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WO1999021882A1 - Polypeptide fixant le hbv - Google Patents

Polypeptide fixant le hbv Download PDF

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Publication number
WO1999021882A1
WO1999021882A1 PCT/AU1998/000889 AU9800889W WO9921882A1 WO 1999021882 A1 WO1999021882 A1 WO 1999021882A1 AU 9800889 W AU9800889 W AU 9800889W WO 9921882 A1 WO9921882 A1 WO 9921882A1
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WO
WIPO (PCT)
Prior art keywords
hbv
binding polypeptide
seq
acid sequence
sequence presented
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU1998/000889
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English (en)
Inventor
Eric James Gowans
Thomas Bernard Macnaughton
Tracey Jane Harvey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
State of Queensland Department of Health
Original Assignee
State of Queensland Department of Health
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Filing date
Publication date
Application filed by State of Queensland Department of Health filed Critical State of Queensland Department of Health
Priority to AU97302/98A priority Critical patent/AU9730298A/en
Publication of WO1999021882A1 publication Critical patent/WO1999021882A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus

Definitions

  • the present invention relates to a novel HBV binding polypeptide, and to nucleic acids encoding the same
  • the invention also relates to a HBV permissive cell- line, HBV therapeutic agents, methods for detecting, preventing and treating HBV infection, methods of purifying and removing HBV from samples, methods of determining the anti-HBV activity of compounds, transge ⁇ ic animals expressing foreign nucleic acid encoding novel HBV binding molecules and methods of vaccine testing utilizing the novel HBV binding polypeptide expressed in a transgenic animal.
  • the Hepadnavindae is a family of enveloped DNA viruses that produce persistent infection of hepatocytes, often resulting in the development of chronic hepatitis, liver failure and hepatocellular carcinoma.
  • Human HBV (HBV) is the prototypic member of the hepadnavirus family
  • a characteristic feature of HBV and other hepadnaviral infections is strong species-specificity, and HBV can infect humans and chimpanzees but not baboons, lower primates or other mammals.
  • the HBV genome contains four overlapping open reading frames (ORF), viz., ORF-
  • ORF-S contains the S, preS2 and preS1 regions that encode the three related envelope glycoproteins (S-HBsAg, M-HBsAg and L-HBsAg) respectively
  • S-HBsAg, M-HBsAg and L-HBsAg The virus envelope proteins can be expected to interact with a cellular receptor and all three envelope proteins have been shown to interact with a range of cellular proteins.
  • the preS1 domain binds to HepG2 cells and liver cell membranes
  • the preS2 domain binds to HepG2 cells, T lymphocytes and liver cell membranes
  • the S domain binds to hepatocytes, fibroblasts, mononuclear cells and Vero cells.
  • HBV particles devoid of M-HBsAg are infectious and the only direct evidence for the contribution of the various domains comes from a study of duck HBV (DHBV) in which recombinant L-DHBsAg particles but not S-DHBsAg particles were able to inhibit DHBV replication in primary duck hepatocytes. (DHBsAg has no equivalent of the M-HBsAg domain)
  • the present invention provides an isolated HBV binding polypeptide comprising the ammo acid sequence presented herein as SEQ ID No:1 or a functional variant thereof.
  • “functional variant” includes portions or fragments of SEQ ID No:1 with HBV binding activity, such as peptides, and larger polypeptides including SEQ ID No.1 with HBV binding activity.
  • Other “functional variants” include analogues and variants of SEQ ID No:1 which maintain their HBV binding activity and include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues. These modifications can also provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase bio-stability.
  • the functional variants can be synthesized directly or obtained by chemical or mechanical disruption of larger molecules, fractioned and then tested for HBV binding activity.
  • Functional variants with useful properties may also be obtained by mutagenesis of a specific region of the nucleotide encoding the polypeptide, followed by expression and testing of the expression product, such as by subjecting the expression product to a HBV binding assay.
  • Functional variants may also be produced by Northern blot analysis of total cellular RNA followed by cloning and sequencing of identified bands derived from different tissues/cells, such as human organs, or by PCR analysis of such RNA also followed by cloning and sequencing. Thus, synthesis or purification of an extremely large number of functional variants is possible.
  • HBV binding activity includes (i) in vivo and in vitro binding of HBV and HBV antigens to the HBV binding polypeptide and (ii) binding of antibodies against the HBV binding polypeptide to prevent the HBV binding polypeptide binding HBV.
  • an “analog” include polypeptides with an amino acid sequence that has at least 50% identity with SEQ ID No:1 , more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID No:1.
  • One analogue according to the present invention with 80% identity to SEQ ID No 1 , is:
  • the HBV binding polypeptide of SEQ ID No-1 represents part of a larger polypeptide
  • the nucleic acid and ammo acid sequences of the complete protein may be determined using the nucleic acid sequence data and the methods disclosed herein
  • the HBV binding polypeptide may be a HBV receptor polypeptide or a HBV accessory molecule polypeptide
  • the HBV binding polypeptide binds HBV but not hepatitis B surface antigen
  • the HBV binding polypeptide may be a cellular protein adapted to retain the HBVpreSI domain inside the cell.
  • the HBV binding polypeptide is a polypeptide with the ammo acid sequence presented herein as SEQ ID No 1
  • polypeptides of the present invention may be synthesized in vitro or may be obtained using a cell-free translation system or a linked transcription-translation system Alternatively, the polypeptides may be directly synthesized or obtained in a polypeptide extract from a cell that does not normally express the polypeptide, but has been transfected or transformed to do so, or is from a transgenic animal, as described below
  • polypeptides may be isolated by any one or more of a number of routine methods such as electrophoresis, blotting, precipitation, immunoprecipitation, dialysis, chromatography or combinations of these and other methods
  • the polypeptide When the polypeptide is a HBV receptor it can be utilized in a system to regulate the binding activity of endogenous HBV receptors, in methods for assaying the regulation of HBV binding polypeptides, or in a model to investigate the regulation of ligand binding proteins
  • polypeptides of the present invention can be attached to sequences designed to provide for some additional property, such as solubility or to provide a means for attaching the sequence to a substrate via for example antibody-antigen interaction.
  • the polypeptides may also be bound to and thus immobilised on a solid support.
  • suitable substrates or supports include polymers, beads (e.g., agarose, polystyrene, sepharose, etc.), latex plates, glass or plastic petri or culture dishes, albumin, and the like.
