US20030235591A1 - Composition used as a therapeutic agent against chronic viral hepatic diseases - Google Patents
Composition used as a therapeutic agent against chronic viral hepatic diseases Download PDFInfo
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- US20030235591A1 US20030235591A1 US10/394,896 US39489603A US2003235591A1 US 20030235591 A1 US20030235591 A1 US 20030235591A1 US 39489603 A US39489603 A US 39489603A US 2003235591 A1 US2003235591 A1 US 2003235591A1
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6018—Lipids, e.g. in lipopeptides
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a composition
- a composition comprising a polypeptide sequence prepared by recombinant DNA techniques and a carrier to provide a curing agent against chronic viral hepatic diseases.
- the invention relates to DNA sequences coding for said polypeptide sequences and to transfected cells for the expression of the same.
- Hepatitis virus A, B, C, D and E are able to trigger the clinical aspect of an acute hepatitis.
- Hepatitis A and E which are transferred enterically, always heal, whereas hepatitis B, C (formerly called parenteral hepatitis Non-A Non-B), and D can progress into a chronic stage of inflammation, which in turn can result in liver cirrhosis and primary hepatocellular carcinoma.
- hepatitis D virus is a RNA virus which is known to be incomplete. Therefore, it needs a helper virus to develop in patients and is found only in individuals infected with HBV. Only very recently the hepatitis C virus has been detected, and an antibody test (anti-HCV) facilitating the diagnosis of chronic hepatitis C infections has been developed. However, there is an increasingly urgent need for a treatment to cure this disease.
- hepatitis B chronic hepatitis B
- Patients are said to be chronic carriers of the hepatitis B virus if the viral DNA persists longer than ten weeks, the HBe-antigen (HBeAg) for more than 12 weeks, or if the hepatitis B surface-antigen (HBsAg) is persistent longer than six months.
- the HB-virion (Dane particle) is composed of different structural proteins, the core proteins and the surface (S) proteins.
- the latter are translation products of an open reading frame encompassing the coding sequence of three S-type domains, each of which starts with an ATG triplet capable of initiating translation in vivo.
- the domains are referred to as preS1, preS2 and S in the order of 5′ to the 3′end of the molecule.
- HBcAg and HBeAg are six protein products derived from this ORF: a glycosylated and a non-glycosylated form of the major protein (gp27 and p24) translated from the S domain only (226 amino acids), a middle protein (281 amino acids) having one or two polysaccharide side chains (gp33 and gp36, respectively), that is encoded by the preS2- and S-region, and finally, both a glycosylated (gp42) and a non-glycosylated (p39) form of the large protein (389-400 amino acids, depending upon the viral serotype), which is formed by translation of preS1, preS2 and S.
- the core proteins are HBcAg and HBeAg, the latter one conceivably being a processing product of HBcAg.
- the Dane particle which is the infectious virion, comprises both core and surface proteins, whereas the filaments consist of a mixture of the six surface antigens.
- the S peptides alone assemble to form the so-called 20 nm particles, which are completely uninfectious.
- CPH chronic persistent hepatitis
- CAH chronic active hepatitis
- interferon ⁇ On chronic hepatitis B remains unclear. Its antiviral activity might protect infected cells from infection or reduce viral transcription, translation and replication in HBV-infected cells. Interferon further has immunomodulatory effects by activating T-cells, macrophages and NK-cells and by inducing the expression of MHC class I proteins.
- Another approach to treat chronic hepatitis B is based on the idea to inhibit replication of the virus, thus impairing its defence sufficiently to render the host immune system capable of eliminating the virus.
- antiviral drugs such as adenine arabinoside and adenine arabinoside monophosphate for treatment of chronically HBV infected individuals.
- adenine arabinoside and adenine arabinoside monophosphate for treatment of chronically HBV infected individuals.
- less than half of the patients responded to this therapy either by sustained or transient seroconversion (HBeAg + to anti-HBe + ).
- a further negative aspect of these antiviral drugs are their immunosuppressive properties.
- interferon ⁇ drugs that have beeen tested for treatment of chronic carriers
- drugs include interferon ⁇ and, acycloguanosine (acyclovir), interleukin 2, steroids, such as prednisolone, and combinations thereof. But none of them could provide better results than treatment with interferon ⁇ . Only a combination therapy, including the initial administration of steroids followed by that of IFN ⁇ may increase the response rate in selected patients.
- chronic carriers of hepatitis B virus are characterized in that HBsAg is detectable in their serum. Therefore, it has been absolutely unforeseeable, that a combination, comprising a T-cell activating epitope of the viral S peptide, according to the present invention, is able to induce an immunization in and a final healing of chronic carriers of hepatitis virus B.
- this goal is achieved by a combination of a) at least one polypeptide sequence mediating the antigenicity of one or more epitopes and b) a carrier, capable of presenting the epitope sequence(s) a), wherein the polypeptide sequence(s) a) can be bound to carrier b) by adsorption, any chemical bonding or secondary valences.
- This invention is furthermore directed to the use of this combination for the production of a medicament for the treatment of chronic viral hepatitis.
- the present invention is further directed to a method for the treatment of chronic viral hepatitis by administering to a patient the above described combination of a) at least one polypeptide sequence mediating the antigenicity of one or more epitopes and b) a carrier capable of presenting the epitope sequence(s) a), wherein the polypeptide sequence(s) a) can be bound to carrier b) by adsorption, any chemical bonding or secondary valences.
- polypeptide sequence a which may be one or more different polypeptides, mediates the antigenicity of a T cell-activating epitope in a direct or indirect way.
- polypeptide sequence(s) a) may be a polypeptide or a combination of two or more polypeptides of hepatitis B virus of any subtype, particularly adw, ayw, adr and ady.
- These peptides derived from hepatitis B virus may be HBV peptides preS1, preS2 or S or the HBV core antigens.
- polypeptide sequence(s) a) are futhermore any of the above-stated polypeptides or a combination of two or more polypeptides which are modified either by amino acid deletions, whereby at least one epitope comprising at least six consecutive amino acid residues must be preserved, or by adding further amino acids either at the N-terminus, the C-terminus or as insertions into the polypeptide sequence(s) a). In each of these cases it is essential, however, that the biologial activity is maintained.
- polypeptide sequence(s) a) is myristylated.
- polypeptide sequences(s) a) is presented on a carrier b).
- This carrier consists of a particular substance which for example may consist of particles of a hydrophobic polymer, of inorganic particles, or of particles of a polysaccharide.
- carrier b) is a second polypeptide sequence which forms particles upon secretion, said particles having preferably a diameter of at least 10 nm.
- the particle forming polypeptide sequence b) is a substantial part of or the complete amino acid sequence of a polypeptide which may be selected from HBV S peptide, HBV core antigen, HAV core antigen, HAV surface antigen, HIV surface antigen and HIV core antigen as well as the surface antigen of polio virus.
- Preferred as the particle-forming carrier b) is HBV S peptide and/or core peptide.
- polypeptide sequence b) When used as the carrier sequence b) the above-stated polypeptides may be modified by arbitrary deletions of amino acids, by substitutions of one or more amino acids or by adding one or more amino acids either at the N-terminus, the C-terminus or by insertion of one or more amino acids into the polypeptide sequence b), provided that the particle-forming capacity is maintained.
- polypeptide sequence b) is myristylated.
- both sequences a) and b) may be linked via one or more of the following interactions: hydrophobic anchoring (mediated by myristic acid), disulfide bridge formation, or both sequences may be connected by a peptide bond to form a fusion peptide.
- hydrophobic anchoring mediated by myristic acid
- disulfide bridge formation or both sequences may be connected by a peptide bond to form a fusion peptide.
- a spacer sequence may be inserted between polypeptide sequence(s) a) and polypeptide sequence b), which spacer sequence is linked to both polypeptides via peptide bonds.
- the present invention furthermore provides a recombinant DNA molecule coding for a combination, that is useful for production of a medicament to treat chronic viral hepatic diseases.
- the recombinant DNA molecule comprises at least one first DNA sequence, optionally a second, a third and/or a fourth DNA sequence wherein
- said at least one first DNA sequence codes for at least one polypeptide sequence a) as defined above,
- DNA sequences are controlled by DNA elements essential for expression, and optionally have a common reading frame.
- the invention further embraces recombinant DNA molecules, which differ from the above-defined recombinant DNA molecules by the fact, that up to 30% of the nucleotides may be substituted.
- a further object of the present application is to provide a host cell transfected with a recombinant DNA molecule coding for the above combination, which is useful for treatment of chronically HBV-infected patients.
- This host cell may be a mammalian, a yeast or a bacterial cell.
- this cell does not produce any human serum proteins or any primate serum proteins other than the polypeptide(s) being comprised within the above combination.
- HBV S peptide refers to the peptide encoded by the entire region of the HBV genome.
- HBV pre-S2 peptide refers to the peptide encoded by the entire pre-S2 and S regions of the HBV genome.
- HBV pre-S1 peptide refers to the polypeptide encoded by the entire pre-S1, pre-S2 and S regions of the HBV genome.
- epipe refers to a sequence of at least six consecutive amino acids encoded by the designated genome region (e.g.
- HBV pre-S2 epitope refers to a sequence of at least six amino acids encoded by the pre-S2 region of the HBV genome).
- T-cell epitope refers to an epitope that interacts with receptors on the surface of T-cells to enhance or otherwise effect an immune response.
- antigenicity means the ability to provoke an immune response (e.g. acting as an antigen), the ability to cause the production of antibodies (e.g. acting as an antigen) and/or the ability to interact with a cell surface receptor so as to enhance an immune response or production of antibodies.
- HBV means any subtype of the virus, particularly adw, ayw, adr and ayr, described in the literature (P. Valenzuela, Nature Vol. 280, p. 815 (1979), Gerlich, EP-A-85 111 361, Neurath, EP-A-85 102 250). Examples of peptide sequences thereof, constituting polypeptide sequence(s) a), which mediate the antigenicity of one or more epitopes, are shown in the Sequence Listing (SEQ ID No. 17-20, 22).
- Preferred embodiments of the present invention are the following combinations:
- Hepatitis A-antigen particles with specific epitopes (determinants) of the hepatitis B S-,pre-S1-, pre-S2-, and/or core-peptides are included in the hepatitis B S-,pre-S1-, pre-S2-, and/or core-peptides.
- Recombinant DNA molecules preferred for the present invention are characterized by the presence of sequences coding for polypeptide sequence(s) a), mediating the antigenicity of one or more T-cell epitopes, and for polypeptide b), which upon secretion forms particles having a diameter of 10 nm or more, both of which are under control of a suitable promoter.
- sequences coding for a) there may be mentioned any of the sequences listed under ID numbers 1 to 24 in the Sequence Listing.
- Examples for the DNA sequence coding for polypeptide sequence b) are represented by any of the ID-sequences 25 to 27 in the Sequence Listing.
- a particular preferred embodiment of the present invention consists in a combination of the epitope sequence ID No. 28 (corresponding to amino acids 9 to 28 of the S1 sequence of HBV) in combination with sequence ID No. 26 and/or 27 as a particle former.
- Hepatitis virus sequences used in the recombinant DNA construct of the present invention can be formed or isolated by any means including isolation and ligation of restriction fragments, chemical synthesis of oligonucleotides using a synthesizer (Cyclon, Bio-Search), and synthesis by the PCR method (T. J. White, N. Arnleim, H. E. Erlich, 1989; The Polymerase Chain Reaction, Technical Focus 5 (6)).
- Preferred recombinant DNA molecules were formed by the ligation of synthetic oligonucleotides to a 5′ XbaI-BglII 3′ fragment (ID number 27) from the S region of the HBV genome, which is derived from a BglII-BglII HBV fragment including the entire pre-S1-pre-S2-S-region, or to the entire S-region. Oligonucleotides used in making such constructs are summarized in Table I below. TABLE I Function Definition SEQ ID No.
- bp 1901-2500 1 core C-terminal deletion, 2 bp 1901-2405 core C-terminal deletion and 3 stop codon inserted, bp 1901-2405 core/precore 10 aa precore, 4 C-terminal deletion, bp 1871-2405 core/precore 10 aa precore, 5 C-terminal deletion and stop codon inserted, bp 1871-2405 core aa ( ⁇ 10-120) 16 core/precore 10 aa precore, 35 complete core, bp 1871-2500
- Table II-2 shows several examples, where the T-cell epitope-coding DNA sequences have been isolated by restriction fragmentation of the HBV genome and have been ligated to the DNA sequence coding for polypeptide sequence b) as defined above.
- TABLE II-2 Function Definition SEQ ID No. core/precore complete, bp 1403-311 ** S2 ay/ad ** S2 (K) ay/ad S2-S, 7 codons 14 deleted, start codon ATG changed to ATA
- Preferred recombinant DNA molecules according to the present invention comprise, apart from the regions coding for polypeptides a) and b), an additional DNA sequence coding for a selection marker. Furthermore, they comprise all usual elements essential for the expression, such as promoter sequence, start codon and a polyadenylation signal.
- suitable promoters are the methallothionein (MT), the U2 and the H2K promoter in case of using mammalian cells as a host cell. If yeast or bacterial cells are to be employed, appropriate yeast and bacterial promoters, such as the GCN4- and the GAL 1/10 promoter or the prokaryotic trp- and tac promoters, respectively, may be used.
- MT methallothionein
- U2 the U2
- H2K promoter in case of using mammalian cells as a host cell.
- yeast or bacterial cells are to be employed, appropriate yeast and bacterial promoters, such as the GCN4- and the GAL 1/10 promoter or the prokaryotic trp- and tac promoters, respectively, may be used.
- the recombinant DNA molecule is inserted into host cells by transfection (in case of mammalian cells), by transformation (in case of yeast and bacterial cells), or by other means.
- transfection in case of mammalian cells
- transformation in case of yeast and bacterial cells
- a host cells of any organism may be used that are capable of transcribing and translating recombinant DNA molecules, such as mammalian, bacterial and yeast cells.
- Suitable mammalian cells are for example VERO cells (a monkey kidney cell line), 3T3-, C127 and L cells (murine fibroblast cell lines), and CHO (Chinese hamster ovary) cells, which are either positive or negative in dehydrofolate reductase.
- VERO cells a monkey kidney cell line
- 3T3-, C127 and L cells murine fibroblast cell lines
- CHO Choinese hamster ovary
- the above-defined first DNA sequence and the above-defined second DNA sequence which code for polypeptide sequence(s) a) and for a polypeptide sequence b), respectively, are present in different recombinant DNA molecules, in which case the host cells are cotransfected with both of these recombinant DNA molecules.
- compositions for particles containing T-cell epitopes for targeting chronic hepatitis carrier FINAL T-CELL-EPITOPE PARTICLE SELECTION PURIFI- CONSTRUCT 1 PROMOTER 2 SYN PCR GENE FORMER GENE CATION 3 12 MT/112/U2 Core(AA ⁇ 10-+87) entire S adw/ayw neo/egpt S/XbaI/BglII 13 MT/112/U2 Core(AA 70-110) entire S adw/ayw neo/egpt S/XbaI/BglII 14 MT/112/U2 Core(AA 80-125) entire S adw/ayw neo/egpt S/XbaI/BglII 15 MT/112/U2 Core(AA 88-120) entire S adw/ayw neo/egpt S/XbaI/Bgl
- Table III gives an overview on how to combine suitable DNA sequences to get DNA constructs according to the present invention. It is to be noted that any constituents disclosed in this table may be combined to provide a DNA sequence which may be taken, if transfected into a host cell, to produce a combination (comprising polypeptides(s) a) and b)) as a medicament for the treatment of chronic viral hepatitis.
- the DNA sequences coding for the T-cell epitope sequences have been prepared synthetically (SYN) with a Biosearch Cyclon synthesizer, by PCR procedure (PCR), or by restriction enzyme fragmentation of the viral genome (GENE).
- the combination of polypeptide sequence(s) a) and a carrier b) may be formulated in any type of a pharmaceutical composition, which furthermore comprises a suitable diluent or pharmaceutical carrier material, such as a buffer solution.
- the administration may be effected by any method, i.e. by parenteral (e.g. intravenous or intramuscular) or oral (e.g. by using typhoid bacterial cells to encapsulate the active substance) administration.
- parenteral e.g. intravenous or intramuscular
- oral e.g. by using typhoid bacterial cells to encapsulate the active substance
- the pharmaceutical preparation comprises the above-described combination in sufficient concentration to elicit a response upon administration.
- FIG. I shows a DNA construct, coding for a promoter, a particle former sequence and a selection gene (described in Example 3/4).
- FIG. II shows a DNA-gene construct containing a promoter, an epitope with the entire HB-S-Ag and a selection gene (described in Example 3/18).
