WO2007128048A1 - Véhicule de livraison d'un vaccin de l'hépatite c - Google Patents

Véhicule de livraison d'un vaccin de l'hépatite c Download PDF

Info

Publication number
WO2007128048A1
WO2007128048A1 PCT/AU2007/000578 AU2007000578W WO2007128048A1 WO 2007128048 A1 WO2007128048 A1 WO 2007128048A1 AU 2007000578 W AU2007000578 W AU 2007000578W WO 2007128048 A1 WO2007128048 A1 WO 2007128048A1
Authority
WO
WIPO (PCT)
Prior art keywords
hcv
delivery vehicle
protein
vaccine delivery
recbv
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/AU2007/000578
Other languages
English (en)
Inventor
Eric James Gowans
John Charles Martyn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Original Assignee
Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2006902253A external-priority patent/AU2006902253A0/en
Application filed by Macfarlane Burnet Institute for Medical Research and Public Health Ltd filed Critical Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Publication of WO2007128048A1 publication Critical patent/WO2007128048A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/855—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • HCV The threat posed to the Australian and other health systems in developed and developing countries of HCV infection continues to grow at an alarming rate.
  • HCV is currently one of the most common notifiable infectious diseases in Australia with 16,000 new cases per annum (Dore et al, JClin Virol 26:171-84, 2003).
  • HCV structural proteins are encoded at the 5 'end of the single long open reading frame in the genome and comprise the core (C) or capsid protein and two envelope glycoproteins, El and E2 (Major and Feinstone, Hepatol 25:1527-1538, 1997).
  • the mature polypeptides are cotranslationally cleaved from the polyprotein precursor by a cellular protease.
  • El and E2 proteins are typical type 1 transmembrane proteins with a large ectodomain and a COOH-terminal hydrophobic domain that anchors the proteins to the endoplasmic reticulum (ER) [Cocquerel et al, J Virol 72:2189-2191, 1998, Cocquerel et al, J Virol 73:2641-2649, 1999].
  • Studies of E1/E2 expression are generally performed using recombinant plasmid or viral systems (Brazzoli et al, Virol 352:438-453, 2005, Martire et al, Virology 250:176-182, 2001).
  • HCV-LP HCV-like particles
  • RecBV baculovirus
  • HCV-LP purified from insect cell lysates are recognised by conformation-dependent monoclonal antibodies and HCV-LP purified from human cell culture supernatants bind the HCV co-receptor CD81, indicating that the E1/E2 proteins form the authentic heterodimer in HCV-LP derived from both cell types.
  • HVRl hypervariable region 1
  • HVRl three peptide mimotopes of HVRl were recognized by serum antibodies from 88% of HCV-positive patients (Puntoriero et al, EMBO J 77:3521-3533, 1998) and two monoclonal antibodies induced by immunization with peptides containing the conserved G - -Q motif at the C-terminus of HVRl were able to capture HCV from 25/31 (81%) patients tested (Li et al, J Virol 75:12412-12420, 2001).
  • a recombinant baculovirus acts as a delivery vehicle for a surface (envelope) antigen from HCV to a subject's immune system in order to generate a humoral response and/or a cell mediated response.
  • the RecBV comprises at least one HCV protein which is incorporated into its envelope and at least one HCV protein which is expressed in a cell of the target subject to which the vaccine delivery vehicle is introduced.
  • the RecBV comprises a protein antigen from another pathogen in order to generate an immune response against that pathogen.
  • the RecBV delivery vehicle may also be used in gene therapy applications.
  • the RecBV delivery vehicle may also be targeted to particular cells via binding to a ligand such as but not limited to inter alia an Fc portion on the delivery vehicle binding to a cell carrying a Fc receptor or an immunoglobulin receptor.
  • a delivery vehicle comprising a recombinant baculovirus (RecBV) having an envelope which contains a surface antigen from Hepatitis C Virus (HCV) or an immunologically cross-reactive homolog thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from HCV and which is expressed in a cell of a target subject.
  • RecBV recombinant baculovirus
  • HCV Hepatitis C Virus
  • a delivery vehicle comprising a RecBV having an envelope which contains an antigen from a pathogen or an immunologically cross-reactive homolog thereof which RecBV induces a cell mediated response by cross presentation.
  • a delivery vehicle comprising a RecBV having an envelope comprising a ligand for a complementary molecule on a cell surface.
  • a delivery vehicle comprising a RecBV carrying genetic material for gene therapy or genetic-based vaccination.
  • a delivery vehicle is provided herein comprising a RecBV having an envelope comprising an agent selected from the list consisting of a surface antigen from HCV, an antigen from a pathogen and a ligand capable of binding to a cell surface molecule which RecBV further comprises a nucleic acid molecule encoding a protein or RNA species and which is expressed in a cell of a target subject.
  • the delivery vehicle described herein may be described as a RecBV delivery vehicle, a virus-like particle (VLP) or a gene therapy facilitating vehicle as well as a vaccine delivery vehicle.
  • VLP virus-like particle
  • the delivery vehicle comprises a RecBV having an envelope which contains a heterologous protein and a nucleic acid molecule encoding a heterologous expression product wherein the heterologous protein is an antigen from a pathogen and/or a ligand to a complementary molecule on a cell surface and wherein the heterologous expression product is a peptide, polypeptide or protein or RNA species produced by a cell of a target subject.
  • the vaccine delivery vehicle aspect is based on the supposition that optimal protection against HCV challenge is a combination of a humoral and cell mediated response and that the immune responses are directed against highly conserved epitopes and hence will protect against infection by any genotype of HCV.
  • a vaccine delivery vehicle comprising a RecBV having an envelope which contains HCV envelope glycoproteins E1/E2 in dimeric form and which RecBV comprises a nucleic acid molecule which encodes at least one HCV protein selected from the list consisting of El, E2, core protein, NS3/4A andNS3m/4A and which protein is expressed in a target mammalian cell wherein said vaccine delivery vehicle when introduced to a subject generates a protective humoral and cell mediated immune response to HCV or an immunologically cross-reactive homolog thereof.
  • the delivery vehicle comprises a RecBV having an envelope containing a portion of Fc which is capable of binding to an FcR receptor (FcR) on the surface of a cell.
  • FcR FcR receptor
  • the baculovirus vector is preferably but not necessarily derived from the vector described by Condreay et al, Proc. Natl. Acad. Sci USA 9(5:127-132, 1999.
  • the HCV genes required for expression in the cells e.g. core, El, E2, NS3 and/or NS4A
  • HCV genes for expression in insect cells e.g. El and/or E2 replace the neo gene as a ScxAl/Csp451 fragment.
  • the donor plasmid is reproduced in Figure 11.
  • the vaccine delivery vector may be targeted to a particular cell via a Toll-like receptor (TLR) such as TLR2, TLR3, TLR4, TLR7, TLR8 and/or TLR9. Proteins which bind to and hence target TLRs are referred to herein as TLR binding proteins.
  • TLR binding proteins Proteins which bind to and hence target TLRs are referred to herein as TLR binding proteins.
  • the vaccine delivery vector may further comprise a TLR binding protein from HCV or another virus which facilitates targeting to a TLR and hence to a particular cell or group or family of cells.
  • TLR binding proteins examples include those listed in Table 2.
  • the baculovirus envelope may contain other targeting moieties such as an IgG constant region to target any antigen-presenting cell with IgG receptors, decay accelerating protein (DAF) which increases resistance to complement, CD40L to target CD40 on DC and/or an Fc portion which binds to Fc receptor (FcR).
  • targeting moieties such as an IgG constant region to target any antigen-presenting cell with IgG receptors, decay accelerating protein (DAF) which increases resistance to complement, CD40L to target CD40 on DC and/or an Fc portion which binds to Fc receptor (FcR).
  • the expression of the HCV protein in, for example, mammalian cells is conveniently controlled via a suitable promoter such as but not limited to a CMV promoter.
  • a suitable promoter such as but not limited to a CMV promoter.
  • the CMV promoter is useful for inducing expression in mammalian cells.
  • a method for vaccinating a subject against HCV infection comprising administering to said subject a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains a surface antigen from HCV or an immunologically cross-reactive homolog thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from HCV and which is expressed by a cell of a target subject, wherein said vaccine delivery vehicle induces a protective humoral and cell mediated immune response to HCV or an immunologically cross-reactive homolog thereof.
