CA2491508A1 - Hcv fusion proteins with modified ns3 domains - Google Patents

Abstract

The invention provides HCV fusion proteins that include a mutated NS3 protease domain, fused to at least one other HCV epitope derived from another region of the HCV polyprotein. The fusions can be used in methods of stimulating a cellular immune response to HCV, such as activating hepatitis C virus (HCV)-specific T cells, including CD4+ and CD8+ T cells. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize a mammal against HCV.

Description

TECHNICAL FIELD
The present invention relates to hepatitis C virus (HCV) constructs. More particularly, the invention relates to HCV fusion proteins with modified NS3 domains. The proteins are capable of stimulating cell-mediated immune responses, such as for priming and/or activating HCV-specific T cells.
BACKGROUND OF THE INVENTION
Hepatitis C virus (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 cluonic carrier state that can result in cirrhosis, liver failure, and hepatocellular carcinoma. See, Alter et al. (1992) N. Engl. J. Med. 327:1899-1905; Resniclc and Koff. (1993) Arch. Intem. Med.
153:1672-1677; Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N.
Engl. J.
Med. 332:1463-1466.
HCV was first identified and characterized as a cause of NANBH by Houghton et al. The viral genomic sequence of HCV is l~nown, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669;
WO
90/11089; and WO 90/14436. HCV has a 9.5 lcb positive-sense, single-stranded RNA
genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Siimnonds et al., J. Gen. ViYOI. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Sciefice (1989) 244:359-362; Choo et al., P~oc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., P~oc. Natl. Acad. Sci. USA (1991) X8:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, as shown in Figure 1, several proteins are encoded by the HCV
genome. The order and nomenclature of the cleavage products of the HCV
polyprotein is as follows:
NH2_C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NSSa-NSSb-COOH. Tilitial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed "core") and two envelope glycoproteins, "El" (also known as E) and "E2" (also known as E2/NS1), as well as nonstructural (NS) proteins that contain the viral enzymes. The NS regions are tenned'NS2, NS3, NS4 and NSS. NS2 is an integral membrane protein with proteolytic activity and, in combination with NS3, cleaves the NS2-NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease serves to process the remaining polyprotein. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NSSa), and an RNA-dependent RNA polyrnerase (NSSb). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease.
Despite extensive advances in the development of pharmaceuticals against certain viruses like HIV, control of acute and chronic HCV infection has had limited success (Hoofnagle and di Bisceglie (1997) N. Engl. J. Med. 336:347-356). In pas-ticular, generation of cellular immune responses, such as strong cytotoxic T
lymphocyte (CTL) responses, is thought to be important for the control and eradication of HCV infections. Thus, there is a need in the art for effective methods of stimulating cellular immune responses to HCV.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods for stimulating a cellular immune response to HCV, such as priming and/or activating T cells which recognize epitopes of HCV polypeptides. This and other obj ects of the invention are provided by one or more of the embodiments described below.
The invention provides HCV fusion proteins useful for stimulating such responses. One embodiment of the invention is directed to an HCV fusion protein that includes an NS3 polypeptide modified to inhibit protease activity, such that cleavage of the fusion is inhibited. The fusion protein includes, in addition to the modified NS3 polypeptide, one or more polypeptides from other regions of an HCV
polyprotein, described in further detail below. These polypeptides are derived from the same HCV isolate as the NS3 polypeptide, 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.
In certain embodiments, the modification to NS3 comprises a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein.
In further embodiments, the protein comprises a modified NS3 polypeptide, an NS4 polypeptide, an NSSa polypeptide, and optionally a core polypeptide.
In additional embodiments, the protein further comprises an NSSb polypeptide, and optionally a core polypeptide.
In yet additional embodiments, the protein further comprises an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.
In further embodiments, the protein fiuther comprises an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.
In additional embodiments, the protein further comprises an E2 polypeptide, and optionally a core polypeptide.
In yet further embodiments, the protein further comprises an E1 polypeptide, an E2 polypeptide, and optionally a core polypeptide.
In further embodiments, the protein comprises an E2 polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.
In additional embodiments, the protein comprises an E1 polypeptide, an E2 polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.
Another embodiment provides a fusion protein that consists essentially of a modified NS3, an NS4, an NSSa, and, optionally, a core polypeptide of an HCV.
In certain embodiments, an NSSb polypeptide is also present.
In the embodiments above, the various regions in the fusion protein need not be in the order in which they naturally occur in the native HCV polyprotein.
Thus, for example, the core polypeptide, if present, may be at the N- and/or C-terminus of the fusion.
In yet additional embodiments, the invention is directed to an immunogenic fusion protein consisting essentially of, in amino terminal to carboxy terminal direction:
(a) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NSSa polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide and an NSSb polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NSSa polypeptide;
(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NSSa polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide and an NSSb polypeptide;
(fJ an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide and an NSSb polypeptide;

(g) an E2 polypeptide axed a modified NS3 polypeptide comprising substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited;
(h) an E1 polypeptide, an E2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited; or (j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited.
In another embodiment, the invention is directed to an immunogenic fusion protein consisting essentially of, in amino terminal to carboxy terminal direction:
(a) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide, and a core polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide, an NSSb polypeptide and a core polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide and a core polypeptide;

(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NSZ
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide and a core polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide, an NSSb polypeptide and a core polypeptide;
(f) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NSSa polypeptide, an NSSb polypeptide and a core polypeptide;
(g) an E2 polypeptide, a modified NS3 polypeptide comprising substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide;
(h) an El polypeptide, an E2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide; or (j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide.
In yet a further embodiment, the invention is directed to a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited when the modified NS3 polypeptide is present in an HCV fusion protein.
Yet another embodiment of the invention provides an isolated polynucleotide which encodes any of the proteins detailed above, recombinant vectors comprising the same, host cells transformed with the vectors, and methods of recombinantly producing the fusion proteins.
The invention also provides compositions comprising any of these fusion proteins, polynucleotides encoding the fusions, or recombinant vectors including the polynucleotides, and a pharmaceutically acceptable Garner.
Yet another embodiment of the invention provides a method of stimulating a cellular irninune response in a vertebrate subject by administering a composition as described herein. In certain embodiments, the composition primes and/or activates T
cells which recognize an epitope of an HCV polypeptide. T cells are contacted with a fusion protein comprising a modified NS3 polypeptide and at least one additional HCV polypeptide. A population of activated T cells recognizes an epitope of the NS3 and/or the additional HCV polypeptide(s).
The invention thus provides methods and reagents for stimulating a cellular immune response to HCV, such as for priming and/or 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 immunizing mammals, including humans, against HCV.
BRIEF DESCRIPTION OF THE FIGURES
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 (SEQ TD
NOS:3-4) of a representative native, unmodified NS3 protease domain.
Figure 3 shows the DNA and corresponding amino acid sequence (SEQ ID
NOS:S-6) of a representative modified fusion protein, with the NS3 protease domain deleted from the N-terminus and including amino acids 1-121 of Core on the C-terminus.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Moleculaf~ Cloning: A Labof~atory Manual (2nd Edition); Methods h2 Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); DNA Cloning, Vols. I and II (D.N. Glover ed.); Oligon.ucleotide Synthesis (M.J. Gait ed.); Nucleic Acid Hyby°idization (B.D. Hames & S.J. Higgins eds.); Afzimal Cell Cultu~~e (R.K. Freshney ed.); Perbal, B., A P~~actical Guide to Moleculaf° Clonifag.
It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture of two or more amtigens, and the like.
The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (I~ Aspartic acid:
Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine:
Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (~

Tyrosine: Tyr (Y) Valine: Val (V) I. Definitions In describing the present invention, the following teens will be employed, and are intended to be defined as indicated below.
The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition.
Both full-length proteins and fragments thereof are encompassed by the definition.
The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity.
These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
An HCV polypeptide is a polypeptide, as defined above, derived from the 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. Geh.
Vif°ol. (1993) 74:2391-2399 (e.g., strains l, 2, 3, 4 etc.), as well as newly identified isolates, and subtypes of these isolates, such as HCVla, HCVlb etc. 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%, when the two sequences are aligned. Thus, for example, the term "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.
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.
hi the case of a modified NS3, an "analog" or "mutein" refers to an NS3 molecule that lacks its native proteolytic activity. In general, the term "analog" refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature, or in the case of modified NS3, non-conservative in nature at the active proteolytic site) and/or deletions, relative to the native molecule, so long as the modifications do not destroy immunogenic activity. The term "mutein" refers to peptides having one or more peptide mimics ("peptoids"), such as those described in International Publication No.
WO 91/04282. Preferably, the analog or mutein has at least the same immunoactivity as the native molecule. Methods for making polypeptide analogs and muteins are lrnown in the art and are described further below.
As explained above, 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. Specifically, amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate;
(2) basic --lysine, argiune, 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. For example, it is reasonably predictable that 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. For example, 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. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.

By "modified NS3" is meant 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.
By "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 irnlnunogenic activity, as measured by the assays described herein.
The term "epitope" as used herein 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. Indeed, viral genomes are in a state of constant flux and contain several variable domains which exhibit relatively high degrees of variability between isolates. Thus the term "epitope" 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 Mappifzg Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, 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 lmown in the art and described in, e.g., U.S. Patent No.
4,708,871;
Geysen et al. (1984) P~oc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al.
(1986) Molec. ITnmuf2ol. 23:709-715. Similarly, 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 Mappifag P~°otocols, 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 fiom the Oxford Molecular Group.
This computer program employs the Hopp/Woods method, Hopp et al., Ps°oc. Natl.
Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.
For a description of various HCV epitopes, see, e.g., Chien et al., Py-oc.
Natl.
Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gast~oefzt. Hepatol.
(1993) 8:533-39; Chien et al., International Publication No. WO 93/00365; Chien, D.Y., International Publication No. WO 94/01778; and U.S. Patent Nos. 6,280,927 and 6,150,087.
As used herein the term "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, ((Jnanue 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). More particularly, 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.
Further, it is believed that 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., Computey~ Prediction of T cell Epitopes, New Generation Vaccines Marcel-Del~l~er, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116) and further that the amphipathic structures have an a-helical configuration (see, e.g., Spouge et al., J.
Ifsamuhol. (1987) 138:204-212; Berlcower et al., J. Imnaufaol. (1986) 136:2498-2503).
Hence, segments of proteins that include T-cell epitopes can be readily predicted using numerous computer programs. (See e.g., Margalit et al., Compute~°
Pj°edictioh 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 wluch are believed to be required for a T-cell epitope.
A~z "immunological response" to ail 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. For purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T
lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T cells ("CTLs").
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 intracellular microbes, or the lysis of cells infected with such microbes.
Both CD8+
and CD4+ T cells are capable of killing HCV-infected cells. A~.zother 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 antiviral cytolcines, chemolcines and other such molecules produced by activated T cells and/or other white blood cells, including those derived from CD4+ and CD8+ T cells, including, but not limited to IFN-y and TNF-a.
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. In addition, 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. Such assays are well known in the art. See, e.g., Erickson et al., J. Im~2urt.ol. (1993) 151:4189-4199; Doe et al., Eur. J. Imr~zuhol. (1994) 24:2369-2376; and the examples below.
Thus, an irmnunological 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 immmle response.
Hence, 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 y8 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. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
By "equivalent antigeuc determinant" is meant an antigenic determinant from different sub-species or strains of HCV, such as from strains 1, 2, 3, etc., of HCV
which antigenic determinants axe not necessarily identical due to sequence variation, but which occur in equivalent positions in the HCV sequence in question. In general the 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 i~2 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.
A "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.
"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function.
Thus, 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. Thus, for example, 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. In general, 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.
A "control element" refers to a polynucleotide sequence which aids in the expression of a coding sequence to which it is linlced. The temp 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 polymerise in a host cell and initiating transcription of a downstream (3' direction) coding sequence operably linlced thereto. For purposes of the present invention, a promoter sequence includes the miumum 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
polymerise. 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 polymerise 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 sequences) or genes) 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 sequences) or genes) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, 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 M13 origin of replication), at least one multiple cloning site, and a "mammalian"
origin of replication (e.g., a SV40 or adenovirus origin of replication).
"Transformation," as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion: for example, transformation by direct uptalce, transfection, infection, and the life. 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.
By "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 macromolecules of the same type. The term "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.
The teen "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. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.