- FIG. III shows a DNA construct presenting a promoter, a T-cell epitope with a particle former residue and a selection gene (described in Example 3/21).
- FIG. IV shows the AST values of chimpanzee 1 during the Hepa-Care treatment (described in Example 10/1).
- FIG. V shows the antigen values of chimpanzee 1 during the Hepa-care treatment (described in Example 10/1)
- FIG. VI shows values of liver enzymes ALT and GGT of chimpanzee 1 booster treated three times with Hepa-Care (described in Example 10/2).
- FIG. VII shows values of liver enzymes ALT, AST, and GGT and of antigen of chimpanzee 2 during the Hepa-Care treatment (described in Example 10/3).
- FIG. VIII shows the liver enzymes as determined for an untreated control chimpanzee (described in Example 10/3).
- FIGS. IX & X show the antigen and antibody titers of patient 1 during the Hepa-Care treatment, respectively (described in Example 11).
- FIGS. XI & XII show the antigen and antibody titers of patient 2 during the Hepa-Care treatment, respectively (described in Example 11).
- FIGS. XIII & XIV show the antigen and antibody titers of patient 2 during the Hepa-Care treatment, respectively (described in Example 11).
- the supernatant of HBV protein producing cultures was collected and split into portions of 2,400 ml. To each portion 144 g of PEG 6000 (Serva) were added and dissolved by stirring at room temperature for 20 minutes and was stirred for another 6 hours at 4° C. The precipitate was separated by centrifugation in 500 ml bottles in a GS 3 rotor at 9,000 rpm (15,000 ⁇ g) for 30 minutes at 10° C. The supernatant was collected and 144 g of PEG 6000 were added and dissolved as described above. The solution was stirred at 4° C. for 3 hours. The precipitate from this solution was harvested as described above except that centrifugation was continued for 60 minutes.
- the plasmid pBPV-342-12 (ATCC 37224) was digested with the endonucleases BglII and BamHI. Three DNA molecules were generated.
- the fragment of interest contains the methallothionein promoter and a pBR322 sequence comprising 4.5 kb and is easily detectable from the other fragments (2.0 kb and 7.6 kb).
- the reaction was performed in a total volume of 200 ⁇ l of reaction buffer at a final concentration of 0.5 ⁇ g/ ⁇ l DNA including 100 units of each restriction enzyme. The completion of the digestion was checked after incubation at 37° C. for three hours by agarose gel electrophoresis at a 0.8% agarose gel. The reaction was stopped by adding 4 ⁇ l 0.5 M EDTA.
- the 4.5 kb fragment was separated from the other fragments by preparative 1.2% agarose gel electrophoresis.
- the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
- the DNA was purified by a phenol-chloroform extraction and two ethanol precipitations.
- a 1.8 kb BamHI-BamHI fragment, containing the HBV-core coding regions was isolated from HBV-containing DNA. This fragment was ligated together with the 4.5 kb fragment containing the MT-promoter and the pBR residue (described in 1).
- the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 ⁇ l/ml ampicillin at volumes of 50 to 300 ⁇ l cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragments.
- the plasmid resulting from (3) above was linearized by digestion with the restriction enzyme EcoRI.
- the reaction was performed in a total volume of 50 ⁇ l and a final concentration of 1 ⁇ g/ ⁇ l plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis.
- the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at ⁇ 80° C. for 30 minutes.
- the precipitated DNA was dissolved in 50 ⁇ l distilled water.
- the plasmid pUC-8-42 (available from Exogene) was digested with the restriction endonucleases EcoRI and ApaI. Two DNA molecules were generated. The fragment of interest contains the U2-promoter comprising 340 bp and is easily detectable from the other fragment (3160 bp). The digestion was performed in a total volume of 200 ⁇ l reaction buffer at a final concentration of 0.5 ⁇ g/ ⁇ l DNA including 100 units of each restriciton enzyme. The completion of the digest was checked after incubation at 37° C. for three hours by agarose gel electrophoresis in a 0.7% agarose gel. The reaction was stopped by adding 4 ⁇ l 0.5 M EDTA.
- the 340 bp fragment was separated from the plasmid DNA by preparative 1.2% agarose gel electrophoresis.
- the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
- the DNA was purified by a phenol/chloroform extraction and two ethanol precipitations.
- the plasmid pSP165 (commercially available from Promega Diotec) containing a polylinker sequence (containing the following restriction sites: EcoRI, SacI, SmaI, AvaI, BanHI, BglII, SalI, PstI, HindIII) was linearized with the restriction enzyme EcoRI. The reaction was performed in a total voume of 50 ⁇ l and a final concentration of 1 ⁇ g/ ⁇ l plasmid DNA. 50 units of EcoRI was added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis.
- the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at ⁇ 80° C. for 30 minutes.
- the precipitated DNA was dissolved in 50 ⁇ l distilled water.
- the sticky ends were converted into blunt ends by reaction with the Mung bean nuclease as follows: to 25 ⁇ l DNA (1 ⁇ g/ ⁇ l concentration) in reaction buffer 20 units of enzyme were added to give a final concentration of 1% glycerol and a final reaction volume of 35 ⁇ l. After an incubation for 30 minutes at 30° C. the DNA was purified by phenol-chloroform extractions followed by two ethanol precipitations. The DNA was dissolved again in 5 ⁇ l of distilled water. The resulting blunt ends were ligated together in 15 ⁇ l reaction volume containing 10 ⁇ more T4 ligase than used above and 2 mM ATP at 14° C. overnight.
- the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 ⁇ g/ml ampicillin at volumes of 50 to 300 ⁇ l cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired U2-promoter fragment. The resulting plasmid was isolated from the bacterial cells and characterized by restriction enzyme analysis.
- the plasmid (described in 7) was digested with BglII and BamHI and a fragment of 13 nucleotides was removed (described in 1).
- the resulting fragment containing the first oligonucleotide 89 (SEQ ID No.: 30), was ligated together with oligonucleotide 101 (SEQ ID No.: 32), a BglII-BamHI fragment.
- SEQ ID No.: 32 oligonucleotide 101
- the new plasmid was proved by a digestion with the endonucleases EcoRI and XbaI, or EcoRI and BglII.
- the new plasmid was then digested with PstI and BamHI. Two molecules were expected, one 2.6 kb fragment, containing a pBR residue, the MT-promoter and the oligonucleotide and a 2.0 kb pBR residue. The 2.6 kb fragment was isolated.
- the plasmid (described in 8) containing the DNA oligonucleotides 89 and 101 (SEQ ID No.: 30 and 32, respectively) was digested with PstI and BglII. Two fragments were expected. A 2.5 kb fragment containing a pBR residue and the MT-promoter and 2.2 kb fragment, containing a pBR residue and both oligos.
- a 2.3 kb BglII-BglII fragment containing the HBV pre-S1, pre-S2 and S coding regions was isolated from HBV-containing DNA.
- the 2.3 kb fragment was ligated together with the 4.5 kb fragment (obtained as described in 1) containing the methallothionein promoter.
- the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plate containing 50 ⁇ g/ml ampicillin at volumes of 50 to 300 ⁇ l cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragment.
- the plasmid resulting from 11 above was digested with the endonucleases BglII and XbaI. Two molecules were expected, one 550 bp fragment and a 6.25 kb fragment which was isolated after agarose gel electrophoresis.
- the 6.25 kb fragment was ligated together with the 270 bp fragment (after digestion with BglII and XbaI and fragment isolation as described above) of the plasmid described in 10, coding for an epitope part of the HBV-core gene.
- the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid. The new plasmid was proved by a digestion with BamHI. Three molecules were expected, a 950 bp, a 450 bp and a 5,150 bp fragment.
- the plasmid (described in 11) was digested with EcoRI and XbaI. Two molecules were expected, one 2,450 bp fragment and a 4,350 bp fragment which was isolated after gel electrophoresis.
- This 4,350 bp fragment was ligated together with the oligo-DNA-nucleotide 39 (SEQ ID No: 29) coding for the entire DNA-sequence of HBV-S-gene from ATG to the XbaI site, wherein the ATG was changed into ATA.
- This “vehicle” plasmid was then digested with PstI and XbaI, two molecules were expected, one 600 bp plasmid residue and a 3,850 bp fragment which was isolated and ligated together with a PstI-XbaI fragment of 2,800 bp (2,700 bp) isolated after digestion of the plasmid described in 10.
- the plasmid (described in 14) was linearized with Eco RI. The reaction was performed in a total volume of 50 ⁇ l and a final concentration of 1 ⁇ g/ ⁇ l plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis.
- the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA and DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at ⁇ 80° C. for 30 minutes.
- the precipitated DNA was dissolved in 50 ⁇ l distilled water.
- Each gene construct described above can be constructed also with the U2-promoter whereby the MT-promoter-containing DNA fragment, after digestion with EcoRI and BglII, is replaced by a DNA fragment containing the U2-promoter isolated after digestion with EcoRI and BglII.
- the fragment containing the egpt selection gene was isolated after digestion of the plasmid PMSG with BamHI and BglII (1.8 kb) and ligated together with a 4.5 kb fragment (BglII-BamHI, described in 1) containing the MT-promoter.
- DNA fragment 400 bp was isolated after gel electrophoresis. It was generated by PCR-method (described in Example 5) by using the specific oligonucleotides 131 and 132 (SEQ ID No.: 33 and 34) as primers.
- the DNA fragemnt was digested with the endonucleases BamHI and XbaI and then purified by gel electrophoresis.
- the isolated PCR-fragment was ligated together with a 6.25 kb fragment which was isolated from the plasmid (described in 13) after digestion with BglII and XbaI. After DNA up-take and bacterial transformation the single bacterial colonies were screened for the desired plasmid.
- the plasmid (described in 17) was finalized by adding a selection gene to the plasmid (described in 15).
- the H2K promoter was isolated as an EcoRI and BglII fragment (2 kb) from pSP65H2 (available from Exogene).
- the 6.250 kb fragment was ligated together with oligo-DNA-nucleotide 23 (SEQ ID No.: 28).
- the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid.
- the new plasmid was proven by a digestion with the endonucleases EcoRI and BglII. Two molecules were expected, one 1,9 kb and one 4.450 kb.
- the plasmid (described in 20) was linearized with EcoRI. The reaction was performed in a total volume of 100 ⁇ l and a final concentration of 0.6 ⁇ g/ ⁇ l plasmid DNA. 60 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis. The reaction was stopped by adding 2 ⁇ l of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 4 volumes of ethanol at ⁇ 80° C. for 1 hour. The precipitated DNA was dissolved in 50 ⁇ l distilled water.
- mammalian cells In order to achieve secretion of substantial amounts of the HBV peptides encoded by constructs of the present invention, mammalian cells must be transfected with a DNA construct of the present invention. The cotransfection was performed in two steps (i.e. a separate transfection for each construct) or in a single step (i.e. one transfection using preparation of both constructs). Cotransfection was confirmed either by use of different selection markers on the two constructs or by detection of secretion of expression products of both constructs by immunoassay.
- a sequence encoding the HBV peptide sequence of the present invention and a separate sequence encoding the entire S or core or HAV protein could be combined in a single construct.
- the polymerase chain reaction allows to amplify specific DNA necleotide sequences of a selected region of a known genomic sequence in vitro by more than a millionfold (Thomas J. White, Norman Arnleim, Henry A. Erlich 1989: The polymerase chain reaction. Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi 1989: Avoiding false positives with PCR. Nature, Vol. 339, pp 237-238).
- DNA isolated from cells or plasmid DNA is treated to separate its complementary strands. These strands are then annealed with an excess of two DNA oligonucleotides (each 20-25 base pairs long) that have been chemically synthesized to match sequences separated by X nucleotides (where X is generally between 50 to 2,000 base pairs).
- the two oligonucleotides serve as specific primers for in vitro DNA synthesis catalysed by DNA polymerase which copies the DNA between the sequences corresponding to the two oligonucleotides. If the two primer oligonucleotides contain the correct sequence it is possible to create new digestion sites at the 5′ and 3′.
- PCR-DNA fragments were amplified with blunt end. To get sticky end (for the ligation procedure) the fragment has to be digested with the desired endonucleases and purified again.
- the PCR-reaction will work for 20 to 30 cycles. One cycle is separated into three steps with different reaction times and different reaction temperatures which is controlled by a PCR-thermo-cycler.
- the first step is “Denaturation” of the matrix-DNA (1 min-95° C.)
- the second step is “Hybridisation” of matrix DNA and primers. (1 min/55° C.) followed by “Polymerisation” (2 min/72° C.).
- the final volume for one assay is 30 ⁇ l for example, which contains the following final concentrations: PCR-buffer (1 ⁇ ), nucleotide-mix with 200 ⁇ M of each of the four nucleotides, 200 ng for 30 ⁇ l of each of the two primers, 0.5 units Taq-Polymerase per 30 ⁇ l aqua bidest.
- the recipient cells (C127 or CHO-cells available from ATCC) were seeded in normal growth medium (DMEN+10% Fetal Calf Serum, Glucose and Glutamine) into petri-dishes (1-2 ⁇ 10 6 cells per dish, ⁇ 10 cm) at day 1. The next day the medium was removed (4 hours before the DNA precipitate was added onto the cells), and the cells were washed twice with 1 ⁇ PBS. Then 8 ml DMEM without FCS were added, 4 hours later the DNA precipitate (prepared as described below) was added to the cells.
- DMEN+10% Fetal Calf Serum, Glucose and Glutamine normal growth medium
- petri-dishes 1-2 ⁇ 10 6 cells per dish, ⁇ 10 cm
- Glycerol-Mix 50 ml 2 ⁇ TBS buffer, 30 ml glycerol, 120 ml distilled water
- the Glycerol-Mix was immediately removed after an incubation at 37° C. for 3 minutes and the cells were washed with 1 ⁇ PBS.
- the cells were cultivated overnight with 8 ml of DMEM with 10% FCS.
- the cells were recovered from the dish by treating with Trypsin-EDTA-Solution (0.025% Trypsin+1 mM EDTA). Afterwards, to remove the Trypsin-EDTA the cells were washed with 1 ⁇ PBS, suspended in DMEM with 10% FCS and distributed into 24 costar-well-plates (cells from one dish into four 24-well-plates).
- selection medium was added (concentration 0.5-1 mg/ml of neomycin or: xanthine (250 ⁇ g/ml), hypoxanthine (15 ⁇ g/ ⁇ l) or adenine (25 ⁇ g/ml), thymidine (10 ⁇ g/ml), aminopterine (2 ⁇ g/ml), mycophenolic acid (25 ⁇ g/ml) for eco-gpt, for example).
- the medium was changed every week. The first growing cell colonies were seen after 2 weeks.
- TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 7.05
- TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 7.05
- 2 ⁇ TBS Hepes 50 mM, NaCl 280 mM, Na 2 HPO 4 1.5 mM, pH 7.05
- the precipitation solution was incubated for 30 minutes at 37° C. and added directly to the cells which were to be transfected.
- the cell supernatant of HB-core-antigen-secreting cells was collected and concentrated by ultrafiltration. The concentrate was cleared by centrifugation at 20,000 rpm for 15 minutes at 4° C. in a Beckman SW28 rotor.
- Particle formaiton was tested by sucrose density centrifugation (0-45% sucrose) in a Beckman SW28 rotor for 24 hours at 28,000 rpm and 4° C. The gradient was fractionated and the single fractions were analyzed by Elisa.
- Table IV shows the Elisa data of the purified HBs-antigen particle produced from any HBV-sequence construct of the present invention including the pre-S1 epitopes and the S region with the anti-pre-S1 monoclonal antibody MA 18/7 and the anti-HBs monoclonal antibody G022.
- Hepa-Care are particles presenting hepatitis B surface antigens (S1 and S) in a specific formulation (ratio 50:50), which are used for the treatment of chronic carriers of hepatitis virus.
- a Hepatitis-B-carrier chimpanzee 1 was treated (intramuscularly) with Hepa-Care at time 0, 4, and 8 weeks with a dosage of 18 ⁇ g per injection.
- liver enzymes were monitored (FIG. IV) as well as the hepatitis-B antigen level (FIG. V).
- liver enzymes were also monitored and shown in FIG. VIII.
Abstract
A combination, comprising at least one polypeptide sequence, mediating the antigenicity of one or more epitopes, and a carrier, capable of presenting this/these polypeptide sequence(s), which are useful for the production of a medicament for the treatment of chronic viral hepatitis, is provided.
Description
- The present invention relates to a composition comprising a polypeptide sequence prepared by recombinant DNA techniques and a carrier to provide a curing agent against chronic viral hepatic diseases. The invention relates to DNA sequences coding for said polypeptide sequences and to transfected cells for the expression of the same.
