WO2000075352A2 - Genomes recombinants vhc/bvdv et utilisation de ceux-ci - Google Patents

Genomes recombinants vhc/bvdv et utilisation de ceux-ci Download PDF

Info

Publication number
WO2000075352A2
WO2000075352A2 PCT/US2000/015527 US0015527W WO0075352A2 WO 2000075352 A2 WO2000075352 A2 WO 2000075352A2 US 0015527 W US0015527 W US 0015527W WO 0075352 A2 WO0075352 A2 WO 0075352A2
Authority
WO
WIPO (PCT)
Prior art keywords
hcv
bvdv
virus
cells
chimeric
Prior art date
Application number
PCT/US2000/015527
Other languages
English (en)
Other versions
WO2000075352A3 (fr
Inventor
Jae-Hwan Nam
Jens Bukh
Suzanne U. Emerson
Robert H. Purcell
Original Assignee
The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US10/009,011 priority Critical patent/US7009044B1/en
Application filed by The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services
Priority to AU53245/00A priority patent/AU5324500A/en
Priority to EP00938165A priority patent/EP1187927A2/fr
Publication of WO2000075352A2 publication Critical patent/WO2000075352A2/fr
Publication of WO2000075352A3 publication Critical patent/WO2000075352A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—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
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24341—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Definitions

