WO2001021807A1 - Proteine enveloppe 2 (e2) du virus de l'hepatite c qui ne possede pas tout ou partie de la region 1 hypervariable (hvr1), acides nucleiques correspondants, virus chimeriques et utilisation de ces derniers - Google Patents

Proteine enveloppe 2 (e2) du virus de l'hepatite c qui ne possede pas tout ou partie de la region 1 hypervariable (hvr1), acides nucleiques correspondants, virus chimeriques et utilisation de ces derniers Download PDF

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WO2001021807A1
WO2001021807A1 PCT/US2000/025987 US0025987W WO0121807A1 WO 2001021807 A1 WO2001021807 A1 WO 2001021807A1 US 0025987 W US0025987 W US 0025987W WO 0121807 A1 WO0121807 A1 WO 0121807A1
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virus
hcv
protein
nucleic acid
envelope
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PCT/US2000/025987
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WO2001021807A8 (fr
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Xavier Forns
Jens Bukh
Suzanne U. Emerson
Robert H. Purcell
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The Government Of The United States Of America As Represented By The Secretary, Department Of Health Services
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Priority to AU76028/00A priority Critical patent/AU7602800A/en
Publication of WO2001021807A1 publication Critical patent/WO2001021807A1/fr
Publication of WO2001021807A8 publication Critical patent/WO2001021807A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24271—Demonstrated in vivo effect

Definitions

  • the present invention relates to nucleic acid molecules that encode a hepatitis C virus (HCV) envelope two protein which lacks all or part of the hypervariable region one (HVR1) of the envelope two (E2) protein.
  • HCV hepatitis C virus
  • the invention further relates to the use of the nucleic acid molecules and their encoded polypeptides as vaccine candidates.
  • HCV Hepatitis C virus
  • HCV has been identified (Bukh et al . , 1993; Simmonds et al., 1993) .
  • the nucleotide and deduced amino acid sequences among isolates within a quasispecies generally differ by ⁇ 2%, whereas those between isolates of different genotypes vary by as much as 35%.
  • HVRl hypervariable region 1
  • HVRl is the region of the genome with the highest degree of genetic variability suggesting that it is under strong immune pressure. Indeed, Ray et al. (1999) recently reported that patients who developed a chronic infection had a higher rate of non-synonymous mutations within the HVRl as compared with the El protein while the reverse was observed in patients who were able to clear the infection. These authors therefore hypothesized that the HVRl region of HCV might act as a decoy antigen by stimulating a strong immune response that is ineffective in clearing viremia.
  • HVRl as an immunologic decoy
  • a hyperimmune rabbit serum raised against the HVRl was demonstrated to be capable of neutralizing HCV in vi tro (Farci et al . , 1996, Shimizu et al . , 1996).
  • the presence of amino acids in the carboxy-terminal half of HVRl which are conserved across genotypes suggests that HVRl might be required for HCV replication.
  • the present invention relates to nucleic acid molecules which lack all or part of the coding sequence of the HVRl region of the envelope 2 (E2) gene of hepatitis C virus (HCV) .
  • the nucleic acid molecule of the invention comprises the genome of an infectious hepatitis C virus in which the HVRl of the envelope 2 gene of the infectious HCV has been deleted.
  • nucleic acid sequence which encodes infectious hepatitis C virus lacking HVRl.
  • Such nucleic acid sequence is referred to throughout the application as “ ⁇ HVRl-infectious nucleic acid sequence .
  • the nucleic acid molecule comprises chimeric genomes of chimeric hepatitis C viruses in which the structural region (core and envelope genes) or the envelope gene of a pestivirus genome, [for example, a bovine viral diarrhea virus (BVDV) ] or a flavivirus genome (for example, a dengue virus or a yellow fever virus) are replaced by the corresponding structural region or El and E2 genes of an HCV in which the HVRl region of HCV E2 has been removed.
  • BVDV bovine viral diarrhea virus
  • flavivirus genome for example, a dengue virus or a yellow fever virus
  • the present invention also relates to the in vitro and in vivo production of ⁇ HVRl-infectious HCV or ⁇ HVRl-chimeric HCV viruses from the ⁇ HVRl-infectious or ⁇ HVRl-chimeric nucleic acid sequences of the invention.
  • the present invention also relates to the use of the ⁇ HVRl viruses of the invention to identify cell lines capable of supporting the replication of the viruses.
  • the invention also relates to the use of the ⁇ HVRl infectious or chimeric nucleic acid sequences of the invention in the production of ⁇ HVRl-infectious HCV or ⁇ HVRl-chimeric HCV respectively, and the use of these virions for the development of inactivated or attenuated vaccines to prevent HCV in a mammal .
  • the DNA construct comprises an
  • HCV E2 gene lacking the HVRl region.
  • Such a " ⁇ HVR1-E2 gene” may also be linked in tandem in the DNA construct with an HCV El gene.
  • the ⁇ HVR1-E2 gene may be further modified at its carboxy-terminus to produce either a secreted or surface expressed ⁇ HVR1-E2 protein.
  • the invention further relates to pharmaceutical compositions and DNA-based vaccines which comprise the nucleic acid molecules of the invention.
  • the invention also relates to methods of preventing or treating HCV in a mammal comprising administering the nucleic acid molecules of the invention to a mammal in an amount effective to stimulate the production of a protective humoral and/or cellular immune response to HCV.
  • the invention also provides a kit for the treatment or prevention of HCV, the kit comprising a DNA molecule of the invention useful as an immunogen in generating a protective immune response to HCV.
  • the invention further relates to the use of the nucleic acid molecules of the invention as immunogens to generate antibodies to the ⁇ HVRl-infectious HCV, the ⁇ HVRl- chimeric HCV or the ⁇ HVR1-E2 protein, preferably neutralizing antibodies.