- At least five different viruses, namely Hepatitis virus A, B, C, D and E, are able to trigger the clinical aspect of an acute hepatitis. Hepatitis A and E, which are transferred enterically, always heal, whereas hepatitis B, C (formerly called parenteral hepatitis Non-A Non-B), and D can progress into a chronic stage of inflammation, which in turn can result in liver cirrhosis and primary hepatocellular carcinoma.
- There is relatively little data available on hepatitis C and D, on methods for the diagnosis and their treatment and on the respective viruses. The hepatitis D virus is a RNA virus which is known to be incomplete. Therefore, it needs a helper virus to develop in patients and is found only in individuals infected with HBV. Only very recently the hepatitis C virus has been detected, and an antibody test (anti-HCV) facilitating the diagnosis of chronic hepatitis C infections has been developed. However, there is an increasingly urgent need for a treatment to cure this disease.
- The same holds true for chronic hepatitis B, a much better studied disease with respect to its recognition by immunological methods, its causative virus and the viral life cycle and DNA sequence. Patients are said to be chronic carriers of the hepatitis B virus if the viral DNA persists longer than ten weeks, the HBe-antigen (HBeAg) for more than 12 weeks, or if the hepatitis B surface-antigen (HBsAg) is persistent longer than six months.
- Roughly three hundred million people are deemed to suffer from chronic hepatitis B, most of them living in the Far East.
- For these people the main risk to be infected appears to be during or immediately after birth, since a chronically infected mother transfers the virus to her newborn. 90 percent of the children infected this way will become chronically infected, too, during later life. In the Western World infection occurs more commonly later in life, during childhood or even adulthood, mainly by a parenteral or sexual transmission. In these cases of hepatitis B infection after birth only five to ten percent of the infected become chronic carriers. The virus transferred, however, is not responsible for distinct reactions shown by infected people either to eliminate the virus or to retain it in the body lifelong. Consequently, it seems to be a matter of the immunological status that determines the future physical condition.
- The HB-virion (Dane particle) is composed of different structural proteins, the core proteins and the surface (S) proteins. The latter are translation products of an open reading frame encompassing the coding sequence of three S-type domains, each of which starts with an ATG triplet capable of initiating translation in vivo. The domains are referred to as preS1, preS2 and S in the order of 5′ to the 3′end of the molecule. There are six protein products derived from this ORF: a glycosylated and a non-glycosylated form of the major protein (gp27 and p24) translated from the S domain only (226 amino acids), a middle protein (281 amino acids) having one or two polysaccharide side chains (gp33 and gp36, respectively), that is encoded by the preS2- and S-region, and finally, both a glycosylated (gp42) and a non-glycosylated (p39) form of the large protein (389-400 amino acids, depending upon the viral serotype), which is formed by translation of preS1, preS2 and S. The core proteins are HBcAg and HBeAg, the latter one conceivably being a processing product of HBcAg.
- The Dane particle, which is the infectious virion, comprises both core and surface proteins, whereas the filaments consist of a mixture of the six surface antigens. The S peptides alone assemble to form the so-called 20 nm particles, which are completely uninfectious.
- Patients infected by the HB virus pass through several stages of the hepatitis, before they are regarded to be chronically HBV-infected. Immediately after infection an infectious stage will follow, characterized by the presence of HBeAg in the serum. Continued HBs antigenaemia in spite of inhibited HBV replication indicates the presence of viral DNA sequences integrated into the cellular genome of the patient. The integrated viral sequences do not enable the host cell to synthesize the complete virus. However, liver cells having HBV-sequences integrated are capable of producing HBsAg only, which in turn is detectable in the serum of the patient and is an indicator for chronic hepatitis B. Most probably the transformed hepatocytes are not lysed by cytotoxic T-cells, but proliferate and induce either chronic persistent hepatitis (CPH) or chronic active hepatitis (CAH), which may then proceed to cirrhosis of the liver or to primary hepatocellular carcinoma resulting in premature death of the patient.
- Recently it has been established that patients who are chronically HBV infected show a defect in endogenous interferon production (Abb et al., 1985; J. Med. Virol 16. 171-176). This was the rationale to treat patients suffering from chronical hepatitis B, as indicated by the presence of HBeAg and HBV-DNA in the serum, with interferon α (IFNα). Controlled trials with large numbers of patients showed that the administration of interferon α resulted in significantly increased elimination of the hepatitis B-virus, when compared to controls. However, persons infected at or around time of birth do not appear to seroconvert in response to this therapy. This phenomenon unfortunately precludes some 75% of carriers from IFNα therapy.
- At present, the exact mode of action of interferon α on chronic hepatitis B remains unclear. Its antiviral activity might protect infected cells from infection or reduce viral transcription, translation and replication in HBV-infected cells. Interferon further has immunomodulatory effects by activating T-cells, macrophages and NK-cells and by inducing the expression of MHC class I proteins.
- Another approach to treat chronic hepatitis B is based on the idea to inhibit replication of the virus, thus impairing its defence sufficiently to render the host immune system capable of eliminating the virus. This led to test antiviral drugs such as adenine arabinoside and adenine arabinoside monophosphate for treatment of chronically HBV infected individuals. However, less than half of the patients responded to this therapy, either by sustained or transient seroconversion (HBeAg+ to anti-HBe+). A further negative aspect of these antiviral drugs are their immunosuppressive properties.
- Other drugs that have beeen tested for treatment of chronic carriers include interferon β and, acycloguanosine (acyclovir),
interleukin 2, steroids, such as prednisolone, and combinations thereof. But none of them could provide better results than treatment with interferon α. Only a combination therapy, including the initial administration of steroids followed by that of IFN α may increase the response rate in selected patients. - It is known from the prior art, that chronically HBV infected chimpanzees can not be cured by treatment with HBsAg (bound to a tetanus toxoid) nor with anti-HBs antibodies. Furthermore, it has been attempted to immunize chronically HBV infected patients by administration of S peptides. This treatment did not even result in anti-HBs antibody formation in these persons.
- Additionally, according to the definition, chronic carriers of hepatitis B virus are characterized in that HBsAg is detectable in their serum. Therefore, it has been absolutely unforeseeable, that a combination, comprising a T-cell activating epitope of the viral S peptide, according to the present invention, is able to induce an immunization in and a final healing of chronic carriers of hepatitis virus B.
- Considering the above-discussed state of the art it is the objective of the present invention to provide an effective therapeutic agent for the treatment of viral chronic hepatic diseases which leads to a complete response (i.e. to the sustained inhibition of HBV-replication, the loss of HBV DNA and DNA polymerase and to a decrease and finally the disappearance of HBeAg and HBsAg in the serum of patients).
- According to the present invention this goal is achieved by a combination of a) at least one polypeptide sequence mediating the antigenicity of one or more epitopes and b) a carrier, capable of presenting the epitope sequence(s) a), wherein the polypeptide sequence(s) a) can be bound to carrier b) by adsorption, any chemical bonding or secondary valences.
- This invention is furthermore directed to the use of this combination for the production of a medicament for the treatment of chronic viral hepatitis.
- The present invention is further directed to a method for the treatment of chronic viral hepatitis by administering to a patient the above described combination of a) at least one polypeptide sequence mediating the antigenicity of one or more epitopes and b) a carrier capable of presenting the epitope sequence(s) a), wherein the polypeptide sequence(s) a) can be bound to carrier b) by adsorption, any chemical bonding or secondary valences.
- It is important that polypeptide sequence a), which may be one or more different polypeptides, mediates the antigenicity of a T cell-activating epitope in a direct or indirect way.
- According to the present invention polypeptide sequence(s) a) may be a polypeptide or a combination of two or more polypeptides of hepatitis B virus of any subtype, particularly adw, ayw, adr and ady.
- These peptides derived from hepatitis B virus may be HBV peptides preS1, preS2 or S or the HBV core antigens.
- Useful as polypeptide sequence(s) a) are futhermore any of the above-stated polypeptides or a combination of two or more polypeptides which are modified either by amino acid deletions, whereby at least one epitope comprising at least six consecutive amino acid residues must be preserved, or by adding further amino acids either at the N-terminus, the C-terminus or as insertions into the polypeptide sequence(s) a). In each of these cases it is essential, however, that the biologial activity is maintained.
- Preferably, polypeptide sequence(s) a) is myristylated.
- In order to display the appropriate pharmacological activity it is necessary that in the combination of the present invention polypeptide sequences(s) a) is presented on a carrier b). This carrier consists of a particular substance which for example may consist of particles of a hydrophobic polymer, of inorganic particles, or of particles of a polysaccharide. Preferably, carrier b) is a second polypeptide sequence which forms particles upon secretion, said particles having preferably a diameter of at least 10 nm.
- It is preferred that the particle forming polypeptide sequence b) is a substantial part of or the complete amino acid sequence of a polypeptide which may be selected from HBV S peptide, HBV core antigen, HAV core antigen, HAV surface antigen, HIV surface antigen and HIV core antigen as well as the surface antigen of polio virus. Preferred as the particle-forming carrier b) is HBV S peptide and/or core peptide.
- When used as the carrier sequence b) the above-stated polypeptides may be modified by arbitrary deletions of amino acids, by substitutions of one or more amino acids or by adding one or more amino acids either at the N-terminus, the C-terminus or by insertion of one or more amino acids into the polypeptide sequence b), provided that the particle-forming capacity is maintained. Preferably, polypeptide sequence b) is myristylated.
- If the carrier b) is a polypeptide sequence, both sequences a) and b) may be linked via one or more of the following interactions: hydrophobic anchoring (mediated by myristic acid), disulfide bridge formation, or both sequences may be connected by a peptide bond to form a fusion peptide. In the latter case optionally a spacer sequence may be inserted between polypeptide sequence(s) a) and polypeptide sequence b), which spacer sequence is linked to both polypeptides via peptide bonds.
- The present invention furthermore provides a recombinant DNA molecule coding for a combination, that is useful for production of a medicament to treat chronic viral hepatic diseases. The recombinant DNA molecule comprises at least one first DNA sequence, optionally a second, a third and/or a fourth DNA sequence wherein
- i) said at least one first DNA sequence codes for at least one polypeptide sequence a) as defined above,
- ii) said second DNA sequence codes for a polypeptide sequence b) according to the above definition of the particle forming peptide,
- iii) said third DNA sequence codes for a spacer sequence, and
- iv) said fourth DNA sequence codes for a selection marker,
- and wherein the DNA sequences are controlled by DNA elements essential for expression, and optionally have a common reading frame.
- On account of the fact, that many amino acids are designated by more than one triplet, there exist several DNA sequences embraced by the present invention, which code for the above-defined peptide sequences a) and b).
- Apart from this, the invention further embraces recombinant DNA molecules, which differ from the above-defined recombinant DNA molecules by the fact, that up to 30% of the nucleotides may be substituted.
- A further object of the present application is to provide a host cell transfected with a recombinant DNA molecule coding for the above combination, which is useful for treatment of chronically HBV-infected patients. This host cell may be a mammalian, a yeast or a bacterial cell. For the purpose of this invention it is preferred, that this cell does not produce any human serum proteins or any primate serum proteins other than the polypeptide(s) being comprised within the above combination.
- The term “HBV S peptide” as used herein refers to the peptide encoded by the entire region of the HBV genome. The term “HBV pre-S2 peptide” as used herein refers to the peptide encoded by the entire pre-S2 and S regions of the HBV genome. The term “HBV pre-S1 peptide” as used herein refers to the polypeptide encoded by the entire pre-S1, pre-S2 and S regions of the HBV genome. The term “epitope” as used herein refers to a sequence of at least six consecutive amino acids encoded by the designated genome region (e.g. a “HBV pre-S2 epitope” refers to a sequence of at least six amino acids encoded by the pre-S2 region of the HBV genome). The term “T-cell epitope” as used herein refers to an epitope that interacts with receptors on the surface of T-cells to enhance or otherwise effect an immune response.
- As used herein “antigenicity” means the ability to provoke an immune response (e.g. acting as an antigen), the ability to cause the production of antibodies (e.g. acting as an antigen) and/or the ability to interact with a cell surface receptor so as to enhance an immune response or production of antibodies.
- The term “HBV” means any subtype of the virus, particularly adw, ayw, adr and ayr, described in the literature (P. Valenzuela, Nature Vol. 280, p. 815 (1979), Gerlich, EP-A-85 111 361, Neurath, EP-A-85 102 250). Examples of peptide sequences thereof, constituting polypeptide sequence(s) a), which mediate the antigenicity of one or more epitopes, are shown in the Sequence Listing (SEQ ID No. 17-20, 22).
- Preferred embodiments of the present invention are the following combinations:
- HB S-antigen particles with specific epitopes (determinants) of the pre-S1-, pre-S2-, and/or core peptides;
- HB core-antigen particles with specific epitopes (determinants) of the pre-S1-, pre-S2-, S-peptide, and/or of the core antigens;
- Hepatitis A-antigen particles with specific epitopes (determinants) of the hepatitis B S-,pre-S1-, pre-S2-, and/or core-peptides.
- Recombinant DNA molecules preferred for the present invention are characterized by the presence of sequences coding for polypeptide sequence(s) a), mediating the antigenicity of one or more T-cell epitopes, and for polypeptide b), which upon secretion forms particles having a diameter of 10 nm or more, both of which are under control of a suitable promoter. As examples for sequences coding for a) there may be mentioned any of the sequences listed under
ID numbers 1 to 24 in the Sequence Listing. Examples for the DNA sequence coding for polypeptide sequence b) are represented by any of the ID-sequences 25 to 27 in the Sequence Listing. - Any of the 24 sequences (
ID numbers 1 to 24) may be combined to any sequence disclosed underID number 25 to 27 in the Sequence Listing, therein both orders a-b and b-a are included. - A particular preferred embodiment of the present invention consists in a combination of the epitope sequence ID No. 28 (corresponding to
amino acids 9 to 28 of the S1 sequence of HBV) in combination with sequence ID No. 26 and/or 27 as a particle former. - Hepatitis virus sequences used in the recombinant DNA construct of the present invention can be formed or isolated by any means including isolation and ligation of restriction fragments, chemical synthesis of oligonucleotides using a synthesizer (Cyclon, Bio-Search), and synthesis by the PCR method (T. J. White, N. Arnleim, H. E. Erlich, 1989; The Polymerase Chain Reaction, Technical Focus 5 (6)).
- Preferred recombinant DNA molecules were formed by the ligation of synthetic oligonucleotides to a 5′ XbaI-
BglII 3′ fragment (ID number 27) from the S region of the HBV genome, which is derived from a BglII-BglII HBV fragment including the entire pre-S1-pre-S2-S-region, or to the entire S-region. Oligonucleotides used in making such constructs are summarized in Table I below.TABLE I Function Definition SEQ ID No. core (adw) aa* 59-87 6 core (adw) aa 2-28 7 core (adw) aa −10-28 8 core (adw) aa 29-58 9 core (adw) aa 1-87 10 core (adw) aa −10-87 11 core (adw) aa 70-110 12 core (adw) aa 80-125 13 core (adw) aa 88-120 15 S1 (ayw) aa 9-28 17 S1 (ayw) aa 83-103 18 S1 (ayw) aa 20-40 19 S1 (ayw) aa 59-94 20 S1 (adw) aa 94-114 21 S1 (adw) aa 70-105 22 S2 (ayw) aa 2-21 23 S2 (ayw) aa 14-33 24 - Other preferred DNA molecules were formed by ligation of core sequences, which are prepared by the PCR method and which code for T-cell epitopes, to the core sequence of HBV (SEQ ID NO 25) functioning as polypeptide sequence b). Oligonucleotides used in preparing these constructs are given in Table II-1.
TABLE II-1 Function Definition SEQ ID No. core complete, bp 1901-2500 1 core C-terminal deletion, 2 bp 1901-2405 core C-terminal deletion and 3 stop codon inserted, bp 1901-2405 core/precore 10 aa precore, 4 C-terminal deletion, bp 1871-2405 core/precore 10 aa precore, 5 C-terminal deletion and stop codon inserted, bp 1871-2405 core aa (−10-120) 16 core/precore 10 aa precore, 35 complete core, bp 1871-2500 - Table II-2 shows several examples, where the T-cell epitope-coding DNA sequences have been isolated by restriction fragmentation of the HBV genome and have been ligated to the DNA sequence coding for polypeptide sequence b) as defined above.
TABLE II-2 Function Definition SEQ ID No. core/precore complete, bp 1403-311 ** S2 ay/ad ** S2 (K) ay/ad S2-S, 7 codons 14 deleted, start codon ATG changed to ATA - In Table II-3 specific recombinant DNA molecules are listed. The procedure for their construction will be described in more detail in the Examples.