  • HBV binding polypeptide comprising an immobilized polypeptide
  • immobilized HBV binding polypeptide include, but are not limited to, affinity chromatography techniques such as those used to concentrate specific molecules which bind to the polypeptide.
  • the immobilized HBV binding polypeptide or portion thereof can be used to identify natural or artificial ligands.
  • HBV binding polypeptide of the present invention bound to a solid support can also be designed and used for virus neutralization testing and/or capture immunoassays in the methods described herein for removal/purification of HBV.
  • a method of detecting the presence of HBV in a sample comprising the steps of: (i) contacting a HBV binding polypeptide comprising the amino acid sequence presented herein as SEQ ID No:1 , or a functional variant thereof, with a sample to form a HBV binding polypeptide-HBV complex; and (ii) detecting said complex.
  • the polypeptide set forth as SEQ ID NO: 1 is one particular polypeptide of the present invention that can be utilized to detect HBV in a sample.
  • One example of a method of detecting HBV in a sample is performed by contacting a fluid or tissue sample from a subject with an amount of the HBV binding polypeptide of the present invention and detecting the binding of the HBV binding polypeptide with the virus.
  • the fluid sample can comprise any body fluid which would contain the virus or a cell containing the virus, such as, but not limited to, blood, plasma, serum, saliva, semen, faeces, or urine. Other possible examples of body fluids include sputum, mucus, gastric juice, and the like.
  • the tissue sample can comprise any tissue obtained from a subject or patient, such as, but not limited to, brain tissue, liver tissue, kidney tissue, heart tissue, lung tissue, placenta tissue, skin tissue, muscle tissue, pancreatic tissue, and so forth. Such tissue samples can be prepared for analysis by disruption and separation into fractions based on size or density, or lysed for analysis of the cellular extracts. Other methods for tissue preparation are common and obvious to a skilled practitioner in the relevant art.
  • the presence of binding is determined by an immunoassay.
  • Immunoassays such as immunofluorescence assays (IFA), ELISAs, and immunoblotting assays can be readily adapted to accomplish the detection of the HBV bound to the HBV binding polypeptide.
  • An ELISA method effective for the detection of the virus can, for example, be as follows: (1 ) bind the HBV binding polypeptide to a substrate; (2) contact the bound HBV binding polypeptide with a fluid or tissue sample containing the virus; (3) contact the above with a specific antibody, which recognises HBV, bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a colour reagent; and (6) observe colour change.
  • a detectable moiety e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme
  • HBV binding polypeptide can be detected by competitive inhibition of HBV binding polypeptide, utilizing monoclonal antibodies (MABs) specifically reactive with the HBV binding polypeptide.
  • MABs monoclonal antibodies
  • sera or other body fluids from the subject is reacted with the HBV binding polypeptide bound to a substrate (e.g. an ELISA 96-weli plate). Excess sera is thoroughly washed away.
  • a labelled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted HBV-HBV binding polypeptide complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control.
  • MABs can also be used for detection directly in samples by IFA for MABs specifically reactive for the HBV binding polypeptide- virus complex.
  • the present invention also provides a competitive inhibition assay comprising the steps of: (i) contacting a HBV binding polypeptide comprising the amino acid sequence presented herein as SEQ ID No:1 , or a functional variant thereof, with a sample to form a HBV binding polypeptide-HBV complex; (ii) removing uncomplexed material; (iii) contacting the above with a labelled monoclonal antibody (MAB) specific for the HBV binding protein; and (iv) comparing the amount of MAB bound in step (iii) with a control to determine the level of inhibition.
  • a competitive inhibition assay comprising the steps of: (i) contacting a HBV binding polypeptide comprising the amino acid sequence presented herein as SEQ ID No:1 , or a functional variant thereof, with a sample to form a HBV binding polypeptide-HBV complex; (ii) removing uncomplexed material; (iii) contacting the above with a labelled monoclonal antibody (MA
  • HBV may also be detected according to the present invention by micro- agglutination.
  • a solid substrate such as latex beads are coated with the HBV binding polypeptide and mixed with a test sample, such as tissue or body fluid, such that HBV in the tissue or body fluids that are specifically reactive with the HBV binding polypeptide become crosslinked with the HBV binding polypeptide, causing agglutination.
  • the agglutinated HBV binding polypeptide- virus complexes form a precipitate, visible with the naked eye or detectable by a spectrophotometer.
  • the present invention also provides an agglutination assay for detecting the presence of HBV in a sample, the assay comprising the steps of: (i) contacting a HBV binding polypeptide comprising the amino acid sequence presented herein as SEQ ID No:1 , or a functional variant thereof bound to a substrate capable of agglutination such as latex beads, with a sample to form a HBV binding polypeptide-HBV complex; and (ii) detecting the precipitated said complex.
  • the sample can be taken directly from a patient or subject or be partially purified prior to being subjected to the methods.
  • the HBV binding polypeptide reacts by binding HBV and more particularly viral amino acid sequences that are preferably associated with virus receptor interaction (the primary reaction). Thereafter, a secondary reaction with an anti-HBV binding polypeptide antibody or anti-HBV antibody bound to, or labelled with, a detectable moiety can be added to enhance the detection of the primary reaction.
  • an antibody or other ligand which is reactive either specifically or non-specifically with a different binding site of the HBV binding polypeptide or the virus will be selected for its ability to react with multiple sites on the complex of HBV binding polypeptide and virus.
  • several molecules of the antibody in the secondary reaction can react with each complex formed by the primary reaction, making the primary reaction more detectable.
  • the detectable moiety can allow visual detection of a precipitate or a colour change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like.
  • detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by colour change).
  • the detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections.
  • the HBV binding polypeptide of the present invention and its use thereof for detecting HBV may be incorporated into a HBV diagnostic kit.
  • the present invention also provides a diagnostic kit comprising a HBV binding polypeptide comprising the amino acid sequence presented herein as SEQ ID No: 1 or a functional variant thereof.
  • the HBV binding polypeptide of the present invention may be used as a therapeutic agent or a vaccine.
  • the present invention also provides a vaccine or therapeutic comprising an isolated HBV binding polypeptide comprising the ammo acid sequence presented herein as SEQ ID No: 1 or a functional variant thereof and a pharmaceutically acceptable carrier
  • Treatment or prevention of HBV infection can be facilitated by competitive inhibition of HBV binding to a cell by administration of the HBV binding polypeptide of the present invention in a pharmaceutically acceptable carrier.
