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WO1997033979A1 - Modele transgenique d'une infection par le virus de l'hepatite c - Google Patents

Modele transgenique d'une infection par le virus de l'hepatite c Download PDF

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Publication number
WO1997033979A1
WO1997033979A1 PCT/US1997/003939 US9703939W WO9733979A1 WO 1997033979 A1 WO1997033979 A1 WO 1997033979A1 US 9703939 W US9703939 W US 9703939W WO 9733979 A1 WO9733979 A1 WO 9733979A1
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hcv
proteins
expression
protein
mammal
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WO1997033979A9 (fr
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Emmett V. Schmidt
T. Jake Liang
Takao Kawamura
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • HCV hepatocellular carcinoma
  • hepatitis C virus The cloning of hepatitis C virus (HCV) has provided an important insight into the viral pathogenesis of hepatocellular carcinoma.
  • the virus is a positive- stranded RNA virus containing a genome of approximately 9,400 nucleotides. This sequence codes for a single, large polyprotein of approximately 3,010 amino acids.
  • HCV is a distant relative of the flaviviruses and pestiviruses.
  • the 5' untranslated region of the HCV virus shares significant homology (45- 49%) with the equivalent region of pestiviruses and is highly conserved (>95%) among all the HCV isolates.
  • HCV encodes a large polyprotein precursor through which individual viral proteins are processed co-translationally and/or post- translationally through the combined actions of host- and viral-encoded proteases.
  • the viral structural proteins are encoded by the N-terminal portion of the genome, followed by the nonstructural genes (NS2-NS5) .
  • Computer sequence comparison and comparative hydropathy plot indicates that the structural proteins are divided into the core capsid protein (C) , the envelope protein (EN1) , and a third protein probably representing a second envelope protein, designated as EN2/NS1 (Houghton et al., 1991, Hepatology 14.:381-388; Hijikata et al., 1991, 88:5547-5551; Weiner et al., 1991, Virology 1£0:842-848) .
  • the C, EN1 and EN2/NS1 glycoproteins have been shown to have molecular weights of 19 Kd, 33 Kd, and 72 Kd, respectively (Harada et al., 1991, J. Virol. 65:3015- 3021)
  • the functions of NS2 and NS4 proteins are unknown at present. Their predicted structures are highly hydrophobic and probably represent membrane-associated proteins.
  • the NS3 protein is probably a helicase/protea ⁇ e, and the NS5 gene, which encodes a large polypeptide, may function as an RNA-dependent RNA polymerase during viral replication.
  • HCV nucleotide sequences A considerable amount of information regarding the HCV nucleotide sequences has accumulated. Sequence comparisons among various HCV isolates reveal significant sequence heterogeneity. Based on this sequence information, three basic groups of HCV strains have been classified (Houghton et al., 1991, Hepatology 14:381- 388) . Several regions of variable and hypervariable sequences have been identified in the putative envelop proteins EN1 and EN2/NS1 (Weiner et al., 1991, Virology i8fJ:842-848; Hijikata et al., 1991, Biochem. Biophys. Res. Comm. 175:220-228) .
  • the hypervariable region in the envelope protein may be a consequence of a strong selection pressure on a protective epitope(s) of either B or T cells. This is reminiscent of the hypervariable neutralizing domain in the V3 loop of the envelop protein of human immunodeficiency virus-l (HIV-1) . This speculation, if confirmed, will provide important information regarding virus-receptor interaction, host- immunological responses and most importantly, the future development of a vaccine against HCV.
  • HCV-1 human immunodeficiency virus-l
  • HCV In contrast to retrovirus infection, the HCV virus replicates through an RNA intermediate (Choo et al. , 1989, Science 244:359-362; Houghton et al., 1991, Hepatology 14.:381-388) .
  • RNA intermediate Choo et al. , 1989, Science 244:359-362; Houghton et al., 1991, Hepatology 14.:381-388
  • HCV may infect mononuclear cells or other cell types. These cells may be important as a source of viral transmission or as a reservoir for latent infection.
  • lymphoid cells infected by HCV may modulate the host immune response to HCV. Since HCV is an RNA virus and replicates without proofreading capacity, significant sequence diversity can result. This virus is therefore capable of mutating rapidly to evade host immune surveillance leading to chronic and persistent infection.
  • the model of the invention is less expensive than larger primate models, and offers the key advantage of a carefully studied immunologic and genetic animal system.
  • Transgenic expression is a cheap and safe method for testing therapeutic compounds, and provides a unique opportunity to assess individual genetic strategies for treatment.
  • Fig. 1 is a schematic representation of the genomic organization of the hepatitis C virus.
  • the encoded structural proteins are represented by “C”, “El”, and M E2"; non-structural proteins are represented by “NS2" through “NS5".
  • Fig. 2 is a map of plasmid pALBHCV
  • Fig. 3 is a map of plasmid pMUPHCV.
  • Fig. 4 is a set of three sequences showing the PCR primers used to amplify HCV genes.
  • Fig. 5 is a Western blot analysis of HCV core protein expression in liver.
  • Fig. 6A and Fig. 6B are pairs of photographs of ethidium bromide-stained gels, and illustrate RT-PCR analysis of HCV mRNA expression in liver.
  • Total RNA was harvested from livers of Tg.MC4 at indicated weeks of age (Fig. 6A) and Tg.AC lines (Fig. 6B) .
  • PCR for HCV upper photograph in Fig. 6A and Fig. 6B
  • 0-actin lower photograph in Fig. 6A and Fig. 6B
  • Fragments were separated on a 1.2% agarose gel and stained by ethidium bromide.
  • Fig. 7 is a Northern blot analysis of HCV mRNA expression in liver.
  • PolyA RNA of liver in Tg.AC lines was separated on a 1% agarose gel containing formaldehyde and transferred to nylon membrane. It was probed by a 32 P-labelled HCV probe. The same membrane was then re- probed by a 32 P-labelled GAPDII probe to control for gel loading.