TABLE II-3 Se- lec- Final T-cell Particle tion construct epitope Former Gene MT-core(−10-120) + core(aa −10-120) S adw/ayw or neo SAg + neo S/Xbal/BglII MT-S1(aa 9-28) − S1(aa 9-28)ay S adw/ayw or egpt# S + egpt S/XbaI/BglII MT-core-neo core/precore core adw neo bp 1403-31 MT-core(1-87) + core(aa 1-87) S adw/ayw or neo HBsAg − neo S/XbaI/BglII - Preferred recombinant DNA molecules according to the present invention comprise, apart from the regions coding for polypeptides a) and b), an additional DNA sequence coding for a selection marker. Furthermore, they comprise all usual elements essential for the expression, such as promoter sequence, start codon and a polyadenylation signal.
- Examples of suitable promoters are the methallothionein (MT), the U2 and the H2K promoter in case of using mammalian cells as a host cell. If yeast or bacterial cells are to be employed, appropriate yeast and bacterial promoters, such as the GCN4- and the
GAL 1/10 promoter or the prokaryotic trp- and tac promoters, respectively, may be used. - In order to produce the combination of polypeptide(s) a) and polypeptide b) according to this application the recombinant DNA molecule is inserted into host cells by transfection (in case of mammalian cells), by transformation (in case of yeast and bacterial cells), or by other means. As a host cells of any organism may be used that are capable of transcribing and translating recombinant DNA molecules, such as mammalian, bacterial and yeast cells.
- Suitable mammalian cells according to this invention are for example VERO cells (a monkey kidney cell line), 3T3-, C127 and L cells (murine fibroblast cell lines), and CHO (Chinese hamster ovary) cells, which are either positive or negative in dehydrofolate reductase.
- According to a specific embodiment of the present invention it is furthermore possible that the above-defined first DNA sequence and the above-defined second DNA sequence, which code for polypeptide sequence(s) a) and for a polypeptide sequence b), respectively, are present in different recombinant DNA molecules, in which case the host cells are cotransfected with both of these recombinant DNA molecules.
TABLE III Possible alternatives of compositions for particles containing T-cell epitopes for targeting chronic hepatitis carrier FINAL T-CELL-EPITOPE PARTICLE SELECTION PURIFI- CONSTRUCT1 PROMOTER2 SYN PCR GENE FORMER GENE CATION3 12 MT/112/U2 Core(AA −10-+87) entire S adw/ayw neo/egpt S/XbaI/BglII 13 MT/112/U2 Core(AA 70-110) entire S adw/ayw neo/egpt S/XbaI/BglII 14 MT/112/U2 Core(AA 80-125) entire S adw/ayw neo/egpt S/XbaI/BglII 15 MT/112/U2 Core(AA 88-120) entire S adw/ayw neo/egpt S/XbaI/BglII 16 MT/112/U2 Core(AA −10 + SATG + entire S adw/ayw neo/egpt AA 2-87) S/XbaI/BglII 17 MT-core MT/112/U2 Core(AA −10-+120) entire S adw/ayw neo/egpt (−10-+120) + S/XbaI/BglII SAg + neo 18 MT-S1 MT/112/U2 S1-(AA 9-28)(ay) entire S adw/ayw neo/egpt (AA 9-28) − S/XbaI/BglII S + egpt 19 MT/112/U2 S1-(AA 83-103)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII 20 MT/112/U2 S1-(AA 20-40)(ay) entire S adw/ayw neo/egpt Materials S/XbaI/BglII and Methods 21 MT/112/U2 S1-(AA 59-94)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII 22 MT/112/U2 S1-(AA 94-114)(ad) entire S adw/ayw neo/egpt S/XbaI/BglII 23 MT/112/U2 S1-(AA 70-105)(ad) entire S adw/ayw neo/egpt S/XbaI/BglII 24 MT/112/U2 S1-(AA 9-28)(ay) core adw neo/egpt 25 MT/112/U2 S2-(AA 2-21)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII 26 MT/112/U2 S2-(AA 14-33)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII 27 MT/112/U2 S2 ayw S ayw/adw neo/egpt 28 MT/112/U2 Adapter S2-(X) S ayw/adw neo/egpt ayw 1 MT/112/U2 Core without pre-core Core(adw) neo/egpt e.g. bp 1901-2500 2 MT/112/U2 Core without pre-core; Core(adw) neo/egpt with deletion of the C-terminus e.g. bp 1901-2405 3 MT/112/U2 Core without pre-Core; Core(adw) neo/egpt with deletion at the C-terminus + stop signal e.g. bp 1901-2405 4 MT-core-neo MT/112/U2 Core and pre core Core with Core(adw) neo/egpt Materials 10 AA pre-core and Methods i.e. bp 1403-31 5 MT/112/U2 Core and pre-core Core(adw) neo/egpt 10 AA; with deletion at the C-terminus e.g. bp 1871-2405 6 MT/112/U2 Core and pre-core with deletion at the C- Core(adw) neo/egpt terminus + stop signal e.g. bp 1871-2405 7 MT/112/U2 Core(AA 59-87) entire S adw/ayw neo/egpt S/XbaI/BglII 8 MT/112/U2 Core(AA 2-28) entire S adw/ayw neo/egpt S/XbaI/BglII 9 MT/112/U2 Core(AA −10-+28) entire S adw/ayw neo/egpt S/XbaI/BglII 10 MT/112/U2 Core(AA 29-58) entire S adw/ayw neo/egpt S/XbaI/BglII 11 MT-core MT/112/U2 Core(AA 1-87) entire S adw/ayw neo/egpt (1-87) + S/XbaI/BglII IIBsAg-neo - Table III gives an overview on how to combine suitable DNA sequences to get DNA constructs according to the present invention. It is to be noted that any constituents disclosed in this table may be combined to provide a DNA sequence which may be taken, if transfected into a host cell, to produce a combination (comprising polypeptides(s) a) and b)) as a medicament for the treatment of chronic viral hepatitis.
- The DNA sequences coding for the T-cell epitope sequences have been prepared synthetically (SYN) with a Biosearch Cyclon synthesizer, by PCR procedure (PCR), or by restriction enzyme fragmentation of the viral genome (GENE).
- For the treatment of patients suffering from chronic viral hepatitis the combination of polypeptide sequence(s) a) and a carrier b) may be formulated in any type of a pharmaceutical composition, which furthermore comprises a suitable diluent or pharmaceutical carrier material, such as a buffer solution.
- The administration may be effected by any method, i.e. by parenteral (e.g. intravenous or intramuscular) or oral (e.g. by using typhoid bacterial cells to encapsulate the active substance) administration.
- The pharmaceutical preparation comprises the above-described combination in sufficient concentration to elicit a response upon administration.
- FIG. I shows a DNA construct, coding for a promoter, a particle former sequence and a selection gene (described in Example 3/4).
- FIG. II shows a DNA-gene construct containing a promoter, an epitope with the entire HB-S-Ag and a selection gene (described in Example 3/18).
- FIG. III shows a DNA construct presenting a promoter, a T-cell epitope with a particle former residue and a selection gene (described in Example 3/21).
- FIG. IV shows the AST values of
chimpanzee 1 during the Hepa-Care treatment (described in Example 10/1). - FIG. V shows the antigen values of
chimpanzee 1 during the Hepa-care treatment (described in Example 10/1) - FIG. VI shows values of liver enzymes ALT and GGT of
chimpanzee 1 booster treated three times with Hepa-Care (described in Example 10/2). - FIG. VII shows values of liver enzymes ALT, AST, and GGT and of antigen of
chimpanzee 2 during the Hepa-Care treatment (described in Example 10/3). - FIG. VIII shows the liver enzymes as determined for an untreated control chimpanzee (described in Example 10/3).
- FIGS. IX & X show the antigen and antibody titers of
patient 1 during the Hepa-Care treatment, respectively (described in Example 11). - FIGS. XI & XII show the antigen and antibody titers of
patient 2 during the Hepa-Care treatment, respectively (described in Example 11). - FIGS. XIII & XIV show the antigen and antibody titers of
patient 2 during the Hepa-Care treatment, respectively (described in Example 11). - The invention is more specifically described by the following examples.
- 1. Fractionated Precipitation with Polyethylene Glycol (PEG)
- The supernatant of HBV protein producing cultures was collected and split into portions of 2,400 ml. To each portion 144 g of PEG 6000 (Serva) were added and dissolved by stirring at room temperature for 20 minutes and was stirred for another 6 hours at 4° C. The precipitate was separated by centrifugation in 500 ml bottles in a
GS 3 rotor at 9,000 rpm (15,000×g) for 30 minutes at 10° C. The supernatant was collected and 144 g of PEG 6000 were added and dissolved as described above. The solution was stirred at 4° C. for 3 hours. The precipitate from this solution was harvested as described above except that centrifugation was continued for 60 minutes. - 2. Gel Chromatography
- The material obtained after PEG precipitation was redissolved in 20 ml PBS and submitted to gel chromatography on A-5m (BioRad). Column dimensions were 25×1000 mm and 480 ml bed volume. In a typical fractionation run 1,000 μg of PEG precipitated HBV protein in 10 to 15 ml was loaded and eluted with PBS at a speed of 6 drops/min (18 ml/h). 3 ml fractions were collected. HBV protein eluted with the first peak. Collected fractions were submitted to a CsCl gradient.
- 3. Sedimentation in CsCl Gradient
- About 30 fractions covering the first peak in column chromatography on A-5m and containing prepurified HBV protein were collected to approximately 100 ml. This solution was adjusted to a density of 1.30 g/cc with CsCl and subsequently transferred to a polyallomer tube fitting into a SW 27/28 rotor (Beckman). A gradient was set by underlaying 4 ml of a CsCl solution of 1.35 g/cc and by overlaying 4 ml of 1.25 g/cc followed by 4 ml of 1.20 g/cc density. This gradient had been run at 28,000 rpm for 50 hours at 10° C. Thereafter the gradient was fractionated, and purified HBV protein floating in the 1.20 g/cc density layer was collected. The solution was desalted by three cycles of dialysis in bags against water.
- 1. With Radioimmunoassay
- In the AUSRIA II-125 “sandwich” radioimmunoassay (commercially available from Abbot), beads coated with guinea pig antibody to Hepatitis B surface antigen (Anti-HBs) were incubated with serum or plasma or purified protein and appropriate controls. Any HBsAg present was bound to the solid phase antibody. After aspiration of the unbound material and washing of the bead, human 125T-Anti-HBs was allowed to react with the antibody-antigen complex on the bead. The beads were then washed to remove unbound125I-Anti-HBs.
- )-Anti-HBs HBsAg
- )-Anti-HBs . HBsAg125I-Anti-HBs
- )-Anti-HBs . HBsAg .125I-Anti-HBs
- The radioactivity remaining on the beads was counted in a gamma scintillation counter.
- 2. With ELISA
- In the Enzygnost HBsAg micro “sandwich” assay (commercially available from Behring), wells-were coated with anti-HBs. Serum plasma or purified protein and appropriate controls were added to the wells and incubated. After washing, peroxidase-labelled antibodies to HBsAg were reacted with the remaining antigenic determinants. The unbound enzyme-linked antibodies are removed by washing and the enzyme activity on the solid phase was determined. The enzymatically catalyzed reaction of hydrogen peroxide and chromogen was stopped by adding diluted sulfuric acid. The colour intensity was proportional to the RBsAg concentration of the sample and was obtained by photometric comparison of the colour intensity of the unknown samples with the colour intensities of the accompanying negative and positive control sera.
- 1. Isolation of the MT-Promoter.
- The plasmid pBPV-342-12 (ATCC 37224) was digested with the endonucleases BglII and BamHI. Three DNA molecules were generated. The fragment of interest contains the methallothionein promoter and a pBR322 sequence comprising 4.5 kb and is easily detectable from the other fragments (2.0 kb and 7.6 kb).
- The reaction was performed in a total volume of 200 μl of reaction buffer at a final concentration of 0.5 μg/μl DNA including 100 units of each restriction enzyme. The completion of the digestion was checked after incubation at 37° C. for three hours by agarose gel electrophoresis at a 0.8% agarose gel. The reaction was stopped by adding 4 μl 0.5 M EDTA.
- The 4.5 kb fragment was separated from the other fragments by preparative 1.2% agarose gel electrophoresis. The DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer. The DNA was purified by a phenol-chloroform extraction and two ethanol precipitations.
- 2. Ligation of a 1.8 kb Fragment Coding for the HBV-Core-Antigen.
- A 1.8 kb BamHI-BamHI fragment, containing the HBV-core coding regions was isolated from HBV-containing DNA. This fragment was ligated together with the 4.5 kb fragment containing the MT-promoter and the pBR residue (described in 1).
- 2 μl of the 1.8 kb fragment were mixed with 3 μl of the 4.5 kb fragment and ligated together in a total volume of 10 μl ligation buffer, containing 2 units T4-DNA ligase and 2 mM ATP at 14° C. overnight.
- The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 μl/ml ampicillin at volumes of 50 to 300 μl cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragments.
- 3. Screening for Desired Plasmid Containing Bacterial Colonies.
- Single colonies were picked with a toothpick and transferred to a LB-ampicillin medium containing tube (5 ml). The tubes were incubated overnight at 37° in a rapidly shaking environment. A mini-plasmid preparation of each grown bacterial suspension was made. The different resulting DNAs were proved by digestion with the restriction endonuclease BglII. Two molecules were expected, a 400 bp fragment and a 5.9 kb fragment. The digestion was analysed by agarose gel electrophoresis. Plasmid DNA was isolated from the bacterial cells.
- 4. Insertion of a Neomycin Selection Marker.
- The plasmid resulting from (3) above was linearized by digestion with the restriction enzyme EcoRI. The reaction was performed in a total volume of 50 μl and a final concentration of 1 μg/μl plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis. The reaction was stopped by adding 1 μl of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at −80° C. for 30 minutes. The precipitated DNA was dissolved in 50 μl distilled water.
- 2 μl of the linearized plasmid was mixed with 3 μl of the DNA fragment containing the methallothionein promoter and the neomycin selection gene (isolated from the plasmid pMT-neo-E (available from ATCC/Exogene) by digestion with the endonuclease EcoRI as a 3.9 kb fragment), and ligated together. Single bacterial colonies were screened for the presence of the desired plasmid.
- 5. Isolation of a Fragment Containing the U2 Promoter Sequence.
- The plasmid pUC-8-42 (available from Exogene) was digested with the restriction endonucleases EcoRI and ApaI. Two DNA molecules were generated. The fragment of interest contains the U2-promoter comprising 340 bp and is easily detectable from the other fragment (3160 bp). The digestion was performed in a total volume of 200 μl reaction buffer at a final concentration of 0.5 μg/μl DNA including 100 units of each restriciton enzyme. The completion of the digest was checked after incubation at 37° C. for three hours by agarose gel electrophoresis in a 0.7% agarose gel. The reaction was stopped by adding 4 μl 0.5 M EDTA. The 340 bp fragment was separated from the plasmid DNA by preparative 1.2% agarose gel electrophoresis. The DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer. The DNA was purified by a phenol/chloroform extraction and two ethanol precipitations.
- 6. Insertion of the Fragment Containing the Promoter Sequence into a Polylinker Plasmid.
- The plasmid pSP165 (commercially available from Promega Diotec) containing a polylinker sequence (containing the following restriction sites: EcoRI, SacI, SmaI, AvaI, BanHI, BglII, SalI, PstI, HindIII) was linearized with the restriction enzyme EcoRI. The reaction was performed in a total voume of 50 μl and a final concentration of 1 μg/μl plasmid DNA. 50 units of EcoRI was added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis. The reaction was stopped by adding 1 μl of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at −80° C. for 30 minutes. The precipitated DNA was dissolved in 50 μl distilled water.
- 2 μl of plasmid DNA was mixed with 10 μl of the fragment DNA containing the U2 promoter sequence, and ligated together in a total volume of 25 μl of ligation buffer containing 2 units T4-DNA ligase and 2 mM ATP at 14° C. overnight. Thereafter, the DNA purified by phenol/chloroform extractions followed by two ethanol precipitations and dissolved in 10 μl distilled water. The resulting sticky ends of EcoRI and ApaI had to be converted into blunt ends and ligated. The sticky ends were converted into blunt ends by reaction with the Mung bean nuclease as follows: to 25 μl DNA (1 μg/μl concentration) in reaction buffer 20 units of enzyme were added to give a final concentration of 1% glycerol and a final reaction volume of 35 μl. After an incubation for 30 minutes at 30° C. the DNA was purified by phenol-chloroform extractions followed by two ethanol precipitations. The DNA was dissolved again in 5 μl of distilled water. The resulting blunt ends were ligated together in 15 μl reaction volume containing 10× more T4 ligase than used above and 2 mM ATP at 14° C. overnight.
