WO2004039950A2 - Activation de lymphocytes specifiques du vhc - Google Patents

Activation de lymphocytes specifiques du vhc Download PDF

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Publication number
WO2004039950A2
WO2004039950A2 PCT/US2003/033610 US0333610W WO2004039950A2 WO 2004039950 A2 WO2004039950 A2 WO 2004039950A2 US 0333610 W US0333610 W US 0333610W WO 2004039950 A2 WO2004039950 A2 WO 2004039950A2
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hcv
cells
polypeptide
polypeptides
fusion protein
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PCT/US2003/033610
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English (en)
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WO2004039950A3 (fr
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Michael Houghton
Steve Coates
Mark Selby
Xavier Paliard
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Chiron Corporation
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Priority to AU2003287188A priority Critical patent/AU2003287188A1/en
Priority to EP03781368A priority patent/EP1576125A4/fr
Priority to CA002505611A priority patent/CA2505611A1/fr
Publication of WO2004039950A2 publication Critical patent/WO2004039950A2/fr
Publication of WO2004039950A3 publication Critical patent/WO2004039950A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA 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
    • A61K2039/51—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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the activation of hepatitis C virus(HCV)-specific T cells. More particularly, the invention relates to the use of multiple HCV polypeptides, either alone or as fusions, to stimulate cell-mediated immune responses, such as to activate HCV-specific T cells.
  • HCV infection is an important health problem with approximately 1% of the world's population infected with the virus. Over 75% of acutely infected individuals eventually progress to a chronic carrier state that can result in cirrhosis, liver failure, and hepatocellular carcinoma. See Alter et al. (1992) N. Engl. J. Med. 327: 1899-1905; Resnick and Koff. (1993) Arch. Intern. Med.
  • the invention provides HCV proteins useful for stimulating immune responses, such as activating HCV-specific T cells.
  • One embodiment provides a fusion protein that comprises HCV polypeptides, wherein the HCV polypeptides consist essentially of an NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide.
  • the fusion protein includes an NS5b polypeptide.
  • At least one of the HCV polypeptides is derived from a different strain of HCV than the other polypeptides.
  • compositions comprising any of these fusion proteins and a pharmaceutically acceptable excipient.
  • the compositions further comprise an adjuvant, a CpG polynucleotide and/or the fusion protein is adsorbed to or entrapped within a microparticle or ISCOM.
  • the compositions can further comprise a polynucleotide encoding an E1E2 complex.
  • the E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle.
  • Another embodiment provides a composition comprising HCV polypeptides and a pharmaceutically acceptable excipient.
  • the HCV polypeptides consist essentially of an NS3, an NS4, an NS5a polypeptide, and optionally a core polypeptide.
  • the composition includes an NS5b polypeptide.
  • the compositions further comprise an adjuvant, a CpG polynucleotide and/or one or more of the HCV polypeptides is adsorbed to or entrapped within a microparticle or ISCOM.
  • the compositions can further comprise a polynucleotide encoding an E1E2 complex.
  • the E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle.
  • one of the HCV polypeptides may be derived from a different strain of HCV than the others.
  • Even another embodiment of the invention provides an isolated and purified polynucleotide which encodes a fusion protein as described above.
  • the fusion proteins further include a polynucleotide encoding an E1E2 complex.
  • Yet another embodiment of the invention provides a composition comprising the polynucleotides described above and a pharmaceutically acceptable excipient.
  • the compositions further comprise an adjuvant and/or the polynucleotide may be adsorbed to or entrapped within a microparticle.
  • the compositions can further comprise a polynucleotide encoding an E1E2 complex.
  • the E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle.
  • the invention provides a composition comprising HCV polynucleotides and a pharmaceutically acceptable excipient, wherein the HCV polynucleotides consist essentially of polynucleotides encoding an NS 3, anNS4, an NS5a polypeptide, and optionally a core polypeptide.
  • the composition also includes a polynucleotide encoding an NS5b polypeptide.
  • the compositions may further comprise an adjuvant and/or one or more of the polynucleotides may be adsorbed to or entrapped within a microparticle.
  • compositions can further comprise a polynucleotide encoding an E1E2 complex.
  • the E1E2 polynucleotide can also be adsorbed to or entrapped withing a microparticle. Additionally, one or more of the polynucleotides may be derived from a different strain of HCV than the others.
  • the invention provides a method of activating T cells which recognize an epitope of an HCV polypeptide.
  • T cells are contacted with any of the fusions, polynucleotides or compositions described above.
  • a population of activated T cells recognizes an epitope of the NS3, NS4, NS5a, NS5b, core and/or E1E2 polypeptide.
  • the regions in the fusions need not be in the order in which they naturally occur in the native HCV polyprotein.
  • the NS5b polypeptide if present, may be at the N- and/or C-terminus of the fusion, or may be located internally.
  • the El polypeptide may precede or follow the E2 polypeptide.
  • the E1E2 polypeptide may also be part of the nonstructural fusion protein or may be provided separately, as an El E2 complex, or as individual polypeptides.
  • the NS3 polypeptide may include a modification to inhibit protease activity, such that cleavage of the fusion is inhibited. Such modifications are described more fully below.
  • the compositions can comprise more than one HCV nonstructural fusion protein, such as a fusion protein with NS3, NS4 and
  • the nonstructural fusion protein consists of, from the amino terminus to the carboxyl terminus, an NS3, an NS4, an NS5a and, optionally, an NS5b polypeptide and the E1E2 complex consists of, from amino terminus to the carboxyl terminus, an El polypeptide and an E2 polypeptide.
  • the various polypeptides are derived from the same HCV isolate, or from different strains and isolates including isolates having any of the various HCV genotypes, to provide increased protection against a broad range of HCV genotypes.
  • Yet another embodiment of the invention provides a method of stimulating an immune response, such as a cellular immune response, in a vertebrate subject by administering a composition as described herein.
  • the composition activates T cells which recognize an epitope of an HCV polypeptide.
  • T cells are contacted with a composition as described above.
  • T cells recognizes an epitope of one or more of the HCV polypeptide(s).
  • the invention thus provides methods and reagents for stimulating immune responses to HCV, such as for activating T cells which recognize epitopes of HCV polypeptides. These methods and reagents are particularly advantageous for identifying epitopes of HCV polypeptides associated with a strong CTL response and for i ⁇ -munizing mammals, including humans, against HCV.
  • Figure 1 is a diagrammatic representation of the HCV genome, depicting the various regions of the HCV polyprotein.
  • Figure 2 depicts the DNA and corresponding amino acid sequence of a representative native NS3 protease domain.
  • Figures 3A-3C shows the nucleotide and corresponding amino acid sequence for the HCV-1 El/E2/p7 region. The numbers shown in the figure are relative to the full-length HCV-1 polyprotein. The El, E2 and p7 regions are shown.
  • Figure 4 is a diagram of plasmid pMHElE2-809, encoding E1E2 809 , a representative E1E2 protein for use with the present invention.
  • Figures 5A-5J depict the DNA and corresponding amino acid sequence of a representative NS345Core fusion protein.
  • the depicted sequence includes amino acids 1242-3011 of the HCV polyprotein (representing polypeptides from NS3, NS4, NS5a and NS5b) with amino acids 1-121 of the HCV polyprotein (representing a polypeptide from the core region) fused to the C-terminus of NS5b. This numbering is relative to the HCV-1 polyprotein.
  • Figure 6 shows a side-by-side comparison of IFN- ⁇ expression generated in animals in response to delivery of alphavirus constructs encoding NS3NS4NS5a.
  • Figure 7 shows IFN- ⁇ expression generated in animals in response to delivery of plasmid DNA encoding NS3NS4NS5a ("naked"), PLG-linked DNA encoding NS NS4NS5a ("PLG), separate DNA plasmids encoding NS5a, NS34a, and NS4ab ("naked"), and PLG-linked DNA encoding NS5a, NS34a, and NS4ab (“PLG”).
  • Figure 8 shows HCV-specific CD8+ and CD4+ responses in vaccinated chimpanzees.
  • Figure 9 depicts the specificity of T cell responses primed by electroporation of plasmid DNA two weeks subsequent to the third immunization.
  • Figure 10 shows the specificity of T cell responses primed by vaccinating chimpanzees with NS345Core 12 ⁇ -ISCOMS two weeks subsequent to the third immunization.