In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O.
in Atlas of P~~otein Sequence and Sty~uctu~~e M.O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances ifz Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waternan algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Paclcage referred to above. For example, 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). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code =
standard;
filter = none; strand ,-- both; cutoff = 60; expect = 10; Matrix = BLOSUM62;

Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein +
Spupdate + PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.
Alternatively, 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., Sambroolc et al., supra; DNA Clohif2g, supra; Nucleic Acid HybridizatiofZ, supra.
By "nucleic acid immunization" is meant the introduction of a nucleic acid molecule encoding one or more selected immunogens into a host cell, for the in vivo expression of the immunogen or immunogens. The nucleic acid molecule can be introduced directly into the recipient subject, such as by injection, inhalation, oral, intra~lasal and mucosal administration, or the lilce, 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.
As used herein, "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 (following infection).
By "vertebrate subject" is meant any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats;
laboratory animals including rodents such as mice, rats and guinea pigs;
birds, including domestic, wild and game birds such as chiclcens, turkeys and other gallinaceous birds, duclcs, geese, and the lilce. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The invention described herein is intended for use in any of the above vertebrate species, since the innnune systems of all of these vertebrates operate similarly.
II. Modes of Carrying out the Invention Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
The present invention pertains to fusion proteins and polynucleotides encoding the same, comprising a modified NS3 polypeptide and at least one other HCV
polypeptide from the HCV polyprotein. The fusion proteins of the present invention can be used to stimulate a cellular immune response, such as to activate HCV-specific T cells, i.e., T cells which recognize epitopes of these polypeptides and/or to elicit the production of helper T cells and/or to stimulate the production of antiviral cytol~ines, chemolcines, and the lilce. Activation of HCV-specific T cells by such fusion proteins provides both ih vitro and ifa vivo model systems for the development of HCV
vaccines, particularly for identifying HCV polypeptide epitopes associated with a response. The fusion proteins can also be used to generate an immune response against HCV in a mammal, for example a CTL response, and/or to prime CD~+ and CD4+ T cells to produce antiviral agents, for either therapeutic or prophylactic purposes.
In order to further an understanding of the invention, a more detailed discussion is provided below regarding fusion proteins for use in the subject compositions, as well as production of the proteins, compositions comprising the same and methods of using the proteins.

Fusion Pf°oteirzs The genomes of HCV strains contain a single open reading frame of approximately 9,000 to 12,000 nucleotides, which is transcribed into a polyprotein.
S As shown in Figure 1 and Table 1, an HCV polyprotein, upon cleavage, produces at least ten distinct products, in the order of NH2-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NSSa-NSSb-COOH. The core polypeptide occurs at positions 1-191, numbered relative to HCV-1 (see, Choo et al. (1991) P~oc.
Natl. Acad. Sci. USA 88:2451-2455, for the HCV-1 genome). This polypeptide is further processed to produce an HCV polypeptide with approximately amino acids 1-173. The envelope polypeptides, E1 and E2, occur at about positions 192-383 and 384-746, respectively. The P7 domain is found at about positions 747-809. NS2 is an integral membrane protein with proteolytic activity and is found at about positions 810-1026 of the polyprotein. NS2, in combination with NS3, (found at about positions 1027-1657), cleaves the NS2-NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease, found at about positions 1027-1207, serves to process the remaining polyprotein. The helicase activity is found at about positions 1193-1657. NS3 liberates an NS3 cofactor (NS4a, found about positions 1658-1711), two proteins (NS4b found at about positions 1712-1972, and NSSa found at about positions 1973-2420), and an RNA-dependent RNA polymerase (NSSb found at about positions 2421-3011). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-Ns4a junction, catalyzed by the NS3 serine protease.
Table 1 omain Approximate Boundaries*

C (core) 1-191 S4a 1658-1711 S4b 1712-1972 NSSa 1973-2420 NSSb 2421-3011 *Numbered relative to HCV-1. See, Choo et al. (1991) Pf°oc. Natl. Acad.
Sci.
USA 88:2451-2455.
Fusion proteins of the invention include an NS3 polypeptide modified to inhibit protease activity, such that further cleavage of the fusion is inhibited. The NS3 polypeptide can be modified by deletion of all or a portion of the NS3 protease domain. Alternatively, proteolytic activity can be inhibited by substitutions of amino acids within active regions of the protease domain. Finally, additions of amino acids to active regions of the domain, such that the catalytic site is modified, will also serve to inhibit proteolytic activity.
As explained above, the protease activity is found at about amino acid positions 1027-1207, numbered relative to the full-length HCV-1 polyprotein (see, Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455), positions 2-182 of Figure 2 (SEQ ID N0:4). The structure of the NS3 protease and active site are known. See, e.g., De Francesco et al., Antivif°. Tlrer. (1998) 3:99-109; Koch et al., Biochemistry (2001) 40:631-640. Thus, deletions or modifications to the native sequence will typically occur at or near the active site of the molecule.
Particularly, it is desirable to modify or make deletions to one or more amino acids occurnng at positions 1- or 2-182, preferably 1- or 2-170, or 1- or 2-155 of Figure 2 (SEQ
ID
N0:4). Preferred modifications are to the catalytic triad at the active site of the protease, i.e., H, D and/or S residues, in order to inactivate the protease.
These residues occur at positions 1083, 1105 and 1165, respectively, numbered relative to the full-length HCV polyprotein (positions 58, 80 and 140, respectively, of Figure 2 (SEQ ID N0:4)). Such modifications will suppress proteolytic cleavage while maintaining T-cell epitopes.
One of skill in the art can readily determine portions of the NS3 protease to delete in order to disrupt activity. The presence or absence of activity can be determined using methods known to those of skill in the art.

For example, protease activity or lacy thereof may be determined using the procedure described below in the examples, as well as using assays well known in the art. See, e.g., Tal~eshita et al., Anal. Biochem. (1997) 247:242-246;
Kalciuchi et al., J.
Biochem. (1997) 122:749-755; Sali et al., Biochemistp (1998) 37:3392-3401; Cho et al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem..
(1999) 266:192-197; Zhang et al., Anal. Biochena. (1999) 270:268-275; Kal~iuchi et al., J.
Viol. Meth. (1999) X0:77-84; Fowler et al., J. Biomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem. (2000) 284:42-48.
The fusion protein of the present invention includes, in addition to the modified NS3 polypeptide, one or more polypeptides from one or more other regions of an HCV polyprotein. In fact, the fusion can include all the regions of the HCV
polyprotein. These polypeptides may derived from the same HCV isolate as the NS3 polypeptide, 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. Additionally, polypeptides can be selected based on the particular viral Glades endemic in specific geograpluc regions where vaccine compositions containing the fusions will be used. It is readily apparent that the subject fusions provide an effective means of treating HCV infection in a wide variety of contexts.
In certain embodiments, the fusion protein comprises a modified NS3 (also referred to herein as NS3*), an NS4 (NS4a and NS4b), an NSSa and, optionally, a core polypeptide of an HCV (NS3*NS4NSSa or NS3*NS4NSSaCore fusion proteins, also termed "NS3*45a" and "NS3*45aCore" herein). These regions need not be in the order in which they naturally occur in the native HCV polyprotein. Thus, for example, the core polypeptide may be at the N- and/or C-terminus of the fusion.
Another embodiment provides a fusion protein that includes an NS3*, an NS4, a~i NSSa, an NSSb, and optionally, a core polypeptide of an HCV
(NS3*NS4NSSaNSSb or NS3*NS4NSSaNSSbCore fusion proteins, also termed "NS3*45ab" and "NS3*45abCore" herein). These regions need not be in the order in which they naturally occur in the native HCV polyprotein. Thus, for example, the core polypeptide may be at the N- and/or C-terminus of the fusion.

Yet other embodiments are directed to a fusion protein comprising an NS3*
combined with an NS2, an NS3* combined with an NS2, p7 and E2, an NS3*
combined with an NS2, p7 and an E1, an NS3* combined with an NS2, p7 and an E1 and an E2, an NS3* combined with an E2, an NS3* combined with an E1 and an E2, all with or without a core polypeptide. As with those fusions described above, these regions need not be in the order in which they occur naturally. Moreover, each of these regions can be derived from the same or a different HCV isolate.
Figure 3 (SEQ ID NOS:S-6) shows a representative modified fusion protein, with the NS3 protease domain deleted from the N-terminus and including amino acids 1-121 of Core on the C-terminus.
The various HCV polypeptides present in the various fusions described above can either be full-length polypeptides or portions thereof. The portions of the HCV
polypeptides malting up the fusion protein comprise at least one epitope, which is recognized by a T cell receptor on an activated T cell, such as 2152-HEYPVGSQL-2160 (SEQ ID NO:1) and/or 2224-AELIEANLLWRQEMG-2238 (SEQ ID N0:2). Epitopes ofNS2, p7, El, E2, NS3, NS4 (NS4a and NS4b), NSSa, NSSb, NS3NS4NSSa, and NS3NS4NSSaNSSb can be identified by several methods. For example, the individual polypeptides or fusion proteins comprising any combination of the above, can be isolated, by, e.g., immunoaffinity 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. By starting with, for example, 100-mer polypeptides, each polypeptide can be tested for the presence of epitopes recognized by a T-cell receptor on an HCV-activated T cell, progressively smaller and overlapping fragments can then be tested from an identified 100-mer to map the epitope of interest.
Epitopes recognized by a T-cell receptor on an HCV-activated T cell can be identified by, for example, 51 Cr release assay (see Example 4) or by lymphoproliferation assay (see Example 6). In a SlCr release assay, target cells can be constructed that display the epitope of interest by cloning a polynucleotide encoding the epitope into an expression vector and transforming the expression vector into the target cells. HCV-specific CD8+ T cells will lyse target cells displaying, for example, one or more epitopes from one or more regions of the HCV polyprotein found in the fusion, and will not lyse cells that do not display such an epitope. In a lynphoproliferation assay, HCV-activated CD4+ T cells will proliferate when cultured with, for example, one or more epitopes from one or more regions of the HCV polyprotein found in the fusion, but not in the absence of an HCV epitopic peptide.
The various HCV polypeptides can occur in any order in the fusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or more of the polypeptides may occur in the fusion protein. Multiple viral strains of HCV occur, and HCV
polypeptides of any of these strains can be used in a fusion protein.
Nucleic acid and amino acid sequences of a number of HCV strains and isolates, including nucleic acid and amino acid sequences of the various regions of the HCV polyprotein, including Core, NS2, p7, E1, E2, NS3, NS4, NSSa, NSSb genes and polypeptides have been determined. For example, isolate HCV J1.1 is described in I~ubo et al. (1989) Japan. Nucl. Acids Res. 17:10367-10372; Talceuchi et al. (1990) Gene 91:287-291; Tal~euchi et al. (1990) J. Gen. Virol. 71:3027-3033; and Talceuchi et al. (1990) Nucl. Acids Res. 18:4626. The complete coding sequences of two independent isolates, HCV-J and BK, are described by Nato et al., (1990) Proc.
Natl.
Acad. Sci. USA 87:9524-9528 and Talcamizawa et al., (1991) J. Virol. 65:1105-respectively.
Publications that describe HCV-1 isolates include Choo et al. (1990) Brit.
Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-and Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:1711-1715. HCV isolates HC-and HC-J4 are described in Okamoto et al. (1991) Japan J. Exp. Med. 60:167-177.
HCV isolates HCT 18~, HCT 23, Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991) Virol. 180:842-848. HCV isolates Pt-1, HCV-Kl and HCV-K2 are described in Enomoto et al. (1990) Biochem. Biophys. Res. Commun.
170:1021-1025. HCV isolates A, C, D & E are described in Tsulciyama-I~ohara et al.
(1991) Virus Genes 5:243-254.

