenance of infection by wild-type virus, and/or eradicating or mitigating wild-type virus infection.
  • permanent cell lines are stably
  • transfected with a hepadnavirus packaging genome whereby such cell lines become capable of permanently expressing products required in trans for packaging and reverse transcription of a second defective genome
  • DHBV duck hepatitis B virus
  • DR direct repeat region, e.g., DR1 or DR2, of a hepadnavirus genome
  • FBS fetal bovine serum
  • GSHBV ground squirrel hepatitis B virus
  • HBV hepatitis B virus (humans)
  • HBcAg core antigen of DHBV, GSHBV, HBV or WHBV
  • NTP nucleoside triphosphate
  • WHBV woodchuck hepatitis B virus DR1 a DHBV packaging genome, having a deletion of the 12 bp direct repeat termed DR1 which is required for complete synthesis of a double-stranded viral DNA molecule
  • Packaging cell line a permanent cell line stably incorporating at least one hepadnavirus packaging genome, which cell line is capable of expressing viral products necessary for in trans
  • Figure 1 Diagrammatic representation of the structural and functional features of DHBV DNA as well as of major transcripts (a); structure of
  • FIG. 1 Immunofluorescent staining of HuH7 cells transfected with particle-defective DHBV DNA. Cells were transfected with the following DNAs:
  • RV-718 (pol-), RV-2650 (core-), Kpn- (pol-env-), or Kpn-Sph- (pol-env-core-), and stained after five days incubation for core ( ⁇ -c) or surface ( ⁇ -S) antigens.
  • ⁇ -c core-
  • ⁇ -S surface antigens.
  • FIG. 3 Southern blot hybridization assays of the appearance of viral replicative DNA in HuH7 cells transfected with DNA of pol-env-, and pol-env-core- viral mutants and ⁇ -DHBV recombinants. Plates (60 mm) of HuH7 cells (2-3 X 10 6 cells/dish) were transfected with DNA of wild-type DHBV (lanes 1,3), wild-type + Kpn-Sph- (pol-env-core-) (lanes 2,4), DR1 (lane 5), ⁇ DR1 + Kpn-+1000 (lane 7), ⁇ DR1 + Kpn-+180 (lane 8), ⁇ DR1 + Kpn-Sph- (pol-env-core-) (lane 9). Southern hybridization analysis was carried out as described in Section 6.4.2, infra, with a DHBV RNA probe of
  • Lane 10 contained 1 picogram cloned DHBV DNA as a hybridization standard. Lane 11 contains ⁇ -HindIII molecular size markers. Exposure times were 4 hours (lanes 1 and 2) or 48 hours (lanes
  • FIG. 1 Immunofluorescent staining of HuH7 cells transfected with the packaging genome, ⁇ DR1.
  • Cells transfected with wild-type DHBV DNA (WT) or ⁇ DR1 DNA ( ⁇ DR1) were stained for core ( ⁇ -C ) or surface ( ⁇ -S) antigens.
  • WT wild-type DHBV DNA
  • ⁇ DR1 ⁇ DR1 DNA
  • PC Phase contrast photograph.
  • Viral DNA was assayed as described for figure 3. Cells were
  • FIG. 6 Assay of transfected HuH7 cell culture supernatants for infectious virus.
  • Supernatant fluids (2 ml) from HuH7 cells transfected with 1.5, 3 or 6 ⁇ g wild-type DHBV DNA (lanes 1, 2, 3, respectively) or 3 ⁇ g of DNA of ⁇ DR1 (lanes 4,5), Kpn- (pol-env-) (lanes 6,7), or Kpn-Sph- (po l- env-core-) (lanes 8,9) were incubated overnight with cultures of primary duck hepatocytes. Infected hepatocytes were incubated for 12 days and total DNA was extracted and assayed for viral replicative DNA forms by Southern blot
  • Lane 10 contained ⁇ -Hindlll molecular size markers. The positions of relaxed circular (RC) and single-stranded
  • HuH7 cells Supernatant fluids from HuH7 cells transfected with viral DNAs were assayed for
  • Viral DNA was obtained from hepatocytes infected by incubation with culture fluids of cell transfected with DNA of wild-type DHBV (lanes 1,2), wild-type + Kpn- (pol-env-) (lanes 3,4), wild-type +
  • FIG. 10 Immunofluorescent staining of primary duck hepatocytes following infection with wild-type DHBV (column 1), and with supernatant fluids recovered from HuH7 cells cotransfected with the defective genomes ⁇ DR1 and 1S (column 2), and stained for presence of core antigen.
  • FIG. 10 The plasmid pHBV ⁇ DR1 Neo, showing the orientation and relative positions of the dimerized ⁇ DR1 sequence, the selectable marker, and certain restriction sites.
  • R EcoRI
  • B BamHI
  • SV SV40 regulatory DNA.
  • FIG. 11 Flow chart representation of the construction steps for preparation of the plasmid pHBV ⁇ DR1Neo.
  • Amp R ampicillin resistance gene
  • restriction sites are as illustrated.
  • Figure 13 Representation of the deduced arrangement of ⁇ DR1 DNA sequences, derived from the plasmid pHBV ⁇ DR1Neo, as integrated into host cellular DNA in HepB1-2 cells.
  • FIG. 14 Southern blot hybridization analysis of nucleic acids isolated from different density gradient fractions of supernatant from Hep B1-2 cell cultures using a nick-translated HBV probe. Supernatant fractions were separated by centrifugation in cesium chloride, and nucleic acids isolated from the fractions, as described in Section 8.2, infra.
  • Transfections were performed with 5 micrograms (lanes 1-3) and 1 microgram (lanes 4-6) of X- plasmid DNA.
  • the present invention relates to
  • the invention relates to two types of defective hepadnavirus genomes and the nucleic acid sequences thereof.
  • the first type (termed herein
  • RNA-defective genome is incapable by itself of supplying all the hepadnaviral functions required for replication, but is able to produce a pregenomic RNA with the appropriate cis-acting signals required for inclusion of the RNA in virions (packaging) and for reverse transcription into DNA.
  • particle-defective genome can be defective in the synthesis of core antigen (HBcAg), viral polymerase, and/or surface antigen (HBsAg).
  • the second type of defective hepadnavirus genome according to the present invention is a genome which can be transcribed into a pregenomic RNA that is incapable of being reverse-transcribed into a double-stranded DNA genome and/or being packaged itself, yet which genome can be transcribed into messenger RNA(s) which encode one or more proteins that are able to supply in trans viral functions necessary for packaging a second pregenomic RNA into virions or for transcribing the second RNA into virion DNA.
  • packaging genomes can provide "helper function," to package particle-defective genomes.
  • Packaging genomes include but are not limited to those hepadnavirus genomes which contain deletions in the direct repeat (DR) regions, DR1 and/or DR2.
  • the particle-defective hepadnavirus genomes of the invention which in a specific embodiment contain heterologous gene sequences, can be packaged by expression of hepadnavirus proteins required in trans for packaging (e.g. core, reverse transcriptase, envelope) which the particle-defective genome itself cannot supply in functional form.
  • hepadnavirus proteins required in trans for packaging e.g. core, reverse transcriptase, envelope
  • Such expression results in the production of a hepadnavirus virion particle containing a particle-defective genome
  • particle-defective virus such in trans packaging function(s) can be provided by expression of genes of one or more nucleic acid vectors (e.g. plasmids) which encode protein(s) supplying such function(s).
  • the particle-defective genomes can be packaged by the expression of one or more hepadnavirus packaging genomes.
  • expression of the packaging genomes supplies the functions needed to permit the particle-defective genome to be included in viral particles that are subsequently released from the cells.