- The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 μg/ml ampicillin at volumes of 50 to 300 μl cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired U2-promoter fragment. The resulting plasmid was isolated from the bacterial cells and characterized by restriction enzyme analysis.
- 7. Ligation of Synthetic Oligo-DNA-Nucleotide 89 (SEQ ID No.: 30) Together with MT-Promoter Fragment (4.5 kb) .
- The 4.5 kb fragment (described in 1) containing the MT-promoter and a pBR residue were ligated together with the synthetic oligonucleotide 89 (SEQ ID No.: 30). The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid. The new plasmid was proved by a digestion with the restriction endonucleases EcoRI and XbaI. Two molecules were expected, one 2.0 kb and one 2.6 kb.
- 8. Ligation of the Synthetic Oligonucleotide 101 (SEQ ID No.: 32) Together with Plasmid (Described in 7).
- The plasmid (described in 7) was digested with BglII and BamHI and a fragment of 13 nucleotides was removed (described in 1). The resulting fragment containing the first oligonucleotide 89 (SEQ ID No.: 30), was ligated together with oligonucleotide 101 (SEQ ID No.: 32), a BglII-BamHI fragment. After DNA up-take single cells were screened for the presence of the desired plasmid. The new plasmid was proved by a digestion with the endonucleases EcoRI and XbaI, or EcoRI and BglII.
- 9. Ligation of Synthetic DNA-Oligonucleotide 99 (SEQ ID No.: 31) to the 4.5 kb Fragment (Described in 1).
- The 4.5 kb fragment (BglII-BamHI) was ligated together with the DNA oligonucleotide 99 (SEQ ID No.: 31). After screening of single bacterial colonies, containing different DNAS, the desired plasmid was characterized by digestion with EcoRI, resulting in two fragments, 1.9 kb and 2.7 kb, and by positive linearization with BglII or BamHI.
- The new plasmid was then digested with PstI and BamHI. Two molecules were expected, one 2.6 kb fragment, containing a pBR residue, the MT-promoter and the oligonucleotide and a 2.0 kb pBR residue. The 2.6 kb fragment was isolated.
- 10. Ligation of the 2.6 kb Fragment of the Plasmid Described in 9, with a Fragment Isolated from Plasmid (Described in 8).
- The plasmid (described in 8) containing the DNA oligonucleotides 89 and 101 (SEQ ID No.: 30 and 32, respectively) was digested with PstI and BglII. Two fragments were expected. A 2.5 kb fragment containing a pBR residue and the MT-promoter and 2.2 kb fragment, containing a pBR residue and both oligos.
- This 2.2 kb fragment was ligated together with the 2.6 kb fragment, containing the pBR residue, the MT-promoter and oligo 99 (SEQ ID No.: 31) described in 8.
- After screening for the desired plasmid, it was characterized by restriction endonuclease digestion with BglII-XbaI. Two fragments were expected, a 270 bp fragment of the oligo-DNA-nucleotides and a 4.5 kb fragment of the MT-promoter and the pBR.
- 11. Ligation of the 2.3 kb HBV BglII-BglII Fragment.
- A 2.3 kb BglII-BglII fragment containing the HBV pre-S1, pre-S2 and S coding regions was isolated from HBV-containing DNA. The 2.3 kb fragment was ligated together with the 4.5 kb fragment (obtained as described in 1) containing the methallothionein promoter.
- 2 μl of the 2.3 kb fragment was mixed with 3 μl of the 4.5 kb fragment and ligated together in a total volume of 10 μl ligation buffer, containing 2 units T4-DNA ligase and 2 mM ATP at 14° C. overnight.
- The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plate containing 50 μg/ml ampicillin at volumes of 50 to 300 μl cell suspension per plate. The agar plates were incubated at 37° C. overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragment.
- 12. Conversion of a Part of the HBV-Gene Sequence with HBV-core epitopes.
- The plasmid resulting from 11 above was digested with the endonucleases BglII and XbaI. Two molecules were expected, one 550 bp fragment and a 6.25 kb fragment which was isolated after agarose gel electrophoresis.
- The 6.25 kb fragment was ligated together with the 270 bp fragment (after digestion with BglII and XbaI and fragment isolation as described above) of the plasmid described in 10, coding for an epitope part of the HBV-core gene.
- The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid. The new plasmid was proved by a digestion with BamHI. Three molecules were expected, a 950 bp, a 450 bp and a 5,150 bp fragment.
- 13. Preparation of a “Vehicle” Plasmid.
- The plasmid (described in 11) was digested with EcoRI and XbaI. Two molecules were expected, one 2,450 bp fragment and a 4,350 bp fragment which was isolated after gel electrophoresis.
- This 4,350 bp fragment was ligated together with the oligo-DNA-nucleotide 39 (SEQ ID No: 29) coding for the entire DNA-sequence of HBV-S-gene from ATG to the XbaI site, wherein the ATG was changed into ATA.
- 14. Core-Epitope Upstream of the Entire HBV-S Gene.
- This “vehicle” plasmid was then digested with PstI and XbaI, two molecules were expected, one 600 bp plasmid residue and a 3,850 bp fragment which was isolated and ligated together with a PstI-XbaI fragment of 2,800 bp (2,700 bp) isolated after digestion of the plasmid described in 10.
- After screening for the desired plasmid, it was characterized by restriction endonuclease digestion with EcoRI and XbaI, EcoRI and BglII and BamHI.
- 15. Insertion of a Selection Marker.
- The plasmid (described in 14) was linearized with Eco RI. The reaction was performed in a total volume of 50 μl and a final concentration of 1 μg/μl plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis.
- The reaction was stopped by adding 1 μl of 0.5 M EDTA and DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at −80° C. for 30 minutes. The precipitated DNA was dissolved in 50 μl distilled water.
- 2 μl of the linearized plasmid was mixed with 3 μl of the DNA fragment containing the methallothionein promoter and the neomycin selection gene (described in 4) and ligated together. Single bacterial colonies were screened for the desired plasmid which was isolated, purified and characterized.
- Each gene construct described above can be constructed also with the U2-promoter whereby the MT-promoter-containing DNA fragment, after digestion with EcoRI and BglII, is replaced by a DNA fragment containing the U2-promoter isolated after digestion with EcoRI and BglII.
- 16. Isolation of theE coli Xanthine Quanine Phosphoribosyl Transferase (egpt) Selection Gene.
- The fragment containing the egpt selection gene was isolated after digestion of the plasmid PMSG with BamHI and BglII (1.8 kb) and ligated together with a 4.5 kb fragment (BglII-BamHI, described in 1) containing the MT-promoter.
- After screening for the desired plasmid it was isolated, purified and finalized by a conversion of the BamHI site into an EcoRI site.
- 17. Isolation of Desired DNA Sequences by PCR-Method.
- One DNA fragment (400 bp) was isolated after gel electrophoresis. It was generated by PCR-method (described in Example 5) by using the specific oligonucleotides 131 and 132 (SEQ ID No.: 33 and 34) as primers.
- The DNA fragemnt was digested with the endonucleases BamHI and XbaI and then purified by gel electrophoresis. The isolated PCR-fragment was ligated together with a 6.25 kb fragment which was isolated from the plasmid (described in 13) after digestion with BglII and XbaI. After DNA up-take and bacterial transformation the single bacterial colonies were screened for the desired plasmid.
- 18. Insertion of a Selection Marker.
- The plasmid (described in 17) was finalized by adding a selection gene to the plasmid (described in 15).
- 19. Isolation of the H2K Promoter.
- The H2K promoter was isolated as an EcoRI and BglII fragment (2 kb) from pSP65H2 (available from Exogene).
- In all constructs described all promoters are replaceable as EcoRI/BglII fragments.
- 20. Conversion of a Part of the HBV-Gene Sequence.
- The plasmid resulting from 11) above was digested with the endonucleases BglII and XbaI. Two molecules were expected, one of which is a 6.250 kb fragment which was isolated after agarose gel electrophoresis.
- The 6.250 kb fragment was ligated together with oligo-DNA-nucleotide 23 (SEQ ID No.: 28). The ligation mixture was added to 150 μl competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid. The new plasmid was proven by a digestion with the endonucleases EcoRI and BglII. Two molecules were expected, one 1,9 kb and one 4.450 kb.
- 21. Insertion of a egpt Selection Marker.
- The plasmid (described in 20) was linearized with EcoRI. The reaction was performed in a total volume of 100 μl and a final concentration of 0.6 μg/μl plasmid DNA. 60 units of EcoRI were added and the digestion was proved after incubation at 37° C. for three hours by agarose gel electrophoresis. The reaction was stopped by adding 2 μl of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 4 volumes of ethanol at −80° C. for 1 hour. The precipitated DNA was dissolved in 50 μl distilled water.
- 2 μl of the linearized plasmid was mixed with 3 μl of the DNA-fragment (3.7 kb) containing the methallothionein promoter and the egpt selection gene (described in 16) by digestion with EcoRI and ligated together. Single colonies were screened for the presence of the desired plasmid. Each of the described gene constructs in Table III are preparable in the same way as described above.
- Transfection of Mammalian Cells with Constructs of the Present Invention
- In order to achieve secretion of substantial amounts of the HBV peptides encoded by constructs of the present invention, mammalian cells must be transfected with a DNA construct of the present invention. The cotransfection was performed in two steps (i.e. a separate transfection for each construct) or in a single step (i.e. one transfection using preparation of both constructs). Cotransfection was confirmed either by use of different selection markers on the two constructs or by detection of secretion of expression products of both constructs by immunoassay.
- Alternatively, a sequence encoding the HBV peptide sequence of the present invention and a separate sequence encoding the entire S or core or HAV protein could be combined in a single construct.
- The polymerase chain reaction allows to amplify specific DNA necleotide sequences of a selected region of a known genomic sequence in vitro by more than a millionfold (Thomas J. White, Norman Arnleim, Henry A. Erlich 1989: The polymerase chain reaction. Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi 1989: Avoiding false positives with PCR. Nature, Vol. 339, pp 237-238).
- DNA isolated from cells or plasmid DNA is treated to separate its complementary strands. These strands are then annealed with an excess of two DNA oligonucleotides (each 20-25 base pairs long) that have been chemically synthesized to match sequences separated by X nucleotides (where X is generally between 50 to 2,000 base pairs).
- The two oligonucleotides serve as specific primers for in vitro DNA synthesis catalysed by DNA polymerase which copies the DNA between the sequences corresponding to the two oligonucleotides. If the two primer oligonucleotides contain the correct sequence it is possible to create new digestion sites at the 5′ and 3′.
- After multiple cycles of reaction, a large amount of a DNA fragment of the desired length was obtained, purified by gel electrophoresis and characterized by restriction enzyme digestion and agarose gel electrophoresis. The amplified, purified DNA fragment was then used to ligate it together with other fragments i.e. plasmid.
- The PCR-DNA fragments were amplified with blunt end. To get sticky end (for the ligation procedure) the fragment has to be digested with the desired endonucleases and purified again.
- The PCR-reaction will work for 20 to 30 cycles. One cycle is separated into three steps with different reaction times and different reaction temperatures which is controlled by a PCR-thermo-cycler. The first step is “Denaturation” of the matrix-DNA (1 min-95° C.), the second step is “Hybridisation” of matrix DNA and primers. (1 min/55° C.) followed by “Polymerisation” (2 min/72° C.).
- The final volume for one assay is 30 μl for example, which contains the following final concentrations: PCR-buffer (1×), nucleotide-mix with 200 μM of each of the four nucleotides, 200 ng for 30 μl of each of the two primers, 0.5 units Taq-Polymerase per 30 μl aqua bidest.
- The recipient cells (C127 or CHO-cells available from ATCC) were seeded in normal growth medium (DMEN+10% Fetal Calf Serum, Glucose and Glutamine) into petri-dishes (1-2×106 cells per dish, φ 10 cm) at
day 1. The next day the medium was removed (4 hours before the DNA precipitate was added onto the cells), and the cells were washed twice with 1×PBS. Then 8 ml DMEM without FCS were added, 4 hours later the DNA precipitate (prepared as described below) was added to the cells. Again after 4 hours the medium was removed, 3 ml of Glycerol-Mix (50ml 2×TBS buffer, 30 ml glycerol, 120 ml distilled water) were added. The Glycerol-Mix was immediately removed after an incubation at 37° C. for 3 minutes and the cells were washed with 1×PBS. The cells were cultivated overnight with 8 ml of DMEM with 10% FCS. - After 48 hours, the cells were recovered from the dish by treating with Trypsin-EDTA-Solution (0.025% Trypsin+1 mM EDTA). Afterwards, to remove the Trypsin-EDTA the cells were washed with 1×PBS, suspended in DMEM with 10% FCS and distributed into 24 costar-well-plates (cells from one dish into four 24-well-plates).
- When the cells had grown well, selection medium was added (concentration 0.5-1 mg/ml of neomycin or: xanthine (250 μg/ml), hypoxanthine (15 μg/μl) or adenine (25 μg/ml), thymidine (10 μg/ml), aminopterine (2 μg/ml), mycophenolic acid (25 μg/ml) for eco-gpt, for example). The medium was changed every week. The first growing cell colonies were seen after 2 weeks.
- To 10 μg of plasmid DNA and 20 μg of carrier-DNA (salmon sperm DNA, calf-thymus DNA) TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.05) was added to a final volume of 440 μl and mixed together with 60 μl 2 M CaCl2. Then the same amount of 2×TBS (Hepes 50 mM, NaCl 280 mM, Na2HPO4 1.5 mM, pH 7.05) was added and mixed well. The precipitation solution was incubated for 30 minutes at 37° C. and added directly to the cells which were to be transfected.
- To the desired concentration of antigen suspended in sterile saline, 1:10,000 volume Thimerosol, 1/10 volume of filter-sterilized 0.2 M KAl(SO4)2φ12 H2O were added. The pH was adjusted to 5.0 with sterile 1 N NaOH and the suspension was stirred at room temperature for 3 hours. The alum-precipitated antigen was recovered by centrifugation for 10 minutes at 2,000 rpm, resuspended in sterile normal saline containing 1:10,000 Thimerosol and aliquoted under sterile conditions.
- The cell supernatant of HB-core-antigen-secreting cells was collected and concentrated by ultrafiltration. The concentrate was cleared by centrifugation at 20,000 rpm for 15 minutes at 4° C. in a Beckman SW28 rotor.
- Particle formaiton was tested by sucrose density centrifugation (0-45% sucrose) in a Beckman SW28 rotor for 24 hours at 28,000 rpm and 4° C. The gradient was fractionated and the single fractions were analyzed by Elisa.
- The following tables give some results of Elisa analysis of immunogenic particles of the present invention as described below:
- Table IV shows the Elisa data of the purified HBs-antigen particle produced from any HBV-sequence construct of the present invention including the pre-S1 epitopes and the S region with the anti-pre-S1
monoclonal antibody MA 18/7 and the anti-HBs monoclonal antibody G022. - Table IV shows the fractions (21) collected after CsCl density gradient.
TABLE IV-1 CsCl-gradient Elisa Measurement (E = 492) Fraction No. Monoclonal Antibody 18/713 0.092 14 0.210 15 0.388 16 1.662 17 2.604 18 0.648 19 0.031 -
TABLE IV-2 CsCl-gradient Elisa Measurement (E = 492) Fraction No. Monoclonal Antibody G022 13 0.136 14 0.426 15 0.822 16 1.970 17 2.954 18 0.967 19 0.076 - Table V shows the Elisa data of the purified HB-core-antigen particles produced from any HB-core-sequence construct of the present invention with polyclonal antibodies against HB-core and with monoclonal antibody G022 against HB-S-Ag.
TABLE V-1 Sucrose Gradient Elisa Measurement (E = 492) Fraction No. Polyclonal Antibodies 6 0.25 7 0.922 8 1.423 9 1.5 10 1.5 11 1.28 12 0.466 -
TABLE V-2 Sucrose Gradient Elisa Measurement (E = 492) Fraction No. Monoclonal Antibody G022 6 0.020 7 0.024 8 0.018 9 0.011 10 0.015 11 0.020 12 0.022 - Hepa-Care are particles presenting hepatitis B surface antigens (S1 and S) in a specific formulation (ratio 50:50), which are used for the treatment of chronic carriers of hepatitis virus.
-
Experiment 1 - A Hepatitis-B-
carrier chimpanzee 1 was treated (intramuscularly) with Hepa-Care attime - The liver enzymes were monitored (FIG. IV) as well as the hepatitis-B antigen level (FIG. V).