  • Yet a further aspect provides a method for vaccinating a subject against a pathogen said method comprising administering to said subject a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains a surface antigen from the pathogen or an immunologically cross-reactive homolog thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from the pathogen and which is expressed by a cell of a target subject, wherein said vaccine delivery vehicle induces a protective humoral and cell mediated immune response to the pathogen or an immunologically cross-reactive homolog thereof.
  • Still another aspect contemplates a method for gene therapy in a subject said method comprising administering to said subject a delivery vehicle comprising a recombinant baculovirus having an envelope which contains a surface ligand capable of binding to a complementary ligand on a target cell and which baculovirus comprises a nucleic acid molecule which encodes a peptide, polypeptide or protein or RNA species and which is expressed by a cell of a target subject, wherein said delivery vehicle facilitate peptide, polypeptide or protein replacement and/or gene regulation.
  • compositions comprising the HCV vaccine or other pathogen vaccine and the use of the RecBV in the manufacture of a medicament to induce a protective immune response against HCV or other pathogen are also described herein as is the use of a RecBV in gene therapy.
  • the word "comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>l [SEQ ID NO: 1], ⁇ 400>2 [SEQ ID NO: 2], etc.
  • Figure 1 is a diagrammatic representation showing the construction of the RecBV-E.
  • A Line diagram showing the organization of the HCV genome (upper) and the region subcloned into the pFBM transfer vector (lower).
  • B Primers used to amplify the E1E2 cDNA with upstream signal sequence.
  • FIGS 2A through C are photographic and tabular representations showing the RecBV- E transduction is more efficient than DNA transfection.
  • Huh7 cells were transduced with RecBV-E or transfected with pFBM-E.
  • A Expression of E2 after RecBV-E infection (upper) or transfection with pFBM-E (lower). The percentage of cells expressing high and low levels of E2 is indicated.
  • B Immunoblot of duplicate samples.
  • C Co- immunoprecipitation of radiolabeled El and E2 using polyclonal anti-HCV E2. The El and E2 bands are indicated by the arrows.
  • Figures 3A and B are photographic and tabular representations showing the efficiency of transduction of primary marmoset hepatocytes with RecBV-E compared to transfection with pFBM-E
  • A Immunofluorescence using polyclonal anti-HCV E2. The percentage of cells expressing high and low levels of E2 is indicated.
  • B Western blot using polyclonal anti-HCV E2.
  • pFBM-E - donor plasmid with E1E2 insert pFBM - donor plasmid without insert, RecBV-ElE2 - recombinant baculovirus encoding E1/E2
  • pFBM-FL - donor plasmid encoding full-length HCV polyprotein.
  • Figures 4A through C are photographic representations showing generation of stable cell lines expressing HCV El and E2 following transduction of the HLl cell line with RecBV- E.
  • A Immunofluorescence of pools of G418-resistant colonies using polyclonal anti-HCV E2 serum. Cells in pools 1 and 2 were transduced at a moi of 10,000 and 1,000, respectively.
  • B Immunofluorescence of clonal stable cell lines (P2C3, P3D1) expressing El and E2, and a clone (P2B6) negative for E1E2. Primary antibody was polyclonal anti- HCV E2.
  • Figures 5A and B are graphical representations of the characteristics of cell lines constitutively expressing E1/E2.
  • A Growth kinetics of 5 stable cell lines (P3C3, P2C3, P3D1, P3B4, P2C6) compared to the parental HLl cell line.
  • B Flow cytometric analysis of apoptosis in cell line P2C6 compared to HLl.
  • the gated area (Ml) of the histogram includes the apoptotic cells in the total population (Rl).
  • the positive control was P2C6 cells treated with 20% ethanol prior to staining.
  • Figures 6A and B are photographic representations showing conformation and localisation of HCV E2 expressed by the stable cell line.
  • A Binding of a conformation- dependent E2 monoclonal antibody, H53, to acetone-fixed cell lines P2C6 and the parental line, HLl (negative control).
  • B Flow cytometric analysis of P2C6 and HLl cell lines following surface staining with polyclonal anti-E2 serum diluted 1:10, 1:20 and 1:40. Normal goat serum (Isotype C) was the negative control.
  • ⁇ is the difference in net mean fluorescence intensity of the P2C6 cell line (grey fill) compared to the HLl cell line (no fill).
  • Figure 7 is a photographic representation showing the persistence of RecBV following transduction of the HLl cell line as determined by a timecourse of GFP expression in HLl cells transduced with RecB V-GFP.
  • the HLl cells were infected with RecBV-eGFP at moi 1,000 and split 1:2 every 5 days post-transduction. Cells were seeded from this flask onto coverslips, incubated for 3 or 4 days, then examined for fluorescence. Cells transduced with RecB V-wt represented the negative control.
  • Figure 8 is a diagrammatical representation of genes encoding expression in insect cells.
  • FIG 9 is a photographical representation of Huh7 cells infected with HCV and stained by immunofluorescence for core antigen expression.
  • the JFHl virus replication system in Huh7 cells was established, as described (Zhong et al, Proc. Natl. Acad. Sa. USA 102:9294-9299, 2005).
  • JFH virus derived from RNA-transfected Huh7 cells, can infect na ⁇ ve Huh7 cells.
  • the virus will be adjusted to 50TCID 5O , incubated at 37 0 C with dilutions of the mouse serum for 1 hour then inoculated into uninfected Huh7 cells. Virus growth will be determined three days later by immunofluorescence of inoculated cells.
  • Figure 10 is a graphical representation showing the organisation of the BVDV genome and position of inserted HCV NS3m/4 gene.
  • Figure 11 is a diagrammatic representation of the baculovirus donor plasmid as described by Condreay et al, 1999 supra.
  • the HCV genes for mammalian expression core, El, E2, NS3 and/or NS4A
  • HCV genes for insect expression (El and/or E2) replace the neogene as a ScxAl/Csp451 fragment.
  • Figure 12 is a graphical representation showing the antibody responses after vaccination of Balb/C mice with RecBV-El/E2. Groups of mice were injected by the IM route with WT or RecBV-El/E2. The antibody titre was determined by ELISA against purified E2 protein or purified WT BV.
  • Figure 13 is a diagrammatic representation of a map of the transfer vectors (A) pFBM- CElE2-gp64+Fc and (B) pFBM-CElE2-hTfR+Fc used to construct the corresponding recombinant baculovirases RecBV:gp64+Fc and RecBV:hTfr+, respectively. Each transfer vector contains a dual expression cassette.
  • Cassette 1 contains the coding sequence of human IgG Fc fused to either the transmembrane (TM) region of the baculovirus gp64 envelope protein (construct A) or the human transferrin receptor (construct B) downstream of the polyhedrin promoter for constitutive expression in insect cells.
  • Cassette 2 is identical in both constructs and contains the coding sequence of the HCV structural proteins (core, El, E2) downstream of the CMV-IE promoter for constitutive expression in mammalian cells.
  • Figures 14A and B are schematic diagrams illustrating the conformation of the Fc molecule in the envelope of the RecBV, pg64 Ig Fc and hTfR Ig Fc. NB.
  • TMl and TM2 represent any suitable transmembrane domain sequences which target proteins to the plasma membrane .
  • Figure 15 is a photographic representation showing that IgG Fc fusion proteins are expressed on the surface of RecBV-infected insect cells. IF of live (unfixed) Sf9 insect cell monolayers infected with RecBVs and stained at 48h pi with goat anti-human IgG Alexa 488 conjugate. Insect cell nuclei were stained red with Syto 61. (A) hTfR-Fc RecBV, (B) gp64-Fc RecBV, (C) parental (non-pseudotyped) RecBV, and (D) mock infection.
  • Figure 16 is a photographic representation showing that IgG Fc fusion proteins are incorporated into the envelope of RecBV particles.
  • RecBV by ELISA RecBV by ELISA. Dilutions of the rabbit anti-human IgG HRP conjugate are shown on the x-axis, while the OD reading is shown on the y-axis. Dilutions were performed in duplicate, and the average represented by solid bars.
  • B Immune EM. RecBV particles were dual labeled with lOnm protein A gold to detect IgG Fc, and anti-gp64 mAb + 5nm protein A gold
  • Panels A, B hTfR-Fc RecBV particles, (Panels C, D) gp64-Fc RecBV particles, (Panel E) parental (non-pseudotyped), (Panel F) RecBV (negative control). Representative lOnm (arrows) and 5nm (arrowheads) protein A gold particles are indicated for each RecBV pseudotype. All magnification bars are 200nm.
  • Figure 17 is a photographic representation showing that IgG Fc fusion proteins are incorporated into the envelope of RecBV particles.
  • A Detection of human IgG Fc on RecBV by ELISA. Dilutions of the rabbit anti-human IgG HRP conjugate are shown on the x-axis, while the OD reading is shown on the y-axis. Dilutions were performed in duplicate, and the average represented by solid bars.