Each of the components of a fusion protein can be obtained from the same HCV strain or isolate or from different HCV strains or isolates. Fusion proteins comprising HCV polypeptides from, for example, the NS3 polypeptide can be derived from a first strain of HCV, and the other HCV polypeptides present can be derived from a second strain of HCV. Alternatively, one or more of the other HCV
polypeptides, for example NS2, NS4, Core, p7, E1 and/or E2, if present, can be derived from a first strain ofHCV, and the remaining HCV polypeptides can be derived from a second strain of HCV. Additionally, each or the HCV
polypeptides present can be derived from different HCV strains.
As explained above, it may be desirable to include polypeptides derived from the core region of the HCV polyprotein in the fusions of the invention. This region occurs at amino acid positions 1-191 of the HCV polyprotein, numbered relative to HCV-1. Either the full-length protein, fragments thereof, such as amino acids 1-160, e.g., amino acids 1-150, 1-140, 1-130, 1-120, for example, amino acids 1-121, 1-122, 1-123...1-151, etc., or smaller fragments containing epitopes of the full-length protein may be used in the subject fusions, such as those epitopes found between amino acids 10-53, amino acids 10-45, amino acids 67-88, amino acids 120-130, or any of the core epitopes identified in, e.g., Houghton et al., U.S. Patent No. 5,350,671;
Chien et al., Ps°oe. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J.
Gastf°oeyat.
Flepatol. (1993) 8:533-39; Chien et al., International Publication No. WO
93/00365;
Chien, D.Y., International Publication No. WO 94/01778; and U.S. Patent Nos.
6,280,927 and 6,150,087. Moreover, a protein resulting from a frameshift in the core region of the polyprotein, such as described in International Publication No.
WO
99/63941, may be used.
If a core polypeptide is present, it can occur at the N- terminus, the C-terminus and/or internal to the fusion. Particularly preferred is a core polypeptide on the C-terminus as this allows for the formation of complexes with certain adjuvants, such as ISCOMs, described further below.
As described above, useful polypeptides in the HCV fusion include T-cell epitopes derived from any of the various regions in the polyprotein. In this regard, E1, E2, p7 and NS2 are known to contain human T-cell epitopes (both CD4+ and CD8+) and including one or more of these epitopes serves to increase vaccine efficacy as well as to increase protective levels against multiple HCV
genotypes.
Moreover, multiple copies of specific, conserved T-cell epitopes can also be used in the fusions, such as a composite of epitopes from different genotypes.
For example, polypeptides from the HCV E1 and/or E2 regions can be used in the fusions of the present invention. E2 exists as multiple species (Spaete et al., Iji~ol.
(1992) 188:819-830; Selby et al., J. Yif-ol. (1996) 70:5177-5182; Gralcoui et al., J.
Ijif°ol.~(1993) 67:1385-1395; Tomei et al., J. Vi~~ol. (1993) 67:4017-4026) and clipping and proteolysis may occur at the N- and C-termini of the E2 polypeptide. Thus, an E2 polypeptide for use herein may comprise amino acids 405-661, e.g., 400, 401, 402... to 661, such as 383 or 384-661, 383 or 384-715, 383 or 384-746, 383 or 384-749 or 383 or 384-809; or 383 or 384 to any C-terminus between 661-809, of an HCV polyprotein, numbered relative to the full-length HCV-1 polyprotein.
Similarly, E1 polypeptides for use herein can comprise amino acids 192-326, 192-330, 192-333, 192-360, 192-363, 192-383, or 192 to any C-terminus between 326-383, of an HCV
polyprotein.
Immunogenic fragments of El and/or E2 which comprise epitopes may be used in the subject fusions. For example, fragments of El polypeptides can comprise from about 5 to nearly the full-length of the molecule, such as 6, 10, 25, 50, 75, 100, 125, 150, 175, 185 or more amino acids of an E1 polypeptide, or any integer between the stated numbers. Similarly, fragments of E2 polypeptides can comprise 6, 10, 25, 50, 75, 100, 150, 200, 250, 300, or 350 amino acids of an E2 polypeptide, or any integer between the stated numbers.
For example, epitopes derived from, e.g., the hypervariable region of E2, such as a region spanning amino acids 384-410 or 390-410, can be included in the fusions.
A particularly effective E2 epitope to incorporate into an E2 polypeptide sequence is one which includes a consensus sequence derived from this region, such as the consensussequence Gly-S er-Ala-Ala-Arg-Thr-Thr-S er-Gly-Phe-V al-S er-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn (SEQ ID N0:7),'which represents a consensus sequence for amino acids 390-410 of the HCV type 1 genome. Additional epitopes of E1 and E2 are l~nown and described in, e.g., Chien et al., International Publication No. WO
93/00365.
Moreover, the E1 and/or E2 polypeptides may lacy all or a portion of the membrane spanning domain. With E1, generally polypeptides terminating with about amino acid position 370 and higher (based on the numbering of HCV-1 E1) will be retained by the ER and hence not secreted into growth media. With E2, polypeptides terminating with about amino acid position 731 and higher (also based on the numbering of the HCV-1 E2 sequence) will be retained by the ER and not secreted.
(See, e.g., International Publication No. WO 96/04301, published February 15, 1996).
It should be noted that these amino acid positions are not absolute and may vary to some degree. Thus, the present invention contemplates the use of El and/or E2 polypeptides which retain the transmembrane binding domain, as well as polypeptides which laclc all or a portion of the transmembrane binding domain, including E1 polypeptides terminating at about amino acids 369 and lower, and E2 polypeptides, terminating at about amino acids 730 and lower. Furthermore, the C-terminal truncation can extend beyond the transmembrane spanning domain towards the N-terminus. Thus, for example, E1 truncations occurring at positions lower than, e.g., 360 and E2 truncations occurring at positions lower than, e.g., 715, are also encompassed by the present invention. All that is necessary is that the truncated E1 and E2 polypeptides remain functional for their intended purpose. However, particularly preferred truncated E1 constructs are those that do not extend beyond about amino acid 300. Most preferred are those terminating at position 360.
Preferred truncated E2 constructs are those with C-terminal truncations that do not extend beyond about amino acid position 715. Particularly preferred E2 truncations are those molecules truncated after any of amino acids 715-730, such as 725.
For a description of various HCV epitopes from these and other HCV regions, see, e.g., Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gast~~eyat. Hepatol. (1993) 8:533-39; Chien et al., International Publication No. WO 93/00365; Cluen, D.Y., International Publication No. WO 94/01778; and U.S. Patent Nos. 6,280,927 and 6,150,087.

