WO2004016738A2 - Procede et composition pour traiter et prevenir une infection par le virus de l'hepatite c - Google Patents

Procede et composition pour traiter et prevenir une infection par le virus de l'hepatite c Download PDF

Info

Publication number
WO2004016738A2
WO2004016738A2 PCT/US2003/022956 US0322956W WO2004016738A2 WO 2004016738 A2 WO2004016738 A2 WO 2004016738A2 US 0322956 W US0322956 W US 0322956W WO 2004016738 A2 WO2004016738 A2 WO 2004016738A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
hcv
composition
nucleic acid
acid encoding
Prior art date
Application number
PCT/US2003/022956
Other languages
English (en)
Other versions
WO2004016738A3 (fr
Inventor
Scott Morham
Kenton Zavitz
Adrian Hobden
Original Assignee
Myriad Genetics, Inc.
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
Application filed by Myriad Genetics, Inc. filed Critical Myriad Genetics, Inc.
Priority to AU2003256678A priority Critical patent/AU2003256678A1/en
Publication of WO2004016738A2 publication Critical patent/WO2004016738A2/fr
Publication of WO2004016738A3 publication Critical patent/WO2004016738A3/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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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

Definitions

  • the invention relates generally to treating and preventing viral infections and associated diseases, and particularly to methods and compositions for treating and preventing hepatitis C infection.
  • Hepatitis C virus is a major cause of liver disease throughout the world. It is estimated that upwards of 10,000 deaths per year are attributable to HCV in the United States. About four million people in the United States have antibody to HCV. It is estimated that well over 150 million people worldwide are chronically infected with HCV. Chronic hepatitis C can cause cirrhosis, liver failure, and liver cancer (hepatocellular carcinoma). There are a variety of subtypes of HCV including at least 6 major genotypes and 50 subtypes. As with many other viruses, HCV is known to mutate quickly, and changes in the envelope protein may help the virus avoid the immune system of the host.
  • Vaccines and immunoglobulin products for preventing HCV are not currently available. Given the rapidly mutating nature of the virus and the variety of variants of Attorney Docket No. 5010.01 WO
  • HCV HCV
  • the only preventative strategies relate to the ability to stop transmission of the virus by screening blood supplies and educating the public regarding high-risk groups and behaviors.
  • Two treatments are available in the United States for those infected with HCV: monotherapy treatment with alpha interferon or combination therapy with alpha interferon and ribavirin. Combination therapy appears to be the most efficacious treatment and therefore is the treatment of choice in the United States.
  • Alpha interferon monotherapy can be effective in treating chronic HCV, but it is not effective against all HCV infections and there can be unwanted side effects associated with this treatment option.
  • the other treatment option is a combination therapy with alpha interferon and ribavirin. Again, there are undesirable side effects associated with this combination therapy.
  • Rous sarcoma virus (RSV), equine infectious anemia virus (EIAV), as well as other retroviruses, were shown to have late domains with characteristic amino acid sequence motifs; the late-domain motifs are critical binding motifs that facilitate the interaction of the viral proteins with host cell proteins. See, e.g., Puffer et al. J. Virol. 72:10218-10221 (1998) and Campbell et al. J. Virol. 71:4425-35 (1997). Unexpectedly, it was discovered that specific hepatitis C proteins have late- Attorney Docket No. 5010.01WO
  • the present invention provides for the treatment and prevention of HCV infection.
  • the invention provides compositions and methods that affect the ability of HCV, or a variant thereof, to utilize the host's cellular machinery for viral budding and egress.
  • the invention relates to the discovery that interfering with the normal ability of viruses to utilize the host cells vesicular trafficking, recycling, and vacuolar sorting machinery for viral propagation can reduce the infectivity of the virus.
  • the invention provides HCV treatment methods and compositions based on the modulation of viral budding. Modulation of the normal HCV budding mechanism can also enhance the host's immune response against the virus.
  • the invention therefore provides compositions and methods for enhancing an immune response against HCV.
  • the invention provides a method of affecting the ability of
  • a composition capable of modulating the budding of the virus is administered to an individual in need of treatment.
  • the composition can be provided as a nucleic acid, a polypeptide, a peptide, a vaccine, a small molecule therapeutic, and the such, as long as the budding modulation composition affects the ability of the virus to bud.
  • a HCV protein that participates in the normal budding pathway of the virus can be provided in a mutant form, e.g., a nucleic acid encoding a mutant protein or a mutant protein itself, to an infected host cell, in order to affect the budding of the virus.
  • the budding of HCV is inhibited partially or completely.
  • the budding of HCV is rendered dysfunctional or abnormal.
  • the invention provides a method of treating an individual infected with HCV by enhancing an immune response against HCV.
  • the method of this embodiment includes the step of administering an effective amount of a composition capable of affecting HCV budding, or inducing HCV altered budding phenotype, to an individual infected with HCV (or in need of prophylaxis).
  • the composition can be Attorney Docket No. 5010.01WO
  • the administered composition is capable of modulating the normal pathway which HCV utilizes for budding and egress. Modulation of the normal HCV pathway for budding and egress can lead to the aberrant production of viral particles. The aberrant production of viral particles can cause an altered budding phenotype. The altered budding phenotype can stimulate an immune response in the host against HCV and HCV infected cells.
  • the present invention provides a method of treating an individual infected with HCV by modulating the ability of a virus to bud from a cellular membrane in a host cell by administering, to a patient in need of therapeutic or prophylactic treatment, a mutant HCV protein encoding a late-domain motif that can affect the ability of the virus to bud or by administering to the patient a nucleic acid encoding such a mutant HCV protein.
  • Such treatments can be used to suppress the symptoms of HCV, i.e., suppressive therapy, or for treating episodic outbreaks, i.e., episodic therapy.
  • a mutant HCV protein that interacts with a host cell's protein(s) to bud from a particular membrane can be provided by nullifying the late- domain motif of a wild-type or modified HCV protein that is capable of mediating the budding process.
  • a nucleic acid encoding a mutant HCV protein sufficient for viral particle assembly but devoid of late domain motifs is incorporated into a plasmid vector which is injected directly into a patient.
  • the nucleic acid can also be delivered into a patient by infecting the patient with a recombinant live vector (e.g., recombinant viral or bacterial vector) carrying the nucleic acid.
  • the nucleic acid is administered to a patient by gene gun or powder jet or an equivalent device thereof.
  • the invention further provides a method of identifying compounds that modulate the activity of a viral protein host cell protein protein-protein interaction that is involved in a viral egress and/or budding pathway. More particularly, the method involves identifying compounds that modulate an interaction between a viral protein having a late- domain motif and a host cell protein that interacts with the late-domain motif containing protein.
  • an assay system is employed to detect compounds and/or compositions that affect the ability of the virus to propagate itself.
  • the assay system targets host cell proteins that are utilized by the virus for budding that interact with the viral late-domain motif containing protein(s).
  • the assay is configured to identify compounds that modulate the activity of a specific protein involved in a viral budding and/or egress pathway.
  • the assay is configured to identify compounds that modulate a protein-protein interaction that is involved in the viral budding and/or egress pathway.
  • the host protein that is modulated is selected from the group consisting of TSG101, NEDD4, a NEDD4-like protein, AIP1, a host cell protein containing a WW domain, or fragments, derivatives, or homologs thereof.
  • the viral late-domain motif containing protein has a YXXL, PXXP, or PPXY late-domain motif, or fragments, derivatives, or homologs thereof.
  • the screening assays of the invention can be accomplished in any manner as long as they examine the affect of a particular treatment on a host cell viral protein protein-protein interaction involved in viral budding (or egress) or a host cell biochemical pathway involved in viral egress.
  • HVC is an enveloped positive strand RNA virus.
  • HCV is a member of the Flaviviridae family and has a genome that encodes a single ORF (polyprotein) of about 3000 amino acids.
  • the HCV genomic structure can be represented as 5' — C — El — E2 — p7— NS2— NS3— NS4A— NS4B— NS4B— NS5A— NS5B— 3' where C encodes the core protein, E encodes the envelope proteins, NS2 encodes a zinc dependent proteinase, NS3 encodes a zinc dependent proteinase/serine protease/helicase, NS4A/B encodes a binding protein, NS5A encodes a protein thought to be involved in ⁇ jS-interferon resistance, and NS5B encodes an RNA dependent RNA polymerase (NS refers to non- structural).
  • the present invention relates to the discovery that HCV contains late-domain motifs that are thought to be involved in the viral budding process.
  • the NS5B protein of HCV contains a late-domain YPDL (or FPDL) motif which is believed to interact with the AP-50 subunit of the AP-2 clathrin associated adapter protein complex to facilitate viral budding.
  • the motif can bind AIP1 and the methods of the invention apply to the use of this protein (Vincent et al. Mol. Cell Biol. 23:1647-1655 (2003).
  • the HCV core protein also contains a late-domain motif similar to the PXXP class that may be involved in viral budding.
  • the PXXP motifs are thought to bind to TSG101 (or another host protein having an src-3 homology domain "SH3").
  • the invention relates to modulating the interaction of late-domain motif containing viral proteins and their respective host cell binding partner protein(s).
  • the HCV core protein (C) has at least one PXXP (t.e., PTDP, PSDP, PNDP, and or PGYP) amino acid motif which is thought to be critical to the budding process.
  • the PXXP (i.e., PTDP, PSDP, PNDP, and/or PGYP) amino acid motif found in the core protein of HCV is thought to be involved in the budding process of HCV infection in a manner similar or analogous to that of the PTAP motif found in the HIV gag protein.
  • the discovery of the PXXP (i.e., PTDP, PSDP, PNDP, and/or PGYP) amino acid motif in the core protein of HCV provides a novel approach for treating and preventing HCV infection.
  • the present invention encompasses methods of treating and preventing HCV infection by providing a variant core protein, or fragment thereof, with a modified PXXP amino acid motif which induces defects in the HCV budding process.
  • the variant core protein can provided to an individual in need of treatment or prophylaxis by a variety of methods.
  • the variant core protein can be provided in vaccine form.
  • the variant core protein can be provided in the form of cells displaying an altered budding phenotype (i.e., cells are treated with an effective amount of a composition of the present invention to induce an altered budding phenotype, and the cells are then administered to an individual in need of such treatment or prophylaxis).
  • Other such methods which are encompassed within the scope of the invention, will be readily apparent to the skilled artisan in view of the disclosure herein.
  • hepatitis C refers to any of the strains and isolates of hepatitis c that have been identified or are identifiable according to known classifications and methods.
  • the term “hepatitis C” or “HCV” is intended to encompass all currenty known strains, types and subtypes of HCV as wells those discovered and classified as HCV in the future. The identification of HCV is well within the purvey of an ordinary skilled artisan. The first known HCV isolate was identified in the USA (see Q.-L.Choo et al. (1989) Science 244:359-362, Q.-L. Choo et al. (1990) Brit. Med. Bull. 46:423-441, Q.-L.
  • HCV strains, types, and subtypes are known with their sequences being deposited in public databases. Examples include, but are not limited to, GenBank accession numbers AF177036, AF169002, D28917, AF169003, D84262, AF238484, Y12083, AB047639, AB047641, D63821, and the such.
  • alpha interferon or “interferon alpha” or “ ⁇ -interferon” refers to the family of interferon proteins that inhibit viral replication, inhibit cellular proliferation, and modulate immune response.
  • alpha interferon encompasses a variety of commercially available alpha interferons, including, but not limited to, Roferon A interferon (Hoffman-La Roche, Nutley, NJ), Berofor alpha 2 (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT), Sumiferon (Sumitomo, Japan), Wellferon interferon alpha-nl (Glaxo- Wellcome Ltd., London, Great Britain).
  • Alpha interferon 2b currently has the broadest approval throughout the world for use in treating HCV.