  • Cysteine Cys (C) Glutamine: Gin (Q)
  • Threonine Thr (T) Tryptophan: Trp (W)
  • HCV polyprotein The polypeptide need not be physically derived from HCV, but may be synthetically or recombinantly produced. Moreover, the polypeptide may be derived from any of the various HCV strains and isolates including isolates having any of the 6 genotypes of HCV described in Simmonds et al., J Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.), as well as newly identified isolates, and subtypes of these isolates, such as HCV la, HCV lb, etc.
  • NS4 polypeptide refers to native NS4 from any of the various HCV strains, as well as NS4 analogs, muteins and immunogenic fragments, as defined further below.
  • El polypeptide a molecule derived from an HCV El region.
  • the mature El region of HCV-1 begins at approximately amino acid 192 of the polyprotein and continues to approximately amino acid 383, numbered relative to the full-length HCV-1 polyprotein. (See, Figures 1 and 3A-3C. Amino acids 192-383 of Figures 3 A-3C correspond to amino acid positions 20-211 of SEQ ID NO:4.) Amino acids at around 173 through approximately 191 (amino acids 1-19 of SEQ ID NO: 4) serve as a signal sequence for El.
  • an "El polypeptide” is meant either a precursor El protein, including the signal sequence, or a mature El polypeptide which lacks this sequence, or even an El polypeptide with a heterologous signal sequence.
  • the El polypeptide includes a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 360-383 (see, International Publication No. WO 96/04301, published February 15, 1996).
  • An El polypeptide, as defined herein, may or may not include the C-terminal anchor sequence or portions thereof.
  • an “E2 polypeptide” is meant a molecule derived from an HCV E2 region.
  • the mature E2 region of HCV-1 begins at approximately amino acid 383-385, numbered relative to the full-length HCV-1 polyprotein. (See, Figures 1 and 3A-3C. Amino acids 383-385 of Figures 3A-3C correspond to amino acid positions 211-213 of SEQ ID NO: 4.) A signal peptide begins at approximately amino acid 364 of the polyprotein.
  • an "E2 polypeptide” is meant either a precursor E2 protein, including the signal sequence, or a mature E2 polypeptide which lacks this sequence, or even an E2 polypeptide with a heterologous signal sequence.
  • the E2 polypeptide includes a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 715-730 and may extend as far as approximately amino acid residue 746 (see, Lin et al, J Virol. (1994) 68:5063-5073).
  • An E2 polypeptide, as defined herein, may or may not include the C-terminal anchor sequence or portions thereof.
  • an E2 polypeptide may also include all or a portion of the p7 region which occurs immediately adjacent to the C-terminus of E2. As shown in Figures 1 and 3A-3C, the p7 region is found at positions 747-809, numbered relative to the full-length HCV-1 polyprotein (amino acid positions 575-637 of SEQ ID
  • HCV E2 HCV E2
  • multiple species of HCV E2 exist (Spaete et al., Virol. (1992) 188:819-830; Selby et al., J Virol. (1996) 70:5177-5182; Grakoui et al., J Virol. (1993) 67:1385-1395; Tomei et al., J Virol. (1993) 67:4017-4026).
  • E2 encompasses any of these species of E2 including, without limitation, species that have deletions of 1-20 or more of the amino acids from the N-terminus of the E2, such as, e.g, deletions of 1, 2, 3, 4, 5....10...15, 16, 17, 18, 19... etc. amino acids.
  • Such E2 species include those beginning at amino acid 387, amino acid 402, amino acid 403, etc.
  • El and E2 regions from HCV-1 are shown in Figures 3A-3C and SEQ ID NO:4.
  • the El and E2 regions are defined with respect to the amino acid number of the polyprotein encoded by the genome of HCV-1 , with the initiator methionine being designated position 1. See, e.g., Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455.
  • the term an "El polypeptide” or an "E2 polypeptide” as used herein is not limited to the HCV-1 sequence.
  • the corresponding El or E2 regions in other HCV isolates can be readily determined by aligning sequences from the isolates in a manner that brings the sequences into maximum alignment. This can be performed with any of a number of computer software packages, such as ALIGN 1.0, available from the University of Virginia, Department of Biochemistry (Attn: Dr. William R. Pearson). See, Pearson et al, Proc. Natl. Acad. Sci. USA (1988) 85:2444-2448.
  • an "El polypeptide” or an “E2 polypeptide” as defined herein is not limited to a polypeptide having the exact sequence depicted in the Figures. Indeed, the HCV genome is in a state of constant flux in vivo and contains several variable domains which exhibit relatively high degrees of variability between isolates. A number of conserved and variable regions are known between these strains and, in general, the amino acid sequences of epitopes derived from these regions will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%, more than 60%, and even more than 80-90% homology, when the two sequences are aligned.
  • El or E2 polypeptide refers to native El or E2 sequences from any of the various HCV strains, as well as analogs, muteins and immunogenic fragments, as defined further below.
  • the complete genotypes of many of these strains are known. See, e.g., U.S. Patent No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799.
  • El polypeptide and E2 polypeptide encompass proteins which include modifications to the native sequence, such as internal deletions, additions and substitutions (generally conservative in nature). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through naturally occurring mutational events. All of these modifications are encompassed in the present invention so long as the modified El and E2 polypeptides function for their intended purpose. Thus, for example, if the El and/or E2 polypeptides are to be used in vaccine compositions, the modifications must be such that immunological activity (i.e., the ability to elicit a humoral or cellular immune response to the polypeptide) is not lost.
  • immunological activity i.e., the ability to elicit a humoral or cellular immune response to the polypeptide
  • E1E2 complex is meant a protein containing at least one El polypeptide and at least one E2 polypeptide, as described above. Such a complex may also include all or a portion of the p7 region which occurs immediately adjacent to the C- terminus of E2. As shown in Figures 1 and 3A-3C, the p7 region is found at positions 747-809, numbered relative to the full-length HCV-1 polyprotein (amino acid positions 575-637 of SEQ ID NO:4). A representative E1E2 complex which includes the p7 protein is termed “E1E2 809 " herein. The mode of association of El and E2 in an E1E2 complex is immaterial. The
  • El and E2 polypeptides may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds.
  • the E1E2 polypeptides of the present invention may be in the form of a fusion protein which includes an immunogenic El polypeptide and an immunogenic E2 polypeptide, as defined above.
  • the fusion may be expressed from a polynucleotide encoding an E1E2 chimera.
  • E1E2 complexes may form spontaneously simply by mixing El and E2 proteins which have been produced individually. Similarly, when co- expressed and secreted into media, the El and E2 proteins can form a complex spontaneously.
  • E1E2 complexes also called aggregates
  • Such aggregates may include one or more El monomers in association with one or more E2 monomers.
  • the number of El and E2 monomers present need not be equal so long as at least one El monomer and one E2 monomer are present.
  • Detection of the presence of an El E2 complex is readily determined using standard protein detection techniques such as polyacrylamide gel electrophoresis and immunological techniques such as immunoprecipitation.
  • the terms “analog” and “mutein” refer to biologically active derivatives of the reference molecule, or fragments of such derivatives, that retain desired activity, such as the ability to stimulate a cell-mediated immune response, as defined below.
  • analog refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy immunogenic activity.
  • mutein refers to peptides having one or more peptide mimics ("peptoids"), such as those described in International Publication No. WO 91/04282.
  • the analog or mutein has at least the same immunoactivity as the native molecule.
  • analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic ⁇ aspartate and glutamate; (2) basic ⁇ lysine, arginine, histidine; (3) non-polar ⁇ alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar — glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity.
  • the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact.
  • modified NS3 an NS3 polypeptide with a modification such that protease activity of the NS3 polypeptide is disrupted.
  • the modification can include one or more amino acid additions, substitutions (generally non-conservative in nature) and/or deletions, relative to the native molecule, wherein the protease activity of the NS3 polypeptide is disrupted. Methods of measuring protease activity are discussed further below.
  • fragment is intended a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure.
  • the fragment can include a C- terminal deletion and/or an N-terminal deletion of the native polypeptide.
  • An "immunogenic fragment" of a particular HCV protein will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, that define an epitope, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains immunogenic activity, as measured by the assays described herein.
  • epitope refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or any integer therebetween), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence.