  • B Immune EM. RecBV particles were dual labeled with lOnm protein A gold to detect IgG Fc 5 and anti-gp64 mAb + 5nm protein A gold.
  • Figure 18 is a graphical representation showing that IgG Fc fusion proteins displayed on the surface of RecBV-infected insect cells are functional according to FACS analysis.
  • (Upper panels) Recombinant Fc molecules detected on the surface of insect cells infected with (A) the gp64-Fc and (B) the hTfR-Fc or (C) the parental BV by flow cytometry using anti-human IgG FITC.
  • Recombinant soluble Fc ⁇ RIIa was incubated with the RecBV- infected cells at 4 0 C, washed and the cells examined by flow cytometry with anti-human Fc ⁇ RIIa (MAb 8.2) followed by anti-mouse IgG FITC (D-pg64-Fc, E-hTfR-Fc and F-parental BV).
  • Figure 19 is a graphical representation showing that IgG Fc fusion proteins displayed on the surface of RecBV bind to the soluble receptor in a reaction that is inhibited by the anti
  • FIG. 20 is a graphical representation of RecBV displaying IgG Fc fusion proteins on their surface bind to cells expressing Fc ⁇ RIIa. RecBV was added to the IIA1.6 cell line expressing the Fc ⁇ RIIa receptor and the level of binding assessed by flow cytometry using anti-pg64. Before and after blocking the Fc ⁇ RIIa with blocking MAb IV-3.
  • Figure 21 is a representation showing that differential binding of RecBV by human monocyte-derived dendritic cells (Mo-DC). RecBV was added to cultures of human Mo- DC at 4 0 C, incubated for Ih, washed and the degree of RecBV binding assessed by flow cytometry using anti-pg64 PE.
  • Mo-DC human monocyte-derived dendritic cells
  • Figure 22 is a representation showing that distinct cellular populations in PBMC bind different BV.
  • Human PBMC were purified by Ficoll-Paque gradient centrifugation and examined for their ability to bind RecBV, as detected by flow cytometry using anti-gp64 PE.
  • the monocyte population was labeled with anti-CD 14 FITC.
  • Different concentrations of the RecBV were added to the purified PBMC, incubated at 4 0 C for Ih, washed and binding examined.
  • Figure 23 is a graphical representation summarizing flow cytometric analyses.
  • Figure 24 is a diagrammatic representation of a RecBV delivery vehicle having different proteins fused to the transmembrane domain of transmembrane domains of pg64, CD4 or hTFR.
  • Figure 25 is a graphical representation of a complement assay showing levels of complement sensitivity of various fusions to IgG Fc. A gp64-Fc fusion particularly showed resistance to complement. DETAILED DESCRIPTION
  • vaccine delivery vehicle means delivery vehicle, delivery vehicle, gene therapy facilitating vehicle, “RecBV delivery vehicle”, “agent”, “reagent”, “compound”, “pharmacologically active agent”, “medicament”, “therapeutic”, “active” and “drug” are used herein to refer to a chemical or biological entity which induces or exhibits a desired effect such as inducing a protective humoral and cell medicated response to HCV or other pathogen and/or which facilitates gene therapy.
  • Reference to a vaccine delivery vehicle in particular includes combinations of two or more vehicles or a vehicle and one or more anti-viral agents.
  • a "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispenzation.
  • a multi-part pharmaceutical pack may have two or more agents separately maintained.
  • this aspect of the present invention includes combination therapy.
  • Combination therapy includes the co-administration of two or more delivery vehicles or a delivery vehicle and an anti-viral agent or immune potentiating agent or a nucleic acid molecule capable of being expressed to produce a peptide, polypeptide or protein or RNA species.
  • RNA species include mRNA, single stranded short or long RNA molecules, RNA hairpins, double stranded short or long RNA molecules and the like.
  • the vaccine delivery vehicle is preferably but not necessarily based on or derived from the baculovirus donor plasmid of Condreay et al, 1999 supra (see Figure 11).
  • the baculo virus donor plasmid may be further modified to express a protein, such as in or in its envelope which facilitates interaction with a TLR such as but not limited to TLR2, TLR3, TLR4, TLR7, TLR8 and/or TLR9.
  • a protein such as in or in its envelope which facilitates interaction with a TLR such as but not limited to TLR2, TLR3, TLR4, TLR7, TLR8 and/or TLR9.
  • TLR such as but not limited to TLR2, TLR3, TLR4, TLR7, TLR8 and/or TLR9.
  • the baculovirus envelope may contain a non-HCV protein in order to target the virus to a particular TLR and therefore a particular cell or group of cells. Examples of suitable targeting proteins are described in Boehme and Compton, J Virol 75
  • the baculovirus envelope may contain other targeting moieties such as an IgG constant region to target any antigen-presenting cell with IgG receptors, decay accelerating protein (DAF) which increases resistance to complement and/or CD40L to target CD40 on DC.
  • the envelope may contain a portion of Fc which binds to FcR or antigen- presenting cells (APCs).
  • a suitable promoter for expression in a mammalian cell of HCV or other proteins, a suitable promoter is required such as but not limited to the CMV promoter. Other promoters active in mammalian cells may also be used.
  • an agent as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological or effect or outcome.
  • Such an effect or outcome includes inducing a humoral and cell mediated response to HCV or other pathogen or for inducing an outcome following the generation of an expression product from the nucleic acid molecule within the RecBV.
  • Outcomes include enabling production of a protein or inhibiting expression by post transcriptional or post translational gene silencing, methylation and/or RNAi- mediated gene silencing.
  • Undesirable effects e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the effective amount is deemed the amount required to induce an immune response against HCV or other pathogen which is protective.
  • a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
  • Treating may involve both active treatment and prophylaxis.
  • the "subject” as used herein refers to an animal, preferably a mammal and more preferably a primate including a lower primate and even more preferably a human who can benefit from the formulations and methods of the present invention.
  • a subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient.
  • the compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.
  • an "animal” includes an avian species such as a poultry bird (including ducks, chicken, turkeys and geese), an aviary bird or game bird.
  • the condition in a non-human animal may not be a naturally occurring but induced such as in an animal model.
  • At least one HCV protein is expressed in a cell of the subject to which it is introduced.
  • the subject includes a mammal, such as a human or test primate animal.
  • at least one of the HCV or other heterologous peptides, polypeptides or proteins or RNA species encoded in the baculovirus is expressed in mammalian cells of the target subject.
  • a "target mammalian cell” is a cell in a mammalian subject to which the delivery vehicle has been administered.
  • the HCV genes for example, may be operably linked to a suitable promoter such as a CMV promoter to permit expression in mammalian cells.
  • heterologous in this context includes any peptide, polypeptide or protein or RNA species which is not naturally present in a baculovirus.
  • an example of a heterologous peptide, polypeptide or protein is an antigen from a pathogen such as HCV.
  • a heterologous expression product includes peptide, polypeptide or protein or RNA species from a pathogen or from a mammalian cell.
  • the animal subjects include humans, non-human primates such as chimpanzees marmosets, baboons, orangutangs, lower primates such as tupia, livestock animals, laboratory test animals, companion animals or captive wild animals.
  • a human is a particularly important target.
  • non-human animal models may be used.
  • test system examples include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, zebrafish, amphibians (including cane toads) and Drosophila species such as Drosophila melanogaster are also contemplated. Instead of a live animal model, a test system may also comprise a tissue culture system.
  • a recombinant baculovirus (RecBV) is used as a vaccine delivery vehicle to introduce to a subject being vaccinated a surface antigen from HCV to generate a humoral response against this antigen.
  • the baculovirus carries nucleic acid which encodes at least one protein from HCV which generate at least a cell mediated immune response and which protein, in a preferred embodiment, is expressed in a mammalian cell of a target subject.
  • HCV baculovirus donor plasmid depicted in Figure 11 (Condreay et al, 1999 supra).
  • HCV genes destined to be expressed in mammalian cells to induce a cell mediated response e.g. core, El, E2, NS3 and/or NS4A
  • HCV genes to be expressed as part of the baculovirus envelope e.g. El and/or E2
  • HCV or non-HCV proteins which target particular TLRs may also be incorporated into the baculovirus envelope. Examples of such proteins are listed in Table 2.