  • U.S. patent no. 4,530,901 (which is hereby incorporated by reference in its entirety) provides a description of the manufacture of alpha interferon 2b.
  • HCV can be detected by testing for antibodies against HCV (anti-HCV) in a patient or suspected carrier's blood serum.
  • Another approach to detecting HCV is to test for HCN R ⁇ A in the serum of a patient or suspected carrier using a PCR based assay.
  • Recombinant immunoblots can also be used to detect HCN. In this test serum is incubated with four recombinant viral proteins that are blotted on nitrocellulose strips. A simple color change indicates antibodies are present in the serum that bind to the viral proteins.
  • detecting HCN are continually being developed and the methods can be employed for diagnosing HCN infection.
  • side effects of alpha interferon treatment include fatigue, muscle aches, headaches, nausea, vomiting, low-grade fever, weight loss, irritability, depression, mild bone marrow suppression, and hair loss.
  • side effects associated with ribavirin treatment refers to anemia, fatigue and irritability, itching, skin rash, nasal congestion, sinusitis, and cough associated with the administration of ribavirin to an individual.
  • the term "alleviate a symptom associated with HCV” refers to a lessening of: fatigue, dark urine, abdominal pain, nausea, tenderness in the upper right quadrant, poor appetite, muscle and joint pain, cirrhosis (with symptoms such as enlarged liver, enlarged spleen, jaundice, muscle wasting excoriations, ascites and ankle swelling).
  • the term also encompasses symptoms not associated with the liver, including, but not limited to, cryoglobulinemia symptoms such as skin rashes, joint and muscle aches, kidney disease, neuropathy, cryoglobulins, rheumatoid factor, low complement levels in serum, glomerulonephritis, and porphyria cutanea tarda.
  • the term is also meant to encompass the alleviation of symptoms associated with disease that are thought to be or might be associated with chronic HCV including seronegative arthritis, keratoconjunctivitis sicca (Sjogren's syndrome), non-Hodgkin's type B-cell lymphomas, fibromyalgia, and lichen planus.
  • the term "individual in need of treatment” encompasses individuals who have symptoms of HCV infection, those who have been diagnosed with HCV, or those in need of prophylaxis.
  • An "individual in need of treatment” can have anti-HCV, HCV R ⁇ A, elevated serum aminotransferase lelels, and/or evidence of chronic hepatitis.
  • ribavirin refers to l-/3-D-ribofuranosyl-lH- 1,2,4- triazole-3 -carboxamide.
  • polypeptide As used herein, the terms "polypeptide,” “protein,” and “peptide” interchangeably to refer to amino acid chains in which the amino acid residues are linked Attorney Docket No. 5010.01WO
  • polypeptide bonds can be of any length of greater than two amino acids.
  • polypeptide protein
  • peptide also encompass various modified forms thereof.
  • modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, prenylated forms, etc. Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.
  • the term "homologue,” when used in connection with a first native protein or fragment thereof that is discovered, according to the present invention, to interact with a second native protein or fragment thereof, means a polypeptide that exhibits an amino acid sequence homology and/or structural resemblance to the first native interacting protein, or to one of the interacting domains of the first native protein such that it is capable of interacting with the second native protein.
  • a protein homologue of a native protein may have an amino acid sequence that is at least 50%, preferably at least 75%, more preferably at least 80%, 85%, 86%, 87%, 88% or 89%, even more preferably at least 90%, 91%, 92%, 93% or 94%, and most preferably 95%, 96%, 97%, 98% or 99% identical to the native protein.
  • homologues may be the ortholog proteins of other species including animals, plants, yeast, bacteria, and the like. Homologues may also be selected by, e.g., mutagenesis in a native protein.
  • homologues may be identified by site-specific mutagenesis in combination with assays for detecting protein-protein interactions, e.g., the yeast two-hybrid system described below, as will be apparent to skilled artisans apprised of the present invention.
  • assays for detecting protein-protein interactions e.g., the yeast two-hybrid system described below, as will be apparent to skilled artisans apprised of the present invention.
  • Other techniques for detecting protein-protein interactions include, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, and the like.
  • derivative when used in connection with a first native protein (or fragment thereof) that is discovered, according to the present invention, to Attorney Docket No. 5010.01WO
  • a second native protein means a modified form of the first native protein prepared by modifying the side chain groups of the first native protein without changing the amino acid sequence of the first native protein.
  • the modified form i.e., the derivative should be capable of interacting with the second native protein.
  • modified forms include glycosylated forms, phosphorylated forms, myristylated forms, ribosylated forms, ubiquitinated forms, prenylated forms, and the like.
  • Derivatives also include hybrid or fusion proteins containing a native protein or a fragment thereof. Methods for preparing such derivative forms should be apparent to skilled artisans.
  • the prepared derivatives can be easily tested for their ability to interact with the native interacting partner using techniques known in the art, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, yeast two-hybrid assays, and the like.
  • interacting means that two protein domains, fragments or complete proteins exhibit sufficient physical affinity to each other so as to bring the two "interacting" protein domains, fragments or proteins physically close to each other.
  • An extreme case of interaction is the formation of a chemical bond that results in continual and stable proximity of the two entities.
  • Interactions that are based solely on physical affinities, although usually more dynamic than chemically bonded interactions, can be equally effective in co-localizing two proteins. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof.
  • the state of proximity between the interaction domains, fragments, proteins or entities may be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities.
  • an "interaction" is exhibited by the binding between the interaction domains, fragments, proteins, or entities. Examples of interactions include specific interactions between antigen and antibody, ligand and receptor, enzyme and substrate, and the like.
  • An "interaction" between two protein domains, fragments or complete proteins can be determined by a number of methods. For example, an interaction can be determined by functional assays such as the two-hybrid systems. Protein-protein Attorney Docket No. 5010.01WO
  • interactions can also be determined by various biophysical and biochemical approaches based on the affinity binding between the two interacting partners.
  • biochemical methods generally known in the art include, but are not limited to, protein affinity chromatography, affinity blotting, immunoprecipitation, and the like.
  • the binding constant for two interacting proteins which reflects the strength or quality of the interaction, can also be determined using methods known in the art. See Phizicky and Fields, Microbiol. Rev., 59:94-123 (1995).
  • protein fragment as used herein means a polypeptide that represents a portion of a protein. When a protein fragment exhibits interactions with another protein or protein fragment, the two entities are said to interact through interaction domains that are contained within the entities.
  • domain means a functional portion, segment or region of a protein, or polypeptide.
  • Interaction domain refers specifically to a portion, segment or region of a protein, polypeptide or protein fragment that is responsible for the physical affinity of that protein, protein fragment or isolated domain for another protein, protein fragment or isolated domain.
  • the term "viral-like particle” refers to nucleocapsids or any HCV protein containing particles which in their wild-type form are capable of being secreted or are capable of budding.
  • TsglOl refers to the TsglOl protein.
  • AP-50 refers to the AP-50 subunit of the AP-2 clathrin associated adapter protein complex.
  • the term "late-domain binding partner” refers to the cellular protein or proteins which interacts with or binds to a viral late-domain motif.
  • the phrase "cells displaying a late-domain phenotype” refers to cells having immature non-infectious viral particles that are incapable of completing the viral budding process, t.e., incapable of budding off the cell surface.
  • the immature non- infectious viral particles are typically tethered to the host cells or to other immature non- infectious viral particles by thin membrane stalks and usually form clusters of Attorney Docket No. 5010.01WO
  • altered budding phenotype refers to the phenotype that a host cell or tissue displays upon modulation of a viral-protein host-protein protein-protein interaction involved in viral budding.
  • HCV altered budding phenotype is in comparison to the normal HCV budding phenotype which is one characterized by normal viral budding and extracellular virus release.
  • the virus will bud normally.
  • viral particles may never reach maturity, i.e., form wild-type viral particles, and/or have an accumulation of virus in a particular cellular compartment.
  • Cells having an HCV altered budding phenotype may also display different markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses. The difference in markers serves as a tag for the immune system to recognize these cells or tissues with the altered phenotype.
  • the HCV altered budding phenotype is readily determined by one of skill in the art. Markers in control host cells, wild-type HCV infected cells, and cells which have had a viral-protein host-protein protein-protein interaction modulated, can be quantitated using standard techniques to determine the altered budding phenotype.
  • a non-limiting example is a comparison of the type and number of viral particles found in, e.g., the cytoplasm, the uninfected host is expected to have no viral particles in the cytoplasm, the wild-type HCV infected host cell is expected to have a given amount of virus in the cytoplasm, and an altered budding phenotype cell is expected to have a substantially different amount of virus in the cytoplasm. Electron microscopy is another useful tool for determining a HCV altered budding phenotype by examination of the cellular Attorney Docket No. 5010.01 WO
  • altered egress phenotype refers to the phenotype that the host cell displays upon modulation of a host cell protein-protein interaction involved in viral egress.
  • the term “altered egress phenotype” is in comparison to the normal egress phenotype which is one characterized by normal viral egress and extracellular virus release.
  • the virus will egress normally.
  • the virus may never reach maturity, i.e., form wild-type virus, or alternatively, have an accumulation of virus in a particular cellular compartment.
  • Altered egress phenotype displaying cells may also display different molecular markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses.
  • the difference in markers can serve as a signal for the immune system to recognize the cells or tissues with the altered phenotype.
  • the altered egress phenotype is readily determined by one of skill in the art. For example, markers in uninfected control host cells, virus infected cells, and infected cells which have had a protein-protein interaction modulated, or a protein modulated, can be quantitated using standard techniques to determine the altered egress phenotype.
  • a non-limiting example is a comparison of the type and number of viral particles found in, e.g., the cytoplasm, the uninfected host is expected to have no viral particles in the cytoplasm, the wild-type virus infected host cell is expected to have a given amount of virus in the cytoplasm, and an altered egress phenotype cell is expected to have a substantially different amount of virus in the cytoplasm. Electron microscopy is another useful tool for determining an altered egress phenotype by examination of the cellular localization of viral particles and proteins. Thus, an altered egress phenotype is recognizable by one of ordinary skill in the art by making such comparisons.
  • the treatment methods of the invention involve administering to an individual an effective amount of a compound or composition capable of affecting the ability of HCV Attorney Docket No. 5010.01WO
  • the step of "administering" can be accomplished by any method capable of delivering the composition or compound to a cell.
  • Examples of such administering steps include gene therapy methods where a nucleic acid encoding a HCV protein, a mutant HCV protein, a peptide, a mutant HCV virus (as in a vaccine), and the such, is administered to an individual in need of treatment.
  • the nucleic acid is designed to have a gene encoding an activity that affects HCV budding. Described in the following section are some methods that may be employed for delivering compositions to a cell to affect HCV budding.
  • the exogenous nucleic acid of the present invention is incorporated into a suitable expression vector and is operably linked to a promoter in the vector such that the promoter can drive the transcription from the exogenous nucleic acid.