  • the invention therefore relates to the use of such antibodies in passive immunoprophylaxis and to pharmaceutical compositions which comprise these antibodies.
  • the invention also relates to transformation of host cells with nucleic acid molecules of the invention to produce host cells which express ⁇ HVRl-infectious HCV, the ⁇ HVRl- chimeric HCV or the ⁇ HVR1-E2 protein.
  • the invention further relates to the use of host cells expressing ⁇ HVRl-infectious HCV, the ⁇ HVRl-chimeric HCV or the ⁇ HVR1-E2 protein as immunogens to stimulate a protective immune response to HCV.
  • the present invention also relates to ⁇ HVRl E2 protein produced from the ⁇ HVR1-E2 gene constructs of the invention or obtained from the ⁇ HVRl-infectious HCVs or ⁇ HVRl-chimeric HCVs of the invention.
  • ⁇ HVR1-E2 proteins may be used as vaccines for immunizing mammals, especially humans, against HCV.
  • Figures LA-IF show the nucleotide sequence (SEQ ID NO: 1) of the infectious hepatitis C virus clone of genotype la [H77C ( ⁇ HVRl) ] which lacks the hypervariable region one (HVRl) of the second envelope protein and Figures 1G-1H show the amino acid sequence (SEQ ID NO: 2) encoded by the clone.
  • the complete sequence of H77C ( ⁇ HVRl) is identical to pCV-H77C but lacks the fragment from nucleotide positions 1491 to 1571 which encodes HVRl (Yanagi et al . , 1997; ATCC accession number PTA-157).
  • Figure 2 shows in vi tro transcription-translation of constructs E1E2-715 (Lanes 1, 3, 4, 5, and 6) and ⁇ HVR1-7 (Lane 8) with rabbit reticulocyte lysates with and without the addition of canine microsomal membranes.
  • Figure 3 shows the results of qualitative reverse transcriptase-nested polymerase chain reaction (RT-PCR) for HCV- RNA, logio HCV GE (genome equivalent) titer (in-house RT-PCR and Amplicor HCV Monitor, Roche Diagnostics) , second generation ELISA for anti-HCV and serum levels of alanine aminotransferase (ALT) in chimpanzee 1590 following transfection with RNA transcripts of the HCV deletion mutant H77C ( ⁇ HVRl).
  • RT-PCR reverse transcriptase-nested polymerase chain reaction
  • Figure 4 shows infection of chimpanzee 96A008 with HCV lacking HVRl.
  • Serum samples were collected weekly from the chimpanzee and monitored for HCV-RNA [in-house RT-nested PCR and HCV Monitor test version 2.0 (Roche)], HCV antibodies (second generation ELISA, Abbott Laboratories) and liver enzyme levels (ALT, Anilytics) .
  • PBMC were collected weekly and tested for HCV-specific proliferative capacity (peripheral CD4) with a panel of recombinant HCV proteins [C22 (core), C33-c (NS3), CIOO (NS3-NS4), and NS5] .
  • peripheral CD8+ T cell response was tested by stimulating PBMC with a large panel of HCV peptides corresponding to known CTL epitopes.
  • Expanded T cells isolated from liver biopsy samples were tested for HCV-specific proliferative responses (Intrahepatic CD4) +, positive; - negative.
  • Liver biopsies were examined also for necroinflammatory changes [0 (normal) , 1+, 2+, 3+, 4+] .
  • the chimpanzee was inoculated intravenously with 90 ml of plasma from chimpanzee 1590 (week 4 after transfection) .
  • At a titer of 10 GE/ml this represented an inoculum of approximately 900 genome equivalents of HCV.
  • FIG. 1A-1F shows the nucleic acid sequence of this infectious HCV clone which was constructed using pCV-H77C (ATCC accession number PTA- 157), an infectious HCV clone of genotype la.
  • the present invention therefore relates in one embodiment to a nucleic acid molecule which comprises the genome of an infectious hepatitis C virus in which the HVRl of the envelope 2 gene of the infectious HCV has been deleted.
  • the E2 protein consists of amino acid 384 to 746 of the HCV polyprotein and the HVRl of the E2 protein consists of amino acid 384 to 410.
  • nucleic acid sequence which encodes infectious hepatitis C virus lacking HVRl.
  • Such nucleic acid sequence is referred to throughout the application as “ ⁇ HVRl-infectious nucleic acid sequence.
  • infectious HCV clones of any genotype infectious HCV clones of different genotypes which have been constructed include those of Kolykhalov et al., (1997) and Yanagi et al. (1997, 1998) [who reported the derivation from HCV strains H77 (genotype la) and HC-J4 (genotype lb) of cDNA clones of HCV that are infectious for chimpanzees], and Yanagi et al. (Yanagi, 1999) [who reported the construction of an infectious cDNA clone from HCV strain HC- J6 (genotype 2a) ] .
  • the infectious HCV clones are infectious HCV clones of genotype la (ATCC accession number PTA- 157), lb (ATCC accession number 209596) or 2a (ATCC accession number PTA-153) .
  • the nucleic acid molecule of the invention comprises the genome of a flavivirus or pestivirus in which one envelope gene of the flavivirus or pestivirus is replaced by the E2 gene of an infectious HCV from which the HVRl region of HCV E2 has been removed.
  • nucleic acid sequence which encodes chimeric HCV lacking the HVRl of the HCV E2 gene.
  • Such nucleic acid sequence is referred to throughout the application as " ⁇ HVRl-chimeric nucleic acid sequence.
  • the Flaviviridae family of viruses which may be used to make the ⁇ HVRl-chimeric nucleic acid sequences include, but are not limited to, dengue virus, bovine viral diarrhea virus, yellow fever virus and Kunjin virus..
  • the ⁇ HVRl-chimeric nucleic acid sequences of the invention are made using the structural region or El and E2 genes of an infectious HCV clone of any genotype.