  • the baculovirus envelope may contain other targeting moieties such as an IgG constant region to target any antigen-presenting cell with IgG receptors, decay accelerating protein (DAF) which increases resistance to complement and/or CD40L to target CD40 on DC.
  • An Fc portion may also be employed as a ligand which binds to FcR on a cell such as an antigen-presenting cell (APC).
  • APC antigen-presenting cell
  • An example of an APC is a dendritic cell (DC).
  • one aspect is directed to a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains a surface antigen from hepatitis C Virus (HCV) or an immunologically cross-reactive homolog thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from HCV and is expressed in a cell of a target subject.
  • HCV hepatitis C Virus
  • Reference to a "immunologically cross-reactive homolog" of HCV includes any virus which exhibits a structural relatedness to HCV such that an antibody to HCV will bind to the other virus and/or where the other virus can induce a CTL positive response to HCV exposed or vaccinated subjects.
  • immunologically cross-reactive homologs of HCV include natural or induced variants of HCV or natural or induced recombinant virus comprising parts or portions of HCV.
  • the vaccine delivery vehicle is capable of inducing humoral and cell-mediated immune protection for any genotype of HCV or a particular strain or group of strains of HCV.
  • a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains a surface antigen from HCV and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from HCV which is expressed in a cell of a target subject.
  • HCV surface antigen from HCV includes any component of HCV which is accessible to the external environment and to which an antibody can bind.
  • the antigen is a surface exposed component of HCV.
  • Particularly preferred examples of surface antigens include HCV envelope glycoproteins such as El or E2 or a translational precursor thereof.
  • a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains a HCV envelope glycoprotein or an antigenic fragment thereof or a translational precursor thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein from HCV and is expressed in a cell of a target subject.
  • HCV envelope glycoproteins are El or E2 or a homo- or hetero-dimeric form thereof.
  • Reference to a "homo-dimer” includes El /El forms or E2/E2 forms.
  • a “hetero- dimer” includes E1/E2 or E2/E1.
  • the aim of the HCV envelope glycoprotein is to provide a stimulatory source for the generation of a humoral response.
  • the HCV envelope glycoprotein may be an "antigenic fragment" thereof meaning that sufficient B-cell epitopes exist to induce a humoral response to HCV envelope glycoproteins.
  • a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope which contains an HCV envelope glycoprotein selected from El, E2, El homo-dimer, E2 homo-dimer, E1/E2 or E2/E1 hetero-dimer and a fragment of El, E2, El homo-dimer, E2 homo-dimer or E1/E2 or
  • E2/E1 hetero-dimer which is capable of inducing a humoral immune response in a subject said baculovirus comprising a nucleic acid molecule which encodes at least one protein from HCV and which is expressed in a cell of the target subject.
  • the target subject is the subject in need of vaccination or treatment.
  • Target subjects include mammals, such as primates and most particularly humans.
  • the protein encoded by the recombinant baculovirus may be the core or capsid protein, an NS protein such as but not limited to NS 3/4 A or a non-functional mutant thereof such as NS3m/4A or an HCV envelope glycoprotein such as El and/or E2. These proteins are produced by cells which are infected by the recombinant delivery vehicle.
  • a vaccine delivery vehicle comprising a recombinant baculovirus having an envelope containing a surface antigen from HCV or an immunologically cross-reactive homolog thereof and which baculovirus comprises a nucleic acid molecule which encodes at least one protein selected from the list consisting of El, E2, core protein, an NS protein and a mutant of a NS protein from HCV that is not immunosuppressive and which protein is expressed in a cell of a target subject.
  • the envelope of the baculovirus may also carry an HCV protein and/or a non-HCV protein to target the baculovirus to a particular TLR and hence to a cell or group or family of cells carrying the TLR.
  • HCV protein and/or a non-HCV protein examples of such TLR-binding proteins are listed in Table 2.
  • the present invention is still further directed to the use of an HCV surface antigens and another HCV protein in the manufacture of a medicament for the induction of humoral and cell mediated protective immunity against HCV.
  • the vaccine delivery vehicle may be formulated in a range of excipients, diluents and/or carriers.
  • a convenient description of a suitable formulation can be found in Remington's Pharmaceutical Sciences, 20th ed. Williams and Wilkins (2000) and The British National Formulary 43rd ed. (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http://bnf.rhn.net), the contents of which are hereby incorporated by reference.
  • the pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed., 1985).
  • the RecBV delivery vehicles of the present invention may also be used in gene therapy and/or for inducing an immune response to non-HCV pathogens or HCV and non-HCV pathogens.
  • Reference to "gene therapy” includes replacing a defective gene, protein replacement or augmentation or for inducing gene silencing via antisense, sense or RNAi suppression.
  • a delivery vehicle comprising a RecBV having an envelope comprising a ligand for a complementary molecule on a cell surface.
  • a ligand for a complementary molecule on a cell surface examples include Fc which binds to FcR, IgG or a portion thereof which binds to IgGR 5 CD40L to target CD40 and DAF.
  • the HCV E1/E2 proteins in the RecBV envelope may also target DC through an interaction between E2 and DC-SIGN or CD81.
  • a delivery vehicle is further provided comprising a RecBV carrying genetic material for gene therapy or genetic-based vaccination.
  • a delivery vehicle comprising a RecBV having an envelope comprising an agent selected from the list consisting of a surface antigen from HCV, an antigen from a pathogen and a ligand capable of binding to a cell surface molecule which RecBV further comprising a nucleic acid molecule encoding a protein or RNA and which is expressed in a cell of a target subject.
  • the delivery vehicle described herein may be described as a RecBV delivery vehicle, a virus-like particle (VLP) or a gene therapy facilitating vehicle.
  • VLP virus-like particle
  • the delivery vehicle comprises a RecBV having an envelope which contains a heterologous protein and a nucleic acid molecule encoding a heterologous expression product wherein the heterologous protein is an antigen from a pathogen and/or a ligand to a complementary molecule on a cell surface and wherein the heterologous expression product is a peptide, polypeptide or protein or RNA species produced by a cell of a target subject.
  • Sf9 insect cells were grown as suspension cultures in polycarbonate Erlenmeyer flasks (Corning, Acton, MA) in Sf-900 II serum-free medium [SFM] (Invitrogen, Carlsbad, CA) containing lO ⁇ g/ml gentamicin.
  • HLl cells an interferon- ⁇ cured derivative of the Huh8 HCV-replicon positive cell line (Blight et al, Science 290:1972-1973, 2000) and COS-I cells were grown in DMEM supplemented with GlutaMAX (Invitrogen), 10% v/v fetal bovine serum (FBS) and penicillin/streptomycin (DMEMlO).
  • the mouse B-cell line IIA1.6 and the stably transformed derivative IIAl. ⁇ -Fc ⁇ RIIa LR which constitutively expresses the Fc ⁇ RIIa receptor were grown in RPMI medium 1640 supplemented with 300 ⁇ g/ml L-glutamine (Invitrogen), 10% v/v fetal bovine serum (FBS), 7 ⁇ M ⁇ -mercaptoethanol, 100units/ml penicillin and lOO ⁇ g/ml streptomycin.
  • a cDNA fragment encoding 36 amino acids at the COOH terminus of the HCV core protein and the complete coding regions of El and E2 were amplified from a full length cDNA clone using primers El for and E2rev ( Figure 1) and PfuTurbo DNA polymerase (Stratagene, La Jolla, CA). It should be noted that this clone is infectious in chimpanzees. Infectious clones are generally used which are infectious in human cells or other mammalian cells.
  • the 1.8 kb product was purified using a High Pure Purification Kit (Roche, Mannheim), digested with HindIII and Xbal, and then gel purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden). The fragment was then ligated into the Hindlll/Xbal site of plasmid pFastBacMam (pFBM) [Condreay et al, 1999 supra] downstream of the CMV-IE promoter, to create plasmid pFBM-E ( Figure 1). Translation of E1/E2 initiates from an ATG codon inserted between the Kozak sequence and the El signal sequence.
  • the E1/E2 insert in pFBM-E was sequenced using BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA) and the plasmid used to construct RecBV-E using the Bacto-Bac system (Invitrogen).
  • pFBM without the insert was used to construct RecBV-wt.
  • pFBM-GFP was used to construct the GFP expressing virus RecBV-GFP. Construction of recombinant baculovirus.