  • Suitable promoters include but are not limited to viral transcription promoters derived from adenovirus, simian virus 40 (SV40) (e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) (e.g., CMV immediate-early promoter), human immunodeficiency virus (HIV) (e.g., long terminal repeat (LTR)), vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSV-2) (e.g., thymidine kinase promoter).
  • tissue-specific promoters may be operably linked to the exogenous gene. In this regard, a CD 4+ T cell-specific promoter will be most desirable.
  • the exogenous nucleic acid is incorporated into a plasmid DNA vector.
  • a plasmid DNA vector Many commercially available expression vectors may be useful for the present invention, including, e.g., pCEP4, pcDNAI, pIND, pSecTag2, pVAXl, pcDNA3.1, pBI-EGFP, pBlueScript, andpDisplay.
  • Various viral vectors may also be used.
  • the viral genome is engineered to eliminate the disease-causing capability, e.g., the ability to replicate in the host cells.
  • the exogenous nucleic acid to be introduced into a patient may be incorporated into the engineered viral genome, e.g., by inserting it into a viral gene that is non-essential to the viral infectivity.
  • Viral vectors are convenient to use as they can be easily introduced into tissue cells by way of infection.
  • the recombinant virus typically is integrated into the genome of the host cell. In rare instances, the recombinant virus may also replicate and remain as extrachromosomal elements.
  • retroviral vectors have been developed for gene therapy. These include vectors derived from oncoretroviruses (e.g., MLV), lentiviruses (e.g., HIV and SIN) and other retroviruses.
  • oncoretroviruses e.g., MLV
  • lentiviruses e.g., HIV and SIN
  • gene therapy vectors have been developed based on murine leukemia virus (See, Cepko, et al., Cell, 37:1053-1062 (1984), Cone and Mulligan, Proc. Natl Acad. Sci. U.S.A., 81:6349-6353 (1984)), mouse mammary tumor virus (See, Salmons et al, Biochem. Biophys. Res.
  • Adeno-associated virus (AAN) vectors have been successfully tested in clinical trials. See e.g., Kay et al, Nature Genet. 24:257-61 (2000).
  • AAV is a naturally occurring defective virus that requires other viruses such as adenoviruses or herpes viruses as helper viruses. See Muzyczka, Curr. Top. Microbiol Immun., 158:97 (1992).
  • a recombinant AAV virus useful as a gene therapy vector is disclosed in U.S. Patent No. 6,153,436, which is incorporated herein by reference.
  • Adenoviral vectors can also be useful for purposes of delivering the exogenous nucleic acid in accordance with the present invention.
  • U.S. Patent No. 6,001,816 discloses an adenoviral vector, which is used to deliver a leptin gene intravenously to a mammal to treat obesity.
  • Other recombinant adenoviral vectors may also be used, which include those disclosed in U.S. Patent Nos. 6,171,855; 6,140,087;
  • viral vectors include recombinant hepatitis viral vectors (See, e.g., U.S. Patent No. 5,981,274), and recombinant entomopox vectors (See, e.g., U.S. Patent Nos. 5,721,352 and 5,753,258).
  • alphavirus vectors may also be desirable.
  • Alphaviruses are positive stranded RNA viruses.
  • the sequence encoding replica structural proteins can be replaced by the exogenous nucleic acid.
  • the alphavirus vectors carrying the exogenous nucleic acid can be targeted to dendritic cells to facilitate the stimulation of cytotoxic T lymphocytes. See Polo et al, Proc. Natl. Acad. Sci. USA, 96:4598-4603 (1996).
  • bacterial vectors can also be used. That is, the exogenous nucleic acid to be delivered can be incorporated into attenuated bacteria such as attenuated salmonella and shigella. See Levine et al, J. Biotechnol, 44:193-196 (1996); Tacket et al, Infect. Immun., 65:3381-3385 (1997); and Sizemore et al, Vaccine, 15:804-807 (1997).
  • the exogenous nucleic acid itself or a plasmid or viral or other expression vector carrying the exogenous nucleic acid can be introduced into a patient by various methods known in the art.
  • the exogenous nucleic acid incorporated into a viral or bacterial vector can be administered to patients by direct infection.
  • the nucleic acid carried by plasmid vectors can be used as DNA or RNA vaccines, i.e., in the form of naked DNA or RNA, that are administered directly into an appropriate tissue or organ of a patient.
  • the naked DNA or RNA can be delivered by injection into skin, muscle or other tissues.
  • a gene gun or an equivalent device thereof can be used for delivery into skin or mucous membrane or other tissues.
  • DNA into mammalian cells by conjugating the DNA to be delivered with a polyelectrolyte to form a complex.
  • the complex may be microinjected into or taken up by cells.
  • exogenous nucleic acid or plasmid DNA vector containing the exogenous gene may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Patent No. 6,090,619; Wu and Wu, J. Biol. Chem., 263: 14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991).
  • U.S. Patent No. 6,090,619 Wu and Wu, J. Biol. Chem., 263: 14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991).
  • 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine, having 3-100 lysine residues), which is itself coupled to an integrin receptor binding moiety (e.g. , a cyclic peptide having the amino acid sequence RGD).
  • a polycation moiety e.g., poly-L-lysine, having 3-100 lysine residues
  • an integrin receptor binding moiety e.g. , a cyclic peptide having the amino acid sequence RGD
  • the exogenous nucleic acid or vectors containing it can also be delivered into cells via amphiphiles. See e.g., U.S. Patent No. 6,071,890.
  • the exogenous nucleic acid or a vector containing the nucleic acid forms a complex with the cationic amphiphile. Cells in a patient's body contacted with the complex can readily absorb the complex.
  • a penetratin is used as a transporter.
  • the homeodomain of Antennapedia, a Drosophila transcription factor can be used as a transporter to deliver a polypeptide of the present invention.
  • any suitable member of the penetratin class of peptides can be used to carry a polypeptide of the present invention into cells.
  • Penetratins are disclosed in, e.g., Derossi et al, Trends Cell Biol, 8:84-87 (1998), which is incorporated herein by reference.
  • Penetratins transport molecules attached thereto across cytoplasm membranes or nucleus membranes efficiently in a receptor- independent, energy-independent, and cell type-independent manner.
  • Methods for using a penetratin as a carrier to deliver oligonucleotides and polypeptides are also disclosed in U.S. Patent No. 6,080,724; Pooga et al, Nat. Biotech., 16:857 (1998); and Schutze et al, J. Immunol, 157:650 (1996), all of which are incorporated herein by reference.
  • U.S. Patent No. 6,080,724 defines the minimal requirements for a penetratin peptide as a peptide of 16 amino acids with 6 to 10 of which being hydrophobic. The amino acid at Attorney Docket No. 5010.01WO
  • the helix 3 of the homeodomain of Drosophila Antennapedia is used as a transporter. More preferably, a peptide having a sequence of the amino acids 43-58 of the homeodomain Antp is employed as a transporter.
  • other naturally occurring homologs of the helix 3 of the homeodomain of Drosophila Antennapedia can also be used. For example, homeodomains of Fushi-tarazu and Engrailed have been shown to be capable of transporting peptides into cells. See Han et al, Mol.
  • penetratin also encompasses peptoid analogs of the penetratin peptides.
  • penetratin peptides and peptoid analogs thereof are covalently linked to a compound to be delivered into cells thus increasing the cellular uptake of the compound.
  • a method for treating and/or preventing HCV infection and symptoms thereof comprising administering to a patient in need of treatment cells displaying HCV altered budding phenotype. That is, cells displaying HCV altered budding phenotype are prepared in vitro and delivered to a patient in need of treatment.
  • antigen preparations obtain from cells displaying an HCV altered budding phenotype can be used. Such antigen preparations can be prepared by an ordinary skilled artisan in apprised of the instant disclosure.
  • cells from a variety of sources may be employed to prepare cells displaying HCV altered budding phenotype, mammalian cells including human cells and others may be preferable.
  • the methods described above for causing, in the body, the formation of cells displaying HCV altered budding phenotype can be used in in vitro procedures to create cells displaying HCV altered budding phenotype.
  • a mutant HCV NS5B or core polypeptide sufficient for viral particle assembly but devoid of the YPDL (or FPDL) motif in the NS5B polypeptide (or devoid of the PXXP motif in the core protein) or devoid of the entire NS5B polypeptide may be Attorney Docket No. 5010.01WO
  • a wild-type or mutant HCV NS5B polypeptide (or HCV core protein) sufficient for viral particle assembly or a nucleic acid encoding such a polypeptide can be administered to cells defective in respect to one or more proteins required for HCV budding.
  • human cells with the AP-2 complex, particularly the AP-50 subunit gene being knocked out may be used for this purpose.
  • dominant-negative mutations may also be used to prevent HCV budding from the cells and an altered budding phenotype when a wild-type or mutant HCV NS5B polypeptides sufficient for viral particle assembly is produced in the cells.
  • Methods for creating AP-2 complex, particularly the AP-50 subunit-deficient cells or cells with dominant-negative mutations are apparent to skilled artisans as well as TSG101 defective cells.
  • a nucleic acid encoding a wild-type or mutant HCV NS5B and/or HCV core polypeptide or vectors containing the nucleic acid can be introduced into cells in vitro using any known techniques such as calcium phosphate precipitation, microinjection, lipofection, electroporation, gene gun, receptor-mediated endocytosis, and the like.
  • the wild-type or mutant HCV NS5B and/or HCV core polypeptides can be introduced into cells in vitro by attaching the polypeptide to a transporter as described above capable of increasing the cell uptake of the polypeptide.
  • the cells displaying or capable of displaying HCV altered budding phenotype can be administered to a patient by, e.g., injection or cell transplantation.
  • the present invention includes compounds and compositions that affect the ability of HCV to bud. Furthermore, the invention provides methods of detecting compounds that affect the ability of HCV to bud. The compounds and methods of detecting them are based on the ability to affect an interaction between a viral late-domain motif containing protein and the host cell partner protein(s) with which it interacts during viral budding.
  • any compounds capable of interfering with the protein-protein interaction between a host cell late-domain motif binding partner and a viral protein containing a late-domain motif may be used.
  • useful compounds include those that interfere with, block, disrupt or destabilize the protein-protein interaction; block or interfere with the formation of a protein complex by, e.g., the AP-2 complex, particularly the AP-50 subunit (or AIP1) and HCV NS5B; or destabilize, disrupt or dissociate an existing protein complex comprising the AP-2 complex, particularly the AP-50 subunit and HCV NS5B, and similarly HCV core protein and TSG101.
  • Such compounds can be selected by various screening assays known in the art.
  • test compounds may be screened in an in vitro assay to select interaction antagonists.
  • An AP-2 complex (particularly the AP-50 subunit)-HCV NS5B protein complex can be contacted with a test compound and disruption or destabilization of the protein complex can be detected.
  • the presence or absence of the protein complex can be detected by an antibody selectively immunoreactive with the protein complex.
  • immunoprecipitation assay can be conducted with the antibody. If the test compound disrupts the protein complex, then the amount of immunoprecipitated protein complex in this assay will be significantly less than that in a control assay in which the same protein complex is not contacted with the test compound.
  • one of the interacting partners with a detectable marker fused thereto is fixed to a solid support.
  • a GST-NS5B fusion protein is attached to a solid support.
  • the other interacting partner with a detectable marker fused thereto e.g., a myc-tagged the AP-2 complex, preferably the AP-50 subunit
  • the immobilized first interacting partner in the presence of one or more test compounds.
  • the myc-tagged AP-2 complex or the myc-tagged AP- 50 subunit is also immobilized, which can be detected using an anti-myc antibody after the binding reaction mixture is washed to remove unbound myc-tagged AP-2 complex or the myc-tagged AP-50 subunit fragment (or AIP1).
  • the assays of the invention include testing for molecules that affect the interaction between TSG101 and HCV core protein.
  • test compounds can also be screened in any in vivo assays to select compounds capable of interfering with the interaction between, e.g., AP-2 complex, particularly the AP-50 subunit (or AIP1) and HCV NS5B.
  • AP-2 complex particularly the AP-50 subunit (or AIP1) and HCV NS5B.
  • Any in vivo assays known in the art useful in selecting compounds capable of interfering with the stability of the protein complexes of the present invention may be used.
  • one of the yeast two-hybrid systems or their analogous or derivative forms is used.
  • suitable two-hybrid systems known in the art include, but are not limited to, those disclosed in U.S. Patent Nos. 5,283,173; 5,525,490; 5,585,245; 5,637,463; 5,695,941; 5,733,726; 5,776,689; 5,885,779; 5,905,025; 6,037,136; 6,057,101; 6,114,111; and Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, NY, 1997, all of which are incorporated herein by reference.