  • the infectious HCV clones are infectious HCV clones of genotype la (ATCC accession number PTA-157), lb (ATCC accession number 209596) or 2a (ATCC accession number PTA-153) .
  • the deletion of HVRl which may be made in the sequence of the invention is at least 5 amino acids in length, preferably 10 amino acids, and most preferably the entire 27 amino acids of HVRl.
  • the nucleic acid molecules of the invention therefore comprise genomes of HCV which lack at least a fragment of the HVRl sequence.
  • the ⁇ HVRl-infectious nucleic acid sequence of the invention may further include one or more mutations such as those described in Example 4 which result in amino acid changes.
  • One mutation is located within the E2 gene and results in a change from leucine to histidine at amino acid position 615.
  • Another mutation is located within the NS3 serine-protease domain and results in a change from arginine to histidine at amino acid position 1143.
  • a third mutation is located within the NS5B RNA-polymerase domain and results in a change from glutamic acid to aspartic acid at amino acid position 2875.
  • the present invention further relates to the in vitro and in vivo production of hepatitis C viruses from the ⁇ HVR1- infectious nucleic acid sequences of the invention, and the production of chimeric viruses from the ⁇ HVRl-chimeric nucleic acid sequences of the invention.
  • the ⁇ HVRl-infectious nucleic acid sequences or the ⁇ HVRl-chimeric nucleic acid sequences of the invention can be inserted into an expression vector that functions in eukaryotic cells.
  • Eukaryotic expression vectors suitable for producing high efficiency gene transfer in vivo are well known to those of ordinary skill in the art and include, but are not limited to, plasmids, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses.
  • sequences contained in the recombinant expression vector can then be transcribed in vitro by methods known to those of ordinary skill in the art in order to produce RNA transcripts which encode the HCV or chimeric viruses of the invention.
  • the HCV or chimeric viruses of the invention may then be produced by transfecting cells by methods known to those of ordinary skill in the art with either the in vitro transcription mixture containing the RNA transcripts or with the recombinant expression vectors containing the nucleic acid sequences described herein.
  • transfection may be done by methods known in the art such as electroporation, precipitation with DEAE-Dextran or calcium phosphate, or incorporation into liposomes.
  • Suitable cells or cell lines for culturing the HCV or chimeric viruses of the invention include, but are not limited to, EBTr and Huh7.
  • the present invention also relates to the use of the ⁇ HVRl-infectious nucleic acid sequences or the ⁇ HVRl-chimeric nucleic acid sequences of the invention to identify cell lines capable of supporting the replication of HCV and the chimeric viruses of the invention.
  • the invention further relates to the use of the ⁇ HVRl- infectious nucleic acid sequences or the ⁇ HVRl-chimeric nucleic acid sequences of the invention to develop inactivated or attenuated vaccines to prevent hepatitis C in a mammal.
  • virions from cell lines infected with the HCV or chimeric viruses of the invention, or transfected with a ⁇ HVRl- infectious nucleic acid sequence or a ⁇ HVRl-chimeric nucleic acid sequence of the invention can be purified from the cells and inactivated by methods known to those of ordinary skill in the art.
  • the inactivated virions can be used to immunize mice, and if neutralizing antibody to HCV is produced, the virions can then be used to immunize chimpanzees to determine whether the antibodies are protective.
  • cells infected with the viruses of the invention may be passaged in cell culture to produce attenuated viruses which can be tested as candidate live vaccines.
  • in assaying the ability of the viruses of the invention to infect mammals one can assay sera or liver of the infected mammal by RT-PCR to determine viral titer.
  • the virulence phenotype of the virus produced by transfection of mammals with the sequences of the invention can be monitored by methods known in the art such as measurement of liver enzyme levels (alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD) ) or by histopathology of liver biopsies .
  • liver enzyme levels alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD)
  • the HCV or chimeric virions can be administered alone or in a suitable diluent, including, but not limited to, water, saline, or some type of buffered medium.
  • the vaccine according to the present invention may be administered to an animal, especially a mammal, and most especially a human, by a variety of routes, including, but not limited to, intradermally, intramuscularly, subcutaneously, or in any combination thereof.
  • formulations or compositions comprising the HCV or chimeric virions of the invention may be used either therapeutically or prophylactically to treat or prevent the signs and symptoms of Hepatitis C.
  • the nucleic acid molecule of the invention comprises an HCV E2 gene lacking the HVRl region.
  • HCV E2 gene may also be linked in tandem in the DNA construct with an HCV El gene.
  • the ⁇ HVR1-E2 gene When contained alone or in tandem with the El gene, the ⁇ HVR1-E2 gene may be further modified at its carboxy- terminus to produce either a secreted or surface expressed ⁇ HVR1-E2 protein.
  • a ⁇ HVR1-E2 gene encoding E2 protein targeted to the cell surface is preferred.
  • Such a construct may be constructed by fusing an endoplasmic reticulum signal sequence to the amino- terminus of the nucleic acid sequence which encodes the truncated E2 gene fused at its carboxy-terminus to a plasma membrane anchor sequence.
  • endoplasmic reticulum (ER) signal sequence is meant a nucleic acid sequence which encodes a continuous stretch of amino acids, typically about 15 to about 25 residues in length, which are known in the art to be generally located at the amino terminus of proteins and are capable of targeting proteins to the endoplasmic reticulum.
  • ER signal sequences are known to those of skill in the art (see, for example, van Heijne, G. J. Mol. Biol., (1985) 184:99-105) and those of skill in the art would understand that even though their amino acid sequences may vary, such ER signal sequences are functionally interchangeable.
  • ER signal sequences which may be used in the chimeric genes of the invention include, but are not limited to, the 20-carboxy-terminal amino acids of the full-length HCV El protein (amino acids 364-383 of the HCV polyprotein) , which serves as the natural signal sequence of the E2 protein or the urine Ig kappa-chain V-J2-C signal peptide sequence contained in the pDisplay vector.