  • a cDNA fragment containing the HCV IRES 3 core, El and E2 genes (nt 1 to nt 2582) were amplified from a full length cDNA clone using primers 5'-ATATGAATTCGCCAGCCCCCGATTGGGG-S' [SEQ ID NO:3] and 5'-AATATCTAGATTAGGCCTCGGCCTGGGCTATCAG-S' [SEQ ID NO:2] and PfuUltraTM high-fidelity DNA polymerase (Stratagene, La Jolla, CA).
  • the 2.6-kb PCR product was purified using a High Pure Purification Kit (Roche, Mannheim), digested with Ec ⁇ R ⁇ and Xbal, and then gel purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden). The fragment was then ligated into the Ec ⁇ BJ/Xbal site of the baculovirus donor plasmid pFastBacMam [pFBM] (Condreay et al, 1999 supra), downstream of the CMV-IE ( Figure 13).
  • a cassette for expression of gp64 TM-IgG Fc or human transferrin receptor (hTfR) TM-IgG Fc fusion proteins in insect cells was inserted into the SexAI/Csp45l site of pFBM-S, replacing the Neo gene, to generate 2 donor plasmids, pFBM-S-gp64 Fc ( Figure 13A) and pFBM-S-hTfR Fc ( Figure 13B), respectively, which were used to construct the corresponding gp64 Fc and hTfR Fc RecBVs using the Bac-to-Bac recombination system (Invitrogen).
  • Baculovirus stocks were purified from the supernatant of Sf9 cells infected 5 days previously. The supernatant was clarified by centrifugation (3,000 rpm, lOmin, 4°C) and the virus filtered through a 0.45 ⁇ m filter unit (Nalge Nunc), then concentrated by ultracentrifugation in a Beckman 60Ti fixed angle rotor (30,000 rpm, 75 min, 18 0 C) and the pellet resuspended in PBS. The virus titre was determined using the BacPAK Baculovirus Rapid Titer Kit (BD Biosciences, Palo Alto, CA).
  • the titers of the RecBV preparations were determined by plaque immunoassay as described previously (Martyn et al, Arch Virol 152(2) -.329-343, 2007) and expressed as plaque forming units per millilitre (pfu/ml). Transduction
  • Monolayer cultures of continuous cell lines or primary marmoset hepatocytes were prepared on glass or plastic (Thermanox, Nalge Nunc) coverslips, respectively, for immunofluorescence (IF), or on 6-well plates for immunoblot or immunoprecipitation (IP) analysis.
  • Huh7 and COS-I cell lines, and primary marmoset hepatocytes were transfected at 60-70% confluency using 6 ⁇ l FuGENE 6 (Roche, Indianapolis, IN) and 2 ⁇ g plasmid/6-well.
  • Acetone fixed coverslip cultures were blocked with 2% v/v FBS in PBS, then incubated in anti-HCV E2 (Virostat, Portland, ME) or monoclonal antibody H53 [9], followed by AlexaFluor 488 specific conjugate (Molecular Probes, Eugene, Oregon). All antibody incubations were for Ih at 37 0 C.
  • the nuclei were stained with propidium iodide (l ⁇ g/ ⁇ l) for 10 min prior to mounting, the samples were examined with a Bio-Rad 1024 confocal microscope and images obtained using LaserSharp 2000 software.
  • IP Immunoprecipitation
  • COS-I cells Twenty-four hours post-transfection, COS-I cells were incubated in met- or cys-free
  • DMEMlO (MP Biomedicals, Aurora, Ohio) for 30min, then pulsed with 150 ⁇ Ci Trans 35S-label (MP Biomedicals) for 4h and chased in complete DMEMlO for 24h.
  • the cells were lysed in ImI RIP buffer (5OmM Tris-HCl pH 7.4, 60OmM KCl, ImM EDTA, ImM
  • microtritre tray (Supplier) were coated with 5 x 10 7 pfu of each baculovirus. IgG on the surface of RecBVs was detected using rabbit anti-human IgG/HRP conjugate (DAKO).
  • DAKO rabbit anti-human IgG/HRP conjugate
  • Virus preparations were loaded on to a carbon and formvar coated grid, incubated with lOnm protein- A gold (Utrecht University, Utrecht, The Netherlands) to label Fc molecules, then fixed with 1% w/v glutaraldehyde (ProSci Tech) as a masking agent, followed by anti-gp64 and 5nm protein- A gold (Utrecht University, Utrecht, The Netherlands).
  • the samples were stained with 1.8% methylcellulose/0.4% uranyl acetate and examined in a JOEL 1010 transmission electron microscope. Images were captured on a MegaView III side-mounted CCD camera (Soft Imaging Systems, USA) and processed for publication in Adobe Photoshop (trade mark).
  • a 96-well plate (Costar) was coated with 5 ⁇ g/ml HSA-Fc ⁇ RIIa then blocked with PBS/1% w/v BSA. The plate was washed 3x with PBS only then dilutions of RecBV added and incubated at 37 0 C for Ih. Control wells contained heat aggregated IgG (HAGG) starting at 50 ⁇ g/ml. The plate was washed 6x with PBS only, then bound virus detect with an anti-human IgG- HRP conjugate followed by TMB substrate. The color development was stopped with 50 ⁇ l IM HCl and the plate read at 450nm.
  • the HLl cell line was infected with RecBV-E at multiplicity of infection (moi) ranging from 104 to 100 as described above; 48h later the cells were selected with medium containing G418 (400 ⁇ g/ml). Colonies of G418-resistant cells were pooled, and E1/E2- positive cells detected by IF. Cloned stable lines were isolated by sorting single cells from each pool using a FAC Star Plus flow cytometer (Becton Dickinson). Two weeks later, cells derived from a single colony were further expanded under G418 selection and confirmed as E1E2 -positive by IF.
  • moi multiplicity of infection
  • Stable cells were seeded into a 6-well plate and replicate wells washed and trypsinised at 24h, 48h, 72h and 96h. A viable cell count was performed and. the doubling time determined in the logarithmic phase of growth. Detection ofE2 on the surface of stable cell lines
  • a single cell suspension of the stable line P2C6 was resuspended in FACS Wash (PBS containing 2% w/v BSA, 2mM EDTA and 0.1% w/v azide).
  • the cells were stained with anti-HCV E2, diluted from 1 : 10 to 1 :40 in FACS Wash or the isotype control, followed by AlexaFluor IgG conjugate (Molecular Probes).
  • the cells were washed, pelleted and resuspended in FACS Wash for analysis.
  • a suspension of the stable line P2C6 was incubated in 20% v/v ethanol for 20min then resuspended in Annexin V-binding buffer (1OmM HEPES pH 7.4, 14OmM NaCl, 2.5mM CaC12) as a positive control.
  • the parental cell line, HLl represented the negative control.
  • Annexin V-PE (BD Biosciences) was added to each sample and incubated for 15min at room temp. 5 ⁇ l of 50 ⁇ g/ml 7-amino actinomycin D (7 -AAD; Sigma) was added to each sample lOmin prior to FACS analysis.
  • a representative stable cell line, P2C6, and the HLl cell line were stained with Annexin V to detect apoptotic cells and the relative percentage of apoptotic cells in the 2 cell lines was determined by flow cytometry (Figure 5B). The percentage of cells in the gated population
  • Annexin V staining of ethanol treated P2C6 cells showed 92.6 % of apoptotic cells.
  • the expression of E1/E2 did not result in an increased rate of apoptosis.
  • E2 expressed in the stable cell line P2C6 was shown to be correctly folded by a specific, though weak, reactivity with a conformation-dependent E2 mAb, H53 (Figure 6A).
  • a proportion of the expressed E2 was detected on the surface of the P2C6 cells by flow cytometry using the polyclonal anti-E2 antibody ( Figure 6B).
  • Detection of E2 on P2C6 cells was detected using a high concentration (1 :10) of the primary antibody and showed a significant increase in the net mean fluorescence intensity compared to parental HLl cells. The level of specific binding progressively decreased using 1:20 and 1:40 dilutions of primary antibody.
  • the RecBV with E1/E2 embedded in the envelope can be expected to target dendritic cells and elicit a strong immune response as a result.
  • mammalian cell-derived West Nile Virus normally binds to DC- SIGNR
  • insect cell-derived virus binds to DC-SIGN, as a result of the differential glycosylation patterns (Davis et al, J Virol 50:1290, 2006) suggesting that the insect cell- derived RecBV will also bind strongly to DC-SIGN.
  • the RecBV with HCV E1/E2 in the envelope is likely to bind to DC through CD81, known to be a cellular receptor for HCV.
  • RecBV-E also elicited an antibody response against the HCV E2 protein, proving that the virus transduced cells in the mouse, resulting in expression of the E1/E2 in the cells (driven by the CMV promoter) that in turn resulted in the development of a humoral immune response.
  • RecBV- E1/E2 elicited a strong antibody reaction against the baculovirus envelope ( Figure 12), which contains gp64.
  • Baculoviruses are enveloped by budding from the plasma membrane, and although E1/E2 is normally localized to the endoplasmic reticulum.
  • the proteins are directed to the plasma membrane by inserting the E1/E2 genes downstream of the leader sequence of the gp64.
  • HCV E1/E2 proteins into the RecBV envelope is achieved by expressing the E1/E2 in a stable insect cell line which can be infected with the virus, or by encoding E1/E2 in the baculovirus genome, in this case, controlled by the polyhedrin promoter.
  • the latter approach is chosen since the expression of constitutively-expressed proteins in stable cell lines is often reduced with time in passage.
  • this strategy has been used successfully in the past to incorporate foreign proteins into the baculovirus envelope (see Kost et al, J Virol 55:1117-1120, 1994).
  • the E1/E2 in the virus envelope is expected to elicit a neutralizing antibody response