  • two chimeric genes are prepared encoding two fusion proteins: one contains a transcription activation domain fused to an interacting protein member of a protein complex of the present invention or an interacting domain of the interacting protein member, while the other fusion protein includes a DNA binding domain fused to another interacting protein member of the protein complex or an interacting domain thereof.
  • AP-2 complex particularly the AP-50 subunit (or AIP1), a mutant form or a binding domain thereof, and HCV NS5B, or a mutant form or a binding domain thereof, are used as test proteins expressed in the form of fusion proteins as described above for purposes of a two-hybrid assay.
  • Similar constructs and assays for TSG101 and HCV core protein can be prepared and used in the Attorney Docket No. 5010.01WO
  • the fusion proteins are expressed in a host cell and allowed to interact with each other in the presence of one or more test compounds.
  • a counterselectable marker is used as a reporter such that a detectable signal (e.g., appearance of color or fluorescence, or cell survival) is present only when the test compound is capable of interfering with the interaction between the two test proteins.
  • a detectable signal e.g., appearance of color or fluorescence, or cell survival
  • the reporters used in various "reverse two- hybrid systems" known in the art may be employed. Reverse two-hybrid systems are disclosed in, e.g., U.S. Patent Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al, Proc. Natl Acad. Sci. USA, 93:10315-10320 (1996); and Vidal et al, Proc. Natl. Acad. Sci. USA, 93:10321-10326 (1996), all of which are incorporated herein by reference.
  • Suitable counterselectable reporters useful in a yeast system include the URA3 gene (encoding orotidine-5' -decarboxylase, which converts 5-fluroorotic acid (5-FOA) to the toxic metabolite 5-fluorouracil), the CAN!
  • the GAL1 gene encoding galactokinase, which catalyzes the conversion of 2-deoxygalactose to toxic 2-deoxygalactose- 1 -phosphate
  • the LYS2 gene encoding ⁇ -aminoadipate reductase, which renders yeast cells unable to grow on a medium containing - aminoadipate as the sole nitrogen source
  • the METIS gene encoding O- acetylhomoserine sulfhydrylase, which confers on yeast cells sensitivity to methyl mercury
  • the CYH2 gene encoding L29 ribosomal protein, which confers sensitivity to cycloheximide
  • any known cytotoxic agents including cytotoxic proteins such as the diphtheria toxin (DTA) catalytic domain can also be used as counterselectable reporters. See U.S. Patent No. 5,733,726. DTA causes the ADP-ribosylation of elongation factor-2 and thus inhibits protein synthesis and causes cell death.
  • Other examples of cytotoxic agents include ricin, Shiga toxin, and exotoxin A of Pseudomonas aeruginosa.
  • yeast cells containing a mutant URA3 gene can be used as host cells (Ura " Foa R phenotype) for the in vivo assay.
  • Such cells lack £/i 43-encoded functional orotidine-5 '- phsphate decarboxylase, an enzyme required for the biosynthesis of uracil.
  • yeast cells are unable to grow on media lacking uracil.
  • the yeast cells cannot convert non-toxic 5-fluoroorotic acid (5-FOA) to a toxic product, 5-fiuorouracil.
  • 5-FOA non-toxic 5-fluoroorotic acid
  • yeast cells are resistant to 5-FOA and can grow on a medium containing 5-FOA.
  • AP-2 complex particularly the AP-50 subunit can be expressed as a fusion protein with a DNA-binding domain of a suitable transcription activator while HCV NS5B is expressed as a fusion protein with a transcription activation domain of a suitable transcription activator.
  • the reporter URA3 gene may be operably linked to a promoter specifically responsive to the association of the transcription activation domain and the DNA-binding domain.
  • an in vivo screening assay can be conducted in the presence of a test compound with the yeast cells being cultured on a medium containing uracil and 5-FOA. If the test compound does not disrupt the interaction between AP-2 complex, particularly the AP-50 subunit and HCV NS5B, active URA3 gene product, i.e., orotidine-5 '-decarboxylase, which converts 5-FOA to toxic 5-fluorouracil, is expressed. As a result, the yeast cells cannot grow.
  • test compound disrupts the interaction between AP-2 complex, particularly the AP-50 subunit and HCV NS5B
  • no active orotidine-5 '-decarboxylase is produced in the host yeast cells. Consequently, the yeast cells will survive and grow on the 5-FOA-containing medium. Therefore, compounds capable of interfering with or dissociating the interaction between AP-2 complex, particularly the AP-50 subunit (or AIP1) and HCV NS5B can thus be identified based on colony formation.
  • the screening assay of the present invention can be applied in a format appropriate for large-scale screening.
  • combinatorial technologies can be employed to construct combinatorial libraries of small organic molecules or small peptides. See generally, e.g., Kenan et al, Trends Biochem. Sc, 19:57-64 (1994); Gallop et al, J. Med. Chem., 37:1233-1251 (1994); Gordon et al, J. Med. Chem., 37:1385-1401 (1994); Ecker et al, Biotechnology, 13:351-360 (1995).
  • Such combinatorial libraries of compounds can be applied to the screening assay of the present invention to isolate specific modulators of particular protein-protein interactions.
  • the random peptides can be co-expressed with the fusion proteins of the present invention in host cells and assayed in vivo. See e.g., Yang et al, Nucl. Acids Res., 23: 1152-1156 (1995). Alternatively, they can be added to the culture medium for uptake by the host cells. Conveniently, yeast mating is used in an in vivo screening assay. For example, haploid cells of a-mating type expressing one fusion protein as described above is mated with haploid cells of alpha-mating type expressing the other fusion protein. Upon mating, the diploid cells are spread on a suitable medium to form a lawn.
  • Drops of test compounds can be deposited onto different areas of the lawn. After culturing the lawn for an appropriate period of time, drops containing a compound capable of modulating the interaction between the particular test proteins in the fusion proteins can be identified by stimulation or inhibition of growth in the vicinity of the drops.
  • the screening assays for selecting compounds capable of interfering with protein- protein interactions can also be fine-tuned by various techniques to adjust the thresholds or sensitivity of the positive and negative selections. Mutations can be introduced into the reporter proteins to adjust their activities.
  • the uptake of test compounds by the host cells can also be adjusted. For example, yeast high uptake mutants such as the ergo mutant strains can facilitate yeast uptake of the test compounds. See Gaber et al, Mol. Cell. Biol, 9:3447-3456 (1989).
  • the uptake of the selection compounds such as 5-FOA, 2-deoxygalactose, cycloheximide, ⁇ -aminoadipate, and the like can also be fine-tuned.
  • test compounds may be screened in the screening assays of the present invention to select compounds capable of interfering with the interaction of the HCV late-domain motif containing protein and the host cell late-domain motif binding partner.
  • AP-2 complex particularly the AP-50 subunit (or AIPl) and HCV NS5B or HCV core protein and TSG101.
  • the test compounds may include, by way of example, proteins (e.g., antibodies, small peptides, artificial or natural proteins), nucleic acids, and derivatives, mimetics and analogs thereof, and small organic molecules having a molecular weight of no greater than 10,000 daltons, more preferably less than 5,000 daltons.
  • the test compounds are provided in library formats known in the art, Attorney Docket No. 5010.01WO
  • recombinant expression libraries e.g., phage display libraries
  • in vitro translation-based libraries e.g., ribosome display libraries
  • the compound capable of interfering with the interaction between AP-2 complex, particularly the AP-50 subunit and HCV NS5B used in the methods of the present invention comprises an amino acid sequence motif of YXiX 2 L and capable of binding the AP-2 complex, particularly the AP-50 subunit (or AIPl), wherein Xi and X 2 are any amino acids.
  • the compound which comprises the amino acid sequence motif YX ⁇ 2 L and is capable of binding the AP-2 complex, particularly the AP-50 subunit can be of any type of chemical compounds so long as the compound is capable of binding the AP-2 complex, particularly the AP-50 subunit.
  • the compound can be a peptide, a modified peptide, an oligonucleotide-peptide hybrid (e.g., PNA), etc.
  • the compound can also include a longer peptide comprising the amino acid sequence motif of YXjX 2 L and capable of binding the AP-2 complex, particularly the AP-50 subunit (or AIPl).
  • the compound may include a peptide of 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids.
  • the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids of a naturally occurring HCV NS5B sequence.
  • the contiguous span has less than 50, preferably less than 40, more preferably less than 30 amino acids.
  • the contiguous span should span the HCV late domain motif which can be the YXXL motif or a variation thereof.
  • the late domain motif in the contiguous span is the YXXL motif.
  • the YX ⁇ X 2 L motif in the compound according to the present invention is within an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least 90 percent or 95 percent identical to a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids, but preferably less than 50 or 40, more preferably less than 30 amino acids, of a naturally occurring HCV late-domain motif containing sequence, which contiguous span of amino acids spans the HCV late domain motif.
  • an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least 90 percent or 95 percent identical to a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids, but preferably less than 50 or 40, more preferably less than 30 amino acids, of a naturally occurring HCV late-domain motif containing sequence, which contiguous span of amino acids spans the HCV late domain motif.
  • the percentage identity is determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77 (1993), which is incorporated into the various BLAST programs. Specifically, the percentage identity is determined by the "BLAST 2 Sequences" tool, which is available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. See Tatusova and Madden, FEMS Microbiol Lett., 174(2):247-50 (1999).
  • the BLASTP 2.1.2 program is employed using default parameters (Matrix: BLOSUM62; gap open: 11; gap extension: l; x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter). It should be understood that such homologue peptides should retain the ability to bind the host protein interacting partner (e.g., TSGIOI for PXXP-like motifs, and the AP-50 subunit or the AP-2 complex for YXXL-like motifs).
  • the host protein interacting partner e.g., TSGIOI for PXXP-like motifs, and the AP-50 subunit or the AP-2 complex for YXXL-like motifs.
  • the homologues can be made by site-directed mutagenesis based on a late domain motif-containing HCV strain polyprotein sequence of HCV or portion thereof.
  • the site- directed mutagenesis can be designed to generate amino acid substitutions, insertions, or deletions. Methods for conducting such mutagenesis should be apparent to skilled artisans in the field of molecular biology.
  • the resultant homologues can be tested for their binding affinity to TSGIOI, the AP-2 complex, particularly the AP-50 subunit (or AIPl).
  • the peptide portion in the compounds according to the present invention can also be in a modified form.
  • modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, prenylated forms, etc.
  • Modifications also include infra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc.
  • modifications may also include cyclization, and branching.
  • Amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide sequence in the compound of the present invention.
  • the compounds may include D- amino acids in place of L-amino acids.
  • various protection groups can also be incorporated into the amino acid residues of the compounds.
  • terminal residues are preferably protected.
  • Carboxyl groups may be protected by esters (e.g., methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl or t- amyl esters, etc.), lower alkoxyl groups (e.g., methoxy, ethoxy, propoxy, butoxy, etc.), aralkyloxy groups (e.g., benzyloxy, etc.), amino groups, lower alkylamino or di(lower alkyl)amino groups.
  • esters e.g., methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl or t- amyl esters, etc.
  • lower alkoxyl groups e.g., methoxy, ethoxy, propoxy, butoxy, etc.
  • aralkyloxy groups
  • lower alkoxy is intended to mean an alkoxy group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. Protection groups for amino groups may include lower alkyl, benzyloxycarbonyl, t- butoxycarbonyl, and sobomyloxycarbonyl. "Lower alkoxy” is intended to mean an alkyl group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. In one example, a 5-oxo-L-prolyl residue may be used in place of a prolyl residue. A 5-oxo-L-prolyl residue is especially desirable at the N-terminus of a peptide compound.
  • peptide mimetics can be designed based on the above-described YXiX 2 L motif-containing compounds according to the present invention.
  • the mimetics must be capable of binding TSGIOI, or the AP-2 complex, particularly the AP-50 subunit (or AIPl).
  • peptoid analogs of the YPDL (or FPDL) motif can be prepared using known methods.
  • Peptoids are oligomeric N-substituted glycines.
  • various side chain groups can be included when forming an N- substituted glycine (peptoid monomer) that mimics a particular amino acid.