  • the truncated HCV envelope protein is a truncated E2 protein
  • the approximately 30 carboxy-terminal amino acids of E2 have been identified to contain an ER retention sequence and its removal and replacement with a plasma membrane anchor sequence is believed to be critical for expression of the truncated E2 protein on the cell surface.
  • the truncated E2 protein contains a truncation of at least the 20 carboxy-terminal amino acids of the full-length E2 protein, more preferably, a truncation of at least the 25 carboxy-terminal amino acids, and most preferably, a truncation of at least about the 30 carboxy-terminal amino acids.
  • plasma membrane anchor sequence as used in the chimeric gene of the invention is meant a nucleic acid sequence which encodes an amino acid sequence that allows for retention of at least part of the protein in the plasma membrane of a cell.
  • a plasma membrane anchor sequence encodes a sequence of hydrophobic amino acids of sufficient length to span the lipid bilayer of the plasma membrane.
  • hydrophobic sequences are known in the art as transmembrane domains and are typically found at the carboxy-terminus of many proteins found on the surface of cells or virions. These transmembrane domains are typically at least 20 to 30 amino acids in length and are followed by charged cytoplasmic domains of varying lengths.
  • the plasma membrane anchor sequence encoded by the coding sequence of the invention may contain in addition to a transmembrane domain of a virion or a protein found on the surface of a cell, a cytoplasmic domain.
  • the encoded plasma membrane anchor sequence is at least twenty amino acids in length, more preferably, from about 20 to about 100 amino acids in length, and most preferably, from about 30 to about 70 amino acids in length.
  • plasma membrane anchor sequences include, but are not limited to, hydrophobic transmembrane domains of receptors such as those for insulin and for a number of growth factors including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) , as well as the transmembrane domains of viral proteins that are anchored in the lipid envelope of the intact virion such as the transmembrane domains of the vesicular stomatitis and rabies virus G proteins.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • Preferred plasma membrane anchor sequences for inclusion in the chimeric genes of the invention are sequences which encode the 50 amino acid transmembrane domain of the PDGF receptor as contained in the pDisplay vector described in the Examples, the - carboxy-terminal 64 and 37 amino acids respectively of the CD4 and decay accelerating factor (DAF) proteins (these sequences constitute the transmembrane and cytoplasmic domains of the CD4 and DAF proteins) and the 49 carboxy-terminal amino acids of the VSV G protein (also constituting the transmembrane and cytoplasmic domains of the VSV G protein) .
  • DAF decay accelerating factor
  • transmembrane domains suitable for use as plasma membrane anchor sequences in the chimeric genes of the invention are known or could be readily identified by carrying out carboxy-terminal deletions of known plasma membrane or viral envelope proteins (see, for example, Men et al (J. Virol. (1991) 65; 1400-1407).
  • a ⁇ HVR1-E2 gene encoding a secreted E2 protein may be constructed by fusing an endoplasmic reticulum signal sequence to the nucleic acid sequence which encodes the truncated E2 gene.
  • the truncated E2 gene contains a truncation of at least the 31 carboxy-terminal amino acids (amino acids 716-746) , and more preferably, a truncation of at least the 85 carboxy-terminal amino acids (amino acids 662-746).
  • the ⁇ HVR1-E2 nucleic acid sequences of the invention may lack all or part of the HVRl sequence.
  • the deletion of HVRl is at least 5 amino acids in length, preferably 10 amino acids, and most preferably the entire 27 amino acids of HVRl.
  • the ⁇ HVR1-E2 nucleic acid sequences of the invention may also contain further truncations at the carboxy-terminus of the E2 gene of HCV, and/or mutations at amino acid position 615 as described above.
  • the present invention therefore relates to insertion of the nucleic acid molecules comprising the ⁇ HVR1-E2 gene of the invention into a suitable expression vector that functions in eukaryotic cells, preferably in mammalian cells.
  • a suitable expression vector that functions in eukaryotic cells, preferably in mammalian cells.
  • the vector is capable of carrying and expressing a chimeric gene of the invention.
  • the expression vector therefore contains at least one promoter and any other sequences necessary or preferred for appropriate transcription and translation of the ⁇ HVR1-E2 gene.
  • Preferred expression vectors include, but are not limited to, plasmid vectors.
  • the invention also relates to the use of expression vectors containing the ⁇ HVR1-E2 nucleic acid molecules of the invention as immunogens to produce protective antibodies to HCV.
  • Direct transfer of the ⁇ HVR1-E2 nucleic acid sequences of the invention to a mammal, preferably a primate, more preferably a human, may be accomplished by injection by needle or by use of other DNA delivery devices such as the gene gun.
  • Possible routes of administration of the expression vector include, but are not limited to, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal and intranasal.
  • nucleic acid molecules comprising ⁇ HVR1-E2 nucleic acid sequences of isolates from multiple genotypes of HCV may be administered together to provide protection against challenge with multiple genotypes of HCV.
  • ⁇ HVR1-E2 nucleic acid sequences may be inserted into a single vector such that a host cell transformed or transfected with the vector will produce multiple envelope proteins.
  • a polycistronic vector in which multiple different ⁇ HVR1-E2 genes may be expressed from a single vector is created by placing expression of each gene under control of an internal ribosomal entry site (IRES) (Molla, a. et al. Nature, 356:255-257 (1992); Gong, S.K. et al. J. of Virol. , 263:1651-1660 (1989)).
  • IRS internal ribosomal entry site
  • copies of different ⁇ HVR1-E2 nucleic acid sequences are inserted into multiple vectors and transformed or transfected into host cells so that multiple envelope proteins can be produced.
  • the expression vectors containing the ⁇ HVR1-E2 nucleic acid sequences of the invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition.
  • Suitable amounts of material to administer for prophylactic and therapeutic purposes will vary depending on the route selected and the immunogen (E2 gene with different deletions) administered.