  • packaging of particle-defective genomes can be accomplished by the introduction of the
  • particle-defective viruses that are produced can then be used for the infection of a hepatocyte or other susceptible cell, in vitro or in vivo, to which cell the particle-defective DNA is delivered and in which it can be expressed, but which viral DNA cannot then bring about another round of hepadnavirus infection due to its defective nature.
  • the particle-defective hepadnavirus genome can be
  • a limited round of hepadnavirus multiplication may be induced in a host cell incorporating a particle-defective genome, by the administration of a conditional replication-defective hepadnavirus (e.g., a helper virus genome under the control of an inducible promoter), which would act as a "helper" virus only under certain conditions.
  • a conditional replication-defective hepadnavirus e.g., a helper virus genome under the control of an inducible promoter
  • Such a limited viral multiplication could provide for further, yet limited and non-pathogenic spread, of the particle-defective virus DNA to other host cells.
  • a conditional viral propagation may be preferred.
  • defective hepadnaviruses can be used as antiviral agents in the treatment of hepadnavirus infection.
  • viruses can be formulated as interfering agents that inhibit propagation or maintenance of wild-type virus.
  • packaged particledefective viruses or hepadnavirus packaging genome products which express immunogenic epitopes (e.g., HBsAg), may be formulated as vaccines for the
  • the present invention is also directed to
  • hepadnavirus genomes which comprise a heterologous gene sequence, viruses containing such genomes , and the therapeutic uses of such viruses.
  • Various regions of the hepadnavirus genome which include but are not limited to those encoding the viral functions that can be complemented in trans by the packaging genomes can be replaced with a heterologous gene sequence, such that the
  • heterologous sequence will be expressed under the control of hepadnaviral or heterologous regulatory sequences when introduced into the appropriate cell.
  • viruses containing these recombinant genomes may be used for genetic therapy of such deficiencies which can be treated by hepatic enzyme production.
  • recombinant hepadnaviruses containing a heterologous gene sequence encoding an immunogenic epitope may be formulated as vaccines for protection against infection by the heterologous organism or for protection against conditions or disorders caused by the presence of an antigen.
  • the present invention is directed to
  • replication-defective hepadnavirus genomes that include but are not limited to particle-defective genomes which may be packaged by functions supplied in trans, and packaging genomes, which are capable of supplying packaging functions in trans.
  • the invention is also directed to nucleic acid sequences encoding the sequence of the particle-defective or packaging genomes.
  • Packaged particle-defective hepadnavirus genomes include but are not limited to hepadnavirus mutants defective in the synthesis of core antigen (HBcAg), viral polymerase, and/or surface antigen (HBsAg).
  • the defect in expression may be due to a deletion encompassing a portion of or the entire coding region for the given protein(s).
  • the packaged particle-defective genomes are defective in the production of one, two, or all three of the above hepadnavirus proteins.
  • such packaging genomes can have deletions in the DR1 (direct repeat 1) or DR2 region.
  • the hepadnavirus genome has a deletion in the DR1 region.
  • the packaging genome can have deletions in both the DR1 and DR2 regions. The DR1 and DR2 regions have been shown to be involved in the initiation of viral DNA synthesis.
  • the types of defective hepadnaviruses whose DNA can be used include but are not limited to hepatitis B virus (HBV), pathogenic in humans and higher primates, ground squirrel hepatitis B virus (GSHBV), duck hepatitis B virus (DHBV), and woodchuck hepatitis B virus (WHBV).
  • HBV hepatitis B virus
  • GSHBV ground squirrel hepatitis B virus
  • DHBV duck hepatitis B virus
  • WHBV woodchuck hepatitis B virus
  • the hepadnavirus DNA may be obtained from hepadnavirus infected hepatocytes or from cloned DNA by known techinques (see, e.g.,
  • the isolated hepadnavirus DNA is treated to generate a mutation that will render the virus
  • the defective viruses may be identified by: (a) nucleic acid hybridization (b) presence or absence of "marker” gene functions, or (c) expression of defective hepadnavirus genes.
  • the wild-type hepadnavirus genomic DNA may be obtained from hepadnaviruses such as HBV, DHBV, GSHBV, and WHBV. These genomic DNAs have been cloned and sequenced (Seeger et al., 1984, J. Virol. 51:367-375; Mandart et al., 1984, J. Virol. 49:782-792; Galibert et al., 1982, 41:51-65; and Valenzuela et al., 1980, In: Animal Virus Genetics, Field, B.N., Jaenisch, R. and Fox, C. F., eds., Academic Press, NY, pp. 57-70). If the cloned hepadnavirus DNA is not readily.
  • hepadnavirus DNA may be obtained from hepatocytes infected with hepadnavirus (See, e.g., Tuttleman et al., 1986, J. Virol. 58:17-25).
  • HBV, GSHBV, DHBV or WHBV DNA may be obtained from purified virus using standard procedures known in the art (See, e.g., Mason et al., 1980, J. Virol. 36:829-836).
  • the hepadnavirus DNA may be obtained from purified virus using standard procedures known in the art (See, e.g., Mason et al., 1980, J. Virol. 36:829-836).
  • the hepadnavirus DNA may be obtained from purified virus using standard procedures known in the art (See, e.g., Mason et al., 1980, J. Virol. 36:829-836).
  • the hepadnavirus DNA may be obtained from purified virus using standard procedures known in the art (See, e.g., Mason et al., 1980, J. Virol. 36:829-836).
  • the hepadnavirus DNA may be obtained from purified virus using standard procedures known in the art (See, e.g.,
  • Nucleotide sequence analysis of the cloned gene can be carried out by various procedures known in the art, e.g., the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al., 1977, Proc. Natl. Acad. Sci.
  • the viral DNA is treated enzymatically or chemically to generate a mutant virus that is replication-defective, yet which retains the signals required in cis for packaging.
  • the viral DNA is treated with one or more restriction enzymes to cleave the DNA at specific sites and then religated. If an appropriate deletion resulting in cohesive DNA termini are
  • nuclease such as nuclease Bal 31, exonuclease III, ⁇ exonuclease, mung bean nuclease, or
  • T4 DNA polymerase exonuclease activity to name but a few, in order to remove portions of the DNA sequence.
  • Enzymatically treated DNA termini can be modified to facilitate ligation of the viral DNA by any of
  • cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation.
  • oligonucleotide sequence which encodes one or more restriction sites can be ligated to the
  • hepadnavirus sequences are then ligated in vitro, either to each other, or to appropriate expression vector sequences.
  • the generation of particle-defective hepadnaviruses involves the
  • restriction enzyme digestion(s) may only remove a portion of the
  • deletions may be generated in one, two, or three of the above-mentioned hepadnavirus genes.
  • particle-defective hepadnavirus genomes may be
  • particle-defective genomes may be deficient in the expression of a viral protein due to transcriptional (e.g., promoter) or translational defects.
  • a hepadnavirus genome may be rendered particle-defective due to a mutation
  • a core protein that is unable to assemble may be produced.
  • Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia), etc.
  • the generation of hepadnavirus packaging genomes can be accomplished by mutation in the DR1 region (see
  • the DR1 region, or a portion thereof, can be deleted by restriction enzyme
  • the particle-defective genomes and packaging genes can, if desired, be inserted into various nucleic acid vectors.
  • the particle-defective genomes or hepadnavirus packaging genomes are inserted into a plasmid cloning vector which is used to transform appropriate host cells in order to replicate the DNA so that many copies of the hepadnavirus sequences of interest are generated. This can be accomplished by ligating the hepadnavirus sequence into a cloning vector which has complementary cohesive termini.
  • any of numerous techniques known in the art may be used to accomplish ligation of the defective hepatitis virus DNA at the desired sites.
  • restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation.