  • the present invention relates to molecular approaches to the production of nucleic acid sequences which comprise the genomes of chimeric hepatitis C virus-bovine viral diarrhea viruses (HCV-BVDV) .
  • the invention also relates to the use of these chimeric nucleic acid sequences to produce chimeric virions in cells and the use of these chimeric virions in HCV antibody neutralization assays, and for the development of vaccines and therapeutics for HCV.
  • Hepatitis C virus has a positive-sense single-strand RNA genome and is a member of the genus Hepacivirus within the Flaviviridae family of viruses (Rice, 1996) .
  • the genome of HCV functions as mRNA from which all viral proteins necessary for propagation are translated.
  • the viral genome of HCV is approximately 9600 nucleotides (nts) in length and consists of a highly conserved 5' untranslated region (UTR) , a single long open reading frame (ORF) of approximately 9,000 nts and a complex 3' UTR.
  • the 5' UTR contains an internal ribosomal entry site (Tsukiyama-Kohara et al., 1992; Honda et al., 1996).
  • the 3' UTR consists of a short variable region, a polypyrimidine tract of variable length and, at the 3' end, a highly conserved region of approximately 100 nucleotides (Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996; Yamada et al., 1996). The last 46 nucleotides of this conserved region were predicted to form a stable stem-loop structure thought to be critical for viral replication (Blight and Rice, 1997; Ito and Lai, 1997; Tsuchihara et al., 1997).
  • the ORF encodes a large polypeptide precursor that is cleaved into at least 10 proteins by host and viral proteinases (Rice, 1996) .
  • the predicted envelope proteins contain several conserved N-linked glycosylation sites and cysteine residues (Okamoto et al., 1992a).
  • the NS3 gene encodes a serine protease and an RNA helicase and the NS5B gene encodes an RNA- dependent RNA polymerase.
  • a remarkable characteristic of HCV is its genetic heterogeneity, which is manifested throughout the genome (Bukh et al . , 1995).
  • the most heterogeneous regions of the genome are found in the envelope genes, in particular the hypervariable region 1 (HVR1) at the N-terminus of E2 (Hijikata et al . , 1991; plante et al . , 1991).
  • HCV circulates as a quasispecies of closely related genomes in an infected individual. Globally, six major HCV genotypes (genotypes 1-6) and multiple subtypes (a, b, c, etc.) have been identified (Bukh et al., 1993; Simmonds et al., 1993).
  • 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% .
  • genotype 1 accounts for the majority of HCV infections but genotypes 2 and 3 each account for 5-15%.
  • the present invention relates to chimeric nucleic acid sequences which comprise the genomes of chimeric hepatitis C virus-bovine viral diarrhea viruses (HCV-BVDV) . More specifically, the chimeric viruses are produced by replacing the structural region or a structural gene of a bovine viral diarrhea virus (BVDV) with the corresponding region or gene of an infectious hepatitis C virus (HCV) .
  • HCV-BVDV chimeric hepatitis C virus-bovine viral diarrhea viruses
  • the present invention also relates to the in vitro and in vivo production of chimeric HCV/BVDV viruses from the chimenc nucleic acid sequences of the invention.
  • the present invention also relates to the use of the chimenc viruses of the invention to identify cell lines capable of supporting the replication of the chimeric viruses.
  • the invention further relates to the use of the chimeric viruses of the invention to screen for neutralizing antibodies to HCV of different genotypes.
  • the invention also relates to the use of the chimenc nucleic acid sequences of the invention in the production of HCV-BVDV virions, and the use of these
  • HCV-BVDV virions for the development of inactivated or attenuated vaccines to prevent HCV-BVDV in a mammal.
  • the invention also relates to the use of the chimeric nucleic acid sequences to study the molecular properties of HCV indirectly in vitro .
  • the present invention also relates to the polypeptides encoded by the chimeric nucleic acid sequences of the invention or fragments thereof.
  • the invention also provides that the chimeric nucleic acid sequences and the chimeric viruses of the invention be supplied in the form of a kit, alone or in the form of a pharmaceutical composition.
  • Fig. 1 Genomic organization of BVDV, HCV and HCV/BVDV chimera.
  • the BVDV and HCV are NADL (14, 21) and H77 strains (12), respectively.
  • the complete BVDV- NADL genome consists of, in 5' to 3' order, 5'NCR (nucleotides 1-385), N pro (nucleotides 386-889), Core (nucleotides 890-1195), E rns (nucleotides 1196-1876), El (nucleotides 1877-2461), E2 (nucleotides 2462-3583), P7 and nonstructural genes (nucleotides 3584-12349) and 3'NCR (nucleotides 12352-12578).
  • Fig. 2 Strategy for the construction of chimeric cDNA, pHCV/BVDV-3, which has core, El and E2 of HCV in the backbone of BVDV.
  • the fusion PCR products were cloned into pBV18-F2 after digestion with SnaB I and Bsm I.
  • the fragments containing fusion PCR products were cloned into pSDMlu-3' after digestion with Cla I and Dra III.
  • Figures 3A-3H show the nucleotide and deduced amino acid sequences of the infectious HCV clone of genotype la.
  • Figures 4A-4H show the nucleotide and deduced amino acid sequences of the infectious clone of genotype lb .
  • Figure 5 shows a Western blot of lysate and supernatant from EBTr (A) cells infected with chimeric HCV-BVDV clone pHCV-BVDV-3 using antibody to HCV El, E2 or core proteins.
  • the present invention relates to nucleic acid sequences which comprise the genomes of chimeric HCV- BVDV.
  • the chimeric viruses are produced by replacing the structural region or a structural gene (or fragment thereof) of a bovine viral diarrhea virus (BVDV) with the corresponding region or gene (or fragment thereof) of an hepatitis C virus (HCV) .
  • the gene borders of the HCV genome, including nucleotide and amino acid locations, have been determined, for example, as depicted in Houghton, M. (1996), and the putative gene borders of the BVDV genome are shown in Figure 1.
  • the chimeric nucleic acid sequence comprises the structural genes from an infectious HCV clone and the nonstructural genes and untranslated regions from an BVDV clone.