Preferably, the above-described fusion proteins, as well as the individual components of these proteins, are produced recombinantly. A polynucleotide encoding these proteins can be introduced into an expression vector which can be expressed in a suitable expression system. A variety of bacterial, yeast, mammalian and insect expression systems are available in the art and any such expression system can be used. Optionally, a polynucleotide encoding these proteins can be translated in a cell-free translation system. Such methods are well known in the art. The proteins also can be constructed by solid phase protein synthesis.
If desired, the fusion proteins, or the individual components of these proteins, also can contain other amino acid sequences, such as amino acid linkers or signal sequences, as well as ligands useful in protein purification, such as glutathione-S-transferase and staphylococcal protein A.
Polyrtucleotides Etacoditzg the Fusion Proteihs Polynucleotides contain less than an entire HCV genome, or alternatively can include the sequence of the entire polyprotein with a mutated NS3 domain, as described above. The polynucleotides can be RNA or single- or double-stranded DNA. Preferably, the polynucleotides are isolated free of other components, such as proteins and lipids. The polynucleotides encode the fusion proteins described above, and thus comprise coding sequences for NS3* and at least one other HCV
polypeptide from a different region of the HCV polyprotein, such as polypeptides derived from NS2, p7, E1, E2, NS4, NSSa, NSSb, core, etc. Polynucleotides of the invention can also comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, or ligands useful in protein purification such as glutathione-S-transferase ~ and staphylococcal protein A.
To aid expression yields, it may be desirable to split the polyprotein into fragments for expression. These fragments can be used in combination in compositions as described herein. Alternatively, these fragments can be joined subsequent to expression. Thus, for example, NS3*NS4Core can be expressed as one construct and NSSaNSSbCore can be expressed as a second construct. Similarly, NS3*NS4NSSa can be expressed as one construct and a second construct with, e.g., NS3*NS4NSSaCore can be expressed as a second construct. For example, NS2p7E2NS3~NS4 can be expressed as a single construct, and NS3(unmodified)NS4NSSb can be expressed as an additional construct for use in the subject compositions. It is to be understood that the above combinations axe merely representative and any combination of fusions can be expressed separately.
Polynucleotides encoding the various HCV polypeptides can be isolated from a genomic library derived from nucleic acid sequences present in, for example, the plasma, serum, or liver homogenate of an HCV infected individual or can be synthesized in the laboratory, for example, using an automatic synthesizer. An amplification method such as PCR, can be used to amplify polynucleotides from either HCV genomic DNA or cDNA encoding therefor.
Polynucleotides can comprise coding sequences for these polypeptides which occur naturally or can be artificial sequences which do not occur in nature.
These polynucleotides can be ligated to form a coding sequence for the fusion proteins using standard molecular biology techniques. If desired, polynucleotides can be cloned into an expression vector and transformed into, for example, bacterial, yeast, insect, or mammalian cells so that the fusion proteins of the invention can be expressed in and isolated from a cell culture.
The expression constructs of the present invention, including the desired fusion, or individual expression constructs comprising the individual components of these fusions, may be used for nucleic acid immunization, to stimulate a cellular immune response, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466.
Genes can be delivered either directly to the vertebrate subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject. For example, the constructs can be delivered as plasmid DNA, e.g., contained within a plasmid, such as pBR322, pUC, or ColEl Additionally, the expression constructs can be paclcaged 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. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim.
Biophys. Acta.
(1991) 1097:1-17; Straubinger et al., inMetIZOds ofEfazysraology (1983), Vol.
101, pp.
512-527.
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.
For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BBL, Grand Island, NY. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.
See, e.g., Szoka et al., P~oc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT
Publication No. WO 90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. The various liposome-nucleic acid complexes are prepared using methods known in the art.
See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp.
512-527; Szolca et al., P~oc. Natl. Acad. Sci. USA (1978) 75:4194-4198;
Papahadjopoulos et al., Biochim. Biop7Zys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629;
Ostro et al., Bioclaem. Biophys. Res. Commufa. (1977) 76:836; Fraley et al., Pf°oc. Natl.
Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Pi°oc. Natl.
Acad. Sci. USA
(1979) 76:145); Fraley et al., J. Biol. Claerya. (1980) 255:10431; Szoka and Papahadjopoulos, PYOC. Natl. Acad. Sci. USA (1978) 75:145; a~ld Schaefer-Bidder et al., Science (1982) 215:166.
The DNA can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Bioclaena. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Patent Nos. 4,663,161 and 4,871,488.
A number of viral based systems have been developed for gene transfer into mammalian cells. For example, 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 paclcaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either irr. vivo or ex vivo. A number of retroviral systems have been described (LJ.S. Patent No. 5,219,740; Miller and Rosman, BioTechyaiques (1989) 7:980-990;
Miller, A.D., Human Gefae Thef°apy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., P~oc. Natl. Acad. Sci. USA (1993) 90:8033-8037;
a~zd Boris-Lawrie and Temin, Cup. Opih. Genet. Develop. (1993) 3:102-109. Briefly, retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses such as FIV, HIV, HIV-1, HIV-2 arid SIV (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, VA 20110-2209), or isolated from known sources using commonly available techniques.
A number of adenovirus vectors have also been described, such as adenovirus Type 2 and Type 5 vectors. Unlilce retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the rislcs associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Viol. (1986) 57:267-274;
Bett et al., J. Yirol. (1993) 67:5911-5921; Mittereder et al., Human Gehe Therapy (1994) 5:717-729; Seth et al., J. Tri~ol. (1994) 68:933-940; Barr et al., Gef~e Tlaef°apy (1994) 1:51-58; Berkner, I~.L. BioTeclz~iques (1988) 6:616-629; and Rich et al., Humaya Gehe TheYapy (1993) 4:461-476).
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., Pf°oc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
Members of the Alphavirus 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. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J.
Pirol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072.
Other vectors can be used, including but not limited to simian virus 40 and cytomegalovirus. Bacterial vectors, such as Salmonella ssp. Ye~sifaia entef°ocolitica, Shigella spp., Vibrio chole~ae, Mycobacteriuan strain BCG, and Listeria moraocytogeraes can be used. Minicluomosomes such as MC and MC1, bacteriophages, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used.
The expression constructs 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 cytol~ine release. Examples of particulate Garners 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., Plaa~m. Res. (1993) 10:362-368; and McGee et al., J. Mic~~oencap. (1996).
A wide variety of other methods can be used to deliver the expression constructs to cells. Such methods include DEAF 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 microinjection. See, e.g., Sambrook et al., supYa, for a discussion of techniques for transforming cells of interest; and Felgner, P.L., Advafaced D~°ug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer.
One particularly effective method of delivering DNA using electroporation is described in International Publication No. WO/0045823.

Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering the expression constructs of the present invention. The particles are coated with the construct to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Patent Nos. 4,945,050;
5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,47,744.
Corrapositions Cofnprisifzg Fusion P~oteiras o~° Polyfaucleotides The invention also provides compositions comprising the fusion proteins or polynucleotides. The compositions may include one or more fusions, so long as one of the fusions includes a mutated NS3 domain as described herein. Compositions of the invention may also 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.
Pharmaceutically acceptable salts 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. 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.
The proteins of the invention can also be adsorbed to, entrapped within or otherwise associated with liposomes and particulate carriers such as PLG. Liposomes and other pas-ticulate carriers are described above.
If desired, co-stimulatory molecules which improve immunogen presentation to lymphocytes, such as B7-1 or B7-2, or cytolcines, lympholcines, and chemokines, including but not limited to cytolcines such as IL-2, modified IL-2 (cys125~ser125), GM-CSF, IL-12, 'y- interferon, IP-10, MIP 1 (3, FLP-3, ribavirin and RANTES, may be included in the composition. Optionally, adjuvants can also be included in a composition. Adjuvants which can be used include, but are not limited to:
(1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, ' aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (PCT Publ. No. WO
90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE ), formulated into submicron particles using a microfluidizer such as Model 1 l0Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (R.AS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); (3) saponin adjuvants, such as QS21 or StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostunulating complexes), which ISCOMs may be devoid of additional detergent (see, e.g., International Publication No. WO 00/07621); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleulcins, such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc. (see, e.g., International Publication No. WO 99/44636), interferons, such as gamma interferon, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E.

coli heat-labile toxin (LT), 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 amino acid at position 72), CT-S 109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. W093/13202 and W092/19265); (7) monophosporyl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB
2220221; EPA 0689454), optionally in the substantial absence of alum (see, e.g., International Publication No. WO 00/56358); (8) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulations (see, e.g., EPA 0835318; EPA
0735898; EPA 0761231); (9) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g., International Publication No. WO 99/52549); (10) an immunostimulatory oligonucleotide such as a CpG oligonucleotide, or a saponin and an immunostimulatory oligonucleotide, such as a CpG oligonucleotide (see, e.g., International Publication No. WO 00/62800); (11) an immunostimulant and a particle of a metal salt (see, e.g., International Publication No. WO 00/23105); (12) a saponin and an oil-in-water emulsion (see, e.g., International Publication No. WO
99/11241;
(13) a saponin (e.g., QS21) + 3dMPL + IL-12 (optionally + a sterol) (see, e.g., International Publication No. WO 98/57659); (14) the MPL derivative RC529; and (15) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Ahun and MF59 are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-z-alanyl-D-isoglutamine (CGP 11637, referred to nor-MDP), N-acetylmuramyl-z-alanyl-D-isoglutaminyl-z-alanine-2-(1'-2'-dipalmitoyl-sf~-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), etc.
Moreover, the fusion protein can be adsorbed to, or entrapped within, an ISCOM. Classic ISCOMs are formed by combination of cholesterol, saponin, phospholipid, and immunogens, such as viral envelope proteins. Generally, irnrnunogens (usually with a hydrophobic region) are solubilized in detergent and added to the reaction mixture, whereby ISCOMs are formed with the immunogen incorporated therein. ISCOM matrix compositions are formed identically, but without viral proteins. Proteins with high positive charge may be electrostatically bound in the ISCOM particles, rather than through hydrophobic forces. For a more detailed general discussion of saponins and ISCOMs, and methods of formulating ISCOMs, see Barr et al. (1998) Adv. Ds°ug Delivery Reviews 32:247-271 (1998).
ISCOMs for use with the present invention are produced using standard techniques, well known in the art, and are described in e.g., U.S. Patent Nos.
4,981,684, 5,178,860, 5,679,354 and 6,027,732; European Publ. Nos. EPA
109,942;
180,564 and 231,039; Coulter et al. (1998) Yaccihe 16:1243. Typically, the term "ISCOM" refers to immunogenic complexes formed between glycosides, such as triterpenoid saponins (particularly Quil A), and antigens which contain a hydrophobic region. See, e.g., European Publ. Nos. EPA 109,942 and 180,564. In this embodiment, the HCV fusions (usually with a hydrophobic region) are solubilized in detergent and added to the reaction mixture, whereby ISCOMs are formed with the fusions incorporated therein. The HCV polypeptide ISCOMs are readily made with HCV polypeptides which show amphipathic properties. However, proteins and peptides which lack the desirable hydrophobic properties may be incorporated into the immunogenic complexes after coupling with peptides having hydrophobic amino acids, fatty acid radicals, alkyl radicals and the like.
As explained in European Publ. No. EPA 231,039, the presence of antigen is not necessary in order to form the basic ISCOM structure (referred to as a matrix or ISCOMATRIX), which may be formed from a sterol, such as cholesterol, a phospholipid, such as phosphatidylethanolamine, and a glycoside, such as Quil A.
Thus, the HCV fusion of interest, rather than being incorporated into the matrix, is present on the outside of the matrix, for example adsorbed to the matrix via electrostatic interactions. For example, HCV fusions with high positive charge may be electrostatically bound to the ISCOM particles, rather than through hydrophobic forces. For a more detailed general discussion of saponins and ISCOMs, and methods of formulating ISCOMs, see Barn et al. (1998) Adv. DYUg Delivery Reviews 32:247-271 (1998).