  • the vaccines of the present invention may be administered once or periodically until a suitable titer of anti-HCV antibodies appear in the blood.
  • a suitable amount of expression vector to be used for prophylactic purposes might be expected to fall in the range of from about 1 ⁇ g to about 5 mg, more preferably from about 100 ⁇ g to about 5 mg, and most preferably from about 1 mg to about 2 mg. Such administration will, of course, occur prior to any sign of HCV infection. Further, one of skill in the art will readily understand that the amount of vector to be used will depend on the size and species of animal the vector is to be administered to.
  • a vaccine of the present invention may be employed in sterile liquid forms such as solutions or suspensions.
  • Any inert carrier is preferably used, such as saline or phosphate- buffered saline, or any such carrier in which the expression vector of the present invention can be suitably suspended.
  • the vaccines may be in the form of single dose preparations or in multi-dose flasks which can be utilized for mass-vaccination programs of both animals and humans.
  • specific adjuvants such as CpG motifs (Krieg, A.K. et al.(1995) Nature 374:546 and Krieg et al . (1996)) J. Lab. Clin. Med. , 128:128) may prove useful with DNA-based vaccines or other vaccines .
  • the DNA-based vaccines will normally exist as physically discrete units suitable as a unitary dosage for animals, especially mammals, and most especially humans, wherein each unit will contain a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent.
  • the dose of said vaccine or inoculum according to the present invention is administered at least once.
  • a second or booster dose may be administered at some time after the initial dose.
  • the need for, and timing of, such booster dose will, of course, be determined within the sound judgment of the administrator of such vaccine or inoculum and according to sound principles well known in the art.
  • such booster dose could reasonably be expected to be advantageous at some time between about 2 weeks to about 6 months following the initial vaccination.
  • Subsequent doses may be administered as indicated.
  • the ⁇ HVR1-E2 nucleic acid sequences of the present invention can also be administered for purposes of therapy, where a mammal, especially a primate, and most especially a human, is already infected, as shown by well-known diagnostic measures.
  • expression vectors containing the chimeric genes of the present invention are used for such therapeutic purposes, much of the same criteria will apply as when they are used as a vaccine, except that inoculation will occur post-infection.
  • the therapeutic agent comprises a pharmaceutical composition containing a sufficient amount of the expression vector so as to elicit a therapeutically effective response in the organism to be treated.
  • the amount of pharmaceutical composition to be administered will, as for vaccines, vary depending on the immunogen contained therein and on the route of administration.
  • the therapeutic agent according to the present invention can thus be administered by subcutaneous, intramuscular, intradermal or intranasal routes.
  • amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation.
  • the actual amounts will vary depending on the route of administration as well as the sex, age, and clinical status of the subject which, in the case of human patients, is to be determined with the sound judgment of the clinician.
  • the therapeutic agent of the present invention can be employed in sterile liquid forms such as solutions or suspensions.
  • An inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the expression vectors of the present invention can be suitably suspended.
  • the therapeutic agents may be in the form of single dose preparations or in multi-dose flasks, which can be utilized for mass-treatment programs of both animals and humans.
  • the expression vectors of the present invention when used as therapeutic agents, they may be administered as a single dose or as a series of doses, depending on the situation as determined by the person conducting the treatment.
  • the invention also relates to transformation of host cells with nucleic acid molecules of the invention to produce host cells which express HVRl-infectious HCV, the ⁇ HVRl-chimeric HCV or the ⁇ HVR1-E2 protein.
  • the invention further relates to the use of host cells expressing ⁇ HVRl-infectious HCV, the ⁇ HVRl-chimeric HCV or the ⁇ HVR1-E2 protein as immunogens to stimulate a protective immune response to HCV.
  • the present invention also relates to
  • ⁇ HVRl E2 protein produced from the ⁇ HVR1-E2 gene constructs of the invention or obtained from the ⁇ HVRl-infectious HCVs or ⁇ HVRl-chimeric HCVs of the invention.
  • the nucleic acids, polypeptides and viruses of the present invention can also be utilized in the production of antibodies against HCV.
  • antibody is herein used to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Examples of antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, F(ab') 2 and F(v) as well as chimeric antibody molecules .
  • polypeptides, viruses and nucleic acid sequences of the present invention can be used in the generation of antibodies that immunoreact (i.e., specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or an active portion thereof) with antigenic determinants on the surface of hepatitis C virus particles.
  • the present invention therefore also relates to antibodies produced following immunization with the nucleic acid sequences, viruses or polypeptides of the present invention.
  • These antibodies are typically produced by immunizing a mammal with an immunogen or vaccine to induce antibody molecules having immunospecificity for polypeptides or viruses produced in response to infection with the nucleic acid sequences of the present invention.
  • the nucleic acid sequences, viruses, or polypeptides of the present invention may be linked to some type of carrier molecule.
  • the resulting antibody molecules are then collected from said mammal.
  • Antibodies produced according to the present invention have the unique advantage of being generated in response to authentic, functional polypeptides produced according to the actual cloned HCV genome.
  • the antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies are readily produced by methods well known in the art. Portions of immunoglobin molecules, such as Fabs, as well as chimeric antibodies, may also be produced by methods well known to those of ordinary skill in the art of generating such antibodies.
  • the antibodies according to the present invention may also be contained in blood, plasma, serum, hybridoma supernatants, and the like. Alternatively, the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, using DEAE Sephadex.
  • the antibodies produced according to the present invention may be further purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, and the like. Antibodies of the IgG class are preferred for purposes of passive protection.
  • the antibodies of the present invention are useful in the prevention and treatment of diseases caused by hepatitis C virus in animals, especially mammals, and most especially humans .
  • the dosage of administered antibodies will vary depending on such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, and the like.
  • antibodies in the range of from about 1 mg/kg body weight to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered if found desirable.