  • additional HCV/BVDV chimeras can be constructed to study HCV infection of cell lines.
  • additional HCV/BVDV chimeras may be made in which only El and E2 genes of the BVDV infectious clone are replaced with the corresponding genes from an HCV clone.
  • Such chimeras can be used to determine whether the core protein of BVDV is critical for encapsidation of the viral RNA.
  • HCV/BVDV chimeras in which either the El or E2 gene of BVDV is replaced by the corresponding gene of HCV may be constructed. Such chimeras can be used to determine the relative importance of El or E2 for infection of cell lines.
  • HCV/BVDV chimeras in which one of the nonstructural genes of BVDV, such as NS3 RNA helicase, NS3 protease, or the NS5B RNA- dependent RNA polymerase are replaced by the corresponding non-structural genes of HCV may be constructed.
  • Such chimeras would, for example, be useful in identifying inhibitors of viral enzyme activity which would be useful as antiviral agents.
  • hypervariable region 1 (HVR1) from multiple HCV genotypes may be combined into one HCV/BVDV chimera.
  • the only limit for constructing this type of chimera is that the viral genome must be able to be packaged.
  • a chimera can be constructed which contain an HVR1 sequence from one HCV genotype. Such chimeras can be used as an inactivated multivalent vaccine or to screen for neutralizing antibodies to multiple HCV genotypes.
  • the HCV/BVDV chimeras of the invention may be constructed using any HCV and BVDV clones.
  • the HCV clones are infectious HCV clones of genotype la (ATCC accession number PTA-
  • the retention of the E rns gene of BVDV in any chimeric is entirely optional.
  • the HCV/BVDV chimeras could be constructed in which, for example, the El or E2 gene of BVDV is replaced by the corresponding El or E2 gene of HCV, it is to be understood that the resultant chimeras may or may not retain the BVDV E rns gene.
  • the present invention further relates to the production of chimeric HCV/BVDV viruses from the HCV/BVDV chimeras of the invention.
  • the chimeric sequences of the invention can be inserted into an expression vector that functions in eukaryotic cells.
  • eukaryotic expression vectors 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 chimeric viruses of the invention.
  • the 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.
  • the method comprises the growing of animal cells in vitro and transfecting the cells with the chimeric nucleic acid of the invention, then determining if the cells show indicia of HCV infection.
  • Such indicia include the detection of viral antigens in the cell, for example, by immunofluorescence procedures well known the art; the detection of viral polypeptides by Western blotting using antibodies specific therefor; and the detection of newly transcribed viral RNA withm the cells via methods such as RT-PCR.
  • the presence of live, infectious virus particles following such tests may also be shown by injection of cell culture medium or cell lysates mto healthy, susceptible animals, with subsequent exhibition of the signs and symptoms of HCV infection.
  • the presence of live, infectious virus particles following such tests may also be shown by serial passaging the chimenc virus cells.
  • Suitable cells or cell lines for cultu ⁇ ng the chimeric viruses of the invention include, but are not limited to, EBTr (A) and Huh7.
  • transfection of cells with the chimeric sequences is carried out in the presence of helper BVDV which is preferably of a noncytopathogenic strain.
  • helper BVDV which is preferably of a noncytopathogenic strain.
  • the cell lines to be infected may already contain a helper BVDV.
  • helper BVDV include, but are not limited to, EBTr (A) .
  • the cell lines to be transfected may be infected with a helper BVDV prior to, or concurrent with, transfection with the chimeric sequences of the invention.
  • the present invention also relates to polypeptides encoded by the chimeric nucleic acid sequences of the invention or fragments thereof.
  • said polypeptide or polypeptides may be fully or partially purified from viruses produced by cells transfected with the chimeric nucleic acid sequences of the invention.
  • the polypeptide or polypeptides may be produced recombinantly from a fragment of the chimeric nucleic acid sequences of the invention.
  • the polypeptides may be chemically synthesized.
  • the present invention also relates to the use of the chimeric sequences of the invention to identify cell lines capable of supporting the replication of the chimeric viruses of the invention.
  • the invention relates to the use of HCV/BVDV chimeras to screen for neutralizing antibodies to HCV of different genotypes.
  • chimeric viruses produced in cell lines infected with the chimeric clones of the invention can be used in neutralization assays to test the neutralizing ability of anti-HCV antibodies.
  • the invention relates to the use of the infectious chimeric clones of the invention to develop inactivated or attenuated vaccines to prevent Hepatitis C in a mammal.
  • chimeric virions from cell lines infected with a chimeric virus of the invention, or transfected with a chimeric 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 HCV-BVDV 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 chimeric viruses of the invention may be passaged in cell culture to produce attenuated viruses which can be tested as candidate live vaccines.
  • assaying the ability of the chimeric 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)
  • mutations may be introduced into the HCV portion of the HCV/BVDV chimeras of the invention in order to enable the production of virions in cell cultures which could then be tested in vivo for improved vaccine properties.
  • multiple chimeras containing HCV structural genes (or fragments thereof, such as the HVR1) from multiple genotypes can be administered to generate multivalent vaccines.
  • the chimeric virions When used as a vaccine, the 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.
  • routes including, but not limited to, intradermally, intramuscularly, subcutaneously, or in any combination thereof.
  • formulations or compositions comprising the chimeric virions of the invention may be used either therapeutically or prophylactically to treat or prevent the signs and symptoms of HCV.