  • There is no critical upper limit to the length of the fragment which may comprise nearly the full- length of the protein sequence, or even a fusion protein comprising two or more epitopes from the HCV polyprotein.
  • An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived.
  • epitopes encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature). Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey.
  • linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715.
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
  • Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the
  • the term "conformational epitope” refers to a portion of a full- length protein, or an analog or mutein thereof, having structural features native to the amino acid sequence encoding the epitope within the fulMength natural protein. Native structural features include, but are not limited to, glycosylation and three dimensional structure.
  • a conformational epitope is produced recombinantly and is expressed in a cell from which it is extractable under conditions which preserve its desired structural features, e.g. without denaturation of the epitope.
  • Such cells include bacteria, yeast, insect, and mammalian cells.
  • T-cell epitope refers to a feature of a peptide structure which is capable of inducing T-cell immunity towards the peptide structure or an associated hapten.
  • T-cell epitopes generally comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC molecules, (Unanue et al., Science (1987) 236:551-557). Conversion of polypeptides to MHC class II-associated linear peptide determinants (generally between 5-14 amino acids in length) is termed "antigen processing" which is carried out by antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • a T-cell epitope is defined by local features of a short peptide structure, such as primary amino acid sequence properties involving charge and hydrophobicity, and certain types of secondary structure, such as helicity, that do not depend on the folding of the entire polypeptide.
  • short peptides capable of recognition by helper T-cells are generally amphipathic structures comprising a hydrophobic side (for interaction with the MHC molecule) and a hydrophilic side (for interacting with the T-cell receptor), (Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G.C. Woodrow et al., (1990) pp.
  • segments of proteins that include T-cell epitopes can be readily predicted using numerous computer programs. (See e.g., Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116).
  • Such programs generally compare the amino acid sequence of a peptide to sequences known to induce a T-cell response, and search for patterns of amino acids which are believed to be required for a T-cell epitope.
  • an "immunological response" to an HCV antigen (including both polypeptide and polynucleotides encoding polypeptides that are expressed in vivo) or composition is the development in a subject of a humoral and/or a cellular immune response to molecules present in the composition of interest.
  • a “humoral immune response” refers to an immune response mediated by antibody molecules
  • a “cellular immune response” is one mediated by T- lymphocytes and/or other white blood cells.
  • TTLs cytolytic T-cells
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of mtracellular microbes, or the lysis of cells infected with such microbes.
  • MHC major histocompatibility complex
  • Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • a “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • a composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface.
  • the cell-mediated immune response is directed at, or near, cells presenting antigen at their surface.
  • antigen-specific T- lymphocytes can be generated to allow for the future protection of an immunized host.
  • the ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T- lymphocytes specific for the antigen in a sensitized subject.
  • assays are well l ⁇ iown in the art. See, e.g., Erickson et al, J Immunol (1993) 111:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.
  • an immunological response as used herein may be one which stimulates the production of CTLs, and/or the production or activation of helper T- cells.
  • the antigen of interest may also elicit an antibody-mediated immune response.
  • an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or ⁇ T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest.
  • These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection or alleviation of symptoms to an immunized host.
  • ADCC antibody dependent cell cytotoxicity
  • Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
  • equivalent antigenic determinant an antigenic determinant from different sub-species or strains of HCV, such as from strains 1, 2, 3, etc., of HCV which antigenic determinants are not necessarily identical due to sequence variation, but which occur in equivalent positions in the HCV sequence in question.
  • amino acid sequences of equivalent antigenic determinants will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, usually more than 40%, such as more than 60%, and even more than 80-90% homology, when the two sequences are aligned.
  • a "coding sequence” or a sequence which "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • nucleic acid molecule or “polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
  • HCV polynucleotide is a polynucleotide that encodes an HCV polypeptide, as defined above.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors, etc., are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, as can transcribed introns, and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • control element refers to a polynucleotide sequence which aids in the expression of a coding sequence to which it is linked.
  • the term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions, including 5'-UTRs and 3'-UTRs and when appropriate, leader sequences and enhancers, which collectively provide for the transcription and translation of a coding sequence in a host cell.
  • a “promoter” as used herein is a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto.
  • a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background.
  • Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • a control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • "Expression cassette” or “expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • the expression cassette includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
  • the expression cassette described herein may be contained within a plasmid construct.
  • the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a Ml 3 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a signal which allows the plasmid construct to exist as single-stranded DNA e.g., a Ml 3 origin of replication
  • at least one multiple cloning site e.g., a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • Transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion: for example, transformation by direct uptake, transfection, infection, and the like. For particular methods of transfection, see further below.
  • the exogenous polynucleotide may be maintained as a nonintegrated vector, for example, an episome, or alternatively, may be integrated into the host genome.
  • a "host cell” is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.
  • isolated is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro- molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • purified as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
  • "Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98%, or more, sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six).
  • BLAST BLAST
  • Another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • nucleic acid immunization is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell, for the in vivo expression of the antigen or antigens.
  • the nucleic acid molecule can be introduced directly into the recipient subject, such as by injection, inhalation, oral, intranasal and mucosal administration, or the like, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule.
  • treatment refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question.
  • Treatment may be effected prophylactically (prior to infection) or therapeutically
  • HCV strains contain a single open reading frame of approximately 9,000 to 12,000 nucleotides, which is transcribed into a polyprotein.
  • an HCV polyprotein upon cleavage, produces at least ten distinct products, in the order of NH 2 - Core-El -E2-p7-NS2-NS3- NS4a-NS4b-NS5a-NS5b-COOH.
  • the core polypeptide occurs at positions 1-191, numbered relative to HCV-1 (see, Choo et al. (1991) Proc. Natl Acad. Sci. USA 88:2451-2455, for the HCV-1 genome).
  • NS3, NS4 (NS4a and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can be identified by several methods.
  • NS3, NS4, NS5a, NS5b polypeptides or fusion proteins comprising any combination of the above, can be isolated, for example, by immunoaffmity purification using a monoclonal antibody for the polypeptide or protein.
  • the isolated protein sequence can then be screened by preparing a series of short peptides by proteolytic cleavage of the purified protein, which together span the entire protein sequence.
  • the NS5a polypeptide can be derived from a first strain of HCV, and the NS3 and NS4 polypeptides can be derived from a second strain of HCV.
  • NS3, NS4 and NS5a polypeptides that are each derived from different HCV strains can also be used in an HCV fusion protein.
  • a fusion protein comprising NS5b at least one of the NS3, NS4, NS5a, and NS5b polypeptides can be derived from a different HCV strain than the other polypeptides.
  • NS3, NS4, NS5a, and NS5b polypeptides that are each derived from different HCV strains can also be used in an NS3NS4NS5aNS5b fusion protein.
  • compositions of the present invention may also include El and E2 polypeptides, complexes of these polypeptides or polynucleotides encoding the same.
  • the El and E2 polypeptides and complexes thereof can be provided independent of the nonstructural fusion protein or can be incorporated into the same fusion.
  • E1E2 complexes can be provided as proteins, or as polynucleotides encoding the same.
  • El , E2 and p7 are known to contain human T-cell epitopes
  • E1E2 complexes may be present as a heterogeneous mixture of molecules, due to clipping and proteolytic cleavage, as described above.
  • a composition including E1E2 complexes may include multiple species of E1E2, such as E1E2 terminating at amino acid 746 (E1E2 746 ), E1E2 terminating at amino acid 809 (E1E2 809 ), or any of the other various El and E2 molecules described above, such as E2 molecules with N-terminal truncations of from 1-20 amino acids, such as E2 species beginning at amino acid 387, amino acid 402, amino acid 403, etc.
  • the constructs can be delivered as plasmid DNA, e.g., contained within a plasmid, such as pBR322, pUC, or ColEl
  • the expression constracts can be packaged in liposomes prior to delivery to the cells.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1 : 1 (mg DNA:micromoles lipid), or more of lipid.
  • Liposomal preparations for use with the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposomes are readily available.
  • N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner et al, Proc. Natl. Acad. Sci. USA (1987) 84:7413- 7416).
  • retroviruses provide a convenient platform for gene delivery systems, such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Patent No.
  • adenoviras vectors such as adenovirus Type 2 and Type 5 vectors.