  • the amount of the HBV binding polypeptide sufficient to treat a HBV infection in a human depends at least partially on the amount of the HBV binding polypeptide, such as a HBV receptor or accessory molecule or the binding domain thereof, on the cells of the human subject. The dose can be determined by optimization procedures apparent to one skilled in the art.
  • the amount of the HBV binding polypeptide will also vary depending upon the weight, size, and health of the human subject, and with the seventy of HBV infection.
  • compositions of a HBV binding polypeptide to block infection with HBV or to block transmission of HBV.
  • fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a non-aqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup.
  • flavouring, preserving, suspending, thickening, or emulsifying agents may be included Tablets and granules are preferred oral administration forms, and these may be coated.
  • the present invention also provides a method of treating a subject infected with HBV, the method comprising administering to the subject a therapeutically effective amount of an isolated HBV binding polypeptide comprising the ammo acid sequence presented herein as SEQ ID No: 1 or a functional variant thereof and a pharmaceutically acceptable carrier.
  • Th e present invention also provides a method of preventing HBV infection in a subject, the method comprising administering to the subject a prophylactically effective amount of an isolated HBV binding polypeptide comprising the ammo acid sequence presented herein as SEQ ID No 1 or a functional variant thereof and a pharmaceutically acceptable carrier.
  • the candidate HBV binding polypeptides can be tested to determine their immunogenicity and specificity for use as a vaccine. Briefly, various concentrations of a putative immunogen are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. Thereafter an animal so inoculated with the immunogen can be exposed to the virus to test the potential vaccine effect of the specific immunogenic fragment. The specificity of an immunogen can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other closely related viruses.
  • immunological response e.g., the production of antibodies or cell mediated immunity
  • Immunogenic amounts of the vaccine antigen can be determined using standard procedures. Briefly, various concentrations of a putative specific immunoreactive epitope are prepared, administered to a subject and the immunological response (e.g., the production of antibodies or cell mediated immunity) of the subject to each concentration is determined.
  • the amounts of antigen administered depend on the subject, e.g. a human or a guinea pig, the condition of the subject and the size of the subject.
  • the present invention provides a vaccine comprising the HBV binding polypeptide of the present invention and a pharmaceutically acceptable carrier.
  • polypeptides include those derived from the polypeptide sequence set forth herein as SEQ ID No:1 and those encoded by the nucleotide sequences set forth herein as SEQ ID No. 2 and No: 3.
  • Such a vaccine would naturally include immunogenic amounts of the HBV binding polypeptide
  • the carrier will depend upon the method of administration and choice of adjuvant, if one is used.
  • An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the. Methods of administration can be by oral or subli ⁇ gual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.
  • the present invention also provides antagonists which specifically bind to the HBV binding polypeptide of the present invention.
  • the antagonist can be an antibody or a chemical which binds the HBV binding polypeptide or portion thereof or otherwise alters the protein or interferes with the interaction of virus and protein.
  • the HBV binding polypeptide is a HBV receptor one can select antagonists which reacts with the binding site of the HBV receptor or a binding domain of a HBV receptor and affects the binding of HBV.
  • Dane particles or empty HBV virions can be utilized as the antagonist.
  • anti-idiotype and anti-anti-idiotype antibodies to both the HBV binding polypeptide and the HBV can be utilized for prophylaxis or therapy.
  • the treatment modality can be selected to minimize any adverse side effects such as immune system recognition and deletion of the desirable HBV receptor expressing cells.
  • the invention also provides a method of screening for compounds which antagonize the binding of HBV using the HBV binding polypeptide of the present invention.
  • the present invention also provides a method of screening compounds for anti- HBV binding activity, comprising contacting the HBV binding polypeptide with a candidate compound and HBV and determining the relative amount of HBV bound to the polypeptide, the relative amount of virus bound to the polypeptide being an indication of the anti-HBV binding activity of the candidate compound.
  • the HBV binding polypeptide may be on a cell which expresses the HBV binding protein.
  • the compounds of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, liquids, and suspensions, or the like, preferably in unit dosage form suitable for delivery of a precise dosage.
  • the compositions will include, as noted above, an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants and diluents.
  • a purified antibody that specifically binds the HBV binding polypeptide is also provided.
  • the antibodies can be polyclonal or monoclonal and can specifically bind a unique epitope of the receptor.
  • "Specifically bind” as used herein describes an antibody or other ligand that does not cross-react substantially with any antigen other than the one specified, in this case, the HBV binding polypeptide of the present invention.
  • Antibodies can be made by many well-known methods. Briefly, viral antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced.
  • the antibody can be bound to a substrate or labelled with a detectable moiety or both bound and labelled.
  • the detectable moieties contemplated with the composition of the present invention can be those listed above in the description of the detection methods, including fluorescent, enzymatic and radioactive markers.
  • HBV infection can also be prevented or treated by administering to a subject an antibody or other ligand reactive with a HBV binding polypeptide, such as a receptor or accessory molecule or binding domain thereof, which blocks the HBV binding domain.
  • the amount of antibody administered will also be dependent upon the amount of natural HBV binding polypeptide on the cells of the subject and can be determined by optimization procedures apparent to one skilled in the art.
  • the present invention also provides a method for detecting a HBV and/or antibodies to the virus utilizing a capture assay. Briefly, to detect antibodies to HBV in a patient sample, antibodies to the patient's immunoglobulin, e.g., anti-lgG (or IgM) are bound to a solid phase substrate and used to capture the patient's immunoglobulin from serum.
  • antibodies to the patient's immunoglobulin e.g., anti-lgG (or IgM) are bound to a solid phase substrate and used to capture the patient's immunoglobulin from serum.
  • Such therapeutics include antibodies directed toward the HBV binding polypeptide, drugs, compounds, or substances which may alter the binding of HBV to its receptor or accessory molecule or a binding domain thereof, fragments of a HBV which bind to a HBV receptor or accessory molecule or a binding domain of a HBV receptor, other natural or synthetic ligands which bind to a HBV receptor or a binding domain of a HBV receptor linked to a drug, compound, or other substance, or antibodies to a HBV receptor or binding domain of a HBV receptor linked to a drug, compound, or other substance.
  • the present invention also provides a cell line which are adapted to express the HBV binding polypeptide of the present invention, preferably on the cell surface, to a level that is elevated relative to normal or endogenous cells.
  • Such cells can be manipulated to contain increased levels of HBV binding polypeptide.