  • the present invention therefore also relates to antibodies reactive with the HCV structural polypeptide (s) contained in the HCV-BVDV virions of the invention where such antibodies are produced following immunization with the HCV-BVDV virions.
  • the antibody molecules of the present invention may be polyclonal or monoclonal and may be useful in the prevention or treatment of diseases caused by HCV in mammals.
  • the invention also provides that the chimeric nucleic acid sequences and the chimeric viruses of the invention be supplied in the form of a kit, alone or in the form of a pharmaceutical composition.
  • Antibodies H79 plasma from patient H obtained in the chronic phase two years after the onset of HCV infection (11); CH1530: serum pool from chimpanzee 1530, obtained in the chronic phase one to two years after the onset of HCV infection. Chimpanzee 1530 became infected with HCV following intrahepatic transfection with pCV-H77C (Yanagi 1997); LMF86 and LMF87: anti-HVRl (Farci 1996), rabbit anti-peptide sera; Mab NS : anti-BVDV NS3 murine monoclonal antibody kindly provided by Dr. E. Dubovi (Cornell University, Ithaca, NY) .
  • HCV/BVDV chimeric clone The C, El and E2 genes originating from an infectious clone of the H77 strain of HCV (pCV-H77C, ref. Yanagi 1997), and the backbone originating from two subgenomic plasmids (pBV18-F2 and pSDMlu-3' ) , used by Vassilev et al. (Vassilev 1997) to generate the infectious clone of the NADL strain of BVDV (pVVNADL) , were used to construct the chimeric cDNA clone pHCV- BVDV-3 (ATCC deposit Number PTA-158) .
  • the chimeric clone includes sequences corresponding to nucleotides 345-2579 (amino acids 2-746) of the pCV-H77C clone of HCV and nucleotides 1-927 (amino acids 1-168) and nucleotides 3622-14578 (amino acids 1067-3988) of the pVVNADL clone of BVDV ( Figure 1) .
  • standard PCR and fusion PCR were performed with pfu polymerase (Strategene) and the oligonucleotides listed in Table 2.
  • PCR fragment was amplified from pCV-H77C with primers Npro-C/H77/S and E2-P7/H77/R, two other fragments were amplified from pBV18-F2 with primers MluI/NADL/S and Npro-C/NADL/R and with primers E2- P7/NADL/S and Bsml/NADL/R, respectively.
  • QIAquick PCR purification kit Qiagen
  • the three PCR products were mixed and a fusion PCR was performed with primers MluI/NADL/S and Bsml/NADL/R.
  • the fusion PCR product was cloned into pBV18-F2 by using SnaBI and Bsml sites (Fig. 2) and multiple clones were screened by sequence analysis. Finally, a clone with the correct sequence was digested with Clal and Drain and the insert was cloned into pSDMlu-3' to generate the full-length chimeric clone, pHCV/BVDV-3 (Fig. 2) .
  • This clone was transformed into JM109 competent cells (Promega) and selected on LB agar plates containing 100 ⁇ g/ml ampicillin (SIGMA). Several colonies were cultured in LB liquid containing ampicillin at 30°C for 18-20 hrs.
  • pHCV/BVDV-3 was linearized with SacII (NEB) and treated with T4 DNA polymerase (GIBCO/BRL) to remove the resulting 3' overhang.
  • Two micrograms of DNA were transcribed at 37°C for 2 hrs in a 100 ⁇ l reaction volume containing 50 U of T7 RNA polymerase (Promega), 10 mM DTT (Promega), 120 U of Rnasin (Promega) and 1 mM rNTPs (GIBCO/BRL) .
  • RNA of each transcription mixture was extracted with the TRIzol system (GIBCO/BRL) and resuspended in 50 ⁇ l of DEPC-treated water, and stored at -80°C.
  • RNA was added to 1 ml of Optimem with 15 ⁇ l of DMRIE-C (GIBCO/BRL) and incubated with cells for 5 hrs. The Optimem was removed and complete medium was added. Cells were cultured in the presence of the appropriate medium (Table 1) and transfected at 80% confluency either in one well of a 12 well plate (Costar) or in a 60 mm dish (Costar) . About 24 hrs prior to immunofluorescent staining, transfected cells were split into 4- or 8- well chamber slides (LAB- TEK) .
  • the supernatant was collected and stored at - 80°C. Cells were scraped with 1 ml of supernatant medium and centrifuged. The pellet was taken through three freezing and thawing cycles to lyse the cells. For homologous passages, lysed cells or supernatant (100 - 500 ⁇ l) were transferred onto new cells of the same type. For heterologous passages, lysed cells or supernatant from EBTr (A) cells were transferred onto different cell lines. Inoculated cells were incubated at 37°C for two hrs followed by the addition of complete medium. Inoculated cells were incubated at 37°C for 4- 12 days.
  • PBS phosphate buffered saline
  • primary antibodies diluted in 10% bovine serum albumin (BSA) in PBS.
  • primary antibodies we used an anti-HCV human plasma sample (H79, 1:100 dilution), an anti-HCV chimpanzee serum (CH1530, 1:100 dilution) and an anti-BVDV NS3 monoclonal antibody (Mab-NS, 1:10 dilution) .
  • NS3 monoclonal antibody and incubated on fixed cells as above, followed by washing and incubation with a mixture of both secondary antibodies. After washing, slides were mounted and examined by fluorescence microscopy (Zeiss) .
  • a T150 flask of EBTr (A) cells was inoculated with virus stock. At days 9 and 13, respectively, supernatant was harvested. A total of 70 ml of supernatant was layered over 20% sucrose in TN buffer
  • EBTr (A) cells 60 mm dish infected with the HCV/BVDV chimeric virus were lysed by adding 300 ⁇ l of M-PER mammalian protein extraction reagent (PIERCE). The cell lysate was cleared of cell debris by low speed centrifugation at 13000 rpm for 5 min. Also, thirty ml of the supernatant from EBTr (A) cells infected with chimeric virus stock was harvested, ultra centrifuged and tested for HCV proteins by immunoblot.
  • PIERCE M-PER mammalian protein extraction reagent
  • the membrane was incubated at 4°C for 16 hrs with CH1530 anti-HCV (1:750 dilution) in a blocking buffer containing 1% BSA in TBST buffer (20mM Tris-HCl, pH 7.5; 150mM NaCl, 0.05% Tween 20). Following washing with
  • the membrane was incubated at room temperature for 1 hr with a 1:5000 dilution of goat anti-human immunoglobulm conjugated to horseradish peroxidase (PIERCE). After washing, the membrane was o incubated with ECL Western blotting detection reagent (Amersham) and exposed to film.
  • PIERCE horseradish peroxidase
  • RNA pellet was resuspended in 10 mM dithiothreitol (DTT) containing 5% (vol/vol) of RNAsin (20-40 U/ ⁇ l)