  • adenovirases persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al, J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Members of the Alphaviras genus such as but not limited to vectors derived from the Sindbis and Semliki Forest viruses, VEE, will also find use as viral vectors for delivering the gene of interest.
  • Sindbis- virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol.
  • vectors can be used, including but not limited to simian virus 40 and cytomegalovirus.
  • Bacterial vectors such as Salmonella ssp. Yersinia enterocolitica, Shigella spp., Vibrio cholerae, Mycobacterium strain BCG, and Listeria monocytogenes can be used.
  • Minichromosomes such as MC and MCI, bacteriophages, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under then- own control in a cell) can also be used.
  • the expression constracts may also be encapsulated, adsorbed to, or associated with, particulate carriers. Such carriers present multiple copies of a selected molecule to the immune system and promote trapping and retention of molecules in local lymph nodes.
  • the particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release.
  • particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al., J. Microencap. (1996).
  • PLG poly(lactides) and poly(lactide-co-glycolides)
  • PLG poly(lactide-co-glycolides)
  • Useful detergents include, but are not limited to, any of the various N-methylglucamides (known as MEGAs), such as heptanoyl-N-methylglucamide (MEGA-7), octanoyl-N- methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9), and decanoyl-N-methyl-glucaniide (MEGA- 10); cholic acid; sodium cholate; deoxycholic acid; sodium deoxycholate; taurocholic acid; sodium taurocholate; taurodeoxycholic acid; sodium taurodeoxycholate; 3-[(3- cholamidopropyl)dimethylammonio] -1-propane-sulfonate (CHAPS); 3-[(3- cholamidopropyl) dimethyla ⁇ rmom ⁇ ]-2-hydroxy-l-propane-sulfonate (CHAPSO); -dodecyl-N,N-di
  • the above detergents are commercially available from e.g., Sigma Chemical Co., St. Louis, MO.
  • Various cationic lipids known in the art can also be used as detergents. See Balasubramaniam et al., 1996, Gene Ther., 3:163-72 and Gao, X., and L. Huang. 1995, Gene Ther., 2:7110-722.
  • a wide variety of other methods can be used to deliver the expression constracts to cells.
  • Such methods include DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyornithine-mediated transfection, or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like.
  • Other useful methods of transfection include electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinj ection.
  • compositions comprising Fusion Proteins or Polynucleotides
  • compositions comprising the fusion proteins or polynucleotides, as well as compositions including the individual components of these fusion proteins or polynucleotides.
  • Compositions of the invention preferably comprise a pharmaceutically acceptable carrier.
  • the carrier should not itself induce the production of antibodies harmful to the host.
  • Pharmaceutically acceptable carriers are well known to those in the art. Such carriers include, but are not limited to, large, slowly metabolized, macromolecules, such as proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like, polylactic acids, polyglycolic acids, polymeric amino acids such as polyglutamic acid, polylysine, and the like, amino acid copolymers, and inactive virus particles.
  • compositions of the invention can also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates.
  • mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates
  • organic acids such as acetates, proprionates, malonates, or benzoates.
  • Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art.
  • compositions of the invention can also contain liquids or excipients, such as water, saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.
  • Liposomes can also be used as a carrier for a composition of the invention, such liposomes are described above.
  • co-stimulatory molecules which improve immunogen presentation to lymphocytes such as B7-1 or B7-2, or cytokines such as GM-CSF, IL-2, and LL- 12, can be included in a composition of the invention.
  • adjuvants can also be included in a composition.
  • ISCOMs may be devoid of additional detergent, see, e.g., International Publication No. WO 00/07621; (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IF A); (5) cytokines, such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-12 (International Publication No.
  • CFA Complete Freund's Adjuvant
  • IF A Incomplete Freund's Adjuvant
  • cytokines such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-12 (International Publication No.
  • coli heat-labile toxin particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63)
  • LT-R72 where arginine is substituted for the wild-type aniino acid at position 72
  • CT-S109 where serine is substituted for the wild-type amino acid at position 109
  • PT-K9/G129 where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129)
  • MPL or 3-O-deacylated MPL see, e.g., GB 2220221
  • EP-A-0689454 optionally in the substantial absence of alum when used with pneumococcal saccharides (see, e.g., International Publication No.
  • WO 00/56358 (8) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g., EP-A-0835318, EP-A-0735898, EP- A-0761231; (9) oligonucleotides comprising CpG motifs (see, e.g., Roman et al. (1997) Nat. Med. 3:849-854; Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94:10833-10837; Davis et al. (1998) J. Immunol. 160:870-876; Chu et al. (1997) J Exp. Med.
  • WO 01/21207 or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (see, e.g., International Publication No. WO 01/21152); (12) a saponin and an immunostimulatory oligonucleotide such as a CpG oligonucleotide (see, e.g., International Publication No. WO 00/62800); (13) an immunostimulant and a particle of metal salt (see, e.g., International Publication No. WO 00/23105); and (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
  • additional non-ionic surfactant such as an octoxynol
  • a saponin and an immunostimulatory oligonucleotide such as a CpG oligonucleotide (see, e.g., International Publication No
  • Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L- teeonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), -acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine ⁇ - ⁇ ' ⁇ 1 - dipalmitoyl-5n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • the HCV proteins can be adsorbed to, or entrapped within, an anti-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • the HCV proteins can be adsorbed to, or entrapped within, an
  • composition may also contain immunostimulatory molecules, either in addition to or in place of the antigen delivery system.
  • Immunostimulatory agents for use herein include, without limitation, monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM).
  • MPL may be formulated into an emulsion to enhance its immunostimulatory affect. See, e.g., Ulrich et al., "MPLr immunostimulat: adjuvant formulations.” in Vaccine Adjuvants: Prepartion Methods and Research Protocols (O'Hagan DT, ed.) Human Press Inc., NJ (2000) pp. 273- 282.
  • MPL has been shown to induce the synthesis and release of cytokines, particularly IL-2 and IFN- ⁇ .
  • Other useful immunostimulatory molecules include
  • LPS and immunostimulatory nucleic acid sequences including but not limited to, unmethylated CpG motifs, such as CpG oligonucleotides.
  • Oligonucleotides containing unmethylated CpG motifs have been shown to induce activation of B cells, NK cells and antigen-presenting cells (APCs), such as monocytes and macrophages. See, e.g., U.S. Patent No. 6,207,646.
  • adjuvants derived from the CpG family of molecules, CpG dinucleotides and synthetic oligonucleotides which comprise CpG motifs see, e.g., Krieg et al. Nature (1995) 374:546 and Davis et al. J. Immunol. (1998) 160:870-876) such as any of the various immunostimulatory CpG oligonucleotides disclosed in U.S. Patent No.
  • CpG molecules include those captured by the formula 5'-X ⁇ X 2 CGX 3 X 4 , where X ! and X 2 are a sequence such as GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT or TpG, and X 3 and X 4 are TpT, CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, or TpG, wherein "p" signifies a phosphate bond.
  • the oligonucleotides do not include a GCG sequence at or near the 5'- and/or 3' terminus.
  • the CpG is preferably flanked on its 5 '-end with two purities (preferably a GpA dinucleotide) or with a purine and a pyrimidine (preferably, GpT), and flanked on its 3 '-end with two pyrimidines, preferably a TpT or TpC dinucleotide.
  • preferred molecules will comprise the sequence GACGTT, GACGTC, GTCGTT or GTCGCT, and these sequences will be flanked by several additional nucleotides. The nucleotides outside of this central core area appear to be extremely amendable to change.
  • the CpG oligonucleotides for use herein may be double- or single- stranded. Double-stranded molecules are more stable in vivo while single-stranded molecules display enhanced immune activity.
  • the phosphate backbone may be modified, such as phosphorodithioate-modified, in order to enhance the immunostimulatory activity of the CpG molecule. As described in U.S. Patent No. 6,207,646, CpG molecules with phosphorothioate backbones preferentially activate B-cells, while those having phosphodiester backbones preferentially activate monocytic (macrophages, dendritic cells and monocytes) and NK cells.
  • One exemplary CpG oligonucleotide for use in the present compositions has the sequence 5'-TCCATGACGTTCCTGACGTT-3' (SEQ LD NO:6).