  • the cleaved ends of the defective hepatitis DNA can be "chewed back" using a nuclease in order to remove portions of the sequence.
  • oligonucleotide sequence which encodes one or more restriction sites can be inserted in a region of the hepadnavirus DNA by ligation to DNA termini.
  • a linker may also be used to generate suitable
  • hepatitis virus sequences can be mutated in vitro or in vivo in order to form new restriction endonuclease sites or destroy preexisting ones, to facilitate in vitro ligation procedures.
  • the genomes of the defective hepatitis viruses may be dimerized using standard procedures known in the art
  • a large excess of the defective hepadnavirus DNA in relation to the vector DNA may be used in the ligation reaction.
  • the hepadnaviral DNA may be obtained in large quantitites by growing
  • transformants isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • genomes have been isolated, they may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding
  • a variety of host-vector systems may be utilized to express the protein-coding sequence.
  • viruses e.g., vaccinia virus, adenovirus, retrovirus, SV40, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA,
  • plasmid DNA or cosmid DNA The expression elements of these vectors vary in their strength and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, (e.g., mouse
  • metallothionien promoter or from viruses that grow in these cells, (e.g., vaccinia virus 75 kb promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted sequences.
  • Specific initiation signals are also required for efficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • Methods used for the insertion of defective hepadnavirus DNA into vectors may include in vitro recombinant DNA and synthetic techniques for such expression vectors as plasmids or bacteriophages or in vivo recombinations (genetic recombination) for virus expression vectors such as vaccinia virus or
  • virus expression vectors such as vaccinia virus, adenovirus, or retrovirus
  • virus expression vectors such as vaccinia virus, adenovirus, or retrovirus
  • particle-defective hepadnavirus or hepadnavirus packaging genome can be modified so that the gene is flanked by virus sequences that allow for genetic recombination in cells infected with the virus
  • hepadnavirus packaging genomes can be identified by three general approaches: (a) nucleic acid
  • the presence of a modified hepadnavirus genome inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the hepadnavirus sequence.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics,
  • recombinants containing the modified hepadnavirus genome can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based on the physical, immunological, or functional properties of the gene product. For example, an ELISA can be used to detect the presence of antigenic determinants reactive with antibodies to HBsAg, HBcAg, or the hepadnaviral polymerase protein.
  • the expression vector comprising the hepadnavirus sequences should then be transferred into an appropriate host. This can be accomplished by any of numerous methods known in the art including but not limited to transformation (e.g., when the vector is a plasmid), phase transduction, calcium phosphate mediated transfection (e.g., mammalian cell virus vectors) or microinjections.
  • a host cell may be chosen which modulates the expression of the inserted sequences, or modifies and proceses the chimeric gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for methallothionein promoters). Furthermore, modifications (e.g., glycosylation) and processing
  • cleavage of protein products may be important for the function of the encoded protein(s).
  • Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • the particle-defective hepadnavirus genome may be packaged into hepadnavirus virion particles (termed herein "packaged particle-defective genomes"). This packaging may be accomplished by supplying
  • nucleic acid vectors which express such hepadnaviral gene products can be used to provide in trans packaging function(s).
  • the particle-defective genome can be packaged by the use of the encoded products of a packaging genome.
  • packaging genome products can be supplied, e.g., by cotransfection of packaging genomes with particle-defective genomes into
  • Virion particles representing packaged particle-defective genomes may then be isolated.
  • the virion particles may be purified using procedures known in the art (for example, see Tuttleman et al., 1986, J. Virol. 58:17-25). Alternatively, the
  • hepadnavirus DNA may be used, e.g., therapeutically, while still cloned into a given vector. 5.1.3. USES FOR DEFECTIVE HEPADNAVIRUS GENOMES
  • products may be used in the prevention and/or
  • vaccines can be formulated from packaging
  • particle-defective genomes (packaged, e.g., by
  • viruses which can be used include but are not limited to HBV, DHBV, GHBV, and WHBV.
  • heterologous gene sequence may be used in vaccine formulations for protection against heterologous pathogens, or in gene therapy approaches.
  • packaged particle-defective genomes may be used as therapeutic agents in the treatment of acute or chronic manifestations of hepatitis resulting from hepadnavirus infection.
  • these defective viruses act by interfering with the replication of wild-type hepadnavirus (see
  • Vaccines for the prevention of hepadnavirus infection may be formulated according to the present invention from defective hepadnaviruses which express an immunogenic epitope, for example, one or more of the epitopes of core antigen and/or surface antigen, which provides protective immunity upon administration to the host.
  • an epitope of the surface antigen is expressed.
  • a vaccine can be formulated from the protein products of
  • empty virion particles i.e., those lacking genomic DNA due to the packaging genome's inability to package its own pregenomic RNA and/or to synthesize its own genomic DNA
  • vaccines i.e., those lacking genomic DNA due to the packaging genome's inability to package its own pregenomic RNA and/or to synthesize its own genomic DNA
  • such empty virions can be harvested from the supernatants of cells
  • the vaccine in another embodiment, can be formulated from packaged
  • particle-defective genomes In this embodiment, an immune response would be generated against epitopes produced by expression of the packaged genome upon introduction into a susceptible cell. Once introduced into such a cell, the particle-defective genome will be expressed, yet will not initiate another round of infection due to its defective nature. In one
  • the vaccine can be formulated from a packaged particle-defective genome whose DNA has deletions in the gene encoding the core antigen.
  • the particle-defective genome can have deletions in the 5' region of the gene encoding the viral polymerase protein, thereby inhibiting the expression of this protein, but not the surface antigen.
  • the hepadnavirus vaccine may be formulated in yet another embodiment of the invention from packaged particle-defective DNA with deletions in both the region encoding the core antigen and the 5' end of the polymerase gene.
  • nucleic acid virion produced by hepadnavirus
  • packaging genome expression in a vaccine formulation can be determined by monitoring the immune response of test animals following immunization with the packaged particle-defective genome or hepadnavirus packaging genome particle.
  • Test animals may include chimpanzees and other primates and eventually human subjects (for HBV immunization).
  • Methods of introduction of the immunogen may include oral, intradermal,
  • the immune response of the test subjects can be analyzed by various approaches such as: (a) the reactivity of the resultant immune serum to
  • the purpose of this embodiment of the invention is to formulate a vaccine in whch the immunogen comprises an epitope of a given hepadnavirus so as to elicit an immune (humoral and/or cell mediated) response to the hepadnavirus epitope that will protect against infection by a given hepadnavirus or against diseases or disorders caused by an hepadnavirus.
  • a vaccine may be univalent or multivalent.
  • Multivalent vaccines can be preared from a singel or a few hepadnavirus virion particles which express one or more hepadnavirus epitopes.
  • multivalent vaccines can be formulated to contain hepadnavirus gene products and/or recombinant viruses when the defective hepadnavirus genome is cloned into a mammalian or insect cell virus vector.
  • the hepadnavirus virion particles can comprise packaged particle-defective genomes, resulting in a virion particle that can undergo only a single round of infection on its own.
  • the immunogenic epitopes are produced by expression of the packaged hepadnavirus genome upon introduction into a susceptible cell.
  • the hepadnavirus virion particle used as immunogen can comprise an "empty shell" devoid of viral DNA (produced by the introduction into a cell and expression therein of an hepadnavirus packaging genome). Unlike the packaged
  • vaccine formulations of the invention include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
  • particles or proteins thereof of the present invention also have potential uses in diagnostic immunoassays, passive immunotherapy, and generation of antiidiotypic antibodies.
  • the generated antibodies may be isolated by standard techniques known in the art (e.g.,
  • immunoassays The antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art, such as those listed supra, may be used for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as
  • the vaccine formulations of the present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of
  • formulations of the present invention can also be used in the production of antiidiotypic antibody.