The ISCOM matrix may be prepared, for example, by mixing together solubilized sterol, glycoside and (optionally) phospholipid. If phospholipids are not used, two dimensional structures are formed. See, e.g., European Publ. No. EPA
231,039. The teen "ISCOM matrix" is used to refer to both the 3-dimensional and 2-dimensional structures. The glycosides to be used are generally glycosides which display amphipathic properties and comprise hydrophobic and hydrophilic regions in the molecule. Preferably saponins are used, such as the saponin extract from Quillaja saponaria Molina and Quil A. Other preferred saponins are aescine from Aesculus hippocastanum (Patt et al. (1960) Arzneinaittelforschurzg 10:273-275 and sapoalbin from Gypsophilla struthium (Vochten et al. (1968) J. Pharnz. Belg. 42:213-226.
In order to prepare the ISCOMs, glycosides are used in at least a critical micelle-forming concentration. In the case of Quil A, this concentration is about 0.03% by weight. The sterols used to produce ISCOMs may be known sterols of animal or vegetable origin, such as cholesterol, lanosterol, lumisterol, stigmasterol and sitosterol. Suitable phospholipids include phosphatidylcholine and phosphatidylethanolamine. Generally, the molar ratio of glycoside (especially when it is Quil A) to sterol (especially when it is cholesterol) to phospholipid is 1:1:0-1, +
20% (preferably not more than +10%) for each figure. This is equivalent to a weight ratio of about 5:1 for the Quil A:cholesterol.
A solubilizing agent may also be present and may be, for example a detergent, urea or guanidine. Generally, a non-ionic, ionic or zwitter-ionic detergent or a cholic acid based detergent, such as sodium desoxycholate, cholate and CTAB
(cetyltriammonium bromide), can be used for this purpose. Examples of suitable detergents include, but are not limited to, octylglucoside, nonyl N-methyl glucamide or decanoyl N-methyl glucamide, alkylphenyl polyoxyethylene ethers such as a polyethylene glycol p-isooctyl-phenylether having 9 to 10 oxyethylene groups (commercialized under the trade name TRITON X-100RTM), acylpolyoxyethylene esters such as acylpolyoxyethylene sorbitane esters (commercialized under the trade name TWEEN 20TM , TWEEN 80TM, and the like). The solubilizing agent is generally removed for formation of the ISCOMs, such as by ultrafiltration, dialysis, ultracentrifugation or chromatography, however, in certain methods, this step is unnecessary. (See, e.g., U.S. Patent No. 4,981,684).
Generally, the ratio of glycoside, such as QuilA, to HCV fusion by weight is in the range of 5:1 to 0.5:1. Preferably the ratio by weight is approximately 3:1 to 1:1, and more preferably the ratio is 2:1.
Once the ISCOMs are formed, they may be formulated into compositions arid administered to animals, as described herein. If desired, the solutions of the irnmunogenic complexes obtained may be lyophilized and then reconstituted before use.
Methods of Pf~oducing HCIT Specific Atatibodies The HCV fusion proteins can be used to produce HCV-specific polyclonal and monoclonal mtibodies. HCV-specific polyclonal and monoclonal antibodies specifically bind to HCV 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 chromatography, preferably affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against HCV-specific epitopes present in the fusion proteins can also be readily produced. Normal B cells from a mammal, such as a mouse, immunized with an HCV fusion protein, can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing HCV-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing HCV-specific antibodies are isolated by another round of screening.
Antibodies, either monoclonal and polyclonal, which are directed against HCV epitopes, are particularly useful for detecting the presence of HCV 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 linlcage so that the antibodies retain their immunoselective activity. Optionally, 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.
HCTV Specific T cells HCV-specific T cells that are activated by the above-described fusions, including the NS3*NS4NSSa fusion protein or NS3*NS4NSSaNSSb fusion protein, with or without a core polypeptide, as well as any of the other various fusions described herein, expressed i~z vivo or ifZ vitf°o, preferably recognize an epitope of an HCV polypeptide such as an NS2, p7, El, E2, NS3, NS4, NSSa or NSSb polypeptide, including an epitope of a fusion of one or more of these peptides with an NS3*, with or without a core polypeptide. HCV-specific T cells can be CD8+ or CD4+.
HCV-specific CD8+ T cells can be cytotoxic T lymphocytes (CTL) which can bill HCV-infected cells that display any of these epitopes complexed with an MHC
class I molecule. HCV-specific CD8+ T cells can be detected by, for example, SlCr release assays (see Example 4). SICr release assays measure the ability of HCV-specific CD8+ T cells to lyse target cells displaying one or more of these epitopes. HCV-specific CD8+ T cells which express antiviral agents, such as IFN-y, are also contemplated herein and can also be detected by immunological methods, preferably by intracellular staining for IFN-y or lilce cytolcine after in vitro stimulation with one or more of the HCV polypeptides, such as but not limited to an NS3, an S NS4, an NSSa, or an NSSb polypeptide (see Example 5).
HCV-specific CD4+ cells activated by the above-described fusions, such as but not limited to an NS3*NS4NSSa or NS3*NS4NSSaNSSb fusion protein, with or without a core polypeptide, expressed ifa vivo or ira vitro, preferably recognize an epitope of an HCV polypeptide, such as but not limited to an NS2, p7, E1, E2, NS3, NS4, NSSa, or NSSb polypeptide, including an epitope of fusions thereof, such as but not limited to an NS3NS4NSSa or NS3NS4NSSaNSSb fusion protein, that is bound to an MHC class II molecule on an HCV-infected cell and proliferate in response to stimulating, e.g., NS3*NS4NSSa or NS3*NS4NSSaNSSb peptides, with or without a core polypeptide.
HCV-specific CD4+ T cells can be detected by a lymphoproliferation assay (see Example 6). Lymphoproliferation assays measure the ability of HCV-specific CD4+ T cells to proliferate in response to, e.g., an NS2, p7, El, E2, NS3, an NS4, an NSSa, and/or an NSSb epitope.
Methods of Activatihg HChSpecific T Cells.
The HCV fusion proteins or polynucleotides can be used to activate HCV-specific T cells either ih vitf°o or in. vivo. Activation of HCV-specific T cells can be used, ifzte~ alia, to provide model systems to optimize CTL responses to HCV
and to provide prophylactic or therapeutic treatment against HCV infection.
For ih vitro activation, proteins are preferably supplied to T cells via a plasmid or a viral vector, such as an adenovirus 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 iy2 vitro by adding, an HCV fusion protein as described herein, such as but not limited to HCV NS3NS4NSSa or NS3NS4NSSaNSSb epitopic peptides, with or without a core polypeptide, to the T
cells. The HCV-specific T cells can then be tested for, irttef- alia, proliferation, the production of IFN-y, and the ability to lyse target cells displaying, for example, NS3NS4NSSa or NS3NS4NSSaNSSb epitopes ih vitro.
In a lymphoproliferation assay (see Example 6), HCV-activated CD4+ T cells proliferate when cultured with an HCV polypeptide, such as but not limited to an NS3, NS4, NSSa, NSSb, NS3NS4NSSa, or NS3NS4NSSaNSSb epitopic peptide, but not in the absence of an epitopic peptide. Thus, particular HCV epitopes, such as NS2, p7, E1, E2, NS3, NS4, NSSa, NSSb, and fusions of these epitopes, such as but not limited to NS3NS4NSSa and NS3NS4NSSaNSSb epitopes that are recognized by HCV-specific CD4+ T cells can be identified using a lymphoproliferation assay.
Similarly, detection of IFN-y in HCV-specific CD4+ and/or CD8+ T cells after i~a vitf°o stimulation with the above-described fusion proteins, can be used to identify, for example, fusion protein epitopes, such as but not limited to NS2, p7, E1, E2, NS3, NS4, NSSa, NSSb, and fusions of these epitopes, such as but not limited to NS3NS4NSSa, and NS3NS4NSSaNSSb epitopes that are particularly effective at stimulating CD4+ andlor CD8+ T cells to produce IFN-y (see Example 5).
Further, SlCr release assays are useful for determining the level of CTL
response to HCV. See Cooper et al. limnunity 10:439-449. For example, HCV-specific CD8+ T cells can be derived from the liver of an HCV infected mammal. These T cells can be tested in 5lCr release assays against target cells displaying, e.g., NS3NS4NSSa NS3NS4NSSaNSSb epitopes. Several target cell populations expressing different NS3NS4NSSa or NS3NS4NSSaNSSb epitopes can be constructed so that each target cell population displays different epitopes of NS3NS4NSSa or NS3NS4NSSaNSSb. The HCV-specific CD8+ cells cm 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 NS3NS4NSSa or NS3NS4NSSaNSSb are responsible for the strongest CTL response to HCV. NS3*NS4NSSa fusion proteins or NS3*NS4NSSaNSSb 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 5lCr release assays.
An HCV fusion protein as described above, or polynucleotide encoding such a fusion protein, can be administered to a marmnal, such as a mouse, baboon, chimpanzee, or human, to activate HCV-specific T cells ifa 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.
Preferably, injection of an HCV polynucleotide is used to activate T cells. In addition to the practical advantages of simplicity of construction and modification, injection of the polynucleotides results in the synthesis of a fusion protein in the host.
Thus, 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 mglkg.
A composition of the invention comprising an HCV fusion protein or polynucleotide is administered in a manner compatible with the particular composition used and in an amount which is effective to activate HCV-specific T
cells as measured by, inter alia, a SlCr release assay, a lymphoproliferation assay, or by intracellular staining for IFN-y. 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. The particular dosages of the polynucleotides or fusion proteins in a composition will depend on many factors including, but not limited to the species, age, and general condition of the mammal to which the composition is administered, and the mode of administration of the composition. An effective amount of the composition of the invention can be readily determined using only routine experimentation. In vitro and in vivo models described above can be employed to identify appropriate doses. The amount of polynucleotide used in the example described below provides general guidance which can be used to optimize the activation of HCV-specific T cells either in vivo or ih vitf°o. Generally, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg of an HCV fusion protein or polynucleotide, with or without a core polypeptide, will be administered to a large mammal, such as a baboon, chimpanzee, or human. If desired, co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions.
Immune responses of the mammal generated by the delivery of a composition of the invention, including activation of HCV-specific T cells, can be enhanced by varying the dosage, route of administration, or boosting regimens.
Compositions of the invention may 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 reinforce an immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose or doses after several months.
III. Experimental Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Those of slcill in the art will readily appreciate that the invention may be practiced in a variety of ways given the teaching of this disclosure.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Production of NS3*NS4NSSaCore Polynucleotides NS3~= in the following examples represents a modified NS3 molecule. A
polynucleotide encoding NS3NS4NSSa (approximately amino acids 1027 to 2399, numbered relative to HCV-1) (also termed "NS345a" herein) is isolated from an HCV. The NS3 portion of the molecule is mutagenzied by mutating the coding sequence for the His, Asp and Ser residues found at the protease active site, such that the resulting molecule codes for amino acids other than His, Asp and Ser at these positions and lacks NS3 protease activity. This construct is fused with a polynucleotide encoding a core polypeptide wluch includes amino acids 1-122 of the full-length polyprotein The core-encoding polynucleotide sequence is fused downstream from the NSSa-encoding portion of the construct such that the resulting fusion protein includes the core polypeptide at its C-terminus. The construct is cloned into plasmid, vaccinia virus, and adenovirus vectors. Additionally, the construct is inserted into a recombinant expression vector and used to transform host cells to produce the NS3*NS4NSSaCore fusion protein.
Protease enzyme activity is determined as follows. An NS4A peptide (KKGSVVIVGRIVLSGKPAIIPKK) (SEQ ID N0:8), and the fusion protein are diluted in 90 ~,1 of reaction buffer (25 mM Tris, pH 7.5, O.15M NaCI, 0.5 mM
EDTA, 10% glycerol, 0.05 n-Dodecyl B-D-Maltoside, 5 mM DTT) and allowed to mix for minutes at room temperature. 90 wl of the mixture is added to a microtiter plate (Costar, Inc., Corning, NY) and 10 ~,1 of HCV substrate (AnaSpec, Inc., San Jose CA) is added. The plate is mixed and read on a Fluostar plate reader. Results are expressed as relative fluorescence units (RFC per minute.

Priming of HCV-specific CTLs in Vaccinated Animals The HCV fusion protein, NS3*NS4NSSaCore, produced as described above is used to produce an HCV fusion-ISCOM as follows. The fusion-ISCOM formulations are prepared by mixing the fusion protein with a preformed ISCOMATRIX (empty ISCOMs) utilizing ionic interactions to maximize association between the antigen and the adjuvant. ISCOMATRIX is prepared essentially as described in Coulter et al.
(1998) Iracci~ze 16:1243.
Rhesus macaques are immunized under anesthesia. Animals are divided into two groups. The first group is infected with 2 x 108 plaque forming units (pfu) (1 x 108 intradermally and 1 x 108 by scarification) of rVVC/E1 at month 0. This group serves as a positive control for CTL priming. Animals from the second group are immunized with 25-100 ~,g of an HCV fusion polypeptide, as described above, that has been adsorbed to an ISCOM, by intramuscular (IM) injection in the left quadriceps at months 0, 1, 2 and 6. Cytotoxic activity is assayed in a standard 5lCr release assay as described in, e.g., Paliard et al. (2000) AIDS Res. Hufn.
Retroviruses 16:273.