  • CpG molecules can readily be tested for their ability to stimulate an immune response using standard techniques, well known in the art. For example, the ability of the molecule to stimulate a humoral and/or cellular immune response is readily determined using the immunoassays described above. Moreover, the antigen and adjuvant compositions can be administered with and without the CpG molecule to determine whether an immune response is enhanced.
  • the HCN proteins may also be encapsulated, adsorbed to, or associated with, particulate carriers, as described above with reference to the HCN polynucleotides.
  • particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al., J. Microencap. (1996).
  • PLG poly(lactide-co-glycolides
  • the HCN proteins can be used to produce HCN-specific polyclonal and monoclonal antibodies.
  • HCN-specific polyclonal and monoclonal antibodies specifically bind to HCN antigens.
  • Polyclonal antibodies can be produced by administering the fusion protein to a mammal, such as a mouse, a rabbit, a goat, or a horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by cliromatography, preferably affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
  • Antibodies either monoclonal and polyclonal, which are directed against HCN epitopes, are particularly useful for detecting the presence of HCN or HCV antigens in a sample, such as a serum sample from an HCV-infected human.
  • An immunoassay for an HCV antigen may utilize one antibody or several antibodies.
  • An immunoassay for an HCV antigen may use, for example, a monoclonal antibody directed towards an HCV epitope, a combination of monoclonal antibodies directed towards epitopes of one HCV polypeptide, monoclonal antibodies directed towards epitopes of different HCV polypeptides, polyclonal antibodies directed towards the same HCV antigen, polyclonal antibodies directed towards different HCV antigens, or a combination of monoclonal and polyclonal antibodies.
  • Immunoassay protocols may be based, for example, upon competition, direct reaction, or sandwich type assays using, for example, labeled antibody.
  • the labels may be, for example, fluorescent, chemiluminescent, or radioactive.
  • the polyclonal or monoclonal antibodies may further be used to isolate HCV particles or antigens by immunoaffinity columns.
  • the antibodies can be affixed to a solid support by, for example, adsorption or by covalent linkage so that the antibodies retain their immunoselective activity.
  • spacer groups may be included so that the antigen binding site of the antibody remains accessible.
  • the immobilized antibodies can then be used to bind HCV particles or antigens from a biological sample, such as blood or plasma.
  • the bound HCV particles or antigens are recovered from the column matrix by, for example, a change in pH.
  • HCV-Specific T cells HCV-specific T cells that are activated by the above-described fusions and
  • E1E2 complexes including the NS3NS4NS5a fusion protein or NS3NS4NS5aNS5b fusion protein, and the E1E2 complexes, expressed in vivo or in vitro, preferably recognize an epitope of an HCV polypeptide such as an El, E2, NS3, NS4, NS5a, NS5b polypeptide, including an epitope of an NS3NS4NS5a fusion protein or an NS3NS4NS5aNS5b fusion protein, or an E1E2 complex.
  • HCV-specific T cells can be CD8 + or CD4 + .
  • HCV-specific CD8 + T cells preferably are cytotoxic T lymphocytes (CTL) which can kill HCV-infected cells that display El, E2, NS3, NS4, NS5a, NS5b epitopes complexed with an MHC class I molecule.
  • CTL cytotoxic T lymphocytes
  • HCV-specific CD8 + T cells may also express interferon- ⁇ (IFN- ⁇ ).
  • IFN- ⁇ interferon- ⁇
  • HCV-specific CD8 + T cells can be detected by, for example, 51 Cr release assays (see the examples).
  • E1E2 complexes and fusions such as an El polypeptide, an E2 polypeptide, an E1
  • HCV-specific CD4 + T cells can be detected by a lymphoproliferation assay (see examples). Lymphoproliferation assays measure the ability of HCV-specific CD4 + T cells to proliferate in response to an El, E2, NS3, an NS4, an NS5a, or an NS5b epitope.
  • HCV proteins or polynucleotides can be used to stimulate an immune response, such as to activate HCV-specific T cells either in vitro or in vivo.
  • HCV-specific T cells can be used, ter alia, to provide model systems to optimize CTL responses to HCV and to provide prophylactic or therapeutic treatment against HCV infection.
  • proteins are preferably supplied to T cells via a plasmid or a viral vector, such as an adenoviras vector, as described above.
  • Polyclonal populations of T cells can be derived from the blood, and preferably from peripheral lymphoid organs, such as lymph nodes, spleen, or thymus, of mammals that have been infected with an HCV. Preferred mammals include mice, chimpanzees, baboons, and humans.
  • the HCV serves to expand the number of activated HCV-specific T cells in the mammal.
  • the HCV-specific T cells derived from the mammal can then be restimulated in vitro by adding, e.g., HCV E1E2 and NS3NS4NS5a or NS3NS4NS5aNS5b epitopic peptides, with or without a core polypeptide, to the T cells.
  • the HCV-specific T cells can then be tested for, ter alia, proliferation, the production of IFN- ⁇ , and the ability to lyse target cells displaying E1E2, NS3NS4NS5a or NS3NS4NS5aNS5b epitopes in vitro.
  • HCV-activated CD4 + T cells proliferate when cultured with an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absence of an epitopic peptide.
  • NS3NS4NS5aNS5b epitopes that are recognized by HCV-specific CD4 + T cells can be identified using a lymphoproliferation assay.
  • detection of IFN- ⁇ in HCV-specific CD8 + T cells after in vitro stimulation with the above-described HCV proteins can be used to identify El, E2, E1E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b epitopes that particularly effective at stimulating CD8 + T cells to produce IFN- ⁇ (see examples).
  • HCV-specific CD8 + T cells can be derived from the liver of an HCV infected mammal. These T cells can be tested in 51 Cr release assays against target cells displaying, e.g., E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes.
  • target cell populations expressing different E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes can be constructed so that each target cell population displays different epitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b.
  • the HCV-specific CD8 + cells can be assayed against each of these target cell populations.
  • the results of the 51 Cr release assays can be used to determine which epitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b are responsible for the strongest CTL response to HCV.
  • E1E2 complexes, NS3NS4NS5a fusion proteins or NS3NS4NS5aNS5b fusion proteins, with or without core polypeptides, which contain the epitopes responsible for the strongest CTL response can then be constructed using the information derived from the 51 Cr release assays.
  • HCV proteins as described above, or polynucleotides encoding such proteins can be administered to a mammal, such as a mouse, baboon, chimpanzee, or human, to stimulate an immune response, such as to activate HCV-specific T cells in vivo.
  • Administration can be by any means known in the art, including parenteral, intranasal, intramuscular or subcutaneous injection, including injection using a biological ballistic gun ("gene gun”), as discussed above.
  • injection of an HCV polynucleotide is used to activate T cells.
  • injection of the polynucleotides results in the synthesis of a fusion protein in the host.
  • these immunogens are presented to the host immune system with native post- translational modifications, structure, and conformation.
  • the polynucleotides are preferably injected intramuscularly to a large mammal, such as a human, at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.
  • a composition of the invention comprising the HCV proteins or polynucleotides is administered in a manner compatible with the particular composition used and in an amount which is effective to stimulate an immune response, such as to activate HCV-specific T cells as measured by, ter alia, a 51 Cr release assay, a lymphoproliferation assay, or by intracellular staining for IFN- ⁇ .
  • the proteins and/or polynucleotides can be administered either to a mammal which is not infected with an HCV or can be administered to an HCV-infected mammal.
  • an HCV fusion and El and E2 polypeptides such as an El E2 complex, an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein or polynucleotide, with or without a core polypeptide, will be administered to a large mammal, such as a baboon, chimpanzee, or human.
  • a large mammal such as a baboon, chimpanzee, or human.
  • co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions.
  • compositions of the invention can be given in a single dose schedule, or preferably in a multiple dose schedule in which a primary course of vaccination includes 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reenforce an immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose or doses after several months.
  • NS3NS4NS5a (approximately amino acids 1027 to 2399, numbered relative to HCV-1) (also termed “NS345a” herein) or NS5a (approximately amino acids 1973 to 2399, numbered relative to HCV-1) was isolated from an HCV.
  • Polynucleotides encoding a methionine residue were ligated to the 5' end of these polynucleotides and the polynucleotides were cloned into plasmid, vaccinia virus, and adenovirus vectors. Immunization Protocols.