  • the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol.
  • immunogenic epitope may be used immunotherapeutically.
  • the immunogenic epitope can be of the core antigen.
  • the epitope can be of the surface antigen.
  • hepatoma by virtue of their ability to stimulate an immune response against hepadnavirus.
  • Experimental evidence indicates a correlation between hepadnavirus infection and the incidence of hepatoma on both an epidemiological and molecular level.
  • the immunogens may be administered alone or concurrently with other therapies (e.g., chemotherapy, radiation therapy, etc.).
  • defective hepadnavirus virion particles or proteins containing an immunogenic epitope may be used as immunostimulators to boost the host's immune system, enhancing cell mediated immunity, and facilitating the clearance of a given infectious agent (e.g., hepatitis A virus, cytomegalovirus).
  • a given infectious agent e.g., hepatitis A virus, cytomegalovirus
  • packaged particle-defective genomes may be administered alone or in conjunction with other therapies in the treatment of diseases that affect the above types of cells (e.g., those caused by hepatitis A virus, malaria parasites, cytomegalovirus,
  • particle-defective hepadnavirus genomes may be used as antiviral agents in the treatment of acute or chronic hepadnavirus infection due to their ability to interfere with the replication of wild-type hepadnaviruses.
  • One embodiment of the invention involves hepadnaviruses that produce defective surface antigen, core antigen, or viral polymerase protein.
  • the particle-defective hepadnavirus is deficient in the synthesis of viral polymerase protein, and encodes both a truncated surface antigen protein and a
  • particle-defective hepadnavirus may be administered alone or concurrently with other antiviral agents which include but are not limited to ⁇ -interferon, and vidarabine phosphate.
  • the present invention also relates to packaged particle-defective genomes comprising a heterologous gene sequence.
  • various regions of the hepadnavirus genome which include but are not limited to those encoding the viral functions that can be complemented in trans by the packaging genomes, can be replaced with a
  • heterologous cloned gene in such a way that the heterologous gene will be expressed under the control of viral or other regulatory sequences when introduced into a given organism.
  • heterologous DNA sequences are inserted into the surface antigen-encoding region of a hepadnavirus.
  • heterologous DNA sequences are inserted into the core antigen region of a hepadnavirus.
  • the heterologous sequences can be expressed under the control of hepadnaviral promoters.
  • heterologous transcriptional regulatory regions can be used, by construction of the
  • these recombinant hepadnaviruses have a number of therapeutic applications.
  • these recombinant hepadnaviruses may be used for gene therapy in the treatment of diseases and disorders affecting the liver, or of disorders which may be treated by enzyme production in the liver (see
  • the heterologous gene may code for a hepatic enzyme (s) or a product which is toxic to a given pathogen that is the causative agent of a disorder.
  • Recombinant hepadnavirus genomes can be generated through the use of recombinant DNA techniques known in the art.
  • the hepadnavirus genomes which can be used include but are not limited to hepatitis B virus
  • HBV ground squirrel hepatitis B virus
  • DHBV duck hepatitis B virus
  • WHBV woodchuck hepatitis B virus
  • recombinant hepadnavirus genomes may be divided into the following three steps solely for the purpose of description: 1) isolation of the heterologous sequence, 2) construction of recombinant hepadnavirus DNA, and 3) expression of recombinant hepadnavirus genomes.
  • heterologous DNA sequence which encodes a functional enzyme whose deficiency is the basis of a disorder can be isolated for use (for non-limiting examples of such enzymes, see Table II infra in Section 5.2.2.1).
  • the DNA sequence may encode a blood coagulation factor for use in gene therapy of inherited disorders of coagulation, since the liver is the normal site of the biosynthesis of such factors.
  • the isolated DNA sequence may encode the whole protein sequence of a given enzyme or a functional portion of the sequence representing the active site.
  • the heterologous DNA sequence may encode a gene product that ameliorates disease.
  • the heterologous DNA sequence directed to the treatment of liver disorders resulting from infection by a pathogenic organism, the
  • heterologous gene sequence may encode a product that is toxic to a pathogen without significant detriment to the host, or which interferes with a pathogen's life cycle, etc. in another embodiment of the
  • DNA sequences may encode an altered gene product of a given pathogen or an "anti-sense" sequence (i.e., one that can hybridize to functional nucleic acid of the pathogen).
  • a further embodiment of the invention directed to vaccine use involves the isolation of a DNA sequence which encodes an epitope of a heterologous organism, which when introduced into an appropriate host, produces protective immunity against such an organism or against a condition or disorder caused by an antigen of the organism.
  • packaged recombinant particle-defective genomes comprising a heterologous gene sequence may be formulated as vaccines to prevent diseases caused by a pathogenic organism containing the heterologous gene sequence.
  • pathogenic organisms include but are not limited to those listed in Table I.
  • Plasmodium spp. (malaria parasites)
  • the heterologous sequence of the recombinant hepadnavirus encodes an epitope of a pathogenic microorganism that is hepatotropic.
  • any DNA sequence which encodes an epitope of a malaria parasite of the genus Plasmodium which is immunogenic in a vertebrate host can be isolated for use according to the present invention.
  • the species of Plasmodium which can serve as DNA sources include but are not limited to the human malaria parasites P. falciparum, P. malariae, P. ovale, P. vivax, and the animal malaria parasites P. berghei, P. yoelii, P. knowlesi, and P. cynomolgi.
  • the antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any antigens or fragments thereof which can be any
  • the heterologous epitope to be expressed is an epitope of the circumsporozoite (CS) protein of a species of Plasmodium. (See, e.g., Dame et al.,
  • recombinant hepadnaviruses include but are not limited to the following: epitopes on the hepatitis A antigen
  • the gene sequences encoding the heterologous epitope to be expressed according to the present invention can be isolated by techniques known in the art including but not limited to purification from genomic DNA of the microorganism, by cDNA synthesis from RNA of the microorganism, by recombinant DNA methods (Maniatis, T., et al., 1982, Molecular
  • the heterologous gene sequence replaces the genes coding for the surface antigen and viral polymerase protein. In another embodiment of the invention, the heterologous gene sequence replaces the gene coding for the surface antigen.
  • the recombinant hepadnavirus DNA in this embodiment can retain pre-S DNA sequences which contain promoters for surface antigen expression. In yet another embodiment of the invention, the
  • heterologous gene replaces the gene coding for the core antigen.
  • the recombinant hepadnavirus DNA of this embodiment can also contain pre-C sequences that promote core antigen expression.
  • hepadnavirus genome and of the heterologous DNA can, by techniques known in the art, be cleaved at
  • restriction endonuclease s
  • the particular strategy for constructing gene fusions will depend on the specifc hepadnavirus sequence to be replaced or inserted into, as well as the heterologous sequence to be inserted.
  • Hepadnavirus genomes containng heterologous gene inserts can be identified by three general approaches:
  • nucleic acid hybridization (a) nucleic acid hybridization, (b) presence or absence of "marker” gene functions, and (C) expression of inserted sequences.
  • first approach the presence of a foreign gene inserted in an hepadnavirus vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the foreign inserted gene.
  • second approach the recombinant vector/host system can be identifed and selected based upon the presence or absence of certain "marker" gene functions caused by the
  • heterologous gene is inserted within the polymerase gene sequence of the
  • recombinants containing the heterologous insert can be identified by the absence of polymerase activity.
  • recombinant containing the heterologous insert can be identified by the absence of polymerase activity.
  • hepadnaviruses can be identified by assaying the foreign gene product expressed by the recombinant.
  • Such assays can be based on the physical,
  • a recombinant hepatitis genome contaiing a heterologous gene sequence that replaces hepatitis viral polymerase and surface antigen genes is described.