  • the core/El/E2 and NS3m/4 expressed as endogenous protein in the transduced mammalian cells, are expected to generate an effective cell mediated immune response.
  • the structural proteins will form virus-like particles (VLPs) that can also be expected to elicit neutralizing antibody responses.
  • NS3/4 is chosen for use in the vaccine because the protein is highly conserved and contains a large number of CD4+ and CD8+ cell epitopes (Wertheimer et al, Hepatol 37:577-589, 2003). Others have suggested that NS3 may also be used as an immunogen.
  • RecBV-J (Japan).
  • the core-p7 region from JFHl is subcloned into the pFastBacMam donor vector downstream of the CMV promoter.
  • JFHl is a genotype 2a isolate that replicates in continuous cell lines.
  • An additional downstream region is inserted encoding the EMCV IRES upstream of the NS3m/4 region from the Australian genotype Ib isolate, to generate a bicistronic transfer vector.
  • the E1/E2 gene from JFHl will then be inserted downstream of the polyhedrin promoter, and the the transfer vector is used to generate RecBV-J.
  • RecBV-A (Australia).
  • the RecBV viruses which has been constructed is based on the HCV genotype Ib (Trowbridge & Gowans, Arch Virol 143:501-511, 1998) and the RecBV-core/El/E2 virus is modified by inserting the EMCV-NS3m/4 region downstream of the existing HCV genes in the transfer vector.
  • the transfer vector is further modified by cloning the genotype Ib E1/E2 genes downstream of the polyhedrin promoter and the RevBV-A generated as described above.
  • RecBV-Am (Australia mutant).
  • the RecBV-A encodes the wild-type HVRl sequence.
  • the wild-type HVRl in RecBV-A is substituted with the seuqence of the G31 mimotope (Puntoriero et al, 1998, supra) as this was the most cross reactive of the mimotopes.
  • the G31-containing-RecBV-Am will express three published neutralizing epitopes that can be expected to elicit antibodies with broad cross reactive, neutralizing activity.
  • the NS3/4 gene in each of the above RecBV encodes a mutant form of NS3 (NS3m/4) containing a C 1125 A mutation in the protease domain that inactivates NS3 protease function. This will ensure that Interferon Regulatory Factor-3 (IRF-3) function is not cleaved because the protease function of wild-type NS3/4 has been shown to block the effector action of IRF-3 (Foy et a!, Science 500:1145-1148, 2003).
  • the NS3m/4 gene downstream of the EMCV or the HCV IRES elements have been constructed and it has been shown that human Mo-DC, transfected with the specific mRNA matured the DC more effectively than did RNA encoding the wild-type protein.
  • RecBV iv) Characterization of RecBV.
  • Each of the above viruses is expected to express the E1/E2 proteins in insect cells (to result in incorporation of the HCV envelope glycoproteins in the RecBV envelope) and the core/El /E2 and NS3m/4 proteins in mammalian cells.
  • the E1/E2 genes encoded downstream of the CMV and polyhedrin promoters, respectively will be homologous.
  • the Sf9 insect cell line is infected with the individual viruses and examine the expression of the El and E2 proteins (from the polyhedrin promoter) by immunofluorescence and immunoblot. The expression of E1/E2 is examined on the plasma membrane of the infected insect cell by flow cytometry as performed in the above Examples.
  • the virus is purified by gradient ultracentrifugation and the infectious fractions tested for E1/E2 by immunoblot. Similarly, expression of the proteins controlled by the CMV promoter 48 hours after transduction of Huh7 cells is confirmed by immunofluorescence and immunoblot.
  • RecBV DNA integration Although the circular, ds, baculovirus DNA does not integrate during replication, it will be vital to exclude integration after intramuscular injection of the RecBV.
  • a previous study (Tjia et al, 1983 supra) used Southern blot hybridization analysis to show that baculovirus DNA did not persist beyond 48 hours in mammalian cells and studies showed that proteins expressed from the RecBV in transduced cells did not persist beyond 18 days post infection (see Examples above). To exclude DNA integration in vivo the mice are killed at intervals after intramuscular injection of the RecBV and the muscle removed.
  • Integrated viral DNA is usually only detected after clonal amplification of a cell which contains the inserted DNA, as infrequent integration events are undetected in the vast excess of normal cellular DNA prior to this. Assuming that all the plasmid DNA detected by PCR is integrated, it is calculated that the risk from mutation by insertional mutagenesis, is approximately 350 times lower than that of the spontaneous rate of mutation. Clearly, the calculated risk will be directly dependent on the amount of residual RecBV DNA, the amount of RecBV DNA is measured and then the relative risk calculated.
  • the injection of RecBV with a ds circular DNA genome poses a lower risk than that after injection of plasmid DNA, some of which is likely to be linear and more likely to result in integration.
  • the baculovirus may also be engineered to contain a TLR-targeting protein either from HCV or a non-HCV source. Examples or suitable sources include those listed in Table 2.
  • the baculovirus envelope may contain other targeting moieties such as an IgG constant region to target any antigen-presenting cell with IgG receptors, decay accelerating protein (DAF) which increases resistance to complement and/or CD40L to target CD40 as DC.
  • DAF decay accelerating protein
  • Vaccination with RecBV-J Balb/C are vaccinated with the RecBV-J by the intramuscular route as described (Facciabene et al, J Virol 75:8663-8673, 2004) using two doses of 10 8 ffu, three weeks apart.
  • the anti-E2 response will be measured at regular intervals by ELISA as described above.
  • the anti-core response is detected using ELISA (Trowbridge et al, J Hepatol 24:532-538, 1996) to act as an internal standard for the efficacy of vaccination with RecBV- J, RecBV-A and RecBV- Am, possible because the protein is highly conserved.
  • mice are vaccinated with RecBV-A.
  • neutralizing antibody is measured against the HCV pseudotyped virus (HCVpp) as described (Drummer et al, CHn Exp Immunol 142:362-369, 1996).
  • HCVpp HCV pseudotyped virus
  • mice are vaccinated with RecBV-Am, and test serum samples for neutralization against Am and A pseudotypes and JFHl virus.
  • Immune capture assay The ability of all serum samples from mice vaccinated with RecBV-JH, RecBV-A or RecBV-Am to preciptate HCV virions in an immune capture assay is examined. This assay uses purified mouse IgG to coat 0.2ml PCR strip tubes which are then blocked prior to the addition of HCV-positive serum. The RNA in hound virus is released, purified and detected by nested RT-PCR.
  • This assay determines the antibodies are capable of recognizing whole virus particles and is particularly relevant for samples form animals which were tested against the HCVpp, but will also extend the information related to the potential cross neutralizing ability of antibodies elicited by the different RecBV, because HCV-positive serum is used from patients infected with different isolates and genotypes.
  • Anti-El /E2 antibody isotype It has been shown that anti-HCV antibodies detected in persistent infection are almost entirely IgGl, whereas most viral antigens induce IgGl and IgG3, with some IgG2 and IgG4. Consequently, it is determined which Ig subclass of the anti-El /E2 antibodies is induced by the RecBV, as it is most likely that the lack of IgG3 during HCV infection is not related to the proteins but to HCV subversion of the immune response. In addition, the induction of IgG2 indicates a preference for the ThI pathway. Examine CMI elicited by RecBV-El/E2 and RecBV-NS3/4 in Balb/C mice
  • CD4 + T-cell response will be evaluated by lymphocyte proliferation ( 3 H-T incorporation) using recombinant HCV protein restimulated spleen cells from the vaccinated mice.
  • CD8 + CTL activity is detected by IFN- ⁇ ELIspot using irradiated, recombinant vaccina virus (recVV)-HCV- infected spleen cells from na ⁇ ve mice as antigen-presenting, stimulatory cells, or with pools of HCV peptides (18mers overlapping by 11).
  • An in vivo CTL assay is also performed spleen cells from na ⁇ ve syngeneic mice is pulsed with pooled HCV-specific peptides.
  • the HCV- peptide-pulsed cells, and unrelated peptide-pulsed controls cells are labeled with high and low concentrations of CFSE, respectively as described (Coles et al, J Immunol 7d ⁇ :834, 2002) then irradiated.
  • the cells are then mixed, injected into the mice by the intravenous route and the cell viability measured 16 hours later by flow cytometry of a spleen cell suspension to identify the differential CFSE populations.
  • These experiments detect CMI to peptides which are restricted by the mouse H-2 2 (Balb/C).
  • the vaccinated female mice are challenged with recVV-HCV. Two weeks are immunization, the mice are injected by the intraperitoneal route with a standard dose of virus (10 7 pfu) and the titre in the ovaries (the preferred site of VV replication) determined four days later by plaque assay.
  • BVDV recombinant BVDV which encodes the wild-type NS3/4 proteins.