Immunization With NS3*NS4NSSaCore Polynucleotides In one immunization protocol, animals are immunized with 50-250 p,g of plasmid DNA encoding an NS3*NS4NSSaCore fusion protein by intramuscular injection into the tibialis anterior. A booster injection of 107 pfu of vaccinia virus (VV)-NSSa (intraperitoneal) or 50-250 ~,g of plasmid control (intramuscular) is provided 6 weeps later.
In another immunization protocol, animals are inj ected intramuscularly in the tibialis anterior with 1010 adenovirus particles encoding an NS3*NS4NSSaCore fusion protein. An intraperitoneal booster injection of 107 pfu of VV-NSSa or an intramuscular booster injection of 1010 adenovirus particles encoding NS3*NS4NSSaCore is provided 6 weeles later.

Activation of HCV-Specific CD8+ T Cells SI Cr Release Assay. A 51 Cr release assay is used to measure the ability of HCV-specific T cells to lyse target cells displaying an NSSa epitope. Spleen cells are pooled from the immunized animals. These cells are restimulated i~z vitro for 6 days with the CTL epitopic peptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO:l) from HCV-NSSa in the presence of IL-2. The spleen cells are 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 are tested. Percent specific lysis is calculated for each effector to target ratio.

Activation of HCV-Specific CD8+ T Cells Which Express IFN-y hZtracellular StairaifZg fog Irate~fef°of2-gamma (IFN y). Intracellular staining for 1FN-y is used to identify the CD8+ T cells that secrete IFN-y after i~2 vitro stimulation with the NSSa epitope p214K9. Spleen cells of individual immunized animals are restimulated in vitYO either with p214K9 or with a non-specific peptide for 6-12 hours in the presence of IL-2 and monensin. The cells are then stained for surface CD8 and for intracellular IFN-y and analyzed by flow cytometry. The percent of CD8+ T
cells which are also positive for IFN-y is then calculated.

Proliferation of HCV-Specific CD4+ T Cells Lyf~zphopYOlife~atiofz assay. Spleen cells from pooled immunized animals are depleted of CD8+ T cells using magnetic beads and are cultured in triplicate with either p222D, an NSSa-epitopic peptide from HCV-NSSa (2224-AELIEANLLWRQEMG-2238; SEQ ID N0:2), or in medium alone. After 72 hours, cells are pulsed with 1 ~, Ci per well of 3H-thymidine and harvested 6-8 hours later. Incorporation of radioactivity is measured after harvesting. The mean cpm is calculated.

Ability of NS3*45aCore-Encoding DNA Vaccine Formulations to prime CTLs Animals are immunized with either 10-250 ~.g of plasmid DNA encoding NS3*45aCore fusion protein as described in Example 3, with PLG-linl~ed DNA
encoding NS3*45aCore (see below), or with DNA encoding NS3*45aCore, delivered via electroporation (see, e.g., W ternational Publication No. WO/0045823 for this delivery technique). The immunizations are followed by a booster injection 6 weeps later of plasmid DNA encoding NS3*45aCore.

PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers are obtained from Boehringer Ingelheim, U.S.A. The PLG polymer is RG505, which has a copolymer ratio of 50/50 and a molecular weight of 65 l~Da (manufacturers data).
Cationic microparticles with adsorbed DNA are prepared using a modified solvent evaporation process, essentially as described in Singh et al., Proc. Natl.
Acad. Sci.
USA (2000) 97:811-816. Briefly, the microparticles are prepared by emulsifying ml of a 5% w/v polymer solution in methylene chloride with 1 ml of PBS at high speed using an II~A homogenizer. The primary emulsion is then added to SOmI of distilled water containing cetyl trimethyl ammonium bromide (CTAB) (0.5% w/v).
This results in the formation of a w/o/w emulsion which is stirred at 6000 rpm for 12 hours at room temperature, allowing the methylene chloride to evaporate. The resulting microparticles are washed twice in distilled water by centrifugation at 10,000 g and freeze dried. Following preparation, washing and collection, DNA
is 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 are then separated by centrifugation, the pellet washed with TE buffer and the microparticles are freeze dried.
CTL activity and IFN-y expression is measured by 5lCr release assay or intracellular staining as described in the examples above.
EXAMPLE ~
Immunization Routes and Replicon particles SINCR (DC+) Encoding for NS3*45aCore Alphavirus replicon particles, for example, SINCR (DC+) are prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603.
Animals are injected with 5 x 106 ILJ SINCR (DC+) replicon particles encoding for NS3*45aCore intramuscularly (IM) as described in Example 3, 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 are followed by a booster injection of vaccinia virus encoding NSSa as described in Example 3. IFN-y expression is measured by intracellular staining as described in Example 5.

Alphavirus Replicon Priming, Followed by Various Boosting Regimes Alphavirus replicon particles, for example, SINCR (DC+) are prepared as described in Polo et al., Py°oc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Animals are primed with SINCR (DC+), 1.5 x 106 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 NSSa, 1010 adenovirus particles encoding NS3*45aCore, 1.5 x 106 IU SINCR (DC+) replicon particles encoding NS3*45aCore, or l0y pfu vaccinia virus encoding NSSa at 6 weeps. IFN-y expression is measured by intracellular staining as described in Example 5.

Alphaviruses Expressing NS3*45aCore Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP) are prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603. Animals are immunized with 1 x 102 to 1 x 106 ICT SINCR (DC+) replicons encoding NS3*45aCore via a combination of delivery routes (2/3 IM
and 1/3 S/C) as well as by S/C alone, or with 1 x 102 to 1 x 106 ICT S1NCR (LP) replicon particles encoding NS3~45aCore via a combination of delivery routes (2/3 IM
and 1/3 S/C) as well as by S/C alone. The immunizations are followed by a booster injection of 10~ pfu vaccinia virus encoding NSSa at 6 weelcs. IFN-y expression is measured by intracellular staining as described in Example 5.
Thus, HCV fusion polypeptides, to stimulate cell-mediated immune responses, are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined herein.