-
Experiment 2 -
Chimpanzee 1 after treatment described above was given a booster treatment atweek -
Experiment 3 -
Chimpanzee 2 was treated with Hepa-Care, but contrary tochimpanzee 1 it was given intravenously. The dosage was 2 mg. The results are shown in FIG. VII. - From a
control chimpanzee 3 the liver enzymes were also monitored and shown in FIG. VIII. - (For Definition See Example 10)
Patient 1 (male, age = 65 years, disease for 2 years): Hepatitis-B parameters: HBsAg pos. anti-HBs neg. HBeAg neg. anti-HBe pos. anti-HBc neg. - was treated (i.m.) with Hepa-Care at
month Patient 2 (female, age = 48 years, disease for 12 years): Hepatitis-B parameters: HBsAg pos. HBeAg neg. anti-HBs neg. anti-HBe pos. anti-HBc pos. - was treated (i.m.) with Hepa-Care at
month Patient 3 (female, age = 41 years, disease for 5 years): Hepatitis-B parameters: HBsAg pos. HBeAg neg. anti-HBs neg. anti-HBe pos. - was treated at
month -
1 40 558 bp Nucleic Acid Single Linear 1 ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG 42 TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA 84 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 126 GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG 168 CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC 210 TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA 252 GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG 294 CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA 336 GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT 378 CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA 420 TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA 462 GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 504 AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA 546 TCT CAA TGT TAG 558 504 bp Nucleic Acid Single Linear 2 ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG 42 TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA 84 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 126 GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG 168 CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC 210 TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA 252 GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG 294 CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA 336 GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT 378 CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA 420 TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA 462 GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGA AGA CGT 504 504 bp Nucleic Acid Single Linear 3 ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG 42 TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA 84 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 126 GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG 168 CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC 210 TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA 252 GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG 294 CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA 336 GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT 378 CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA 420 TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA 462 GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 504 534 bp Nucleic Acid Single Linear 4 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC 126 ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG 168 CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC 210 TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT 252 AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT 294 GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG 336 TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT 378 GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA 420 GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG 462 GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT 504 AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 534 534 bp Nucleic Acid Single Linear 5 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC 126 ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG 168 CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC 210 TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT 252 AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT 294 GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG 336 TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT 378 GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA 420 GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG 462 GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT 504 AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 534 87 bp Nucleic Acid Single Linear 6 ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG 42 GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT GTA GTA 84 AAT 87 81 bp Nucleic Acid Single Linear 7 GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA 42 CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 81 114 bp Nucleic Acid Single Linear 8 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 114 90 bp Nucleic Acid Single Linear 9 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 42 GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG 84 CAA GGT 90 261 bp Nucleic Acid Single Linear 10 ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG 42 TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG 84 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 126 GAG TCT CCT GAG CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG 168 CAA GGT ATC CTC TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC 210 TGG GTG GGC AAT AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT 252 GTA GTA AAT 261 291 bp Nucleic Acid Single Linear 11 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTC AGG GAT CTC CTA GAC 126 ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG 168 CTA TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GGT ATC CTC 210 TGC TGG GGG GAA TGG ATG ACT CTA GCT ACC TGG GTG GGC AAT 252 AAT TTG GAA GAT CCA GCA TCT AGG GAC CTT GTA GTA AAT 291 123 bp Nucleic Acid Single Linear 12 ACC TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT 42 CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC 84 AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT 123 138 bp Nucleic Acid Single Linear 13 GCA TCC AGA GAT CTA GTA GTC AAT TAT GTT AAT ACT AAC ATG 42 GGT TTA AAG ATC AGG CAA CTA TTG TGG TTT CAT ATA TCT TGC 84 CTT ACT TTT GGA AGA GAG ACT GTA CTT GAA TAT TTG GTC TCT 126 TTC GGA GTG TGG 138 822 bp Nucleic Acid Single Linear 14 ATG CAG TGG AAT TCC AGA ACC TTC CAC CAA ACT CTG CAA GAT 42 CCC AGA GTG AGA GGC CTG TAT TTC CCT GCT GGT GGC TCC AGT 84 TCA GGA ACA GTA AAC CCT GTT CTG ACT ACT GCC TCT CCC TTA 126 TCG TCA ATC TTC TCG AGG ATA GAG AAC ATC ACA TCA GGA TTC 168 CTA GGA CCC CTT CTC GTG TTA CAG GCG GGG TTT TTC TTG TTG 210 ACA AGA ATC CTC ACA ATA CCG CAG AGT CTA GAC TCG TGG TGG 252 ACT TCT CTC AAT TTT CTA GGG GGA ACT ACC GTG TGT CTT GGC 294 CAA AAT TCG CAG TCC TCA ACC TCC AAT CAC TCA CCA ACC TCT 336 TGT CCT CCA ACT TGT CCT GGT TAT CGC TGG ATG TGT CTG CGG 378 CGT TTT ATC ATC TTC CTC TTC ATC CTG CTG CTA TGC CTC ATC 420 TTC TTG TTG GTT CTT CTG GAC TAT CAA GGT ATG TTG CCC GTT 462 TGT CCT CTA ATT CCA GGA TCC TCA ACA ACC AGC ACG GGA CCA 504 TGC CGG ACC TGC ATG ACT ACT GCT CAA GGA ACC TCT ATG TAT 546 CCC TCC TGT TGC TGT ACC AAA CCT TCG GAC GGA AAT TGC ACC 588 TGT ATT CCC ATC CCA TCA TCC TGG GCT TTC GGA AAA TTC CTA 630 TGG GAG TGG GCC TCA GCC CGT TTC TCC TGG CTC AGT TTA CTA 672 GTG CCA TTT GTT CAG TGG TTC GTA GGG CTT TCC CCC ACT GTT 714 TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TGG GGG CCA AGT 756 CTG TAC AGC ATC TTG AGT CCC TTT TTA CCG CTG TTA CCA ATT 798 TTC TTT TGT CTT TGG GTA TAC ATT 822 99 bp Nucleic Acid Single Linear 15 TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG 42 TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA 84 CTT GAA TAT TTG GTC 99 390 bp Nucleic Acid Single Linear 16 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC 126 ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG 168 CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC 210 TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT 252 AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT 294 GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG 336 TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT 378 GAA TAT TTG GTC 390 60 bp Nucleic Acid Single Linear 17 AAT CCT CTG GGA TTC TTT CCC GAT CAC CAG TTG GAT CCA GCC 42 TTC AGA GCA AAC ACC GCA 60 63 bp Nucleic Acid Single Linear 18 CCT GCC TCC ACC AAT CGC CAG TCA GGA AGG CAG CCT ACC CCG 42 CTG TCT CCA CCT TTG AGA AAC 63 63 bp Nucleic Acid Single Linear 19 GAT CCA GCC TTC AGA GCA AAC ACC GCA AAT CCA GAT TGG GAC 42 TTC AAT CCC AAC AAG GAC ACC 63 108 bp Nucleic Acid Single Linear 20 CCG CAC GGA GGC CTT TTG GGG TGG AGC CCT CAG GCT CAG GGC 42 ATA CTA CAA ACT TTG CCA GCA AAT CCG CCT CCT GCC TCC ACC 84 AAT CGC CAG TCA GGA AGG CAG CCT 108 63 bp Nucleic Acid Single Linear 21 CCT GCC TCC ACC AAT CGG CAG TCA GGA AGG CAG CCT ACT CCC 42 ATC TCT CCA CCT CTA AGA GAC 63 108 bp Nucleic Acid Single Linear 22 CCA CAC GGC GGT ATT TTG GGG TGG AGC CCT CAG GCT CAG GGC 42 ATA TTG ACC ACA GTG TCA ACA ATT CCT CCT CCT GCC TCC ACC 84 AAT CGG CAG TCA GGA AGG CAG CCT 108 60 bp Nucleic Acid Single Linear 23 CAG TGG AAT TCC AGA ACC TTC CAC CAA ACT CTG CAA GAT CCC 42 AGA GTG AGA GGC CTG TAT 60 60 bp Nucleic Acid Single Linear 24 GAT CCC AGA GTG AGA GGC CTG TAT TTC CCT GCT GGT GGC TCC 42 AGT TCA GGA ACA GTA AAC 60 558 bp Nucleic Acid Single Linear 25 ATG GAC ATT GAC CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG 42 TTA CTC TCG TTT TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA 84 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 126 GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG 168 CAA GCC ATT CTC TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC 210 TGG GTG GGT AAT AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA 252 GTA GTC AAT TAT GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG 294 CAA CTA TTG TGG TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA 336 GAG ACT GTA CTT GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT 378 CGC ACT CCT CCA GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA 420 TCA ACA CTT CCG GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA 462 GGC AGG TCC CCT AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT 504 AGA TCT CAA TCG CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA 546 TCT CAA TGT TAG 558 678 bp Nucleic Acid Single Linear 26 ATA GAG AAC ATC ACA TCA GGA TTC CTA GGA CCC CTT CTC GTG 42 TTA CAG GCG GGG TTT TTC TTG TTG ACA AGA ATC CTC ACA ATA 84 CCG CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA 126 GGG GGA ACT ACC GTG TGT CTT GGC CAA AAT TCG CAG TCC TCA 168 ACC TCC AAT CAC TCA CCA ACC TCT TGT CCT CCA ACT TGT CCT 210 GGT TAT CGC TGG ATG TGT CTG CGG CGT TTT ATC ATC TTC CTC 252 TTC ATC CTG CTG CTA TGC CTC ATC TTC TTG TTG GTT CTT CTG 294 GAC TAT CAA GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA 336 TCC TCA ACA ACC AGC ACG GGA CCA TGC CGG ACC TGC ATG ACT 378 ACT GCT CAA GGA ACC TCT ATG TAT CCC TCC TGT TGC TGT ACC 420 AAA CCT TCG GAC GGA AAT TGC ACC TGT ATT CCC ATC CCA TCA 462 TCC TGG GCT TTC GGA AAA TTC CTA TGG GAG TGG GCC TCA GCC 504 CGT TTC TCC TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG 546 TTC GTA GGG CTT TCC CCC ACT GTT TGG CTT TCA GTT ATA TGG 588 ATG ATG TGG TAT TGG GGG CCA AGT CTG TAC AGC ATC TTG AGT 630 CCC TTT TTA CCG CTG TTA CCA ATT TTC TTT TGT CTT TGG GTA 672 TAC ATT 678 585 bp Nucleic Acid Single Linear 27 CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG GGA TCT 42 CCC GTG TGT CTT GGC CAA AAT TCG CAG TCC CCA ACC TCC AAT 84 CAC TCA CCA ACC TCC TGT CCT CCA ATT TGT CCT GGT TAT CGC 126 TGG ATG TGT CTG CGG CGT TTT ATC ATA TTC CTC TTC ATC CTG 168 CTG CTA TGC CTC ATC TTC TTA TTG GTT CTT CTG GAT TAT CAA 210 GGT ATG TTG CCC GTT TGT CCT CTA ATT CCA GGA TCA ACA ACA 252 ACC AGT ACG GGA CCA TGC AAA ACC TGC ACG ACT CCT GCT CAA 294 GGC AAC TCT ATG TTT CCC TCA TGT TGC TGT ACA AAA CCT ACG 336 GAT GGA AAT TGC ACC TGT ATT CCC ATC CCA TCG TCC TGG GCT 378 TTC GCA AAA TAC CTA TGG GAG TGG GCC TCA GTC CGT TTC TCT 420 TGG CTC AGT TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GGG 462 CTT TCC CCC ACT GTT TGG CTT TCA GCT ATA TGG ATG ATG TGG 504 TAT TGG GGG CCA AGT CTG TAC AGC ATC GTG AGT CCC TTT ATA 546 CCG CTG TTA CCA ATT TTC TTT TGT CTC TGG GTA TAC ATT 585 106 bp Nucleic Acid Single Linear 28 GAT CTT TAA CAT GGA GAA CAA TCC TCT GGG ATT CTT TCC CGA 42 TCA CCA GTT GGA TCC AGC CTT CAG AGC AAA CAC CGC AAA TCC 84 AGA TTG GGA CTT CAA TCC CAG T 106 115 bp Nucleic Acid Single Linear 29 AAT TCT AGA CTC GAG TCT GAA CAT AGA GAA CAT CAC ATC AGG 42 ATT CCT AGG ACC CCT TCT CGT GTT ACA GGC GGG GTT TTT CTT 84 GTT GAC AAG AAT CCT CAC AAT ACC GCA GAG C 115 108 bp Nucleic Acid Single Linear 30 GAT CTT TTA AAG GGA TCC TCT GCT GGG GGG AAT GGA TGA CTC 42 TAG CTA CCT GGG TGG GCA ATA ATT TGG AAG ATC CAG CAT CTA 84 GGG ACC TTG TAG TAA ATC TAG ACA 108 106 bp Nucleic Acid Single Linear 31 GAT CTC CGG GAA TTC CTG GGG CAT GGA CAT TGA CCC TTA TAA 42 AGA ATT TGG AGC TAC TGT GGA GTT ACT CTC GTT TTT GCC TTC 84 TGA CTT CTT TCC TTC CGT CAG G 106 89 bp Nucleic Acid Single Linear 32 GAT CTC CTA GAC ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA 42 GAG TCT CCT GAG CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG 84 CAA GG 89 25 bp Nucleic Acid Single Linear 33 TTG GAT CCT CCA ACC TGT GCC TTG G 25 25 bp Nucleic Acid Single Linear 34 CCT CTA GAA CCA AAT ATT CAA GTA C 25 588 bp Nucleic Acid Single Linear 35 TCC AAC CTG TGC CTT GGG TGG CTT TGG GGC ATG GAC ATT GAC 42 CCT TAT AAA GAA TTT GGA GCT ACT GTG GAG TTA CTC TCG TTT 84 TTG CCT TCT GAC TTC TTT CCT TCC GTA CGA GAT CTC CTA GAC 126 ACC GCC TCA GCT CTG TAT CGA GAA GCC TTA GAG TCT CCT GAG 168 CAT TGC TCA CCT CAC CAT ACT GCA CTC AGG CAA GCC ATT CTC 210 TGC TGG GGG GAA TTG ATG ACT CTA GCT ACC TGG GTG GGT AAT 252 AAT TTG CAA GAT CCA GCA TCC AGA GAT CTA GTA GTC AAT TAT 294 GTT AAT ACT AAC ATG GGT TTA AAG ATC AGG CAA CTA TTG TGG 336 TTT CAT ATA TCT TGC CTT ACT TTT GGA AGA GAG ACT GTA CTT 378 GAA TAT TTG GTC TCT TTC GGA GTG TGG ATT CGC ACT CCT CCA 420 GCC TAT AGA CCA CCA AAT GCC CCT ATG TTA TCA ACA CTT CCG 462 GAA ACT ACT GTT GTT AGA CGA CGG GAC CGA GGC AGG TCC CCT 504 AGA AGA AGA ACT CCC TCG CCT CGC AGA CGT AGA TCT CAA TCG 546 CCG CGT CGC AGA AGA TCT CAA TCT CGG GAA TCT CAA TGT TAG 588 20 Amino Acids Amino Acid Linear 36 Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe 5 10 15 Arg Ala Asn Thr Ala 20 21 Amino Acids Amino Acid Linear 37 Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile 5 10 15 Ser Pro Pro Leu Arg Asn 20 21 Amino Acids Amino Acid Linear 38 Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe 5 10 15 Asn Pro Asn Lys Asp Thr 20 36 Amino Acids Amino Acid Linear 39 Pro His Gly Gly Leu Leu Gly Trp Ser Pro Gln Ala Gln Gly Ile 5 10 15 Leu Glu Thr Leu Pro Ala Asn Pro Pro Pro Ala Ser Thr Asn Arg 20 25 30 Gln Ser Gly Arg Gln Pro 35 36 Amino Acids Amino Acid Linear 40 Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala Gln Gly Ile 5 10 15 Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr Asn Arg 20 25 30 Gln Ser Gly Arg Gln Pro 35
Claims (21)
1. Use of a composition for the production of a medicament for the treatment of chronic viral hepatitis, said composition comprising a combination of
a) at least one polypeptide sequence having one or more antigenic T cell-activating epitopes and
b) a carrier capable of presenting the epitope sequence(s) a), wherein the polypeptide sequence(s) a) is bound to carrier b) by covalent or hydrophobic bonding.
2. Use according to claim 1 wherein said polypeptide sequence(s) a) is a polypeptide of hepatitis B virus.
3. Use according to claim 2 wherein said polypeptide sequence(s) a) is the amino acid sequence of one or more members selected from the group comprising the HB viral pre-S1, pre-S2 and S peptides and the HB core antigens.
4. Use according to one of claims 1 to 3 wherein said polypeptide sequence(s) a) may be a native polypeptide sequence modified:
i) by having arbitrary deletions, whereby an epitope comprising at least six consecutive amino acid residues is preserved,
ii) by having substitutions of one or several amino acids, or
iii) by carrying an additional amino acid sequence either at its N-terminus, at its C-terminus or as an insertion into the polypeptide sequence(s) a).