  • These cells can be manipulated in many ways including direct addition of HBV binding polypeptide to cells with subsequent incorporation by mass action into the lipid bilayer of the cell.
  • the manipulated cells of the present invention can include cells originally non-permissive for HBV infection as well as permissive cells made more permissive.
  • the present invention also provides a method of delivering a desired gene or nucleic acid into a cell expressing the HBV binding polypeptide, such as a receptor or accessory molecule or binding domain thereof, comprising infecting the cell with a non-virulent (modified) HBV having the desired nucleic acid molecule inserted into the HBV genome.
  • the present invention also provides a method of augmenting the above method, comprising the step of increasing the amount of HBV binding polypeptide expressed on the cell surface and infecting the cell with a HBV having the desired nucleic acid molecule inserted into the HBV genome.
  • the identification of HBV binding polypeptide as taught by the present invention, enables methods of gene therapy with HBV as the vector system.
  • the desired human DNA fragment can be inserted into a host cell, e.g., one with sufficient levels of HBV binding polypeptide on the cell surface.
  • the present invention also provides a method of isolating HBV from a sample, comprising contacting the sample with an isolated HBV binding polypeptide and separating the bound HBV from the unbound impurities in the sample, thereby separating the HBV from impurities in the sample.
  • the purification of HBV can be accomplished by the use of immobilized HBV binding polypeptide that specifically bind the target HBV. Once a complex of the HBV binding polypeptide and virus is formed, the impurities in a sample can be separated using known techniques, such as column purification and centrifugation.
  • Th e present invention provides a method for removing HBV from a blood sample comprising binding the HBV in the blood with an isolated HBV binding polypeptide and separating the bound virus from the blood, thereby removing the HBV from the blood sample
  • the method of the present invention utilizes the HBV binding polypeptide, e g., the polypeptide of SEQ ID NO: 1 or a functional variant thereof, to bind to the virus.
  • the bound complex can be removed from the blood sample by preparing a column with the immobilized HBV binding polypeptide. The sample is then passed through the column, thereby removing HBV from the sample utilizing the binding affinity of HBV for the HBV binding polypeptide.
  • the immobilized HBV binding polypeptide can be mixed with the sample and the bound virus-binding protein complex removed by centrifugation, or by having the binding protein attached to a magnetic bead, followed by removal of the complex and bead by magnet.
  • the present invention also provides isolated nucleic acid molecules encoding a HBV binding polypeptide comprising the ammo acid sequence presented herein as SEQ ID No: 1 or a functional variant thereof.
  • nucleic acids of the present invention include variants such as allelic variants and nucleic acids that have been derived from the nucleic acid sequences of the present invention including altered sequences that have been manipulated to encode functional variants of the HBV binding polypeptide of the present invention.
  • nucleic acids presented herein as SEQ ID No:2 and 3 encode HBV binding polypeptides which are a portion of a larger protein.
  • the complete nucleic acid sequence may be obtained using the information contained herein and techniques apparent to one skilled in the art including chromosome walking and RACE. Briefly, chromosome walking involves the use of the nucleic acid sequences herein to generate probes which are then used to screen a genomic library under high stringency conditions. Isolated clones are sequenced and then a portion of that clone, not present in the nucleotide fragments already known, is used to reprobe the genomic library. This procedure can be repeated until the entire sequence is determined.
  • the nucleic acid molecule of the present invention includes at least a portion of the nucleic acid sequence presented herein as SEQ ID No: 2.
  • the nucleic acid molecule includes at least a portion of the nucleic acid sequence presented herein as SEQ ID No: 3.
  • the nucleic acids of the invention can be double-stranded or can be in denatured (single-stranded) form.
  • the invention includes DNA having a sequence including or comprising that set forth in SEQ ID No: 2 or No: 3 and their complement, and RNAs which correspond to the DNA.
  • nucleic acid that encodes a HBV binding polypeptide comprising a HBV receptor binding domain or accessory molecule or a HBV receptor regulatory domain.
  • Such regulatory domains can be manipulated through recombinant techniques to alter their activity and or effect on other regions of the HBV binding polypeptide.
  • the nucleic acids of the present invention include positive and negative strand RNA as well as DNA and includes genomic and sub-genomic nucleic acids present in an organism.
  • the nucleic acids contemplated by the present invention include cDNA encoding the HBV binding polypeptide, the genomic DNA fragments containing the relevant introns and exons, as well as any upstream or downstream regulatory regions, the mRNA encoded by either the cDNA or the genomic DNA, and any nucleic acid which can selectively hybridize to or encode the HBV binding polypeptides of the present invention.
  • the present invention also provides isolated nucleic acid molecules encoding the HBV binding polypeptide that selectively hybridize with at least a portion of the nucleic acids set forth herein as SEQ ID No 2 or No. 3 or their complement.
  • the term "selectively hybridize” excludes the occasional randomly hybridizing nucleic acids under at least moderate stringency conditions.
  • the selectively hybridizing nucleic acids can be used, for example, as probes or primers for detecting the presence of the HBV binding polypeptide coding gene or messenger RNA, or a homologue thereof, that has the nucleic acid to which the primer or probe hybridizes.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55°C, can be used, e.g., 5x SSC, 0.1% SDS, 0 25% milk, and no formamide.
  • Moderate stringency hybridization conditions correspond to a higher T m , e g , 40% formamide, with 5x or 6x SCC
  • High stringency hybridization conditions correspond to the highest T m , e.g. , 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order RNA RNA, DNA RNA, DNA.DNA
  • equations for calculating T m have been derived and are known to those skilled in the art.
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20 nucleotides.
  • the selectively hybridizing nucleic acids of the invention may have at least 60% , 70% , 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes.
  • the nucleic acids can be a coding sequence for the polypeptide of SEQ ID No: 1 or a functional variant thereof, or can be used as probes or primers for detecting the presence of the HBV binding polypeptide.
  • nucleic acids in other species that encode HBV binding polypeptides which can be utilized to prevent or treat HBV infections in other species.
  • the purified HBV receptor for chimpanzee HBV can be isolated and utilized in a composition to prevent or treat infection or to block transmission of the virus in a chimpanzee utilizing methods for preparing the composition and optimization procedures for therapy described herein.
  • the selectively hybridizable nucleic acids of the present invention may be used for gene therapy comprising the step of administering to a subject a selectively hybridizable nucleic acid whereby the selectively hybridizable nucleic acid hybridizes to a nucleic acid, such as mRNA, encoding the HBV binding polypeptide of the present invention, thus preventing its expression.