  • RT was performed with avian myeloblastosis virus reverse transcriptase (Promega) and the external anti-sense primer (see below) and PCR was performed with AmpliTaq Gold DNA polymerase (Perkin Elmer) as described (Bukh 1998a) . Specificity was
  • the genome equivalent (GE) titer of HCV, BVDV and HCV/BVDV in positive samples was determined by RT-nested PCR on 10-fold serial dilutions of the extracted RNA (Bukh 1998a) .
  • One GE was defined
  • -_ as the number of genomes present in the highest dilution positive in RT-nested PCR.
  • the sensitivity of the RT- PCR assays for HCV/BVDV was established by comparison with the HCV titer determined by using HCV primers with established optimal sensitivity.
  • the consensus sequence of the chimeric HCV/BVDV genome was determined by direct sequencing of overlapping PCR products obtained by long RT-nested PCR on supernatant from infected EBTr (A) cells .
  • Chimeric virus stocks Medium harvested from EBTr (A) cells infected with serially passaged chimeric virus was frozen at - 80°C and thawed only once.
  • the medium was aspirated and 200 ⁇ l of chimeric virus diluted in 10% Boyt DMEM was added and the cells were gently rocked for 2 hours at room temperature.
  • Cells were then overlaid with 2 ml/well of 0.5% low melting point agarose in minimal essential medium containing 2% fetal calf serum, 20 mM glutamine and 250 ⁇ g of gentamicin sulfate/ml. After the agar solidified, the slides were incubated at 37° C and 5% C0 2 .
  • Focus neutralization assay The assay was performed exactly as for the focus assay except the 200 ⁇ l inoculum consisted of 100 ⁇ l of chimeric virus diluted in 10% DMEM, 20 ⁇ l undiluted test or control serum, and 80 ⁇ l 10% DMEM. Each 200 ⁇ l sample was incubated at 4° C in ice overnight prior to inoculation of cells. Sera included fetal calf serum (Boyt) and rabbit pre-immune serum as negative controls, hyperimmune rabbit antisera raised to peptides spanning the HVR1 region of the H27 strain of HCV (Farci, 1996), and goat anti-BVDV (VMRD Pullman, WA) prepared without azide. All sera had been heat- inactivated at 56° C for 30 minutes.
  • RNA genomes transcribed from the chimeric virus cDNA pHCV-BVDV-3 were transfected into four bovine cell lines, including two independently derived lines of embryonic bovine trachea cells (EBTr) .
  • a Western blot of material pelleted from the medium by ultracentrifugation revealed anti-HCV reactive bands consistent in size with core, El and E2 proteins of HCV (Figure 5).
  • the chimeric genomes concentrated by high-speed centrifugation, banded in a sucrose gradient at a density of 1.119 to 1.128 g/ml, suggesting that they were in enveloped virus particles.
  • the sucrose banding pattern coupled with the Western blot data, suggest that the chimeric genome was enveloped in a particle containing significant amounts of HCV protems .
  • EBTr (A) cells Although the proportion of cells producing HCV protems increased in EBTr (A) cells, it remained low in the MDBK, BT, and EBTr(B) cell lines, suggesting that the virus was not spreading in these cells. In order to determine if these cells were making infectious virus, a homologous transmission was attempted by removing supernatant from each transfected culture and adding it to a new culture of the same cell line. The only successful transmission was from the transfected EBTr (A) cells to naive EBTr (A) cells (Table 3). Therefore, although the chimeric virus genome could replicate m all four cell lines and produced HCV protems, only in the EBTr (A) cells was vi ⁇ on morphogenesis coupled with availability of a receptor conducive to infection.
  • the EBTr (A) cells were obtained from the ATCC (ATCC ascession number CLL44) and were not listed as being contaminated with BVDV (9) .
  • one of the antibodies used to check replication of the chimera was raised against the NS3 protein of the NADL strain of BVDV.
  • this antibody stained, on average, 30-40 percent of uninfected EBTr (A) cells but did not stain EBTr (B) cells, MDBK or BT cells. Therefore, it appeared that EBTr (A) cells either were persistently infected with a noncytopathogenic strain of BVDV, or were harboring a BVDV replicon, or were producing a protein that cross-reacted with the anti-NS3 antibody.
  • EBTr (A) cell line was contaminated with a transmissible agent, most likely a noncytopathogenic strain of BVDV.
  • RT-PCR primers designed to amplify known BVDV strains were able to amplify a cDNA fragment from uninoculated EBTr (A) cultures (titer: 10 6 GE/ml). The sequence of the cDNA was determined and found to match that of the CP-7 strain of BVDV (18) .
  • Chimeric virus produced in EBTr (A) cells was examined for its susceptibility to neutralization by anti-serum to BVDV as compared to neutralization by anti-sera raised against the hypervariable region 1 (HVR1) of the same HCV strain as was in the chimera. Dilutions of chimeric virus were incubated overnight with anti-BVDV, anti-HCV or control sera and the number of infectious particles remaining was determined by the focus assay (Table 4) . The number of foci in the rabbit and bovine serum controls decreased in parallel with the dilution factor, y indicating that the assay was linear and reliable. The anti-HCV sera did not neutralize the chimera.
  • anti-BVDV eliminated all foci at each dilution, suggesting that each and every infectious particle contained BVDV glycoproteins and that they were probably serving as the viral receptor for binding to the bovine cells. Therefore, the chimeric virus was actually also a pseudotype since the virion contained glycoproteins contributed by a helper virus.
  • the neutralizing titer may not have been high enough. An earlier bleeding from rabbit LMF87 was able to neutralize 64 chimpanzee infectious doses of the H77 strain of HCV in both of two attempts when tested in chimpanzees.
  • the anti-HVRl serum we used was from a later bleeding and had not been similarly tested for neutralizing antibodies.
  • the HCV glycoproteins might not bmd to bovine cells and entry into these cells might be totally independent of HCV glycoprotein.
  • the HCV E2 glycoprotein might not have folded properly to function or to be recognized by the antibody.
  • the anti-HVRl serum had titers of 1:1600 and 1:3200 for rabbits LMF86 and LMF87 respectively but the antibody detected by this assay is not necessarily neutralizing antibody.