  • HCV IRES-NS3/4 (wild-type) gene is inserted downstream of the NS5B gene in the BVDV genome.
  • the NS5A/5B cleavage site in the BVDV polyprotein recognized by the BVDV NS3 is substituted with the HCV 5A/5B cleavage site that is recognized by the NS3/4A protease. This will ensure that continued expression of the HCV NS 3/4 A is required for replication of the mutant BVDV ( Figure 10).
  • the virus is titrated in sheep to determine a 50% infectivity dose and vaccinated sheep will be challenged with 10- 100ID 50 .
  • EXAMPLE 8 Construction of recombinant baculovirus vectors designed to display human IgG Fc
  • RecBvs were constructed and characterized displaying the Fc region of human IgG in their envelope. It was proposed that the baculovirus vectors displaying Fc would more efficiently transduce cell lines expressing Fc receptors and antigen-presenting cells, in a process analogous to the uptake of immune complexes, compared to the wild- type (wt) baculovirus vector.
  • the aim of the study was to assess the relative levels of binding of the RecB Vs and WtB V to cells expressing the Fc receptor (cell lines and APCs) and determine if the levels of expression of the HCV structural proteins (core, El and E2) following transduction of mammalian cells showed a corresponding increase.
  • cytoplasmic domain at the N-terminus of the transferrin receptor (aa 1-67) may lead to exclusion of the hTfR-IgG Fc fusion protein from the BV envelope in the process of budding.
  • cytoplasmic N-terminal domain of the TfR sequence from aa 1-53 was deleted in the construct, as this truncation was previously reported to have no effect on expression of the TfR in the type II orientation (Zerial et al, EMBO J5:1543-1550, 1986).
  • IgG Fc is expressed as a homodimer on the plasma membrane of RecBV-infected insect cells and on the envelope of RecBV particles.
  • Human IgG Fc was expressed as a fusion protein with either the gp64 TM or the hTfR TM in RecBV-infected, but not wtBV-infected, Sf9 insect cells, as determined by IF ( Figure 15). Analysis of live (unfixed) Sf9 insect cells infected with RecBVs showed that the expressed Fc fusion proteins were localised to the plasma membrane ( Figures 15 A, 15B) while the wtBV-infected and mock-infected cells were negative ( Figures 15C, 15D). Minor differences in the distribution of Ig Fc were noted between gp64 and hTfR RecBV (Fig 3 A, 3B) but it was clear that the proteins were expressed on the plasma membrane.
  • the proportion of 35:70kDa proteins was approximately 1 :1 in the gp64 Fc Ig RecBV-infected cells but was approx 100:1 in the hTfR FcIg RecBV-infected cells ( Figure 16). Monomeric and dimeric Fc fusion proteins were also detected in RecBV, but not wtBV, particles ( Figure 16) but in this case, the 35:70kDa proportion was similar in both virus preparations and was considered to be approximately 2:1. As the concentration of the two RecBV preparations was adjusted to contain 2 x 10 8 pfu, the data in the right panel ( Figure 16), show that each virus preparation appeared to contain similar levels of Ig-Fc and similar ratios of the 35:70kDa fusion proteins. However, the ratio of the p35:p70 proteins may simply reflect the degree of denaturation achieved in this denaturing gel system and thus the level of the p70 dimer may actually be much higher.
  • the results of this part of the study show that the RecBV gp64 Ig Fc and RecBV hTfR Ig Fc both direct the expression of human Ig Fc on the plasma membrane of infected Sf9 insect cells and that semi-purified preparations of the viruses contain dimeric forms of the Ig Fc and may also contain monomeric forms. Ig Fc is expressed on the envelope of RecBV.
  • IgG Fc displayed on the plasma membrane of RecBV-infected insect cells binds specifically to Fc ⁇ RIIa.
  • RecBV bind soluble Fc ⁇ RIIa and bind to cells which express surface Fc ⁇ RIIa.
  • Ig Fc The inclusion of Ig Fc into the envelope of RecBV should impart the particles with the ability to bind soluble Fc ⁇ RIIa protein and also result in increased binding and uptake of the RecBV to cells which express the Fc ⁇ RIIa.
  • dilutions of RecBV or wtBV were added to the wells of a microtitre plate, previously coated with soluble Fc ⁇ RIIa, and bound virus detected with an anti-human IgG HRP conjugate.
  • BSA-coated plates were used as negative controls and heat aggregated IgG (HAGG) included as a positive control.
  • the II A 1.6 cell line which was constructed to express the Fc ⁇ RIIa in a constitutive manner, was transduced and the MFI resulting from detection of bound virus was then determined by flow cytometry (Figure 20).
  • the parental cell line failed to bind RecBV hTfR-Fc, whereas the ILAl .6 line bound the virus in a dose-dependent manner.
  • the RecBV hTfR-Fc-IIA1.6 interaction was inhibited by the prior addition of MAb IV-3 ( Figure 20), in a similar manner to that described above to soluble RecBV hTfR-Fc.
  • hTfR-Fc RecBV binding is Fc ⁇ R dependent.
  • the binding of gp64-Fc and hTfR-Fc RecBVs to primary cells which express Fc ⁇ Rs was also assessed (Figure 21).
  • Immature human CD14 + monocyte-derived dendritic cells (Mo- DC) were prepared, mixed with virus as described (Martyn et al, 2007 supra) and bound virus detected by flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • Virus was incubated with PBMC as described above and binding assessed by flow cytometry (Figure 22) to two major cell populations viz. granulocytes, determined by FSC characteristics and monocytes, determined by staining with anti-CD 14.
  • Figure 22 The results of this experiment showed that the parental virus was able to bind to a proportion of cells, determined to be monocytes, in a dose dependent manner (Rl in panel A, Figure 22) and to a minor proportion of granulocytes (Ll in panel A)-compare these data with the no virus control (see inset).
  • the gp64 Fc RecBV was able to bind to a high proportion of granulocytes (Ll in panel B) and monocytes (Rl in panel B), but the level of binding was not increased with higher concentrations of virus, suggesting that the gp64 Fc RecBV receptors were already saturated at a moi of 20.
  • the hTfR Fc RecBV was not only able to bind to granulocytes and monocytes at low moi (Panel C) but showed increased levels of binding related to increased virus moi.
  • RecBVs displaying IgG Fc exhibit enhanced expression of HCV structural proteins compared to wild-type baculovirus following transduction of cells expressing Fc receptors.
  • the increased binding efficiency of the gp64-Fc RecBV and hTfR-Fc RecBV also resulted in an increase in the levels of expression of the HCV proteins driven by the CMV promoter.
  • This Example provides a RecBVs displaying functional IgG Fc in the envelope in type I and type II orientations. These RecBVs are improved baculoviral vectors able to enhance binding to cell lines and APCs expressing Fc receptors in vitro, thereby delivering encoded genes and targeting expression to these cells. These viral vectors have application for the delivery of genes in vivo for the purpose of vaccination or gene therapy.
  • RecBVas a virus-like particle to generate humoral and cell mediated immune responses
  • a RecBV is generated which contains foreign (heterologous) proteins inserted into the virus envelope which is a VLP that, in addition to inducing humoral immune responses, also induces cell mediated immune responses as a result of cross presentation.
  • Cross presentation is described as the process of exogenous proteins being taken up by antigen presenting cells, processed and presented in the context of MHC class 1.
  • VLPs constructed in this way are used for vaccination purposes either with or without the addition of a suitable adjuvant which may help to induce MHC class I-restricted responses. This is useful as there are currently no adjuvants which are licensed to elicit cell mediated responses in humans.
  • proteins fused to the transmembrane domain of the transmembrane domains of pg64, CD4 or hTfR are shown as examples.
  • These proteins represent any of the HCV structural proteins (core, El and E2) or the nonstructural proteins (NS2, NS3, NS4, NS5) but the conserved highly immunogenic core protein might be preferred as might be the NS3 protein which is not only conserved, but also because cellular immune responses to this protein are associated with recovery from acute phase HCV infection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne généralement un véhicule de livraison utile en vaccination et thérapie génique. En particulier, le véhicule de livraison induit une réponse immunitaire, humoral et par l'intermédiaire de cellules, protectrice contre le Virus de l'hépatite C (HCV) ou d'autres pathogènes ou peut être utilisé pour faciliter une thérapie génique. L'invention concerne également des méthodes pour vacciner contre le HCV ou d'autres pathogènes ou pour faciliter une thérapie génique et des compositions qui comprennent le véhicule de livraison.
PCT/AU2007/000578 2006-05-02 2007-05-02 Véhicule de livraison d'un vaccin de l'hépatite c Ceased WO2007128048A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2006902253A AU2006902253A0 (en) 2006-05-02 A Vaccine
AU2006902253 2006-05-02