  • mice were immunized with 50 ⁇ g of plasmid DNA encoding either NS5a or encoding an NS3NS4NS5a fusion protein by intramuscular injection into the tibialis anterior.
  • a booster injection of 10 7 pfu of vaccinia viras (W)-NS5a (intraperitoneal) or 50 ⁇ g of plasmid control (intramuscular) was provided 6 weeks later.
  • mice were injected intramuscularly in the tibialis anterior with 10 10 adenovirus particles encoding an NS3NS4NS5a fusion protein.
  • An intraperitoneal booster injection of 10 7 pfu of W-NS5a or an intramuscular booster injection of 10 10 adenoviras particles encoding NS3NS4NS5a was provided 6 weeks later.
  • HCV-specific T cells to lyse target cells displaying an NS5a epitope.
  • Spleen cells were pooled from the immunized animals. These cells were restimulated in vitro for 6 days with the CTL epitopic peptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO:l) from HCN-NS5a in the presence of IL-2. The spleen cells were then assayed for cytotoxic activity in a standard 51 Cr release assay against peptide-sensitized target cells (L929) expressing class I, but not class II MHC molecules, as described in Weiss (1980) J. Biol. Chem. 255:9912-9917.
  • Ratios of effector (T cells) to target (B cells) of 60:1, 20:1, and 7:1 were tested. Percent specific lysis was calculated for each effector to target ratio.
  • the results of the assays are shown in Tables 1 and 2.
  • Table 1 demonstrates that immunization with plasmid DNA encoding an NS3NS4NS5a fusion protein activates CD8 + T cells which recognize and lyse target cells displaying an NS5a epitope.
  • the NS5a polypeptide of the NS3NS4NS5a fusion protein was able to activate T cells even though the NS5a polypeptide was present in a fusion protein.
  • Table 2 demonstrates that delivery of the NS3NS4NS5a fusion protein to mice by means of an adenovirus vector also activates CD8 + T cells which recognize and lyse target cells displaying an HCN ⁇ S5a epitope.
  • immunization with either "naked" (plasmid) DNA encoding an NS3NS4NS5a fusion protein or adenovirus vector-encoded fusion protein can be used to activate HCN- specific T cells.
  • Immunization with D ⁇ A encoding an ⁇ S3 ⁇ S4 ⁇ S5a fusion protein activates HCV-specific CD8 + T cells which express IFN- ⁇ .
  • Intracellular Staining for Interferon-gamma IFN- ⁇ . Intracellular staining for IFN- ⁇ was used to identify the CD8 + T cells that secrete IFN- ⁇ after in vitro stimulation with the NS5a epitope p214K9. Spleen cells of individual immunized mice were restimulated in vitro either with p214K9 or with a non-specific peptide for 6-12 hours in the presence of IL-2 and monensin. The cells were then stained for surface CD8 and for intracellular IFN- ⁇ and analyzed by flow cytometay. The percent of CD8 + T cells which were also positive for JFN- ⁇ was then calculated. The results of these assays are shown in Tables 1 and 2. Table 1 demonstrates that CD8 + T cells activated in response to immunization with plasmid DNA encoding an NS3NS4NS5a fusion protein also express IFN- ⁇ . Immunization with an IFN- ⁇ .
  • NS3NS4NS5a fusion protein encoded in an adenoviras also results in CD8 + HCV- specific T cells which express JFN- ⁇ , although to a lesser extent than immunization with a plasmid-encoded NS3NS4NS5a fusion protein (Table 2).
  • Target cells were pulsed with p214K9 or media alone and labeled with 51 Cr.
  • p214K9 is a CTL epitopic peptide (2152-HEYPVGSQL-2160, SEQ ID NO:l) from HCV-NS5a '-' refers to the absence of peptide
  • Target cells were pulsed with p214K9 or p214J and labeled with 51 Cr.
  • p214K9 is a CTL epitopic peptide (2152-HEYPVGSQL-2160, SEQ LD NO:l) from HCV-NS5a
  • P214J is a control peptide (10 mer) from HCV-NS5a
  • Lymphoproliferation assay Spleen cells from pooled immunized mice were depleted of CD8 + T cells using magnetic beads and were cultured in triplicate with either p222D, an NS5a-e ⁇ itopic peptide from HCV-NS5a (2224- AELIEANLLWRQEMG-2238; SEQ LD NO:2), or in medium alone. After 72 hours, cells were pulsed with l ⁇ Ci per well of 3 H-thymidine and harvested 6-8 hours later. Incorporation of radioactivity was measured after harvesting. The mean cpm was calculated.
  • p222D is a CD4+ epitopic peptide (aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 1
  • p222D is a CD4+ epitopic peptide (aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO:2) from HCV-NS5a
  • NS345a fusion protein as described in Example 1, with PLG-linked DNA encoding NS345a, described below, or with DNA encoding NS345a, delivered via electroporation (see, e.g., U.S. Patent Nos. 6,132,419; 6,451,002, 6,418,341, 6233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, for this delivery technique).
  • the immunizations were followed by a booster injection 6 weeks later of 1 x 10 7 pfu vaccinia viras encoding NS5a, plasmid DNA encoding NS345a or plasmid DNA encoding NS5a each as described in Example 1.
  • PLG-delivered DNA The polylactide-co-glycolide (PLG) polymers were obtained from Boehringer Ingelheim, U.S.A.
  • the PLG polymer used in this study was RG505, which has a copolymer ratio of 50/50 and a molecular weight of 65 kDa (manufacturers data).
  • Cationic microparticles with adsorbed DNA were prepared using a modified solvent evaporation process, essentially as described in Singh et al., Proc. Natl. Acad. Sci. USA (2000) 97:811-816.
  • the microparticles were prepared by emulsifying 10 ml of a 5% w/v polymer solution in methylene chloride with 1 ml of PBS at high speed using an IKA homogenizer. The primary emulsion was then added to 50ml of distilled water containing cetyl trimethyl ammonium bromide (CTAB) (0.5% w/v). This resulted in the formation of a w/o/w emulsion which was stirred at 6000 rpm for 12 hours at room temperature, allowing the methylene chloride to evaporate. The resulting microparticles were washed twice in distilled water by centrifugation at 10,000 g and freeze dried.
  • CTAB cetyl trimethyl ammonium bromide
  • DNA was adsorbed onto the microparticles by incubating 100 mg of cationic microparticles in a lmg/ml solution of DNA at 4 C for 6 hours. The microparticles were then separated by centrifugation, the pellet washed with TE buffer and the microparticles were freeze dried.
  • CTL activity and IFN- ⁇ expression were measured by 51 Cr release assay or intracellular staining as described in examples 2 and 3 respectively. The results are shown in Table 5.
  • Results demonstrate that immunization using plasmid DNA encoding for NS345a to prime mice results in activation of CD8+ HCV specific T cells.
  • Alphavirus replicon particles for example, SINCR (DC+) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were injected with 5 x 10 6 IU SINCR (DC+) replicon particles encoding for NS345a intramuscularly (IM) as described in Example 1, or subcutaneously (S/C) at the base of the tail (BoT) and foot pad (FP), or with a combination of 2/3 of the DNA delivered via IM administration and 1/3 via a BoT route. The immunizations were followed by a booster injection of vaccinia virus encoding NS5a as described in Example 1.
  • IM intramuscularly
  • S/C subcutaneously
  • FP foot pad
  • IFN- ⁇ expression was measured by intracellular staining as described in Example 3. The results are shown in Table 6. The results demonstrate that immunization via SINCR (DC+) replicon particles encoding for NS345a by a variety of routes results in CD8+ HCV specific T cells which express IFN- ⁇ .
  • Alphavirus replicon particles for example, SINCR (DC+) and SINCR (LP) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with 1 x 10 3 to 1 x 10 7 IU of SINCR (DC+) or SINCR (LP) replicon particles encoding for NS345a, by intramuscular injection into the tibialis anterior, followed by a booster injection of 10 7 pfu vaccinia viras encoding NS5a at 6 weeks.
  • IFN- ⁇ expression was measured by intracellular staining as described in Example 3.
  • Administration of an increase in the number of SINCR (DC+) replicon particles encoding NS345a resulted in an increase in % of CD8+ T cells expressing IFN- ⁇ .
  • Alphavirus replicon priming followed by various boosting regimes.
  • Alphavirus replicon particles for example, SINCR (DC+) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603.