SEQUENCE LISTING
<110> Chiron Corporation <120> HCV FUSION PROTEINS WITH MODIFIED NS3 DOMAINS
<130> PP19545.004 (2300-19545.40) <150> 60/394,510 <151> 2002-07-08 <150> 60/393,694 <l5l> 2002-07-02 <150> 09/721,479 <151> 2000-11-22 <150> 60/167,502 <151> 1999-11-24 <160> 8 <170> PatentIn version 3.2 <210> 1 <211> 9 <212> PRT
<213> Artificial <220>
<223> epitope recognized by a Tcell receptor <400> 1 His Glu Tyr Pro Val Gly Ser G1n Leu <210> 2 <211> 15 <212> PRT
<213> Artificial <220>
<223> epitope recognized by a Tcell receptor <400> 2 Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly l 5 10 15 <210> 3 <211> 546 <212> DNA
<213> Artificial <220>
<223> DNA sequence of a representative native, unmodified NS3 protease domain <400> 3 atggcg cccatcacg gcgtacgcc cagcagaca aggggcctc ctaggg 48 MetAla ProIleThr AlaTyrAla GlnGlnThr ArgGlyLeu LeuGly tgcata atcaccagc ctaactggc cgggacaaa aaccaagtg gagggt 96 CysIle TleThrSer LeuThrGly ArgAspLys AsnGlnVal GluGly gaggtc cagattgtg tcaactget gcccaaacc ttcctggca acgtgc 144 GluVal GlnIleVal SerThrAla AlaGlnThr PheLeuAla ThrCys atcaat ggggtgtgc tggactgtc taccacggg gccggaacg aggacc 192 IleAsn GlyValCys TrpThrVal TyrHisGly AlaGlyThr ArgThr atcgcg tcacccaag ggtcctgtc atccagatg tataccaat gtagac 240 IleAla SerProLys GlyProVal IleGlnMet TyrThrAsn ValAsp caagac cttgtgggc tggcccget ccgcaaggt agccgatca ttgaca 288 GlnAsp LeuValG1y TrpProAla ProGlnG1y SerArgSer LeuThr ccctgc acttgcggc tcctcggac ctttacctg gtcacgagg cacgcc 336 ProCys ThrCysGly SerSerAsp LeuTyrLeu Va1ThrArg HisAla gatgtc attcccgtg cgccggcgg ggtgatagc aggggcagc ctgctg 384 AspVal I1eProVal ArgArgArg G1yAspSer ArgGlySer LeuLeu tcgccc cggcccatt tcctacttg aaaggctcc tcggggggt ccgctg 432 SerPro ArgProIle SerTyrLeu LysGlySer SerGlyGly ProLeu ttgtgc cccgcgggg cacgccgtg ggcatattt agggccgcg gtgtgc 480 LeuCys ProAlaGly HisAlaVal GlyIlePhe ArgAlaAla ValCys acccgt ggagtgget aaggcggtg gactttatc cctgtggag aaccta 528 ThrArg GlyValAla LysAlaVal AspPheTle ProValGlu AsnLeu gagaca accatgagg tcc 546 GluThr ThrMetArg Ser <210> 4 <211> 182 <212> PRT
<213> Artificial <220>
<223> amino acid sequence of a representative native unmodified NS3 protease domain <400> 4 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn G1y Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro G1n Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala 100 105 1l0 Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly G1y Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Tle Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe I1e Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser <210> 5 <211> 5676 <212> DNA
<213> Artificial <220>
G223> DNA sequence of a representative modified fusion protein, with the NS3 protease domain deleted from the N-terminus and including amino acids l-l21 of Core on the C-terminus <400> 5 atg get gca tat gca get cag ggc tat aag gtg cta gta ctc aac ccc 48 Met Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro tct gtt get gca aca ctg ggc ttt ggt get tac atg tcc aag get cat 96 Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His ggg atc gat cct aac atc agg acc ggg gtg aga aca att acc act ggc 144 Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly agc ccc atc acg tac tcc acc tac ggc aag ttc ctt gcc gac ggc ggg 192 Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly tgc tcg ggg ggc get tat gac ata ata att tgt gac gag tgc cac tcc 240 Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser acg gat gcc aca tcc atc ttg ggc att ggc act gtc ctt gac caa gca 288 Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala gag act gcg ggg gcg aga ctg gtt gtg ctc gcc acc gcc acc cct ccg 336 Glu Thr A1a Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro ggc tcc gtc act gtg ccc cat ccc aac atc gag gag gtt get ctg tcc 384 Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser 115 7,20 125 acc acc gga gag atc cct ttt tac ggc aag get atc ccc ctc gaa gta 432 Thr Thr Gly G1u Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val atc aag ggg ggg aga cat ctc atc ttc tgt cat tca aag aag aag tgc 480 Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys gac gaa ctc gcc gca aag ctg gtc gca ttg ggc atc aat gcc gtg gcc 528 Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala tac tac cgc ggt ctt gac gtg tcc gtc atc ccg acc agc ggc gat gtt 576 Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val gtc gtc gtg gca acc gat gcc ctc atg acc ggc tat acc ggc gac ttc 624 Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe gac tcg gtg ata gac tgc aat acg tgt gtc acc cag aca gtc gat ttc 672 Asp Ser Va1 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe agc ctt gac cct acc ttc acc att gag aca atc acg ctc ccc caa gat 720 Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp get gtc tcc cgc act caa cgt cgg ggc agg act ggc agg ggg aag cca 768 Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg G1y Lys Pro ggc atc tac aga ttt gtg gca ccg ggg gag cgc ccc tcc ggc atg ttc 816 Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe gac tcg tcc gtc ctc tgt gag tgc tat gac gca ggc tgt get tgg tat 864 Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr 275 ~ 280 285 gag ctc acg ccc gcc gag act aca gtt agg cta cga gcg tac atg aac 912 Glu Leu Thr Pro Ala Glu Thr Thr Va1 Arg Leu Arg Ala Tyr Met Asn acc ccg ggg ctt ccc gtg tgc cag gac cat ctt gaa ttt tgg gag ggc 960 Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly gtc ttt aca ggc ctc act cat ata gat gcc cac ttt cta tcc cag aca 1008 Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr aag cag agt ggg gag aac ctt cct tac ctg gta gcg tac caa gcc acc 1056 Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Va1 Ala Tyr Gln Ala Thr-gtg tgc get agg get caa gcc cct ccc cca tcg tgg gac cag atg tgg 1104 Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp aag tgt ttg att cgc ctc aag ccc acc ctc cat ggg cca aca ccc ctg 1152 Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu cta tac aga ctg ggc get gtt cag aat gaa atc acc ctg acg cac cca 1200 Leu Tyr Arg Leu Gly Ala Val G1n Asn Glu Ile Thr Leu Thr His Pro gtc acc aaa tac atc atg aca tgc atg tcg gcc gac ctg gag gtc gtc 1248 Vai Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val acg agc acc tgg gtg ctc gtt ggc ggc gtc ctg get get ttg gcc gcg 1296 Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu A1a Ala tat tgc ctg tca aca ggc tgc gtg gtc ata gtg ggc agg gtc gtc ttg 1344 Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu tcc ggg aag ccg gca atc ata cct gac agg gaa gtc ctc tac cga gag 1392 Ser G1y Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu ttc gat gag atg gaa gag tgc tct cag cac tta ccg tac atc gag caa 1440 Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln ggg atg atg ctc gcc gag cag ttc aag cag aag gcc ctc ggc ctc ctg 1488 Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu cag acc gcg tcc cgt cag gca gag gtt atc gcc cct get gtc cag acc 1536 Gln Thr Ala Ser Arg Gln Ala Glu Va1 Ile Ala Pro Ala Val Gln Thr aac tgg caa aaa ctc gag acc ttc tgg gcg aag cat atg tgg aac ttc 1584 Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe atcagt gggatacaa tacttg gcgggcttgtca acgctgcct ggtaac 1632 I1eSer GlyIleGln TyrLeu AlaGlyLeuSer ThrLeuPro G1yAsn cccgcc attgettca ttgatg gettttacaget getgtcacc agccca 1680 ProAla IleAlaSer LeuMet AlaPheThrAla AlaValThr SerPro ctaacc actagccaa accctc ctcttcaacata ttggggggg tgggtg 1728 LeuThr ThrSerGln ThrLeu LeuPheAsnIle LeuGlyGly TrpVal getgcc cagctcgcc gccccc ggtgccgetact gcctttgtg ggcget 1776 AlaAla GlnLeuAla AlaPro GlyAlaAlaThr AlaPheVal GlyAla ggctta getggcgcc gccatc ggcagtgttgga ctggggaag gtcctc 1824 GlyLeu AlaGlyAla AlaIle GlySerValGly LeuGlyLys ValLeu atagac atccttgca gggtat ggcgcgggcgtg gcgggaget cttgtg 1872 IleAsp IleLeuAla GlyTyr GlyAlaGlyVal AlaGlyAla LeuVa1 gcattc aagatcatg agcggt gaggtcccctcc acggaggac ctggtc 1920 AlaPhe LysTleMet SerGly GluValProSer ThrGluAsp LeuVal aatcta ctgcccgcc atcctc tcgcccggagcc ctcgtagtc ggcgtg 1968 AsnLeu LeuProAla IleLeu SerProGlyAla LeuValVal GlyVal gtctgt gcagcaata ctgcgc cggcacgttggc ccgggcgag ggggca 2016 ValCys AlaAlaIle LeuArg ArgHisVa1Gly ProGlyGlu GlyA1a gtgcag tggatgaac cggctg atagccttcgcc tcccggggg aaccat 2064 ValGln TrpMetAsn ArgLeu IleA1aPheA1a SerArgGly AsnHis gtttcc cccacgcac tacgtg ccggagagcgat gcagetgcc cgcgtc 2112 Va1Ser ProThrHis TyrVal ProGluSerAsp AlaAlaAla ArgVal actgcc atactcagc agcctc actgtaacccag ctcctgagg cgactg 2160 ThrA1a IleLeuSer SerLeu ThrValThrGln LeuLeuArg ArgLeu caccag tggataagc tcggag tgtaccactcca tgctccggt tcctgg 2208 HisGln TrpIleSer SerGlu CysThrThrPro CysSerGly SerTrp ctaagg gacatctgg gactgg atatgcgaggtg ttgagcgac tttaag 2256 LeuArg AspIleTrp AspTrp IleCysGluVal LeuSerAsp PheLys acctgg ctaaaaget aagctc atgccacagctg cctgggatc cccttt 2304 ThrTrp LeuLysAla LysLeu MetProGlnLeu ProGlyIle ProPhe gtgtcc tgccagcgc gggtat aagggggtctgg cgaggggac ggcatc 2352 Val Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile atg cac act cgc tgc cac tgt gga get gag atc act gga cat gtc aaa 2400 Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys aac ggg acg atg agg atc gtc ggt cct agg acc tgc agg aac atg tgg 2448 Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp agt ggg acc ttc ccc att aat gcc tac acc acg ggc ccc tgt acc ccc 2496 Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro ctt cct gcg ccg aac tac acg ttc gcg cta tgg agg gtg tct gca gag 2544 Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser A1a Glu gaa tac gtg gag ata agg cag gtg ggg gac ttc cac tac gtg acg ggt 2592 Glu Tyr Val Glu Ile Arg Gln Val Gly Asp Phe His Tyr Val Thr Gly 850 ~ 855 860 atg act act gac aat ctt aaa tgc ccg tgc cag gtc cca tcg ccc gaa 2640 Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser Pro Glu ttt ttc aca gaa ttg gac ggg gtg cgc cta cat agg ttt gcg ccc ccc 2688 Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro tgc aag ccc ttg ctg cgg gag gag gta tca ttc aga gta gga ctc cac 2736 Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His gaa tac ccg gta ggg tcg caa tta cct tgc gag ccc gaa ccg gac gtg 2784 Glu Tyr Pro Va1 Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val gcc gtg ttg acg tcc atg ctc act gat ccc tcc cat ata aca gca gag 2832 Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Tle Thr Ala Glu gcg gcc ggg cga agg ttg gcg agg gga tca ccc ccc tct gtg gcc agc 2880 Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser tcc tcg get agc cag cta tcc get cca tct ctc aag gca act tgc acc 2928 Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr get aac cat gac tcc cct gat get gag ctc ata gag gcc aac ctc cta 2976 Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu tgg agg cag gag atg ggc ggc aac atc acc agg gtt gag tca gaa aac 3024 Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn aaa gtg gtg att ctg gac tcc ttc gat ccg ctt gtg gcg gag gag gac 3072 Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp gagcgggag atctccgtaccc gcagaa atcctgcgg aagtctcgg aga 3120 GluArgGlu IleSerValPro AlaGlu IleLeuArg LysSerArg Arg ttcgcccag gccctgcccgtt tgggcg cggccggac tataacccc ccg 3168 PheAlaGln AlaLeuProVal TrpAla ArgProAsp TyrAsnPro Pro cta'gtggag acgtggaaaaag cccgac tacgaacca cctgtggtc cat 3216 LeuValGlu ThrTrpLysLys ProAsp TyrGluPro ProValVal His ggctgcccg cttccacctcca aagtcc cctcctgtg cctccgcct cgg 3264 GlyCysPro LeuProProPro LysSer ProProVal ProProPro Arg aagaagcgg acggtggtcctc actgaa tcaacccta tctactgcc ttg 3312 LysLysArg ThrValValLeu ThrGlu SerThrLeu SerThrAla Leu gccgagctc gccaccagaagc tttggc agctcctca acttccggc att 3360 AlaG1uLeu AlaThrArgSer PheGly SerSerSer ThrSerGly Ile acgggcgac aatacgacaaca tcctct gagcccgcc ccttctggc tgc 3408 ThrGlyAsp AsnThrThrThr SerSer GluProAla ProSerGly Cys ccccccgac tccgacgetgag tcctat tcctccatg ccccccctg gag 3456 ProProAsp SerAspAlaGlu SerTyr SerSerMet ProProLeu Glu ggggagcct ggggatccggat cttagc gacgggtca tggtcaacg gtc 3504 GlyGluPro GlyAspProAsp LeuSer AspGlySer TrpSerThr Val agt agtgag gccaacgcg gaggat gtcgtgtgctgc tcaatgtcttac 3552 Ser SerGlu AlaAsnAla GluAsp Va1ValCysCys SerMetSerTyr tct tggaca ggcgcactc gtcacc ccgtgcgccgcg gaagaacagaaa 3600 Ser TrpThr GlyAlaLeu Va1Thr ProCysAlaAla G1uGluGlnLys ctg cccatc aatgcacta agcaac tcgttgctacgt caccacaatttg 3648 Leu ProIle AsnAlaLeu SerAsn SerLeuLeuArg HisHisAsnLeu gtg tattcc accacctca cgcagt gettgccaaagg cagaagaaagtc 3696 Val TyrSer ThrThrSer ArgSer AlaCysGlnArg GlnLysLysVal aca tttgac agactgcaa gttctg gacagccattac caggacgtactc 3744 Thr PheAsp ArgLeuGln ValLeu AspSerHisTyr GlnAspValLeu aag gaggtt aaagcagcg gcgtca aaagtgaagget aacttgctatcc 3792 Lys GluVal LysAlaAla AlaSer LysValLysAla AsnLeuLeuSer gta gag gaa get tgc agc ctg acg ccc cca cac tca gcc aaa tcc aag 3840 Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys ttt ggt tat ggg gca aaa gac gtc cgt tgc cat gcc aga aag gcc gta 3888 Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val acc cac atc aac tcc gtg tgg aaa gac ctt ctg gaa gac aat gta aca 3936 Thr His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr cca ata gac act acc atc atg get aag aac gag gtt ttc tgc gtt cag 3984 Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln cct gag aag ggg ggt cgt aag cca get cgt ctc atc gtg ttc ccc gat 4032 Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp ctg ggc gtg ogc gtg tgc gaa aag atg get ttg tac gac gtg gtt aca 4080 Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr aag ctc ccc ttg gcc gtg atg gga~agc tcc tac gga ttc caa tac tca 4128 Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser cca gga cag cgg gtt gaa ttc ctc gtg caa gcg tgg aag tcc aag aaa 4176 Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys acc cca atg ggg ttc tcg tat gat acc cgc tgc ttt gac tcc aca gtc 4224 Thr Pro Met G1y Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val act gag agc gac atc cgt acg gag gag gca atc tac caa tgt tgt gac 4272 Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp ctc gac ccc caa gcc cgc gtg gcc atc aag tco ctc acc gag agg ctt 4320 Leu Asp Pro Gln Ala Arg Val A1a Ile Lys Ser Leu Thr Glu Arg Leu 1425 1430 1435 ' 1440 tat gtt ggg ggc cct ctt acc aat tca agg ggg gag aac tgc ggc tat 4368 Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr cgc agg tgc cgc gcg agc ggc gta ctg aca act agc tgt ggt aac acc 4416 Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr ctc act tgc tac atc aag gcc cgg gca gcc tgt cga gcc gca ggg ctc 4464 Leu Thr Cys Tyr Ile Lys Ala Arg Ala Ala Cys Arg A1a Ala Gly Leu cag gac tgc acc atg ctc gtg tgt ggc gac gac tta gtc gtt atc tgt 4512 Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Va1 Val Ile Cys gaa agc gcg ggg gtc cag gag gac gcg gcg agc ctg aga gcc ttc acg 4560 Glu Ser Ala Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr gag get atg acc agg tac tcc gcc ccc cct ggg gac ccc cca caa cca 4608 Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro gaa tac gac ttg gag ctc ata aca tca tgc tcc tcc aac gtg tca gtc 4656 Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val gcc cac gac ggc get gga aag agg gtc tac tac ctc acc cgt gac cct 4704 Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro aca acc ccc ctc gcg aga get gcg tgg gag aca gca aga cac act cca 4752 Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro gtc aat tcc tgg cta ggc aac ata atc atg ttt gcc ccc aca ctg tgg 4800 Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp gcg agg atg ata ctg atg acc cat ttc ttt agc gtc ctt ata gcc agg 4848 Ala Arg Met I1e Leu Met Thr His Phe Phe Ser Val Leu Ile Ala Arg gac cag ctt gaa cag gcc ctc gat tgc gag atc tac ggg gcc tgc tac 4896 Asp Gln Leu Glu Gln A1a Leu Asp Cys Glu Ile Tyr Gly Ala Cys Tyr tcc ata gaa cca ctg gat cta cct cca atc att caa aga ctc cat ggc 4944 Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly ctc agc gca ttt tca ctc cac agt tac tct cca ggt gaa atc aat agg 4992 Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu I1e Asn Arg gtg gcc gca tgc ctc aga aaa ctt ggg gta ccg ccc ttg cga get tgg 5040 Val Ala Ala Cys Leu Arg Lys Leu Gly Va1 Pro Pro Leu Arg Ala Trp aga cac cgg gcc cgg agc gtc cgc get agg ctt ctg gcc aga gga ggc 5088 Arg His Arg A1a Arg Ser Val Arg A1a Arg Leu Leu Ala Arg Gly Gly agg get gcc ata tgt ggc aag tac ctc ttc aac tgg gca gta aga aca 5136 Arg Ala Ala I1e Cys G1y Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr aag ctc aaa ctc act cca ata gcg gcc get ggc cag ctg gac ttg tcc 5184 Lys Leu Lys Leu Thr Pro Ile Ala A1a Ala Gly G1n Leu Asp Leu Ser ggc tgg ttc acg get ggc tac agc ggg gga gac att tat cac agc gtg 5232 Gly Trp Phe Thr Ala Gly Tyr Ser G1y Gly Asp Ile Tyr His Ser Val tct cat gcc cgg ccc cgc tgg atc tgg ttt tgc cta ctc ctg ctt get 5280 1~

SerHis AlaArgProArg TrpIleTrp PheCys LeuLeuLeuLeu Ala gcaggg gtaggcatctac ctcctcccc aaccga atgagcacgaat cct 5328 AlaGly ValGlyIleTyr LeuLeuPro AsnArg MetSerThrAsn Pro aaacct caaagaaagacc aaacgtaac accaac cggcggccgcag gac 5376 LysPro GlnArgLysThr LysArgAsn ThrAsn ArgArgProGln Asp gtcaag ttcccgggtggc ggtcagatc gttggt ggagtttacttg ttg 5424 ValLys PheProGlyGly GlyGlnIle ValGly GlyValTyrLeu Leu ccgcgc aggggccctaga ttgggtgtg cgcgcg acgagaaagact tcc 5472 ProArg ArgGlyProArg LeuGlyVal ArgAla ThrArgLysThr Ser gagcgg tcgcaacctcga ggtagacgt cagcct atccccaagget cgt 5520 GluArg SerGlnProArg GlyArgArg GlnPro IleProLysAla Arg cggccc gagggcaggacc tgggetcag cccggg tacccttggccc ctc 5568 ArgPro GluGlyArgThr TrpAlaGln ProGly,TyrProTrpPro Leu tatggc aatgagggctgc gggtgggcg ggatgg ctcctgtctccc cgt 5616 TyrGly AsnGluG1yCys GlyTrpAla GlyTrp LeuLeuSerPro Arg ggctct cggcctagctgg ggccccaca gacccc cggcgtaggtcg cgc 5664 GlySer ArgProSerTrp G1yProThr AspPro ArgArgArgSer Arg aatttg ggtaag 5676 AsnLeu GlyLys <210>

<211> 892 <212>
PRT

<213>
Artificial <220>
<223> amino acid sequence of a representative modified fusion protein, with the NS3 protease domain deleted from the N-terminus and including amino acids 1-121 of Core on the C-terminus <400> 6 Met Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly 50 55 ~ 60 Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser 65 70 75 8p Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn I1e Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Tle Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys i45 150 155 160 Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro G1n Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Va1 Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala G1u Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro G1y Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu I1e Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val 405 4l0 415 Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg G1n Ala Glu Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gln Lys Leu G1u Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu A1a Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu Tle Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala I1e Leu Ser Pro G1y Ala Leu Va1 Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Tle Ala Phe Ala Ser Arg G1y Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe 755 76b 765 Val Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Tle Met His Thr Arg Cys His Cys Gly Ala G1u Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile Arg Gln Va1 Gly Asp Phe His Tyr Val Thr Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser Pro Glu 865 870 875 gg0 Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys A1a Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg G1n G1u Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser'Phe Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val G1u Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His G1y Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr Val Val Leu Thr G1u Ser Thr Leu Ser Thr Ala Leu A1a Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Va1 Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys Ala Ala Ala Ser Lys Va1 Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Thr His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro G1u Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr IS

Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe G1n Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Tle Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Va1 Ile Cys Glu Ser Ala G1y Val Gln Glu Asp Ala Ala Ser Leu Arg A1a Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Sex Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu A1a Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile, Ile Met Phe Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Va1 Leu I1e Ala Arg Asp Gln Leu Glu Gln A1a Leu Asp Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu A1a Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp Phe Thr A1a Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His Ala Arg Pro Arg Trp Tle Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val G1y Gly Val Tyr Leu Leu Pro Arg Ar,g Gly Pro Arg Leu Gly Va1 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro I1e Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala G1n Pro Gly Tyr Pro Trp Pro Leu T'yr Gly Asn Glu G1y Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys <210> 7 <211> 21 <212> PRT
<213> Artificial <220>
<223> E2 epitope consensus sequence <400> 7 Gly Ser Ala Ala Arg Thr Thr Ser Gly Phe Val Ser Leu Phe Ala Pro Gly Ala Lys Gln Asn <210> 8 <211> 23 <212> PRT
<213> Artificial <220>
<223> NS4A peptide <400> 8 Lys Lys Gly Ser Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Tle Pro Lys Lys 1$

Claims (24)

We claim:

1. An immunogenic fusion protein comprising (a) a modified NS3 polypeptide comprising at least one amino acid substitution to the HCV NS3 region, such that protease activity is inhibited, and (b) at least one polypeptide derived from a region of the HCV polyprotein other than the NS3 region.

2. The fusion protein of claim 1, wherein the modification comprises a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein.

3. The fusion protein of either of claims 1 or 2, wherein the protein comprises a modified NS3 polypeptide, an NS4 polypeptide, an NS5a polypeptide, and optionally a core polypeptide.

4. The fusion protein of claim 3, wherein the protein further comprises an NS5b polypeptide, and optionally a core polypeptide.

5. The fusion protein of claim 3, wherein the protein further comprises an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.

6. The fusion protein of claim 3, wherein the protein further comprises an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.

7. The fusion protein of claim 3, wherein the protein further comprises an E2 polypeptide, and optionally a core polypeptide.

8. The fusion protein of claim 3, wherein the protein further comprises an E1 polypeptide, an E2 polypeptide, and optionally a core polypeptide.

9. The fusion protein of claim 1, wherein the protein comprises an E2 polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.

10. The fusion protein of claim 1, wherein the protein comprises an E1 polypeptide, an E2 polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.

11. The fusion protein of any of claims 1-10, wherein the polypeptides of (a) and (b) are derived from the same HCV isolate.

12. The fusion protein of any of claims 1-10, wherein at least one of the polypeptides present in the fusion is derived from a different isolate that the modified NS3 polypeptide.

13. An immunogenic fusion protein consisting essentially of, in amino terminal to carboxy terminal direction:
(a) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NS5a polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide and an NS5b polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NS5a polypeptide;
(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, and an NS5a polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide and an NS5b polypeptide;
(f) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide and an NS5b polypeptide;
(g) an E2 polypeptide and a modified NS3 polypeptide comprising substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited;
(h) an E1 polypeptide, an E2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited; or (j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited.

14. An immunogenic fusion protein consisting essentially of, in amino terminal to carboxy terminal direction:

(a) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide, and a core polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide, an NS5b polypeptide and a core polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide and a core polypeptide;
(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide and a core polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide, an NS5b polypeptide and a core polypeptide;
(f) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, an NS4 polypeptide, an NS5a polypeptide, an NS5b polypeptide and a core polypeptide;
(g) an E2 polypeptide, a modified NS3 polypeptide comprising substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered tgcagctctc ccctgggcca ctaac relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide;
(h) an E1 polypeptide, an E2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide; or (j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited, and a core polypeptide.

15. A modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such that protease activity is inhibited when the modified NS3 polypeptide is present in an HCV fusion protein.

16. A composition comprising an immunogenic fusion protein according to any of claims 1-14 or a modified NS3 polypeptide according to claim 15, in combination with a pharmaceutically acceptable excipient.

17. A method of stimulating a cellular immune response in a vertebrate subject comprising administering a therapeutically effective amount of the composition of claim 16.

18. Use of a composition according to claim 15 in a method for stimulating a cellular immune response in a vertebrate subject.

19. Use of an immunogenic fusion protein according to any of claims 1-14 or a modified NS3 polypeptide according to claim 15, in the manufacture of a medicament for stimulating a cellular immune response in a vertebrate subject.

20. A method for producing a composition comprising combining the immunogenic fusion protein according to any of claims 1-14 or a modified NS3 polypeptide according to claim 15, with a pharmaceutically acceptable excipient.

21. A polynucleotide comprising a coding sequence encoding a fusion protein according to any of claims 1-14 or a modified NS3 polypeptide according to claim 15.

22. A recombinant vector comprising:
(a) the polynucleotide of claim 21; and (b) at least one control element operably linked to said polynucleotide, whereby said coding sequence can be transcribed and translated in a host cell.

23. A host cell comprising the recombinant vector of claim 22.

24. A method for producing an immunogenic fusion protein or a modified polypeptide, said method comprising culturing a population of host cells according to claim 23 wider conditions for producing said protein.