  • a nucleic acid such as mRNA
  • compositions including at least two nucleic acids which selectively hybridize with different regions of the target nucieic acid so as to amplify a desired region
  • the target region can range between 70% complementary bases and full complementarity
  • nucleic acids of the invention are also contemplated, provided the essential structure and function of the polypeptide encoded by the nucleic acids is maintained. Likewise, portions used as primers or probes can have substitutions provided enough complementary bases exist for selective hybridization
  • nucieic acids described herein or more particularly portions thereof can be used to detect the nucleic acid of the present invention in samples by methods such as the polymerase chain reaction, ligase chain reaction, hybridization, and the like. Alternatively, these sequences can be utilized to produce an antigenic protein or protein portion, or an active protein or protein portion.
  • portions of the nucleic acids described herein can be selected to selectively hybridize with homologous nucleic acids present in other animals or humans.
  • Such a nucleotide sequence shared with other organisms can be used, for example, to simultaneously detect related sequences for cloning of homologues of the nucleic acid of the present invention encoding a HBV binding polypeptide of the present invention.
  • Vectors comprising the nucieic acids of the present invention are also provided.
  • the vectors of the invention can be in a host capable of expressing the HBV polypeptide of the present invention.
  • the present invention provides a vector comprising a nucleic acid encoding the HBV binding polypeptide of the present invention or the nucleic acids set forth herein as SEQ ID No: 2 or No: 3. Additionally, the present invention provides a vector comprising a nucleic acid molecule complementary to or capable of selectively hybridizing with the nucleic acid comprising the nucleotide sequences set forth herein as SEQ ID No: 2 or No: 3 or a portion thereof.
  • An alternative coding sequence for the HBV binding polypeptide can also be expressed.
  • E coli expression vectors There are numerous E coli expression vectors known to one of ordinary skill in the art useful for the expression of the HBV binding polypeptide or portion thereof of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacte ⁇ aceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacte ⁇ aceae such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta- lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ⁇ bosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an ammo terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen.
  • the carboxy-termmal extension of the HBV binding polypeptide can be removed using standard ohgonucleotide mutagenesis procedures.
  • yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion system exhibit correct disulfide pairing. Second, post- transiational glycosylation is efficiently carried out by yeast secretory systems. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.
  • Mammalian cells permit the expression of proteins in an environment that favours important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
  • Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal.
  • the vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or methotrexate resistance, or other genes or phenotypes suitable for use as selectable markers.
  • the HBV binding polypeptide coding sequence can be introduced into a Chinese Hamster Ovary cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers Presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the antigen coding sequence can be confirmed by Southern and Northern blot analysis, respectively.
  • suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transc ⁇ ptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus
  • the vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate mediated transfection or electroporation may be used for other cellular hosts.
  • Alternative vectors for the expression of the HBV binding polypeptide in mammalian cells include those similar to those developed for the expression of human gamma-interferon, tissue plasmmogen activator, clotting Factor VIII, HBV surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS-7).
  • the nucleic acid sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned, to ensure the functioning of " an expression control sequence.
  • These expression vectors are typically replicabie in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences.
  • Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced.
  • such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.
  • host cells expressing a foreign gene or nucleic acid encoding a HBV binding polypeptide, such as a receptor or accessory molecule or a binding domain thereof.
  • Such cells include prokaryotic cells such as E. coli, or eukaryotic cells, such as COS-1 cells.
  • Foreign genes and nucleic acids can be introduced into these cells by a number of techniques, including, but not limited to, transfection, transformation, electroporation, injection, microinjection, and the like.
  • transfection includes techniques such as calcium phosphate co-precipitation, DEAE-Dextran mediated transfection, and lipofection.
  • Viral vectors may also be utilized to introduce foreign genes into host cells. Cells expressing the foreign gene may therefore express the polypeptide encoded by the foreign gene on the cell surface. Such cells may therefore be infectable by HBV and utilized either as models for studying infection of cells by HBV, or as cells producing HBV post- infection.
  • Transgenic animals expressing the HBV binding polypeptide of the present invention are also provided.
  • a non-human transgenic animal expressing a nucleic acid encoding a HBV binding polypeptide having the polypeptide sequence set forth herein as SEQ ID No: 1 or a functional variant thereof, but not expressing an endogenous active HBV binding polypeptide is provided.
  • the foreign nucleic acid expressed in the animal is preferably a nucleic acid sequence encoding the polypeptide of SEQ ID No:1 or a functional variant thereof or the sequence set forth herein as SEQ ID No: 2 or No: 3.
  • Another embodiment of the present invention is a transgenic animal expressing the sequence encoding the HBV binding polypeptide encoded by a nucleic acid that selectively hybridizes with the sequences set forth herein as SEQ ID No: 2 or No: 3.
  • Uses contemplated for the transgenic animals of the present invention can be, but are not limited to, methods to screen drugs, vaccines, or other compounds or substances for their anti-HBV binding activity, methods to screen drugs, vaccines, or other compounds or substances for their anti-HBV infection activity, methods to screen drugs, vaccines, or other compounds or substances for their HBV therapeutic activity, or as a model animal which can be used to produce HBV after being previously infected with HBV.
  • the nucleic acid used for generating a transgenic animal of the invention includes, but is not limited to, a cDNA fragment encoding a HBV binding polypeptide or a genomic sequence encoding a HBV binding polypeptide.
  • a genomic sequence may contain introns as well as exons, upstream and/or downstream regulatory sequences, and other functional and/or structural regions.
  • Nucleic acids used for generating such a transgenic animal may be circular or linear molecules, and may be introduced into the animal with or without additional nucleic acids.
  • additional nucieic acids include, but are not limited to, plasmid, phage, cosmid, viral, or mammalian cloning vectors, and the like.
  • the nucleic acid may be introduced into a zygote or fertilized egg of a female animal containing two pronuclei, or embryonic stem cells prior to introducing the nucleic acid into an embryo, zygote, or fertilized egg of a female animal containing two pronuclei.
  • the nucleic acid may be introduced into embryonic stem cells by transfection, retroviral infection, electroporation, injection, microinjection, and the like. After introduction of the foreign nucleic acid into the embryo, the embryo is transferred to the oviduct of a foster, pseudopregnant mother, and upon subsequent implantation into the uterus, the embryo may develop to term.