Publications (1)

Publication Number Publication Date
WO2007128048A1 true WO2007128048A1 (fr) 2007-11-15

Family

ID=38667321

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2007/000578 Ceased WO2007128048A1 (fr) 2006-05-02 2007-05-02 Véhicule de livraison d'un vaccin de l'hépatite c

Country Status (1)

Country Link
WO (1) WO2007128048A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652450A (zh) * 2015-07-10 2019-04-19 陕西诺威利华生物科技有限公司 一种猪用疫苗载体

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021338A1 (fr) * 1996-11-08 1998-05-22 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Synthese et purification de particules de type virus de l'hepatite c
WO2002022155A1 (fr) * 2000-09-13 2002-03-21 Hawaii Biotechnology Group, Inc. Composition immunogene de l'hepatite c et procedes d'utilisation de cette derniere
US20040001853A1 (en) * 2002-06-20 2004-01-01 Rajan George Chimeric antigens for eliciting an immune response
US20050013828A1 (en) * 2002-06-20 2005-01-20 Virexx Medical Corp. Chimeric antigens for eliciting an immune response

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021338A1 (fr) * 1996-11-08 1998-05-22 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Synthese et purification de particules de type virus de l'hepatite c
WO2002022155A1 (fr) * 2000-09-13 2002-03-21 Hawaii Biotechnology Group, Inc. Composition immunogene de l'hepatite c et procedes d'utilisation de cette derniere
US20040001853A1 (en) * 2002-06-20 2004-01-01 Rajan George Chimeric antigens for eliciting an immune response
US20050013828A1 (en) * 2002-06-20 2005-01-20 Virexx Medical Corp. Chimeric antigens for eliciting an immune response

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AOUBALA M. ET AL.: "The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis", JOURNAL OF GENERAL VIROLOGY, vol. 82, 2001, pages 1637 - 1646 *
BARTENSCHLAGER R.: "The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy", JOURNAL OF VIRAL HEPATITIS, vol. 6, May 1999 (1999-05-01), pages 165 - 181, XP001029119 *
BAUMERT T.F. ET AL.: "Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells", JOURNAL OF VIROLOGY, vol. 72, no. 5, May 1998 (1998-05-01), pages 3827 - 3836, XP002167716 *
JEONG S.H. ET AL.: "Immunization with Hepatitis C Virus-Like Particles Induces Humoral and Cellular Immune Responses in Nonhuman Primates", JOURNAL OF VIROLOGY, vol. 78, no. 13, July 2004 (2004-07-01), pages 6995 - 7003 *
OVERTON H. ET AL.: "Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays", JOURNAL OF GENERAL VIROLOGY, vol. 76, 1995, pages 3009 - 3019, XP002158506 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652450A (zh) * 2015-07-10 2019-04-19 陕西诺威利华生物科技有限公司 一种猪用疫苗载体

Similar Documents

Publication Publication Date Title
TWI364294B (en) Chimeric antigens for breaking host tolerance to foreign antigens
US8603468B2 (en) Neutralization of HCV
Saito et al. Plasmid DNA-based immunization for hepatitis C virus structural proteins: immune responses in mice
US20110223185A1 (en) Chimeric hepatitis c virus antigens for eliciting an immune response
US9339535B2 (en) Vaccines and immunopotentiating compositions and methods for making and using them
KR102236137B1 (ko) 수포성 구내염 바이러스의 변형된 기질 단백질
JP2003093081A (ja) Ｃ型肝炎ウイルスアシアロ糖タンパク質
US20040001853A1 (en) Chimeric antigens for eliciting an immune response
JP6942309B2 (ja) フラビウイルスウイルス様粒子
KR20140132332A (ko) 프라임 부스트 백신용 수포성 구내염 바이러스
JP2002517200A (ja) フラビウイルス感染の予防のための核酸ワクチン
Ying et al. Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle
EP3013852A1 (fr) Procédés et compositions pour des vaccins contre le virus de la dengue
JP2005532064A (ja) 改変したｎｓ３ドメインを有するｈｃｖ融合タンパク質
WO2001021807A1 (fr) Proteine enveloppe 2 (e2) du virus de l'hepatite c qui ne possede pas tout ou partie de la region 1 hypervariable (hvr1), acides nucleiques correspondants, virus chimeriques et utilisation de ces derniers
JP5710254B2 (ja) 外来エピトープを含む組換え偽ウイルスの発現および分泌を可能とするポリヌクレオチド、それらの製造および使用
WO1998028004A1 (fr) Particule de l'hepatite delta contenant un immunogene de proteine de fusion
CN105330730A (zh) 一种丙型肝炎病毒重组蛋白的制备及其应用
CN105254756B (zh) 一种针对丙型肝炎病毒包膜蛋白e2的单克隆抗体及应用
Zhu et al. The wild-type hepatitis C virus core inhibits initiation of antigen-specific T-and B-cell immune responses in BALB/c mice
US20050277192A1 (en) Hepatitis C virus nonstructural protein 4A (NS4A) is an enhancer element
JP2002501737A (ja) Ｃ型肝炎ウイルスの非構造蛋白質による遺伝的免疫感作
US7078500B1 (en) Genetic immunization with nonstructural proteins of hepatitis C virus
US20230330218A1 (en) Hepatitis c virus modified e2 glycoprotein and uses thereof as vaccines
KR100902817B1 (ko) 플라스미드 ｄｎａ 및 재조합 아데노 바이러스를 이용한ｃ형 간염 바이러스에 대한 방어면역력을 증진시키는 백신

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07718825

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07718825

Country of ref document: EP

Kind code of ref document: A1