  • Fig. 8 is a Western blot analysis of HCV core protein expression in liver. Liver extracts of Tg. C4 and Tg.AC lines were separated on an 15% SDS- polyacrylamide gel and transferred to PVDF membrane. It was probed using an anti-HCV core monoclonal antibody. It was then re-probed using an anti-actin antibody to control for gel loading. Controls included lysates of insect cells expressing HCV proteins (Bac+) , and insect cells expressing 3-glucuronidase (Bac-) .
  • Fig. 9 is a series of twelve photomicrographs illustrating immunohistochemical analysis of HCV proteins in liver.
  • the livers of Tg.MC4 (A, D) , Tg. AC1 (B, E, F, H-L) and wild type (C, G) were fixed and sections were stained using rabbit anti-core antibody (C-F) , rabbit anti-E2 antibody (G-L) , and rabbit prei mune serum (A, B) .
  • Photomicrographs were obtained with magnification of 200X (A-D, G) , 100X (E, F, H-K) and 800X (L) .
  • Core antibody staining was predominantly cytoplasmic although the arrows in 9F indicate positive core antibody staining in occasional nuclei compared with negative core staining in adjacent nuclei.
  • HCV Structural Genes The genomic organization of HCV is shown in Fig. 1.
  • the structural genes cluster around the 5' region of the viral genome. Comparative hydropathy plots and computer prediction of secondary structure of these gene products have defined the junctions and domains of these proteins (Kato et al., 1991, Proc. Natl. Acad. Sci., USA 82:9524-9528; Taka izawa et al., 1991, J. Virol. 65:1105- 1113) .
  • At least three structural proteins can be predicted from the sequences: core capsid (C) , ENl, and EN2/NS1.
  • HCV cDNA clone type lb from a Japanese patient with chronic hepatitis, was generously provided by Dr. Kunitada Shimotohno, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104, Japan. We cloned a fragment containing the entire core, El and E2 sequences (Stul-Tthllll fragment - GenEMBL HPCJCG nucleotides 280 to 2834) into the EcoR I and Stu I sites of pFASTBac 1 (Gibco-BRL Gaithersburg, D) . HCV cDNA clone can be obtained from any of a number of available strains, using standard methods.
  • This baculovirus construct was used for high level expression HCV structural proteins; infected SF9 cells were shown to express HCV proteins of appropriate size. This cell lysate was used as protein control in subsequent studies.
  • the N-terminal primers are designed with sequences conforming to Kozak's rules (Kozak, 1984, Nucleic Acids Res. jl2.:857-872) , and an in-frame AUG codon (Primers 1, 3, and 5).
  • the C-terminal primers (primers 2, 4, and 6) encode two consecutive TAG stop codons.
  • the HCV cDNA was placed under the control of the murine albumin enhancer/promoter (Fig. 2; plasmid pALBHCV) and under the control of regulatory sequences of the Mouse Urinary Protein (MUP) promoter region (Fig. 3; plasmid pMUPHCV) .
  • the DNA fragments used in the transgenic experiments were obtained by digesting pALBHCV with Nhel and Nsil to remove the pGEM7 sequences, and by digesting pMUPHCV with Aatll to remove the pSVSport vector sequences.
  • the rationale for selecting these regulatory elements. particularly the MUP enhancer/promoter, is discussed below.
  • Alternative means of directing expression of HCV proteins in the liver could include, but are not limited to, placing the HCV sequences under the control of liver-specific promoters such as those associated with the genes for fatty acid synthase, glutamine synthetase, apoVLDL-II, apolipoprotein E, human apolipoprotein E/C-I, apolipoprotein C-III, metallothionein-I, transthyretin, apolipoprotein B, and alpha-1-antitrypsin.
  • HCV sequences could also be placed under the control of inducible promoters.
  • Transgenic mice were made using the Aatll-Asel fragment of pMUPHCV and the Pvul-Nsil fragment of pAl HCV. Fertilized oocytes from FVB inbred mice (T. onic; Germantown, NY) were microinjected at the single-cell stage and candidate transgenic offspring were obtained using standard methods. Transgenic mice containing the HCV transgenic constructs were identified by Southern blots using DNA isolated from individual mouse tails. Transgenic lines were produced by breeding heterozygous transgenic mice to inbred FVB mice.
  • RNA from the transgenic construct encoding HCV was identified in the first generation of mice.
  • MUPHCV mice and AlbHCV mice with the highest levels of viral RNA were studied for expression of HCV core protein to demonstrate that the viral structural proteins were present (Fig. 5) . This immunoblot provided clear evidence that the AlbHCV transgenic mice were making HCV core protein in their livers.
  • PolyA RNA was purified from the cesium-purified RNAs using oligo(dT) magnetic particles (Promega;
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Liver samples were homogenized in 3 volumes of RIPA buffer + 1% sodium dodecyl sulphate (SDS) (0.15 M NaCl, 50 M Tris pH 8.0, 0.5% sodium deoxycholate, 1% SDS, and 1% NP40) . After centrifugation to clear cell debris, the protein lysates were electrophoresed in 15% SDS-polyacrylamide gel and transferred to PVDF membrane (Millipore; Bedford, MA) by electroblotting. The blot was probed with anti-core monoclonal antibody (C7-50A7; a generous gift of Dr.
  • SDS sodium dodecyl sulphate
  • Jack Wands and developed with horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (Amersham) using an enhanced chemiluminesence kit (DuPont NEN; Boston, MA) .
  • the membrane was re-probed with anti-actin monoclonal antibody (Amersham; Arlington Heights, II) to control for protein loading.
  • mice Livers from mice were fixed in 4% paraformaldehyde in phosphate buffered saline, sectioned and subjected to immunohistochemistry using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) . Rabbit polyclonal antibodies against HCV core and E2 (obtained from Dr. Richard Lesniewski of the Abbott Laboratories) were used as primary antibodies in dilution of 1:100. As negative controls, nonimmune rabbit serum or BSA were used. For secondary antibody, biotinylated goat anti- rabbit IgG was used at a dilution of 1:500. The slides were counterstained with hematoxylin. Transgenic lines Potential founder mice were analyzed for the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) . Rabbit polyclonal antibodies against HCV core and E2 (obtained from Dr. Richard Lesniewski of the Abbott Laboratories) were used as primary antibodies in