  • This packaged recombinant particle-defective genome requires for its propagation the concurrent introduction of a "helper" hepatitis virus packaging genome into a given cell line.
  • Such packaging genomes an example of whose construction is detailed in Section 6.3, are capable of providing in trans viral functions necessary for packaging an appropriate pregenomic RNA into virions and
  • the gene product should be analyzed. This can be achieved by assays based on the physical, immunological or functional properties of the product known in the art.
  • the peptide or protein encoded by the heterologous DNA may be isolated and purifed by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • Recombinant particle-defective genomes and their recombinant hepadnaviruses i.e., packaged recombinant particle-defective genomes which contain a
  • heterologous gene sequence can be therapeutically valuable.
  • the stable incorporation by host cells e.g., hepatocytes
  • a recombinant hepadnavirus genome containing a heterologous gene sequence capable of expression by the host cells can be of great value in the treatment of diseases and disorders.
  • hepadnavirus DNA into host cells (e.g., hepatocytes), for the purposes of gene therapy.
  • host cells e.g., hepatocytes
  • the technique used should provide for the stable trnasfer of the hepadnavirus sequence to host cells, which may include but are not limited to hepatocytes, lymphocytes, and leukocytes, so that the recombinant hepadnavirus sequence is expressible by the above mentioned cells, and so that the necessary
  • hepadnavirus DNA is packaged into virions (e.g., through the use of helper packaging genome
  • such a virion particle can then provide for stable transfer of its contained recombinant DNA to a host cell through the noraml route of
  • virion particles which may be used include but are not limited to intrahepatic, oral, intravenous,
  • recombinant hepadnaviruses may be used to treat disorders of metabolic hepatic dysfunction.
  • these recombinant hepadnaviruses may be used in the treatment of liver disorders which are due to inherited deficiencies of hepatic enzymes.
  • Such genetic disorders include but are not limited to those listed in Table II (for a detailed discussion of some of these disorders, see Sharp, 1985, In
  • recombinant hepadnavirus genomes include but are not limited to complement components, medium chain acyl CoA dehydrogenase, low density lipoprotein receptor, insulin, digestive enzymes, etc.
  • the liver is the site of biosynthesis of factors involved in coagulation such as fibrinogen, factors VIII, IX, X,
  • XI, XII, and XIII can be supplied by expression of a recombinant hepadnavirus particle-defective genome.
  • a recombinant hepadnavirus directed to the treatment of alcoholic liver disease, a recombinant hepadnavirus can be used which expresses a mutant alcohol dehydrogenase that has a lower K m for alcohol, thus accelerating the production of
  • acetaldehyde the agent responsible for making the alcohol consumer very ill, thus inhibiting drinking behavior.
  • Another embodiment of the invention involves treating patients with liver disorders resulting from infections by pathogenic microorganisms, with
  • hepadnaviruses can contain a heterologous gene which is expressed as a product which ameliorates disease, is toxic to the pathogen without significant detriment to the host, or interferes with the pathogen's life cycle, etc.
  • Pathogens which cause disorders which may be treated with recombinant hepadnaviruses according to this embodiment of the invention include but are not limited to those pathogens listed in Table I, supra.
  • the heterologous gene may code for an altered gene product of the pathogenic organism. Alternativly, it is possible to construct a
  • hepadnavirus that expresses a sequence which is "anti-sense" to the nucleic acid of a
  • hepatocyte pathogen hepatocyte pathogen.
  • a sequence which is complementary to the pathogen's RNA or DNA, can hybridize to and inactivate such RNA or DNA,
  • an inhibitor or degradative enzyme for the product, or an inhibitor of its snythesis may be expressed.
  • An antisense oligonucleotide can be expressed by recombinant hepadnavirus DNA for the purpose of blocking specific gene transcription.
  • recombinant hepadnavirus virions or proteins which comprise an immunogenic epitope of a heterologous organism are formulated for vaccine use.
  • vaccines can be used to provide protection against infection by a heterologous pathogenic organism, including but not limited to those listed in Table I, supra, or for protection against conditions or
  • Such vaccine formulations can comprise live
  • hepadnavirus recombinants may be packaged by use of an approriate hepadnavirus packaging genome or other source of hepadnavirus protein, and the resulting virion formulated for use as a vaccine, or the expressed protein containng the heterologous epitope may be purified for use in a subunit vaccine.
  • the vaccine formulations of the invention can be of use in animals and/or humans.
  • expressed by a recombinant hepadnavirus, in its live vaccine formulation can be determined by monitoring the immune response of test animals following
  • the immune response of test animals can be monitored following immunization with the isolated heterologous product of the
  • hepadnavirus which can be formulated with an appropriate adjuvant to enhance the immunological response.
  • Suitable adjuvants include, but are not limited to, mineral gels, e.g., aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG
  • Test animals may include chimpanzees and other
  • hepadnavirus is HBV.
  • Other hepadnaviruses such as HBV.
  • DHBV, GSHBV, and WHBV which have been constructed to contain an epitope of a heterologous organism may also be used in vaccine formulations to immunize ducks, ground squirrels or woodchucks respectively, against infection by the heterologous organism.
  • Methods of introduction of the immunogen may include oral, intradermal, intramuscular, intraperitoneal,
  • the immune response of the test subjects can be analyzed by various approaches such as: (a) the reactivity of the resultant immune serum to the native antigen or a fragment thereof containing the
  • heterologous epitope or to the isolated naturally occurring heterologous organism, as assayed by known techniques, e.g., enzyme-linked immunosorbant assay
  • recombinant hepadnaviruses which express an epitope of a heterologous organism are formulated for live vaccine use.
  • the protein products of such a recombinant virus can be formulated for use in subunit vaccines.
  • the live vaccine or subunit vaccine may be univalent or multivalent.
  • the vaccine formulations of the invention are of use in humans and/or animals.
  • the purpose of this embodiment of the invention is to formulate a vaccine in which the immunogen is a packaged recombinant particle-defective genome, comprising a sequence encoding an epitope of a
  • heterologous organism which can be expressed in vivo so as to elicit an immune (humoral and/or cell
  • Such a live vaccine will allow introduction and expression of the particle-defective genome within a susceptible cell, but no subsequent round of infection will occur.
  • the live vaccine formulation can be univalent or multivalent.
  • Multivalent vaccines can be prepared from a single or a few packaged recombinant
  • particle-defective genomes which encode one or more heterologous epitopes, which may be of different organisms.
  • a single recombinant hepadnavirus genome may encode more than one epitope of the same or different antigens.
  • live vaccine formulations of the invention include but are not limited to oral, intradermal,
  • heterologous peptide or protein may be used as an immunogen in subunit vaccine formulations, which may be multivalent.
  • subunit vaccines comprise solely the relevant immunogenic material necessary to immunize a host.
  • these heterologous proteins which may be recombinant fusion proteins, may be purified from recombinant hepadnaviruses that express the heterologous epitopes using procedures established in the art.
  • the purified protein(s) should be adjusted to an appropriate concentration, formulated with any suitable vaccine adjuvant and packaged for use.
  • Suitable adjuvants include, but are not limited to those described supra in Section 5.2.2.2.1.
  • the immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.
  • vaccine formulations described above include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and
  • hepadnaviruses of the present invention also have potential uses in diagnostic immunoassays, passive immunotherapy, and generation of antiidiotypic
  • the generated antibodies may be isolated by standard techniques known in the art (e.g.,
  • immunoassays to detect the presence of viruses, bacteria, or parasites of medical or veterinary importance in human or animal tissues, blood, serum, etc.
  • the antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used for this purpose including but not limited to competitive and
  • the vaccine formulations of the present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of
  • formulations of the present invention can also be used in the production of antiidiotypic antibody.