  • the transgenic animal of the invention can be used in a method of testing the efficacy of a HBV vaccine.
  • This method comprises administering the potential vaccine to a transgenic animal which expresses the introduced nucleic acid encoding a HBV binding polypeptide of the present invention, such as a receptor or accessory molecule or a binding domain thereof, and determining whether the transgenic animal is protected from infection with HBV.
  • Protection of the transgenic animal from infection by HBV may be determined in a number of ways, including, but not limited to, detecting the presence of virus in the serum, spinal fluid, plasma, blood, mucus, gastric fluids, faeces, urine, and other fluids, brain tissue, liver tissue, kidney tissue, heart tissue, lung tissue, placenta tissue, skin tissue, muscle tissue, pancreatic tissue, and other tissues. Detection of virus is contemplated to distinguish between detection of virus inoculum introduced into the animal and detection of replicating virus produced as a result of a failure of a potential vaccine to prevent infection.
  • Methods of detection for the presence of replicating virus include, but are not limited to, PCR, ELISA, IFA, Southern blotting, Western blotting, Northern blotting, plaque assay, immunocytochemical techniques and serological profiling, such as assaying for anti-HBc antibodies or HBsAg.
  • a transgenic animal of the invention can be used in a method of producing HBV, comprising generating a transgenic animal expressing a foreign nucleic acid encoding a HBV binding polypeptide followed by productive infection of the animal with introduced HBV.
  • HBV replicated by cells that express the introduced foreign nucleic acid and become infected with HBV can be harvested by any of a number of methods known to a skilled practitioner in the art. Harvesting the replicating HBV from a transgenic animal expressing the HBV binding polypeptide may therefore provide a source of newly synthesized HBV for other clinical (e.g., diagnostic) or research procedures, or for vaccines.
  • Figure 1 SDS-PAGE analysis of the ten 35 S methionme-labelled, in vitro translated, cellular proteins used in the in vitro binding assay.
  • the radiolabelled proteins ranged in size from 23kDa to 35kDa.
  • the molecular weight markers were generated from a commercial standard (BioRad).
  • FIG. 1 SDS-PAGE analysis of the 35 S-meth ⁇ on ⁇ ne-labelled in vitro translated proteins bound to baculovirus-expressed recombmant GST-preS1 coated Sepharose beads. Lanes 1 -10 represent the proteins derived from clones 1 -10. Lane M represents a binding reaction using a control in vitro translation mixture in which RNA was omitted.
  • Figure 3 Comparison of the relative level of binding of radiolabelled cellular proteins 1 -10 to GST-preS1 coated beads. The results from the in vitro binding assay described in Figure 2 were quantitated using ImageQuant; (+ to ++++) represents the level of intensity of the interaction of each clone observed using the yeast two hybrid system.
  • Figures 4a and 4b The predicted hydrophilicity plots and secondary structure of proteins #1 (4a) and #6 (4b). The plots were generated using the Kyte-Doolittle scale and a hydrophilicity window size of 7. The secondary structures (alpha helices, beta sheets etc) were examined using the Robson-Gamier and Chou- Fasman methods, with the MacVector 3.5 protein analysis toolbox.
  • Figure 5 SDS-PAGE analysis of the polypeptide profile of the purified HBsAg (lane 1 ) and purified HBV (lane 2) preparations. After gel electrophoresis, the separated polypeptides were stained with silver. The molecular weight markers (lane 3) were pre-stained low range standards (BioRad).
  • Figure 6 Analysis of the in vitro HBV competition assay. 35S methionine- labelled in vitro translated proteins #1 and #6 were incubated respectively with HBV or HBsAg prior to incubation with the GST-preS1 coated Sepharose beads. Radiolabelled protein bound to the beads was analysed by SDS-PAGE and examined by phospho maging.
  • FIG. 7 Analysis of the binding reaction between GST-protein 6 and HBV by PCR and gel electrophoresis.
  • GST- (lanes 1 and 2) or GST-protein 6- (lanes 3 and 4) coated bead were incubated with partially purified HBV virions.
  • HBV virions bound to the beads after washing (lanes 1 and 3) and in the supernatant fluids prior to washing the beads (lanes 2 and 4) was then determined by PCR; a negative (no DNA) and positive control (pKSHBV DNA) were examined by PCR in parallel and the results are shown in lanes 5 and 6 respectively.
  • preS1 region of the L-HBsAg gene was amplified by PCR using the forward and reverse primers, preSI F and preSI R, which contain an EcoRI and Pstl site respectively for ease of cloning.
  • the sequence of the primers with the restriction enzyme site underlined is preS1 F: 5'CGCGAATTCATGGGGACGAATCTTTCT3'; preSI R 5'CGGCTGCAGCTAGGCCTGAGGATGACTGTC3'. These bind to nucleotide positions 2850 and 3173 on the HBV genome (4).
  • the 309 bp product was cioned into the respective restriction enzyme sites of plasmid pGBT9 (Clontech) to ensure that this region was in frame with the GAL4 DNA-binding domain and this plasmid was subsequently named pGBT9/preS1.
  • the human liver cDNA library which is fused with the GAL4 activation domain of the pGADIO vector, was also obtained from Clontech.
  • the region representing the cellular gene insert in a number of reactive clones was amplified by PCR using primers, pGADIOUP and pGADI ODOWN, which anneal to the GAL4 activation domain.
  • the forward primer, pGADIOUP contained a T7 RNA polymerase promoter region which was subsequently removed from the PCR product by digestion with the restriction enzyme Smal.
  • the sequence of the pGADIOUP and pGADI ODOWN primers is
  • the plasmid, bacMP which was used to construct the GST-preS1 recombinant baculovirus (bacMPV) for the expression of the GST-preS1 fusion protein " in Spodoptera frugiperda (Sf9) cells, was constructed by inserting a PCR cDNA fragment, encoding the preS1 domain into the EcoRI site of pAcSG2T-tag (13), generated with the primers preSI BacF
  • the forward primer contains a BamHI restriction enzyme site which is also present within the preS1 region itself, the amplicon was inserted into the EcoRI site of pAcSG2T-tag by blunt-end ligation.
  • the screening procedure used was a modification of the method described (2); Saccharomyces cerevisiae HF7C or SFY526 (Clontech) was grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or synthetic minimal medium (0.67% yeast nitrogen base, 2% dextrose and appropriate auxotrophic supplements). Both yeast host strains carry a lacZ reporter gene under the control of GAL4-binding sites, while the HF7C strain contains a second reporter gene (HIS3) under the control of GAL4-responsive elements. The yeast strain HF7C was used to screen the liver cDNA library.