5. Use according to one of claims 1 to 4 wherein said polypeptide sequence(s) a) is myristylated.
6. Use according to any one of claims 1 to 5 wherein said polypeptide sequence(s) a) is produced by expression of a recombinant DNA molecule.
7. Use according to one of claims 1 to 6 wherein said carrier b) is a polysaccharide, a hydrophobic polymer or an inorganic molecule having particle form.
8. Use according to one of claims 1 to 7 wherein said carrier b) is a second polypeptide sequence.
9. Use according to claim 8 wherein said polypeptide sequence b) upon secretion forms particles having a diameter of at least 10 nm.
10. Use according to claim 8 or 9 wherein said polypeptide sequence b) is a substantial part of or the complete amino acid sequence of a polypeptide selected from a group consisting of the HBV S-peptide, the HBV core-, the HAV core- and the HIV core-antigen as well as the surface antigens of poliovirus, HAV or HIV.
11. Use according to one of claims 8 to 10 wherein said polypeptide sequence b) may be modified
i) by having arbitary deletions, whereby the particle forming capacity is preserved,
ii) by having substitutions of one or several amino acids, or
iii) by carrying an additional amino acid sequence either at its N-terminus, at its C-terminus or as an insertion into the polypeptide sequence b).
12. Use according to one of claims 8 to 11 wherein said polypeptide sequence b) is myristylated.
13. Use according to any one of claims 8 to 12 wherein said polypeptide sequence b) is produced by expression of a recombinant DNA expression.
14. Use according to one of claims 1 to 6 or 8 to 13 wherein said polypeptide sequences a) and b) are linked via disulfide bridges.
15. Use according to one of claims 1 to 6 or 8 to 14 wherein said polypeptide sequences a) and b) are linked via “hydrophobic anchoring” (mediated by myristic acid).
16. Use according to one of claims 1 to 6 or 8 to 15 wherein said polypeptide sequences a) and b) are linked by a peptide bond, wherein optionally a spacer sequence is inserted in between polypeptide sequence(s) a) and polypeptide sequence b), said spacer-sequence being linked via peptide bonds to polypeptide sequences a) and b).
17. Use according to claim 16 wherein said polypeptide sequence(s) a) and said polypeptide sequence b) are encoded in tandem on the same recombinant DNA molecule.
18. Use according to claim 17 wherein said polypeptide sequence(s) a) and said polypeptide sequence b) are expressed from a single open reading frame on said DNA molecule, and are optionally separated by a spacer polypeptide sequence also encoded in the single open reading frame.
19. Use as claimed in any one of claims 1 to 18 wherein said composition is for administration by intravenous or intramuscular injection.
20. Method of treatment of viral hepatitis characterised in that a composition as defined in any one of claims 1 to 19 is administered in a patient.
21. A method as claimed in claim 20 for the treatment of viral hepatitis B.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/394,896 US20030235591A1 (en) | 1990-12-19 | 2003-03-21 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EPEP90124775.9 | 1990-12-19 | ||
EP90124775A EP0491077A1 (en) | 1990-12-19 | 1990-12-19 | A composition used as a therapeutic agent against chronic viral hepatic diseases |
US08/075,520 US6020167A (en) | 1990-12-19 | 1991-12-19 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
US30480799A | 1999-05-04 | 1999-05-04 | |
US10/394,896 US20030235591A1 (en) | 1990-12-19 | 2003-03-21 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US30480799A Continuation | 1990-12-19 | 1999-05-04 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030235591A1 true US20030235591A1 (en) | 2003-12-25 |
Family
ID=8204863
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/075,520 Expired - Lifetime US6020167A (en) | 1990-12-19 | 1991-12-19 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
US10/394,896 Abandoned US20030235591A1 (en) | 1990-12-19 | 2003-03-21 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/075,520 Expired - Lifetime US6020167A (en) | 1990-12-19 | 1991-12-19 | Composition used as a therapeutic agent against chronic viral hepatic diseases |
Country Status (15)
Country | Link |
---|---|
US (2) | US6020167A (en) |
EP (2) | EP0491077A1 (en) |
JP (2) | JP3857305B2 (en) |
KR (1) | KR0156587B1 (en) |
AT (1) | ATE170220T1 (en) |
AU (1) | AU657935B2 (en) |
CA (1) | CA2098021C (en) |
CZ (1) | CZ290233B6 (en) |
DE (1) | DE69130071T3 (en) |
DK (1) | DK0563093T4 (en) |
ES (1) | ES2121000T5 (en) |
HK (1) | HK1002901A1 (en) |
HU (1) | HU216301B (en) |
SK (1) | SK280551B6 (en) |
WO (1) | WO1992011368A1 (en) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0300213A1 (en) * | 1987-06-22 | 1989-01-25 | Hexal-Pharma Gentechnik GmbH & Co. KG | Hepatitis a viral peptide particle immunogens |
EP0491077A1 (en) * | 1990-12-19 | 1992-06-24 | Medeva Holdings B.V. | A composition used as a therapeutic agent against chronic viral hepatic diseases |
DE4107612A1 (en) | 1991-03-09 | 1992-09-10 | Behringwerke Ag | RECOMBINANT PROTEINS WITH THE IMMUNE REACTIVITY OF HEPATITIS B VIRUS E ANTIGENS (HBEAG), METHODS FOR THEIR PRODUCTION AND THEIR USE IN IMMUNOASSAYS AND VACCINE SUBSTANCES |
US7611713B2 (en) | 1993-03-05 | 2009-11-03 | Pharmexa Inc. | Inducing cellular immune responses to hepatitis B virus using peptide compositions |
US6689363B1 (en) | 1992-01-29 | 2004-02-10 | Epimmune Inc. | Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions |
US6509149B2 (en) * | 1995-06-06 | 2003-01-21 | Hybridon, Inc. | HPV-specific oligonucleotides |
EP0912195B1 (en) * | 1996-06-25 | 2009-01-21 | Pepscan Systems B.V. | Vaccine comprising antigens bound to carriers through labile bonds |
US7091324B2 (en) | 1998-11-05 | 2006-08-15 | Board Of Trustees Of Leland Stanford Junior University | Prevention and treatment of HCV infection employing antibodies directed against conformational epitopes |
CN1268345C (en) | 2000-03-29 | 2006-08-09 | 乔治敦大学 | Method of treating hepatitis delta viral infection |
IL142802A (en) * | 2000-04-27 | 2015-01-29 | Enzo Therapeutics Inc | Use of one or more hbv antigens for the preparation of oral pharmaceutical compositions for treating a subject having an active hbv infection or hepatocellular carcinoma |
US6787526B1 (en) | 2000-05-26 | 2004-09-07 | Idenix Pharmaceuticals, Inc. | Methods of treating hepatitis delta virus infection with β-L-2′-deoxy-nucleosides |
US7022830B2 (en) * | 2000-08-17 | 2006-04-04 | Tripep Ab | Hepatitis C virus codon optimized non-structural NS3/4A fusion gene |
CA2437503A1 (en) * | 2001-02-05 | 2002-08-15 | Stressgen Biotechnologies Corp. | Hepatitis b virus treatment |
US7351413B2 (en) * | 2002-02-21 | 2008-04-01 | Lorantis, Limited | Stabilized HBc chimer particles as immunogens for chronic hepatitis |
CN1305905C (en) * | 2002-03-22 | 2007-03-21 | Aprogen株式会社 | Humanized antibody and process for preparing same |
DE10339927A1 (en) * | 2003-08-29 | 2005-03-24 | Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH | Composition for the prophylaxis / therapy of HBV infections and HBV-mediated diseases |
JP4486647B2 (en) * | 2003-11-12 | 2010-06-23 | エイチビーヴイ セラノスティカ アクチエボラーグ | Methods and means related to hepatitis B infection |
GB0326416D0 (en) | 2003-11-12 | 2003-12-17 | Karolinska Innovations Ab | Methods and means relating to hepatitis B infection |
US9603921B2 (en) * | 2004-10-27 | 2017-03-28 | Janssen Vaccines Ag | Virosome particles comprising antigens from influenza virus and hepatitis b virus |
US8071561B2 (en) * | 2007-08-16 | 2011-12-06 | Chrontech Pharma Ab | Immunogen platform |
WO2009092396A1 (en) | 2008-01-25 | 2009-07-30 | Universitätsklinikum Heidelberg | Hydrophobic modified pres-derived peptides of hepatitis b virus (hbv) and their use as hbv and hdv entry inhibitors |
CN101485672B (en) * | 2008-12-05 | 2010-08-25 | 广东粤龙药业有限公司 | Application of hylotelephin in preparing medicament for treating human HBeAg-positive chronic hepatitis B |
NZ598000A (en) | 2009-08-07 | 2013-10-25 | Transgene Sa | Composition for treating hbv infection |
US10076570B2 (en) | 2009-08-07 | 2018-09-18 | Transgene S.A. | Composition for treating HBV infection |
Citations (48)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3636191A (en) * | 1969-10-08 | 1972-01-18 | Cancer Res Inst | Vaccine against viral hepatitis and process |
US4415491A (en) * | 1980-01-14 | 1983-11-15 | The Regents Of The University Of California | Synthetic vaccine peptide epitomes of hepatitis B surface antigen |
US4428941A (en) * | 1979-08-03 | 1984-01-31 | Institut Pasteur | Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby |
US4563423A (en) * | 1981-09-02 | 1986-01-07 | Biogen N.V. | Products displaying the antigenicity of hepatitis B virus e antigens and methods of producing those antigens |
US4599230A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US4599231A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US4639371A (en) * | 1984-10-02 | 1987-01-27 | New York Blood Center, Inc. | Hepatitis B antigenic compositions and vaccines against hepatitis B derived therefrom |
US4649192A (en) * | 1985-05-30 | 1987-03-10 | Smith Kline-Rit | Method for the isolation and purification of hepatitis B surface antigen using polysorbate |
US4683136A (en) * | 1985-03-06 | 1987-07-28 | Scripps Clinic And Research Foundation | Proteinaceous antigens with conformation-independent and conformation-dependent determinants |
US4696898A (en) * | 1984-01-16 | 1987-09-29 | Integrated Genetics, Inc. | Vector encoding hepatitis B surface antigen |
US4710463A (en) * | 1978-12-22 | 1987-12-01 | Biogen N.V. | Recombinant DNA molecules capable of expressing HBV core and surface antigens |
US4722940A (en) * | 1984-08-23 | 1988-02-02 | Georgia Tech Research Corporation | Aminoalkyl phenyl selenides for the treatment of hypertension and nervous system dysfunctions |
US4741901A (en) * | 1981-12-03 | 1988-05-03 | Genentech, Inc. | Preparation of polypeptides in vertebrate cell culture |
US4742158A (en) * | 1986-04-25 | 1988-05-03 | Merck & Co., Inc. | Purification of hepatitis pre-S antigens by polymerized serum albumin affinity binding |
US4777240A (en) * | 1984-03-08 | 1988-10-11 | Scripps Clinic And Research Foundation | SV40 expression vector containing HBxAg as an expression marker |
US4816564A (en) * | 1986-01-31 | 1989-03-28 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
US4818527A (en) * | 1986-12-09 | 1989-04-04 | Scripps Clinic And Research Foundation | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US4847080A (en) * | 1984-03-07 | 1989-07-11 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipide vesicle carriers |
US4861588A (en) * | 1985-02-05 | 1989-08-29 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US4882145A (en) * | 1986-12-09 | 1989-11-21 | Scripps Clinic And Research Foundation | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US4883865A (en) * | 1987-09-30 | 1989-11-28 | Merck & Co. Inc. | Recovery of pres2+s antigen |
US4895800A (en) * | 1985-11-26 | 1990-01-23 | Phillips Petroleum Company | Yeast production of hepatitis B surface antigen |
US4935235A (en) * | 1979-05-24 | 1990-06-19 | The Regents Of The University Of California | Non-passageable viruses |
US4942125A (en) * | 1984-09-07 | 1990-07-17 | Scripps Clinic And Research Foundation | SV40 expression vector containing HBxAg as an expression marker |
US4945046A (en) * | 1986-06-03 | 1990-07-31 | The Green Cross Corporation | Yeast promoter and process for preparing heterologous protein |
US4959323A (en) * | 1985-11-04 | 1990-09-25 | Mt. Sinai School Of Medicine Of The City University Of New York | Production and use of pre S polypeptides of hepatitis B virus |
US4963483A (en) * | 1987-10-13 | 1990-10-16 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
US4977092A (en) * | 1985-06-26 | 1990-12-11 | Amgen | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells |
US5011915A (en) * | 1987-10-26 | 1991-04-30 | Merck & Co., Inc. | Process for purifying recombinant hepatitis antigens |
US5024938A (en) * | 1982-08-20 | 1991-06-18 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Recombinant DNA inserted with hepatitis B virus gene, mammalian cells transformed with cloned viral DNA, and production of hepatitis B virus proteins |
US5039522A (en) * | 1988-01-29 | 1991-08-13 | New York Blood Center, Inc. | Immunogens containing peptides with an attached hydrophobic tail for adsorption to hepatitis B virus surface antigen |
US5068185A (en) * | 1986-07-10 | 1991-11-26 | Merck & Co., Inc. | Trains of yeast for the expression of heterologous genes |
US5098704A (en) * | 1984-06-18 | 1992-03-24 | Chiron Corporation | Hepatitis surface antigen particle vaccine |
US5102989A (en) * | 1991-03-15 | 1992-04-07 | Merck & Co., Inc. | Method of stabilizing recombinant hepatitis B virus surface proteins from recombinant host cells |
US5143726A (en) * | 1986-12-09 | 1992-09-01 | The Scripps Research Institute | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US5158769A (en) * | 1984-03-07 | 1992-10-27 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US5196194A (en) * | 1979-05-24 | 1993-03-23 | The Regents Of The University Of California | Vaccines containing Hepatitis B S-protein |
US5198348A (en) * | 1982-08-30 | 1993-03-30 | Amgen Inc. | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells |
US5204096A (en) * | 1984-03-07 | 1993-04-20 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US5242812A (en) * | 1989-02-07 | 1993-09-07 | Bio-Technology General Corp. | Method for production and purification of hepatitis B vaccine |
US5314808A (en) * | 1980-04-22 | 1994-05-24 | Institut Pasteur | Method for the transformation of cells, particularly eukaryotes by a DNA originating from viruses of hepatitis, more particularly from virus of a B viral hepatitis, and preparations containing the expression products of said DNAs |
US5324513A (en) * | 1984-03-07 | 1994-06-28 | Institut Pasteur | Composition useful for the fabrication of vaccines |
USRE34705E (en) * | 1979-08-30 | 1994-08-23 | Institut Pasteur | Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby |
US5436139A (en) * | 1979-05-24 | 1995-07-25 | The Regents Of The University Of California | Non-passageable virus |
US5462863A (en) * | 1989-02-09 | 1995-10-31 | Development Center For Biotechnology | Isolation of Hepatitis B surface antigen from transformed yeast cells |
US5792163A (en) * | 1996-01-03 | 1998-08-11 | Hitzig; Gary | Linear punch |
US5837249A (en) * | 1985-04-19 | 1998-11-17 | The Wistar Institute | Method for generating an immunogenic T cell response protective against a virus |
US5840303A (en) * | 1991-08-26 | 1998-11-24 | The Scripps Research Foundation | Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus |
Family Cites Families (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US34705A (en) * | 1862-03-18 | Improvement in tompions for fire-arms | ||
EP0182442B2 (en) * | 1978-12-22 | 1996-04-03 | Biogen, Inc. | Recombinant DNA molecules and their method of production |