  • the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol.
  • the invention is illustrated by way of a duck model system in which two types of defective duck hapatitis B virus genomes are constructed.
  • the first type, particle-defective DHBV genomes contain deletions in the genes encoding the polymerase protein, core antigen, and/or surface antige (see figure 1). These genomes are incapable of replication by themselves, but are able in each case to produce a pregenomic RNA with appropriate
  • the second type of defective DHBV genomes, packaging genomes contain deletions in the DR1 region of the DHBV genome. These mutant genomes are capable of providing in trans all viral functions necessary for packaging an appropriate pregenomic RNA into virions and for transcribing the RNA into virion DNA. The mutant genomes however fail to be themselves
  • HuH7 human hepatoma cell line
  • DMEM Dulbecco's Modified Eagle's Media
  • F12 GEBCO, Grand Island, NY
  • FBS fetal bovine serum
  • hepatocyte cultures were isolated from the livers of two week old Pekin ducks (obtained from Dr. W. Mason, Fox Chase Cancer Center, Philadelphia, PA) by
  • the primary hepatocytes were propagated in serum free L15 medium (GIBCO, Grand Island, NY) plus 10 mM sodium bicarbonate, 1.5% (v/v) dimethyl sulfoxide (DMSO), 5% FBS as well as
  • DHBV sequences used in the construction of defective DHBV genomes were obtained from DHBV genomic DNA cloned into a plasmid pSP65 vector at the EcoRI site, p5.2 Galxl, which was derived from a virion DNA clone used by Mandart et al. for the determination of the DHBV DNA nucleotide sequence (Mandart et al., 1984, J. Virol. 49:782-792).
  • Tris-borate pH 8.2, 1 mM EDTA or in 0.8% or 1.5% agarose gels in Tris-acetate buffer (0.02 M
  • Tris-acetate 0.05 M sodium acetate, 1 mm EDTA, pH
  • DNA fragments were visualized under ultraviolet light by negative shadowing over a PEI
  • DNA fragments were recovered by electroelution at room temperature for 2 hours at 200 V in TBE buffer, or were recovered from low-melt agarose gels by phenol extraction of the liquefied gel.
  • Oligonucleotides were snythesized on the 0.2 micromole scale, on an Applied Biosystems Inc. model
  • oligonucleotides were separated and purified by gel electrophoresis using a polyacrylamide/8 M urea gel in the TBE buffer and subsequently visualized by negative shadowing over a
  • the particle-defective DHBV genome containig deletions in regions encoding the polymerase and surface antigen (i.e., envelope) genes (pol-, env-) was generated by the digestion of the wild-type genome with Kpnl, treatment with T4 polymerase in the
  • the pol-env-core- mutant DNA (termed Kpn-Sph-) (figure 1b) with deletions in regions encoding all three viral genes was produced by first digesting the DHBV genome with Kpnl, resulting in the loss of the Kpnl restriction site and generation of the Kpn- genome. The Kpn- genome was subsequently digested with Sphl, then as before, treated with T4 polymerase in the presence of all four dNTPs. The plasmid was then closed with T4 DNA ligase.
  • All of the particle-defective DHBV genomes were dimerized using one of two strategies.
  • Linearized DNAs were identifed in 1% agarose gels by ethidium bromide fluorescense and recovered by electroelution in the TBE buffer at room temperature for 2 hours at 150 V.
  • the isolated linearized plasmid was then treated with bacterial alkaline phosphatase and joined with the corresponding EcoRI-cut monomer genomes purified on a 5% nondenaturing polyacrylamide gel.
  • the second stragegy of dimerization employed joining the EcoRI-cut gel-purified monomer in a 3 to 10-fold excess with EcoRI-bacterial aklaline phosphatase treated pSP65 DNA.
  • Wild-type dimer plasmids can be prepared by either of these two methods from wild-type HBV or DHBV genomic DNA.
  • the defective DHBV genome cloned into pSP65 was amplified by transformation into E. coli HB101 cells.
  • the dimer clones were subsequently identified by restriction analysis of plasmid DNAs.
  • a portion of the wild-type DHBV genome which includes the DR1 region was excised from the recombinant plasmid using the enzymes Aflll and AccI (cleaving at nucleotide numbers 2526 and 2577, respectively) and was replaced by a double-stranded synthetic DNA segment precisely lacking the 12 base pairs comprising DR1.
  • the proximal portion of the DHBV genome was inserted as a BamHI fragment into the unique BamHI site.
  • the ⁇ DR1 genome like the attenuated DHBV
  • the ⁇ DR1 genome cloned into pSP65 was amplified by transformation into E. coli HB101 cells.
  • the dimer clones were identified by the presence of a new Hinfl site at the position of the DR1 deletion.
  • HBcAg and/or HBsAg were fixed at -20°C in 95% (v/v) ethanol:5% (v/v) glacial acetic acid.
  • HBcAg and/or HBsAg was detected by indirect immunofluorescence using a rabbit antiserum to HBcAg or HBsAg (provided by Dr. W. Mason, Fox Chase Cancer Center, Philadelphia, PA) and rhodamine conjugated goat anti-rabbit IgG (Tuttleman, et al., 1986, J.
  • particle-defective DHBV DNA with deletions in regions encoding the surface antigen and polymerase protein (Kpn- DNA) and in those with deletions in regions encoding the core antigen, surface antigen, and
  • Kpn-Sph- DNA polymerase protein
  • RV-2650 mutant DNA was able to produce surface antigen
  • the particle-defective DHBV with deletions in the region encoding the polymerase protein (RV-718), and the Kpn- genome were able to produce nucleocapsid (core) proteins as expected.
  • Kpn-Sph- encodes both a
  • the packaging genome is deleted of a specific viral sequence, a 12 base pair direct repeat called DR1, which is required for complete synthesis of a double-stranded viral DNA.
  • the packaging genome was transfected into HuH7 cells by the calcium phosphate precipitation method (Graham and van der Eb, 1973, Virology 52:456-467). Five days after transfection, cells were washed with HBS (12 mM
  • Viral core particles which contain all the intermediates in DHBV DNA synthesis, where then isolated by the following procedure. The supernatant was removed and magnesium acetate (MgAC 2 ) was added to a final concentration of 6 mM.
  • MgAC 2 magnesium acetate
  • Tris-hydrochloride pH 7.9, 6 mM MgAc 2 and 100 M g/ml
  • the precipitate was collected by centrifugation at 4°C in a microfuge, and dissolved in 0.3 ml of buffer containing 50 mM Tris-HCl, 5 mM MgCl 2 , 0.1 M
  • the reaction mixture was adjusted to contain 15 mM EDTA, 0.5% SDS, and 500/(g/ml pronase. After incubation at 37°C for 30 minutes, the sample was extracted with an equal volume of phenol. Nucleic acids were precipitated from the aqueous fraction with ethanol, dissolved in 10 mM Tris-hydrochloride (pH
  • the filter was washed briefly in 2 x SSC then air dried and exposed to an ultraviolet light source (240-390 nm) for 5 minutes to covanently cross-link DNA to the membrane.
  • an ultraviolet light source 240-390 nm
  • the filter was hybridized at 50°C with a 32 P-labelled
  • DHBV ribonucleotide probe of plus strand polarity DHBV ribonucleotide probe of plus strand polarity.
  • particle-defective DHBV genomes may also complement one another.
  • core- mutant DNA was cotransfected with either the pol- mutant or Kpn-
  • HuH7 human hepatoma cells can produce virions that are capable of infecting primary duck hepatocytes.
  • a 100 mm dish was seeded with
  • the culture medium was removed from the cells and distributed equally onto 3 x 60 mm dishes of primary duck hepatocytes prepared 48 hours earlier.