  • Yeast cells were transformed with pGBT9/preS1 and the pGAD10/cDNA library using a modified version of the lithium-acetate method previously published by Gietz et al (5) and Ito et al (6), and selected for histidine, leucine and tryptophan prototrophy. After 6-8 days at 30°C, the colonies were assayed for ⁇ -Galactosidase ( ⁇ -Gal) activity by replica plating the yeast transformants onto filter paper.
  • ⁇ -Galactosidase ⁇ -Gal
  • the filters were snap frozen in liquid nitrogen for 20 sec and incubated for 1 -12 h at 30°C in a buffer containing 4mM 5-bromo-4-chloro- 3-indolyl- ⁇ -D-galactopyranoside (X-Gal). Positive interactions were detected by the appearance of blue colonies and were verified by isolation of these colonies, replating and retesting for ⁇ -Gal activity
  • the pGADI O plasmids were isolated from positive yeast transformants by culture in leuc e-deficient medium that resulted in spontaneous loss of the pGBT9-de ⁇ ved plasmids.
  • the pGAD10/cDNA plasmid DNA was purified by CsCI gradient cent ⁇ fugation to permit sequence analysis using the Matchmaker sequencing primer 2 (Clontech) that anneals to the GAL4 activation domain. Sequencing was performed using the dideoxynucleotide chain termination sequencing method (ABI PRISM, Perkm Elmer) and the resulting sequences were compared against the databases of EMBlJGenBank by the BLAST program via the Australian National Genome Information Service
  • the DNA sequences of clones from the cDNA library that were reactive in the yeast two-hybrid system were cloned into pBluesc ⁇ pt KS+ and pcDNA3 as described above for use as a template for transcription of RNA
  • the pBluesc ⁇ pt KS and pcDNA3 constructs were linearised with Sad I and NotI respectively, purified by phenol/chloroform extraction followed by ethanol precipitation then added to the transcription reaction (10). 1 -10 ⁇ l of the transcribed RNA was denatured at 67°C for 7 mm then added to the rabbit reticulocyte lysate system (Promega). The translation mixture (as described by the manufacturer) was incubated at 30°C for
  • a highly efficient system for obtaining recombinant baculovirus was used (7)
  • the system is based on the co-transfection of digested baculovirus DNA, BacPak ⁇ , with a baculovirus transfer vector carrying the gene of interest; digestion of BacPak ⁇ with Aocl inactivates the essential gene downstream of the polyhedrin expression locus resulting in the inability of the DNA to direct the synthesis of infectious virus after transfection.
  • subsequent recombination with the baculovirus . transfer vector restores the essential gene sequence and results in the production of recombinant virus.
  • Replacement of the polyhedrin gene in the BacPak ⁇ DNA with ⁇ -galactosidase allowed blue-white selection of the recombinant virus at the same time.
  • bacMP 1 -2 ⁇ g was mixed with 100-200ng of Aocl-digested BacPak ⁇ DNA in 30 ⁇ l of HBS buffer (20mM Hepes pH7.3, 150mM NaCI) and added to 30 ⁇ l of HBS buffer containing 10 ⁇ l DOTAP (Boehringer). The transfection mixture was then added to a 35mm petri dish containing a monolayer of 1.2 x 10
  • Recombinant plaques were identified by the addition of 1 ml of phosphate- buffered saline (PBS) containing 0.5mg of X-gal (Promega) and incubated overnight at 28°C.
  • the X-gal solution was replaced with 1 ml of PBS containing 0.2% neutral red (GIBCO) for 1 h at 28°C, the neutral red removed and the dishes were inverted and stored at room temperature for 12-48h until the plaques became visible.
  • White plaques were picked, resuspe ⁇ ded in 100 ⁇ l of sterile water, vortexed to disperse the agarose through the solution and the virus amplified by infecting
  • TCF and cell lysates were harvested and GST-preS1 protein purified using glutathione Sepharose 4B beads (Pharmacia) then analysed by SDS-PAGE.
  • Recombinant baculovirus stocks for the expression of GST-preS1 protein were prepared and a high titre recombinant virus stock of bacMPV was used to infect Sf9 insect cells.
  • Cell culture supernatants were harvested 4 - 5 days post infection and the secreted GST-preS1 protein absorbed with 0.3 - 0.5 mi of glutathione Sepharose 4B beads for 2-4 h at 4°C.
  • Hepatitis B virions were purified from HBV-i ⁇ fected patients with a high serum level
  • HBsAg was purified from HH1 cells, a Mycoplasma-free cell line derived from PLC/PRF/5, which stably expresses HBsAg. Briefly, the particles in the TCF were concentrated by pelleting through a 2 ml cushion of 20% sucrose [in 10mM Tris-HCI pH 7.4, 100mM NaCI, 1 mM EDTA (THE)] in a SW41Ti rotor (Beckman) at 230 000 g for 5 h at 4°C. Each HBsAg pellet was resuspended overnight in 50 ⁇ l of TNE at 4°C. The HBsAg preparations were then pooled and diluted to 2 ml with a CsCI solution to yield a
  • GST-preS1 protein was expressed and purified as described above.
  • a recombinant baculovirus which expressed only GST (a gift from Dr. Alexander
  • 35 dialysed [ S]-labelled proteins were incubated with 1 -2 ⁇ g of either GST or GST- preSI protein overnight at 4°C in binding buffer (20mM Tris-HCI pH 8.0, 300mM NaCI, 0.05% Nonidet-P40, 10% BSA, 2 ⁇ g/ml Leupeptin, 1mM Pefabloc, 1 U/5ml Aprotinin).
  • the beads were then pelleted at low speed (1000g), washed four times in RIPA buffer [50mM Tris-HCI pH 7.5, 300mM NaCI, 0.5% NP40, 0.5% sodium deoxycholate, 0.1 % sodium dodecyl sulphate (SDS)] supplemented with protease inhibitors.
  • the pellets were solubilised in sample buffer (10% glycerol, 1 % ⁇ - mercaptoethanol, 2% SDS, 50mM Tris pH6.8, 0.05% bromophenol blue), the samples separated by SDS-PAGE and analysed by phosphorimaging (Molecular Dynamics).