  • the functionality of the HCV glycoproteins would best be proved by infecting cells which are not susceptible to infection by BVDV due to an absence of the BVDV receptor.
  • Huh 7 cells were chosen as an experimental system to test for functional HCV glycoproteins because they are a human cell line which grows well and is of hepatocyte origin.
  • the virus did not spread, suggesting that in Huh 7 cells , as in the MDBK and BT cells, virions either were not assembled or were not released from cells. Most likely, the CP-7 virus could not provide the required helper function because it could net enter the cells. This suggested that at least one of the HCV glycoproteins was functioning as a receptor or receptor- component for entry into Huh 7 cells. However, entry into Huh 7 cells was not blocked by preincubation with rabbit LMF87 anti-HVRl serum but was totally blocked by anti-BVDV serum (Table 5) .
  • HCV glycoproteins were functioning in association with one or both of the BVDV glycoproteins, which would have been incorporated during growth in the persistently infected EBTr (A) cells.
  • the failure to neutralize with the anti-HVRl serum might be due to low neutralizing titers, to mediation of virus entry into these cells by El rather than E2 or to interference with antibody binding to the HVR1 because of the BVDV glycoproteins.
  • H72 strain of HCV (Chl530 and 1494) or vaccinated with a
  • DNA vaccine expressing the E2 glycoprotein of HCV were also tested. The results suggested that two of the four samples tested contained neutralizing antibodies to HCV.
  • the sample incubated with the human H79 plasma did not infect any cells indicating complete neutralization while the sample incubated with plasma from the DNA vaccinated chimp infected only 1/3 to 1/4 as many cells as the control suggesting it also had neutralizing antibody but at a lower titer. Since the DNA vaccine expressed only the E2 glycoprotein, this protein must be involved in binding to Huh 7 cells.
  • the plasma from chimp 1530 contained antibodies to the HCV envelope proteins as measured by ELISA or immunofluoresence microscopy but apparently, these were not neutralizing antibodies. Chimpanzee 1494 did not have demonstrable antiodies against the HCV glycoproteins so its failure to neutralize was not unexpected. Therefore, the chimera should be very useful for screening samples for neutralizing antibodies and discriminating between those that neutralize as compared to those that just bind.
  • Huh 7 cells were used for infection but the virus had been grown in EBTr (A) cells; viruses were undiluted.
  • a chimeric genome consisting of HCV structural genes and BVDV nonstrucural genes and untranslated regions was able to replicate in cell lines of bovine and human origin.
  • the HCV glycoproteins and core protein were efficiently expressed from this genome.
  • Virion particles incorporating the chimeric genome were formed only in the presence of an endogenous BVDV helper virus that provided El and/or E2 BVDV glycoproteins to each infectious particle. In the presence of helper virus, this chimera replicated to high titers and significant amounts of HCV glycoprotein were released from the cells.
  • the HCV glycoproteins on the virions are believed to mediate entry of the chimeric virus into cultured hepatocytes (Huh 7 cells) where the genome replicated via the BVDV non-structural proteins.
  • chimeric virus replicated to such high levels and such large quantities of HCV glycoproteins were synthesized, it would be feasible to test purified chimeric virions as a candidate inactivated vaccine.
  • Purified chimeric virions can be tested first in mice and if antibody to HCV is produced, the virions will be tested in chimpanzees to determine if the candidate vaccine is efficacious.
  • EBTr (A) cells were able to infect Huh 7 cells and were neutralized by some anti-HCV positive plasmas (Table 6) suggests that such chimeric viruses could be used to screen for neutralizing antibodies to HCV as well as to screen other cell lines for HCV receptors.
  • HCV-BVDV chimeras can serve as a useful tool for studying the molecular biology of HCV.
  • the glycoprotein genes from the five other genotypes of HCV can be similarly inserted into the BVDV backbone in order to provide an assay for antibodies to each genotype. Additional chimeras are being constructed in which the core protein of BVDV is included so that only the glycoproteins of HCV are introduced. If BVDV core is critical for encapsidation of the RNA, it may be possible to generate chimeric viruses in the absence of helper. It will also be revealing to determine if the
  • HCV contribution to the chimera can be localized to either El or E2 alone. Such a chimera will be tested for its ability to infect EBTr (A) and Huh 7 cells.
  • chimeras in which the BVDV nonstructural genes such as p7 or NS4B or NS5A are replaced with the corresponding genes of HCV may also be generated to determine if they are functional in cell culture.
  • Rice CM. Flaviviridae The viruses and their replication. Field Virology, Third Edition Lippincott-Raven Publishers, Philadelphia 1996; 931- 959. 20. Houghton M. Hepatitis C viruses. Field Virology, Third Edition Lippincott-Raven Publishers, Philadelphia 1996; 1035-1058.
  • Variable and hypervariable domains are found in the regions of HCV corresponding to the Flavivirus envelope and NSl proteins and the Pestivirus envelope glycoproteins.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des approches moléculaires de la production de séquences d'acide nucléique contenant les génomes recombinants des virus de l'hépatite C/virus de la diarrhée virale bovine (VHC-BVDV). L'invention concerne également l'utilisation de ces séquences recombinantes d'acide nucléique pour produire des virions recombinants dans des cellules, et l'utilisation de ces virions recombinants dans des méthodes de neutralisation d'anticorps de VHC, et pour la mise au point de vaccins et de traitements dirigés contre le VHC.
PCT/US2000/015527 1999-06-04 2000-06-02 Genomes recombinants vhc/bvdv et utilisation de ceux-ci WO2000075352A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/009,011 US7009044B1 (en) 1999-06-04 2000-02-06 HCV/BVDV chimeric genomes and uses thereof
AU53245/00A AU5324500A (en) 1999-06-04 2000-06-02 Hcv/bvdv chimeric genomes and uses thereof
EP00938165A EP1187927A2 (fr) 1999-06-04 2000-06-02 Genomes recombinants vhc/bvdv et utilisation de ceux-ci