  • HCV transgene by Southern blot and PCR analyses of tail DNA.
  • Four founders containing MUPHCV sequences and eleven founders containing AlbHCV were identified and designated as Tg.MCl-4 and Tg.ACl-11.
  • Transgenes were not successfully transmitted in Tg.MC3, AC2, AC4, and AC7 lines.
  • the Tg.ACl line contained two transgene insertion sites.
  • two sublines, each containing one of the transgenes, were identified by
  • HCV mRNA was detected in seven of ten Tg.AC lines (Fig. 6B) .
  • the expression levels were generally higher in the Tg.AC lines than seen in Tg. MC4.
  • the highest level of expression was detected in Tg.AC3.
  • Northern blots confirmed the result of RT-PCR (Fig. 7) .
  • Tg.AC3 A strong signal was detected in Tg.AC3 and weaker expression was seen in Tg.ACl-0, Tg.ACl-1 and Tg.ACl-2 ines.
  • HCV core protein was confirmed on Western blots of liver lysates extracts using a monoclonal anti-core antibody (Fig. 8) .
  • Lysates of insect cells expressing the HCV structural proteins revealed a prominent 22 kd band. The same band was clearly visualized in Tg.AC3, ACl-O, ACl-1 and AC1-2. Expression levels of protein correlated well with mRNA levels. A prominent 50 kD band was also detected in Tg.AC3 and may represent unprocessed polyprotein. This band was also evident in the lysate of insect cells.
  • the product of the transgene can be expressed as a self antigen, to which the host animal is tolerant.
  • immunologically mediated responses which are important in a viral infectious process cannot be studied.
  • an adult-specific promoter Alternatively, but less desirably, an inducible promoter could have been used. Most of the inducible promoters permit low but significant expression of the protein product of the transgene, which is probably sufficient to be recognized as a self antigen.
  • the mouse major urinary proteins are a family of proteins synthesized at high levels in liver and sweat glands, and excreted in urine by kidney or in sweat by the sweat glands after the rodent reaches puberty (Hastie et al., 1979, Cell 17:449-457).
  • MUPs There are several types of MUPs, some of which are exclusively produced by hepatocytes. These genes are silent £n utero when the predominate intrathymic education of the lymphoid precursors takes place.
  • the enhancer and promoter elements of these liver-specific MUPs represent ideal regulatory sequences for the expression of a transgene in an adult-specific manner. This promoter has been successfully used to express the SV40 T antigen in transgenic mice for such a purpose, and the expression was indeed adult- and liver-specific (Held et al., 1989, EMBO J. 8:183-191) .
  • mice of the invention can be used to assess the immunobiology and pathogenesis of hepatocellular injury associated with HCV infection. For example. spleen cells primed with recombinant vaccinia virus containing HCV structural genes can be transferred into the transgenic animals to elucidate this complex process. Thus, the mice can be used to study the question of whether immune mechanisms can contribute to cellular damage caused by viral infections, and whether either antibodies or cytotoxic lymphocytes may play an adverse role in the pathogensis of HCV infection. HCV-specific T helper and cytotoxic lymphocytes have been demonstrated in the liver and peripheral blood of chronically infected individuals, and these cellular components of immune responses are also directed against multiple viral proteins. These effector cells presumably participate in the lysis of virus-infected hepatocytes and clearance of virus. However, if this response is incomplete, then the virus will persist and cytokine-mediated inflammatory responses may contribute to hepatic damage.
  • transgenic mice Animal models using transgenic mice have been successfully used to address similar issues in other viral infections, such as hepatitis B virus infections. Since transgenic mice expressing an antigen in utero become tolerant to the antigen, pathogenic effects of cloned viral products can be examined in the absence of an immune response. The apparent co-localization of core and E2 in the mice of the invention may suggest that the structural proteins can form complexes with the endoplasmic reticular membrane as part of an early step in virion assembly. These findings are consistent with observations made in various tissue culture systems. In contrast to one study (Dubuisson et al. (1994) , J.
  • transgenic mice of the invention can be made using not just microinjection, but any suitable method, such as homologous recombination into embryonic stem cells.
  • liver-specific promoter/enhancer sequences can be used, e.g., metallothioneien, serum amyloid P component (SAP) promoter (Zhao et al. , J.Biochem. , 111:736-738. 1992), or ⁇ l-antitrypsin promoter (Sifers et al.. Nucleic acid Research, i5:1459-1475, 1987.
  • SAP serum amyloid P component
  • ⁇ l-antitrypsin promoter Sifers et al.. Nucleic acid Research, i5:1459-1475, 1987.
  • other adult-specific promoter/enhancer sequences can be used, e.g., the ⁇ 2-u globulin promoter (Da Costa Soares et al., Mol.Cell.Biol. , 7:3749-3758, 1987).
  • HCV DNA the example described herein employed type lb HCV cDNA from a particular patient; it is of course contemplated that the HCV DNA can be obtained from any suitable source, e.g. , other patients, and that any HCV strain can be used as the source of DNA. These various strains will be expected to exhibit fairly wide sequence variability, as HCV is a virus which exhibits a high mutation rate. It is also contemplated that mutations can be intentionally introduced into the HCV sequences used to make the transgenic mice of the invention. What is claimed is:

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Abstract

Mammifère transgénique dont les hépatocytes expriment une ou plusieurs protéines C, E1 ou E2 du virus de l'hépatite C.
PCT/US1997/003939 1996-03-13 1997-03-13 Modele transgenique d'une infection par le virus de l'hepatite c Ceased WO1997033979A1 (fr)

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JP53282297A JP2001508283A (ja) 1996-03-13 1997-03-13 Ｃ型肝炎ウイルス感染のトランスジェニックモデル

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000252A3 (fr) * 2000-06-27 2002-06-13 Chiron Corp Tolerance et virus de l'hepatite c chronique
EP0919122A4 (fr) * 1996-07-24 2004-11-17 Tokyo Metropolitan Inst Of Med Modeles animaux de l'hepatite c

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003037081A1 (fr) * 2001-10-30 2003-05-08 Tokyo Metropolitan Organization For Medical Research Animal transgenique a gene hcv

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF VIROLOGY, 1996, Vol. 141, No. 5, KATO et al., "Inactivation of Hepatitis C Virus cDNA Transgene by Hypermethylation in Transgenic Mice", pages 951-958. *
JOURNAL OF GENERAL VIROLOGY, 1995, Vol. 76, No. 12, KOIKE et al., "Expression of Hepatitis C Virus Envelope Proteins in Transgenic Mice", pages 3031-3038. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0919122A4 (fr) * 1996-07-24 2004-11-17 Tokyo Metropolitan Inst Of Med Modeles animaux de l'hepatite c
WO2002000252A3 (fr) * 2000-06-27 2002-06-13 Chiron Corp Tolerance et virus de l'hepatite c chronique

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