IL63224A (en) * | 1980-07-17 | 1985-05-31 | Scripps Clinic Res | Synthetic peptide specific antigenic determinant and method of manufacturing antigenic materials therefrom |
GR76274B (en) * | 1981-08-04 | 1984-08-04 | Univ California | |
JPS58194897A (en) * | 1982-05-07 | 1983-11-12 | Takeda Chem Ind Ltd | Novel dna, its preparation and host transformed with the same |
JPS5974985A (en) * | 1982-10-19 | 1984-04-27 | Takeda Chem Ind Ltd | Novel dna, its preparation and transformant having the same |
CA1341302C (en) * | 1983-02-22 | 2001-10-09 | Rae Lyn Burke | Yeast expression systems with vectors having gapdh or pyk promoters and synthesis of foreign protein |
FR2559159B1 (en) * | 1984-02-02 | 1986-09-12 | Inst Nat Sante Rech Med | VIRAL VECTORS FOR THE CLONING AND EXPRESSION OF A PROTEIN IN A EUKARYOTIC CELL, COMPRISING AT LEAST ONE PART OF THE GENOME OF A RETROVIRUS; TRANSFECTED EUKARYOTIC CELLS; PROCESS USING SUCH CELLS AND PROTEINS OBTAINED |
FR2560890B1 (en) * | 1984-03-07 | 1987-10-16 | Grp Genie Genetique | COMPOSITION USEFUL FOR THE MANUFACTURE OF VACCINES CONTAINING PARTICLES CARRYING THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND THE POLYMERIZED HUMAN SERUM ALBUMIN RECEPTOR, ANIMAL CELLS CAPABLE OF PRODUCING SUCH PARTICLES |
AU579148B2 (en) * | 1984-03-09 | 1988-11-17 | Scripps Clinic And Research Foundation | Synthetic hepatitis b virus vaccine including both t cell and b cell determinants |
EP0401941A3 (en) * | 1984-07-11 | 1991-04-17 | Takeda Chemical Industries, Ltd. | Hepatitis b virus surface antigen and production thereof |
GB8421282D0 (en) * | 1984-08-22 | 1984-09-26 | Connaught Lab | Multispecific antigenic proteins |
US4722840A (en) * | 1984-09-12 | 1988-02-02 | Chiron Corporation | Hybrid particle immunogens |
DE3584866D1 (en) * | 1984-09-12 | 1992-01-23 | Chiron Corp | HYBRID PARTICLE IMMUNOGENES. |
EP0180012A1 (en) * | 1984-10-27 | 1986-05-07 | Wolfram H. Prof. Dr. Gerlich | Immunogenic polypeptide sequence of hepatits B virus |
CA1324094C (en) * | 1985-04-03 | 1993-11-09 | Dino Dina | Hepatitis a virus vaccines |
FI861417A0 (en) * | 1985-04-15 | 1986-04-01 | Endotronics Inc | HEPATITIS B YTANTIGEN FRAMSTAELLD MED REKOMBINANT-DNA-TEKNIK, VACCIN, DIAGNOSTISKT MEDEL OCH CELLINJER SAMT FOERFARANDEN FOER FRAMSTAELLNING DAERAV. |
US4639271A (en) * | 1985-04-24 | 1987-01-27 | Moore Business Forms, Inc. | Chromogenic mixtures |
FR2581394B1 (en) * | 1985-05-02 | 1988-08-05 | Grp Genie Genetique | PARTICLES HAVING IMMUNOGENIC PROPERTIES OF THE HBS ANTIGEN AND CARRYING A FOREIGN ANTIGENIC SITE TO THE EPITOPES CARRIED BY THE HBS ANTIGEN, ANIMAL VECTORS AND CELLS FOR THE PRODUCTION OF SUCH PARTICLES AND COMPOSITIONS CONTAINING SUCH PARTICLES FOR THE PRODUCTION |
JPH084507B2 (en) * | 1985-10-03 | 1996-01-24 | 武田薬品工業株式会社 | Novel DNA and polypeptide |
IE59389B1 (en) * | 1986-05-23 | 1994-12-23 | Merck & Co Inc | Method for producing hepatitis b virus core antigen (HBcAg) in yeast |
WO1987007896A1 (en) * | 1986-06-20 | 1987-12-30 | Scripps Clinic And Research Foundation | T and b cell epitopes of the pre-s region of hepatitis b virus surface antigen |
IL79740A0 (en) * | 1986-08-17 | 1986-11-30 | Yeda Res & Dev | Hepatitis vaccine |
EP0278940A3 (en) * | 1987-01-30 | 1988-12-07 | Smithkline Biologicals S.A. | Hepatitis b virus surface antigens and hybrid antigens containing them |
EP0300213A1 (en) * | 1987-06-22 | 1989-01-25 | Hexal-Pharma Gentechnik GmbH & Co. KG | Hepatitis a viral peptide particle immunogens |
EP1088830A3 (en) * | 1987-06-22 | 2004-04-07 | Medeva Holdings B.V. | Hepatitis b surface antigen particles |
NZ229260A (en) * | 1988-06-03 | 1994-02-25 | Merck & Co Inc | Hepatitis b virus, expression cassette for pre-s domain, host cells and |
GB8903313D0 (en) * | 1989-02-14 | 1989-04-05 | Wellcome Found | Conjugates |
ATE159031T1 (en) * | 1989-07-25 | 1997-10-15 | Smithkline Biolog | ANTIGENS AND METHODS FOR THE PRODUCTION THEREOF |
CA2025481A1 (en) * | 1989-09-19 | 1991-03-20 | Peter J. Kniskern | Vaccine for aids and hepatitis b |
EP0491077A1 (en) * | 1990-12-19 | 1992-06-24 | Medeva Holdings B.V. | A composition used as a therapeutic agent against chronic viral hepatic diseases |
IL101653A0 (en) * | 1991-04-29 | 1992-12-30 | Merck & Co Inc | Multiple hepatitis b virus surface proteins which form particles |
CA2067003A1 (en) * | 1991-04-29 | 1992-10-30 | Peter J. Kniskern | Hbsag escape mutant vaccine |
JP2501186Y2 (en) * | 1992-03-31 | 1996-06-12 | 株式会社栗本鐵工所 | Hammer mill |
CN1063343C (en) * | 1993-04-27 | 2001-03-21 | 中国科学院上海生物化学研究所 | Hepatitis B surface antigen protein having ammonia end with pre-surface antigen determinant |
CN1094311A (en) * | 1993-04-27 | 1994-11-02 | 中国科学院上海生物化学研究所 | The hepatitis b surface antigen protein of ammonia end band front surface antigenic determinant |
CN1059927C (en) * | 1994-03-10 | 2000-12-27 | 中国科学院上海生物化学研究所 | Reconstituted hepatitis B vaccine with corboxy end having anterior surface antigen 1 determinant |
-
1990
- 1990-12-19 EP EP90124775A patent/EP0491077A1/en not_active Withdrawn
-
1991
- 1991-12-19 WO PCT/EP1991/002460 patent/WO1992011368A1/en active IP Right Grant
- 1991-12-19 HU HU9301794A patent/HU216301B/en unknown
- 1991-12-19 SK SK636-93A patent/SK280551B6/en unknown
- 1991-12-19 ES ES92900527T patent/ES2121000T5/en not_active Expired - Lifetime
- 1991-12-19 CA CA002098021A patent/CA2098021C/en not_active Expired - Lifetime
- 1991-12-19 DE DE69130071T patent/DE69130071T3/en not_active Expired - Lifetime
- 1991-12-19 EP EP92900527A patent/EP0563093B2/en not_active Expired - Lifetime
- 1991-12-19 DK DK92900527T patent/DK0563093T4/en active
- 1991-12-19 AT AT92900527T patent/ATE170220T1/en not_active IP Right Cessation
- 1991-12-19 US US08/075,520 patent/US6020167A/en not_active Expired - Lifetime
- 1991-12-19 CZ CZ19931186A patent/CZ290233B6/en not_active IP Right Cessation
- 1991-12-19 AU AU90817/91A patent/AU657935B2/en not_active Ceased
- 1991-12-19 JP JP50100592A patent/JP3857305B2/en not_active Expired - Lifetime
-
1993
- 1993-06-18 KR KR1019930701862A patent/KR0156587B1/en not_active IP Right Cessation
-
1998
- 1998-03-10 HK HK98101997A patent/HK1002901A1/en not_active IP Right Cessation
-
2000
- 2000-06-07 JP JP2000175584A patent/JP2001019643A/en active Pending
-
2003
- 2003-03-21 US US10/394,896 patent/US20030235591A1/en not_active Abandoned
Patent Citations (53)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3636191A (en) * | 1969-10-08 | 1972-01-18 | Cancer Res Inst | Vaccine against viral hepatitis and process |
US4710463A (en) * | 1978-12-22 | 1987-12-01 | Biogen N.V. | Recombinant DNA molecules capable of expressing HBV core and surface antigens |
US4935235A (en) * | 1979-05-24 | 1990-06-19 | The Regents Of The University Of California | Non-passageable viruses |
US5196194A (en) * | 1979-05-24 | 1993-03-23 | The Regents Of The University Of California | Vaccines containing Hepatitis B S-protein |
US5436139A (en) * | 1979-05-24 | 1995-07-25 | The Regents Of The University Of California | Non-passageable virus |
US4428941A (en) * | 1979-08-03 | 1984-01-31 | Institut Pasteur | Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby |
USRE34705E (en) * | 1979-08-30 | 1994-08-23 | Institut Pasteur | Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby |
US4415491A (en) * | 1980-01-14 | 1983-11-15 | The Regents Of The University Of California | Synthetic vaccine peptide epitomes of hepatitis B surface antigen |
US5314808A (en) * | 1980-04-22 | 1994-05-24 | Institut Pasteur | Method for the transformation of cells, particularly eukaryotes by a DNA originating from viruses of hepatitis, more particularly from virus of a B viral hepatitis, and preparations containing the expression products of said DNAs |
US5591638A (en) * | 1980-04-22 | 1997-01-07 | Institut Pasteur And Institut Nationale De La Sante Et De La Recherche Medicale | Method for the transformation of cells, particularly eukaryotes by a DNA originating from viruses of hepatitis, more particularly from virus of B viral hepatitis, and preparations containing the expression products of said DNAs |
US4758507A (en) * | 1981-09-02 | 1988-07-19 | Biogen N.P. | Products displaying the antigenicity of hepatitis B virus e antigens and methods of producing those antigens |
US4563423A (en) * | 1981-09-02 | 1986-01-07 | Biogen N.V. | Products displaying the antigenicity of hepatitis B virus e antigens and methods of producing those antigens |
US4741901A (en) * | 1981-12-03 | 1988-05-03 | Genentech, Inc. | Preparation of polypeptides in vertebrate cell culture |
US5024938A (en) * | 1982-08-20 | 1991-06-18 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Recombinant DNA inserted with hepatitis B virus gene, mammalian cells transformed with cloned viral DNA, and production of hepatitis B virus proteins |
US5198348A (en) * | 1982-08-30 | 1993-03-30 | Amgen Inc. | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells |
US4696898A (en) * | 1984-01-16 | 1987-09-29 | Integrated Genetics, Inc. | Vector encoding hepatitis B surface antigen |
US5620844A (en) * | 1984-03-07 | 1997-04-15 | New York Blood, Inc. | Assays for detecting hepatitis B virus envelope antigens or antibodies thereto and diagnostic test kits for use in performing the assays |
US4847080A (en) * | 1984-03-07 | 1989-07-11 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipide vesicle carriers |
US5565548A (en) * | 1984-03-07 | 1996-10-15 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens and synthetic lipid vesicle carriers |
US5324513A (en) * | 1984-03-07 | 1994-06-28 | Institut Pasteur | Composition useful for the fabrication of vaccines |
US5204096A (en) * | 1984-03-07 | 1993-04-20 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US5158769A (en) * | 1984-03-07 | 1992-10-27 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US4777240A (en) * | 1984-03-08 | 1988-10-11 | Scripps Clinic And Research Foundation | SV40 expression vector containing HBxAg as an expression marker |
US4599230A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US4599231A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
US5098704A (en) * | 1984-06-18 | 1992-03-24 | Chiron Corporation | Hepatitis surface antigen particle vaccine |
US4722940A (en) * | 1984-08-23 | 1988-02-02 | Georgia Tech Research Corporation | Aminoalkyl phenyl selenides for the treatment of hypertension and nervous system dysfunctions |
US4942125A (en) * | 1984-09-07 | 1990-07-17 | Scripps Clinic And Research Foundation | SV40 expression vector containing HBxAg as an expression marker |
US4639371A (en) * | 1984-10-02 | 1987-01-27 | New York Blood Center, Inc. | Hepatitis B antigenic compositions and vaccines against hepatitis B derived therefrom |
US4861588A (en) * | 1985-02-05 | 1989-08-29 | New York Blood Center, Inc. | Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers |
US4683136A (en) * | 1985-03-06 | 1987-07-28 | Scripps Clinic And Research Foundation | Proteinaceous antigens with conformation-independent and conformation-dependent determinants |
US5837249A (en) * | 1985-04-19 | 1998-11-17 | The Wistar Institute | Method for generating an immunogenic T cell response protective against a virus |
US4649192A (en) * | 1985-05-30 | 1987-03-10 | Smith Kline-Rit | Method for the isolation and purification of hepatitis B surface antigen using polysorbate |
US4977092A (en) * | 1985-06-26 | 1990-12-11 | Amgen | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells |
US4959323A (en) * | 1985-11-04 | 1990-09-25 | Mt. Sinai School Of Medicine Of The City University Of New York | Production and use of pre S polypeptides of hepatitis B virus |
US4895800A (en) * | 1985-11-26 | 1990-01-23 | Phillips Petroleum Company | Yeast production of hepatitis B surface antigen |
US4816564A (en) * | 1986-01-31 | 1989-03-28 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
US5133961A (en) * | 1986-01-31 | 1992-07-28 | Merck & Co., Inc. | Vaccines comprising yeast-derived hepatits b virus polypeptides |
US4742158A (en) * | 1986-04-25 | 1988-05-03 | Merck & Co., Inc. | Purification of hepatitis pre-S antigens by polymerized serum albumin affinity binding |
US4945046A (en) * | 1986-06-03 | 1990-07-31 | The Green Cross Corporation | Yeast promoter and process for preparing heterologous protein |
US5068185A (en) * | 1986-07-10 | 1991-11-26 | Merck & Co., Inc. | Trains of yeast for the expression of heterologous genes |
US5143726A (en) * | 1986-12-09 | 1992-09-01 | The Scripps Research Institute | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US4882145A (en) * | 1986-12-09 | 1989-11-21 | Scripps Clinic And Research Foundation | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US4818527A (en) * | 1986-12-09 | 1989-04-04 | Scripps Clinic And Research Foundation | T cell epitopes of the hepatitis B virus nucleocapsid protein |
US4883865A (en) * | 1987-09-30 | 1989-11-28 | Merck & Co. Inc. | Recovery of pres2+s antigen |
US4963483A (en) * | 1987-10-13 | 1990-10-16 | Merck & Co., Inc. | Method for producing hepatitis B virus proteins in yeast |
US5011915A (en) * | 1987-10-26 | 1991-04-30 | Merck & Co., Inc. | Process for purifying recombinant hepatitis antigens |
US5039522A (en) * | 1988-01-29 | 1991-08-13 | New York Blood Center, Inc. | Immunogens containing peptides with an attached hydrophobic tail for adsorption to hepatitis B virus surface antigen |
US5242812A (en) * | 1989-02-07 | 1993-09-07 | Bio-Technology General Corp. | Method for production and purification of hepatitis B vaccine |
US5462863A (en) * | 1989-02-09 | 1995-10-31 | Development Center For Biotechnology | Isolation of Hepatitis B surface antigen from transformed yeast cells |
US5102989A (en) * | 1991-03-15 | 1992-04-07 | Merck & Co., Inc. | Method of stabilizing recombinant hepatitis B virus surface proteins from recombinant host cells |
US5840303A (en) * | 1991-08-26 | 1998-11-24 | The Scripps Research Foundation | Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus |
US5792163A (en) * | 1996-01-03 | 1998-08-11 | Hitzig; Gary | Linear punch |
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DE69130071T2 (en) | 1999-01-07 |
HUT66437A (en) | 1994-11-28 |
HU9301794D0 (en) | 1993-10-28 |
CZ118693A3 (en) | 1994-02-16 |
EP0563093A1 (en) | 1993-10-06 |
EP0491077A1 (en) | 1992-06-24 |
HK1002901A1 (en) | 1998-09-25 |
AU657935B2 (en) | 1995-03-30 |
ATE170220T1 (en) | 1998-09-15 |
CA2098021A1 (en) | 1992-06-20 |
EP0563093B1 (en) | 1998-08-26 |
HU216301B (en) | 1999-06-28 |
EP0563093B2 (en) | 2007-10-03 |
ES2121000T5 (en) | 2008-04-16 |
DK0563093T4 (en) | 2008-01-21 |
JP3857305B2 (en) | 2006-12-13 |
DK0563093T3 (en) | 1999-02-08 |
SK280551B6 (en) | 2000-03-13 |
DE69130071D1 (en) | 1998-10-01 |
CZ290233B6 (en) | 2002-06-12 |
SK63693A3 (en) | 1993-10-06 |
DE69130071T3 (en) | 2008-02-21 |
KR0156587B1 (en) | 1998-10-15 |
CA2098021C (en) | 2000-04-25 |
ES2121000T3 (en) | 1998-11-16 |
US6020167A (en) | 2000-02-01 |
AU9081791A (en) | 1992-07-22 |
JP2001019643A (en) | 2001-01-23 |
WO1992011368A1 (en) | 1992-07-09 |
JPH06507303A (en) | 1994-08-25 |
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