  • Such antibodies will normally be administered by intravenous or intramuscular route as an inoculum.
  • the antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of any existing infection.
  • nucleic acid molecules, viruses, polypeptides and antibodies of the invention may be supplied in the form of a kit, alone or in the form of a pharmaceutical composition. All scientific publication and/or patents cited herein are specifically incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.
  • Deletion of the nucleotide sequence encoding the HVRl region was performed by fusion PCR using pCV-H77C, the infectious cDNA clone of HCV genotype la (Yanagi et al . , 1997;ATCC accession number PTA-157) .
  • E2-Pstl 5'-ACG CGT CTG CAG CTT AAT GGC CCA GGA CGC GAT GCT TG-3'
  • HVRl-mutS 5' -CTT GTA CCA TCA ATT ACA CCA TAT TC-3'
  • HVRl-mutR 5' -GAT AGT GCC AAT GCC TAT ACG GG-3'
  • FUSION 1 5' -CGT ATA GGC ATT GGC ACT ATC CTT GTA CCA TCA ATT ACA CC-3'
  • FUSION2 5' -GGT GTA ATT GAT GGT ACA AGG ATA GTG CCA ATG CCT ATA CG-3'
  • E2-661-HindIII 5' -ACG CGT AAG CTT CTA TTA CTC GGA CCT GTC CCT GTC TTC CAG-3' ⁇ ⁇ Restriction sites within primers are underlined
  • the sequences encoding the El protein (aa 192-383) and a truncated E2 protein (aa 411-715) lacking the HVRl region (aa 384-410) were PCR amplified from pCV-H77C.
  • cDNA 50 ng of cDNA were added to a master mix containing 4 ⁇ l of 5X Advantage KlenTaq Buffer, 1.25 ⁇ l of dNTP (10 mM) , 1 ⁇ l of 10 ⁇ M sense primer [El-Bglll (SEQ ID NO: 4) for El and E2-411S(+10) (SEQ ID NO: 6) for E2], 1 ⁇ l of lO ⁇ M antisense primer [E1383R (+10) for El and E2-Pstl for E2] and 1 ⁇ l of the Advantage KlenTaq polymerase mixture. Cycling conditions were 99°C for 1 min followed by 25 cycles of 99°C for 35 sec, 67°C for 30 sec, 68°C for 3 min 30 sec.
  • a fusion PCR was performed. Briefly, 2.5 ⁇ l of each PCR product were added to a master mix containing 10 ⁇ l of 10X Pfu buffer, 2 ⁇ l of 10 mM dNTP, 5 ⁇ l of 10 ⁇ M sense primer (El-Bglll) (SEQ ID NO: 4), 5 ⁇ l of 10 ⁇ M antisense primer (E2-Pstl) (SEQ ID NO: 3), 1 ⁇ l of Pfu and 72 ⁇ l of H 2 0. Cycling conditions were 95 °C for 1 min, 67 °C for 1 min, 72 °C for 3 min 30 sec (30 cycles) .
  • the fusion PCR product was digested with Bglll and Pstl and cloned into the expression vector pDisplay (Invitrogen) .
  • a clone with an insert of the correct size ( ⁇ HVR1-7) was selected and sequenced. Sequence analysis of both strands of DNA confirmed that this clone contained the expected sequence of El and a truncated E2 lacking the HVRl, i.e., ⁇ HVR1-7 encodes amino acids 192-383 of the El protein fused to amino acids 411-715 of the E2 protein.
  • Construction of an infectious cDNA clone that was full-length except for the HVRl was performed by digestion of the expression vector ⁇ HVR1-7 with Muni (which cuts at nucleotide positions 1254 and 1983 of pCV-H77C) and cloning of the resulting fragment into the digested HCV cDNA clone H77C. Briefly, a 3 ⁇ g sample of the infectious HCV cDNA clone H77C was digested with Muni for 3 hours and the enzyme was then inactivated at 65 °C for 20 min.
  • the digested cDNA clone lacking a portion of the El and E2 regions (nucleotides 1255 to 1983 of pCV-H77C) , was dephosphorylated with calf intestinal alkaline phosphatase.
  • a sample of ⁇ HVR1-7 was then digested with Muni for 3 hours. After inactivation of the Muni enzyme, the digested fragment was ligated into the digested pCV-H77C using standard procedures (Forns et al . , 1999).
  • a clone containing the correct insert was selected, retransformed and large-scale plasmid DNA was prepared as previously described (Yanagi et al . , 1997).
  • the complete sequence of the HVRl deletion mutant [H77C ( ⁇ HVRl) ] was the expected one, that is, identical to pCV-H77C but lacking the fragment from nucleotide positions 1491 to 1571 which encodes HVRl.
  • E1E2-715 were used for in vi tro transcription-translation.
  • ⁇ HVR1-7 plasmid contained the nucleotide sequence encoding El
  • the control plasmid contained the nucleotide sequence encoding El (aa 192-383) a carboxy-truncated form of E2 (aa 384-715) of pCV-H77C inserted between a leader sequence which targeted the HCV proteins to the secretory pathway and the transmembrane domain of PDGFR which anchored the HCV E2 proteins to the plasma membrane in the expression vector pDisplay. Reactions were performed in 25 ⁇ l of the TNT Coupled Reticulocyte Lysate System (Promega) containing [S 35 ] methionine, with or without the addition of canine microsomal membranes at 30 °C for 90 min. Total translation products were separated in 12% SDS/PAGE and identified by autoradiography .
  • Huh7 cells grown in 4-well tissue culture chambers were transfected with plasmids E1E2-715 and ⁇ HVR1-7 described above. Immuno-fluorescence analysis was performed 48 hours after transfection. Live cells were incubated for 30 minutes with a 1:100 dilution of rabbit hyperimmune serum ( Lmf86) raised against a peptide encoding the carboxy-terminal 21 amino acids of HVRl (aa 390-410 of pCV-H77C) or a rabbit hyperimmune serum ⁇ FOR-1 ) raised against a peptide within E2, but outside the HVRl (aa 517-535 of pCV-H77C) .