  • mice were primed with SINCR (DC+), 1.5 x 10 6 IU replicon particles encoding NS345a, by intramuscular injection into the tibialis anterior, followed by a booster of either 10-100 ⁇ g of plasmid DNA encoding for NS5a, 10 10 adenovirus particles encoding NS345a, 1.5 x 10 6 IU SINCR (DC+) replicon particles encoding NS345a, or 10 7 pfu vaccinia virus encoding NS5a at 6 weeks.
  • DC+ SINCR
  • IFN- ⁇ expression was measured by intracellular staining as described in Example 3. The results are shown in Table 7. The results demonstrate that boosting with vaccinia virus encoding NS5a DNA results in the strongest generation of CD8+ HCV specific T cells which express IFN- ⁇ . Boosting with plasmid encoding NS5a DNA also results in a good response, while lesser responses are noted with adenovirus NS345a or SINCR DC+ boosted animals.
  • Alphaviruses expressing NS345a Alphaviruses expressing NS345a
  • Alphavirus replicon particles for example, SINCR (DC+) and SINCR (LP) were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with 1 x 10 2 to 1 x 10 6 IU SINCR (DC+) replicons encoding NS345a via a combination of delivery routes (2/3 DVI and 1/3 S/C) as well as by S/C alone, or with 1 x 10 2 to 1 x 10 6 IU SINCR (LP) replicon particles encoding NS345a via a combination of delivery routes (2/3 IM and 1/3 S/C) as well as by S/C alone.
  • DC+ SINCR
  • LP SINCR
  • the immunizations were followed by a booster injection of 10 7 pfu vaccinia virus encoding NS5a at 6 weeks.
  • mice were immunized with either 10-100 ⁇ g of plasmid DNA encoding NS5a as described in Example 1 or with PLG-linked DNA encoding NS5a as described in Example 5.
  • the immunizations were followed by a booster inj ection at 6 weeks of either 10-100 ⁇ g of plasmid DNA encoding for NS5a, 10 10 adenoviras particles encoding NS345a, 1.5 x 10 6 IU SINCR (DC+) replicon particles encoding NS345a, or 10 7 pfu vaccinia viras encoding NS5a.
  • CTL activity and IFN- ⁇ expression were measured by the methods described in Examples 2 and 3.
  • mice were immunized with 100 ⁇ g plasmid DNA encoding for NS345a or with 100 ⁇ g PLG-linked DNA encoding NS345a. Additionally, separate DNA plasmids encoding NS5a, NS34a, and NS4ab (33.3 ⁇ g each) were administered concurrently to another group of mice. Finally, PLG-linked DNA encoding NS5a, NS34a, andNS4ab (33.3 ⁇ g each) were administered concurrently to another group of mice. The irnmunizations were followed by a booster injection of lxl 0 7 pfu vaccinia virus encoding NS5a, 6 weeks post first immunization. IFN- ⁇ expression was measured by the method described in Example 3. The results are shown in Figure 7. The results demonstrate a particularly vigorous response in the activation of CD 8+ HCV specific T cells when the DNA is broken down into smaller sub units and linked to PLG.
  • mice Groups of 10 C57 black mice were immunized IM at 0, 21 and 60 days with the formulations shown in Table 10.
  • the NS345Core 121 -PLGdss group received a vaccine dose of 50 ⁇ l in each leg whereas the other vaccine groups received a vaccine dose of 50 ⁇ l in one leg.
  • NS345Core 121 -ISCOMS were comprised of amino acids 1242 to 3011 and 1- 121 and the HCV polyprotein, numbered relative to HCV- land were adsorbed to ISCOMS with a ratio of protein to QH of approximately 8:1, using standard techniques. See, e.g., International Publication No. WO 01/37869A.
  • Core-ISCOMS including an HCV core protein fragment from the region spanning amino acid positions 1-191 of the HCV polyprotein, numbered relative to HCV-1, with a ratio of protein to QH of 1:1, were produced using standard techniques. See, e.g., International Publication No. WO 01/37869A.
  • NS345Core 121 was formulated in 0.1% SDS in PBS and contained DTT. Protein was diluted in PBS and mixed 1 : 1 with MF59 (see, Ott et al., "MF59 ⁇ Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines" in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M.F. and Newman, MJ. eds.) Plenum Press, New York (1995) pp. 277-296; and U.S. Patent No. 6,299,884) prior to immunization.
  • NS345Core 121 -PLGdss PLG microparticles produced as described above were treated with 3-(trimethylsilyl)-l-propanesulfonic acid (DSS) to enhance adsorption of antigen.
  • DSS 3-(trimethylsilyl)-l-propanesulfonic acid
  • NS345Core 121 was adsorbed thereto using standard techniques (see, International Publication No. WO 00/050006).
  • the NS345Core m -PLGdss was mixed with MF59 prior to immunization.
  • NS345Core I21 -ISCOMS produced antibody response only to NS5 in immunized C57 black mice. Higher levels of antibodies to NS5 were produced in mice immunized with NS345Core 121 adjuvanted with MF59, however no antibody response to core, NS3 or NS4 was produced with this adjuvant either. Mice immunized with Core-ISCOMS produced antibodies to core. In contrast, NS345Core I21 -PLGdss immunized mice produced significantly higher antibodies to NS5 thanNS345Core 121 -ISCOMS. In addition, NS345Core I21 -PLGdss immunized mice produced antibodies to NS3 and some antibody response to core, but no antibodies to NS4.
  • a Groups of 10 C57 black mice were immunized IM at 0, 21 and 60 days. Serum was obtained after the last immunization.
  • the NS345 Core m -PLGdss group received vaccine dose of 50 ⁇ l in each leg whereas the otlier vaccine groups received vaccine dose of 50 ⁇ l in one leg.
  • the ratio of protein to QH was approximately 8:1. "The ratio of protein to QH was approximately 1:1.
  • mice Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days with the formulations shown in Tables 11 and 12.
  • the NS345 and NS345Core 121 protein concentration was 10 ⁇ g per dose, and for those in Table 12, the concentration was 5 ⁇ g per dose.
  • PLG-NS345 amino acids 1242 to 3011 of the HCV polyprotein
  • PLG-NS345Core 121 amino acids 1242-3011 and 1-121 of the HCV polyprotein
  • PLG microparticles were prepared and NS345 or NS345Core 121 were adsorbed thereto using standard techniques, as described above.
  • E1E2 DNA was produced as follows. Mammalian expression plasmid pMH-ElE2-809 ( Figure 4, ATCC Deposit No. PTA-3643) encodes an E1E2 fusion protein which includes amino acids 192-809 of HCN la (see, Choo et al., Proc. Natl. Acad. Sci. r ⁇ -4 (1991) 88:2451-2455). Chinese Hamster Ovary (CHO) cells were used for expression of the HCN E1E2 sequence from ⁇ MH-ElE2-809.
  • CHO DG44 cells were used. These cells, described by Uraub et al., Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220, were derived from CHO K-l cells and were made dihydrofolate reductase (dhfr) deficient by virtue of a double deletion in the dhfr gene. DG44 cells were transfected with pMH-ElE2-809. The transfected cells were grown in selective medium such that only those cells expressing the dhfr gene could grow (Sambrook et al., supra). Isolated CHO colonies were picked ( ⁇ 800 colonies) into individual wells of a 96-well plate.
  • dhfr dihydrofolate reductase
  • PLG-NS345Core 121 + E1E2 DNA PLG-NS345Core 121 and E1E2 DNA were produced as described above.
  • PLG-NS345 or PLG-NS345Core 121 + MF59 PLG-NS345 or PLG-
  • NS345Core 121 was combined with MF59 as described above.
  • NS345 or NS345Core 121 was adsorbed to PLG as described above.
  • the CpG molecule used was 5'-TCCATGACGTTCCTGACGTT-3' and this was treated with CTAB, as described above.
  • NS345Core 12 ⁇ /MF59/MPL and NS345Core /MF59/CpG formulations produced very high antibody titers to NS345Core 12 ⁇ .
  • /MF59/Chol/QS21 formulations produced moderate antibody titers to NS345Core m .
  • the other formulations produced very low or no antibody titers to NS345Core 12 ⁇ .
  • mice Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days. Serum was obtained after the last immunization.
  • the NS345 or NS345Core 121 protein concentration was 10 ⁇ g per dose.