  • the hepatocytes were infected for 20 hours at 37°C with the HuH7 culture medium. Cells were harvested as described by
  • FIG. 6 shows the appearance of viral DNA in the cultures, indicating that infectious virus had been present in the HuH7 culture
  • HuH7 cells are capable of producing infectious DHBV from wild-type cloned DHBV DNA and, as expected, these cells failed to produce infectious virus from ⁇ DR1, Kpn-, or
  • Genome S1 was specifically designed to be defective for envelope production yet fully
  • Polymerase function was maintained in the S1 mutant to permit enhanced replication of viral DNA and enhanced production of viral gene products relative to a particle defective genome defective in both envelope and polymerase functions (e.g. Kpn-).
  • the S1 particle-defective genome was obtained by the S1 particle-defective genome.
  • 1S particle-defective genome designated pSPDHBV.1S
  • pSPDHBV.1S 1S particle-defective genome
  • the dimer-containing plasmid pSPDHBV.1S was transfected in HuH7 cells (Section 6.1.1., supra) by calcium precipitation. Transfected cells were assayed by Southern blot hybridization for viral replicative forms using the procedure described in Section 6.4.2., and the results are shown in Figure 8(b), lanes 1 and
  • HuH7 cells transfected with the 1S defective genome contain substantially wild-type levels of both the relaxed circular (RC) and single-stranded (SS) replicative forms of viral DNA. Immunostaining analysis of the transfected HuH7 cells (not shown) revealed
  • HuH7 cells are capable of producing infectious
  • infectious particles are capable of
  • DHBV sequences necessary for transcription, packaging, and reverse transcription of the vector pre-genome must be present in the defective transducing genome. Since the mutant genomes pol-env- (Kpn-), pol-env-core- (Kpn-Sph-), pol-, and core- can be complemented in trans for DNA synthesis by ⁇ DR1, they must contain these necessary sequences. However, some of these mutations, which lie in viral open reading frames, appear to interfere in a dominant fashion with the synthesis of DNA and infectious particles by wild-type virus. An example of such interference is shown in figure 3 (lanes 1,2 or 3,4). When the Kpn-Sph- mutant
  • DHBV genomes containing heterologous gene sequences A deletion was made in a region of the DHBV genome which encodes the surface antigen and viral polymerase proteins. Different sized fragments of bacteriophage ⁇ genomic DNA were subsequently inserted into this region.
  • HuH7 human hepatoma cell line
  • Dr. C Koiki Department of Gene Research, Cancer Institute, Tokyo, Japan
  • DHBV sequences used in the construction of ⁇ -DHBV recombinants were obtained from DHBV genomic DNA cloned into a plasmid pSP65 vector at the EcoRI site. % genomic DNA was purchased from New England Biolabs (Beverly, MA). Restriction enzymes, T4 DNA ligase, T4 DNA polymerase, and the Klenow fragment of E. coli DNA Polymerase I were purchased from New England Biolabs (Beverly, MA). Restriction enzymes, T4 DNA ligase, T4 DNA polymerase, and the Klenow fragment of E. coli DNA Polymerase I were purchased from New
  • DNA fragments were separated and purified by gel electrophoresis in a 5% polyacrylamide gel in TBE buffer (0.01 M
  • ⁇ -DHBV recombinants were generated by deleting sequences encoding the polymerase protein and surface antigen by digestion of the wild-type DHBV genome with Kpnl.
  • the linearized plasmid was filled in with the Klenow fragment of E. coli DNA Polymerase I.
  • Fragments of bacteriophage ⁇ were subsequently ligated into the plasmid (at nucleotide number 1290) with T4 DNA ligase.
  • the ⁇ -DHBV recombinants containing a 180 bp Haelll ) ⁇ fragment or a 1000 bp Haelll ⁇ fragment, were termed Kpn-+180, and Kpn-+1000, respectively.
  • the ⁇ -DHBV recombinant cloned into SP65 was amplified by transformation into E. coli HB101 cells.
  • the dimer clones were subsequently identified by restriction analysis of plasmid DNAs.
  • ⁇ -DHBV recombinant expression was monitored by Southern blot hybridization using procedures described in Section 6.4.2. As shown in figure 3, lane 8, the ⁇ -DHBV recombinant containing the 180 bp Haelll fragment of ⁇ (Kpn-+180) was packaged into virions, however, the packaging occurred at a level
  • Transient expression systems are constrained by inherent limitations, primarily the efficiency of transfection. For this reason, it is desireable to incorporate one or more of the defective hepadnaviral genomes of the invention into permanent,
  • a stably transfected permanent cell line containing the correct genetic information is capable of substantially continuous production of viral particles.
  • a packaging cell line is stably
  • transfected with genetic information to provide complementation, in trans, of viral functions required by a second defective hepadnaviral genome transfected into the packaging cell line. More specifically, transfection of a particle defective genome into a packaging cell line results in supply of the helper functions required in trans by the "packaging" genome to enable the input genome to be packaged and reverse transcribed to produce infectious defective viral particles carrying the particle defective genome.
  • the packaging genome present within the packaging cell line, is not itself recovered in the form of
  • infectious particles since the packaging genome lacks the cis acting sequences necessary for its own
  • particle defective hepadnaviral genomes carrying a heterologous gene sequence can be propagated and recovered in the form of infectious viral particles following
  • the invention further relates to the
  • a producer cell line may contain stably incorporated therein both a packaging genome and a particle defective genome.
  • the producer cell line is capable of packaging and producing packaged particle defective genomes in a substantially continuous manner, and obviates the need for repeated transfections.
  • the particles are recoverable from the supernatant of cultured transfected cells using known methods .
  • the HepG2 cell line is a human hepatoma cell line available in many laboratories.
  • the HepG2 cells retain many morphological features which are
  • the HepG2 line has previously been demonstrated to be capable of producing wild-type virus following transfection with wild-type HBV DNA in dimer form. See, e.g.. Sells et al., Proc. Nat. Acad. Sci., U.S.A. 84:1005, 1987.
  • cells of the HepG2 cell line contain no endogenous HBV DNA sequences.
  • the plasmid vector For transfection of the HepG2 cell line with the packaging genome ⁇ DR1, the plasmid vector
  • pHBV ⁇ DR1Neo ( Figure 10) was constructed.
  • the vector pHBV ⁇ DR1Neo comprises a dimer form of the human HBV ⁇ DR1 packaging genome, and further comprises an antibiotic (neomycin) resistance gene under control of an SV40 promoter.
  • a reproducible method for constructing pHBV DR1Neo using established laboratory procedures is illustrated schematically in Figure 11 and described as follows.
  • a 479 base pair Xmnl-BglI fragment carrying ampicillin resistance is excised from the E. coli plasmid pSP65, and the plasmid backbone filled in with the Klenow fragment of DNA polymerase I and blunt end ligated to a 2057 bp Clal-Xml fragment carrying chloramphenicol resistance derived from pBR328.
  • the resulting plasmid construct was used to transform E. coli strain HB101 to chloramphenicol resistance.
  • the construct pSPCAM has no restriction sites for the restriction enzyme Fspl, and carries a polylinker region derived from pSP65.
  • a Hinc-Xbal fragment in the polylinker region of pSPCAM was then replaced with a 266bp fragment corresponding to positions 1726 to 1992 of the wild-type HBV genome, which fragment was obtained as a Dral-Xbal fragment from wild-type HBV dimer plasmid.
  • the resulting CAM R construct was used to transform E. coli strain HB101 to chloramphenicol resistance.
  • sequence at positions 1826 to 1837, inclusive was excised as a Fspl-Styl fragment, which fragment comprises nucleotide positions 1804 to 1884.