  • rabbit hyperimmune serum Lmf86
  • HVRl aa 390-410 of pCV-H77C
  • a rabbit hyperimmune serum ⁇ FOR-1 rabbit hyperimmune serum
  • Serum samples were collected weekly from the chimpanzee and monitored for serum levels of alanine aminotransferase (ALT) , anti-HCV antibodies [second generation ELISA] and HCV-RNA [HCV Monitor test (Roche) and in-house RT- nested PCR (Bukh et al . , 1998)]. Sequence analysis of the recovered virus was performed at different time points during follow-up. In short, genomic regions were amplified in RT- nested PCR (Bukh et al . , 1998) with primers specific for the H77 strain of HCV.
  • ALT alanine aminotransferase
  • HCV-RNA HCV Monitor test
  • both products were fused by an amplification reaction that contained a mixture of both external primers (El-Bglll (SEQ ID NO: 4) and E2-Pstl (SEQ ID NO: 3) and the two fusion primers SEQ ID NO: 9 and SEQ ID NO: 10) (Table 1), by using Advantage KlenTaq Polymerase mix.
  • the fusion product was digested with Bglll and Pstl and cloned into the digested pDisplay vector to produce ⁇ HVRl-mut5.
  • Clones E1E2-715, ⁇ HVR1-7 and ⁇ HVRl-mut5 were used to transfect Huh7 cells and to determine their pattern of reactivity against a panel of 5 rabbit hyperimmune sera and 12 human monoclonal antibodies (Cardoso et al . , 1998, Inchauspe et al. 1998) by immunofluorescence in live and fixed/permeabilized cells .
  • E2- 661 encoding the ER signal sequence of El (aa 364-383 of pCV- H77C) and a carboxy-terminal truncated E2 protein (aa 384-661 of pCV-H77C) ;
  • E2-661 ⁇ HVRl encoding the same protein with a deletion of the HVRl region (aa 384-410 of pCV-H77C) ;
  • E2- 661 ⁇ HVRl-mut encoding the same protein with a deletion of the HVRl and the replacement of leucine for histidine at amino acid position 615.
  • amplification was performed from templates E1E2-715, ⁇ HVR1-7 and ⁇ HVRl-mut5, respectively, with primers shown in Table 1.
  • PCR products were cloned into the expression vector pcDNA3.1(-) (Invitrogen) .
  • Clones E2-661, E2- 661 ⁇ HVRl, and E2-661 ⁇ HVRl-mut were used to express soluble E2 protein in vi tro . Briefly, one microgram of each of the plasmids was used for in vi tro transcription-translation. Reactions were performed in 25 ⁇ l of the TNT Coupled Reticulocyte Lysate System (Promega) containing [ ⁇ 35 ] methionine, with the addition of canine microsomal membranes at 30 °C for 90 min.
  • expression vectors E1E2-715 (encoding the complete El protein and a cell-surface E2 protein lacking the carboxy-terminal 31 amino acids of E2) and ⁇ HVR1-7 (encoding the complete El protein and a cell-surface E2 lacking the HVRl and carboxy-terminus 31 amino acids) were assayed for in vi tro protein synthesis.
  • vi tro transcription- translation was performed with the TNT Coupled Reticulolysate System using [S 35 ] methionine .
  • translation products of the expected size were obtained for E1E2-715 and ⁇ HVR1-7 ( Figure 2) .
  • HVRl region could be targeted to the cell surface
  • Huh7 cells were transfected with E1E2-715 and ⁇ HVR1-7. After transfection, fixed/permeabilized cells and live cells were analyzed for expression of E2 by immunofluorescence . Up to 20% of cells transfected with E1E2-715 were positive when stained with a rabbit hyperimmune serum lmf 86 or FOR-1 . Both fixed /permeabilized and live cells were stained. A similar proportion of cells transfected with ⁇ HVR1-7 was positive when stained with rabbit hyperimmune serum FOR-1 (raised against aa 517-535) . Again, both fixed/permeabilized and live cells were stained.
  • RNA transcripts were injected into the liver of chimpanzee 1590.
  • HCV-RNA was detected by RT-nested PCR at week one post- inoculation (p.i.) and the animal remained HCV-RNA positive throughout the entire follow-up period of 22 weeks ( Figure 3) .
  • the quantitative HCV Monitor test was negative until week 10 p.i.
  • the genome titer during the first 7 weeks was D 10 genome equivalent (GE)/ml. However, beginning at week 8, the titer progressively increased over time (Table 2 and Figure 3) .
  • Serum liver enzyme values remained normal during the follow-up.
  • the second generation anti-HCV test remained negative at least through week 22 p.i.
  • the follow-up period for chimpanzee 1590 has been extended to 72 weeks. Chimpanzee 1590 remained HCV-RNA positive throughout the 72 week follow-up period. Thus, the chimpanzee developed a chronic infection. In addition, the genome titer reached 10 3 - 10 4 GE/ml at weeks 11-14 (see Table 2) and remained at 10 3 - 10 4 GE/ml throughout the remainder of the 72 week period.
  • the relative genome titer of HCV lacking HVRl was also assessed in serum, PMBC, and liver obtained from the transfected chimpanzee. HCV RNA was not detected in PBMCs, but the HCV titers determined from liver tissue at weeks 51 and 52 p.i. were higher than the titers determined from the serum.
  • the 72 week follow-up period also revealed that the transfected chimpanzee became positive for antibodies to second generation ELISA HCV at week 37 and third generation RIBA (Chiron) confirmed the presence of antibodies to C22 (core) , C33- c (NS3), and clOO (NS3-NS-4).
  • CD4+ and CD8+ T cell responses were also measured.