  • Peptoid monomers can be linked together to form an oligomeric N-substituted glycines - peptoid.
  • Peptoids are easy to synthesize in large amounts.
  • the backbone linkage of peptoids are resistant to hydrolytic enzymes.
  • peptoid analogs corresponding to any peptides can be produced with improved characterics. See Simon et al, Proc. Natl. Acad. Sci.
  • peptoid analogs of the above-described YX ⁇ X 2 L motif-containing compounds of the present invention can be made using methods known in the art.
  • the prepared peptoid analogs can be tested for their binding affinity to AP-2 complex, particularly the AP-50 subunit. They can also be tested in anti-viral assays for their ability to inhibit virus budding from infected host cells and ability to inhibit virus propagation. In particular, they can be tested for ability to suppress HCV budding from infected cells and inhibit HCV propagation.
  • Mimetics of the compounds of the present invention can also be selected by rational drug design and/or virtual screening.
  • Methods known in the art for rational drug design can be used in the present invention. See, e.g., Hodgson et al, Bio/Technology, 9:19-21 (1991); U.S. Patent Nos. 5,800,998 and 5,891,628, all of which are incorporated herein by reference.
  • Structural information on the AP-2 complex, particularly the AP-50 subunit and/or the binding complex formed by the AP-2 complex, particularly the AP-50 subunit and the YPDL (or FPDL) motif are obtained.
  • the interacting complex can be studied using various biophysics techniques including, e.g.
  • structural information can also be obtained from protein complexes formed by the AP-2 complex, particularly the AP-50 subunit and a variation of the YPDL (or FPDL) motif. Similar methodologies can be applied to TSGIOI and the HCV core protein PXXP motifs. Computer programs are employed to select compounds based on structural models of the binding complex formed by the AP-2 complex, particularly the AP-50 subunit (or AIPl) and the HCV YPDL (or FPDL) motif. In addition, once an effective compound is identified, structural analogs or mimetics thereof can be produced based on rational drug design with the aim of improving drug efficacy and stability, and reducing side effects.
  • understanding of the interaction between TSGIOI, or the AP-2 complex, particularly the AP-50 subunit and compounds of the present invention can also be derived from mutagenesis analysis using yeast two-hybrid system or other methods for detection protein-protein interaction.
  • various mutations can be introduced into the interacting proteins and the effect of the mutations on protein-protein interaction Attorney Docket No. 5010.01WO
  • mutations including amino acid substitutions, deletions and insertions can be introduced into the protein sequence of TSGIOI, or the AP-2 complex, particularly the AP-50 subunit domain and/or a compound of the present invention using conventional recombinant DNA technologies.
  • it is particularly desirable to decipher the protein binding sites.
  • Mutations are preferably designed based on knowledge of the three-dimensional structure of the interacting proteins.
  • mutations are introduced to alter charged amino acids or hydrophobic amino acids exposed on the surface of the proteins, since ionic interactions and hydrophobic interactions are often involved in protein-protein interactions.
  • the "alanine scanning mutagenesis" technique is used.
  • a structural model can be established by a modeling process that may incorporate data from NMR analysis, X-ray diffraction data, alanine scanning, specfroscopic techniques and the like.
  • Various techniques including Attorney Docket No. 5010.01WO
  • a template can be formed based on the established model.
  • Various compounds can then be designed by linking various chemical groups or moieties to the template.
  • Various moieties of the template can also be replaced.
  • These rationally designed compounds are further tested. In this manner, pharmacologically acceptable and stable compounds with improved efficacy and reduced side effect can be developed.
  • the compounds identified in accordance with the present invention can be incorporated into a pharmaceutical formulation suitable for administration to an individual.
  • the mimetics including peptoid analogs can exhibit optimal binding affinity to the AP-2 complex, particularly the AP-50 subunit, AIPl (or TSGIOI) or animal orthologs thereof.
  • the AP-2 complex particularly the AP-50 subunit -binding characteristics of the mimetics.
  • the AP-2 complex particularly the AP-50 subunit protein (or TSGIOI) or a fragment thereof can be recombinantly expressed, purified, and contacted with the mimetics to be tested. Binding can be determined using a surface plasmon resonance biosensor. See e.g., Panayotou et al, Mol. Cell. Biol, 13:3567-3576 (1993). Other methods known in the art for estimating and determining binding constants in protein- protein interactions can also be employed.
  • binding constant is then estimated based on the concentrations of the bound protein and the eluted protein.
  • the method of sedimentation through gradients monitors the rate of sedimentation of a mixture of proteins through gradients of glycerol or sucrose. At concentrations above the binding constant, the two interacting members sediment as a complex. Thus, binding constant can be calculated based on the concentrations.
  • Suitable methods known in the art for estimating binding constant include but are not limited to gel filtration column such as nonequilibrium "small-zone” gel filtration columns (See e.g., Gill et al, J. Mol. Biol, 220:307-324 (1991)), the Hummel-Dreyer method of equilibrium gel filtration (See e.g., Hummel and Dreyer, Biochim. Biophys. Ada, 63:530-532 (1962)) and large-zone equilibrium gel filtration (See e.g., Gilbert and Kellett, J. Biol.
  • the compounds capable of interfering with the interaction between the host cell late-domain motif binding partner (i.e., AP-2 complex, particularly the AP-50 subunit, AIPl, or TSG101) and the HCV late-domain motif containing protein (NS5B or core protein) can be delivered into cells by direct cell internalization, receptor mediated endocytosis, or via a "fransporter.” It is noted that a compound preferably is delivered into patient's cells in order to achieve optimal results.
  • peptidic transporters as described above such as penetratins are employed. Fusion proteins can be conveniently made by recombinant expression to contain a fransporter peptide covalently linked by a peptide bond to a peptide having the Y K.jL motif.
  • conventional methods can be used to chemically synthesize a transporter peptide or a peptide of the present invention or both.
  • an adjuvant capable of stimulating immune response is also administered to a patient who is treated with cells displaying HCV altered budding phenotype or with a compound (nucleic acids, polypeptides or small organic compounds) capable of causing the formation, in a patient's body, of cells displaying HCV altered budding phenotype.
  • adjuvant means any substance that is not a component of HCV but is capable of stimulating or enhancing immune responses to an immunogen administered to a patient.
  • the disruption or interference with the protein-protein interaction between the host cellular late-domain motif binding partner protein (AP-2 complex, particularly the AP-50 subunit or TSGIOI) and a HCV late-domain containing protein (NS5B or core) is contemplated in itself be effective in treating and/or preventing HCV infection and symptoms thereof by way of inhibiting HCV viral budding and propagation.
  • the disruption or interference with the protein-protein interaction between the host cellular late-domain motif binding partner protein (AP-2 complex, particularly the AP-50 subunit, or TSGIOI) and the HCV late-domain motif containing protein (i.e., NS5B or core protein) may also induce the formation of cells displaying HCV altered budding phenotype which act as immunogens in eliciting immune responses in the patient.
  • AP-2 complex particularly the AP-50 subunit, or TSGIOI
  • HCV late-domain motif containing protein i.e., NS5B or core protein
  • the compounds administered to a patient capable of disrupting or interfering with the protein-protein interaction between the host cell late-domain motif binding partner (the AP-2 complex, particularly the AP-50 subunit, or TSGIOI) and the HCV late-domain motif containing protein (HCV NS5B or core protein) can also stimulate immune responses in the patient against HCV viruses, particularly cytotoxic T lymphocytes (CTL) response.
  • the host cell late-domain motif binding partner the AP-2 complex, particularly the AP-50 subunit, or TSGIOI
  • HCV late-domain motif containing protein HCV late-domain motif containing protein
  • CTL cytotoxic T lymphocytes
  • an adjuvant capable of enhancing the patient's immune response particularly cytotoxic T lymphocytes (CTL) response
  • CTL cytotoxic T lymphocytes
  • a compound capable of disrupting or interfering with the protein-protein interaction between the host cell late- domain motif binding partner i.e., AP-2 complex, particularly the AP-50 subunit, or TSG 101
  • AP-2 complex particularly the AP-50 subunit, or TSG 101
  • HCV late-domain motif containing protein NS5B or core protein
  • an adjuvant capable of enhancing patient immune response would also significantly boost the prophylactic and/or therapeutic effect when coupled with a mutant HCV NS5B or core polypeptide that is sufficient for viral particle assembly but causes an altered budding phenotype in cells, or a nucleic acid encoding the mutant HCV NS5B or core polypeptide, or with cells displaying HCV altered budding phenotype.
  • any adjuvant may be used so long as the adjuvant is capable of stimulating immune response against cells displaying HCV altered budding phenotype or against the HCV viral proteins in such cells, and thus can enhance immune response against HCV viruses in a patient.
  • alum has long been used as an adjuvant for human use.
  • suitable adjuvant is MF59. See Minutello et al, Vaccine, 17:99-104 (1999).
  • Another useful adjuvant in development that may also be used is LTR72, a mutant of E. coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. See Giuliani et al, J. Exp.
  • Polyphosphazine adjuvant may also be used.
  • cytokines and lymphokines may also be used as adjuvants along with the cells, polypeptides, nucleic acids and/or a compound capable of disrupting or interfering with the protein-protein interaction between TsglOl and HCV core protein or HCV NS5B and AP-50.
  • cytokines and lymphokines include, but are not limited to, interleukins such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, interferons such as alpha-interferon and gamma-interferon, tumor necrosis factor (TNF), platelet derived growth factor (PDGF), GCSF, granulocyte-macrophage colony- stimulating factor (GM-CSF), epidermal growth factor (EGF), and the like.
  • TNF tumor necrosis factor
  • PDGF platelet derived growth factor
  • GCSF granulocyte-macrophage colony- stimulating factor
  • EGF epidermal growth factor
  • adjuvants capable of stimulating cellular immune responses particularly cytotoxic T lymphocytes (CTL) responses, are used.
  • CTL cytotoxic T lymphocytes
  • a synergistic effect on the induction of HCV-specific CTL response can be achieved when adjuvants capable of stimulating CTL response are used in the presence of cells displaying HCV altered budding phenotype. This is especially beneficial to the prevention and treatment of HCN Attorney Docket No. 5010.01WO
  • Thl cells cytokines secreted by helper T cells called Thl cells have been shown to promote cellular responses.
  • Thl type cytokines include interleukin-2 (IL- 2), interleukin-4, and interleukin-12 (IL-12), interleukin-18, among others.
  • fusion proteins having one of such Thl type cytokines e.g., IL-2) fused to the Fc portion of immunoglobulin G (IgG) may also be used. See e.g., Barouch et al, Science, 290:486- 492 (2000), which is incorporated herein by reference.
  • chemokines capable of stimulating cell-mediated immune responses include interferons such as alpha- interferon, beta-interferon and gamma-interferon.
  • Certain chemokines may also be used to enhance cellular immune responses. Such chemokines are small molecules that attract T cells to infected tissues.
  • certain short bacterial immunostimulatory DNA sequences ISSs have also been discovered as CTL response-promoting adjuvants that potently stimulate immune responses to co-administered antigens. See Roman et al, Nat. Med., 3:849-854 (1997), which is incorporated herein by reference.
  • noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides can also be used in the present invention as adjuvants to enhance cellular immunity against cells displaying HCV altered budding phenotype.
  • One or more adjuvants can be used for purposes of the present invention.
  • the adjuvant(s) can be administered in the same composition as the cells or compounds as described above. Alternatively, the adjuvant(s) can also be given to a patient separately.
  • the present invention also provides compositions comprising cells displaying HCV altered budding phenotype and one or more adjuvants capable of enhancing immune responses in a patient.
  • the compositions include cells displaying HCV altered budding phenotype and one or more adjuvants capable of enhancing cellular immune response, cell-mediated immune response or CTL response.
  • Suitable adjuvants are proteins or peptides. Such adjuvants can be administered to patients in the form of Attorney Docket No. 5010.01WO
  • nucleic acids e.g., DNA, RNA, and the like