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13781799P 1999-06-04 1999-06-04
US60/137,817 1999-06-04

Publications (2)

Publication Number Publication Date
WO2000075352A2 true WO2000075352A2 (fr) 2000-12-14
WO2000075352A3 WO2000075352A3 (fr) 2001-11-15

Family

ID=22479160

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/015527 WO2000075352A2 (fr) 1999-06-04 2000-06-02 Genomes recombinants vhc/bvdv et utilisation de ceux-ci

Country Status (3)

Country Link
EP (1) EP1187927A2 (fr)
AU (1) AU5324500A (fr)
WO (1) WO2000075352A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004024904A2 (fr) 2002-09-13 2004-03-25 Institut National De La Sante Et De La Recherche Medicale (Inserm) Pseudo-particules d'hepacivirus infectieuses renfermant des proteines fonctionnelles d'enveloppe e1, e2
WO2010060114A1 (fr) * 2008-11-24 2010-05-27 Acrometrix Pestivirus chimérique avec insertion dans une région non traduite en 3' (3'-ntr) avec réplication stable et résistance à l’arnase
CN103924006A (zh) * 2014-04-21 2014-07-16 江苏省农业科学院 一种用于检测HoBi样瘟病毒核苷酸片段的引物序列
US9775894B2 (en) 2013-07-09 2017-10-03 University Of Washington Through Its Center For Commercialization Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055366A1 (fr) * 1998-04-24 1999-11-04 Washington University Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055366A1 (fr) * 1998-04-24 1999-11-04 Washington University Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FROLOV I ET AL: "CIS-ACTING RNA ELEMENTS REQUIRED FOR REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS-HEPATITIS C VIRUS 5' NONTRANSLATED REGION CHIMERAS" RNA,CAMBRIGDE UNIVERSITY PRESS, CAMBRIDGE,GB, vol. 4, no. 11, 25 November 1998 (1998-11-25), pages 1418-1435, XP000952790 ISSN: 1355-8382 *
H LU ET AL: "Poliovirus chimeras replicating under the translation control of genetic elements of HCV reveal unusual properties of the IRES of HCV" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,NATIONAL ACADEMY OF SCIENCE. WASHINGTON,US, vol. 93, February 1996 (1996-02), pages 1412-1417, XP002919370 ISSN: 0027-8424 *
LAI VC, ZHONG W, SKELTON A, INGRAVALLO P, VASSILEV V, DONIS RO, HONG Z, LAU JY: "Generation and characterization of a hepatitis C virus NS3 protease-dependent bovine viral diarrhea virus." JOURNAL OF VIROLOGY., vol. 74, no. 14, July 2000 (2000-07), pages 6339-6347, XP000952808 THE AMERICAN SOCIETY FOR MICROBIOLOGY., US ISSN: 0022-538X *
MEYERS G ET AL: "RECOVERY OF CYTOPATHOGENIC AND NONCYTOPATHOGENIC BOVINE VIRAL DIARRHEA VIRUSES FROM CDNA CONSTRUCTS" JOURNAL OF VIROLOGY,THE AMERICAN SOCIETY FOR MICROBIOLOGY,US, vol. 70, no. 12, December 1996 (1996-12), pages 8606-8613, XP000952807 ISSN: 0022-538X *
VENUGOPAL K. & GOULD E.A.: "Towards a new generation of Flavivirus vaccines" VACCINE, vol. 2, no. 11, 1994, pages 966-975, XP002919372 GUILDFORD GB *
YU H ET AL: "SEQUENCE AND STRUCTURAL ELEMENTS AT THE 3' TERMINUS OF BOVINE VIRALDIARRHEA VIRUS GENOMIC RNA: FUNCTIONAL ROLE DURING RNA REPLICATION" JOURNAL OF VIROLOGY,THE AMERICAN SOCIETY FOR MICROBIOLOGY,US, vol. 73, no. 5, May 1999 (1999-05), pages 3638-3648, XP000946998 ISSN: 0022-538X *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004024904A2 (fr) 2002-09-13 2004-03-25 Institut National De La Sante Et De La Recherche Medicale (Inserm) Pseudo-particules d'hepacivirus infectieuses renfermant des proteines fonctionnelles d'enveloppe e1, e2
WO2010060114A1 (fr) * 2008-11-24 2010-05-27 Acrometrix Pestivirus chimérique avec insertion dans une région non traduite en 3' (3'-ntr) avec réplication stable et résistance à l’arnase
US8932606B2 (en) 2008-11-24 2015-01-13 Life Technologies Corporation Chimeric pestivirus with insertion in 3′ nontranslated region (3′NTR) with stable replication and rnase resistance
US8986673B2 (en) 2008-11-24 2015-03-24 Life Technologies Corporation Replication stable and RNase resistant chimeras of pestivirus with insertion in 3′ nontranslated region (3′NTR)
US9775894B2 (en) 2013-07-09 2017-10-03 University Of Washington Through Its Center For Commercialization Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling
US10434164B2 (en) 2013-07-09 2019-10-08 University Of Washington Through Its Center For Commercialization Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling
US11324817B2 (en) 2013-07-09 2022-05-10 University Of Washington Through Its Center For Commercialization Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling
US12023375B2 (en) 2013-07-09 2024-07-02 University Of Washington Methods and compositions for activation of innate immune responses through RIG-I like receptor signaling
CN103924006A (zh) * 2014-04-21 2014-07-16 江苏省农业科学院 一种用于检测HoBi样瘟病毒核苷酸片段的引物序列