  • sample buffer 10% glycerol, 1 % ⁇ - mercaptoethanol, 2% SDS, 50mM Tris pH6.8, 0.05% bromophenol blue
  • HBV competition assay To determine if HBV could compete with the interaction between protein #6 and the GST-preS1 protein in a dose-dependent manner, the HBV competition assay described above was employed using various dilutions of purified HBV, i.e., 1/2.5, 1/5, 1/10, 1/20, 1/50, 1/100. The dilutions were prepared relative to the original concentration of HBV used in the competition assay and were preincubated with [ 35 S]-labelled protein #6 prior to incubation of the protein with the GST-preS1 coated beads. The samples were then separated by SDS-PAGE and examined by phosphorimaging (results not shown).
  • the mRNA was purified from the HepG2 and HeLa cell RNA using the PolyATract mRNA isolation system IV (Promega) according to the manufacturers protocol.
  • RNA was also isolated from normal human liver tissue by grinding 0.2g into a fine powder in liquid nitrogen, with a mortar and pestle, and the RNA extracted as described above. Twenty ⁇ g samples of total RNA were analysed by northern blot hybridization as described (1 ), except that the hybridization was carried out at 50°C
  • RNA polymerase from plasmid pBluescript KS+, containing the corresponding cDNA, linearized with Sacll.
  • yeast two-hybrid system can identify genuine protein-protein interactions, the system can also generate artefacts which appear to be positive. Thus, to confirm the above results in a more rigorous system, the specificity of the interaction of the preSI region with the proteins expressed from the ten cDNA clones was examined in an in vitro binding assay using a GST-preS1 fusion protein.
  • the ten cDNA clones were excised from pGADIO and recioned into plasmids suitable for the in vitro transcription of RNA (as described in materials and methods). This was then used for the in vitro translation of radiolabelled protein and the products were examined by SDS-PAGE.
  • the in vitro translated proteins ranged in size from 23kDa to 60kDa ( Figure 1).
  • the results demonstrated an increase in binding between protein #6 and the GST-preS1 protein concomitant with decreased levels of HBV.
  • the SDS- PAGE results were then quantitated using ImageQuant, and the data were used to generate a dose response curve (not shown).
  • the results indicate that HBV competes for the interaction between protein #6 and the GST-preS1 coated beads in a dose dependent manner.
  • the HBV DNA present in each sample was detected ( Figure 7) by PCR using primers DAW1 and DAW2 (a gift from Dr Ming Qiao, Institute of Medical and Veterinary Science, Sydney) which are designed to amplify a 384-bp region from the core gene (9)
  • the plasmid, pKSHBVA (14) was used as a positive control.
  • a HBV DNA product of 384 bp was detected in DNA purified from the GST- prote ⁇ n6, but not in the sample from the GST protein alone (lanes 3 and 1 respectively)
  • a PCR product of similar size was apparent in the positive control (lane 6), and in the supernatants of each reaction, suggesting that an excess of HBV virions was present
  • Sequence ID No 1 is the ammo acid sequence of the protein #6
  • Sequence ID No: 2 is the nucleotide sequence (5'-3') of the clone encoding protein #6
  • sequence ID No. 3 is the nucleotide sequence (5'-3') of a 3' RACE product obtained from the nucleotide sequence listed herein as Sequence ID No: 2
  • RACE was carried out according to the instructions supplied with the Boeh ⁇ nger Mannheim 573 * RACE kit
  • First-strand cDNA was synthesized using 1 mg of normal human liver RNA in a reaction mixture containing reverse transc ⁇ ptase, dNTPs and the clone #6 specific primer 3-R-46-1 (ATTGTGAGAAAGTGCTCACCATCTA) in buffer (50mM Tris-HCI, 8mM MgCI 2 , 30mM KCI, 1 mM DTT, pH 8 5)
  • the reaction was incubated for 60 minutes at 55°C, followed by heat inactivation of the reverse transcriptase at 65°C for 10 minutes.
  • cDNA was then directly amplified by PCR using primers 3-R-46-1 and oligo-dT (GACTCGAGTCGACATCGA I I I I I I I I I I I I I I I I ) designed to anneal to the . natural poly(A) tail of mRNAs.
  • PCR was carried out for a total of 30 cycles under the following conditions - annealing at 55°C 30 seconds, elongation at 72°C for 40 seconds, and denaturation at 94°C for 15 seconds, with a final elongation step of 10 minutes.
  • PCR products were visualised by agarose gel electrophoresis prior to subsequent characterisation.
  • gp180 a host cell glycoprotei ⁇ that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022-15028. 9. Qiao, M., Macnaughton, T. B., and Gowans, E. J. (1994). Adsorption and penetration of hepatitis B virus in a ⁇ onpermissive cell line. Virology 201 ,356-363
  • ACCATCTCAA TTTCTTTCAT TTATACATCG TTTTGCCTTC TTTTATGTA
  • GGTCATTAAC ATCCTCCCCC AGTCCCTAAC ATCACATTGT CCTGCGTGGC CCAGTAATTG TAGGAGGGGG TCAGGGATTG TAGTGTAACA GGACGCACCG
  • ATCTCTGCAA GGGAGGTGTT ACAGCTGGTT CTGAGCCGCT TGCCTTGTGA TAGAGACGTT CCCTCCACAA TGTCGACCAA GACTCGGCGA ACGGAACACT

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Abstract

L'invention porte sur un polypeptide isolé fixant le virus de l'hépatite B (HBV) qui comprend la séquence d'acides aminés présentée comme (SEQ ID N° 1) ou comme une variante focntionnelle de celle-ci; sur des acides nucléiques codant pour ce polypeptide et des procédés permettant d'utiliser ces polypeptides et acides nucléiques. L'invention concerne également des vaccins,des thérapies et des animaux transgéniques non humains refermant de tels polypeptides et acides nucléiques.
PCT/AU1998/000889 1997-10-27 1998-10-27 Polypeptide fixant le hbv Ceased WO1999021882A1 (fr)

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AU97302/98A AU9730298A (en) 1997-10-27 1998-10-27 Hbv binding polypeptide

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AUPP0041A AUPP004197A0 (en) 1997-10-27 1997-10-27 Novel hbv binding protein and portions thereof
AUPP0041 1997-10-27

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WO1999021882A1 true WO1999021882A1 (fr) 1999-05-06

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CN107325176A (zh) * 2017-06-16 2017-11-07 神威药业集团有限公司 源于血红蛋白的免疫活性人胎盘多肽

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