  • administering an adjuvant to a patient or a paraphrase thereof also encompasses administering a nucleic acid encoding a protein adjuvant and capable of expressing the adjuvant in a patient's body. Methods for administering nucleic acids are described above.
  • the present invention provides a composition
  • a composition comprising at least (1) a first nucleic acid encoding a mutant HCV NS5B or core polypeptide sufficient for viral particle assembly but devoid of late domain motifs, and (2) a second nucleic acid encoding an adjuvant capable of stimulating cytotoxic T lymphocyte (CTL) response.
  • the two nucleic acids can be incorporated into separate expression vectors.
  • the two nucleic acids are carried within the same expression vector.
  • each of the two nucleic acids is operably linked to a transcriptional promoter, which drives the transcription from the nucleic acid linked to the promoter.
  • the nucleic acid encoding a mutant HCV NS5B or core polypeptide sufficient for viral particle assembly but devoid of late domain motifs is capable of expressing the mutant HCV NS5B or core polypeptide.
  • the nucleic acid may contain one or more mutations that decrease the effect of an inhibitory/instability sequence that is present in the corresponding nucleotide sequence of the native HCV NS5B or core nucleic acid.
  • compositions according to the present invention are distinct from other known or commercially available approaches for treating or preventing HCV infection and symptoms thereof. Therefore, it may be desirable to employ combination therapies to combine a method of the present invention with other prophylactic or treatment methods so as to increase the prophylactic or therapeutic effect.
  • prophylactic materials in the methods of the present invention and in other methods may be combined in the same pharmaceutical composition or administered separately.
  • one or more adjuvants described above may be included in the same composition or administered separately in the combination therapy approaches.
  • such other anti-HCV agents should be pharmaceutically compatible with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention.
  • pharmaceutically compatible it is intended that the other anti-viral agent(s) will not interact or react with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention, directly or indirectly, in such a way as to substantially adversely affect the effect of the freatment, or to cause any significant adverse side reaction in the patient.
  • the active agents are administered to a patient in a pharmaceutical composition, which typically includes one or more pharmaceutically acceptable carriers that are inherently nontoxic and non- therapeutic.
  • the pharmaceutical composition according to the present invention may be administered to a subject needing treatment or prevention through any appropriate routes such as parenteral, oral, mucosal or topical administration.
  • the active agents of this invention are administered at a therapeutically or prophylactically effective amount to achieve the desired therapeutic and/or prophylactic effect without causing any serious adverse effects in the patient treated.
  • the toxicity profile and therapeutic or prophylactic efficacy of the active agents can be determined by standard pharmaceutical procedures in suitable cell models or animal models or human clinical trials.
  • the LD 50 represents the dose lethal to about 50% of a tested population.
  • the ED 50 is a parameter indicating the dose therapeutically or prophylactically effective in about 50% of a tested population.
  • Both LD5 0 and ED 50 can be determined in cell models and animal models.
  • the IC 50 may also be obtained in cell models and Attorney Docket No. 5010.01 WO
  • the dosage range for human use should be designed such that the range centers around the ED 50 and/or IC 50 , but significantly below the LD 50 obtained from cell or animal models.
  • the cells displaying HCV altered budding phenotype may be effective at an amount of from about 10 cells to about 20x10 6 per dosage with an administration frequency of once per year, once per month up to once per day.
  • the cells can be injected in a composition having a suitable pharmaceutically acceptable carrier.
  • a suitable pharmaceutically acceptable carrier may include cells suspended in a standard sterile cell culture medium, preferably devoid of serum.
  • the cells may be simply suspended in a saline solution, e.g., PBS, or other standard cell transplantation carrier before administration.
  • the composition should contain one or more agents for maintaining optimal isotonicity, isomocity and pH. Suitable adjuvant(s) may also be included in the composition.
  • the cell-containing composition can be administered to a patient through any suitable routes, e.g., by parenteral injection or transplantation. Preferably, the cells are injected intravenously.
  • the nucleic acid can be administered in an amount of from about 0.1 microgram to about 5000 milligram, preferably from about 1 microgram to about 500 milligram per dosage. The appropriate amount can be administered daily, weekly, monthly, bimonthly, semi-annually or annually.
  • the nucleic acid can be administered to a patient in a manner as described above in Section 1 or by a standard procedure known in the art, as will be apparent to skilled artisans.
  • mutant HCV NS5B and core polypeptides and the other active peptidic or small organic compounds of the present invention capable of causing the formation of cells displaying HCV altered budding phenotype can be effective at an amount of from about 0.01 microgram to about 5000 mg per day, preferably from about 1 Attorney Docket No. 5010.01WO
  • microgram to about 2500 mg per day can vary with the body weight of the patient treated and the state of disease conditions.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time.
  • the suitable dosage unit for each administration of the compounds of the present invention can be, e.g., from about 0.01 microgram to about 2000 mg, preferably from about 1 microgram to about 1000 mg.
  • a therapeutically or prophylactically effective amount of another anti-HCV compound can be administered in a separate pharmaceutical composition, or alternatively included in the pharmaceutical composition that contains a compound according to the present invention.
  • the pharmacology and toxicology of many of such other anti-HCV compounds are known in the art. See e.g., Physicians Desk Reference, Medical Economics, Montvale, NJ; and The Merck Index, Merck & Co., Rahway, NJ.
  • the therapeutically or prophylactically effective amounts and suitable unit dosage ranges and route of administration of such compounds used in art can be equally applicable in the present invention.
  • the therapeutically or prophylactically effective amount for each active agent can vary with factors including but not limited to the activity of the agent used, stability of the active agent in the patient's body, the severity of the conditions to be alleviated, the purpose of the treatment (prophylactic vs. therapeutic), the total weight of the patient treated, the route of administration, the ease of absorption, distribution, and excretion of the active agent by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan.
  • the amount of administration can also be adjusted as the various factors change over time.
  • the active agents according to this invention can be administered to patients to be treated through any suitable routes of administration.
  • the active agents are delivered to the patient parenterally, i.e., by intravenous, intramuscular, infraperiotoneal, intracisternal, subcutaneous, or intraarticular injection or infusion.
  • the non-cell active agents can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use.
  • Lyophilized compositions may include pharmaceutically acceptable carriers such as gelatin, DL-lactic and glycolic acids copolymer, D-mannitol, etc.
  • diluent containing, e.g., carboxymethylcellulose sodium, D-mannitol, polysorbate 80, glycerine, and water may be employed.
  • Lyophilized forms may be stored in, e.g., a dual chamber syringe with one chamber containing the lyophilized composition and the other chamber containing the diluent.
  • the active ingredient(s) can also be incorporated into sterile lyophilized microspheres for sustained release.
  • Methods for making such microspheres are generally known in the art. See U.S. Patent Nos. 4,652,441; 4,728,721; 4,849,228; 4,917,893; 4,954,298; 5,330,767; 5,476,663; 5,480,656; 5,575,987; 5,631,020; 5,631,021; 5,643,607; and 5,716,640 (all of which are incorporated by reference).
  • the pharmaceutical composition can include, in addition to a therapeutically or prophylactically effective amount of an active agent of the present invention, a buffering agent, an isotonicity adjusting agent, a preservative, and/or an anti-absorbent.
  • suitable buffering agent include, but are not limited to, citrate, phosphate, tartrate, succinate, adipate, maleate, lactate and acetate buffers, sodium bicarbonate, and sodium carbonate, or a mixture thereof.
  • the buffering agent adjusts the pH of the solution to within the range of 5-8.
  • Suitable isotonicity adjusting agents include sodium chloride, glycerol, mannitol, and sorbitol, or a mixture thereof.
  • a preservative e.g., anti-microbial agent
  • useful preservatives may include benzyl alcohol, a paraben and phenol or a mixture thereof.
  • compositions such as human serum albumin, gelatin or a mixture thereof may be used as anti- absorbents.
  • conventional solvents, surfactants, stabilizers, pH balancing buffers, and antioxidants can all be used in the parenteral formulations, including but not limited to dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, and the like.
  • the parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes. Attorney Docket No. 5010.01WO
  • the active agents can also be delivered orally in enclosed gelatin capsules or compressed tablets.
  • Capsules and tablets can be prepared in any conventional techniques.
  • the active agents can be incorporated into a formulation which includes pharmaceutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum fragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint).
  • Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets.
  • liquid carriers such as fatty oil can also be included in capsules.
  • oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing the active compounds used in this invention.
  • Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included.
  • the active agents can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil.
  • Topical formulations are generally known in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, drops and aerosols.
  • topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like.
  • a special form of topical adminisfration is delivery by a transdermal patch.
  • the active agents e.g., cells, nucleic acids, polypeptides and small organic molecules
  • the active agents can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant the active Attorney Docket No. 5010.01WO
  • a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material.
  • Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co-L-lactic acid) are suitable for this invention. See, e.g., Phillips et al., J. Pharmaceut. Sci, 73:1718-1720 (1984).
  • microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the active agents.
  • liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Active compounds can be enclosed within such micelles.
  • Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, and Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., both of which are inco ⁇ orated herein by reference.
  • Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomes can reduce the toxicity of the active agents, and increase their stability.
  • Example 1 Analysis of the YPDL (or FPDL) Motif.
  • a yeast two-hybrid assay is utilized to determine the effect of amino acid substitution mutations in the YPDL (or FPDL) motif of HCV NS5B on the interaction between AP-2 complex, particularly the AP-50 subunit and NS5B.
  • a yeast two-hybrid activation domain-AP-50 construct is prepared from a DNA fragment encompassing the Attorney Docket No. 5010.01WO
  • the yeast two-hybrid DNA binding domain-HCV NS5B construct can be prepared from a fragment corresponding to the HCV NS5B polypeptide derived from a HCV strain NS5B protein.
  • Any amino acid substitution mutations can be introduced by PCR into the HCV NS5B sequence in the yeast two-hybrid DNA binding domain-HCV NS5B construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of mutations that can be introduced into the DNA binding domain-HCV NS5B construct are summarized in Table I below.
  • the effect of the mutations can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 frpl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-AP-2 complex (preferably the AP-50 subunit) construct and one of the binding domain-mutant NS5B constructs or the binding domain-wild type NS5B construct.
  • ⁇ -Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive ⁇ -Gal activity indicates that the NS5B wild type or mutant protein interacts with the AP-2 complex, particularly the AP-50 subunit. All binding domain constructs are also tested for self-activation of ⁇ -Gal activity.
  • Interactions between AP-2 complex (preferably the AP-50 subunit) and wild-type NS5B (WT) or the NS5B YPDL (or FPDL) mutants are further quantitated by performing liquid culture ⁇ -galactosidase assays.
  • Cultures are grown overnight in synthetic media (-Leu, -T ⁇ , + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KCl, 6mM MgSO 4 , 360mM Na 2 HPO 4 , 240 mM NaH 2 PO 4 , 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm).
  • 6X Z-buffer 60mM KCl, 6mM MgSO 4 , 360mM Na 2 HPO 4 , 240 mM NaH 2 PO 4 , 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40.