  • the excised fragment was replaced with a homologous synthetic fragment in which nucleotides 1826 to 1837 were precisely deleted, and in which Fspl and Styl sites were precisely regenerated for inserting the synthetic fragment into the plasmid construct in proper orientation. Removal of nucleotides 1826 to 1837 deletes the DR1 sequence within the construct.
  • a FspI-EcoRI fragment containing nucleotides from positions 1804 to 3182 was removed.
  • the fragment was first ligated to a 5' fragment of HBV derived by Hind III-Fsp I digestion of wild-type HBV dimer plasmid, and then into a pBR322 backbone, in a trimolecular reaction as shown in Figure 11.
  • the pBR322 backbone was prepared by
  • the ⁇ DR1 genome was then dimerized by partially digesting the recombinant monomer with
  • a Clal-SalI fragment was excised, which fragment lies outside of and does not interfere with the tandem ⁇ DR1 genomes in the dimer.
  • the fragment is replaced with a Clal-SalI fragment containing a bacterial neomycin resistance gene under the control of an SV40 promoter.
  • Source plasmids wherein a bacterial antibiotic resistance gene is controlled by an SV40 promoter are available and well known. See, e.g. Southern and Berg, 1982, J. Mol. Applied Genet., 1:327. The resulting vector was designated pHBV ⁇ DR1Neo.
  • vectors may be prepared by available methods which comprise a defective viral genome dimer and a selectable marker.
  • the vector pHBV ⁇ DRINeo is described herein as exemplary of a suitable vector.
  • the permanent human hepatoma cell line HepG2 was transfected with the plasmid pHBV ⁇ DR1Neo.
  • HepG2 line Medium was changed the morning prior to transfection, and transfection was carried out by the calcium phosphate precipitation method. Eight (8) micrograms of plasmid DNA was transfected into each of three 10 centimeter dishes, and the medium changed after 8 hours. Two days following transfection, the cells were trypsinized from confluent plates and seeded out at densities of 350,000 cells per plate,
  • Hepatitis B surface antigen assay was carried out using a commercially available solid phase radioimmunoassay kit (Connaught
  • HepBl-2 This clone, designated HepBl-2, was expanded by recloning. Subsequent retesting of HepBl-2 for both HBsAg and HBeAg production tested strongly positive.
  • Immunofluorescent staining analysis for HBsAg was carried out on fixed HepBl-2 cells.
  • HepG2 cells were examined using the same procedure. In each instance, cells were fixed on glass cover slips using cold 95% ethanol/5% glacial acetic acid. The plates were left in the fix solution at -20°C for two days, then washed with
  • PBS phospate-buffered saline
  • cover slips were then incubated with a solution of goat polyclonal anti-HB surface antigen (DAKO, Santa Barbara, Cal.) for 2 hours at 37°C, washed in PBS, then incubated with fluorescein-conjugated rabbit anti-goat antiserum (DAKO) for a further 2 hours at
  • HepBl-2 cells exhibited a uniform pattern of cytoplasmic immunofluorescence in all cells tested, suggesting that all cells of the HepBl-2 clonal expansion were derived from the same original clone.
  • the particles are of correct diameter (approximately
  • HBsAg using radioimmunoassay.
  • a peak of antigen was detected at a density of approximately 1.20 g/ml in the CsCl density gradient, which corresponds to the expected sedimentation density of Dane particles.
  • the particles produced by the HepBl-2 cell line were analyzed by various methods to determine whether the particles contained infectious HBV DNA.
  • packaging genome into HuH7 cells failed to produce relaxed circular DNA, but apparently produced minor but detectable quantities of single stranded (-) strand DNA, as shown in Figure 3, lane 5.
  • the minor amount of detectable ssDNA may be due to anamolous initiation of first-strand DNA synthesis. It was anticipated that, due to the similar mutation in the DR1 repeat region present in the plasmid DNA used to prepare the packaging cell line HepBl-2, the particles produced by the packaging cell line would consist predominantly of pregenomic RNA species unable to complete replication of dsDNA.
  • the supernatant was fractionated in a cesium chloride gradient. Nucleic acids were then isolated from the different fractions by treatment with 15mM EDTA, 0.5% sodium dodecyl sulfate, and 200 microgram/ml Proteinase K (Boehringer Mannheim) at
  • fractions were analyzed in blot hybridization assays using a nick translated wild-type HBV probe. As shown in Figure 14, 3.0 kilobase species corresponding in size to an HBV genome were not detected in any of the fractions tested. Weakly hybridizing species were detected in fractions 1.18 - 1.25 g/ml and 1.26 - 1.32 g/ml of approximate 3.5 kilobase and 5 kilobase size, and a large smear that extends from 3.5 kilobases downward was observed in the density fraction
  • the cell line would be capable of providing packaging functions in trans to both package and reverse transcribe the RNA from a second defective HBV genome introduced into the cells.
  • the packaging cell line should fully complement in trans all viral functions required by a particle-defective HBV genome. Transfection of the particle-defective genome into the packaging cell line should result in production of a packaged virus.
  • the defective genome designated X-, was produced by restricting wild-type HBV genome with Xhol to cleave the genome at
  • nucleotide position 129 The cohesive ends generated by Xhol digestion were filled in with Klenow fragment, and a 10 bp synthetic linker containing a SalI
  • restriction site was blunt end ligated into the Xhol site.
  • the resulting X- defective genome was dimerized into a pSP65 plasmid backbone, as described
  • HepG2 cells were then transfected with the X- dimer plasmid using two different procedures.
  • the first procedure using calcium phosphate
  • transfected cells for analysis 3, 4, and 5 days following transfection.
  • Plasmid DNA was mixed with 150 microliters of water in a polystyrene tube. 150 microliters of Lipofectin was then added and the tube was gently shaken to mix the DNA. Recipient cells were washed twice with DMEM (less serum). To each dish was added 9 ml Optimem (Bethesda Research
  • the DNA mixture which was then mildly cloudly, was applied dropwise to the cells.
  • the DNA mixture was left on the cells for 24 hours, after which period the cells were washed 5 times with PBS, then cultured in DMEM plus 10% fetal calf serum.
  • the cells were again washed 5 times with PBS the next day, and after an additional day, were washed 3 more times with PBS.
  • the medium was then collected on days 4, 5, and 6 following transfection, and the recovered medium pooled at 4°C.
  • Figure 15 shows the results of Southern blot hybridization of supernatants from two transfections of HepBl-2 cells with X- dimer plasmid. In one transfection, 5 micrograms of plasmid DNA were
  • 9 kilobase size and larger represent residual input plasmid DNA. These larger species are observed throughout the density gradient, and restriction analysis confirms their identity.
  • the 3.0 kb species were excised from a low melting point agarose gel (Seakem, FMC) and following melting of the gel slice at 65°C for 4 minutes, digested with the enzymes SalI and Ncol at 37°C for two hours in the presence of 1 microgram of carrier mouse DNA. The digestion products were then analyzed in Southern blot analysis with a nick-translated HBV probe. Precisely as expected from a packaged mutant X- genome, hybridizing fragments of 1900 bp and 1200 bp were observed.
  • a low melting point agarose gel Seakem, FMC
  • producer cell lines may be prepared by transfecting permanent cell lines such as HepG2 with more than one defective hepadnaviral genome.
  • the packaging cell line HepBl-2 is further stably transfected with a particle defective genome, like X-, the resulting cell line may constitute a permanent producer of the packaged form of the defective genome.
  • every cell in the transfected population as opposed to the low percentage of cotransfectants available in the context of a transient expression system, becomes a producer of the particle defective genome. In this manner, greater titers of defective hepadnaviral particles could be recovered without repeated transfection steps.
  • the producer cell line designated HepBl-2 has been deposited with the American type Culture

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