  • An HCV-specific proliferative CD4+ T cell response to C22 (core), C33-c (NS3) , clOO (NS3-NS4 ) and/or NS5 antigens of HCV was detected in the peripheral blood mononuclear cells (PBMC) beginning at week one post-infection and throughout the follow-up period. During most weeks, however, only a mono- specific response to the core was detected.
  • PBMC peripheral blood mononuclear cells
  • CTL Peripheral CD8+ T cell responses were tested at weeks 5, 9, 14, and 18 by an in vi tro peptide stimulation assay. CTL were detected with two NS4 peptides at weeks 9 and 14 and with one of these at week 18. A transient and monospecific (NS5) proliferative T cell response was detected in the liver at weeks 14 and 15. Although serum liver enzyme values remained normal during the entire follow-up, necroinflammatory changes indicative of hepatitis were detected in liver biopsies during weeks 34-40.
  • the single new mutation identified at week 24 was located within the serine protease domain of NS ' 3 (corresponding to aa position 1456 of H77C; threonine to methionine) .
  • a na ⁇ ve chimpanzee (#96A008) was inoculated intravenously with 90 ml of plasma taken at week 4 from chimpanzee 1590, before appearance of consenus mutations in that chimpanzee (Table 3) .
  • the virological and immunological correlates of HCV infection in chimpanzee 96A009 are shown in
  • Serum HCV-RNA was first detected at week 2 p.i., with a genome titer of 10 1 GE/ml and the titer increased to peak levels of 10-10 5 GE/ml during weeks 3-9. The infection was resolved at week 18 p.i. Antibodies to HCV were not detected.
  • a peripheral mono-specific proliferative CD4+ T cell response against core (C22) was detected during weeks 4-13 p.i.; a multi-specific response against NS3 (C33-c) , NS3-NS4 (clOO) and NS5 was detected at week 18.
  • the chimpanzee mounted an early and multi- specific peripheral CD8+ T cell response to a total of seven different epitopes (representing core, NS3, NS4 and NS5) at the weeks tested (weeks 4, 7, 11, 15 and 19) .
  • a multi- specific (C33-c, clOO and NS5) sustained proliferative CD4+ T cell response was detected in the liver from week 8 p.i.
  • Liver enzyme values were marginally elevated during weeks 10-18 p.i. and necroinflammatory changes were detected in liver biopsies during weeks 11-19 p.i.
  • Huh7 cells were transfected with E1E2-715

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Abstract

La présente invention concerne des molécules d'acide nucléique codant le virus de l'hépatite C, le virus de l'hépatite C chimérique ou la protéine enveloppe 2 du virus de l'hépatite C qui ne possède pas tout ou partie de la région 1 hypervariable de la protéine enveloppe 2. L'invention concerne également l'utilisation de ces molécules d'acide nucléique et leurs polypeptides codés comme vaccins possibles.
PCT/US2000/025987 1999-09-23 2000-09-22 Proteine enveloppe 2 (e2) du virus de l'hepatite c qui ne possede pas tout ou partie de la region 1 hypervariable (hvr1), acides nucleiques correspondants, virus chimeriques et utilisation de ces derniers WO2001021807A1 (fr)

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EP1326625A4 (fr) * 2000-09-13 2005-05-04 Hawaii Biotech Inc Composition immunogene de l'hepatite c et procedes d'utilisation de cette derniere
WO2004024904A3 (fr) * 2002-09-13 2004-06-03 Inst Nat Sante Rech Med Pseudo-particules d'hepacivirus infectieuses renfermant des proteines fonctionnelles d'enveloppe e1, e2
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US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US7361503B2 (en) 2003-11-12 2008-04-22 Albert Einstein College Of Medicine Of Yeshiva University Sequences encoding hepatitis C virus glycoproteins
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JP2010501594A (ja) * 2006-08-25 2010-01-21 ザ・マクファーレーン・バーネット・インスティテュート・フォー・メディカル・リサーチ・アンド・パブリック・ヘルス・リミテッド 組換えｈｃｖｅ２糖タンパク質
AU2007288129B2 (en) * 2006-08-25 2013-03-07 The Macfarlane Burnet Institute For Medical Research And Public Health Limited Recombinant HCV E2 glycoprotein
US9598467B2 (en) 2006-08-25 2017-03-21 The Macfarlane Burnet Institute For Medical Research And Public Health Limited Recombinant HCV E2 glycoprotein
US8535686B2 (en) 2006-08-25 2013-09-17 The Macfarlane Burnet Institute For Medical Research And Public Health Limited Recombinant HCV E2 glycoprotein
JP2014240427A (ja) * 2006-08-25 2014-12-25 ザ・マクファーレーン・バーネット・インスティテュート・フォー・メディカル・リサーチ・アンド・パブリック・ヘルス・リミテッド 組換えｈｃｖｅ２糖タンパク質
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region
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US8618275B2 (en) 2007-05-18 2013-12-31 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 5A
US8569472B2 (en) 2007-12-20 2013-10-29 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 6A
WO2009146902A1 (fr) * 2008-06-03 2009-12-10 Okairòs Ag Vaccin pour la prévention et la thérapie d'infections par le vhc
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US20140128447A1 (en) * 2010-11-30 2014-05-08 The Trustees Of Columbia University In The City Of New York Novel non-primate hepacivirus
WO2018176075A1 (fr) * 2017-03-27 2018-10-04 The University Of Queensland Flavivirus chimériques spécifiques des insectes
US11572390B2 (en) 2017-03-27 2023-02-07 The University Of Queensland Chimeric insect-specific flaviviruses
WO2021226664A1 (fr) * 2020-05-11 2021-11-18 Macfarlane Burnet Institute For Medical Research And Public Health Limited Vaccin contre l'hépatite c à base d'acide nucléique comprenant un polypeptide e2 à domaine variable délété

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