  • Full length AP-2 complex (preferably the AP-50 subunit) bound wild-type NS5B in the two-hybrid liquid culture assay, will result in higher levels of ⁇ -galactosidase activity (e.g. , greater than 10- fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background).
  • Different NS5B point mutants can be used to test whether the AP-2 complex (preferably the AP-50 subunit) binding interaction requires the YPDL late domain motif within HCV NS5B, and will result in reduced ⁇ -galactosidase activity.
  • a fusion protein with a GST tag fused to the HCV NS5B late domain can be recombinantly expressed and purified by chromatography.
  • a NS5B peptide containing selected regions of the protein can be synthesized chemically by standard peptide synthesis methods.
  • the peptide can be purified by conventional protein purification techniques, e.g., by chromatography.
  • a blocking buffer (SuperBlock; Pierce-Endogen, Rockford, IL).
  • 100 ⁇ l of a mixture containing Drosophila S2 cell lysate myc-tagged AP-2 complex (preferably the AP-50 subunit) and a specific amount of the NS5B polypeptide (or fragments thereof) is applied to the wells of the plate. This mixture is allowed to react for 2 hr at room temperature to form NS5B: AP-2 complex (preferably the AP-50 subunit) protein-protein complexes.
  • Plates are then washed 4 x lOO ⁇ l with 1 x PBST solution (Invitrogen; Carlsbad, CA). After washing, lOO ⁇ l of l ⁇ g/ml solution of anti-myc monoclonal antibody (Clone 9E10; Roche Molecular Biochemicals; Indianapolis, IN) in 1 x PBST is added to the wells of the plate to detect the myc-epitope tag on AP-2 (preferably the AP-50 subunit) protein.
  • AP-2 preferably the AP-50 subunit
  • HRP horseradish peroxidase
  • Goat anti-mouse IgG Goat anti-mouse IgG
  • the plates are then washed again with 4 x lOO ⁇ l with 1 x PBST solution and 100 ⁇ l of fluorescent substrate (QuantaBlu; Pierce-Endogen, Rockford, IL) is added to all wells. After 30 min, 100 ⁇ l of stop solution is added to each well to inhibit the function of HRP.
  • Plates are then analyzed on a Packard Fusion instrument at an excitation wavelength of 325 nm and an emission wavelength of 420nm.
  • the presence of fluorescent signals indicates binding of AP-2 complex (preferably the AP-50 subunit) to the fixed HCV NS5B.
  • the absence of fluorescent signals indicates that the YXXL-containing short peptide is capable of disrupting the interaction between AP-2 complex (preferably the AP-50 subunit) and HCV NS5B.
  • Different concentrations of the NS5B late domain peptide are tested, and the relative intensities of the fluorescence signals obtained at different concentrations are plotted against the peptide concentrations.
  • Example 3 Yeast Screen To Identify Small Molecule Inhibitors Of The Interaction Between HCV NS5B And AP-2 complex (preferably the AP-50 subunit).
  • Beta-galactosidase is used as a reporter enzyme to signal the interaction between yeast two-hybrid protein pairs expressed from plasmids in Saccharomyces cerevisiae.
  • Yeast strain MY209 (ade2 his3 leu2 trpl cyh2 ura3::GALlp-lacZ gal4 gal80 lys2 GALlv-HIS3) bearing the plasmids
  • A LEU2 CEN4 ARS1 ⁇ E
  • Hip-SV40NLS- GAL4 (768-881)- AP-2 complex (preferably the AP-50 subunit) -PGKlt AmpR ColEl_o ⁇ ) and B (TRP1 CEN4 ARS ADHl ⁇ -GAL4(l-141)-HCV-NS5B- ADHlX AmpR ColEl m) is cultured in synthetic complete media lacking leucine and fryptophan (SC - Leu -T ⁇ ) overnight at 30°C.
  • This culture is diluted to 0.01 OD 630 units/ml using SC - Leu -T ⁇ media.
  • the diluted MY209 culture is dispensed into 96-well microplates. Compounds from a library of small molecules are added to the microplates; the final concentration of test compounds is approximately 60 ⁇ M.
  • the assay plates are incubated at 30°C overnight. The following day an aliquot of concentrated substrate/lysis buffer is added to each well and the plates incubated at 37°C for 1-2 hours. At an appropriate time an aliquot of stop solution is added to each well to halt the beta-galactosidase reaction. For all microplates an absorbance reading is obtained to assay the generation of product from the enzyme subsfrate.
  • ⁇ CV NS5B and AP-2 complex results in inhibition of the beta-galactosidase signal generated by MY209. Additional testing eliminates compounds that decreased expression of beta-galactosidase by affecting yeast cell growth and nonspecific inhibitors that affected the beta-galactosidase signal generated by the interaction of an unrelated protein pair.
  • a hit i.e., a compound which inhibits the interaction between the viral and cellular proteins
  • the compound is identified and subjected to further testing wherein the compounds are assayed at several concentrations to determine an IC 50 value, this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor.
  • IC 50 value this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor.
  • a homogeneous biochemical assay can be utilized for the detection of compounds that disrupt the interaction between a host cell protein and a viral protein containing a late-domain sequence.
  • GST-host protein tagged components of the interaction
  • Biotin- viral-protein or peptide fragment thereof
  • cognate labels GST-Europium [donor fluor] and streptavidin-APC [acceptor chromophore]
  • the donor europium
  • APC acceptor chromophore
  • the ratio of the APC emission at 665nm and the europium emission at 620nm is known as FRET (Fluorescence Resonance Energy Transfer) and can be utilized as a direct measure of protein-protein interaction.
  • FRET Fluorescence Resonance Energy Transfer
  • the europium and APC are not brought into close proximity and thus excitation of europium at 360nm results only in a fluorescence emission at 620nm; detection of fluorescence emission at 620nm would indicate that APC did not receive any excitation at this wavelength.
  • Detection reagents LANCE Eu-W1024-labelled Anti-GST Antibody (PerkinElmer Life Sciences) is provided as a 3.13 ⁇ M stock and is stored at 4 °C and used at a final concentration of 2nM in the assay.
  • Sfreptavidin conjugated to SureLightTM- Allophycocyanin (APC) (PerkinElmer Life Sciences) is provided as a lyophilizate and should be reconstituted with diH2O to a final concentration of lmg/ml and stored at 4 °C in the dark.
  • SureLightTM-APC is used at a final concentration of 1 ⁇ g/ml ( ⁇ 10nM) in the assay.
  • Second pass read establishes APC signal by excitation at 360nm and emission detection at 665nm (same settings as Attorney Docket No. 5010.01WO
  • FRET Fluorescence Activated protein
  • GST-host-protein, Biotin-viral-protein, LANCE-Eu and SureLightTM-APC are added concomitantly to 50mM HEPES, pH 7.0, 0.1%o BSA, to final concentrations of 33nM, 330nM, 2nM and 1 ⁇ g/ml, respectively.
  • 100 ⁇ l is aliquoted to each well of a 96-well plate. Compound addition can follow immediately and at least 30 minutes of incubation at room temperature is required prior to reading plate.
  • Example 5 Analysis of the PTDP (and/or PSDP, PNDP, PGYP) Motif.
  • a yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PTDP (and/or PSDP, PNDP, PGYP) motif of HCV core protein on the interaction between TsglOl and HCV core protein.
  • a yeast two-hybrid activation domain-TsglOl construct is prepared from a DNA fragment encompassing the full-length coding sequence for TsglOl (GenBank Accession No. U82130) by PCR from a human fetal brain cDNA library and is subsequently cloned into the EcoRI/Pstl sites of the activation domain parent plasmid GADpN2 (LEU2, CEN4, ARS 1 , ADH lp-
  • the yeast two-hybrid DNA binding domain-HCV core protein construct can be prepared from a fragment corresponding to the HCV core protein peptide derived from a HCV strain core protein. This fragment can be obtained by PCR from a plasmid Attorney Docket No. 5010.01WO
  • Any amino acid substitution mutations can be introduced by PCR into the HCV core protein sequence in the yeast two-hybrid DNA binding domain-HCV core protein construct described above.
  • the mutations can be verified by DNA sequence analysis.
  • Non-limiting examples of mutations that can be introduced into the DNA binding domain-HCV core protein construct are summarized in Tables II-IV below.
  • the effect of the mutations (and the WT) can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 frpl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-TsglOl construct and one of the binding domain-mutant HCV core protein constructs or the binding domain- wild type core protein construct.
  • Filter lift assays for ⁇ -Gal activity can be conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive ⁇ -Gal activity indicates that the core protein wild-type or mutant protein interacts with TsglOl. All binding domain constructs can also be tested for self-activation of ⁇ -Gal activity.
  • TSGIOI and wild-type HCV core protein (WT) or the core protein mutants can be further quantitated by performing liquid culture ⁇ -galactosidase assays.
  • Cultures are grown overnight in synthetic media (-Leu, -T ⁇ , + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/subsfrate solution in 6X Z-buffer (60mM KCl, 6mM MgSO 4 , 360mM ⁇ a 2 HPO 4 , 240 mM NaH 2 PO 4 , 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40).
  • HCV core protein point mutants can be used to test whether the TsglOl binding interaction requires the core protein late domain motif within HCV core protein, and will result in reduced ⁇ -galactosidase activity.
  • a fusion protein with a GST tag fused to the HCV core protein late-domain can be recombinantly expressed and purified by chromatography.
  • a HCV core protein fragment peptide containing can be synthesized chemically by standard peptide synthesis methods.
  • the peptide can be purified by conventional protein purification techniques, e.g., by chromatography.
  • a blocking buffer SuperBlock; Pierce-Endogen, Rockford, IL
  • Plates are then washed 4 x lOO ⁇ l with 1 x PBST solution (Invitrogen; Carlsbad, CA). After washing, lOO ⁇ l of l ⁇ g/ml solution of anti-myc monoclonal antibody (Clone 9E10; Roche Molecular Biochemicals; Indianapolis, IN) in 1 x PBST is added to the wells of the plate to detect the myc-epitope tag on the TsglOl protein.
  • 1 PBST solution Invitrogen; Carlsbad, CA
  • lOO ⁇ l of l ⁇ g/ml solution of anti-myc monoclonal antibody (Clone 9E10; Roche Molecular Biochemicals; Indianapolis, IN) in 1 x PBST is added to the wells of the plate to detect the myc-epitope tag on the TsglOl protein.
  • Plates are then washed again with 4 x lOO ⁇ l with 1 x PBST solution and lOO ⁇ l of l ⁇ g/ml solution of horseradish peroxidase (HRP) conjugated Goat anti-mouse IgG (Jackson Immunoresearch Labs; West Grove, Pennsylvania) in 1 x PBST was added to the wells of the plate to detect bound mouse anti-myc antibodies.
  • HRP horseradish peroxidase
  • Goat anti-mouse IgG Jackson Immunoresearch Labs; West Grove, Pennsylvania
  • the plates are then washed again with 4 x lOO ⁇ l with 1 x PBST solution and 100 ⁇ l of fluorescent substrate (QuantaBlu; Attorney Docket No. 5010.01 WO
  • Example 7 Yeast Screen To Identify Small Molecule Inhibitors Of The Interaction Between HCV Core Protein And TsglOl.
  • Beta-galactosidase can be used as a reporter enzyme to signal the interaction between yeast two-hybrid protein pairs expressed from plasmids in Saccharomyces cerevisiae.
  • Yeast strain MY209 (ade2 his3 leu2 trpl cyh2 ura3::GALlp-lacZ gal4 gal80 lys2::GALlp-HIS3) bearing the plasmids Mp364 (LEU2 CEN4 ARS1 -4E>Hip-SV40NLS- GAL4 (16&-S8l)-Tsgl01 (l-390)-PGKlt AmpR ColEl_o ⁇ ) and Mp206 (TRP1 CEN4 ARSADHl ⁇ >-GAL4(l-141)-HCV-coreprotein-ADHltAmpR ColEl_o ⁇ ) is cultured in synthetic complete media lacking leucine and tryptophan (SC -Leu -T ⁇ ) overnight at 30°C
  • This culture is diluted to 0.01 OD 63 o units/ml using SC -Leu -T ⁇ media.
  • the diluted MY209 culture is dispensed into 96-well microplates. Compounds from a library of small molecules are added to the microplates; the final concentration of test compounds is approximately 60 ⁇ M.
  • the assay plates are incubated at 30°C overnight. The following day an aliquot of concentrated substrate/lysis buffer is added to each well and the plates incubated at 37°C for 1-2 hours. At an appropriate time an aliquot of stop solution is added to each well to halt the beta-galactosidase reaction. For all microplates an absorbance reading is obtained to assay the generation of product from the enzyme subsfrate.
  • the presence of putative inhibitors of the interaction between ⁇ CV core Attorney Docket No. 5010.01WO
  • a hit i.e., a compound which inhibits the interaction between the viral and cellular proteins
  • the compound is identified and subjected to further testing wherein the compounds are assayed at several concentrations to determine an IC 50 value, this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés pour prévenir et traiter une infection par le virus de l'hépatite C et des symptômes de cette infection. Ces procédés consistent à introduire chez un patient des cellules présentant un phénotype de bourgeonnement modifié du virus de l'hépatite C ou à administrer à un patient des acides nucléiques, des polypeptides et de petits composés organiques afin d'induire la formation de cellules présentant un phénotype de bourgeonnement modifié du virus de l'hépatite C dans le corps de ce patient.
PCT/US2003/022956 2002-07-19 2003-07-21 Procede et composition pour traiter et prevenir une infection par le virus de l'hepatite c WO2004016738A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003256678A AU2003256678A1 (en) 2002-07-19 2003-07-21 Method and composition for treating and preventing hepatitis c infection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US39726702P 2002-07-19 2002-07-19
US60/397,267 2002-07-19