Also Published As

Publication number Publication date
EP1187927A2 (fr) 2002-03-20
WO2000075352A3 (fr) 2001-11-15
AU5324500A (en) 2000-12-28

Similar Documents

Publication Publication Date Title
Zhao et al. Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection
Moradpour et al. Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins
AU746965B2 (en) Cloned genomes of infectious hepatitis C viruses and uses thereof
Aizaki et al. Full‐length complementary DNA of hepatitis C virus genome from an infectious blood sample
EP1694694B1 (fr) Virus de l'hepatite c capable de replication et procedes d'utilisation
US9057048B2 (en) Infectious hepatitis C virus—high producing HCV variants and use thereof
US7655406B2 (en) Nucleotide sequences coding for the non-structural proteins of the hepatitis C virus
US7678569B2 (en) Cloned genome of infectious hepatitis C virus strain HC-TN and uses thereof
US20070141668A1 (en) Cloned genome of infectious hepatitis C virus of genotype 2A and uses thereof
US8367401B2 (en) Replication competent hepatitis C virus and methods of use
EP1185664B1 (fr) Genome clone du virus de l'hepatite c de genotype 2a et son utilisation
Yagi et al. Identification of novel HCV subgenome replicating persistently in chronic active hepatitis C patients
Prabhu et al. Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone
WO2000075352A2 (fr) Genomes recombinants vhc/bvdv et utilisation de ceux-ci
US7009044B1 (en) HCV/BVDV chimeric genomes and uses thereof
Lim et al. Inducible system in human hepatoma cell lines for hepatitis C virus production
Nam et al. High-level expression of hepatitis C virus (HCV) structural proteins by a chimeric HCV/BVDV genome propagated as a BVDV pseudotype
Hwang et al. Emergence of mutation clusters in the HCV genome during sequential viral passages in Sip-L expressing cells
Kurihara et al. A highly hydrophobic domain within hypervariable region 1 may be related to the entry of hepatitis C virus into cultured human hepatoma cells
WO2001022984A1 (fr) Agents de liaison de proteine hcv e2 utilises pour le traitement de l'infection par le virus de l'hepatite c
Oniangue-Ndza Development and characterization of subgenomic and full-length genome replicons based on the sequence of HCV AD78 strain
Murata Real time RT-PCR analysis To monitor the effect of cytokines on neo-resistant replicon RNA, TGF-B, IFN-c, or BMP-4 was added in the media of replicon cells seeded on 6-well plate (4 X 104
WO2000037650A1 (fr) Virus de l'hepatite y

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 2000938165

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000938165

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 10009011

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2000938165

Country of ref document: EP