  • mice Groups of 10 C57 black mice were immunized IM at 0, 30 and 60 days. Serum was obtained after the last immunization.
  • the NS345 or NS345Core 121 protein concentration was 5 ⁇ g per dose.
  • mice Groups of 8 C57 black mice were immunized IM at 0, 30 and 60 days with the formulations shown in Table 13 and are as described above. Spleens were obtained after the last immunization.
  • the NS345Core 121 protein concentration was 25 ⁇ g per dose.
  • the NS34a, NS5b and core doses were 3 ⁇ g each.
  • NS345Core 121 /Alum/CpG, PLG-NS345Core 121 /PLG/CpG, NS34a+NS5B+ Core/MF59/CpG and PLG-NS345Core 121 /MF59/CpG formulations demonstrated strong LPA responses to NS5, NS34 and core antigens.
  • the NS345Core 121 /MF59 formulation also produced a strong LPA response to NS5 and NS34. Core was not tested.
  • NS345Core 121 /MF59/CpG formulation may not have been administered properly in that no LPA response was observed in this experiment.
  • an LPA was observed to this formulation.
  • mice Groups of 8 C57 black mice were immunized once IM with the formulations shown in Table 14, produced as described above. Draining lymph nodes were obtained.
  • the NS345Core 121 protein concentration was 25 ⁇ g per dose.
  • mice Groups of 8 C57 black mice were immunized IM at 0, 30 and 60 days. Spleens were obtained after the last immunization.
  • the NS345Core 121 protein concentration was 25 ⁇ g per dose.
  • the NS34a, NS5B and Core doses were 3 ⁇ g each.
  • mice Groups of 8 C57 black mice were immunized once IM. Draining lymph nodes were obtained. The NS345Core ⁇ 21 protein concentration was 25 ⁇ g per dose.
  • HCV proteins The safety and immunogenicity of HCV proteins completed with the adjuvant, Iscomatrix, was studied in Rhesus macaques. Three groups made up of four animals each were immunized IM as detailed below at week 0, 4 and 8 weeks. Vaccines were prepared as described above. The ISCOMS used lacked QH-A.
  • the HCV Core-ISCOM vaccine produced no CTL positive responses in any of the 4 immunized macaques after the second or third immunizations.
  • At least two of four macaques produced a strong LPA response after the second immunizations, but only one remained positive after the third immunization.
  • Two of four macaques produced positive CD4 intracellular staining after the second immunization and four of four after the third immunization.
  • the HCV NS345Core 121 -ISCOM vaccine after the second immunization produced CTL positive responses to peptide pools representing two or more HCV proteins in three of four macaques (two of these macaques had responses to peptide pools from NS3, NS5a and NS5b, one to peptide pools from core and NS3).
  • CD8 positive ⁇ -interferon and/or TNF- ⁇ intracellular staining to peptide pools representing two or more HCV proteins was positive in at least three of four macaques.
  • One of four macaques produced a strong LPA response.
  • At least three of four macaques produced CD4 positive intracellular staining to two or more HCV proteins.
  • the HCV Core-ISCOM + NS5b-ISCOM vaccine produced a CTL positive response to NS5b in one of the 4 immunized macaques after the second immunization which did not remain positive after the third immunizaton.
  • CD8 positive intracellular positive staining was observed in one of four animals post second. Two of four macaques produced a strong LPA response after the second immunization which did not remain positive after the third immunization. Two other macaques did develop a strong LPA response after the third immunization.
  • Three or four developed positive CD4 intracellular staining One developed positive CD 8 intracellular staining.
  • HCV Core-ISCOMS The immunogenicity of HCV Core-ISCOMS, NS345Core 121 -ISCOMS and Core-ISCOMS + NS5b-ISCOMS as assessed by EIA antibody response is shown in Table 18. As can be seen, all three vaccines produced an antibody response by the third immunization to their corresponding HCV proteins, except for the NS345Core 121 -ISCOM vaccine.
  • the NS345Core 121 -ISCOM vaccine produced antibody responses to NS3, NS4 and a very strong antibody response to NS5, but no antibody response to HCV core.
  • Formulation 1 20 ⁇ g E1E2 polypeptide + MF59 + 500 ⁇ g CpG (produced as described above);
  • Formulation 2 1 mg NS345Core I2I -ISCOM (produced as described above); Formulation 3: 6 mg each of CTAB-PLG-E1E2 (bp 574-2427, encoding amino acids 192-809 of the HCV polyprotein, numbered relative to HCV-1); CTAB- PLG-NS34a (bp 3079-5133, encoding amino acids 1027-1711 of the HCV polyprotein, numbered relative to HCV-1); CTAB-PLG-NS34ab (bp 4972-5916, encoding amino acids 1658-1972 of the HCV polyprotein, numbered relative to HCV-1); CTAB-PLG-NS5a (bp 5917-7260, encoding amino acids 1973-2420 of the HCV polyprotein, numbered relative to HCV-1);
  • Formulation 4 6 mg each of E1E2 DNA, NS34a DNA, NS34ab DNA and NS5a DNA, having the same coordinates as described above, delivered without PLG via electroporation (see, e.g., U.S. Patent Nos. 6,132,419; 6,451,002, 6,418,341, 6233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, for this delivery technique). Results are shown in Figures 8-10. As can be seen, in Figure 8, all vaccines were capable of priming CD4+ and CD8+ cells specific to HCV. Thus, all vaccines were successful at inducing a T cell response to HCV. Determination of the results for the PLG-DNA from formulation 3 at two weeks subsequent to the fourth vaccination is in progress.
  • ⁇ lectroporated IM with 1.5 mg of each plasmid at 0, 0.7, 2 and 5 months.
  • HCV polypeptides and polynucleotides either alone or as fusions, to stimulate cell-mediated immune responses, are disclosed.

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Abstract

L'invention concerne un procédé d'activation de lymphocytes T spécifiques du virus de l'hépatite C (VHC), notamment des lymphocytes T CD4+ et CD8+. Les lymphocytes T spécifiques du VHC sont activés au moyen de protéines de fusion comprenant des polypeptides du VHC NS3, NS4, NS5a, et NS5b, des polynucléotides codant lesdites protéines de fusion, ou des compositions polypeptidiques ou polynucléotidiques contenant les composants individuels de ces fusions. Le procédé de l'invention peut être utilisé dans des systèmes modèles pour développer des compositions immunogènes spécifiques du VHC, ainsi que pour immuniser un mammifère contre le VHC.
PCT/US2003/033610 2002-10-25 2003-10-24 Activation de lymphocytes specifiques du vhc WO2004039950A2 (fr)

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WO2007026190A3 (fr) * 2004-07-18 2007-05-03 Csl Ltd Formulations a base de complexes immunostimulants et d'oligonucleotides permettant d'induire des reponses d'interferon-gamma ameliorees
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
WO2011163558A1 (fr) * 2010-06-25 2011-12-29 Abbott Laboratories Matériaux et procédés pour le dosage d'anticorps anti-virus de l'hépatite c (hvc)
US8216590B2 (en) 2006-08-25 2012-07-10 Novartis Ag HCV fusion polypeptides
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EP1765386A4 (fr) * 2004-05-17 2008-07-30 Novartis Vaccines & Diagnostic Domaine ns5 du virus de l'hepatite c tronque et proteines hybrides comprenant ce dernier
AU2005245909B2 (en) * 2004-05-17 2010-05-27 Novartis Vaccines And Diagnostics, Inc. Truncated hepatitis C virus NS5 domain and fusion proteins comprising same
EP1765386A2 (fr) * 2004-05-17 2007-03-28 Novartis Vaccines and Diagnostics, Inc. Domaine ns5 du virus de l'hepatite c tronque et proteines hybrides comprenant ce dernier
EP2484374A1 (fr) * 2004-07-18 2012-08-08 CSL Limited Formulations à base de complexes immunostimulants et d'oligonucléotides permettant d'induire des réponses d'Interféron-Gamma ameliorées
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EP1576125A4 (fr) 2008-09-03
AU2003287188A8 (en) 2004-05-25
CA2505611A1 (fr) 2004-05-13
AU2003287188A1 (en) 2004-05-25
WO2004039950A3 (fr) 2007-11-22
EP1576125A2 (fr) 2005-09-21

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