Publications (2)

Publication Number Publication Date
WO2004016738A2 true WO2004016738A2 (fr) 2004-02-26
WO2004016738A3 WO2004016738A3 (fr) 2004-06-17

Family

ID=31888187

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/022956 WO2004016738A2 (fr) 2002-07-19 2003-07-21 Procede et composition pour traiter et prevenir une infection par le virus de l'hepatite c

Country Status (2)

Country Link
AU (1) AU2003256678A1 (fr)
WO (1) WO2004016738A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006011686A1 (fr) * 2004-07-30 2006-02-02 Japan Tobacco Inc. Procédé de recherche par criblage d'un composé empêchant l'infection de cellules par le virus de l'hépatite c (vhc)
EP1888775A1 (fr) * 2005-05-27 2008-02-20 Creagene Inc. Procédé de mesure efficace des lymphocytes t cytotoxiques chez l'homme et chez des animaux non saillis
WO2008080302A1 (fr) * 2006-12-28 2008-07-10 Beijing Yanhuang Qilin Biotechnology Development Co., Ltd. NOUVELLE COMBINAISON D'UNE PROTÉINE DE FUSION CYTOKINE (IL-2/Fc) ET COMPOSÉ 861 DE MÉDECINE TRADITIONNELLE CHINOISE PERMETTANT D'AMÉLIORER LA RÉPONSE IMMUNITAIRE DU VACCIN CONTRE L'HÉPATITE B ET DE ROMPRE L'IMMUNOTOLÉRANCE DU VIRUS DE L'HÉPATIT
CN108707190A (zh) * 2018-04-18 2018-10-26 中国科学院武汉病毒研究所 猪瘟病毒ns5b蛋白保真度突变体及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6228576B1 (en) * 1997-12-11 2001-05-08 Smithkline Beecham Corporation Hepatitis C virus NS5B truncated protein and methods thereof to identify antiviral compounds
US6461845B1 (en) * 1995-09-27 2002-10-08 Emory University Recombinant hepatitis C virus RNA replicase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6461845B1 (en) * 1995-09-27 2002-10-08 Emory University Recombinant hepatitis C virus RNA replicase
US6228576B1 (en) * 1997-12-11 2001-05-08 Smithkline Beecham Corporation Hepatitis C virus NS5B truncated protein and methods thereof to identify antiviral compounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EGGER D. ET AL.: 'Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex' J. VIROL. vol. 76, no. 12, June 2002, pages 5974 - 5984, XP002973294 *
KOLYKHALOV A.A. ET AL.: 'Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo' J. VIROL. vol. 74, no. 4, February 2000, pages 2046 - 2051, XP002973293 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006011686A1 (fr) * 2004-07-30 2006-02-02 Japan Tobacco Inc. Procédé de recherche par criblage d'un composé empêchant l'infection de cellules par le virus de l'hépatite c (vhc)
EP1790983A1 (fr) * 2004-07-30 2007-05-30 Japan Tobacco, Inc. Procédé de recherche par criblage d'un compose empêchant l'infection de cellules par le virus de l'hépatite c (vhc)
EP1790983A4 (fr) * 2004-07-30 2008-05-07 Japan Tobacco Inc Procédé de recherche par criblage d'un compose empêchant l'infection de cellules par le virus de l'hépatite c (vhc)
EP1888775A1 (fr) * 2005-05-27 2008-02-20 Creagene Inc. Procédé de mesure efficace des lymphocytes t cytotoxiques chez l'homme et chez des animaux non saillis
EP1888775A4 (fr) * 2005-05-27 2009-03-25 Creagene Inc Procédé de mesure efficace des lymphocytes t cytotoxiques chez l'homme et chez des animaux non saillis
WO2008080302A1 (fr) * 2006-12-28 2008-07-10 Beijing Yanhuang Qilin Biotechnology Development Co., Ltd. NOUVELLE COMBINAISON D'UNE PROTÉINE DE FUSION CYTOKINE (IL-2/Fc) ET COMPOSÉ 861 DE MÉDECINE TRADITIONNELLE CHINOISE PERMETTANT D'AMÉLIORER LA RÉPONSE IMMUNITAIRE DU VACCIN CONTRE L'HÉPATITE B ET DE ROMPRE L'IMMUNOTOLÉRANCE DU VIRUS DE L'HÉPATIT
CN108707190A (zh) * 2018-04-18 2018-10-26 中国科学院武汉病毒研究所 猪瘟病毒ns5b蛋白保真度突变体及其应用
CN108707190B (zh) * 2018-04-18 2021-07-13 中国科学院武汉病毒研究所 猪瘟病毒ns5b蛋白保真度突变体及其应用

Also Published As

Publication number Publication date
AU2003256678A1 (en) 2004-03-03
AU2003256678A8 (en) 2004-03-03
WO2004016738A3 (fr) 2004-06-17

Similar Documents

Publication Publication Date Title
US8663648B2 (en) Agents for treatment of HCV and methods of use
EP1156817B1 (fr) Regulation des taux de proteines dans des organismes eucaryotiques
US20070213271A1 (en) Composition and method for treating hiv infection
ES2633470T3 (es) Métodos y composiciones de generación de una respuesta inmune mediante la inducción de CD40 y adaptadores de receptores de reconocimiento de patrones
US20100256040A1 (en) Composition and method for treating viral infection
US20070253968A1 (en) Identification of peptides that facilitate uptake and cytoplasmic and/or nuclear transport of proteins, DNA and viruses
JP2009269918A (ja) Ｔｓｇ１０１−ｇａｇ相互作用およびその使用
AU753031B2 (en) Functional fragments of HIV-1 VPR protein and methods of using the same
US7943146B2 (en) Immunizing compositions comprising HIV-1 proviral constructs with an inactive p6 gag TSG101 UEV binding domain capable of producing budding-defective viral particles that remain tethered to the cell surface
WO2004009032A2 (fr) Methode et composition utilisees pour traiter et prevenir l'infection par le virus de l'hepatite b et les symptomes associes
WO2004016738A2 (fr) Procede et composition pour traiter et prevenir une infection par le virus de l'hepatite c
WO2004009028A2 (fr) Methodes et composition permettant de traiter et de prevenir l'infection grippale et les symptomes associes
US6960431B2 (en) Therapeutic compositions and methods for treating viral infection
US20030105277A1 (en) Compositions and therapeutic methods for viral infection
WO2004094605A2 (fr) Methodes et compositions permettant d'inhiber les voies de signalisation des stat
WO2004009027A2 (fr) Procede et composition pour traiter et prevenir l'infection par le virus de l'herpes
US6218103B1 (en) Herpes simplex virus US3 and ICP4 as inhibitors of apoptosis
US6162440A (en) EBNA2 peptides and methods of using same
WO2002034770A1 (fr) Identification d"epitopes du vhc restreints par hla-dr
US7094401B1 (en) HCV core protein binding agents for treatment of hepatitis C virus infection

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 BZ CA CH CN CO CR CU CZ DE DK DM DZ EC 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 NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM 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 ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP